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Sharma, Anmol, Pawan Gupta, and Pranav Kumar Prabhakar. "Endogenous Repair System of Oxidative Damage of DNA." Current Chemical Biology 13, no. 2 (July 12, 2019): 110–19. http://dx.doi.org/10.2174/2212796813666190221152908.
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DNA is one of the most important biomolecules of living cells which carries genetic information from generation to generation. Many endogenous and exogenous agents may disrupt the structure of DNA. Change in the cellular genome can lead to errors in replication, transcription and in protein synthesis. DNA damage occurs naturally or result from a metabolic and hydrolytic process which release some very active chemical entities like free radicals, Reactive Oxygen Species (ROS), Reactive Nitrogen Intermediate (RNI), Reactive Carbonyl Species (RCS), lipid peroxidation products and alkylating agents. Superoxide radical, hydroxyl radical and hydrogen peroxide cause a significant threat to cellular integrity by damaging the DNA, lipids, proteins and other biomolecules. Oxidative stress may be explained as a disturbance in the number of free radicals and our system’s ability to neutralize these free radicals. Imbalances in the normal redox potential can also lead to toxic effects via the generation of peroxides. Oxidation of DNA bases leads to the base damage, nick in the strand and break in the strand either single or double strand. Oxidative stress can also cause modifications in normal mechanisms of cell signaling. DNA mutation can result in a number of genetic abnormalities such as cancer, heart failure, Alzheimer’s disease, and depression. Human body has special protection in the form of antioxidant molecules and enzymes against these free radicals. Generation of ROS and its neutralization must be regulated to protect cells and signalling biomolecules from the deleterious effect of oxidative stress with the involvement of antioxidant systems, enzymes, and specific proteins. DNA repair system is a complex system which helps in the identification, removal of the wrong nucleotide and repairs them and as a result, the cell will produce correct and functional protein and active enzyme.
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Motherby, Helma, Mary Kube, Nikolaus Friedrichs, Bahram Nadjari, Kristiane Knops, Andreas Donner, Betty Baschiera, Peter Dalquen, and Alfred Böcking. "Immunocytochemistry and DNA-Image Cytometry in Diagnostic Effusion Cytology I. Prevalence of Markers in Tumour Cell Positive and Negative Smears." Analytical Cellular Pathology 19, no. 1 (1999): 7–20. http://dx.doi.org/10.1155/1999/459158.
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To determine the prevalence of immunocytochemical positivities for a panel of antibodies in benign and malignant cells in effusions with known follow‐up in order to use these as diagnostic markers. Besides their ability to identify malignant epithelial cells their contribution to the differential diagnosis between carcinomatoses and mesotheliomas was investigated. 101 tumour cell positive and 53 negative effusions were stained with 12 different antibodies. Results were scored semiquantitatively per cell type. Furthermore, DNA‐image cytometry was performed. While prevalence of Ber‐EP4 positivity was 95.4% in metastatic carcinoma cells, it was 0% in those from mesotheliomas. No cell type reacted with this marker in benign effusions (0%). Ber‐EP4 correctly differentiated between metastatic carcinoma and mesothelioma in 98.0%. Prevalence of DNA‐aneuploidy was 95.4% in metastatic carcinomas, 57.1% in mesotheliomas and 0% in reactive effusions. Combining immunocytochemistry (Ber‐EP4 positivity) and DNA‐image cytometry (aneuploidy) results in a 100% detection of metastatic carcinomatoses and 57.1% of mesotheliomas. Both markers furthermore allowed a correct differentiation of these entities in 98%.
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Centio, Anders, Montserrat Estruch, Kristian Reckzeh, Kumar Sanjiv, Camilla Vittori, Sophia Engelhard, Ulrika Warpman Berglund, Thomas Helleday, and Kim Theilgaard-Mönch. "Inhibition of Oxidized Nucleotide Sanitation By TH1579 and Conventional Chemotherapy Cooperatively Enhance Oxidative DNA Damage and Survival in AML." Molecular Cancer Therapeutics 21, no. 5 (February 28, 2022): 703–14. http://dx.doi.org/10.1158/1535-7163.mct-21-0185.
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Abstract Currently, the majority of patients with acute myeloid leukemia (AML) still die of their disease due to primary resistance or relapse toward conventional reactive oxygen species (ROS)- and DNA damage–inducing chemotherapy regimens. Herein, we explored the therapeutic potential to enhance chemotherapy response in AML, by targeting the ROS scavenger enzyme MutT homolog 1 (MTH1, NUDT1), which protects cellular integrity through prevention of fatal chemotherapy-induced oxidative DNA damage. We demonstrate that MTH1 is a potential druggable target expressed by the majority of patients with AML and the inv(16)/KITD816Y AML mouse model mimicking the genetics of patients with AML exhibiting poor response to standard chemotherapy (i.e., anthracycline & cytarabine). Strikingly, combinatorial treatment of inv(16)/KITD816Y AML cells with the MTH1 inhibitor TH1579 and ROS- and DNA damage-inducing standard chemotherapy induced growth arrest and incorporated oxidized nucleotides into DNA leading to significantly increased DNA damage. Consistently, TH1579 and chemotherapy synergistically inhibited growth of clonogenic inv(16)/KITD816Y AML cells without substantially inhibiting normal clonogenic bone marrow cells. In addition, combinatorial treatment of inv(16)/KITD816Y AML mice with TH1579 and chemotherapy significantly reduced AML burden and prolonged survival compared with untreated or single treated mice. In conclusion, our study provides a rationale for future clinical studies combining standard AML chemotherapy with TH1579 to boost standard chemotherapy response in patients with AML. Moreover, other cancer entities treated with ROS- and DNA damage–inducing chemo- or radiotherapies might benefit therapeutically from complementary treatment with TH1579.
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Dhir, Hardika, Monica Choudhury, Ketki Patil, Candice Cheung, Adriana Bodlak, Danny Pardo, Asana Adams, Stefano Travaglino, Jose Araque Rojas, and S. Balakrishna Pai. "Interception of Signaling Circuits of Esophageal Adenocarcinoma Cells by Resveratrol Reveals Molecular and Immunomodulatory Signatures." Cancers 13, no. 22 (November 19, 2021): 5811. http://dx.doi.org/10.3390/cancers13225811.
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Deregulation of signaling pathways due to mutations sets the cell on a path to neoplasia. Therefore, recent reports of increased mutations observed in esophageal tissue reflects the enhanced risk of tumor formation. In fact, adenocarcinoma of the esophagus has been on the rise lately. Increase in mortality due to a paucity of efficacious drugs for this cancer prompted us to discover molecular signatures to combat this malady. To this end, we chose resveratrol—a polyphenol with anticancer property—and studied its impact on three esophageal adenocarcinoma cell lines (OE33, OE19 and FLO-1) by multilevel profiling. Here, we show the impact of resveratrol on the viability of the three adenocarcinoma esophageal cell systems studied, at the cellular level. Furthermore, an analysis at the molecular level revealed that the action was through the programmed cell death pathway, resulting in an increase in apoptotic and caspase-positive cells. The impact on reactive oxygen species (ROS) and a decrease in Bcl2 levels were also observed. Moreover, proteomic profiling highlighted pivotal differentially regulated signaling molecules. The phenotypic effect observed in resveratrol-treated esophageal cells could be due to the stoichiometry per se of the fold changes observed in entities of key signaling pathways. Notably, the downregulation of Ku80 and other pivotal entities by resveratrol could be harnessed for chemo-radiation therapy to prevent DNA break repair after radiation therapy. Additionally, multilevel profiling has shed light on molecular and immune-modulatory signatures with implications for discovering novel treatments, including chemo-immunotherapy, for esophageal adenocarcinomas which are known to be aggressive cancers.
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Senoner, Thomas, and Wolfgang Dichtl. "Oxidative Stress in Cardiovascular Diseases: Still a Therapeutic Target?" Nutrients 11, no. 9 (September 4, 2019): 2090. http://dx.doi.org/10.3390/nu11092090.
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Cardiovascular diseases (CVD) are complex entities with heterogenous pathophysiologic mechanisms and increased oxidative stress has been viewed as one of the potential common etiologies. A fine balance between the presence of reactive oxygen species (ROS) and antioxidants is essential for the proper normal functioning of the cell. A basal concentration of ROS is indispensable for the manifestation of cellular functions, whereas excessive levels of ROS cause damage to cellular macromolecules such as DNA, lipids and proteins, eventually leading to necrosis and apoptotic cell death. CVD is the main cause of death worldwide with several conditions being affected by oxidative stress. Increased ROS lead to decreased nitric oxide availability and vasoconstriction, promoting arterial hypertension. ROS also negatively influence myocardial calcium handling, causing arrhythmia, and augment cardiac remodeling by inducing hypertrophic signaling and apoptosis. Finally, ROS have also been shown to promote atherosclerotic plaque formation. This review aims at giving an introduction into oxidative stress in CVD, with special focus on endothelial dysfunction, and then examining in detail the role of oxidative stress in the most prevalent of these diseases. Finally, potential nutraceuticals and diets that might be beneficial in diminishing the burden of oxidative stress in CVD are presented.
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Deaton, C. "Effects of airway inflammation, ozone and exercise on the pulmonary antioxidant capacity of the horse: A war of nutrition." BSAP Occasional Publication 32 (2004): 129–31. http://dx.doi.org/10.1017/s0263967x00041318.
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Within the body there is continual production of entities known as Reactive Oxygen Species (ROS). These include radical derivatives of oxygen that contain at least one unpaired electron and include species such as the superoxide and hydroxyl radicals. ROS also include nonradical derivatives of oxygen that are capable of oxidising biomolecules such as hydrogen peroxide, ozone and nitrogen dioxide. ROS are formed from processes such as the respiratory burst of phagocytes and from mitochondrial oxidative phosphorylation, so production is often increased by situations that elevate oxygen utilisation such as exercise. ROS may also act as “signalling” species within the body. Controlled production of ROS is therefore essential for normal cellular function and health, especially with respect to the functioning of the immune system. However, uncontrolled production of ROS can result in cell damage and death, the induction and propagation of inflammation and DNA damage. Thus, the body has evolved intricate and elaborate enzymatic and non–enzymatic antioxidant defences to control and buffer excess ROS production. In situations where the antioxidant defences are overwhelmed either due to their depletion, malfunction or simply due to excessive ROS bombardment, oxidative stress and oxidative damage are likely to occur.
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Gautam, Nandini, Anil K. Mantha, and Sunil Mittal. "Essential Oils and Their Constituents as Anticancer Agents: A Mechanistic View." BioMed Research International 2014 (2014): 1–23. http://dx.doi.org/10.1155/2014/154106.
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Exploring natural plant products as an option to find new chemical entities as anticancer agents is one of the fastest growing areas of research. Recently, in the last decade, essential oils (EOs) have been under study for their use in cancer therapy and the present review is an attempt to collect and document the available studies indicating EOs and their constituents as anticancer agents. This review enlists nearly 130 studies of EOs from various plant species and their constituents that have been studied so far for their anticancer potential and these studies have been classified asin vitroandin vivostudies for EOs and their constituents. This review also highlights in-depth various mechanisms of action of different EOs and their constituents reported in the treatment strategies for different types of cancer. The current review indicates that EOs and their constituents act by multiple pathways and mechanisms involving apoptosis, cell cycle arrest, antimetastatic and antiangiogenic, increased levels of reactive oxygen and nitrogen species (ROS/RNS), DNA repair modulation, and others to demonstrate their antiproliferative activity in the cancer cell. The effect of EOs and their constituents on tumour suppressor proteins (p53 and Akt), transcription factors (NF-κB and AP-1), MAPK-pathway, and detoxification enzymes like SOD, catalase, glutathione peroxidase, and glutathione reductase has also been discussed.
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Münch, A., D. Teichmann, P. Kuzman, D. Spille, E. Perez, S. May, W. Mueller, et al. "P05.05.B A new IDH-wildtype glioma subtype characterized by highly diffuse growth pattern, distinct epigenetic profile and relatively favorable prognosis." Neuro-Oncology 24, Supplement_2 (September 1, 2022): ii37. http://dx.doi.org/10.1093/neuonc/noac174.124.
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Abstract Background DNA methylation profiling has emerges as a powerful approach to CNS tumor classification and the discovery of novel, molecularly distinct entities. With the release of the 12.5 version of the Heidelberg Brain Tumor Classifier, some unclassifiable cases can be assigned to novel methylation classes. We retrospectively reviewed our databases and identified 16 previously unclassifiable cases, all of which belong to the provisional methylation class “adult-type diffuse high-grade glioma, IDH-wildtype, subtype F (HGG_F)”. Material and Methods We clinically, radiologically and morphologically characterized 16 HGG_F cases and compared them to 347 glioblastomas. We additionally analyzed copy-number alterations and performed DNA exome sequencing. Results Median age at diagnosis of the 12 males and 4 females was 65 years. Upon initial diagnostic workup, specimens were classified as CNS tissue with reactive changes (n=3) or suspicious for the infiltration zone of a diffuse glioma (n = 13). None of the cases demonstrated endothelial proliferation or necrosis and 10/16 tumors had flat copy number profiles. Radiological characteristics were reminiscent of gliomatosis cerebri in eight cases and 9/9 cases had normal FET-PET scans. Whole-exome sequencing revealed genetic alterations frequently found in IDH-wildtype glioblastomas, including TERT promoter mutations in 11/14 (78.6%) and PIK3 mutations (10/14, 71.4%). Outcome was significantly better compared to TCGA IDH-wildtype glioblastomas with a median progression-free survival of 58 months and overall survival of 73 months (both p<0.001). Conclusion We provide evidence that TERT promoter mutations in diffusely infiltrating gliomas without further morphological or molecular signs of high-grade glioma should be interpreted in the context of the clinico-radiological presentation as well as epigenetic prolife and may not be suitable as standalone diagnostic marker for glioblastoma, IDH wildtype.
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Kattoor, Jayasree, and Meherbano M. Kamal. "The gray zone squamous lesions: ASC-US / ASC-H." Cytojournal 19 (April 30, 2022): 30. http://dx.doi.org/10.25259/cmas_03_10_2021.
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The unequivocal and easily recognizable entities of LSIL and HSIL pose no diagnostic problems for a trained eye. However, when the defining morphologic features are either qualitatively or quantitatively insufficient, it is then that the borderline category of “Atypical Squamous cells” (ASC) may have to be used. Scant and suboptimal preparations (mainly in conventional smears) are the common causes that hinder confident decision-making. The binary classification of the ASC category has been retained in The Bethesda System 2014. It includes ASC of undetermined significance (ASC-US) when the atypia is seen in mature cells and ASC-cannot rule out high-grade lesion (ASC-H) when borderline changes are seen in less mature, smaller metaplastic cells or smaller basaloid cells. There are many criticisms of the ASC category. The major one is its subjective and inconsistent applications and the low interobserver and intraobserver reproducibility. However, studies have shown that if we eliminate ASC-US, the LSIL rate will increase. If ASC-H is eliminated, the chances of detecting true lesions are reduced. Hence, there are strong reasons to retain the ASC category. The usual problems leading to the categorization of such cells as atypical are hyperchromasia beyond that acceptable as reactive change; abnormal chromatin pattern that is not overt dyskaryosis; minor variations in nuclear shape; and membrane outlines. Qualifying the atypical cells precisely in one of the categories has bearing on the clinical management and follow-up of the patient. Surveillance of women under the ASC-US category is either by repeat smear at 6 months and 1 year or by reflex human papillomaviruses DNA testing. Women with a Pap smear interpretation of ASC-H are directed to undergo immediate colposcopy. This article describes in detail the morphologic features of the ASC category, doubts about the correct interpretation of the chromatin pattern of the cells in question, and the differential diagnosis between normal, reactive, or inflammatory conditions, and LSIL/HSIL.
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Kanakis, C. E., K. Gvozdjan, L. Sereseanu, D. Rodheim, and P. J. DeChristopher. "Fatal, Rapidly Progressive EBV-Associated Warm Autoantibody Hemolytic Anemia (WAIHA): A Rare Case Study." American Journal of Clinical Pathology 156, Supplement_1 (October 1, 2021): S158. http://dx.doi.org/10.1093/ajcp/aqab191.337.
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Abstract Introduction/Objective Separately WAIHA and Epstein-Barr Virus-infectious mononucleosis (EBV-IM) are well- studied clinical entities. EBV-IM cases are commonly linked with cold autoagglutinins (IgMs with anti-i specificity), but uncommonly associated with WAIHA. More rarely, EBV-IM can be associated with rapidly fatal AIHA. Prompt and appropriate diagnostic Blood Bank testing for WAIHA in EBV-infected patients should include work-ups to prevent missing the possibility of treating complicating WAIHA. Methods/Case Report A 24-year-old woman presented to the ED with a month of cough/cold symptoms, night sweats, cervical lymphadenopathy, and imaging showing diffuse thoracic lymphadenopathy. DNA/PCR testing was positive for active EBV infection and a lymph node biopsy was diagnostic for EBV lymphadenitis. Increasing respiratory distress developed, coupled with renal and liver insufficiency, hypotension, and possible DIC, and (suspected septic) shock. On hospital day (HD) 5, altered mental status worsened, cardiac arrest occurred requiring resuscitation and vasopressors, and acidosis trended rapidly higher. The patient rapidly decompensated and she was compassionately extubated, expiring on HD 6. There were significant laboratory interval changes in the final 20 hours of life. New positive DATs (with anti-IgG and - C3b/d), a pan-reactive warm autoantibody of broad specificity (in plasma & eluate), onset of acute hemolysis (nadir Hgb to 4.6 g/dL with net drop of 4.7 g/dL & LDH 23, 582 U/L), coagulopathy (peak PT 96.6 sec) and evidnece of organ failure. Blood Bank testing, received on HD 5 at 21:45, was resulted in the EMR on HD 6 at 00:30 and the patient expired at 05:15--further illustrating the rapid clinical course presented in this case. Results (if a Case Study enter NA) N/A Conclusion This patient died of complications directly associated with acute, immune-mediated intravascular hemolysis leading to high-output cardiac failure and resulting in multi-organ failure. With a relative paucity of published EBV-IM WAIHA associations, the rapidly evolving unexpected hematologic sequelae observed complicated the clinical recognition and treatment of severe WAIHA during the course of treatment for clinically suspected DIC. The rapid progression of this patients’ symptoms and laboratory values did not allow for adequate recognition of WAIHA-related pathogenesis and appropriate interventions.
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Thurner, Lorenz, Sylvia Hartmann, Theresa Bock, Natalie Fadle, Maria Kemele, Evi Regitz, Moritz Bewarder, et al. "Identification of Posttranvslationally Modified Neoantigens As Targets of B Cell Receptors of Burkitt Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 1588. http://dx.doi.org/10.1182/blood-2018-99-114209.
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Abstract Introduction: Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma. Methods: BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities. Results: The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma cell line with the reactive BCR induced proliferaton. The analysis of the ICGC MMML-Seq lymphoma databases (representing different cohorts) showed a higher expression of Bystin in Burkitt lymphoma compared to other aggressive B-cell lymphomas. However, the mutation and methylation status of Bystin and HSP40 in the ICGC MMML-seq cohort did not provide any direct indication of the origin of their immunogenic post-translational modifications. Conclusions: A subgroup of sporadic Burkitt lymphoma has autoreactive BCRs with specific affinity against posttranslationally modified self antigens, demonstrating a new aspect in the pathogenesis of Burkitt lymphoma. Specific secondary modifications, as sumolytation of Bystin or acetylation of HSP40 appear to evoke the immunogenicity. Future studies will focus on the functional consequences of the antigen/BCR interaction on Burkitt cells. Furthermore, the causes of these posttranslationally modified neoantigens will be investigated in more detail. Disclosures Stilgenbauer: Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding.
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Arakaki, Hideki, Yuki Osada, Satoshi Takanashi, Chisako Ito, Yoshinobu Aisa, and Tomonori Nakazato. "Oxidative Stress Is Associated with Poor Prognosis in Patients with Follicular Lymphoma." Blood 128, no. 22 (December 2, 2016): 1787. http://dx.doi.org/10.1182/blood.v128.22.1787.1787.
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Abstract [Introduction] Follicular lymphoma (FL) is an indolent non-Hodgkinfs lymphoma (NHL), with a highly variable clinical course that can range from indolent to rapidly progressive disease, including the transformation to aggressive NHL. Oxidative stress caused by the increased production of reactive oxygen species (ROS) or decreased efficacy of the antioxidant system is implicated in the pathogenesis of various disease entities, such as atherosclerosis, cardiovascular disease, renal failure, malignant tumors, and autoimmune diseases. Recent observations suggested that oxidative stress is closely related to all aspects of cancer. Oxidative stress markers are prognostically important in various cancers including diffuse large B-cell lymphoma (DLBCL). However, the prognostic role of oxidative stress in FL is still unknown. The objective of this study is to evaluate the role of oxidative stress in patients with FL. [Methods] 8-hydroxy-2-deoxyguanosine (8-OHdG), which originates from damaged DNA repaired by non-specific endonucleases and specific glycosylates and is eliminated into urine, is widely used as a sensitive biomarker of oxidative stress. Urinary 8-OHdG levels have been reported to be elevated in patients with various malignancies. In the present study, urinary 8-OHdG levels were prospectively examined in 30 patients with FL by using a novel automatic oxidative stress analyzer, ICR-001. We also evaluated serum d-ROMs (the derivatives of Reactive Oxygen Metabolites) levels by using the Free Radical Analytical System 4 (FRAS 4, Wismerll Co. Ltd., Tokyo, Japan). The d-ROMs test has been successfully used to evaluate oxidative stress in a very large number of studies on humans and animals. The d-ROMs test essentially determines the concentration of hydroperoxides in the blood, which are substances that belong to a broad class of reactive oxygen metabolites. The d-ROMs concentration is expressed in Carratelli Units (1 CARR U = 0.08mg hydrogen peroxide/dl). The study protocol and sampling were approved by the Institutional Review Board of Yokohama Municipal Citizen's Hospital, and it was carried out in accordance with the Declaration of Helsinki. [Results] Median age at diagnosis was 70 years (range, 43-84 years) and 53% were male. According to the WHO pathological grading, grade 1 FL was observed in 16 patients (53%), grade 2 FL in 8 patients (27%), and grade 3 FL in 6 patients (20%). FLIPI scores were 0 to 1 in 27%, 2 in 27%, and 3 to 5 in 46%. The urinary 8-OHdG levels in patients with FL (N=30) were elevated compared with normal controls (N=20) (19.7+/-10.3 vs. 13.7+/-3.4 ng/mg/Cr, P=0.01). In 30 FL patients, patients with high urinary 8-OHdG levels (over 23.9 ng/mg/Cr) had significantly shorter overall survival (OS) than those with low urinary 8-OHdG levels (under 23.9 ng/mg/Cr) (3-year OS, 33.3% versus 90.5%, respectively; P<0.001) (Figure. 1). The serum d-ROMs levels in patients with FL were significantly elevated compared with normal controls (543.9+/-199.4 vs. 281.1+/-25.9 CARR U, P<0.001). Patients with high serum d-ROMs levels (over 519 CARR U) had significantly shorter overall survival (OS) than those with low serum d-ROMs levels (under 519 CARR U) (3-year OS, 41.7% versus 94.4%, respectively; P<0.001) (Figure. 2). In univariate analysis, high 8-OHdG levels, high d-ROMs levels, high PS, high LDH levels, high tumor burden (GELF criteria), high beta-2 microglobulin levels, and high FLIPI score were associated with poor OS in patients with FL. In multivariate analysis, parameters having independent adverse significance for OS were: high serum d-ROMs levels (over 519 CARR U) (p=0.01, HR 15.68), high LDH levels (p=0.02, HR 12.25). [Conclusion] In the present study we demonstrated that elevated urinary 8-OHdG levels and serum d-ROMs levels are associated with poor prognosis in patients with FL. In particular, our data proved that a high serum d-ROMs level is an independent prognostic factor for survival in patients with FL. These results suggest that oxidative stress may have an important role in FL and also may be a useful prognostic biomarker. Since our results are based on a small-sized analysis, further large prospective studies are warranted to verify this conclusion. Disclosures Nakazato: Mundipharma KK: Research Funding.
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Sekiguchi, Nodoka, Kazuyuki Matsuda, Kayoko Momose, Hideki Makishima, Toshiro Ito, and Fumihiro Ishida. "STAT3 Gene Mutations and the Association with Pure Red Cell Aplasia in Large Granular Lymphocyte Leukemia." Blood 120, no. 21 (November 16, 2012): 2668. http://dx.doi.org/10.1182/blood.v120.21.2668.2668.
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Abstract Abstract 2668 Large granular lymphocyte leukemia (LGL-L) is a proliferative disorder of cytotoxic T cells or NK cells frequently complicated with cytopenia and autoimmune phenomena. In the current WHO classification, T-cell large granular lymphocyte leukemia (T LGL-L), and chronic lymphoproliferative disorders of NK cells (CLPD-NK) are included in this category. Aggressive NK cell leukemia (ANKL) and Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disease of childhood (EBV-T LPD) are also categorized as entities in the classification based on the clinical characteristics and EBV-positivity, and their morphologies closely resemble LGL. There has been controversy concerning the nature of these diseases, whether they are reactive processes or neoplasms. Recently, recurrent somatic mutations in the src homology (SH) 2 domain of the signal transducer and activator of transcription 3 (STAT3) gene have been found in T LGL-L and CLPD-NK, leading to constitutive activation of STAT3 and dysregulation of genes downstream of STAT3. Since LGL-L consists of various disorders and is suggested to differ based on racial backgrounds, these findings prompted us to investigate mutations in STAT3 in a Japanese cohort of LGL-L. The study included a total of 36 patients (pts) with LGL. Genes of exons 19 to 24 in STAT3 were amplified by PCR and sequenced directly using genomic DNA isolated from peripheral blood mononuclear cells. Since Y604F and D661Y mutations in STAT3 gene were representative and frequently recognized, allele specific PCR (AS-PCR) assays for these mutations were also performed. Pts consisted of 18 with T LGL-L (14 with αβ T cell receptor (TCR) type and 4 with γΔ TCR type), 11 with CLPD-NK, 5 with ANKL, and 2 with EBV-T LPD; one pt with αβ TCR type and 1 with γΔ TCR type, as well as 1 with reactive NK lymphocytosis used as control. By direct sequencing, two mutations, Y640F and D661Y, were identified. Y640F was recognized in 1 pt with αβ T LGL-L and D661Y in 3 pts with CLPD-NK. By AS-PCR, 10 additional pts were found to be positive for mutations of either Y604F or D661Y. A pt with T LGL-L was positive for both mutations by AS-PCR, although no mutations were detected by direct sequencing. A pt with CLPD-NK was positive for Y640F by AS-PCR in addition to D661Y recognized by direct sequencing. All 4 pts positive for the mutations by direct sequencing were confirmed to be positive by AS-PCR. In total, 7 pts with αβ TCR type T LGL-L and three T LGL-L pts with γΔ TCR type were positive for mutations. All these mutations were heterozygous. Mutations in SH2 domains of the STAT3 gene were not found in ANKL or EBV T-LPD pts by either direct sequencing or AS-PCR. Samples from fifty healthy controls were examined by AS-PCR and all were negative for Y604F or D661Y mutations. Reactive NKL lymphocytosis and three cell lines, Jurkat, NKL and NK92, were also negative for the mutations. The frequencies of STAT3 mutations in T LGL-L and CLPD-NK were 55.6% and 27.3%, respectively (P = 0.25). Among the pts with T LGL-L and CLPD-NK, the frequency of pure red cell aplasia (PRCA) was significantly higher in pts with the mutations (8/13) than in those without the mutations (3/16) (P = 0.03). In three T LGL-L pts positive for the mutations examined on subsequent occasions, the mutation became undetectable after cyclosporine A treatment in one pt, and was persistently found at stable amounts in two other pts by quantitative PCR. Our results indicate that mutations in the SH2 domain of the STAT3 gene frequently occur in T LGL-L and CLPD-NK in a Japanese cohort and these mutations are closely associated with PRCA and treatment requirements. STAT3 mutation thus likely contributes to the pathogenesis of T LGL-L and CLPD-NK, while EBV-associated LGL diseases, such as ANKL or EBV T-LPD, might be driven by mechanisms other than STAT3-associated pathways. Disclosures: No relevant conflicts of interest to declare.
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Nakazato, Tomonori, Chisako Ito, and Yoshinobu Aisa. "Oxidative Stress Is Associated with Poor Prognosis in Patients with Malignant Lymphoma." Blood 124, no. 21 (December 6, 2014): 1653. http://dx.doi.org/10.1182/blood.v124.21.1653.1653.
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Abstract [Introduction] Oxidative stress caused by the increased production of reactive oxygen species (ROS) or decreased efficacy of the antioxidant system is implicated in the pathogenesis of various disease entities, such as atherosclerosis, cardiovascular disease, renal failure, malignant tumors, and autoimmune diseases. Recent observations suggested that oxidative stress is closely related to all aspects of cancer. Oxidative stress markers are prognostically important in various cancers. However, the prognostic role of oxidative stress in hematologic malignancies (HM) is still unknown. The objective of this study is to evaluate the role of oxidative stress in patients with HM. [Methods] 8-hydroxy-2-deoxyguanosine (8-OHdG), which originates from damaged DNA repaired by non-specific endonucleases and specific glycosylates and is eliminated into urine, is widely used as a sensitive biomarker of oxidative stress. Urinary 8-OHdG levels have been reported to be elevated in patients with various malignancies. In the present study, urinary 8-OHdG levels were examined in 196 patients with HM (112 malignant lymphoma, 34 multiple myeloma, 28 myelodysplastic syndrome, 15 acute myeloid leukemia, 3 acute lymphoblastic leukemia, 4 chronic myeloid leukemia) by using a novel automatic oxidative stress analyzer, ICR-001. The study protocol and sampling were approved by the Institutional Review Board of Yokohama Municipal Citizen's Hospital. [Results] The urinary 8-OHdG levels in patients with HM were elevated compared with normal controls (23.2+/-18.3 vs. 13.7+/-3.4ng/mg/Cr, P=0.02). In particular, urinary 8-OHdG levels were significantly elevated in patients with malignant lymphoma (25.9+/-31.4ng/mg/Cr, P=0.02). In 112 lymphoma patients, patients with high urinary 8-OHdG levels (over 25.0ng/mg/Cr) had significantly shorter overall survival (OS) than those with low urinary 8-OHdG levels (under 25.0ng/mg/Cr) (2-year OS, 44.0% versus 83.4%, respectively; P<0.001) (Figure. 1). Among lymphoma patients, urinary 8-OHdG levels were significantly higher in patients with diffuse large B-cell lymphoma (DLBCL) (31.04+/-36.54ng/mg/Cr, P=0.01). In 55 DLBCL patients, high urinary 8-OHdG levels were also associated with shorter OS (2-year OS, 31.1% versus 88.9%, respectively; P<0.001) (Figure. 2). In univariate analysis, a high 8-OHdG level, high PS, a high LDH level, advanced stage, a high sIL2R level, and R-IPI poor risk were associated with poor OS in patients with DLBCL. In multivariate analysis, parameters having independent adverse significance for OS were: a high urinary 8-OHdG level (over 25.0ng/mg/Cr) (p=0.01, HR 4.97), high PS (2-4) (p=0.01, HR 6.61). [Conclusion] In the present study we demonstrated that many patients with HM have elevated urinary 8-OHdG levels, and these elevated levels are associated with a poor prognosis in patients with ML. In particular, our data proved that a high urinary 8-OHdG level is an independent prognostic factor for survival in patients with DLBCL. These results suggest that oxidative stress may have an important role in ML and also may be a useful prognostic biomarker. Since our results are based on a small-sized analysis, further large prospective studies are warranted to verify this conclusion. Disclosures No relevant conflicts of interest to declare.
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Zheng, Yongwei, Alexander W. Wang, Mei Yu, Anand Padmanabhan, Benjamin E. Tourdot, Debra K. Newman, Gilbert C. White, Richard H. Aster, Renren Wen, and Demin Wang. "Role Of B Cell Tolerance In PF4/Heparin Antibody Production." Blood 122, no. 21 (November 15, 2013): 2396. http://dx.doi.org/10.1182/blood.v122.21.2396.2396.
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Abstract Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis. Disclosures: White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.
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Nakazato, Tomonori, Chisako Ito, Kengo Shimazaki, Hideki Arakaki, and Yoshinobu Aisa. "Evaluation of Oxidative Stress Markers in Hematopoietic Stem Cell Transplantation Patients." Blood 126, no. 23 (December 3, 2015): 1925. http://dx.doi.org/10.1182/blood.v126.23.1925.1925.
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Abstract [Introduction] Oxidative stress caused by the increased production of reactive oxygen species (ROS) or decreased efficacy of the antioxidant system is implicated in the pathogenesis of various disease entities, such as atherosclerosis, cardiovascular disease, renal failure, malignant tumors, and autoimmune diseases. Chemotherapy and radiation therapy are associated with increased formation of ROS. Conditioning regimens preceding hematopoietic stem cell transplantation (HSCT) usually consist of high-dose chemotherapy and/or total body irradiation (TBI). A limited number of studies demonstrated that the conditioning therapy given to HSCT patients creates a high oxidative stress and decreases the antioxidant defense system. The objective of this study was to look for further evidence of oxidative stress status in HSCT patients. [Methods] In this study, urine samples were collected from 32 HSCT patients before and after conditioning therapy and from 15 healthy controls. The patients included 19 male and 13 female with a median age of 58 years (27-68 years). Twenty patients received allogeneic HSCT (9 uBMT, 1 rPBSCT and 10 CBT) and 12 patients received autologous PBSCT. The conditioning regimens for allo-HSCT included TBI12Gy+Ara-C+G-CSF (n=3), TBI12Gy+Ara-C+CY (n=1), TBI12Gy+VP-16+CY (n=1), Flu+L-PAM+TBI3Gy (n=6), Flu+BU+TBI3Gy (n=1), Flu+Ara-C+G-CSF+BU+TBI4Gy (n=3), Flu+L-PAM (n=2), and BU+CY (n=3). The diagnosis included AML (n=9), MDS (n=5), DLBCL (n=1), FL (n=2), T-ALL (n=1) and MM (n=2). Fifteen patients (75%) had high-risk disease at transplantation. Twelve multiple myeloma (MM) patients received L-PAM 200mg/m2 + autologous PBSCT. We measured urinary 8-hydroxydeoxyguanosine (8-OHdG) by competitive immunochromatography using a novel automatic oxidative stress analyzer, ICR-001 (Techno-Medica). 8-OHdG, which originates from damaged DNA repaired by non-specific endonucleases and specific glycosylates and is eliminated into urine, is widely used as a sensitive biomarker of oxidative stress. [Results] Urinary 8-OHdG significantly increased immediately after conditioning therapy. In 20 allo-HSCT patients, urinary 8-OHdG levels on day 0 were significantly higher than pre-conditioning levels and healthy controls (mean: 458.2 vs. 90.9 vs. 15.7 ng/mgCr). In 12 auto-HSCT patients, urinary 8-OHdG levels on day 0 were also higher than pre-conditioning levels (mean: 273.6 vs. 107.2 ng/mgCr). No significant correlation was found between urinary 8-OHdG levels and serum ferritin levels during pre- and post-transplant period. Allo-HSCT patients with high urinary 8-OHdG levels on day 0 (over 300ng/mg/Cr; N=10) had significantly shorter survival than those with low urinary 8-OHdG levels on day 0 (under 300ng/mg/Cr; N=10) (1-year OS, 11.1% vs. 80.0%, respectively; P=0.01) (Figure. 1). On the other hand, post-conditioning urinary 8-OHdG levels were not associated with outcome in auto-HSCT patients. In univariate analysis, a high 8-OHdG level on day 0, CBT, and RIC regimen were associated with poor OS in allo-HSCT patients. [Conclusion] In the present study we demonstrated that conditioning therapy results in increased oxidative stress in HSCT patients. In particular, our data proved that high urinary 8-OHdG levels on day 0 were associated with poor prognosis in allo-HSCT patients. These results suggest that oxidative stress may have an important role in allo-HSCT and also may be a useful prognostic biomarker. Since our results are based on a small-sized analysis, further large prospective studies are warranted to verify this conclusion. Disclosures No relevant conflicts of interest to declare.
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Nieborowska-Skorska, Margaret, Piotr Kopinski, Regina Ray, Grazyna Hoser, Danielle Ngaba, Sylwia Flis, Kimberly Cramer, et al. "Targeting Rac2 - Mitochondrial Respiratory Chain Complex III Signaling to Prevent Genomic Instability in Leukemia Stem and Progenitor Cells." Blood 118, no. 21 (November 18, 2011): 2736. http://dx.doi.org/10.1182/blood.v118.21.2736.2736.
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Abstract Abstract 2736 BCR-ABL1 –positive chronic myeloid leukemia in chronic phase (CML-CP) is a leukemia stem cell (LSC)-derived but leukemia progenitor cell (LPC)-driven disease, which may eventually develop resistance to the tyrosine kinase inhibitors (TKIs) and progress to fatal CML blast phase (CML-BP). In CML-CP, LSCs and LPCs reside in the CD34+CD38- and CD34+CD38+ populations, respectively. In addition, majority of LSCs and LPCs belong to quiescent (CFSEmax) and proliferative (CFSElow) populations, respectively. Quiescent LSCs are intrinsically insensitive to TKIs, and LPCs can acquire resistance to TKIs. In the TKI era, these cells may eventually initiate the disease relapse and progression to CML-BP, which is associated with genomic instability manifested by accumulation of a new or additional TKI-resistant BCR-ABL1 kinase mutations and chromosomal aberrations. We reported that BCR-ABL1 –positive leukemia cells contain high levels of the reactive oxygen species (ROS)-induced oxidative DNA damage resulting in genomic instability (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). These studies highlighted the importance of identification of the origin of leukemia cell lineage accumulating genomic instability and the mechanisms responsible for generation of ROS-mediated oxidative DNA damage. Here we show that LSC-enriched CD34+CD38- cells and quiescent LSCs, and also LPC-enriched CD34+CD38+ and proliferating CML-CP cells contain higher levels of ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and oxidative DNA lesions (8-oxoG and DNA double-strand breaks) than corresponding cells from healthy donors. Surprisingly, the most primitive quiescent LSCs accumulated the highest levels of ROS and oxidative DNA damage. On the basis of these observations and the studies in murine hematopoietic 32Dcl3 cells expressing TKI-resistant BCR-ABL1 kinase variants (Y253F, T315I, H396P), we would predict that primitive CML-CP cells carrying these mutations would also contain high levels of ROS and oxidative DNA damage. Moreover, inhibition of BCR-ABL1 kinase with imatinib exerted only modest, if any, effect on ROS and oxidative DNA damage in LSCs/LPCs in the presence of growth factors (GFs). Among numerous signaling proteins activated in CML cells, Rac GTPases were potential candidates to regulate production of ROS. Importantly, Rac was stimulated in leukemia cells expressing non-mutated BCR-ABL1 and TKI-resistant kinase mutants and it remained active in CML-CP cells treated with imatinib in the presence of GFs. We used Rac dominant-negative mutant (RacT17N), Rac specific inhibitors (NSC23766 and EHT1864) and Rac1, Rac2 and Rac3 knockout cells to document that Rac2 GTPase is responsible for elevation of ROS and oxidative DNA damage in LSC-enriched CD34+CD38- cells, quiescent LSCs, and also in LPCs. Active Rac2 reduced mitochondrial membrane potential (ΔΨm) and slowed the electron flow between mitochondrial respiratory chain (MRC) complexes I-II and I-III leading to overproduction of ROS. Using cells depleted of functional mitochondria (Rho0 cells), applying specific probes to measure mitochondrial ROS (MitosoxRed and mitochondria matrix-targeted circularly permuted yellow fluorescence protein = mt-cpYFP) and employing a specific inhibitor of mitochondrial ROS (MitoQ) we determined that mitochondria are the main source of ROS causing oxidative DNA damage in CD34+CD38- and quiescent LSCs and in LPCs. Furthermore, using selective inhibitors of various MRC complexes we pinpointed complex III as major producer of ROS in LSCs and LPCs. This conclusion is supported by the observation that BCR-ABL1 –positive cells with genetically inactivated complex III, but not complex I, displayed diminished capability to generate ROS. Targeting Rac2 GTPase by RacT17N and reduction of mitochondrial ROS by mitochondrial-targeted catalase and by mitochondrial-targeted ROS-scavenging peptide aptamers prevented genomic instability. Altogether, Rac2 - MRC-cIII pathway is a major source of ROS-mediated oxidative DNA damage resulting in genomic instability in LSCs and LPCs, which could be targeted to prevent the relapse and malignant progression of CML. We also postulate that similar mechanisms cause genomic instability in FLT3(ITD)-positive acute myeloid leukemia cells and in JAK2(V617F)-positive polycythemia vera cells. Disclosures: Holyoake: Novartis: Consultancy, Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Ricci, Craig, Viktor Pastukh, Mahmood Mozaffari, and Stephen W. Schaffer. "Insulin withdrawal induces apoptosis via a free radical-mediated mechanismThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute." Canadian Journal of Physiology and Pharmacology 85, no. 3-4 (March 2007): 455–64. http://dx.doi.org/10.1139/y07-029.
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Diabetes is characterized by chronic hyperglycemia as well as insulin deficiency or resistance. However, the majority of research has focused on the consequences of hyperglycemia in development of diabetic complications, whereas the effects of insulin deficiency or resistance, independent of hyperglycemia, have received little attention. Since insulin is a well known cytoprotective factor, we hypothesized that its removal could significantly impact cell survival. To examine this possibility, cultured neonatal cardiomyocytes were subjected to insulin withdrawal and examined for apoptosis. Insulin deficient cells succumbed to apoptosis, an effect associated with impaired PI3-kinase/Akt signaling and reduction in the Bcl-2 to Bax ratio. Perhaps more importantly, superoxide generation was altered in cells subjected to insulin withdrawal. Removal of insulin caused a significant increase in reactive oxygen species production and resulted in oxidative mitochondrial DNA damage the latter effect is associated with impaired expression of mitochondrially encoded proteins that make up the electron transport chain. Significantly, the effects of insulin withdrawal could be mitigated by treatment with the antioxidant, Tiron. Collectively, these data demonstrate that insulin deficiency leads to apoptosis and suggest a role for oxidative mitochondrial DNA damage in this cascade.
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Bolton, Elisabeth, Mirle Schemionek, Hans-Urlich Klein, Grazyna Hoser, Sylwia Flis, Thoralf Lange, Linda Kerstiens, et al. "Genomic Instability in CML-CP originates From the Most Primitive Imatinib-Refractory Leukemia Stem Cells." Blood 120, no. 21 (November 16, 2012): 909. http://dx.doi.org/10.1182/blood.v120.21.909.909.
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Abstract Abstract 909 Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in the appearance of clones carrying BCR-ABL1 kinase mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or those harboring additional chromosomal aberrations, eventually leading to disease relapse and/or malignant progression to blast phase (CML-BP) [Skorski, T., Leukemia and Lymphoma, 2011]. We found that Lin−CD34+CD38− human leukemia stem cells (huLSCs), including the quiescent sub-population, and Lin−CD34+CD38+ human leukemia progenitor cells (huLPCs) accumulate high levels of reactive oxygen species (ROS) resulting in numerous oxidative DNA lesions such as 8-oxoguanine (8-oxoG) and DNA double-strand breaks (DSBs) [Nieborowska-Skorska, Blood, 2012]. huLSCs and huLPCs treated with TKIs continue to exhibit ROS-induced oxidative DNA damage suggesting the persistence of genomic instability in TKI-treated patients. Furthermore, genomic instability in TKI-refractory huLSCs and TKI-sensitive huLPCs may have a varying impact on disease progression and determining novel treatment modalities. To determine if TKI-refractory huLSCs are a source of genomic instability we employed a tetracycline-inducible murine model of CML-CP: SCLtTA/p210BCR-ABL1. Mice exhibiting CML-CP -like disease demonstrated splenomegaly, leukocytosis, and expansion of mature Gr1+/CD11b+ cells. ROS were elevated in Lin−c-Kit+Sca-1+ cells (muLSCs), but not Lin−c-Kit+Sca-1− cells (muLPCs), which was associated with higher mRNA expression of BCR-ABL1 in muLSCs. In addition, ROS levels were directly proportional to BCR-ABL1 kinase expression in transduced CD34+ human hematopoietic cells, thus confirming the “dosage-dependent” effect of BCR-ABL1 on ROS. Among the Lin−c-Kit+Sca-1+ cells, enhanced ROS were detected in TKI-refractory quiescent muLSCs, in CD34−Flt3− long-term and CD34+Flt3− short-term muLSCs, and also in CD34+Flt3+ multipotent progenitors. High levels of ROS in muLSCs were accompanied by aberrant expression of genes regulating ROS metabolism (mitochondrial electron transport, oxidative phosphorylation, hydrogen peroxide synthesis, and detoxification). In addition, muLSCs, including the quiescent sub-population, displayed high levels of oxidative DNA lesions (8-oxoG, and DSBs). ROS-induced oxidative DNA damage in muLSCs was accompanied by genomic instability in CML-CP –like mice, which accumulated a broad range of genetic aberrations recapitulating the heterogeneity of sporadic mutations detected in TKI-naive CML-CP patients. These aberrations include TKI-resistant BCR-ABL1 kinase mutations, deletions in Ikzf1 and Trp53 and additions in Zfp423 and Idh1 genes, which have been associated with CML-CP relapse and progression to CML-BP. Imatinib caused only modest inhibition of ROS and oxidative DNA damage in TKI-refractory muLSCs. In concordance, CML-CP –like mice treated with imatinib continued to accumulate genomic aberrations. Since BCR-ABL1(K1172R) kinase-dead mutant expressed in CD34+ human hematopoietic cells did not enhance ROS, it suggests that BCR-ABL1 kinase-independent mechanisms contribute to genomic instability. In summary, we postulate that ROS-induced oxidative DNA damage resulting in genetic instability may originate in the most primitive TKI-refractory huLSCs in TKI-naive and TKI-treated patients. Disclosures: Lange: Novartis: Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Glencer, Alexa, Alexander Borowsky, Hidetoshi Mori, Michael Campbell, Olivier Harismendy, Janet Stein, Prachi Ghule, et al. "Abstract P1-05-01: The tumor immune microenvironment and HER2 landscape of high-risk ductal carcinoma in situ: The DEFENSE study." Cancer Research 82, no. 4_Supplement (February 15, 2022): P1–05–01—P1–05–01. http://dx.doi.org/10.1158/1538-7445.sabcs21-p1-05-01.
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Abstract Introduction: Ductal carcinoma in situ (DCIS) of the breast is a premalignant lesion representing a spectrum of biology from indolent to aggressive. A minority of women present with clinically high-risk features associated with poor outcome. Yet even in patients with biologically aggressive DCIS, the risk of breast cancer mortality is only 3.3% compared to 30-40% in patients with biologically aggressive invasive cancer of the same size.1 We hypothesize that the tumor immune microenvironment could play a proactive role in preventing invasion in high grade clinically high-risk DCIS and that HER2 status, including specific HER2 isoform expression and post-translational modification of HER2, could impact progression. Methods: DCIS: Elaboration of Factors from Enlarged lesions that Nevertheless remain Stage 0 Entities (DEFENSE) is a study of high-risk DCIS, defined as having at least two of the following characteristics: large (>5cm), high grade, hormone receptor-negative status and/or HER2+ status. Slides obtained from FFPE tissue blocks were stained with fluorescence-based multiplex immunohistochemistry (mIHC) panels and imaged to characterize immune infiltrate within the ducts and the stromal compartments. mIHC was also used to detect extracellular and intracellular domains of HER2 with imaging analysis performed to identify HER2 isoforms of HER2+ specimens. Isoforms were characterized as 1) full-length/extracellular domain (ECD) intact, 2) pure/complete loss of ECD (p95 isoform), 3) subclonal populations of both full-length ECD and p95 and 4) gradient/representing partial loss of ECD per cell (reflecting post-translational cleavage). Clinical characteristics were correlated with molecular profile, tumor immune infiltrates, and HER2 isoform. Finally, expression profiling with a 44k array was conducted by Agendia, and MammaPrint and BluePrint results were generated. Results: Of 92 total patients, median age is 46 years. The average DCIS lesion size is 8.2cm, and 33% are hormone receptor negative (HR-). Based upon initial analysis, mIHC demonstrates significant heterogeneity in immune infiltrate populations of pathologically identical DCIS specimens and within regions of the same specimen. High-grade HR- disease has highly reactive stroma, characterized by dense CD3+, CD34+, and CD68+ infiltrate within the stromal compartment. HER2 testing of the first 51 cases demonstrates a high rate of positivity of 67% (34/51). Of those tested for HER2 isoform expression (n=21), none had homogeneous intact full-length HER2. Six (29%) demonstrate the pure p95 isoform, 3 (14%) demonstrate the subclonal isoform, and 12 (57%) demonstrate a gradient HER2 isoform phenotype. Preliminary data from expression profiling shows that the HER2+ cases are also HER2 intrinsic sub-type by BluePrint. Across all of the high-risk DCIS cases, all were scored as MammaPrint high risk, either Luminal B or HER2-type, with only 1 basal and no Luminal A. Additional analyses are ongoing, including completion of testing for the whole data set as well whole exome DNA sequencing and SMART-3SEQ RNA sequencing. Conclusions: Clinically high-risk and pathologically homogenous DCIS lesions demonstrate significant immune infiltrate heterogeneity. Nearly 70% of these large clinically high-risk DCIS lesions are HER2+ with HER2 isoforms most commonly representing either partial or complete loss of the HER2 ECD. This is significantly higher than what is reported for invasive HER2+ breast cancer. A comparison to size and molecularly matched invasive cancers, including from the I-SPY 2 trial, is underway in an effort to elucidate how molecularly aggressive lesions remain in situ despite their large size. References:1 Narod SA et al (2015). Breast Cancer Mortality After a Diagnosis of Ductal Carcinoma in Situ. JAMAOncol. 1(7): 888-96. Citation Format: Alexa Glencer, Alexander Borowsky, Hidetoshi Mori, Michael Campbell, Olivier Harismendy, Janet Stein, Prachi Ghule, Mark Evans, Robert West, Gillian Hirst, Nicole Schindler, Phoebe Miller, Kyra Lee, Donald Weaver, Laura Esserman. The tumor immune microenvironment and HER2 landscape of high-risk ductal carcinoma in situ: The DEFENSE study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-01.
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Maciocia, Paul, Margarida Neves, Shimobi Onuoha, Patrycja Wawrzyniecka, Giuseppe Gritti, Manuela Tosi, Alessia Moioli, Martin Pule, and Teresa Marafioti. "Analysis of T-Cell Receptor Beta-Constant Region Expression for Rapid Assessment of T-Cell Clonality." Blood 132, Supplement 1 (November 29, 2018): 2867. http://dx.doi.org/10.1182/blood-2018-99-112569.
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Abstract Introduction T-cell malignancies are heterogenous disorders, representing approximately 10-15% of cases of non-Hodgkin's lymphoma and 15% of acute lymphoblastic leukaemia. Diagnosis may be complicated both by the fact that non-malignant oligoclonal T-cell proliferations often mimic T-cell cancers, and due to the lack of consistent markers of malignancy which discriminate healthy and malignant T-cells. B-cell lymphoproliferations, by contrast, can be reliably identified as clonal by confirmation of restricted kappa or lambda light chain expression. We have recently described an analogous method for evaluation of T-cell clonality, by evaluating expression of one of two virtually identical and mutually exclusive alleles at the T-cell receptor beta constant region: TRBC1 or TRBC2. We demonstrated that approximately 35% of normal T-cells and T-cell malignancies expressed TRBC1, using a TRBC1-specific antibody suitable for use in fresh/ frozen tissues. We have now developed a novel antibody with TRBC2 specificity, which recognises an intracellular, non-surface exposed epitope and can be used in both fresh and formalin-fixed paraffin-embedded (FFPE) tissues. Here we demonstrate TRBC1/2 assessment can be used to identify T-cell clonality using immunohistochemistry on FFPE tissues, and confirm our results using reference methods including Sanger and massively parallel sequencing. Methods/ Results To generate anti-TRBC2 antibody, we immunised New Zealand rabbits with a peptide comprising the intracellular portion of the TCR beta-2 chain (VKRKDSRG). Protein A-purified immunoglobulin from the sera of immunised rabbits was tested for reactivity to TRBC1 (VKRKDF) and TRBC2 peptides by ELISA. Strong TRBC2-specificity with limited TRBC1 binding was seen. Immunoglobulin was then depleted of residual TRBC1 reactivity by depletion on TRBC1 peptide-coated agarose beads. Following this, no reactivity to TRBC1 peptide could be detected by ELISA. We then stained fresh frozen and FFPE samples of pelleted TRBC1 and TRBC2 cell lines with the novel, polyclonal anti-TRBC2 antibody. We confirmed that anti-TRBC2 antibody stained only TRBC2 and not TRBC1 cell lines, and that the antibody performed equivalently in fresh or FFPE preparations. We then showed that in a range of normal tissues, including tonsil, spleen and others, a staining pattern restricted to only a proportion of T-cells was seen. In bone marrow sections, some staining of megakaryocytes was also seen, as has been previously demonstrated for other T-cell markers such as linker for activation of T-cells (LAT). No other non-T-cell staining was noted. We obtained paired FFPE and frozen samples of several TCR+ T-cell malignancies. We performed staining for TRBC1 and TRBC2 on both frozen and FFPE samples: TRBC1 staining was performed only on frozen samples. In addition, we extracted DNA from these tissues and performed PCR for TCR beta VDJ rearrangement using standard BIOMED protocols. Clonal proliferations were identified by heteroduplex gel analysis and capillary electrophoresis (Sanger) sequencing was performed on dominant bands. In addition, pooled PCR products were subjected to massively parallel sequencing to quantify clone frequency and to confirm VDJ identity. We demonstrated mutual exclusivity between samples staining positive for TRBC1 and TRBC2, and concordance between TRBC1/2 status identified by Sanger sequencing, NGS and antibody-based methods. Further, in a number of TCR+ malignancies, non-T-cell haematological tumours and reactive lymphoproliferations, where only FFPE material was available, we used anti-TRBC2 antibody to evaluate TRBC2 expression. We demonstrated that in non-T cell tumours, a limited pattern of staining was seen, restricted to a proportion of infiltrating normal T-cells only. T-cell tumours, by contrast, were uniformly TRBC2-negative or positive, across a range of entities. 18/29 (62%) of T-cell neoplasms were TRBC2-positive. Conclusions We have generated a new TRBC2-specific polyclonal reagent suitable for use in FFPE tissues, which displays minimal cross-reactivity against other cell types. A monoclonal reagent is in preparation. We suggest that TRBC1/2 assessment can thus be used to rapidly identify clonal T-cell lymphoproliferations and may be a helpful addition to the diagnostic haematopathology toolkit. Disclosures Maciocia: Autolus: Equity Ownership, Patents & Royalties: UCLB. Neves:Autolus Ltd: Employment. Onuoha:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Pule:UCLB: Patents & Royalties; Autolus: Employment, Equity Ownership.
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Corrales-Medina, Fernando F., Daniela E. Egas Bejar, Christa Manton, Blake Johnson, Mary E. Irwin, Robert Z. Orlowski, and Joya Chandra. "Unique Apoptotic Effects Of Panobinostat and Marizomib In Acute Myeloid Leukemia and Bortezomib-Resistant Models." Blood 122, no. 21 (November 15, 2013): 3837. http://dx.doi.org/10.1182/blood.v122.21.3837.3837.
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Abstract Panobinostat, a potent pan-histone deacetylase inhibitor (HDACi) is emerging as a valuable therapeutic option for cancer treatment. Compared with the FDA-approved vorinostat, it displays a more potent and broader spectrum of inhibitory activity (inhibiting all class I, II and IV HDACs) at clinically achievable concentrations. Combinatorial therapies with proteasome inhibitors (PI) have been recently evaluated. Marizomib, a second-generation irreversible PI, exhibits excellent pre-clinical activity against numerous hematologic malignancies. Indeed, published work from our laboratory has shown that marizomib is more effective than the FDA approved PI bortezomib in inhibiting the activity of the proteasome. Additionally, marizomib has been shown to induce higher caspase-8 and reactive oxygen species (ROS) dependent cell death than bortezomib, alone and in combination with HDACi in acute lymphoblastic leukemia (ALL) models. Despite progress in the treatment of acute myeloid leukemia (AML), 40% of patients die from disease recurrence or treatment toxicities; therefore, targeted approaches allowing for low drug doses are needed to increase clinical efficacy. The objectives of this study were to (1) determine if a panobinostat/PI regimen displayed synergy; (2) whether cell death by this combination triggers an unique profile of caspase activation; and (3) assess utility of this combination in a bortezomib refractory setting. Human-derived AML cell lines, ML-1 and AML3, were exposed to increasing concentrations of HDACi, (panobinostat or vorinostat), and PI (bortezomib or marizomib) alone and in combination. Panobinostat had an IC50 within the nanomolar range at 24 hours of treatment; in contrast, an IC50 was not achievable with vorinostat until after 48 hours of treatment. Marizomib was found to have a much lower IC50 and higher DNA fragmentation capacity than bortezomib. Calcusyn software was used to determine synergistic combinations. Synergistic cytotoxicity was observed with the combination treatment of panobinostat with both PIs. No synergy was observed with combinations involving vorinostat. Interestingly, the panobinostat + marizomib combination demonstrated earlier and higher ROS induction. To assess the differences in apoptotic mechanisms between these agents, caspase activities were measured. The combination of panobinostat + marizomib induced an earlier and 2.5x higher induction of caspase-3 activation than the combination of panobinostat + bortezomib. Caspase-8 and caspase-9 dependence, were evaluated using pre-treatment with specific caspase-8 (IETD-fmk) and caspase-9 (LEHD-fmk) inhibitors. Caspase-8 inhibition decreased the cytotoxicity of the panobinostat + marizomib combination compared to control, whereas no difference was observed on cells treated with panobinostat + bortezomib. Western blotting for cleaved caspase-8 corroborated these data. Caspase-9 inhibitors did not significantly protect against DNA fragmentation in any of the combinations. These AML results were consistent with our prior published work using ALL cells, highlighting a role for caspase-8 in sensitivity to synergistic combinations of HDACi and PI. Bortezomib resistance is an emerging problem in the treatment of hematological malignancies. Therefore, RPMI-8226vr10 (bortezomib-resistant multiple myeloma) cells were treated with equimolar doses of either of the HDACi; panobinostat was able to induce higher DNA fragmentation than vorinostat. When panobinostat was combined with either PI, the combination with marizomib caused higher DNA fragmentation than the bortezomib combination. Because caspase-2 has been implicated in panobinostat cytotoxicity in other systems, caspase-2 cleavage was assessed by western blotting assays. At 12 hours of treatment, panobinostat + marizomib caused stronger caspase-2 cleavage than the panobinostat + bortezomib combination, suggesting that marizomib may uniquely augment the caspase-2 activating capacity of panobinostat, providing insight into a potential mechanism of bortezomib resistance. Further experiments will focus on the molecular mechanisms of panobinostat and marizomib combinations and how efficacy could be further optimized. Overall, these data support the use of these novel anticancer agents in hematological malignancies. Disclosures: Orlowski: Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Resverlogix: Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity’s Board of Directors or advisory committees.
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Lin, Jianhong, Chun Yang, Ariel Kwart, Jianjun Zhao, Mehmet K. Samur, Weihong Zhang, Purushothama Nanjappa, et al. "Apurinic/Apyrimidinic Endonuclease 1 Induced Genomic Instability Causes T-Cell Acute Lymphoblastic Leukemia in Zebrafish." Blood 126, no. 23 (December 3, 2015): 1431. http://dx.doi.org/10.1182/blood.v126.23.1431.1431.
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Abstract Genomic instability is not only a hallmark of cancer, but potentially a primary mechanism for its occurrence. DNA repair mechanisms play a protective role during DNA damage induced by both normal metabolic activities and environmental factors such as reactive oxygen species (ROS), UV light and γ-irradiation. Genes related to DNA repair are usually considered as tumor suppressors. However, incomplete repair may induce severe genomic instability, leading eventually to transformation. Apurinic/apyrimidinic endonuclease 1 (APEX1), a gene involved in DNA repair with an important role in the base excision repair pathway, leads to transformation of normal cells in vitro. To investigate the role of APEX1 in tumor initiation in vivo, we generated a novel transgenic zebrafish model to overexpress APEX1 in fish. Specifically, pDestTol2A2_ubi:loxP-EGFP-loxP-APEX1-mCherry plasmid was injected into single cell embryos derived from the TP53 mutant line Tp53M214K/M214K to generate a stable conditional inducible transgenic zebrafish line: Tg:APEX1fl/- mCherry Tp53M214K/M214K. To activate APEX1 expression in vivo, this line was mated with Tg:HSP70-Cre+/+ fish. Compound zebrafish Tg: APEX1fl/- mCherry,Tp53+/M214K ,HSP70-Cre+/- carrying a Cre-activatable APEX1 knock-in allele were heated at 24hpf, and induction of APEX1 expression was monitored by downstream reporter - mCherry expression. Ten to twelve months post-fertilization, Tg:APEX1fl/- mCherry,Tp53+/M214K ,HSP70-Cre+/- fish developed abnormal swelling. Flow cytometry analysis of fish kidney marrow and peripheral blood showed dramatically increased precursor populations in scatter analysis. Histopathologic analysis showed that multiple organs were infiltrated with malignant lymphoblastic cells. None of the control fish Tg: GFP,Tp53+/M214K ,HSP70-Cre+/- developed tumors during their life span. Zebrafish with T-ALL have heterozygous Tp53+/M214K background, but the expression of p21, mdm2 and bax in Tp53+/M214K fish is exactly the same as in Tp53+/+ fish; and Tp53+/M214K zebrafish themselves have not developed tumors during their life span. RNA from lymphoblastic cells was evaluated by qRT-PCR and showed increased expression of CD3, LCK and Tal indicating a T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). We have performed whole genomic DNA sequencing in extracted DNA from fish tumor cells and compared it with their normal counterpart and observed multiple copy number changes and mutations. We have now begun to see the development of other tumors in other organs including the eye and the testis. To uncover the molecular mechanism of tumorigenesis induced by APEX1, we have performed Mass Spectrometry analysis on APEX1 pulled down from 293T and AG08498 cells ectopically expressing APEX1. Besides verified binding proteins, such as PCNA, we also identified Ku70 and Ku80 binding to APEX1 directly. Further immunoflurescent staining and confocal microscopy of 293T cells also found co-localization of APEX1 and Ku70/Ku80. Those two proteins initiate Non-Homologous End Joining (NHEJ) repair and start the error-prone double strand repair and DNA damage. These results indicate that excessive repair activity may induce DNA damage and genomic instability. In summary, this is the first demonstration where overexpression of a DNA repair gene is responsible for induction of genomic instability leading to malignant transformation. It provides new insight into the process of tumorigenesis and development of both therapy as well as preventive strategies. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
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Bolton, Elisabeth, Linda Kamp, Hardik Modi, Ravi Bhatia, Steffen Koschmieder, and Tomasz Skorski. "CML-CP Mouse Model of Genomic Instability." Blood 116, no. 21 (November 19, 2010): 1210. http://dx.doi.org/10.1182/blood.v116.21.1210.1210.
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Abstract Abstract 1210 Background: BCR-ABL1 transforms hematopoietic stem cells to induce chronic myeloid leukemia in chronic phase (CML-CP). Although CML is stem cell-derived, it is a progenitor cell-driven disease. In CML-CP, leukemia stem cells (LSCs) are characterized by elevated BCR-ABL1 expression in comparison to leukemia progenitor cells (LPCs). Increased expression of BCR-ABL1 kinase is also associated with progression from CML-CP to CML-blast phase. Previously we showed that BCR-ABL1 kinase stimulates reactive oxygen species (ROS)-dependent DNA damage resulting in genomic instability in vitro, which was responsible for acquired imatinib-resistance and accumulation of chromosomal aberrations (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). Result: To examine the effects of BCR-ABL1 expression on genomic instability during in vivo leukemogenesis we employed an inducible transgenic mouse model of CML-CP with targeted expression of p210BCR-ABL1 in hematopoietic stem and progenitor cells (Koschmieder et al., Blood, 2005). Mice exhibiting CML-CP-like disease resulting from BCR-ABL1 induction demonstrated splenomegaly, leukocytosis, and Gr1+/CD11b+ myeloid expansion in bone marrow, spleen and peripheral blood, as detected by FACS analysis. BCR-ABL1 mRNA expression was higher in Lin-c-Kit+Sca1+ stem-enriched cells than in Lin-c-Kit+Sca1- progenitor-enriched cells, thus reminiscent of CML-CP (LSCs>LPCs). BCR-ABL1 increased levels of ROS (hydrogen peroxide, hydroxyl radical) and oxidative DNA lesions (8-oxoG) in LSC-enriched Lin-c-Kit+Sca1+ cells. Preliminary data also suggested that quiescent (CFSEmax) Lin-c-Kit+Sca1+ cells from BCR-ABL1-induced mice exhibited greater ROS (superoxide) production than non-induced counter parts. Moreover, higher levels of ROS were detected in BCR-ABL1-positive Lin-c-Kit+Sca1+ stem-enriched population in comparison to BCR-ABL1-positive Lin-c-Kit+Sca1- progenitor population, suggesting a dosage-dependent effect of BCR-ABL1. To confirm that BCR-ABL1 exerts a dosage-dependent effect on ROS-induced oxidative DNA damage, we showed that the levels of ROS, 8-oxoG and DNA double-strand breaks were proportional to BCR-ABL1 kinase expression in murine 32Dc13 and human CD34+ cells. Conclusion: In summary, this mouse model recapitulates the BCR-ABL1 expression profile attributed to stem and progenitor populations in human CML-CP. It also shows that the BCR-ABL1-positive, stem cell-enriched Lin-c-Kit+Sca1+ population displays elevated levels of ROS and oxidative DNA damage in comparison to normal counterparts, which makes it suitable to study the mechanisms of genomic instability in LSCs. Single nucleotide polymorphism (SNP) arrays will shed more light on the genomic instability of this BCR-ABL1-induced transgenic model of CML-CP. Disclosures: Koschmieder: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.
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Cichocki, Frank, Karrune Woan, Cheng-Ying Wu, Bruce R. Blazar, Ryan Bjordahl, Bahram Valamehr, and Jeffrey S. Miller. "NK Cells Lacking CD38 Are Resistant to Oxidative Stress-Induced Death." Blood 134, Supplement_1 (November 13, 2019): 3215. http://dx.doi.org/10.1182/blood-2019-124490.
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Cytolytic effector lymphocytes must contend with unfavorable microenvironments when infiltrating sites of infection or malignancy. Tumor cells typically have high levels of oxidative stress and produce reactive oxygen species (ROS) that suppress the cytotoxic functions of both natural killer (NK) cells and CD8+ T cells. Levels of activated granulocytes that release ROS are also elevated in cancer patients. Free radicals, such as ROS, cause detrimental cellular effects including protein oxidation, lipid peroxidation and DNA damage. Chronic viral infections, including CMV, are also associated with increased oxidative stress. We hypothesized that adaptive NK cells, which arise specifically in response to CMV, could have properties that allow these cells to persist and retain function in high oxidative stress environments. Adaptive NK cells are present in the peripheral blood of many otherwise healthy CMV seropositive individuals and expand in response to CMV reactivation in hematopoietic cell transplant (HCT) patients. Mounting evidence suggests that CMV peptides presented by HLA-E on infected cells can trigger the expansion of adaptive NK cells expressing the activating receptor NKG2C. The majority of NKG2C-positive adaptive NK cells co-express the terminal maturation marker CD57. Work by our group and others has shown that adaptive NK cells exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) and interferon (IFN)-γ production relative to canonical NK cells, appear to persist long-term and have metabolic attributes similar to memory CD8+ T cells. We also reported clinical correlations between adaptive NK cell numbers and reduced relapse risk in HCT patients with hematologic malignancies. Here, we show that CD38 expression is markedly reduced on adaptive NK cells from CMV seropositive individuals. This observation was first made from analyses of RNA-seq data comparing adaptive and canonical NK cells and was validated by flow cytometry (Figure 1A). CD38 is expressed both intracellularly and on the plasma membrane and functions as an NADase, degrading nicotinamide adenine dinucleotide (NAD+) into ADP-ribose and nicotinamide. NAD+ is a necessary cofactor for the sirturin family of protein deacetylases, which protect against oxidative stress. We hypothesized that CD38 downregulation in adaptive NK cells could be associated with more resistance to oxidative stress-induced cell death through increased NAD+ levels and sirturin activity. To determine whether there was a connection between CD38 expression and resistance to oxidative stress, we isolated NK cells from the peripheral blood of CMV seropositive donors and cultured them overnight with or without 15 mM H2O2 (hydrogen peroxide), known to induce oxidative stress and cell death. We found that NKG2C+ adaptive NK cells were markedly more resistant to oxidative stress-induced cell death compared to NKG2C-negative canonical NK cells as determined by annexin V and a fixable amine-reactive dye (LIVE/DEAD) that can permeate damaged membranes of dead cells and react with interior amines (Figure 1B). Similar results were observed in assays where NK cells from CMV seropositive donors were co-cultured with neutrophils pre-activated with phorbol 12-myristate 13-acetate (PMA) to induce the release of reactive oxygen species. To determine whether CD38 expression is directly associated with the NK cell response to oxidative stress, we generated induced pluripotent stem cell (iPSC) lacking CD38 through CRISPR/Cas9 gene editing that were differentiated into NK cells and tested for their ability to resist oxidative stress-induced death. Compared to control iPSC-derived NK (iNK) cells that express high levels of CD38 (Figure 1C), a substantially larger percentage of CD38 knockout iNK cells were viable when cultured overnight with H2O2 (Figure 1D). Our results have implications for adoptive immunotherapy to treat patients with cancer where a major goal is to manufacture cytotoxic cells that can persist and function in a tumor environment that contains high levels of oxidative radicals. We are exploring other cell stressors of high translational relevance such as freeze/thaw stress in adaptive and CD38 knockout cells that will be critical for cell therapy platforms. Disclosures Cichocki: Fate Therapeutics, Inc: Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bjordahl:Fate Therapeutics, Inc.: Employment. Valamehr:Fate Therapeutics, Inc: Employment. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: NK cells
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Jing, Xu-Bin, Xian-Bin Cai, Hui Hu, Su-Zuan Chen, Bin-Ming Chen, and Ju-Yu Cai. "Reactive oxygen species and mitochondrial membrane potential are modulated during CDDP-induced apoptosis in EC-109 cellsThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process." Biochemistry and Cell Biology 85, no. 2 (April 2007): 265–71. http://dx.doi.org/10.1139/o07-014.
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cis-Diamminedichloroplatinum (CDDP), commonly know as cisplatin, is a well known DNA-damaging agent, which is highly active in suppressing the proliferation of tumor cells. However, it is not clear that CDDP can induce growth inhibition of esophagus cancer cells. Using the cell line EC-109 from the esophagus, we found that CDDP would induce apoptotic responses. The addition of CDDP to cells led to the inhibition of growth in a time- and dose-dependent manner. CDDP generated reactive oxygen species (ROSs) in cells, which brought about a reduction in the intracellular mitochondrial transmembrane potential (Δψm), leading to apoptosis. Our findings demonstrate that ROSs, and the resulting oxidative stress, play a pivotal role in apoptosis. Preincubation of EC-109 cells with the hydrogen-peroxide-scavenging enzyme catalase partially inhibited the following: (i) the production of ROS; (ii) the disruption of the Δψm; and (iii) apoptosis. These results indicate that the enhancement of the generation of ROS and the disruption of Δψm are events involved in the apoptotic pathway of EC-109 induced by CDDP.
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Devin, Julie, Elena Viziteu, Laurie Herviou, Anja Seckinger, Grandmougin Camille, Hartmut Goldschmidt, Laure Vincent, et al. "Inhibition of SUV39H Methyltransferase As a Potent Therapeutic Target in Multiple Myeloma." Blood 126, no. 23 (December 3, 2015): 1771. http://dx.doi.org/10.1182/blood.v126.23.1771.1771.
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Abstract Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the SUV39H1 histone methyltransferase. SUV39H1 and SUV39H2 are regulators of chromatin organization. SUV39H1-dependent trimethylation of H3K9 is essential for maintenance of both pericentromeric and telomeric heterochromatin. SUV39H1 deficiency reduced cell viability severely and is associated to heterochromatin decompaction, loss of silencing, genome instability, and a wide range of defects in cell cycle, cell growth, and meiosis. SUV39H1-mediated H3K9me has been linked to gene silencing of the tumor suppressor genes, such as p15INK4B and E-cadherin, in acute myeloid leukemia (AML). Therefore, it is highly possible that the default function of SUV39H1 is to maintain genome stability by limiting the acute activation of oncogenes while its dysregulation could cause tumor formation. We reported that high SUV39H1 expression, in MM cells, is associated with a poor prognosis in two independent cohorts of patients (Heidelberg-Montpellier cohort - N=206 and UAMS-TT2 cohort - N=345). SUV39H1 expression was downregulated by conditional shRNA expression through lentiviral delivery. SUV39H1 knock down significantly inhibits H3K9me3, growth of myeloma cells, induces apoptosis, cell cycle deregulation, reactive oxygen species production and spontaneous accumulation of DNA double strand breaks. According to these results, SUV39H1 depletion sensitizes myeloma cells to melphalan. Chaetocin is a selective inhibitor of SUV39H1. We identified that chaetocin has anti-myeloma effects at low nanomolar doses (range: 4 to 17 nM), on 11 different human myeloma cell lines, that are representative of the molecular heterogeneity of the patients, in association with H3K9 trimethylation inhibition. Furthermore, this significant toxicity of chaetocin in MM was confirmed on primary myeloma cells of 5 patients cocultured with their bone marrow microenvironment without significant toxicity on normal bone marrow cells and hematopoietic stem cells. Interestingly, the IC50 doses of chaetocin in MM were 50 fold lower compared to results published in AML, suggesting H3K9 histone methyltransferases could be a potent therapeutic target in MM. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Goldschmidt:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau. Hose:EngMab AG: Research Funding; Takeda: Other: Travel grant.
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Rozovski, Uri, David M. Harris, Ping Li, Zhiming Liu, Taghi Manshouri, Ivo Veletic, Preetesh Jain, et al. "Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL." Blood 134, Supplement_1 (November 13, 2019): 3024. http://dx.doi.org/10.1182/blood-2019-124183.
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Human pentraxins are a family of proteins with a unique pentameric structure. Unlike C-reactive protein (CRP), serum amyloid P (SAP) and pentraxin-3 (PTX3) play an opposite role in tissue remodeling. PTX3 induces whereas SAP inhibits the differentiation of CD14+ monocytes into fiborcytes. While in patients with CLL CRP levels are high and were found to be associated with poor overall survival (OS) (Herishanu et al. Ann Med 2017), little is known about the plasma levels or clinical significance of other pentraxins in CLL. Therefore, we obtained plasma sample from 36 randomly chosen treatment-naïve CLL patients and 12 age-matched healthy individuals and, using an enzyme linked immuno-sorbent assay, found that PTX3, CRP and SAP plasma levels were significantly higher in CLL patients than in healthy individuals (P<.001, P.002 and P<.001, respectively). Because PTX3 induces differentiation of CD14+ monocytes into fiborcytes known to induce bone marrow (BM) fibrosis in myelofibrosis (MF) (Verstovsek et al. J Exp Med, 2016), and because a retrospective analysis of 176 CLL patients (Tadmor et al. Cancer, 2013) detected reticulin fibrosis in CLL patients' BM and showed that the degree of BM fibrosis was associated with thrombocytopenia, anemia, elevated β2M, 11q- (P<.0015), and OS (P<.0001), we analyzed BM biopsy specimens of 8 randomly selected CLL patients and 3 healthy donors. Using fluorescent immuno-histochemistry we detected grade 1 to 2 reticulin fibrosis and an increased number of fibrocytes co-expressing CD45, CD68 and pro-collagen-I in 8 of 8 CLL patients' but not in normal donors' BM sections. Whereas CRP and SAP are produced by the liver, PTX3 was found to be released from macrophages and endothelial cells. Because in patients with CLL is characterized by an increased number of circulating CLL cells, we wondered whether CLL cells produce PTX3. Using flow cytometry we found that approximately 50% of CD5+/CD19+ CLL cells co-expressed intracellular PTX3, and using Western immunoblotting we detected PTX3 protein in 7 out of 7 CLL patients' cell extracts but not in normal B cells. Therefore we sought to determine why CLL cells produce PTX3. In MF, characterized by constitutive activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, high PTX3 levels constitute an independent indicator of disease burden, clonal expansion and OS (Veletic, et al. Br J Haemat 2019). In CLL STAT3 is constitutively phosphorylated on serine 727 residues and activates genes known to be activated by phosphotyrosine STAT3. Therefore we postulated that phosphoserine STAT3 induces the production of PTX3 in CLL cells. To test our hypothesis we performed western immunoblotting and found PTX3 protein in 6 of 6 CLL patients' cell extracts, and by using flow cytometry we found that intracellular phosphoserine STAT3 and PTX3 are co-expressed in 77% of CLL cells. Then, using chromatin immunoprecipitation (ChIP) we found that STAT3 protein co-immunoprecipitated with DNA of PTX3 and with DNA of the STAT3-target genes C-Myc, ROR1, VEGF, and STAT3. Sequence analysis of the PTX3 gene promoter identified four γ-interferon activation sequence (GAS)-like elements, known as putative STAT3-binding sites spanning within 630 base pairs upstream the PTX3 start codon. To determine which putative binding site binds STAT3 we designed three different probes. Using ChIP we found that STAT3 protein co-immunoprecipitated all 3 STAT3-putative binding sites at high affinity. Similar results, obtained with IL-6-stimulated MM1 cells, further confirmed these findings. Then, using an electromobility shift assay (EMSA) of CLL cell nuclear extracts from 3 different patients we detected STAT3-PTX3 DNA complexes with DNA probes, designed to detect the PTX3 gene promoter binding sites, and found that anti-STAT3 antibodies significantly attenuated the binding, confirming that STAT3 binds to the PTX3 gene promoter. To further confirm these data, we transfected CLL cells with STAT3-short hairpin (sh)RNA and found that STAT3-shRNA significantly downregulated STAT3 and PTX3 mRNA levels. In conclusion, we found that constitutively activated STAT3 activates the PTX3 gene and induces the production of high levels of PTX3 in patients with CLL. PTX3, known to induce differentiation of CD14+ monocytes into fibrocytes, enhances the production of BM fibrocytes that induce BM reticulin fibrosis in patients with CLL. Disclosures Burger: Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; BeiGene: Research Funding; AstraZeneca: Honoraria. Bose:Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Constellation: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; CTI BioPharma: Research Funding. Thompson:Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Research Funding. Jain:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding. Wierda:Cyclcel: Research Funding; AbbVie: Research Funding; Juno Therapeutics: Research Funding; KITE pharma: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Acerta Pharma Inc: Research Funding; Loxo Oncology Inc.: Research Funding; Sunesis: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Xencor: Research Funding; Janssen: Research Funding; Gilead Sciences: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding.
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Mongkolrattanakul, Pannawat, Jackrapong Bruminhent, Tanaya Siripoon, Punlop Wiwattanathum, Suchin Worawichawong, and Susarak Kantachuvesiri. "1382. A Prospective Epidemiological Study BK Polyomavirus DNAuria and DNAemia within the First Year after Kidney Transplantation." Open Forum Infectious Diseases 8, Supplement_1 (November 1, 2021): S777. http://dx.doi.org/10.1093/ofid/ofab466.1574.
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Abstract Background Screening and early detection for the preceding BK polyomavirus (BKV) DNAuria and DNAemia to prevent the occurrence of BK polyomavirus BKV-associated nephropathy (BKPyVAN) among kidney transplant (KT) recipients has not been universally utilized and never assessed in a setting where the resource is limited. Therefore, we aimed to investigate this entity’s incidence, risk factors, and outcome with this intervention at our institution. Methods A prospective study of KT recipients at a tertiary care transplant center in Bangkok, Thailand, was conducted between January 2019 and March 2020. All patients underwent preemptive monitoring of urine and plasma BKV DNA load, measured by quantitative real-time PCR at 1, 2, 3, 6, 9, and 12 months post-KT. Low- and high-level BKV DNAuria was defined as urine BKV DNA load of < and > 7log10 copies/mL, respectively. Low- and high-level BKV DNAemia was defined as plasma BKV DNA load of < and > 4log10 copies/mL, respectively. The incidences were calculated by Kaplan-Meier analysis. The chi-square or student’s T-test compared clinical characteristics between those with and without high-level BKV DNAuria as appropriate. Risk factors of high-level BKV DNAuria were analyzed using Cox proportional hazard model. Results Among 99 evaluable KT recipients, a mean (SD) age was 42 (11) years, 64.6% were male, and 69.6% received an induction immunosuppressive therapy. Within 12 months post-KT, the incidences of low-level BKV DNAuria, high-level BKV DNAuria, low-level BKV DNAemia, and high-level BKV DNAemia were 22.63%, 13.14%, 9.49%, and 5.11%, respectively. High panel reactive antibody (PRA) was associated with high-level BKV DNAuria at 6 and 12 months, (HR 1.02 [95% CI (1.00-1.04)], P=0.019) and (HR 1.02 [95% CI (1.00-1.04)], P=0.023), respectively. Underlying diabetes mellitus was associated with high-level BKV DNAuria (HR 3.49 [95% CI (1.28-9.51)], P=0.015) at six months; however, not at 12 months. There was no allograft rejection directly related to a reduction of immunosuppression for BKV infection observed. Conclusion BKPyV infection is also prevalent among KT recipients in a resource-limited setting, however, without unfavorable consequence. Those with high-level PRA and underlying diabetes could be at risk of high-level BKV DNAuria after KT. Disclosures All Authors: No reported disclosures
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Cea, Michele, Antonia Cagnetta, Yu-Tzu Tai, Mariateresa Fulciniti, Teru Hideshima, Dharminder Chauhan, Sun-Young Kong, et al. "Targeting NAD+ Salvage Pathway Induces Autophagy in Multiple Myeloma Cells." Blood 118, no. 21 (November 18, 2011): 2920. http://dx.doi.org/10.1182/blood.v118.21.2920.2920.
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Abstract Abstract 2920 Background: Nicotinamide adenine dinucleotide (NAD+) is a coenzyme crucially involved in several cellular functions, including energy metabolism, reactive oxygen species scavenging, DNA repair, and gene expression. Intracellular NAD+ stores are continuously replenished through pathways whose activity depends on the tissue and availability of substrates. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. During neoplastic transformation, Nampt is upregulated to compensate for increased metabolic demands. It promotes myeloid and lymphoid differentiation and increases specific cytokine production (TNF- α, IL-6 and VEGF). Importantly, cancer and leukaemia cells appear to be more sensitive to Nampt inhibitor drugs than normal cells. The reasons for this selectivity are not fully understood, but may include aberrant metabolic demands and increased reliance on NAD+-dependent enzymes. Promising results obtained with Nampt inhibitors (such as FK866) in preclinical cancer models suggest that Nampt activity represents an innovative therapeutic target for novel anticancer agents. Methods: A panel of eighteen different MM cell lines, both sensitive and resistant to conventional and novel anti-myeloma drugs, as well as patient MM cells were used in the study. The mechanism of action of FK866 was investigated by Annexin-V/propidium iodide staining, thymidine incorporation, Western-blotting, and with lentivirus-mediated shRNAs. For the autophagy assay, EGFP-LC3+ cells were treated with FK866 and the number of GFP-LC3 punctae was analyzed and quantified by fluorescence microscopy and flow cytometry, respectivley. Intracellular NAD+ content was measured using a biochemical assay. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assay. Results: To study the role of Nampt in MM cells, we performed a protein analysis of this enzyme in eighteen MM cell lines. Nampt is constitutively activated in all cell lines tested. Moreover, patient MM cells highly express this enzyme whereas normal cells lack this protein. Indeed, the Nampt inhibitor FK866 decreased MM cell line viability in a dose and time dependent manner, with an IC50 ranging from 3–30nM. Similar results were observed in patient MM cells. Importantly, FK866 did not inhibit viability of normal peripheral blood mononuclear cells. Tritiated thymidine uptake assay confirmed the antiproliferative effects of FK866 in MM cell lines and patient cells. To examine the mechansim of action, we showed that intracellular NAD+ levels decreased with FK866 treatment at 24 and 48 hours. Furthermore, knock-down of Nampt by small interfering RNAs caused significant inhibition of MM cell growth. FK866 triggered anti-MM activity in our models of MM in the bone marrow (BM) microenvironment, confirming its ability to overcome the proliferative advantage conferred by the BM milieu. FK866 treatment also inhibited angiogenesis via suppression of pivotal MM pathways PI3K/AKT and ERK. In further studies to delineate its mechanisms of action, no activation of apoptosis was observed in treated-cells. Instead FK866 treatment resulted in a marked increase in autophagy, evidenced by autophagic vacuoles in the cytoplasm and proteolitic processing of endogenous LC3-I to LC3-II. FK866 inhibited mTOR signaling and triggered increased formation of EGFP-LC3 punctae, confirming involvement of autophagic cell death. Finally, combined FK866 with bortezomib (CI < 0.6), melphalan (CI < 0.9), and dexamethasone (CI < 0.8), induced synergistic cytotoxicity against MM cells. Conclusion: Our data therefore show a pivotal role of Nampt in MM cell growth, survival and drug resistance. The ability of FK866 to inhibit Nampt activity strongly supports its clinical evaluation to improve patient outcome in MM. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.
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Cosío, Silvia Gutiérrez, Esteban Ballestar, Carlos Santamaría, Belen Blanco, Luis Ignacio Sánchez Abarca, Teresa Caballero Velázquez, Carmen Herrero Sánchez, et al. "Effect of 5-Azacytidine (5-AzaC) In the Expression of PRAME In Acute Myeloid Leukemia (AML)." Blood 116, no. 21 (November 19, 2010): 3615. http://dx.doi.org/10.1182/blood.v116.21.3615.3615.
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Abstract Abstract 3615 Introduction: Preferentially expressed antigen of melanoma (PRAME) was first isolated as a human melanoma antigen by cDNA expression cloning using melanoma-reactive cytotoxic Tcells (CTL). PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies and solid tumors. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). In addition, several authors have suggested that PRAME could be used as a target for anticancer T-cell therapy. PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies. Methods: We analyzed PRAME mRNA expression of blast cells from AML patients at diagnosis versus CD34+ stem cells from healthy donors by RT-PCR without treatment or after exposure to 1mM 5-azaC during the four days of culture and correlated the expression of PRAME with the methylation status of the promoter. Results: PRAME is significantly over-expressed in blasts from AML patients (n=11) compared with normal CD34+ cells (n=8) ((700±1102 vs. 1.8±2.5 p=0.002). Interestingly, we found an inverse correlation between PRAME expression and the degree of methylation in the promoter among both AML samples and healthy donors (r=-0.77 p=0.010). In order to evaluate the effect of 5-azaC on PRAME gene expression, we treated blast cells and CD34+ cells from healthy donors with the drug and we observed that the exposure to the drug induced a decrease in the percentage of methylation in the promoter and subsequently increased the expression of PRAME but, interestingly, the higher the basal methylation of the promoter the more intense the effect of the drug among AML cells. By contrast, CD34+ cells from healthy donors were resistant to the effect of the drug so that no significant changes were observed neither in terms of methylation status of the promoter nor in the expression of PRAME prior to or after exposure to the drug among healthy donors. Conclusions: The promoter region is highly methylated in normal CD34+ cells compared to AML cells and this pattern correlates with a higher expression of PRAME in blasts. Furthermore, the level of PRAME methylation was reduced in AML patients after exposure to 5-azaC which correlated with an increase in the expression of PRAME. By contrast, the effect of 5-azaC on the methylation pattern of the promoter was significantly lower in CD34+ cells from healthy donors. Disclosures: Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: Membership on an entity's Board of Directors or advisory committees; millennium: Membership on an entity's Board of Directors or advisory committees. Off Label Use: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is to increase PRAME expression in blasts from AML patients and generate CTL CD8+ specific response against tumor cells.
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Das, Deepika Sharma, Ze Tian, Arghya Ray, Durgadevi Ravillah, Yan Song, Paul G. Richardson, Bryan Oronsky, Jan Scicinski, Dharminder Chauhan, and Kenneth C. Anderson. "Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001." Blood 124, no. 21 (December 6, 2014): 4712. http://dx.doi.org/10.1182/blood.v124.21.4712.4712.
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Abstract Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA. Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance. Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.
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Zanetti, Costanza, Joscha Ender, Mark Hartmann, Joschka Hey, Parimala Sonika Godavarthy, Eva Weissenberger, Rahul Kumar, et al. "The Influence of the Age of the Bone Marrow Microenvironment on Leukaemia Progression." Blood 134, Supplement_1 (November 13, 2019): 2748. http://dx.doi.org/10.1182/blood-2019-122797.
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B-cell acute lymphoblastic leukaemia (B-ALL) occurs most commonly in children, while chronic myeloid leukaemia (CML) is more frequent in adults. So far, the myeloid bias of haematopoiesis in elderly people has been considered the main reason for these differences in leukaemia phenotype in different age groups. However, differential contribution of a young versus an old bone marrow (BM) microenvironment (BMM) to leukaemia development may have been underrecognized given the fact that the BMM undergoes constant remodelling during a lifetime. Using a murine transduction/transplantation model of BCR-ABL1+ B-ALL and CML, we recapitulated the human phenotype, whereby young (3-4 weeks old) recipient mice, which received the same number and type of leukaemia-initiating cells as their old (>30 weeks old) counterparts, died significantly earlier of B-ALL, while showing prolonged survival in the CML model. Engraftment of CML-initiating cells in young bone marrow (BM) was decreased and myeloid progenitors in young mice were reduced in the CML model compared to old mice. In contrast, homing of B-ALL-initiating cells to a young BMM was increased, and by in vivo 2-photon microscopy we observed that B-ALL-initiating cells homed to locations closer to bone and vessels and migrated faster in young compared to old mice. In in vitro coculture assays we showed that BCR-ABL1+ B-ALL cells proliferate, migrate and adhere significantly more in the presence of young BM macrophages or in conditioned medium from these macrophages, while CML cells showed stronger adhesion and proliferation in the presence of old BM macrophages. Old macrophages showed hallmarks of ageing such as an increase of reactive oxygen species, increased DNA damage, proliferative defects and mitochondrial alterations compared to young macrophages. In addition, genome-wide profiling of chromatin accessibility using ATAC-seq revealed strong differences between young and old bone marrow-derived macrophages and an enrichment of inflammatory response gene sets, which includes IL-6/Jak/Stat3 signalling, as well as the C-X-C motif chemokine (CXCL) 13. Cxcl13 is a chemoattractant for B cells and we showed its expression to be upregulated in young versus old BM macrophages and to have higher levels in a young versus an old BMM. Inhibition or knockdown of Cxcl13 in BMM-derived macrophages led to a decrease in proliferation of cocultured B-ALL cells and impaired migration of leukaemia cells towards young BMM-derived macrophages. Consistently, deficiency of Cxcr5, the receptor for Cxcl13, on B-ALL-initiating cells prolonged murine survival in our B-ALL model. In support of our murine data, decreased CXCR5 expression was associated with improved outcome in human hyperdiploid B-ALL and revealed a trend towards improved outcome in BCR-ABL1+ B-ALL and other subtypes. Taken together, our study shows that the age of the BMM and, in particular, BM macrophages influence the leukemia phenotype. The CXCL13-CXCR5 axis may act as prognostic marker or an attractive target for the treatment of B-ALL. Disclosures Kumar: Merck: Research Funding; European Patent No. 16187926.7: Other: USE OF FIBRONECTIN OR ILK INHIBITORS FOR USE IN THE TREATMENT OF LEUKEMIA. Stock:Agios: Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding. Lipka:InfectoPharm GmbH: Employment. Krause:Merck KGaA: Research Funding; Patent: Patents & Royalties: European Patents No. 16187926.7-1401, EP18184430.9-.
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Kirschner, Martin M., Mirle Schemionek, Claudia Schubert, Nicolas Chatain, Stephanie Sontag, Susanne Isfort, Nadina Ortiz-Brüchle, et al. "Dissecting Genomic Aberrations In CML and Bcr-Abl Negative Myeloproliferative Neoplasms By The Use Of Multiplex-PCR and Parallel Resequencing." Blood 122, no. 21 (November 15, 2013): 1612. http://dx.doi.org/10.1182/blood.v122.21.1612.1612.
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Abstract Introduction Next-Generation Sequencing holds the promise of comprehensive analysis of molecular aberrations in human malignancies and therapeutic approaches individually tailored to each patient. Methods We investigated the use of a multiplex-PCR (TruseqAmplicon Cancer Panel, Illumina) of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes to identify mutations in a cohort of patients with myeloproliferativeneoplasms (MPN). After signed informed consent, samples from 59 patients with MPN (19 MF [8 PMF, 11 post-PV/ET-MF], 14 PV, 10 ET, 10 CML, 4 HES, and 2 SM), two patients with reactive erythrocytosis, and two anonymized healthy controls as well as six myeloid cell lines (K562, HEL, HMC1, SUPB15, HL60, U937) were analyzed on a Miseq sequencer (Illumina), using 250 ng of genomic DNA from peripheral blood -derived cells. Results Altogether, the quality of the sequencing runs was very good, with Q30 values above 90%. 151 bidirectional cycles were performed, yielding between 2 and 6 Gigabases of sequencing data.Healthy donor and reactive erythrocytosis samples showed several SNPs but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of a TP53 frameshift mutation (c.405_406insC; in 98% of transcripts) in K562, JAK2 V617F (100%) and TP53 M133K (99%) mutations in HEL, two heterozygous KIT mutations (V560G in 51% and D816V in 52%) and a TP53 C277F (16%) mutation in HMC1, while SUP-B15, HL60, and U937 showed no abnormality in the tested gene set.JAK2 V617F was present in all PV, 4 of 10 ET, and 14 of 19 MF patients.The JAK2 V617F allele burden was significantly higher in MF than ET (p=0.026) but not PV (71+/-27% vs. 33+/-22% vs. 55+/-29%, respectively). Further analysis detected a previously described G12V NRAS mutation (13% of transcripts) in a patient with JAK2 V617F negative PMF and an additional IDH1 R132H mutation (24%) in a JAK2 V617F positive (46%) MF patient with 20% basophils and hyperhistaminemia. Another JAK2 V617F positive (31%) MF sample showed an E255G ABL mutation (10%). One patient with JAK2 V617F negative ET showed an ERBB2 A847D sequence variant (50%). Moreover, an S935N CSF1R mutation (17%) and a V125G IDH1 mutation (9%) were each detected in one case of PV, but the biological relevance remains unclear so far. Four patients with CML-CP (n=3) or –AP (n=1) showed subclones with sequence variants in the HNF1A gene, with two S304P changes (9 and 10% of transcripts) and two 872delC mutations (6 and 5%), the latter of which have already been implicated in colon cancer. Two patients with CML-CP showed KIT mutations (a V532I mutation and a known oncogenic mutation V530I). This latter patient also harbored the known E255K ABL mutation – leading to imatinib resistance. Interestingly, this patient showed a good response to dasatinib (which is also active against KIT) but not to bosutinib (which has no activity against KIT). These data suggest that HNF1A and KIT may play a role in CML pathogenesis. One patient with lymphoid BC/Ph+ ALL who had a T315I ABL mutation and was treated with ponatinib, was found to harbor a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib had led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation may have caused ponatinib resistance in this patient. Additionally, other not yet defined aberrations may have been responsible for the observed resistance. Finally, while both SM patients were negative for KIT D816V, one of them harbored a KRAS 436G>A(146A>T) mutation (34%) which is a known oncogene in colorectal cancer and may thus also play a role in SM pathogenesis. We are currently generating induced pluripotent stem cells from patients harboring selected mutations described above in order to better be able to study the functional properties of genetically unstable malignant stem cell populations. Conclusion Amplicon-based next-generation sequencing may uncover additional oncogenic mutations in patients with MPN, potentially explaining therapy resistance and opening new therapeutic options for individual patients. Disclosures: Off Label Use: Two individual patients mentioned that were treated with ponatinib or bosutinib within compassionate use trials before these drugs were approved for the indication. Bruemmendorf:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria. Koschmieder:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.
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Thompson, Meghan C., Matthew S. Davids, Nitin Jain, Jacob D. Soumerai, Praveen Ramakrishnan Geethakumari, Amy Gubits, Denice Hickman, et al. "Phase 1 and Dose Expansion Study of APR-246 in Combination with Ibrutinib or Venetoclax-Based Therapy in Subjects with TP53-Mutant Relapsed and/or Refractory Non-Hodgkin Lymphomas (NHL) Including Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL)." Blood 136, Supplement 1 (November 5, 2020): 15–16. http://dx.doi.org/10.1182/blood-2020-139022.
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Background: Outcomes for CLL pts with TP53 aberrancy including deletion of 17p (del17p) or TP53 mutations treated with chemo+/-immunotherapy (CIT) have been historically poor. Despite improvements, pts with TP53 aberrancy treated with novel agent-based regimens such as ibrutinib (ibr), acalabrutinib and venetoclax (ven), still have inferior outcomes as compared to pts with intact TP53. In 5 year follow-up data from relapsed/refractory (R/R) CLL pts treated with ibr, pts with del17p had a shorter median progression free survival (PFS) and overall survival (OS) (26 and 57 months) vs. the overall cohort (median PFS 51 months, OS not reached, O'Brien et al Blood 2018). Additionally, for R/R CLL pts treated with 24 months of ven and rituximab (VR), del17p and/or a TP53 mutation was associated with an increased risk of CLL progression after stopping ven (p=0.01, Kater et al JCO 2019). In addition, pts with mantle cell lymphoma (MCL) with TP53 mutations have a poor response to CIT and autologous stem cell transplantation. In a series of MCL pts who discontinued ibr, 75% who discontinued for progression harbored TP53 alterations (Jain et al Br J Haematol 2018). These studies highlight an unmet need for improved treatments for CLL and MCL pts with TP53 mutations. APR-246 is a novel small molecule that is converted to methylene quinuclidinone (MQ), a reactive electrophile that forms a covalent bond with the p53 core domain to reactivate mutant p53 and restore wild type p53 function and apoptotic activity (Zhang et al Cell Death Disease 2018). Additionally, APR-246 has been shown to deplete glutathione and induce reactive oxygen species (Liu et al Nat Commun 2017). Clinical activity has been demonstrated in phase II studies of APR-246 + azacitadine in TP53 mutant MDS (ORR 88%, CR 61%, Sallman et al ASH 2019; ORR 75%, CR 57%, Cluzeau T et al EHA 2020). A phase I trial of APR-246 in combination with venetoclax is ongoing in AML (NCT04214860). APR-246 induces apoptosis in TP53 mutated CLL cells (Jaskova et al, Leuk Res 2020) and single agent activity in CLL has been described in the APR-246 first-in-human clinical trial (Lehmann S et al JCO 2012; Deneberg S et al Blood Canc J 2016). Study Design and Methods: This is a phase 1, open-label, 3+3 dose de-escalation and dose expansion study investigating APR-246 in combination with (cohort 1) ibrutinib or (cohort 2) VR in pts with R/R TP53 mutant CLL or MCL (Figure 1). The safety lead-in portion of the study includes two safety cohorts of CLL pts with TP53 mutations: APR-246 in combination with (1) ibrutinib (ibr), n~28 pts or (2) APR-246 in combination with VR, n=~28 pts. Treatment will be administered according to Figure 2. Eligible pts must have a TP53 mutation. Based on the results of an integrated assessment of the safety, tolerability and preliminary clinical activity in the safety lead-in cohorts, ibr and/or VR will be selected for further study in combination with APR-246 in a dose expansion portion of the study which will include pts with TP53-mutant (1) R/R CLL (n≤20) and (2) R/R MCL (≤40). The primary study endpoints will be (1) the occurrence of DLTs according to the NCI CTCAE, version 5.0, (2) the frequency of treated-emergent adverse events (AE) and SAE, and (3) the recommended phase 2 dose (RP2D) of APR-246 in combination with ibr or VR. Secondary study endpoints include pharmacokinetic parameters, the complete response rate, objective response rate, duration of response and PFS for APR-246 in combination with ibr or VR. Correlative studies are planned to examine the effect of APR-246 combinations on the p53 pathway and examine genome and transcriptome correlates of response and resistance. Patient samples will be collected at multiple timepoints to measure p53 protein expression by immunoblot of protein lysates from mononuclear cells. Specifically, p53, BCL2, BAX, NOXA and PUMA levels will be examined to assess the effect of APR-246 + ibr or VR on the p53 pathway. These studies will be complemented by BH3 profiling, a functional technique to assess the propensity of the tumor cells to undergo apoptosis and their dependence on specific anti-apoptotic proteins. Additionally, intracellular flow cytometry will evaluate the effect of the combinations on key markers within the p53 pathway. DNA and RNA sequencing will be performed to identify potential biomarkers of response. This study is planned to be open for enrollment by September 2020 at the first study site and is planned to open at up to 10 study sites. Disclosures Davids: Bristol Myers Squibb: Research Funding; Pharmacyclics: Consultancy, Research Funding; Surface Oncology: Research Funding; Merck: Consultancy; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Research to Practice: Honoraria; Zentalis: Consultancy; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; AbbVie: Consultancy; Adaptive Biotechnologies: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Janssen: Consultancy; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Sunesis: Consultancy. Jain:Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Aprea Therapeutics: Research Funding; Precision Bioscienes: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Fate Therapeutics: Research Funding; BMS: Research Funding; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; TG Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Soumerai:AstraZeneca: Consultancy; AbbVie: Consultancy; TG Therapeutics: Research Funding; Beigene: Consultancy, Research Funding; BostonGene: Research Funding; Genentech/Roche: Research Funding; GlaxoSmithKine: Research Funding; Verastem: Consultancy. Gubits:Aprea Therapeutics: Current Employment. Hickman:Aprea Therapeutics: Current Employment. Wennborg:Aprea Therapeutics: Current Employment, Current equity holder in publicly-traded company. Attar:Aprea Therapeutics: Current Employment. Abdel-Wahab:Merck: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy. Mato:TG Therapeutics: Consultancy, Other: DSMB, Research Funding; Adaptive: Consultancy, Research Funding; BeiGene: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; LOXO: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding.
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Klose, Nathan, Michael R. Tallack, Graham Magor, Peter Mollee, and Andrew C. Perkins. "Rapid Molecular Diagnosis Of JAK2V617F Negative MPN By Targeted Deep Sequencing Using The Ion Torrent PGM." Blood 122, no. 21 (November 15, 2013): 4093. http://dx.doi.org/10.1182/blood.v122.21.4093.4093.
Full textAbstract:
Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells, increased risk of thrombotic complications and slow progression to myelofibrosis or, less often, leukemia. Activation of the JAK-STAT signaling pathway is a common underlying feature of these diseases and JAK kinase inhibitors are efficacious in the more advanced forms of disease. Most cases of polycythemia vera (PV) and approximately 60% of essential thrombocythemia (ET) and primary myelofibrosis (MF) harbor a point mutation in JAK2 (V617F) which leads to constitutive JAK-STAT signaling and factor independent cell growth. The remaining 40% of cases of MF and ET harbor a broad range of mutations in many genes including those involved in cytokine receptor signaling, other components or the JAK-STAT pathway or epigenetic regulators. This poses a challenge for rapid molecular diagnosis. Also, since ET is essentially a diagnosis of exclusion of reactive causes of thrombocytosis, many cases of chronic ‘ET’ may not be clonal hematological neoplasms but reactive conditions. We have developed a rapid deep sequencing pipeline to detect mutations in 65 genes which have been implicated in MPN through previous reports of human mutations, mouse models of MPN, or other known components of hematopoietic cytokine receptor signaling. We used 10ng of DNA from blood to amplify and sequence all the exons of these 65 genes using Ampliseq and Ion Torrent PGM. Using 318 PGM chips and 8-fold multiplexing we achieved on average 200 fold coverage of the target exome. The bioinformatics of SNP validation and rapid generation of reports will be presented. From a pilot study of 30 cases referred for molecular diagnosis, we have detected the likely causative mutation in approximately 80% of ET and MF where JAK2 is wild type. Many of these mutations are known to be causative in MPN, including those in MPL, ASXL1, SET2, SH2B3 (LNK), EZH2, CBL, DNMT3A and other genes. We have identified a novel inherited mutation in a family with MPN and validated it in BAF3 factor-independency assays. We have identified further novel mutations in JAK3, EED, DNMT3A, APC and two phosphatases involved in silencing activated JAK-STAT pathway components. The biological significance of these is under investigation and progress will be reported. In many cases we find evidence for clonal evolution involving secondary mutations in epigenetic modifying proteins on top of driver mutations in the JAK-STAT pathway. In short, targeted exome re-sequencing using Ampliseq and Ion Torrent PGM provides a rapid and relatively cheap method for molecular diagnosis and characterization of most cases of MPN. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.
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Figueroa, Mary, Megan R. Rodriguez, Marcos Estecio, Yue Lu, Seyed Javad Moghaddam, Elias J. Jabbour, Marina Konopleva, Courtney D. DiNardo, and Joya Chandra. "Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation." Blood 138, Supplement 1 (November 5, 2021): 3331. http://dx.doi.org/10.1182/blood-2021-145944.
Full textAbstract:
Abstract Acute myeloid leukemia (AML) patients with any history of cigarette smoking have worse survival outcomes compared to patients that have never smoked. The molecular basis of cigarette smoking or cigarette smoke exposure (CSE) impacting AML progression or treatment response is unknown. Altered DNA methylation from smoking persists decades after quitting and has been followed in peripheral blood mononuclear cells (PBMCs) but have not been evaluated in the context of AML. Smoking also causes oxidative stress in PBMCs, but this has yet to be studied in AML patients with a history of smoking. To model how smoking worsens AML progression, we created a CSE model using a cigarette smoking robot where NOD-SCID mice received 2 hours of CSE 5 days/week or air alone. After 2 weeks of CSE, 100,000 luciferase-tagged, human AML cells were injected via tail-vein and leukemic burden was monitored by bioluminescent imaging. Two cell lines, MOLM13 and MOLM14 carrying the oncogenic fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation, when introduced into CSE mice had enhanced leukemic progression within one week (p-value <0.0001 and <0.001 respectively). MOLM14-bearing mice showed increased leukemic burden 24 days post injection (p-value<0.05). DNA methylation was evaluated by bisulfite sequencing of splenocytes from CSE mice, which was mapped to the human (AML) or mouse (host) genome. Over 200 significant changes in DNA methylation of gene promoters was seen, including hypomethylation of aryl-hydrocarbon receptor repressor (AHRR), an established hallmark of smoking in humans. Indicating that our CSE model recapitulates DNA methylation from smoking in humans. Additionally, GATA-2, a critical protein for hematopoietic differentiation known to be frequently mutated in AML, was also amongst the top hypomethylated genes. We quarried TCGA to understand the implications of altered DNA methylation in AML patients. In AML patients, low GATA-2 methylation showed decreased survival compared to those with high GATA-2 methylation (N=42/group, p-value: 0.000138). The discovery of GATA-2 methylation in smoking models and its attribution as a poor prognostic indicator in AML is novel, which underscores a need to understand the role of GATA-2 methylation in AML. Reactive oxygen species (ROS) have been implicated in AML progression, drug resistance, and are elevated in otherwise healthy smokers. No significant differences were seen in intracellular ROS in spleen or PBMCs of CSE mice; however, we found more than a two-fold increase of heme oxygenase 1 (HO-1) (p-value:0.0505), a protein involved in antioxidant responses and AML treatment resistance. There was also increased BCL-2 protein expression and a decrease in AHRR expression (p-value: 0.0098) in CSE mouse samples. This suggests that CSE causes oxidative stress and increases pro-leukemic signaling. To address if CSE impacted treatment efficacy, we treated mice with daunorubicin (2 mg/kg, twice weekly via tail-vein) once evidence of engraftment was detected. We found a trend towards increased leukemic burden compared to non-smoking mice which approached statistical significance. To study the direct impact of CSE on AML, without exposure to the tumor environment, AML cells were treated in vitro with a commercially available cigarette smoke condensate (CSC) that contains the chemicals from cigarette smoke. To mimic the in vivo CSE, MOLM13 cells received two weeks of CSC treatment before being injected into mice. At 3, 10, and 17 days post injection, mice with CSC-treated AML had enhanced leukemic burden (p-value <0.0001, <0.0001, and <0.001). This model revealed that chemicals in cigarette smoke can directly promote AML. On day 14 of CSC treatment, mirroring when the cells were injected into mice, both FLT3-ITD cell lines had increased ROS levels and or glutathione as measured by flow cytometry; this is indicative of CSC-induced oxidative stress. Cumulatively, these data define novel changes in DNA methylation and redox from smoking or tobacco products with strong potential to drive AML progression and therapy resistance. Future studies will determine if blocking these redox or methylation events can reverse the accelerated AML growth and will aid in the creation of a tailored treatment strategy for AML patients with any history of smoking. Disclosures Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Konopleva: Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; KisoJi: Research Funding; Stemline Therapeutics: Research Funding; Agios: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding. DiNardo: Novartis: Honoraria; Agios/Servier: Consultancy, Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Takeda: Honoraria; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding.
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Molenaar, Remco, Tomas Radivoyevitch, Mohammed Khurshed, Eiju Negoro, Bartlomiej P. Przychodzen, Hetty E. Carraway, Cornelis J. van Noorden, and Jaroslaw P. Maciejewski. "Clinical Effects of IDH1/2-Mutant Inhibitors in IDH1/2-Mutated Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients: Suggestions from Ex Vivo Experiments." Blood 128, no. 22 (December 2, 2016): 4308. http://dx.doi.org/10.1182/blood.v128.22.4308.4308.
Full textAbstract:
Abstract Background Isocitrate dehydrogenase 1 and 2 mutations (IDH1/2MT) occur in up to 20% of MDS, MDS/MPN overlap and (secondary) AML, 80% of secondary glioma and 20% of cholangiocarcinoma patients. IDH1/2MT confer an improved prognosis in glioma and cholangiocarcinoma patients, but not AML patients, compared with IDH1/2 wild-type (IDH1/2WT)counterparts (Fig 1). IDH1/2MT enzymes produce D-2-hydroxyglutarate (D-2HG)at the expense of NADPH, an important cellular antioxidant. IDH1/2MT are ancestral in ~30% of AML/MDS cases, rendering them attractive therapeutic targets because all cancer cells carry IDH1/2MT and this limits the chance of therapy resistance. Inhibitors of IDH1/2MT were recently developed and are currently in clinical trials. IDH1MT inhibitors protect IDH1MT solid tumor cells against irradiation and cisplatin. Thus, IDH1/2MT inhibitors and cytotoxic drugs should not be used together in patients with IDH1/2MT solid tumors. It remains unknown whether IDH1/2MT inhibitors can be safely used as adjuvants to cytotoxic drugs in patients with myeloid malignancies. Methods Primary IDH1/2MT and IDH1/2WT AML and MDS cells (n=20) were cultured and treated with D-2HG, the IDH1/2MT inhibitors, the antioxidant N-acetyl cysteine (NAC) and/or irradiation, cisplatin, 5-azacytidine, cytarabine, daunorubicin or decitabine. We determined cell death, ROS levels and DNA double strand break numbers. In addition, maximal cellular IDH1/2 and glucose-6-phosphate dehydrogenase (G6PD) activity was determined. Results After treatment with irradiation, cisplatin, 5-azacytidine, cytarabine, daunorubicin or decitabine, we did not observe more reactive oxygen species (ROS), DNA double strand breaks or cell death in IDH1/2MT primary AML/MDS cells versus IDH1/2WT primary AML/MDS cells (Fig 2). Accordingly, IDH1/2MT inhibitors or the ROS scavenger NAC had no effect on these aforementioned outcomes in IDH1/2MT primary AML/MDS cells. These results contrast the situation in glioma and carcinoma cells, where increased ROS levels, DNA double strand breaks and cell death levels were observed in irradiated or cisplatin-treated IDH1/2MT cancer cells, and where IDH1/2MT inhibitors or NAC reverse these effects (Fig 1). IDH1/2 MT were associated with a striking reduction in the IDH1/2-mediated NADPH production capacity in primary AML cells and solid tumor cells. However, IDH1/2 are not important NADPH providers in primary AML/MDS cells and the contribution of G6PD to the total NADPH production pool is ~4 times larger than IDH1/2 (Fig 3). In contrast, IDH1/2 provide 65% of all NADPH in glioma. Administration of D-2HG, the metabolite produced by IDH1/2MT enzymes, decreased IDH1/2-mediated NADPH production capacity in primary AML/MDS cells and solid tumor cells and is a likely cause of the decreased IDH1/2-mediated NADPH production in IDH1/2MT cells. Discussion Our results offer an explanation for the relatively longer survival of patients with IDH1/2MT glioma and cholangiocarcinoma and the unchanged survival of patients with IDH1/2MT AML or MDS. A plausible mechanism is the strong production of the antioxidant NAPDH by IDH1/2 in solid tumor, but not AML/MDS cells. These data imply that concomitant use of IDH1/2MT inhibitors and cytotoxic therapy in patients with IDH1/2MT solid tumors may limit therapy efficacy. However, in patients with IDH1/2MT AML or MDS, IDH1/2MT inhibitors can probably be used safely as adjuvants to AML/MDS standard of care 5-azacytidine, cytarabine, daunorubicin and/or decitabine therapies, or whole-body irradiation in the context of bone marrow transplantations. These data are crucial for the rational design of clinical trials with IDH1/2MT inhibitors and complete understanding of their outcomes. Figure 1 Increased survival of IDH1/2MT patients (A) in a glioblastoma population, (B) but not in a myeloid neoplasm population sequenced and analyzed by our groups. (C) IDH1MT radiosensitize carcinoma cells and (D) IDH1MT inhibitors radioprotect carcinoma cells. Figure 1. Increased survival of IDH1/2MT patients (A) in a glioblastoma population, (B) but not in a myeloid neoplasm population sequenced and analyzed by our groups. (C) IDH1MT radiosensitize carcinoma cells and (D) IDH1MT inhibitors radioprotect carcinoma cells. Figure 2 Primary IDH1/2MT AML/MDS cells are not sensitized to (A) 5-azacitidine, (B) cytarabine, (C) daunorubicin or (D) decitabine, compared with primary IDH1/2WT AML/MDS cells. Figure 2. Primary IDH1/2MT AML/MDS cells are not sensitized to (A) 5-azacitidine, (B) cytarabine, (C) daunorubicin or (D) decitabine, compared with primary IDH1/2WT AML/MDS cells. Figure 3 G6PD (A, C, E, G) and IDH1 activity (B, D, F, H) staining of IDH1WT (A, B, E, F) and IDH1MT (C, D, G, H) primary AML cells (A-D) and glioblastoma (E-H) cryostat sections. The amount of blue color directly reflects enzyme activity (production of NADPH). Figure 3. G6PD (A, C, E, G) and IDH1 activity (B, D, F, H) staining of IDH1WT (A, B, E, F) and IDH1MT (C, D, G, H) primary AML cells (A-D) and glioblastoma (E-H) cryostat sections. The amount of blue color directly reflects enzyme activity (production of NADPH). Disclosures Carraway: Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.
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Oksvold, Morten P., Ulrika Warpman Berglund, Helge Gad, Baoyan Bai, Trond Stokke, Idun D. Rein, Therese Pham, et al. "Karonudib Has Potent Anti-Tumor Effects in Preclinical Models of B-Cell Lymphoma." Blood 136, Supplement 1 (November 5, 2020): 18–19. http://dx.doi.org/10.1182/blood-2020-140943.
Full textAbstract:
Although chemo-immunotherapy has improved survival in B-cell lymphoma patients, refractory and relapsed disease still represents a major challenge, urging for development of new therapeutics. A new approach is to target nucleotide metabolism. Karonudib (TH1579), was developed to inhibit MutT-homologue-1/Nudix hydrolase 1 (MTH1/NUDT1), an enzyme that prevents oxidized nucleotides to be incorporated into DNA. Under normal conditions with low reactive oxygen species (ROS) burden, MTH1 is not essential for cell survival. This contrasts cancer cells which frequently upregulate MTH1 to compensate for increased ROS with corresponding higher oxidized nucleotide levels, and therefore become more susceptible for MTH1 inhibition. Here, our aim was to perform preclinical testing of karonudib in B-cell lymphoma. Using two different gene expression datasets, we demonstrate that NUDT1, the gene encoding MTH1, was highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma as compared to follicular lymphoma and peripheral blood B cells from healthy donors, hence demonstrating a rationale for targeting MTH1 in aggressive B-cell lymphoma. We tested the efficacy of karonudib (0.06-1 µM) in vitro in a range of B-cell lymphoma cell lines using CellTiterGlo and by flow cytometry detection of active caspase-3 and TUNEL to identify apoptotic cells. Karonudib strongly reduced viability in all B-cell lymphoma cell lines tested (n = 12) and induced apoptosis at concentrations well tolerated by peripheral blood B cells from healthy donors. Cell cycle analysis and microscopy revealed that most cells arrested in prometaphase in the presence of karonudib. Failed spindle assembly led to mitotic arrest and subsequent apoptosis. Prometaphase arrest was seen in TP53 mutant as well as in TP53 wild type cell lines, confirming that karonudib induced apoptosis independent of TP53 mutational status. To test the efficacy of karonudib in vivo, we utilized two different lymphoma xenograft models, including an ABC DLBCL patient-derived model. Mice were treated with karonudib (90 mg/kg) or vehicle b.i.d, three times a week and tumor growth was monitored by in vivo imaging system or MR. In both models, karonudib as single agent completely controlled tumor growth, and significantly prolonged survival (p<0.0001, as compared to control mice). The specificity of MTH1 inhibitors has been debated and TH588, the first generation MTH1 inhibitor, was recently shown to bind b-tubulin and induce mitotic arrest in MTH1 knock out cell lines (Patterson et al, Cell Syst 2019). To elucidate the mechanism of karonudib in B-cell lymphoma, we generated MTH1 knock out cells using CRISPR/Cas9, and compared the functional effects of karonudib in these clones with the original lymphoma cells. We demonstrated on-target effect of the inhibitor as the MTH1 knock out clones were less sensitive towards karonudib. However, MTH1 knock out clones exhibited a similar cell cycle arrest as the wild type cells after karonudib treatment. This clearly indicates that karonudib can induce cell cycle arrest independent of MTH1, and hence has a dual mechanism of action. Our preclinical data suggest that karonudib is a promising drug with potential therapeutic use in B-cell lymphoma, and may be particular effective in aggressive lymphoma types. Disclosures Warpman Berglund: Oxcia AB: Other: shareholders; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Gad:Oxcia: Other: shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Pham:Oxcia AB: Other: Shareholder. Sanjiv:Oxcia AB: Other: Shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Helleday:Oxcia AB: Other: Shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
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Oved, Joseph H., Caitlin W. Elgarten, Scott G. Daniel, Lidiya Denu, Michael Silverman, Kyle Bittinger, Timothy S. Olson, and Michele P. Lambert. "Pediatric Patients with Immune Thrombocytopenic Purpura Have a Dysbiotic Gut Microbiome at Time of Diagnosis." Blood 138, Supplement 1 (November 5, 2021): 3169. http://dx.doi.org/10.1182/blood-2021-154180.
Full textAbstract:
Abstract Background: Pediatric immune thrombocytopenia purpura (ITP) is the most common cause of autoimmune cytopenias in children and results in autoimmune destruction of platelets, leading to significantly decreased circulating platelets, increased bleeding risk and fatigue. The underlying mechanism of action is thought to be auto-antibody driven, though auto-reactive T cells have also been implicated in some cases. Given the proximity of many cases of ITP to either recent viral infection or vaccine administration, a leading hypothesis is that the environmental exposure mimics a platelet produced epitope causing platelet clearance. Despite the correlation of viral infection and/or inflammatory cascades in the propagation of ITP, the gut microbiome in newly diagnosed pediatric patients has not previously been interrogated. The gut microbiome has previously been shown to modulate the host inflammatory milieu and dysbiosis has been associated with other auto-immune disorders. Thus, we assessed the gut microbiome in newly diagnosed pediatric ITP patients to determine if a similar dysbiosis is present. Methods: Stool samples were collected from 32 pediatric patients (0-18 years) at the Children's Hospital of Philadelphia. 17 patients were newly diagnosed with ITP, 7 patients had ITP for greater than 3 months at time of stool collection and 8 patients had newly diagnosed severe acquired aplastic anemia. Stool samples were kept on ice until processing at our PENN/CHOP Microbiome Core (not longer than 24 hours/sample). Age matched healthy control samples were provided by the Microbiome Core. Shotgun libraries were generated from 0.5 ng DNA using the Nextera XT Library Prep kit and libraries were sequenced on an Illumina HiSeq 2500 in High Output mode to produce paired-end 125 bp sequence reads. Shotgun metagenomic data were analyzed using Sunbeam, a user-extendable bioinformatics pipeline that we developed for this purpose.Diversity within samples were assessed by the number of OTUs at a rarefaction level of 1,000 sequences and the Shannon index. Sample similarity were assessed by Bray-Curtis and Jaccard distances, which were then visualized using principle coordinates analysis. Results: Subjects included 17 patients with newly diagnosed ITP (i.e. stool sample collected < 3 months from diagnosis), 7 patients with ITP > 3 months and 8 patients with newly diagnosed severe acquired aplastic anemia. Patient samples were assessed for gut microbial diversity and richness. As seen in figure 1a, gut microbial diversity in newly diagnosed pediatric patients with ITP was significantly decreased (p = 0.024) while gut microbial richness trended to a concomitant decrease as well (p = 0.093). Interestingly, healthy controls had similar gut microbiomes to one another than newly diagnosed ITP patients had to one another (Fig 1b). These alterations in the gut microbiome were not seen in pediatric patients with newly diagnosed aplastic anemia or with ITP diagnosed > 3 months prior to collection (data not shown). These findings indicate that while there is a dysbiotic gut microbiome in patients with newly diagnosed ITP, there is no dysbiosis present in other pediatric autoimmune cytopenias. Furthermore, the dysbiosis corrects by 3 months post-ITP diagnosis. When we assess specific bacterial families and subgroups we do not find a predominant species that is increased or decreased but rather there are global changes amongst many bacterial species. We did find a trend of lower Alistipes species and increased Eschrechia coli in newly diagnosed ITP patients which is similar to other autoimmune disorders with shifted gut microbiota. Functional pathways analyses showed similar global alterations in vital pathways such as decreased aminoacyl tRNA biosynthesis and homologous recombination. Conclusion: Pediatric patients with newly diagnosed ITP but not other autoimmune cytopenias have gut microbial dysbiosis with perturbations in many bacterial species that may be a cause of, or a result of the underlying mechanism of pathogenesis. Further studies will help determine the role of dysbiosis in the pathobiology of ITP and determine whether intervention alters the duration of disease. Figure 1 Figure 1. Disclosures Lambert: Bayer: Consultancy; Astra Zeneca: Research Funding; Shionogi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ClinGen, ISTH, ASH, GW University: Honoraria; PDSA: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dova: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Yao, Yao, Woojun D. Park, Eugenio Morelli, Mehmet Kemal Samur, Nicholas P. Kwiatkowski, Yan Xu, Chandraditya Chakraborty, et al. "Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition." Blood 136, Supplement 1 (November 5, 2020): 1–2. http://dx.doi.org/10.1182/blood-2020-142592.
Full textAbstract:
Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.
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Drasar, Emma, Jie Jiang, Kate Gardner, Jo Howard, Tom Vulliamy, Nisha Vasavda, and Swee Lay Thein. "Telomere Lengths Correlate With Inflammatory Markers In Sickle Cell Disease." Blood 122, no. 21 (November 15, 2013): 2230. http://dx.doi.org/10.1182/blood.v122.21.2230.2230.
Full textAbstract:
Abstract Telomeres are DNA protein structures that protect chromosome ends from degradation and fusion and are essential for maintenance of genomic integrity. Shortened telomere length has been found in DNA from patients with inherited conditions associated with premature cellular aging and acquired disorders such as cardiovascular heart disease. In vitro studies have shown that telomeres are highly susceptible to oxidative stress. We hypothesize that the elevated level of reactive oxygen species and oxidative stress generated by ongoing hemolysis in sickle cell disease (SCD) could predispose to shorter telomeres in white blood cells (WBCs), and that telomere length could be a marker of SCD severity. The study population included 126 healthy controls of mixed ethnicities and 426 patients of African descent with SCD of mixed genotypes (291 HbSS, 112 HbSC, 16 HbSβ+ thalassaemia and 7 HbSβ0 thalassemia) recruited from King’s College and Guy’s and St Thomas’ hospitals (King’s College Hospital Local Research Ethics Committee protocol 07/H0606/165). Ages ranged from 6 to 86 years for the control group and 17 to 81 years for the SCD group. Clinical and steady state laboratory data were collected from the electronic patient records and sickle cell database. Telomere length measurement was performed on DNA extracted from peripheral blood leucocytes using a monochrome multiplex quantitative polymerase chain reaction technique adapted from the method as described by Richard Cawthon (Cawthon 2009). The method compares telomere repeat sequence copy number (T) to albumin (a single copy gene, S) copy number within the same DNA sample. Three reference DNA samples were included in each run as internal quality controls. The sample DNAs were assayed in duplicate and the standard curve DNAs, in triplicate. Polymerase chain reaction was performed using a Rotorgene 6000 (Corbett Life Sciences) after which data were analysed using Rotorgene 6000 series software version 1.7. For each sample, telomere length was expressed as telomere to single copy gene ratio (TSR) which is based on the delta Ct (Cttelomere / Ctsingle-gene) derived from the standard curve. To make comparable the results from different runs, the results were approved only if the relative TSRs of the validation reference samples fell within a 5% variation. Regression analysis was performed correcting for age, gender, alpha genotype, and hydroxyurea (HU) therapy at the time of sample collection. Laboratory variables were only available for the SCD group. 57/301 (19%) of sickle cell anemia (SCA, including HbSS and HbSβ0) patients had received HU treatment for at least 3 months prior to sample collection. TSRs for the controls, study group and sub-groups was significantly negatively associated with age as has previously been shown in healthy controls and disease states (Fig 1) (p<0.0001 in all groups). Contrary to our expectations, mean TSR was significantly longer (p<0.0001) in patients with SCD (mean 2.37 range 0.14 to 4.87) compared to controls (mean 1.80 range 0.87 to 4.17). Further, within the SCD group, TSR was significantly longer (p<0.0001) in patients with SCA (mean 2.44 range 0.14 to 4.87) compared to those with HbSC (2.19 range 0.35 to 3.46). The association with sickle genotype persisted with regression analysis (p = 0.001). We postulate that the longer telomeres in patients with SCD are due to increased telomerase activity related to the underlying inflammation. This suggestion is supported by the: 1) positive association of TSR with WBC (R = 0.19 p = 0.001) and neutrophil count (R = 0.14 p = 0.02) which persisted with regression analysis (WBC, p = 0.003 and neutrophil count, p = 0.049); 2) longer TSRs in patients with SCA when compared to patients with HbSC disease who have less inflammation; 3) significantly shorter telomeres in patients on HU therapy compared to the untreated group (regression analysis p = 0.004), we propose that this is due to the anti-inflammatory effects of HU via suppression of WBC count and down-regulation of cytokines. These results could have significant implications for our understanding of the pathophysiology of SCD, particularly the role that inflammation plays in chronic organ damage in this patient group. To validate the hypothesis, future work would include correlation of telomere length with both telomerase activity and organ damage in patients with SCD. Disclosures: Howard: Sangart: Membership on an entity’s Board of Directors or advisory committees.
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Peggs, Karl S., Kirsty Thomson, Edward Samuel, Gemma Dyer, Julie Armoogum, Kwok Pang, Ronjon Chakraverty, Stephen Mackinnon, and Mark W. Lowdell. "Adoptive Transfer of CMV-Specific Donor T Cells Following Allogeneic Transplantation Leads to Rapid and Safe Systemic Reconstitution." Blood 114, no. 22 (November 20, 2009): 44. http://dx.doi.org/10.1182/blood.v114.22.44.44.
Full textAbstract:
Abstract Abstract 44 Reactivation of CMV remains a significant problem following allogeneic hematopoietic stem cell transplantation. Antiviral drug therapy is effective but toxic, and resistant strains of CMV are increasingly being reported. Virus-specific T lymphocytes are necessary for the control of viral reactivation. Adoptive transfer of donor derived CMV-specific T cells has been reported previously but most methods to produce such cells have involved several weeks of in vitro culture or have produced a therapeutic product restricted to CD8 T cells. The current method involves a short incubation of donor peripheral blood mononuclear cells with either CMV-pp65 protein (20 hours) or a pool of peptides from pp65 (6 hours) with subsequent isolation of interferon-gamma secreting cells by CliniMACS using IFNψ capture microbeads (Miltenyi Biotec). This technique permits rapid isolation of an enriched IFNψ secreting T cell product, manufactured to clinical grade, which is then cryopreserved in dosed aliquots for subsequent infusion. Here we report the outcome of a single arm phase I/II in which CMV-T cells given pre-emptively at first detection (qPCR) of CMV DNA in peripheral blood, or at day +40-60 as prophylaxis. CMV replication was monitored by weekly PCR and reconstitution of CMV-specific T cells by pentamer labelling and/or IFNψ secretion assay. Conventional antiviral drug therapy was instituted if the viral load rose above institutional threshold. 30 recipients of T cell depleted low intensity transplants from HLA-matched CMV-seropositive related donors were enrolled between 2006 and 2008. Donors underwent a second, short apheresis procedure approximately 15 days after collection of the mobilised HPC-A for the collection of CMV-T cells. 26 clinical-grade products were produced to full cGMP standards; four donors were unsuitable or withdrew. The mean yield of cells following enrichment was 41.7% with a median purity of 43.9% (range 1.4-81.8). Adequate CMV-T cells were isolated from all donors. Both pp65 and peptide stimulated products contained both CD4 and CD8 reactive T cells. Median dose of CMV-specific CD4 T cells was 2840/kg and of CMV-specific CD8 was 630/kg. Eighteen patients received a single dose of 1×10^4 CD3+/kg; 13 were CMV seropositive; 11 were treated pre-emptively and 7 prophylactically. 83% had received T cell deplete regimens. Within 2 weeks of infusion in vivo expansion of CMV-T cells was observed in 17 of 18 patients. One patient required 4 weeks to generate detectable CMV-T cell in his peripheral blood. TCR-BV usage of the CMV-T cells post infusion matched that of the cells which had been infused. The 7 patients who had cells infused prophylactically all showed expansions of CMV-T cells in the absence of detectable viral DNA in peripheral blood. Subsequent low level CMV-reactivation was seen in one of these and was associated with rapid CMV-T cell expansion with clearance of virus without anti-viral drug therapy. One developed subsequent extensive chronic GvHD and required antiviral treatment for multiple reactivation episodes following introduction of steroids. Of the 11 patients treated pre-emptively, 9 received antiviral therapy for the initial reactivation, although in 7 patients this was required for only 7-15 days. (compared to a median of 21 days in historical controls). Three patients had a further CMV reactivation event. One followed prednisolone therapy for acute grade II GvHD. The second was the patient who had shown poor T cell expansion post infusion and had required prolonged anti-viral therapy (33 days) for the initial CMV reactivation. The third patient received no treatment and cleared virus following a further in vivo expansion of CMV-reactive T cells, suggesting the presence of a functional memory population. GVHD incidence and severity was no worse than seen in comparable historical controls. 3 patients suffered grade 2-3 acute GvHD. 3/17 evaluable patients developed extensive chronic GvHD (2 were recipients of T replete grafts). 16/18 patients were alive at the end of the 6 month monitoring period and CMV-reactive T cells were detectable in all 16. CMV-specific donor T cells can be readily produced to cGMP compliance which can be safely infused and lead to early immune reconstitution in at-risk patients. Cells expand in response to subsequent CMV-reactivation and patients appear to require fewer anti-viral treatment episodes which is being tested in an ongoing phase III trial. Disclosures: Lowdell: Cell Medica Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Kapp, Friedrich G., Julie R. Perlin, Eirini Trompouki, Charlotte M. Niemeyer, Wolfgang Driever, and Leonard I. Zon. "Protection from UV-Light Is an Evolutionarily Conserved Feature of the Hematopoietic Niche." Blood 128, no. 22 (December 2, 2016): 2663. http://dx.doi.org/10.1182/blood.v128.22.2663.2663.
Full textAbstract:
Abstract Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow cavity in all mammals, but other locations are used in non-mammalian animals, such as the kidney marrow in zebrafish. The niche location is thought to be selected based on unique advantages for HSPC function and protection in each organism. Studying the zebrafish HSPC niches during development and adulthood, we observed that melanocytes occupy the location above the kidney prior to the arrival of HSPCs. To elucidate whether these particular melanocytes play a role for HSPCs, we compared HSPCs in wildtype and nacre-mutant embryos (that lack all melanocytes) by cmyb in situ at different time-points. There were no obvious differences, suggesting that melanocytes are dispensable for steady state hematopoiesis. Since the position of the melanocytes above the HSPCs was very consistent in different strains and at various ages, we asked whether the melanocytes protect the HSPC niche from UV light. Indeed, after irradiation with UV light, levels of UV-induced DNA damage were approximately 50% higher in HSPCs isolated from kidneys of unpigmented embryos compared to their pigmented siblings as assessed by immunocytochemistry with a cyclobutane pyrimidine dimer antibody (TDM-2). In addition to acquiring more DNA damage, the unpigmented embryos also showed a significantly more pronounced decrease of HSPCs as assessed by cmyb in situ: 55% of control embryos had high cmyb staining. Upon irradiation 44% of irradiated pigmented embryos retained high c-myb staining compared to only 28% of irradiated unpigmented embryos (total embryos analyzed: n= 29, 27 and 36 respectively; p=0.01 between the latter two groups). The reduction of HSPCs could not be rescued in unpigmented p53-mutant embryos, indicating a p53-independent mechanism of HSPC loss after UV irradiation. The protective effect of melanocytes could either be due to a physical shielding of the HSPCs or due to a paracrine mechanism, such as secretion of cytokines or uptake of reactive oxygen species. The paracrine mechanism could be dependent on melanin. To address this question, we focused the path of light at a different angle in which the melanocytes would not be able to block the UV light from reaching the kidney marrow. This abrogated the protective effect of pigment containing melanocytes and caused a reduction of HSPCs by cmyb in situ to the levels of unpigmented siblings: pigmented embryos irradiated at an angle now also had high cmyb staining in only 28% of the embryos (n= 25). This suggests that the melanocytes only provide shade for the HSPCs. We then asked if this protective pigmentation pattern is specific to zebrafish and traced the origin of this trait by comparative histology. We found that other teleost fish but also evolutionarily older fish such as sturgeon and polypterus have melanocytes covering the kidney marrow. Even in lamprey, an ancient jawless vertebrate, the HSPC niche (located in the supraspinal organ) is covered by melanocytes, which allows us to date the evolutionary origin of UV protection back to at least 500 million years. We performed histological studies in frogs and found that the HSPCs reside under a melanocyte shield in the tadpole stage, while it is known that in adult frogs the HSPCs move into the bone marrow cavity. Considering that all terrestrial animals such as reptiles, birds and mammals host hematopoiesis in the bone marrow, we suggest that during the transition from aquatic to terrestrial life, the HSPCs were driven out of their melanocyte covered niche due to the high level of irradiation and relocated into a more protective niche, the bone marrow. Disclosures Zon: Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
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Hirsch, Cassandra M., Michael Clemente, Peter Chomczynski, Bartlomiej P. Przychodzen, Yasunobu Nagata, Vera Adema, Louis Williams, et al. "Polyclonal Immune Response in T-LGL Leads to Clonal Expansions Preceding Occurrence of STAT3 Mutations Further Solidifying Clonal Dominance." Blood 132, Supplement 1 (November 29, 2018): 516. http://dx.doi.org/10.1182/blood-2018-99-116307.
Full textAbstract:
Abstract T cell large granular lymphocyte leukemia (T-LGLL) characterized by excessive clonal cytotoxic T cell proliferation, is often accompanied by cytopenias and is thought to result from immune-mediated suppression, either by misguided immune antigenic recognition or mimicry triggered by auto-, tumor or viral antigens. Irrespective of the mode of evolution, T-LGLL progresses via: i) purely reactive clonal outgrowth in the context of a polyclonal immune response or: ii) a transforming event such as a STAT3 mutation (STAT3MT) within the immunodominant clone. Studies of TCR rearrangement using DNA based NGS of the TCR Vβ complementarity-determining region 3 (TCR VB CDR3) facilitate analyses of T cell clonality, as the CDR3-based "biological barcodes" allow for clonal quantification. We used serial CDR3 clonotyping accompanied by clonal STAT3 mutant burden measurements to recapitulate the pathological cascade in the course of T-LGLL. We asked if a polyclonal/oligoclonal immune response is the primary process that is clonally "imprinted" by STAT3MT, which would likely arise with the most proliferative clone. We were interested in the dynamics of underlying clonal behavior during treatment. Our initial cohort included 207 well characterized LGL patients. Most presented with anemia (46%), and/or neutropenia (46%). VB expansion was present in 94% of cases, with an average LGL count of 2317k/uL .STAT3MT were found by targeted deep sequencing in 38% of patients in 4 common hotspots: 42% Y640F, 34% D661Y, 11% D661V, and 8% N647I, with an average clonal burden of 28%. Multiple STAT3MT variants were found in some patients. From this cohort, serial samples obtained at presentation and throughout clinical course were obtained from 20 representative cases (10 STAT3MT and 10 STAT3WT), and subjected to analysis of clonal dynamics, including simultaneous deep TCR VB and STAT3 NGS. Patients were sequenced at an average of 4 time points (range 2-8). At least one major clonotype was identified in all patients, and multiple major clonotypes were identified in half. Analyses of clonal architecture revealed that STAT3 clones arose with VB expanded clones. In all cases, the TCR clonal burden was greater than that of STAT3MT demonstrating for the first time that STAT3MT is not the ancestral event for clonal expansion; rather, it evolves within the pre-expanded immunodominant clone. More than half of the patients were treated with immunosuppressive therapy (IST) achieving an approximate 40% response rate. Distinct patterns of clonal dynamics were seen following treatment (see Figure). In some patients, both the STAT3 (if present) and the major TCR clone decreased upon successful therapy. A correlation between a specific IST treatment and a clonal burden decrease was not found. In a subset of patients, the clones persisted despite a hematologic response, suggesting the major clonotype was functionally silenced. We also observed a common phenomenon of TCR "clonotype switching", whereby therapy contracts one major clonotype, while another previously "minor" clonotype emerges. These newly expanded clones did not harbor STAT3MT, and most patients with "switching" were resistant to IST therapy. Multiple clonotypes were present at initial sampling in a few patients without STAT3MT and persisted at the same rate in subsequent samplings, precluding identification of a clear immunodominant clonotype. A stable or increasing clonal burden of both STAT3MT and VB CDR3 was seen in IST non-responders. In sum, STAT3MT were found to be a secondary event to the clonal expansion of the TCR VB clonotype, as a response within an already expanded clonotype and not the initiator of clonal expansion. The dynamics of both the STAT3MT and the TCR VB clonotype can be assessed over disease course and treatment regimens and demonstrate additional clinical ultility when applied to larger prospective clinical trials. The difficulty in finding a direct correlation between response to specific IST and a decrease in TCR VB clonal burden may be due to variable time frames between samplings, heterogeneity of IST regimens and response assessment, or large asymptomatic clonotypes. Prior to this study, the association of remission and elimination of immunodominant clonotypes was unclear. Our results suggest that clonal elimination is not necessary for complete clinical response; rather, the clone can be contracted to a manageable clonal burden or silenced. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.
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Bader, Cameron S., Henry Barreras, Casey O. Lightbourn, Sabrina N. Copsel, Dietlinde Wolf, Jingjing Meng, Jeonghyun Ahn, et al. "The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation." Blood 134, Supplement_1 (November 13, 2019): 3202. http://dx.doi.org/10.1182/blood-2019-130197.
Full textAbstract:
Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.
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Pettengell, Ruth, Pier Luigi Zinzani, Geetha Narayanan, Fernando Hurtado de Mendoza, Raghunadharao Digumarti, Henry Gomez, Bertrand Coiffier, et al. "Phase 3 Trial of Pixantrone Dimaleate Compared with Other Agents as Third-Line, Single-Agent Treatment of Relapsed Aggressive Non-Hodgkin Lymphoma (EXTEND): End of Study Results." Blood 116, no. 21 (November 19, 2010): 2833. http://dx.doi.org/10.1182/blood.v116.21.2833.2833.
Full textAbstract:
Abstract Abstract 2833 Introduction: No therapy with reliable, durable efficacy exists for patients with aggressive non-Hodgkin lymphoma (aNHL) who relapse following at least two lines of therapy. Pixantrone dimaleate (PIX) is a novel aza-anthracenedione, structurally similar to anthracyclines and mitoxantrone, and forms stable DNA adducts. Unlike anthracyclines and mitoxantrone, PIX does not bind iron, minimally promotes reactive oxygen species formation, and is substantially less cardiotoxic in preclinical models. On the basis of promising early clinical activity and acceptable safety, we conducted a phase 3 trial with PIX in patients with aNHL and ≥2 relapses. Patients and Methods: This randomized, multicenter, controlled, open-label study enrolled patients with aNHL (de novo or transformed) with ≥1 prior anthracycline-containing regimen and ≥2 relapses. Patients were randomized to PIX 85 mg/m2 on days 1, 8, and 15 of a 28-day cycle, for up to 6 cycles, or to investigator's choice of single-agent comparator (COMP): vinorelbine, oxaliplatin, ifosfamide, etoposide, mitoxantrone, or, in the US only, gemcitabine or rituximab. Both groups were followed for 18 months after last treatment. The primary endpoint, CR/CRu rate in ITT population, was assessed by an independent assessment panel. Other efficacy endpoints were overall response rate (ORR), duration of response, progression-free survival (PFS), and overall survival (OS). Final end-of-study results are reported here. Results: Planned enrollment was set at 320 patients; however, due to slow accrual, a total of 140 patients were randomized (n=70 per group) to this study. Median number of treatment cycles for PIX group was 4 vs 3 for COMP. Median duration of treatment for patients in the PIX group was about one month longer than in the COMP group (3.8 vs 2.6 months). The end-of-study CR/CRu rate was 24% (16% CR, 8%CRu) for PIX vs 7% (no CRs, 7% CRu only) for COMP (P = 0.009); ORR (CR/CRu/PR) was 40% vs 14% (P = 0.001). After treatment ended, 3 patients in the PIX group achieved CR with no subsequent therapy. Two of the 3 patients converted from SD to CR and 1 from PR to CRu. Median CR/CRu duration was 9.6 months for PIX vs 4.0 months for COMP (P = 0.081). For survival endpoints, there was a 40% improvement in PFS with a median of 5.3 months vs 2.6 months (HR = 0.60, log-rank P = 0.005) and a 21% improvement in OS with a median of 10.2 months for PIX vs 7.6 months for COMP (HR = 0.79, log-rank P = 0.251). Exploratory subgroup analyses of CR/CRu and ORR were consistently higher for PIX patients and suggest stronger efficacy with the subgroups sex (female), <3 prior regimens, no prior rituximab, and ≥1 yr from 1st to 2nd line therapy. During treatment, neutropenia and leukopenia were the most common (≥10%) grade 3/4 adverse events and the incidence of febrile neutropenia was 7.4% for PIX vs 3% for COMP. Percentage of patients with cardiac disorder SAEs was 8.8% for PIX vs 4.5% for COMP. There were 11 grade 1/2 and 2 grade 3 LVEF events for PIX vs 7 grade 1/2 for COMP. Conclusions: In this phase 3 study, patients treated with PIX achieved superior efficacy when compared with other agents, as assessed by CR/CRu rate, ORR, and PFS, and had a positive trend in OS. Pixantrone had a tolerable safety profile in heavily pretreated patients with relapsed aggressive NHL. Disclosures: Pettengell: Cell Therapeutics, Inc: Honoraria. Coiffier:Cell Therapeutics, Inc: Advisory Board. Schiller:Sunesis: Consultancy; Celgene, Genzyme, Millenium, CTI, Antisoma, Novartis: Research Funding. Rizzieri:Cell Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees. Cernohous:Cell Therapeutics, Inc: Employment. Wang:Cell Therapeutics, Inc: Employment. Singer:Cell Therapeutics, Inc: Employment, Membership on an entity's Board of Directors or advisory committees; DiaKine Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees.
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Talati, Chetasi, Ling Zhang, Ghada Shaheen, Andrew Kuykendall, Markus Ball, Qing Zhang, Jeffrey E. Lancet, et al. "Monocyte Subset Analysis Accurately Distinguishes Chronic Myelomonocytic Leukemia (CMML) from Myelodysplastic Syndromes (MDS) and Is Associated with a Favorable MDS Prognosis." Blood 128, no. 22 (December 2, 2016): 2009. http://dx.doi.org/10.1182/blood.v128.22.2009.2009.
Full textAbstract:
Abstract Background: The WHO requires a sustained peripheral monocytosis (≥1x109cells/L) for the diagnosis of CMML. However, a peripheral monocytosis is not pathognomonic because monocytosis is observed in other hematologic neoplasms and benign reactive conditions. A recent study demonstrated that CMML is uniquely represented by the expansion of classical monocytes (CD14+/CD16-) (Selimoglu-Buet et al, Blood 20151). Further, measuring the relative fraction of classical monocytes, by itself, was capable of distinguishing CMML from reactive conditions and a mixed cohort of hematologic neoplasms. In this study, we aimed to validate these findings in a clinical and genetically annotated cohort of CMML and other hematologic malignancies with a focus on MDS, and normal age-matched controls. Methods: We profiled monocyte subsets in patients with a suspected diagnosis of CMML or MDS as previously described1 after obtaining institutional review board approval. Clinical demographics and genotyping of patient samples (52 gene TruSight panel, Illumina) were collected via retrospective chart review. Descriptive statistics were used to summarize clinical demographics, genotyping, and their association to classical monocytosis (CM). Receiver Operator Curves (ROC) were generated to test the sensitivity and specificity of the monocyte analysis and all calculated p-values were two-sided. Results: From October 2015 to May 2016 monocyte subsets were profiled in 159 genetically defined cases. The diagnosis of patients in our cohort included CMML (n=29), MDS (n=86), other myeloid malignancies (n=26), and reactive conditions (n=18). Within CMML cases the median age at diagnosis was 70 years, median hemoglobin, platelets, and monocyte counts were 10.9 g/dL, 102x109cells/L, and 2.05x109cells/L, respectively. As previously reported, CM was evident in all CMML cases and was capable of distinguishing CMML from normal age-matched controls. ROC analysis confirmed that the assay was capable of differentiating between these groups (AUC of 0.9592, p<0.001) (Figure 1A). Further, CM was also capable of discriminating CMML from MDS (AUC 0.8793, p <0.0001 (Figure 1B). However, no difference in CM was evident between French American British or WHO-defined CMML subtypes. There were also no differences in CM between lower and higher risk disease as defined by established cytogenetic risk stratification or prognostic scoring systems validated in CMML. Exposure to hypomethylating agent did not affect the pattern of CM. When comparing cases based on the presence of splicing gene mutations, DNA methylation gene mutations, ASXL1 or signaling gene mutations, no difference in classical monocytes was identified. To explore the impact of CM in MDS, we identified 24 MDS cases that had "CMML-like" CM (CM ≥ 94%) and 60 MDS cases with normal monocyte subsets (Figure 2). There were no differences in age, hemoglobin, platelets, or presence of splenomegaly. However, CMML-like MDS cases were associated with an increased WBC (3.815x109 cells/L vs. 2.34x109 cells/L), increased neutrophils (1.73x109 cells/L vs. 1.07x109 cells/L, p=0.02), and increased absolute monocyte counts (355X109 cells/L vs. 120x109 cells/L, p=0.02) (Figure 2). Furthermore, the MDS cohort without classical monocytosis was more frequently associated with poor risk cytogenetics (Odds ratio (OR) 3.429, 95% CI 1.032-10.08, p=0.04) and was more likely to be higher-risk as defined by the IPSS-R (OR 8.767, 95% CI 1.088-70.69, p=0.0174). Analysis of mutated genes identified SF3B1 to be present at greater frequency in the CMML-like MDS subgroup (OR 3.457, 95% CI 1.074-11.21, p=0.0486) while the frequency of other commonly mutated genes in CMML were not significantly different (Figure 2). Conclusions: Our study demonstrates that classical monocytes can discriminate CMML from normal age-matched controls, validating a previous study. We additionally demonstrate that CM is capable of discriminating CMML from a large MDS cohort. Further, we identified two MDS subgroups that can be differentiated by the fraction of classical monocytes and are clinically distinguished by a favorable prognosis and higher frequency of SF3B1 mutation. Our data suggest that analysis of monocyte subsets should be incorporated in the diagnostic algorithm of CMML and that the clinical significance of CM in MDS merits further investigation. Disclosures Lancet: ERYtech: Consultancy; Biopath Holdings: Consultancy; Baxalta: Consultancy; Amgen: Consultancy; Jazz Pharmaceuticals: Consultancy; Boehringer-Ingelheim: Consultancy; Kalo Bios: Consultancy; Pfizer: Research Funding; Quantum First: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Research Funding; Seattle Genetics: Consultancy. Komrokji:Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Padron:KALOBIOS: Research Funding; CTI: Honoraria, Research Funding; Incyte: Research Funding; Novartis: Honoraria.
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Stewart, Peter, Jana Gazdova, Nikos Darzentas, Dorte Wren, Paula Proszek, Grazia Fazio, Simona Songia, et al. "Euroclonality-NGS DNA Capture Panel for Integrated Analysis of IG/TR Rearrangements, Translocations, Copy Number and Sequence Variation in Lymphoproliferative Disorders." Blood 134, Supplement_1 (November 13, 2019): 888. http://dx.doi.org/10.1182/blood-2019-127822.
Full textAbstract:
Introduction: Current diagnostic standards for lymphoproliferative disorders include detection of clonal immunoglobulin (IG) and/or T cell receptor (TR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. These analyses frequently require a series of separate tests such as clonality PCR, fluorescence in situ hybridisation and/or immunohistochemistry, MLPA or SNParrays and sequencing. The EuroClonality-NGS DNA capture (EuroClonality-NDC) panel, developed by the EuroClonality-NGS Working Group, was designed to characterise all these alterations by capturing variable, diversity and joining IG and TR genes along with additional clinically relevant genes for CNA and mutation analysis. Methods: Well characterised B and T cell lines (n=14) representing a diverse repertoire of IG/TR rearrangements were used as a proficiency assessment to ensure 7 testing EuroClonality centres achieved optimal sequencing performance using the EuroClonality-NDC optimised and standardised protocol. A set of 56 IG/TR rearrangements across the 14 cell lines were compiled based on detection by Sanger, amplicon-NGS and capture-NGS sequencing technologies. For clinical validation of the NGS panel, clinical samples representing both B and T cell malignancies (n=280), with ≥ 5% tumour infiltration were collected from 10 European laboratories, with 88 (31%) being formalin fixed paraffin-embedded samples. Samples were distributed to the 7 centres for library preparation, hybridisation with the EuroClonality-NDC panel and sequencing on a NextSeq 500, using the EuroClonality-NDC standard protocol. Sequencing data were analysed using a customised version of ARResT/Interrogate, with independent review of the results by 2 centres. All cases exhibiting discordance between the benchmark and capture NGS results were submitted to an internal review committee comprising members of all participating centres. Results: All 7 testing centres detected all 56 rearrangements of the proficiency assessment and continued through to the validation phase. A total of 10/280 (3.5%) samples were removed from the validation analysis due to NGS failures (n=1), tumour infiltration < 5% (n=7), and sample misidentification (n=2). The EuroClonality-NDC panel detected B cell clonality (i.e. detection of at least one clonal rearrangement at IGH, IGK or IGL loci) in 189/197 (96%) B cell malignancies. Seven of the 8 discordant cases were post-germinal centre malignancies exhibiting Ig somatic hypermutation. The EuroClonality-NDC panel detected T cell clonality (i.e. detection of at least one clonal rearrangement at TRA, TRB, TRD or TRG loci) in 70/73 (96%) T cell malignancies. In all 3 discordant cases analysis of benchmark PCR data was not able to detect clonality at any TR loci. Next, we examined whether the EuroClonality-NDC panel could detect clonality at each of the individual loci, resulting in sensitivity values of 95% or higher for all IG/TR loci, with the exception of those where limited benchmark data were available, i.e. IGL (n=3) and TRA (n=7). The specificity of the panel was assessed on benign reactive lesions (n=21) that did not contain clonal IG/TR rearrangements based on BIOMED-2/EuroClonality PCR results; no clonality was observed by EuroClonality-NDC in any of the 21 cases. Limit of detection (LOD) assessment to detect IG/TR rearrangements was performed using cell line blends with each of the 7 centres receiving blended cell lines diluted to 10%, 5.0%, 2.5% and 1.25%. Across all 7 centres the overall detection rate was 100%, 94.1%, 76.5% and 32.4% respectively, giving an overall LOD of 5%. Sufficient data were available in 239 samples for the analysis of translocations. The correct translocation was detected in 137 out of 145 cases, resulting in a sensitivity of 95%. Table 1 shows how translocations identified by the EuroClonality-NDC protocol were restricted to disease subtypes known to harbour those types of translocations. Analysis of CNA and somatic mutations in all samples is underway and will be presented at the meeting. Conclusions: The EuroClonality-NDC panel, with an optimised laboratory protocol and bioinformatics pipeline, detects IG and TR rearrangements and translocations with high sensitivity and specificity with a LOD ≤ 5% and provides a single end-to-end workflow for the simultaneous detection of IG/TR rearrangements, translocations, CNA and sequence variants. Table. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Ferrero:Gilead: Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees; Servier: Speakers Bureau. van den Brand:Gilead: Speakers Bureau. Groenen:Gilead: Speakers Bureau. Brüggemann:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy. Langerak:Gilead: Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; Janssen: Speakers Bureau. Gonzalez:Roche: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.
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Schmohl, Jörg Uwe, Martin Felices, Jeffrey S. Miller, and Dan Vallera. "Engineering of Trispecific Molecule That Induces Natural Killer Cell Expansion While Mediating CD133-Specific Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)." Blood 128, no. 22 (December 2, 2016): 3518. http://dx.doi.org/10.1182/blood.v128.22.3518.3518.
Full textAbstract:
Abstract Background: Selective cancer stem cell (CSC) elimination in solid as well as in and hematologic malignancies is a critical goal in immunotherapy since CSC cause drug refractory relapse. Furthermore this small cell group is known for tumor initiation and self-renewal capabilities and was already described to be a valuable target in breast, head and neck as well as in ovarian cancer. CSCs are also important for the development of hematologic malignancies and might be an excellent cell to target, currently not addressed through current therapies, in order to prevent relapse. The design of state-of-art anti-cancer immune engagers should address three important issues including ADCC, CSC targeting, and rapid effector cell expansion that sustains a potent anti-cancer response. In order to improve the current conventional bispecific immune-engager platform, we synthesized a 16133 BiKE consisting of scFvs binding FcγRIII (CD16) on Natural Killer (NK) cells and CD133 on CSC and then introduced a modified IL-15 crosslinker capable of stimulating NK effector cells. Methods: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE) (Figure 1A). The construct was tested for specificity using flow cytometry, cytotoxicity using chromium release assays, and lytic degranulation. IL-15-mediated expansion was measured using flow based proliferation assays. Also, Interferon (IFN)-γ release was measured by flow cytometry since it is important in the anti-cancer response. Results: The new TriKE showed specific and efficient induction of NK cell related cytotoxicity as seen in 51chromium release assays with Caco-2 cells, which express high levels of CD133. Activity was superior compared to 16133 BiKE as seen in effector:target ratios of 20:1 (21.9 ± 0.8% vs. 17.9 ± 2.2%), 10:1 (9.4 ± 0.3% vs. 7.9 ± 2.4%) and 5:1 (4.3 ± 0.2% vs. 5.4 ± 1.5%) (Figure 1B). Proliferation and NK expansion with the 1615133 TriKE was far greater than that achieved with the BiKE devoid of IL-15 as tested with purified NK cells exposed to both drugs for 7 days and stained with a reactive dye (Proliferation index 1.7 ± 0.3 vs. 1.2 ± 0.01, p=<0.0001, n=5). Drug binding and induction of cytotoxic degranulation was CD133+ specific as proved with Caco-2 and Raji cells as positive and negative controls (respectively). In Caco-2 cell targets the BiKE as well as the TriKE showed significant superior activity compared to, NCI IL-15, anti-CD16 scFv and anti-CD133 scFv controls (CD107a expression 37.5 ± 0.2% and 36.9 ± 0.2% vs. 19.6 ± 0.1, 18.3 ± 0.7, 12.6 ± 0.4, p<0.001, n=3), (Figure 1C). NK cell related cytokine release measured via IFN-γ detection was higher in the TriKE compared the BiKE (38.3 ± 0.2 % vs. 13.1 ± 0.3 %, p<0.001, n=3) and higher than all other controls. NK cell related cytokine release studies showed that although IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18 (38.3 ± 0.2 % vs. 60 ± 0.2 %, p<0.001, n=3) indicating that the release induced with the TriKE was not at supraphysiologic levels. Conclusion: 1615133 TriKE showed specific and improved anti-cancer activity over BiKE and provides a self-sustaining mechanism via induction of IL-15 signaling on NK cells. Inclusion of IL-15 might be a promising platform technology since CD133 can be substituted by other promising tumor associated antigens to create a highly specific and efficient drug. By improving NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse inducing CSC with an encouraging safety profile. Disclosures Miller: Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees.