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Montazersaheb, Soheila, Mohammad Saeid Hejazi, and Hojjatollah Nozad Charoudeh. "Potential of Peptide Nucleic Acids in Future Therapeutic Applications." Advanced Pharmaceutical Bulletin 8, no. 4 (November 29, 2018): 551–63. http://dx.doi.org/10.15171/apb.2018.064.
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Peptide nucleic acids (PNA) are synthetic analog of DNA with a repeating N-(2-aminoethyl)-glycine peptide backbone connected to purine and pyrimidine nucleobases via a linker. Considering the unique properties of PNA, including resistance to enzymatic digestion, higher biostability combined with great hybridization affinity toward DNA and RNA, it has attracted great attention toward PNA- based technology as a promising approach for gene alteration. However, an important challenge in utilizing PNA is poor intracellular uptake. Therefore, some strategies have been developed to enhance the delivery of PNA in order to reach cognate site. Although PNAs primarily demonstrated to act as an antisense and antigene agents for inhibition of transcription and translation of target genes, more therapeutic applications such as splicing modulation and gene editing are also used to produce specific genome modifications. Hence, several approaches based on PNAs technology have been designed for these purposes. This review briefly presents the properties and characteristics of PNA as well as different gene modulation mechanisms. Thereafter, current status of successful therapeutic applications of PNA as gene therapeutic intervention in different research areas with special interest in medical application in particular, anti-cancer therapy are discussed. Then it focuses on possible use of PNA as anti-mir agent and PNA-based strategies against clinically important bacteria.
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Gaetani, Simona, Federica Monaco, Massimo Bracci, Veronica Ciarapica, Giulia Impollonia, Matteo Valentino, Marco Tomasetti, Lory Santarelli, and Monica Amati. "DNA damage response in workers exposed to low-dose ionising radiation." Occupational and Environmental Medicine 75, no. 10 (August 7, 2018): 724–29. http://dx.doi.org/10.1136/oemed-2018-105094.
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ObjectiveMedical personnel using radiation for diagnosis and therapeutic purposes are potentially at risk of cancer development. In this study, the effect of ionising radiation (IR) exposure was evaluated as DNA damage response (DDR) in the circulating cells of occupationally exposed subjects.MethodsThe study population consisted of IR-exposed workers included both in group B (effective dose ranging between 0.04 and 6 mSv/year) and group A (probable effective dose exceeding 6 mSv/year), and the control group consisted of healthy individuals who had never been occupationally exposed to IR or other known carcinogenic agents. DNA damage (single-strand breaks, oxidised purine and pyrimidine bases) and DNA repair (t1/2, half time to repair DNA damage, amount of repaired DNA and DNA repair activity) were measured in lymphocytes using the comet assay. To evaluate the influence of IR doses and genetic predisposition to cancer, the enrolled population was stratified according to IR exposure level and family history of cancer.ResultsIncreased DNA repair activity was found in IR-exposed group, and only subjects highly exposed to IR doses accumulated DNA damage in their circulating cells, thus supporting the hypothesis of ‘radiation hormesis’. A significant increase in DNA damage accumulation and a reduced 8-oxoguanine glycosylase 1-dependent DNA repair activity were found in IR-exposed subjects with cancer cases across their family.ConclusionOur results indicate that chronic exposure to a low dose of IR in occupational settings induces DDR in exposed subjects and may be mutagenic in workers with family history of cancer, suggesting that periodic surveillance might be advisable, along with exposure monitoring.
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Campagnaro, Gustavo D., and Harry P. Koning. "Purine and pyrimidine transporters of pathogenic protozoa – conduits for therapeutic agents." Medicinal Research Reviews 40, no. 5 (March 7, 2020): 1679–714. http://dx.doi.org/10.1002/med.21667.
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Burnstock, Geoffrey. "Purine and purinergic receptors." Brain and Neuroscience Advances 2 (January 2018): 239821281881749. http://dx.doi.org/10.1177/2398212818817494.
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Adenosine 5′-triphosphate acts as an extracellular signalling molecule (purinergic signalling), as well as an intracellular energy source. Adenosine 5′-triphosphate receptors have been cloned and characterised. P1 receptors are selective for adenosine, a breakdown product of adenosine 5′-triphosphate after degradation by ectonucleotidases. Four subtypes are recognised, A1, A2A, A2B and A3 receptors. P2 receptors are activated by purine and by pyrimidine nucleotides. P2X receptors are ligand-gated ion channel receptors (seven subunits (P2X1-7)), which form trimers as both homomultimers and heteromultimers. P2Y receptors are G protein-coupled receptors (eight subtypes (P2Y1/2/4/6/11/12/13/14)). There is both purinergic short-term signalling and long-term (trophic) signalling. The cloning of P2X-like receptors in primitive invertebrates suggests that adenosine 5′-triphosphate is an early evolutionary extracellular signalling molecule. Selective purinoceptor agonists and antagonists with therapeutic potential have been developed for a wide range of diseases, including thrombosis and stroke, dry eye, atherosclerosis, kidney failure, osteoporosis, bladder incontinence, colitis, neurodegenerative diseases and cancer.
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Cacciari, Barbara, Romeo Romagnoli, Arianna Romani, Alessandro Trentini, and Stefania Hanau. "Thio-substituted derivatives of 4-amino-pyrazolo[3,4-d]pyrimidine-6-thiol as antiproliferative agents." Future Medicinal Chemistry 13, no. 18 (September 2021): 1515–30. http://dx.doi.org/10.4155/fmc-2021-0131.
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The current study was designed to identify new compounds as potential antiproliferative drug candidates. Synthesis of heteroaromatic bicyclic and monocyclic derivatives as purine bioisosters was employed. Their antiproliferative activity was studied against U937 cancer cells. The most effective compounds were evaluated for their selectivity against cancer cells, the possible mechanism of cell death, and their interference with DNA replication. Among the synthesized compounds, only three (4b, 4j and 4l) demonstrated a value of IC50 less than 20 μM. However, two of them (4b and 4l) were specific against cancer cells, with 4l presenting high selectivity. The presence of substituted pyrazolo[3,4- d]pyrimidine core is as essential for this activity as the presence of substituents at the thiol function in 6-position.
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Pal, Sharmistha, Jakub P. Kaplan, Sylwia A. Stopka, Michael S. Regan, Bradley R. Hunsel, Benjamin H. Kann, Nathalie Y. R. Agar, et al. "DDRE-32. THERAPEUTIC TARGETING OF A NOVEL METABOLIC ADDICTION IN DIFFUSE MIDLINE GLIOMA." Neuro-Oncology Advances 3, Supplement_1 (March 1, 2021): i13. http://dx.doi.org/10.1093/noajnl/vdab024.054.
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Abstract Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer that is in need of urgent “outside the box” therapeutic approaches. Recent studies show that tumor cells adapt to stresses created by oncogenic mutations and these oncogene-induced adaptations create vulnerabilities that can be exploited to therapeutic ends. To uncover these oncogene-induced vulnerabilities in DMGs we conducted a genome-wide CRIPSR knockout screen in three DMG lines. The top common DMG dependency pathway that we discovered is de novo pyrimidine biosynthesis. Under normal conditions pyrimidine nucleotide needs are met through the salvage pathway. However, in DMG tumorigenesis, pyrimidine nucleotide synthesis is rewired such that the cells become dependent on the de novo biosynthesis pathway. De novo pyrimidine synthesis is catalyzed by CAD, DHODH and UMPS; all three genes are identified as dependencies in our screen and have been validated using shRNA mediated gene knockdown. Interestingly, DMG cells did not exhibit a dependency on the de novo purine biosynthesis pathway. Using a small molecule inhibitor of DHODH, BAY2402234 [currently studied in phase I trial for myeloid malignancies (NCT03404726)], we have demonstrated and validated, (i) efficacy and specificity of de novo pyrimidine synthesis inhibition in vitro in DMG cells; (ii) de novo pyrimidine addiction is not attributable to cell proliferation; (iii) DHODH inhibition induces apoptosis by hindering replication and inciting DNA damage; (iv) DHODH and ATR inhibition act synergistically to induce DMG cell death; and (v) critical in vivo efficacy. The in vivo experiment documents that BAY2402234 crosses the blood-brain barrier, is present in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in intracranial DMG tumors in mice, and prolongs survival of orthotopic DMG tumor bearing mice. Taken together, our studies have identified a novel metabolic vulnerability that can be translated for the treatment of DMG patients.
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Kawano, Yawara, Yuki Inada, Takayuki Sasano, Nao Nishimura, Hiroyuki Hata, and Masao Matsuoka. "The Purine Metabolic Enzyme AMPD1 Is a Novel Therapeutic Target for Multiple Myeloma." Blood 132, Supplement 1 (November 29, 2018): 5614. http://dx.doi.org/10.1182/blood-2018-99-118603.
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Abstract Introduction. Novel agents have improved the prognosis of multiple myeloma. However, side effects of novel agents have been a huge issue for especially elderly and frail patients. Additionally, despite the high remission rate by novel agents, multiple myeloma is still an incurable disease. In order to improve those issues, it is necessary to develop a new therapeutic strategy which is highly specific to myeloma cells and which targets a different pathway from the present anti-myeloma agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and myeloma cells, and to examine whether it can be a novel target for multiple myeloma therapy. Materials and methods. A public available gene expression website GenomicScape (http://www.genomicscape.com/) and Genevestigator (https://genevestigator.com/gv/index.jsp) were utilized in order to study genes specifically expressed in human plasma cells and myeloma cells. To examine gene expression in myeloma cell lines, we utilized the gene expression data set of multiple cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE: http://www.broadinstitute.org/ccle). AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression was analyzed by westernblot and immunohistochemistry. Genes co-expressed with AMPD1 in human myeloma cells were identified using public available gene expression datasets (GSE 4581, GSE 9782). Molecular pathways associated with genes co-expressed with AMPD1 were analyzed using Molecular Signatures Database (http://software.broadinstitute.org/gsea/msigdb/index.jsp). Cell viability of myeloma cell lines and peripheral blood mononuclear cells (PBMCs) treated by AMPD inhibitors (compound #3, #4) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed using 7AAD dye and flow-cytometry. Results. Public gene expression screening analysis identified several genes specifically expressed in human plasma cells and myeloma cells. Among the identified genes, we focused on AMPD1, which has not been previously studied in multiple myeloma. CCLE analysis and RT-PCR analysis showed that AMPD1 is specifically expressed in bone marrow plasma cells, myeloma cell lines and patient derived myeloma cells. AMPD1 protein expression was limited to myeloma cell lines, human bone marrow myeloma cells and extramedullary plasmacytomas. Genes co-expressed with AMPD1 in human myeloma cells were associated with hypoxic pathways. Myeloma cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to cell lines cultured under normoxic condition. AMPD inhibitors induced cell death in myeloma cell lines from around 50 uM, while the effect against PBMCs were minimal. AMPD inhibitors were more effective against myeloma cell lines under hypoxic condition compared to normoxic condition, reflecting the higher AMPD1 expression under hypoxia. Conclusions. AMPD1 is a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), freeing ammonia during the process. AMPD1 has been previously reported that its expression is limited to skeletal muscles. This is the first report so far that among leukocytes and hematological malignancies, AMPD1 is specifically expressed in bone marrow plasma cells and myeloma cells. We also showed that AMPD1 expression in myeloma cells are increased under hypoxia. This indicates that AMPD1 plays a significant role in myeloma cells surviving under hypoxic conditions such as the bone marrow microenvironment. AMPD inhibitors showed cytotoxicity on myeloma cell lines in vitro, while PBMCs were not affected. Additionally, AMPD inhibitors were more effective under hypoxic condition, suggesting that AMPD1 inhibition works more specifically in the bone marrow microenvironment. Our report raise the possibility that AMPD1 inhibition can be a novel therapeutic strategy for multiple myeloma. Detailed analysis of myeloma cell death by AMPD1 inhibition is now undergoing. Disclosures Matsuoka: Bristol Myers Squibb: Research Funding.
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Sek, Kevin, Christina Mølck, Gregory Stewart, Lev Kats, Phillip Darcy, and Paul Beavis. "Targeting Adenosine Receptor Signaling in Cancer Immunotherapy." International Journal of Molecular Sciences 19, no. 12 (December 2, 2018): 3837. http://dx.doi.org/10.3390/ijms19123837.
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The immune system plays a major role in the surveillance and control of malignant cells, with the presence of tumor infiltrating lymphocytes (TILs) correlating with better patient prognosis in multiple tumor types. The development of ‘checkpoint blockade’ and adoptive cellular therapy has revolutionized the landscape of cancer treatment and highlights the potential of utilizing the patient’s own immune system to eradicate cancer. One mechanism of tumor-mediated immunosuppression that has gained attention as a potential therapeutic target is the purinergic signaling axis, whereby the production of the purine nucleoside adenosine in the tumor microenvironment can potently suppress T and NK cell function. The production of extracellular adenosine is mediated by the cell surface ectoenzymes CD73, CD39, and CD38 and therapeutic agents have been developed to target these as well as the downstream adenosine receptors (A1R, A2AR, A2BR, A3R) to enhance anti-tumor immune responses. This review will discuss the role of adenosine and adenosine receptor signaling in tumor and immune cells with a focus on their cell-specific function and their potential as targets in cancer immunotherapy.
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Zarou, Martha M., Alexei Vazquez, and G. Vignir Helgason. "Folate metabolism: a re-emerging therapeutic target in haematological cancers." Leukemia 35, no. 6 (March 11, 2021): 1539–51. http://dx.doi.org/10.1038/s41375-021-01189-2.
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AbstractFolate-mediated one carbon (1C) metabolism supports a series of processes that are essential for the cell. Through a number of interlinked reactions happening in the cytosol and mitochondria of the cell, folate metabolism contributes to de novo purine and thymidylate synthesis, to the methionine cycle and redox defence. Targeting the folate metabolism gave rise to modern chemotherapy, through the introduction of antifolates to treat paediatric leukaemia. Since then, antifolates, such as methotrexate and pralatrexate have been used to treat a series of blood cancers in clinic. However, traditional antifolates have many deleterious side effects in normal proliferating tissue, highlighting the urgent need for novel strategies to more selectively target 1C metabolism. Notably, mitochondrial 1C enzymes have been shown to be significantly upregulated in various cancers, making them attractive targets for the development of new chemotherapeutic agents. In this article, we present a detailed overview of folate-mediated 1C metabolism, its importance on cellular level and discuss how targeting folate metabolism has been exploited in blood cancers. Additionally, we explore possible therapeutic strategies that could overcome the limitations of traditional antifolates.
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Wang, Shufeng, Xin Yang, Feng Liu, Xinzheng Wang, Xuemin Zhang, Kun He, and Hongxia Wang. "Comprehensive Metabolomic Analysis Reveals Dynamic Metabolic Reprogramming in Hep3B Cells with Aflatoxin B1 Exposure." Toxins 13, no. 6 (May 27, 2021): 384. http://dx.doi.org/10.3390/toxins13060384.
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Hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure have been recognized as independent risk factors for the occurrence and development of hepatocellular carcinoma (HCC), but their combined impacts and the potential metabolic mechanisms remain poorly characterized. Here, a comprehensive non-targeted metabolomic study was performed following AFB1 exposed to Hep3B cells at two different doses: 16 μM and 32 μM. The metabolites were identified and quantified by an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)-based strategy. A total of 2679 metabolites were identified, and 392 differential metabolites were quantified among three groups. Pathway analysis indicated that dynamic metabolic reprogramming was induced by AFB1 and various pathways changed significantly, including purine and pyrimidine metabolism, hexosamine pathway and sialylation, fatty acid synthesis and oxidation, glycerophospholipid metabolism, tricarboxylic acid (TCA) cycle, glycolysis, and amino acid metabolism. To the best of our knowledge, the alteration of purine and pyrimidine metabolism and decrease of hexosamine pathways and sialylation with AFB1 exposure have not been reported. The results indicated that our metabolomic strategy is powerful to investigate the metabolome change of any stimulates due to its high sensitivity, high resolution, rapid separation, and good metabolome coverage. Besides, these findings provide an overview of the metabolic mechanisms of the AFB1 combined with HBV and new insight into the toxicological mechanism of AFB1. Thus, targeting these metabolic pathways may be an approach to prevent carcinogen-induced cancer, and these findings may provide potential drug targets for therapeutic intervention.
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Kalinichenko, Е. N. "Synthesis of Nucleosides`s Analogues and their Application as Chemotherapeutic Agents." Eurasian Chemico-Technological Journal 15, no. 3 (May 13, 2013): 189. http://dx.doi.org/10.18321/ectj222.
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Application of the chemical-enzyme approach which is a combination of chemical synthesis and enzyme reaction has allowed to develop original effective methods of synthesis both new compounds and known nucleoside antibiotics which have been inaccessible earlier. Among them preparations for the treatment of oncohaematological diseases like: Cytarabin, Leukladin (Cladribine), Fludarabel, ara-adenosine (Vidarabine), ara-guanosine and produced from the last by chemical method a new medicine for the treatment of T-cellular lymphomas – Nelarabine. Results on the comparative study of antileukemic activity of purine nucleosides using the model of the lymphoid leukemia L1210 are presented. It is shown that the model of mouse lymphoid leukemia L1210 in vivo is not useful for results extrapolation for human if specific activity of modified nucleoside shows itself in therapy of hair-cellular leucosis (HCL), acute myeloid leukemia (AML) and especially for T-cellular leucosis. From commercially available derivative of 2'-fluoro-arabinofuranose developed an original method for the synthesis of antitumor drug Clofarabine for treatment of acute lymphoblastic leukaemia (ALL) in pediatric patients. Highly effective technology producing pharmaceutical substance Pemetrexed has been developed. At present new chemical-therapeutic agent Pemetrexed, which is structural analog of folic acid containing 7-deasaguanine is introduced into clinic practice. It is first agent specially developed for treatment of malignant pleural mesothelioma. It is worth to note that possibilities of the Pemetrexed’s usage for treatment of other tumor diseases are not exhausted still. It is interesting to develop new method of synthesis of modified pyrimidine nucleoside Azacytidine possesses both cytostatic action and ability to inhibit DNA methyltransferases.
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Li, Lu, Sheng Nie, Chen Ren, Yanqin Li, and Dehua Wu. "The incidence, risk factors and outcomes of chemotherapy related acute kidney injury in China." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e24161-e24161. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e24161.
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e24161 Background: Nephrotoxicity of chemotherapeutic agents remains a significant complication limiting the efficacy of the treatment. However, comprehensive data on the epidemiology and outcomes of chemotherapy related acute kidney Injury in China is lacking. Methods: We conducted a nationwide cohort study of hospitalized patients from 25 general and children’s hospitals in China during 2013-2015. Patient-level data were obtained from the electronic hospitalization information system, prescription database and laboratory databases of all cancer patients who received chemotherapy and had at least two serum creatinine tests within any 7-day window during the hospitalization. AKI was defined and staged according to Kidney Disease Improving Global Outcomes criteria. The incidence of AKI in patients with various type of cancer and chemotherapeutic agents was examined. The outcomes of AKI, including in-hospital mortality, death after discharge, kidney recovery, and length of stay, were also assessed. Results: A total of 23,232 cancer patients, including 3,120 children ( < 18 years old), 16,310 adult (19-65 years old) and 3,802 elderly patients ( > 65 years old), were analyzed. Platinum compounds and pyrimidine analogues were the most common used chemotherapy agents for cancer patients. The overall incidence of AKI was 4.9%. Patients with urinary system malignancy (12.3%), hematological malignancy (10.2%) and nerve motor system malignancy (4.1%) have the highest incidence of HA-AKI. The top three types of chemotherapy drugs with the highest incidence of AKI were Purine analogues (30.1%), folic acid analogues (15.3%) and combinations of antineoplastic agents (14.1%). The nephrotoxicity of chemotherapy drugs was different among age groups. AKI is associated with a higher risk of in-hospital mortality and death after discharge. Conclusions: The risk of AKI in cancer patients varied in different age group, type of cancer and chemotherapeutic agents.
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Reddy, PurraBuchi, M. B. Madhusudana Reddy, Ramakrishna Reddy, Santosh S. Chhajed, and Pramodkumar P. Gupta. "Computational modelling and analysis of Pyrimidine analogues as EGFR inhibitor in search of anticancer agents." Biomedicine 41, no. 1 (April 2, 2021): 130–38. http://dx.doi.org/10.51248/.v41i1.548.
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Introduction and Aim:Epidermal Growth Factor Receptor tyrosine kinase is a well-known and widely studied cancer therapeutic target protein. Based on the reported anticancer activity of pyrimidines, a series of 13 compounds are designed. In the present studythe EGFR kinase domain is targeted with the designed 13 compounds.
Materials and Methods:With missing residue in the kinase domain of EGFR crystallized structure, the domain is modelled using homology modelling, evaluated, energy-based optimization is carried out using OPLS in Gromacs. The default bindingsite was considered from the known EGFR kinase domain – Erlotinibcomplex crystallized structure. The molecular docking is carried out using AutodockVina, Insilico toxicity profiling and enrichment analysis of pathway is studied using Swiss-ADME and Enrich R.
Results:Compounds 7, 9, 10 and 12 revealed a binding energy of -8.8, -8.3, -8.3 and -8.4 Kcal/mol and makes two h-bonds with MET-769. All the 13 compounds are under the range of Lipnski drug likeness, with high GI-absorption rate. Considering the metabolic enzyme activity, the entire series of compounds are predicted to inhibit the metabolizing enzyme CYP1A2, CYP2D6 and CYP3A4. Compounds 2, 3, 7, 8 and 13 acts as a substrate to CYP2C19 and compound 1, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 act as a substrate to CYP2C9.
Conclusion:The inhibition of metabolizing enzyme may affect the poor metabolizing and slowing down the excretion time of molecules from the body. The current in-silico molecular docking, in-silico PKPD study of compounds suggesting that they can be developed as putative lead compounds for developing new anti-cancer drugs.
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Rosenbach, Hannah, Julian Victor, Manuel Etzkorn, Gerhard Steger, Detlev Riesner, and Ingrid Span. "Molecular Features and Metal Ions That Influence 10-23 DNAzyme Activity." Molecules 25, no. 13 (July 7, 2020): 3100. http://dx.doi.org/10.3390/molecules25133100.
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Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.
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Kandimalla, E. R., F. G. Zhu, L. Bhagat, D. Yu, and S. Agrawal. "Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpG DNAs." Biochemical Society Transactions 31, no. 3 (June 1, 2003): 654–58. http://dx.doi.org/10.1042/bst0310654.
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Bacterial and synthetic DNA containing unmethylated 2´-deoxyribo(cytidine-phosphate-guanosine) (CpG) dinucleotides in specific sequence contexts activate the vertebrate innate immune system. A molecular pattern recognition receptor, Toll-like receptor 9 (TLR9), recognizes CpG DNA and initiates the signalling cascade, although a direct interaction between CpG DNA and TLR9 has not been demonstrated yet. TLR9 in different species exhibits sequence specificity. Our extensive structure–immunostimulatory activity relationship studies showed that a number of synthetic pyrimidine (Y) and purine (R) nucleotides are recognized by the receptor as substitutes for the natural nucleotides deoxycytidine and deoxyguanosine in a CpG dinucleotide. These studies permitted development of synthetic YpG, CpR and YpR immunostimulatory motifs, and showed divergent nucleotide motif recognition pattern of the receptor. Surprisingly, we found that synthetic immunostimulatory motifs produce different cytokine induction profiles compared with natural CpG motifs. Importantly, we also found that some of these synthetic immunostimulatory motifs show optimal activity in both mouse and human systems without the need to change sequences, suggesting an overriding of the species-dependent specificity of the receptor by the use of synthetic motifs. In the present paper, we review current understanding of structural recognition and functional modulation of TLR9 receptor by second-generation synthetic CpG DNAs and their potential application as wide-spectrum therapeutic agents.
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Tiacci, Enrico, Valentina Pettirossi, Gianluca Schiavoni, and Brunangelo Falini. "Genomics of Hairy Cell Leukemia." Journal of Clinical Oncology 35, no. 9 (March 20, 2017): 1002–10. http://dx.doi.org/10.1200/jco.2016.71.1556.
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Hairy cell leukemia (HCL) is a chronic mature B-cell neoplasm with unique clinicopathologic features and an initial exquisite sensitivity to chemotherapy with purine analogs; however, the disease relapses, often repeatedly. The enigmatic pathogenesis of HCL was recently clarified by the discovery of its underlying genetic cause, the BRAF-V600E kinase-activating mutation, which is somatically and clonally present in almost all patients through the entire disease spectrum and clinical course. By aberrantly activating the RAF-MEK-ERK signaling pathway, BRAF-V600E shapes key biologic features of HCL, including its specific expression signature, hairy morphology, and antiapoptotic behavior. Accompanying mutations of the KLF2 transcription factor or the CDKN1B/p27 cell cycle inhibitor are recurrent in 16% of patients with HCL and likely cooperate with BRAF-V600E in HCL pathogenesis. Conversely, BRAF-V600E is absent in other B-cell neoplasms, including mimickers of HCL that require different treatments (eg, HCL-variant and splenic marginal zone lymphoma). Thus, testing for BRAF-V600E allows for a genetics-based differential diagnosis between HCL and HCL-like tumors, even noninvasively in routine blood samples. BRAF-V600E also represents a new therapeutic target. Patients’ leukemic cells exposed ex vivo to BRAF inhibitors are spoiled of their HCL identity and then undergo apoptosis. In clinical trials of patients with HCL who have experienced multiple relapses after purine analogs or who are refractory to purine analogs, a short course of the oral BRAF inhibitor vemurafenib produced an almost 100% response rate, including complete remission rates of 35% to 42%, without myelotoxicity. To further improve on these results, it will be important to clarify the mechanisms of incomplete leukemic cell eradication by vemurafenib and to explore chemotherapy-free combinations of a BRAF inhibitor with other targeted agents (eg, a MEK inhibitor and/or an anti-CD20 monoclonal antibody).
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Kapadiya, Khushal M., and Ranjan C. Khunt. "Discovery of Hybrid Purine-quinoline Molecules and Their Cytotoxic Evaluation." Letters in Drug Design & Discovery 16, no. 1 (November 1, 2018): 21–28. http://dx.doi.org/10.2174/1570180815666180419151742.
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Background: Apart from the “hit drugs”, there are many others being studied for their potent activity against several hostilities. To date, anticancer research has been exploited on the inherent versatility and active core skeleton of the compounds. Literature suggests that nitrogen rich molecules are most active and found in their potent cancer activity. Purine-based compounds such as olomoucine and roscovitine, which contain other heterobicyclic ring systems, are useful for the cell proliferation inhibitors in the treatment of many types of cancer. Methods: We put forward the novel purine based compounds, aryl amino-quinoline-purine by a two-step procedure. In the first step, nitrogen rich molecule was synthesized by the coupling of 2,6- dichloropurine with 3-aminoquinoline in an acidic reaction conditions at the C-6 position of purine. Aryl amines were introduced at the C-2 position by acid catalyst and using polar solvent at comparatively higher reaction conditions to furnish the desired products. Results: Stereochemical aspect was introduced for the identification of attachment of 3- aminoquinoline at the C-2/ C-6 position of purine and it was concluded by the spectral analysis (HMBC spectrum). The spectral data revealed that the first chloro-amine coupling was directed at the C-6 position rather than C-2 and the second chloro-amine coupling by various aryl amines were directed at the C-2 position. The applications of synthesized compounds were identified by their cytotoxic study against NCI-60 cell-lines. Out of nine selected molecules by NCI, 5a has shown promising response in a single dose study and GI50 value, 7.57 µM indicated that it has 7.57% lethality over HOP-92 cell-line (non-small cell lung cancer panel). Conclusion: Two straightforward novelties were introduced, first stereochemical identification for chloro-amine coupling in purine either at the C-2 or C-6 position on the basis of HMBC spectrum. And a second type of uniqueness was to identify better anti-cancer agents out of synthesized scaffolds. Overall study shows that compound 5a is a novel therapeutic agent after modification for the treatment of non-small cell lung and it satisfied determined threshold growth inhibition criteria at a single dose level.
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Young, James D. "The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey." Biochemical Society Transactions 44, no. 3 (June 9, 2016): 869–76. http://dx.doi.org/10.1042/bst20160038.
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Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na+-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins.
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Grote, Michaela, Steffi Noll, Bernhard Noll, Bernd Johannsen, and Werner Kraus. "Syntheses of novel modified acyclic purine and pyrimidine nucleosides as potential substrates of herpes simplex virus type-1 thymidine kinase for monitoring gene expression." Canadian Journal of Chemistry 82, no. 4 (April 1, 2004): 513–23. http://dx.doi.org/10.1139/v04-005.
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Suicide gene therapy with the herpes simplex virus type-1 thymidine kinase gene (HSV-1 tk) is considered to be a promising approach to the treatment of cancer. Making use of the lower specificity of the viral enzyme compared to human thymidine kinase, the therapy involves the administration of antiviral agents (e.g., ganciclovir) as prodrugs to induce enzymatic cell death in those cells that express the transferred gene. 18F-labelled derivatives have been described for monitoring location, duration, and magnitude of the viral kinase enzyme activity by positron emission tomography (PET). Since an optimal radiotracer has not been developed, novel substances were synthesized for monitoring gene expression. A group of 13 nucleoside analogues were synthesized, among them N1-methyl-9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (5) and N1-methyl-9-[(4-hydroxy)-3-hydroxymethylbutyl]guanine (7) as methyl analogues of ganciclovir and penciclovir and their related fluoro compounds (6, 8). Further novel derivatives include N6-methyl-9-[(1,3-dihydroxy-2-propoxy)methyl]-, N6-methyl-9-[(4-hydroxy)-3-hydroxymethylbutyl]adenine (9, 10), as well as the uracil derivatives 5-hydroxy-1-[(1,3-dihydroxy-2-propoxy)methyl]uracil (11), 6-methyl-1-[(1,3-dihydroxy-2-propoxy)-methyl]uracil (12), and its 3-fluoro-derivative (13).Key words: fluorinated nucleoside analogues, gene therapy, PET, thymidine kinase.
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van der Westhuyzen, Christiaan W., Richard K. Haynes, Jenny-Lee Panayides, Ian Wiid, and Christopher J. Parkinson. "Anti-Mycobacterial Peroxides: A New Class of Agents for Development Against Tuberculosis." Medicinal Chemistry 16, no. 3 (April 17, 2020): 392–402. http://dx.doi.org/10.2174/1573406415666190430143535.
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Background: With few exceptions, existing tuberculosis drugs were developed many years ago and resistance profiles have emerged. This has created a need for new drugs with discrete modes of action. There is evidence that tuberculosis (like other bacteria) is susceptible to oxidative pressure and this has yet to be properly utilised as a therapeutic approach in a manner similar to that which has proven highly successful in malaria therapy. Objective: To develop an alternative approach to the incorporation of bacterial siderophores that results in the creation of antitubercular peroxidic leads for subsequent development as novel agents against tuberculosis. Methods: Eight novel peroxides were prepared and the antitubercular activity (H37Rv) was compared to existing artemisinin derivatives in vitro. The potential for toxicity was evaluated against the L6 rat skeletal myoblast and HeLa cervical cancer lines in vitro. Results: The addition of a pyrimidinyl residue to an artemisinin or, preferably, a tetraoxane peroxidic structure results in antitubercular activity in vitro. The same effect is not observed in the absence of the pyrimidine or with other heteroaromatic substituents. Conclusion: The incorporation of a pyrimidinyl residue adjacent to the peroxidic function in an organic peroxide results in anti-tubercular activity in an otherwise inactive peroxidic compound. This will be a useful approach for creating oxidative drugs to target tuberculosis.
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Wiederkehr, D., R. Casciano, L. Stern, J. Zheng, and J. Baladi. "Therapeutic care in metastatic renal cell carcinoma during the follow-up phase of the RECORD-1 phase III trial." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e17531-e17531. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e17531.
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e17531 Background: Following drug discontinuation for progression or adverse event in a clinical trial for relapsed or stage IV kidney cancer, supportive care including surgery, palliative radiotherapy, or bisphosphonates continue to be recommended by National Comprehensive Cancer Network (NCCN). However, published data on active therapeutic agents given to patients following study drug discontinuation in recent clinical trials is limited. Methods: World Health Organization Anatomical Therapeutic Chemical codes or therapeutic names, captured from the follow-up phase in a phase III clinical trial (RECORD-1) of patients with metastatic renal cell carcinoma (mRCC) patients, were used to describe antineoplastic therapies following discontinuation of study drug. Prior to trial, patients had progressed on at least one VEGFr-TKI therapy. Results: Of the 130 patients with follow-up after discontinuation of study drug, 78.5% received at least one of the following: corticosteroids, radiotherapy, protein kinase inhibitors, mTOR inhibitor, pyrimidine analogues, monoclonal antibodies, interferons, and investigational drugs. Among patients who received an active agent, nearly three-quarters (73.5%) utilized targeted therapy (protein kinase inhibitors, mTOR inhibitor, monoclonal antibodies). Conclusions: In a clinical trial setting with mRCC patients who have received several classes of systemic therapy, care delivered following study drug discontinuation often includes an active antineoplastic agent, despite the limited supportive evidence in this setting. While the placebo control with supportive care in a double-blind phase is acceptable to evaluate the efficacy and safety of a therapy for regulatory approval purposes, decision makers must also consider how these data may inform comparisons with the usual alternatives available to and used by physicians and patients in the non-trial setting. [Table: see text]
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Raje, Noopur, Shaji Kumar, Teru Hideshima, Kenji Ishitsuka, Hiroshi Yasui, Norihiko Shiraishi, Tanyel Kiziltepe, Shweta Chhetri, Nikhil C. Munshi, and Kenneth C. Anderson. "Didox Induced Apoptosis Occurs by Inhibiting DNA Synthesis and Repair Via Down-Regulation of Ribonucleotide Reductase M1 in Multiple Myeloma (MM)." Blood 106, no. 11 (November 16, 2005): 5153. http://dx.doi.org/10.1182/blood.v106.11.5153.5153.
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Abstract Ribonucleotide reductase is the rate-limiting enzyme of deoxynucleoside triphosphate synthesis and is therefore an excellent target for cancer chemotherapy. Inhibition of ribonucleotide reductase results in inhibition of DNA synthesis and has anti-neoplastic effects. Several ribonucleotide reductase inhibitors (RRIs) are already in clinical practice, including Hydroxyurea and Gemcitabine. Here we examine the anti-MM activity of a novel RRI, Didox. Our data shows that Didox has potent cytotoxicity against MM cells in 48-hour cultures. Apoptosis induced in MM cell lines by Didox (200mmol/l), evidenced by increased sub-G1 cell fraction, is caspase dependent, confirmed by blocking experiments with Z-VAD-FMK. Unlike other RRIs which mainly target the pyrimidine metabolism pathway (hydroxyurea and gemcitabine), didox targets both the purine and pyrimidine metabolism pathways in MM, as demonstrated by transcriptional profiling using the Affymetrix U133A 2.0 gene chip. Specifically, our data shows a ≥ 2 fold downregulation of genes in these pathways as early as 12 hours after exposure to Didox. Importantly, ribonucleotide reductase (RR) M1 component transcript was downregulated, associated with downregulation of this enzyme by Western Blot analysis, and inhibition of DNA synthesis. Furthermore, genes involved in DNA repair mechanisms were also downregulated after exposure to Didox, demonstrated by heirachial clustering of gene chip data and further validation by western blotting. Specifically RAD 51 homologue, which is involved in nucleotide excision repair and recombination repair, was downregulated in MM cells both transcriptionally as well as at the protein level. This was accompanied by downregulation of the BCL family protein including Bcl-2, Bclxl, and XIAP both at the transcriptional and post transcriptional levels. Since Didox acts on MM cells by inhibiting DNA synthesis and repair, combination studies with melphalan, an alkylating agent commonly used in MM, were next performed. Our data shows a strong in vitro synergism, with combination indices of <0.7 by the Chou Talahay method. When combined with DNA damaging agents like doxorubicin, additive cytotoxicity was noted. These studies therefore provide the preclinical rationale for evaluation of Didox, alone and in combination with alkylating agents and anthracyclines, to improve patient outcome in MM.
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Kawano, Yawara, Takayuki Sasano, Saki Kushima, Yuki Inada, Nao Nishimura, Hiroyuki Hata, and Masao Matsuoka. "Targeting the Plasma Cell Specific Purine Metabolic Enzyme, AMPD1, Induces Multiple Myeloma Cell Death Accompanying NAD Depletion." Blood 134, Supplement_1 (November 13, 2019): 3097. http://dx.doi.org/10.1182/blood-2019-125756.
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Introduction. Novel agents have improved the prognosis of multiple myeloma (MM) patients. However, MM is still an incurable disease. In order to achieve a cure, it is necessary to develop novel therapeutic agents that are highly specific to MM cells and target different pathways from the present anti-MM agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and MM cells, and to examine whether it can be a novel target for MM therapy. Materials and methods. Public available gene expression websites, GenomicScape and Genevestigator were utilized to study genes specifically expressed in human plasma cells and MM cells. To examine gene expression in MM cell lines, gene expression data set from the Cancer Cell Line Encyclopedia (CCLE) was analyzed. AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression in MM cell lines and patient derived MM cells were examined by western blot and immunohistochemistry. Genes co-expressed with AMPD1 in MM cells were identified using public available gene expression datasets. Molecular pathway analysis was conducted using Molecular Signatures Database. Cell viability of MM cell lines, peripheral blood mononuclear cells (PBMCs) and patient derived bone marrow mononuclear cells treated by AMPD1 inhibitor (compound #3) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed by flow-cytometry after staining with 7AAD. Intracellular NAD and NADH concentrations in MM cell lines were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Z-VAD-FMK (Caspase inhibitor) and Nec-1 (RIP1 kinase inhibitor) were used in combination with compound #3 to study the mechanism of AMPD1 inhibition induced MM cell death. Results. We identified several genes specifically expressed in human plasma cells and MM cells using public available gene expression websites. Among the identified genes, we focused on AMPD1, a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), since this gene has not been previously studied in MM. We found that AMPD1 gene expression was limited to MM cell lines and patient derived MM cells through CCLE analysis and RT-PCR. AMPD1 protein expression was detected only in MM cell lines, bone marrow MM cells and extramedullary plasmacytomas. Genes associated with AMPD1 expression were related to hypoxic pathways. MM cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to those cultured under normoxia, indicating that AMPD1 plays a significant role in MM cell surviving under hypoxic condition such as the bone marrow microenvironment. Compound #3 induced cell death in MM cell lines and patient derived MM cells, while toxicity against PBMCs and non-MM cells were minimal. Compound #3 was more effective against MM cell lines cultured under hypoxia compared to those under normoxia, reflecting the higher AMPD1 expression under hypoxia. Since, AMPD1 is associated with purine metabolism, we analyzed the intracellular concentration of NAD and NADH, which are major cellular metabolites, in MM cell lines post compound #3 treatment. Marked decrease of NAD concentration and NAD / NADH ratio was observed in compound #3 treated MM cell lines compared to control, demonstrating that AMPD1 inhibition depletes intracellular NAD leading to MM cell death. Compound #3 treated MM cell lines showed increase in Annexin V- PI+ and Annexin V+ PI+ fractions. Additionally, Z-VAD-FMK and Nec-1 treatment did not reverse cell death induced by compound #3, indicating a distinct mechanism of cytotoxicity by compound #3 from other anti-MM agents inducing apoptosis and necroptosis. Conclusions. This is the first report so far that AMPD1 is specifically expressed in MM cells. AMPD1 inhibitor showed specific cytotoxicity against MM cells, while toxicities against non-MM cells were minimal. Additionally, AMPD1 inhibitor was more effective under hypoxic condition, suggesting that AMPD1 inhibition works more efficiently in the bone marrow microenvironment. Considering the specificity against MM cells and its distinct mechanism of action from the present anti-MM agents, AMPD1 inhibition is a potent novel therapeutic strategy for MM. Disclosures Matsuoka: Bristol-Myers Squibb Corp.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.
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Stankovic, Tatjana, Nicholas Davies, Louise J. Tee, Andrew D. Beggs, and Malcolm Taylor. "Identification of Novel Therapeutic Targets in Atm-Deficient Lymphomas Using a Whole Genome CRISPR/CAS-9 Screen." Blood 134, Supplement_1 (November 13, 2019): 1504. http://dx.doi.org/10.1182/blood-2019-129974.
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ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells. We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated. Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions. Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs. A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719. These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours. Disclosures No relevant conflicts of interest to declare.
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Khedkar, Harshita Nivrutti, Yu-Chi Wang, Vijesh Kumar Yadav, Prateeti Srivastava, Bashir Lawal, Ntlotlang Mokgautsi, Maryam Rachmawati Sumitra, Alexander T. H. Wu, and Hsu-Shan Huang. "In-Silico Evaluation of Genetic Alterations in Ovarian Carcinoma and Therapeutic Efficacy of NSC777201, as a Novel Multi-Target Agent for TTK, NEK2, and CDK1." International Journal of Molecular Sciences 22, no. 11 (May 31, 2021): 5895. http://dx.doi.org/10.3390/ijms22115895.
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Ovarian cancer is often detected at the advanced stages at the time of initial diagnosis. Early-stage diagnosis is difficult due to its asymptomatic nature, where less than 30% of 5-year survival has been noticed. The underlying molecular events associated with the disease’s pathogenesis have yet to be fully elucidated. Thus, the identification of prognostic biomarkers as well as developing novel therapeutic agents for targeting these markers become relevant. Herein, we identified 264 differentially expressed genes (DEGs) common in four ovarian cancer datasets (GSE14407, GSE18520, GSE26712, GSE54388), respectively. We constructed a protein-protein interaction (PPI) interaction network with the overexpressed genes (72 genes) and performed gene enrichment analysis. In the PPI networks, three proteins; TTK Protein Kinase (TTK), NIMA Related Kinase 2 (NEK2), and cyclin-dependent kinase (CDK1) with higher node degrees were further evaluated as therapeutic targets for our novel multi-target small molecule NSC777201. We found that the upregulated DEGs were enriched in KEGG and gene ontologies associated with ovarian cancer progression, female gamete association, otic vesicle development, regulation of chromosome segregation, and therapeutic failure. In addition to the PPI network, ingenuity pathway analysis also implicate TTK, NEK2, and CDK1 in the elevated salvage pyrimidine and pyridoxal pathways in ovarian cancer. The TTK, NEK2, and CDK1 are over-expressed, demonstrating a high frequency of genetic alterations, and are associated with poor prognosis of ovarian cancer cohorts. Interestingly, NSC777201 demonstrated anti-proliferative and cytotoxic activities (GI50 = 1.6 µM~1.82 µM and TGI50 = 3.5 µM~3.63 µM) against the NCI panels of ovarian cancer cell lines and exhibited a robust interaction with stronger affinities for TTK, NEK2, and CDK1, than do the standard drug, paclitaxel. NSC777201 displayed desirable properties of a drug-like candidate and thus could be considered as a novel small molecule for treating ovarian carcinoma.
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Ghatalia, Pooja, Eddy Shih-Hsin Yang, Dongquan Chen, Shi Wei, Tiffiny Cooper, Sunil Sudarshan, Gurudatta Naik, and Guru Sonpavde. "Kinase gene expression profiling of metastatic tumor tissue to prioritize therapeutic targets in clear cell renal cell carcinoma." Journal of Clinical Oncology 33, no. 7_suppl (March 1, 2015): 476. http://dx.doi.org/10.1200/jco.2015.33.7_suppl.476.
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476 Background: Kinase inhibitors are the mainstay of therapy for patients (pts) with metastatic clear cell renal cell carcinoma (ccRCC). Since kinases are actionable and lethal metastatic tumor tissue has not been comprehensively studied, we hypothesized that paired intra-patient kinase gene expression analysis in primary tumor (T), matched normal kidney (N) and metastatic tumor tissue (M) may help identify drivers of metastasis and potential therapeutic targets. Methods: Total mRNA isolated from macrodissected formalin fixed paraffin embedded tissue from T, N, and M from 35 pts (total 105 samples) with ccRCC underwent expression profiling for 519 kinase genes using the nCounter System by Nanostring Technologies. Digital raw counts of mRNA abundance were normalized using internal positive and negative controls as well as 7 housekeeping genes. The mean signals from N, T, and M sites were used to calculate change in gene expression, and a p value by t test <0.05 was considered significant. To identify genes unique to metastasis, kinases with significant expression in the M compared to N or T were selected. Functional analysis was conducted to identify key signaling pathways by using Ingenuity Pathway Analysis. Results: The median age of pts was 56 years and 30 were male. Fifteen pts had metastatic disease at presentation and the remaining developed subsequent metastasis. Clustering analysis for all 105 samples separated normal tissues from tumor or metastasis. The top pathways involved in the kinases overexpressed in M compared to N or T involved Pyridoxal 5'-phosphate Salvage, Salvage Pathways of Pyrimidine Ribonucleotides, NF-kB Signaling, NGF Signaling and Cell Cycle Control of Chromosomal Replication. The top kinases over-expressed in M compared to T include: EPHB2, AURKA, GUCY2C, GSG2, IKBKE, MELK, CSK, CHEK2, CDC7, and MAP3K8. Conclusions: Multiple pathways and kinase genes wereover-expressed in metastatic ccRCC, which suggests a major role for agents targeting metabolic pathways and inhibitors of kinases not currently recognized as therapeutic targets. These data warrant validation and suggest potential drivers of metastasis and actionable therapeutic targets in metastatic ccRCC.
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Yao, Yixin, Yang Liu, Hui Guo, Makhdum Ahmed, Shabnam Bhuiyan, Krystle Nomie, Liang Zhang, and Michael Wang. "Metabolic Profiling Identifies De Novo Nucleotide Synthesis As a Potential Metabolic Vulnerability for Targeted Therapy Against Mantle Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 2945. http://dx.doi.org/10.1182/blood-2018-99-112192.
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Abstract Introduction: Cancer cells exhibit dramatic alterations in cellular metabolism, such as enhanced de novo nucleotide synthesis, to support cell growth, proliferation and survival. The abundance of the nucleotide pool as well as the level and activity of different rate-limiting enzymes belonging to the nucleotide synthetic pathway limit the maximal proliferative capacity of cells. Maintenance of an adequate pool of deoxyribonucleotide triphosphates is essential for DNA replication and DNA repair, and consequently, the genetic integrity of nuclear and mitochondrial genomes. We and others have demonstrated that mantle cell lymphoma (MCL) undergoes metabolic reprogramming to progress and develop resistance to targeted therapy; however, the contribution of de novo nucleotide synthesis to the development and progression of MCL remains poorly understood. In contrast, oncogenic Myc is demonstrated to be highly upregulated in a subset of MCL. In addition to its pro-glycolysis, pro-biogenesis and pro-tumor growth functions, oncogenic levels of Myc induce the expression of multiple genes involved in the nucleotide biosynthetic pathway (e.g., IMPDH2, CTPS1, and CAD). Myc-induced glutamine metabolism also increases the abundance and activity of different rate-limiting enzymes that produce the molecular precursors required for de novo nucleotide synthesis. The γ-nitrogen amide group of glutamine is an indispensable donor of nitrogen for de novo synthesis of both nucleobases purine and pyrimidine. Here, we hypothesize that a subset of MCL depends on de novo nucleotide synthesis for anabolic cell growth and cancer progression due to aberrant Myc expression and Myc-induced glutaminolysis. Methods: Primary MCL biopsy, apheresis, and blood specimens as well as MCL cell lines were utilized for metabolic and functional analyses. Liquid Chromatography Mass Spectrometry (LC-MS) metabolomics was employed to measure the steady-state level of metabolites. Western-blotting and real-time qPCR were utilized determine protein and gene expression levels. BrdU incorporation and the Cell-Trace Violet Cell Proliferation Assay were employed to assess DNA synthesis and cell proliferation. Cell viability was measured with the Cell Titer-Glo Cell Viability Assay. Pharmacological agents were employed to inhibit either de novo nucleotide synthesis or glutaminolysis. Results: Metabolomics profiling of steady-state levels of intracellular metabolites showed significant increases in N-carbamoyl aspartate/dihydroorotate and 5-phosphoribosyl-1-pyrophosphate (PRPP), which are critical intermediates in de novo pyrimidine and purine synthesis, respectively, as well as CTP, dUTP, dCTP, in a subset of MCL, indicating remarkably upregulated de novo nucleotide synthesis. The protein and mRNA levels of c-Myc and its target genes involved in the metabolism of nucleotides (IMPDH2, CTPS1, CAD) were significantly increased. Inhibition of pyrimidine synthesis with DON (6-diazo-5-oxo-L-norleucine), a CTPS1 inhibitor, dramatically reduced the pool of pyridine nucleotides, leading to remarkable apoptosis and halted cell proliferation of a subset of MCL cell lines. Consistent with c-Myc overexpression, increased glutamine uptake was also observed in a subset of MCL cell lines. Glutamine deprivation or pharmacological inhibition of glutamine metabolism showed a similar effect on the inhibition of pyrimidine synthesis as DON (6-diazo-5-oxo-L-norleucine), manifested by a significant reduction of pyrimidine nucleoside triphosphate levels, a dramatic increase in apoptosis, and retarded cell proliferation of a subset of MCL cell lines. Conclusions: Our preliminary results indicate that de novo nucleotide synthesis is upregulated in a subset of MCL with aberrant c-Myc expression. The expression of genes involved in nucleotide metabolism as well as glutaminolysis is also elevated in these cancer cells. Disruption of de novo nucleotide synthesis or glutaminolysis induces apoptosis and suppresses proliferation of a subset of MCL. Myc does not possess enzymatic activity and is considered "undruggable"; therefore, the inhibition of Myc target genes such as those involved in de novo nucleotide synthesis and glutaminolysis presents a promising alternative approach. Taken together, MCL dependency on de novo nucleotide synthesis may represent a metabolic vulnerability for targeted therapy for MCL. Disclosures Wang: AstraZeneca: Consultancy, Research Funding; Juno: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Dava Oncology: Honoraria; MoreHealth: Consultancy; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Metzger, A. S., P. Lara, D. Lau, P. Mack, P. Gumerlock, D. Gandara, and A. M. Davies. "Phase I study of two different schedules of bortezomib (BORT) and pemetrexed (PEM) in advanced solid tumors." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 17051. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.17051.
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17051 Background: BORT(PS-341) is a small molecule proteasome inhibitor while PEM, a multi-targeted antifolate, inhibits multiple enzymes involved in purine/pyrimidine formation. Both are approved anti-cancer agents. We examined the safety and tolerability of two different schedules of BORT and PEM in patients with advanced solid tumors. Methods: Two dose escalation trials (Arm A and Arm B) were conducted simultaneously. PEM was given every 21 days in both arms (500–600 mg/m2 IV). Arm A: BORT was given D1,4,8 &11 (0.7–1.3 mg/m2). Arm B: BORT was given D1 & 8 (1.0–1.6 mg/m2). All patients received vitamin B12, folic acid and decadron. Dose limiting toxicity (DLT) was defined as: grade (GR) 4 platelets (plts) (≤25K); GR 3 plts (25K-49,999K) with bleeding, requirement for transfusion, or lasting > 7 days; febrile neutropenia; GR3 ANC (ANC ≤1.0 × 109) with documented infection, or any ≥ GR3 non-heme toxicity. Results: 18 patients have been treated on 3 of the 4 planned dose levels. Tumor types included lung (12), prostate (2), breast (1), thymus (1), head & neck (1) and adenoid cystic carcinoma (1). Pt. characteristics: Median age 65 years; Sex M/F = 7/11; Performance Status ≤1/2 = 18/0. There have been no DLTs in either arm (Dose level 3-Arm A: PEM 500 mg/m2, BORT 1.3 mg/m2; Arm B:PEM 500 mg/m2, BORT 1.6 mg/m2). Most common GR3/4 toxicities were: neutropenia 27% and lymphopenia 11%. Of 17 evaluable patients: 1 had partial response, 11 had stable disease, 5 had progressive disease. Mean number of cycles: 4. Arm A had more doses held during the first cycle than Arm B (6 doses held vs. 1 dose held). Accrual to the final dose level in both arms is ongoing. Conclusions: 1) PEM in combination with BORT is feasible and tolerable. 2) Thus far, there are no differences in toxicity between the arms. 3) A randomized, multi-institutional phase II study will examine the efficacy of these two schedules in patients with NSCLC. No significant financial relationships to disclose.
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Wu, Xiaowei, Ziyi Fu, Han Ge, Xiaoan Liu, Yiqin Xia, Minghui Li, Yue Huang, Jinhui Peng, Shui Wang, and Hui Xie. "tRNA-derived small RNA fragments (tRFs) as potential biomarkers for doxorubicin resistance in breast cancer." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e12000-e12000. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e12000.
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e12000 Background: It has been confirmed that tRNA-derived small RNA fragments (tRFs) participate in various kinds of biological activities. However, the function of tRFs in chemotherapy resistance in breast cancer still remains unclear. The purpose of this study is to elucidate the relation between tRFs expression character and chemotherapy resistance in breast cancer. Methods: Drug sensitivity was validated by using Cell Counting Kit-8 in ADR-sensitive breast cancer cells (MCF-7) and ADR-resistant cells (MCF-7/ADR). Next-generation sequencing technology was conducted to identify differentially expressed tRFs between MCF-7 and MCF-7/ADR breast cancer cell lines; quantitative RT-PCR (qRT-PCR) were used to validate the differentially expressed tRFs in MCF-7 and MCF-7/ADR cell lines and the serums of chemotherapy sensitive and resistant patients. Then the dysregulated tRFs were identified through Gene ontology (GO), Pathway analysis and network analysis. Finally, receiver operated-characteristic curve (ROC) analysis was carried out to assess the predictive power of the dysregulated tRFs as biomarkers for chemotherapy resistance.The growth and sensitivity of breast tumor to Adriamycin were assessed by micro-CT in vivo. Results: There were 588 differentially expressed tRFs between MCF-7 and MCF-7/ADR cell lines (fold change > 2).tRF-2023, which was significantly downregulated in chemotherapeutic resistance breast cancer tissues and cells, could induce the sensitivity of breast cancer cells to Adriamycin both in vivo and in vitro. Bio-informatic analysis showed that tRF-2023 was mainly involved in flavonoid glucuronidation and cellular gluuronidation, and the target genes of tRF-2023 were involved in RNA polymerase, purine and pyrimidine metabolism, and p53 signaling pathway, which might illuminate the function of tRF-2023 in chemotherapy resistance of breast cancer. The result of ROC also showed that tRF-2023 was involved in breast cancer resistance. Conclusions: Our study provides a comprehensive analysis of tRFs in breast cancer resistance. It is helpful in revealing the significance of tRFs during the development of chemotherapy resistance in breast cancer. We also identified that tRF-2023 could be a new potential biomarker and therapeutic target for chemotherapy-resistant breast cancer.
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Babgi, Bandar A., Jalal H. Alsayari, Bambar Davaasuren, Abdul-Hamid Emwas, Mariusz Jaremko, Magda H. Abdellattif, and Mostafa A. Hussien. "Synthesis, Structural Studies, and Anticancer Properties of [CuBr(PPh3)2(4,6-Dimethyl-2-Thiopyrimidine-κS]." Crystals 11, no. 6 (June 16, 2021): 688. http://dx.doi.org/10.3390/cryst11060688.
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CuBr(PPh3)2(4,6-dimethylpyrimidine-2-thione) (Cu-L) was synthesized by stirring CuBr(PPh3)3 and 4,6-dimethylpyrimidine-2-thione in dichloromethane. The crystal structure of Cu-L was obtained, and indicated that the complex adopts a distorted tetrahedral structure with several intramolecular hydrogen bonds. Moreover, a centrosymmetric dimer is formed by the intermolecular hydrogen bonding of the bromine acceptor created by symmetry operation 1−x, 1−y, 1−z to the methyl group (D3 = C42) of the pyrimidine–thione ligand. HSA-binding of Cu-L and its ligand were evaluated, revealing that Cu-L binds to HSA differently than its ligand. The HSA-bindings were modeled by molecular docking, which suggested that Cu-L binds to the II A domain while L binds between the I B and II A domains. Anticancer activities toward OVCAR-3 and HeLa cell lines were tested and indicated the significance of the copper center in enhancing the cytotoxic effect; negligible toxicities for L and Cu-L were observed towards a non-cancer cell line. The current study highlights the potential of copper(I)-phosphine complexes containing thione ligands as therapeutic agents.
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Wadler, Scott, Snehal Damle, Hilda Haynes, Ron Kaleya, Richard Schechner, Robert Berkenblit, Robert D. Ladner, and Anthony Murgo. "Phase II/Pharmacodynamic Trial of Dose-Intensive, Weekly Parenteral Hydroxyurea and Fluorouracil Administered With Interferon Alfa-2a in Patients With Refractory Malignancies of the Gastrointestinal Tract." Journal of Clinical Oncology 17, no. 6 (June 1999): 1771. http://dx.doi.org/10.1200/jco.1999.17.6.1771.
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PURPOSE: Combined depletion of pyrimidine and purine DNA precursors has resulted in therapeutic synergism in vitro. The aims of the current study were to test this strategy in patients with refractory tumors and to assess its effects on selected nucleotide pools. PATIENTS AND METHODS: A single-institution phase II trial was initiated in patients with advanced carcinomas of the stomach and pancreas. Patients had measurable disease and had no prior chemotherapy except adjuvant fluorouracil (5FU) or gemcitabine. 5FU was administered by CADD + pump at 2.6 g/m2 intravenously by 24-hour infusion on days 1, 8, 15, 22, 29, and 36. Parenteral hydroxyurea (HU) was administered at 4.3 g/m2 as a 24-hour infusion concurrently with 5FU. Interferon alfa-2a (IFN-α2a) was administered at 9 million units subcutaneously on days 1, 3, and 5 each week. No drug was administered in weeks 7 and 8. Pharmacodynamic studies were performed to assess drug effects on levels of deoxyuridine triphosphate (dUTP) and thymidine triphosphate (TTP) pools in peripheral-blood mononuclear cells (PBMCs) before and 6 hours after treatment using a highly sensitive DNA polymerase assay. RESULTS: There were 53 patients enrolled onto the study (gastric carcinoma, 31; pancreatic carcinoma, 22). The median age was 61 years, with 22% of patients ≥ 70 years old. The predominant grade 3 to 4 toxicities were leukopenia (49%), granulocytopenia (55%), and thrombocytopenia (22%). Severe diarrhea occurred in 12%, mucositis in 0%, and vomiting in 10% of patients. Patients ≥ 70 years had no greater incidence of toxicities. Among the 30 assessable patients with gastric carcinoma, there were two (7%) complete responders and 11 (37%) partial responders (median duration, 7 months). Among the 21 assessable patients with pancreatic carcinoma, there was one responder. Median survival among all patients with gastric carcinoma was 10 months and 13 months for patients with pancreatic carcinoma. Twenty-three patients had samples studied for levels of dUTP and TTP. There was no change in the levels of TTP before and after treatment. Furthermore, dUTP was detected in only five of 28 samples after treatment with no increase in the dUTP/TTP ratio. CONCLUSION: Combination therapy with high-dose, weekly infusional HU and 5FU with IFN-α2a modulation was well-tolerated with activity in gastric cancer. Patients ≥ 70 years tolerated therapy as well as younger patients. This was the first study to correlate levels of TTP and dUTP after treatment with clinical outcome. In PBMCs used as a surrogate tissue, HU abrogated the 5FU-induced increase in dUTP levels without reversing the overall efficacy of the regimen.
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Sobh, Amin, Charlotte Kaestner, Alberto Riva, and Jonathan D. Licht. "Identification of Genetic Vulnerabilities and Synthetic-Lethal Targets in NSD2-High Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 3757. http://dx.doi.org/10.1182/blood-2019-125951.
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The histone methyltransferase NSD2 is overexpressed in 15-20% of multiple myeloma (MM) patients due to the t(4;14) chromosomal translocation. NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. Despite strong efforts by academia and industry, there remains no tool compound or drug that inhibits NSD2 enzymatic activity. Therefore we explored the molecular and biochemical consequences of NSD2 overexpression in MM cells and investigated genetic vulnerabilities associated with high NSD2 levels as well as synthetic-lethal drug-gene interactions in t(4;14)-positive MM cells. We utilized the well-studied isogenic pair of human MM cells derived from the t(4;14)-positive KMS-11 cell line, where NSD2 is disrupted by knocking out either the translocated overexpressed allele (TKO; NSD2-low) or the wild-type non-translocated allele (NTKO; NSD2-high). Untargeted metabolomic profiling of TKO and NTKO cells revealed that NSD2 overexpression substantially alters nucleotide metabolism. NSD2-high cells exhibited an increase in purine synthesis and a decrease in pyrimidine synthesis. In addition, a genome-wide loss-of-function CRISPR gene editing screen using the 76,000 guide Brunello library uncovered genes differentially essential between NSD2-high and low MM cells. Intriguingly, one of the genes whose disruption is selectively lethal in NSD2-high cells encodes Adenylate Kinase 2 (AK2), an enzyme involved in purine metabolism, indicating that defective nucleotide metabolism associated with increased NSD2 expression introduces genetic vulnerabilities that can be therapeutically exploited. We further explored liabilities that can improve therapeutic outcomes in t(4;14) MM. A genome-wide CRISPR screen was performed in KMS-11 MM cells to identify mechanisms of sensitivity and resistance to dexamethasone, a steroid commonly used in MM treatment. As expected, inactivating the gene encoding the glucocorticoid receptor (GR) resulted in remarkable dexamethasone tolerance. We then identified and validated multiple genes/pathways that can alter response of MM cells to dexamethasone when disrupted. For example, inactivation of interleukin 10 (IL10) signaling by disrupting either subunit of the IL10 receptor or components of the downstream JAK/STAT pathway considerably enhances dexamethasone sensitivity. In addition, disruption of many components of heparan sulfate or glycosaminoglycans synthesis pathways, whose targeting has been previously shown to increase sensitivity to conventional MM chemotherapeutic agents, increases susceptibility to dexamethasone. By contrast, genetic perturbations leading to dexamethasone resistance correspond to GR co-chaperones including FKBP4 and PTGES3 and transcriptional coactivators like the nuclear receptor transactivator 1 (NCOA1). Interestingly, disruption of genes encoding proteins implicated in RNA stability and translation such as the N6-methyladenosine (m6A)-containing RNA binding protein YTHDF2 and the PAN2-PAN3 deadenylase complex results in dexamethasone resistance. How these proteins affect the expression of pro or anti-apoptotic genes in response to dexamethasone is under investigation. Our work reveals insight into novel molecular-based treatment options for t(4;14) MM that are independent on direct NSD2 inhibition which remains unsuccessful. Disclosures No relevant conflicts of interest to declare.
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Jayanthan, Aarthi, Anjali Singh, Victor A. Lewis, and Aru Narendran. "Preclinical Studies of Cytotoxicity, Drug Synergy and Biological Correlates of Clofarabine Against Infant Leukemia Cells." Blood 116, no. 21 (November 19, 2010): 4342. http://dx.doi.org/10.1182/blood.v116.21.4342.4342.
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Abstract Abstract 4342 Preclinical Studies of Cytotoxicity, Drug Synergy and Biological Correlates of Clofarabine Against Infant Leukemia Cells. Infants with leukemia, specifically those who relapse on frontline therapy, are extremely difficult to cure and are candidates for novel therapies that induce remission, allowing them to proceed to transplantation. The unique molecular, growth and chemoresistance properties of infant acute lymphoblastic leukemia (iALL) allow for focused preclinical studies within this group. Clofarabine (Clolar), an anti-neoplastic purine nucleoside analog, has shown significant efficacy in older children with refractory lymphoblastic leukemia. Its safety profile has more recently been established in Phase I and II single-agent trials. In addition to its anti-metabolite action, clofarabine appears to disrupt the integrity of mitochondrial membranes and activates pathways of programmed cell death, adding to its theoretical potential to synergize with agents that interfere with mitochondrial integrity. Methods: Primary leukemic cells, cell lines derived from iALL patients and cell lines carrying the molecular abnormalities commonly found in iALL, were used in this study (n=10). Karyotypic abnormalities of these cells include: t(11;19) (q23;p13), t(9;11)(p21;q23) and t(4;11)(q21;q23). With respect to Flt-3 expression, cell lines demonstrating wild-type, internal tandem duplication (ITD) and over-expression phenotypes were also included in this panel. Primary infant AML samples (n=2) and the infant AML cell line TIB-202 (THP-1) containing the t(9;11)(p21;q23) rearrangement and MLL-AF9 fusion gene were also included. ALL cell lines derived from pediatric patients (n= 5) were evaluated in parallel. Stromal cells established from normal bone marrow specimens and peripheral blood mononuclear cells were evaluated under identical conditions for assessment of non-specific toxicity. An increasing concentration of clofarabine was added to leukemic and control cells (104 cells per well, in 96 well plates) cell lines. Over the following four days, cell growth inhibition was measured by the Alamar blue assay. For drug combination studies, leukemia cells were incubated with a panel of conventional and targeted therapeutic agents (n=12) alone or in combination with clofarabine (IC10 or IC25 concentrations). Growth inhibition under each condition was measured and combination indices were calculated according to established methods. Induction of apoptosis and the release of mitochondrial mediators were measured by Western blot analysis. Alteration in mitochondrial integrity was evaluated by immunocytochemistry for fluorescent labeled anti-mitochondrial Hsp70 and real-time imaging. Results and Discussion: Clofarabine inhibited growth of all iALL cells tested with IC50 values ranging from 0.1 μ M to 0.01 μ M. Primary iAML cells were found to be most sensitive to clofarabine. For iALL cell lines the highest IC50 value was found in Bel-1 cells, expressing a t(4;11)(q21;q23) karyotype. Drug combination studies showed significant synergy with 17-AAG (Hsp90 inhibitor, CI 0.7), sorafenib (CI 0.12), bortezomib (CI 0.3) and rapamycin (CI 0.2). No drug combinability was noted, with conventional alkylating agents and antimetabolites. Interestingly, the therapeutic opioid methadone (D,L-methadone hydrochloride), used extensively in the treatment of cancer pain and opioid addiction, showed significant synergy with clofarabine at low concentrations (CI 0.74, range 0.66 – 0.79 μ M). Incubation of cells with clofarabine (IC25) for 48 hours resulted in detectable activation of caspase 9 and cleavage of PARP. We demonstrate the ability of clofarabine to induce cytotoxicity against a panel of leukemia cells that carry the molecular aberrations and growth properties seen in iALL. We also present data on the biological correlates and synergistic effects of clofarabine with other anti-leukemic agents. Of particular interest is the synergy with methadone, which has been shown previously to affect mitochondrial activity in leukemia cells. Data presented in study provide key initial data to construct effective xenograft studies and to formulate a clofarabine based treatment protocol for iALL in the near future. Disclosures: Narendran: Genzyme Canada: Research Funding.
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Puissant, Alexandre, Nina Fenouille, Christopher F. Bassil, Issam Ben-Sahra, Gabriela Alexe, Lina Benajiba, Azucena Ramos, et al. "Identification of CKMT1B As a New Target in EVI1-Positive AML." Blood 126, no. 23 (December 3, 2015): 3674. http://dx.doi.org/10.1182/blood.v126.23.3674.3674.
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Abstract Recent investigations into the relationship between cancer and cellular metabolism have revealed the strong dependency of different cancers on a diverse array of metabolic pathways. For instance, a variety of cancers depend on the PI3K/AKT pathway for a wide range of glucose-related mechanisms, including AKT-mediated membrane translocation of glucose transporters, activation of the glycolytic enzymes hexokinase and phosphofructokinase, and up-regulation of de novo fatty acid synthesis. A major area of therapeutic and biological interest in the study of cancer metabolism is the process by which these metabolic pathways become deregulated in the first place en route to, or as a result of, the development of cancer. The deregulation of metabolic pathways can occur through alterations in the cellular landscape brought about by mutations in metabolic enzymes (e.g., IDH1/IDH2 and SDH), aberrant expression of transcription factors, such as the proto-oncogene MYC, or the loss of tumor suppressors, such as p53. Through gene expression and metabolic profiling analyses, we found that the transcription factor EVI-1, whose overexpression in acute and chronic myeloid leukemia (AML and CML) is correlated with poor patient outcome, induced key metabolic perturbations in hematopoietic progenitor cells. These result in a decrease of the mitochondrial oxygen consumption rate, a blockade of the de novo purine and pyrimidine synthesis, and an increase in glycolysis. Using a library of pooled shRNAs targeting genes involved in each of these pathways, we then established a direct link between EVI-1 expression and the development of a heretofore undescribed cellular dependency on the overexpression of the ATP-buffering mitochondrial creatine kinase protein CKMT1B. We showed that EVI-1 directly promotes CKMT1B expression through repression of the master regulator of myeloid differentiation RUNX1. Alteration of the CKMT1B-dependent pathway, either with shRNA or with the small molecule cyclocreatine, impairs production of intracellular phospho-creatine, which in turn alters cell viability specifically in EVI-1-positive (n = 8) versus EVI-1-negative AML cell lines (n = 8), and in EVI-1-positive (n = 5) versus EVI-1-negative (n = 17) primary AML blasts. This decrease in cell viability is associated with the activation of an erythroid differentiation program concomitant with a downregulation of the immature lineage marker c-KIT, both induced by GSK3A/B inhibition. Overexpression of a constitutively activated form of GSK3B (S9A) or GSK3A (S21A) impairs the cell differentiation induced by CKMT1B inhibition. Finally, suppression of CKMT1B alleviates leukemic burden in vivo in two AML mouse models: i) an orthotopic model of transplanted human EVI1-positive AML cells and ii) a syngeneic model of transplanted murine NrasG12D + Evi1 AML cells. This new interplay between EVI-1 and the creatine pathway uncovers CKMT1B as a new target of interest in EVI-1-positive AML, a high-risk subtype of AML for which current treatment regimens remain inadequate. Disclosures DeAngelo: Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Agios: Consultancy; Celgene: Consultancy. Stone:Celgene: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Novartis: Research Funding; Celator: Consultancy; Merck: Consultancy; Roche/Genetech: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.
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Rogalinska, Malgorzata, Jerzy Blonski, Pawel Goralski, Oxana Komina, Arleta Kazmierczuk, Ewa Wawrzyniak, Aleksandra Kotkowska, et al. "Usefulness of Differential Scanning Calorimetry for Monitoring Ex Vivo the Changes In Responses of CLL Cells to Anti-Cancer Drugs: Development of Personalized Therapy." Blood 116, no. 21 (November 19, 2010): 4635. http://dx.doi.org/10.1182/blood.v116.21.4635.4635.
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Abstract Abstract 4635 Objective: Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia among elderly people in Europe and North America. CLL is considered as a cancer involving mainly deregulated apoptosis. A strong difference between patients in the disease progression, response to anti-cancer therapy, and outcome is a major challenge in elaboration of efficacious treatment. Therefore, it is important to develop personalized therapeutic protocols based on the individual patient's sensitivity to medication. The aim of our ex vivo studies was to monitor the changes in responses of leukemic cells to anti-cancer agent(s) and correlate results obtained by DSC and other supplementary techniques with patients clinical response. Methods: Leukemic cells were obtained from peripheral blood of 14 patients prior to the onset of CC (cladribine + cyclophosphamide) therapy. Primary tumour cell samples were exposed ex vivo to CM or FM, as well as to ROSC alone for 48 hrs. We monitored the induction and progress of apoptosis in CLL cells exposed ex vivo to purine analogs combined with mafosfamide i.e. CM (cladribine + mafosfamide), FM (fludarabine + mafosfamide), and additionally to R-roscovitine (ROSC), an inhibitor of cyclin-dependent kinases, by a few techniques, i.e. cell viability test, apoptosis assay, differential scanning calorimetry (DSC), immunoblotting and determination of caspase-3/7 activity in the culture medium. The characteristic changes in chromatin complex induced upon the above treatments were detected by DSC, a simple thermal technique. This study received approval of the Medical University of Lodz Ethical Committee (RNN/196/07/KE). Informed consent was obtained from all the patients. Results: Marked differences in the individual patients’ sensitivity to the agents used were registered. A decrease or even loss of transition at 95±5°C in thermal scans of nuclear fraction preparations occurred in the treated cells in which apoptosis was induced. Remarkably, the changes in thermal profiles coincided with an accumulation of apoptotic cells, a decrease of the number of viable cells, and the changes in cellular levels as well as functional status of apoptosis-related proteins (caspase-9, Mcl-1) and CLL prognostic factor – kinase Zap-70. However, no significant changes were observed in thermal profiles of nuclei originating from CLL cells resistant to the treatment. Our studies revealed that the ex vivo exposure of CLL cells to CM combination, equivalent to clinically used CC regimen, was of prognostic value. Among the DSC profiles of nuclear preparations isolated from leukemic cells treated ex vivo with CM 70% of the cases examined indicated the decrease or loss of transition at about 95 ± 5°C. Moreover, we noticed a statistically significant dependence between the patient's response to CC treatment and thermal transition reduction at 95 ± 5°C (p=0.034). Interestingly, the results of the examined group did not show any correlation between disease advance (Rai stage) or cytogenetic abnormalities versus the clinical patient's response to the used treatment. Conclusion: Our results demonstrate the advantage of DSC in the evaluation of the treatment efficacy and indicate that its application could facilitate the choice of highly effective therapy for individual patients. Disclosures: No relevant conflicts of interest to declare.
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Badoux, Xavier C., Michael Keating, Susan O'Brien, Alessandra Ferrajoli, Jan A. Burger, Stefan Faderl, Guillermo Garcia-Manero, Tapan Kadia, Lynne V. Abruzzo, and William G. Wierda. "Patients with Relapsed CLL and 17p Deletion by FISH Have Very Poor Survival Outcomes." Blood 114, no. 22 (November 20, 2009): 1248. http://dx.doi.org/10.1182/blood.v114.22.1248.1248.
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Abstract Abstract 1248 Poster Board I-270 Background There has recently been substantial work to identify factors that correlate with clinical outcome for patients with CLL. Identifying and validating factors may give understanding and insight into the biology of the disease and may help guide management of patients. TP53 is a gene located on the short arm of chromosome 17 (17p). The protein product of TP53, p53, is a transcription factor that regulates cell cycle and functions as a tumor suppressor. Therefore, p53 is critically important for the survival of CLL cells. In addition, p53 is critical for the therapeutic response to chemotherapy, particularly for alkylating agents and purine analogues. Thus patients with loss (17p del) or mutation of TP53 have inferior response to standard treatments, shorter remission duration, and shorter overall survival. We previously reported outcomes for untreated patients with de novo loss of 17p identified by FISH (Tam et al. Blood 114:957, 2009). In that analysis, there were patients with 17p del who did not require treatment and were observed for a prolonged period, however, it was clear that once treatment was required, outcomes markedly deteriorated. The current analysis summarizes outcomes for previously treated patients with 17p del who went on salvage therapy at MD Anderson Cancer Center. Methods and Results We identified 95 previously treated patients who were evaluated at MDACC, had 17p del by FISH, and went on a salvage therapy from March 2004 to May 2009 (Table). There were 10 patients known to have de novo 17p del prior to frontline therapy and 9 patients with acquired mutations (76 unknown). In addition to 17p del, 18 also had 11q del, 17 had +12, 53 had 13q del; 26 had no additional, 53 had 1 additional, 13 had 2 additional, and 3 had 3 additional chromosome abnormalities by FISH analysis. The median number of prior treatments was 3 (range 1-10); prior treatment regimens in hierarchical order consisted of multi-agent-based chemotherapy (N=18); fludarabine combination-based (N=59); single purine analogue or alkylating agent-based (N=13); and monoclonal antibody- (mAb) or steroid-based (N=4). For this analysis follow-up began from the first salvage therapy after discovery of 17p del by FISH. Salvage therapies included alemtuzumab or alemtuzumab with rituximab (N=15); other mAb-based (N=12); lenalidomide-based (N=8); fludarabine-based combinations (N=24); combined oxaliplatine, fludarabine, cytarabine, rituximab (OFAR) (N=28), and others not fitting any of these categories (N=8). The outcomes for these patients were very poor (Table). The complete remission (CR) rate with salvage therapy was 6% and the overall response rate (ORR) was 36%. The median follow-up was 10 months, time to treatment failure was 4.1 months, and overall survival was 14 months. Sixteen patients proceeded on to allogeneic stem cell transplant with a median OS of 27 months. Conclusion Previously treated patients with 17p del have extremely poor prognosis and outcomes based on low response rate to salvage treatment, short time to treatment failure and overall survival, including with alemtuzumab-containing treatment. This is a population who clearly needs new and active treatment regimens that have a p53-independent mechanism of action. Experience with allogeneic stem cell transplant in this population is limited and requires further investigation. Disclosures No relevant conflicts of interest to declare.
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Tedeschi, Alessandra, Giulia Benevolo, Gloria Casaluci Margiotta, Simone Ferrero, Paola Picardi, Marzia Varettoni, Claudia Baratè, et al. "Bendamustine and Rituximab Combination Is Safe and Effective As Salvage Regimen in Waldenstrom's Macroglobulinemia." Blood 124, no. 21 (December 6, 2014): 3072. http://dx.doi.org/10.1182/blood.v124.21.3072.3072.
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Abstract Introduction: Accordingto National Comprehensive Cancer Network (NCCN) and ESMO guidelines on Waldenstrom’s Macroglobulinemia (WM) bendamustine may be considered as a therapeutic option in first line treatment or in relapsed refractory disease. Even though there are only two clinical trials including a limited number of patients addressing the role of bendamustine and rituximab (BR) treatment in WM. Patients and Methods: To define the efficacy and tolerability of BR combination as salvage regimen in WM patients, we retrospectively analyzed the outcome of symptomatic refractory relapsed patients treated with BR in 14 Italian centres. All patients receiving at least one day of treatment were included in the study. Treatment consisted of: R 375 mg/sqm iv day 1 and B iv days 1, 2. Therapy was administered every 4 weeks up to 6 courses. Results: Seventy-one patients are included in the study. As regards B dosage; 45 patients (63%) received the highest dose of 90 mg/sqm while 22 (31%) were treated with 70 mg/sqm. The 4 patients (6%) with a cumulative illness rating scale ≥ 6, received the lowest dose of 50 mg/sqm. At treatment, median age was 72 years (49-88), sex ratio M/F 46/25. Mediannumber of prior regimens was 2 (range 1-6). Twenty-four patients (34%) presented with refractory disease. The majority (90%) of patients had been previously treated with alkylating agents, 30% had also received purine analogues based treatments. Previous R was administered in the 77% of cases. The main reason (62%) for starting treatment was anemia followed by adenopathy and/or splenomegaly (35%). Median IgM level at treatment was 3815 mg/dL.Overall 361 courses of BR treatment were administered, median number 6 (range 1-6) with 47 (66%) of patients completing the 6 planned courses. Toxicity was discontinuation cause in 10 patients (14%): 4 infection, 1 fatal, 6 myelosuppression. In the remaining 14 treatment was discontinued for clinical clinical decision after disease reassessment. No difference in terms of treatment discontinuation was observed according to B dosage and age. Overall response rate (ORR) was 80.3% including: 7% complete remissions (CR), 15.5 % very good partial remissions (VGPR), 52.2% partial remissions (PR) and 5.6% of minor responses. A stable disease was observed in 16.9% of patients. One (1.4%) disease progression and one death were recorded. A progressive decrease of IgM level was observed during follow-up leading to an amelioration of response in 4 cases leading to a final ORR of 84.5%. None of the clinical and biological characteristics considered (age, sex, disease status, previous lines of treatment, previous fludarabine, bulky disease, Hb and IgM level, beta 2 microglobulin, B dosage) had an impact on ORR achievement. A better quality of response (CR plus VGPR) was observed in patients with an IgM level < 3000 mg/dL and in those treated with the higher dosage of B (90 mg/sqm). After a median follow-up of 19 months (3-54) 11 of the 57 responding patients met the criteria for disease progression. No difference was observed when patients were stratified according to the quality of response. B dosage did not impact disease progression. Considering that most of the patients received prophylactic growth factors, grade 3-4 neutropenia developed in only 13% of courses, 36% of patients. Dose modification or delayed treatment administration was necessary in 4 and 10% of courses respectively. During treatment we recorded 14 episodes of FUO and 5 major infections, leading death in one case. After a median follow up of 19 months none of the patients developed secondary myelodisplastic syndrome, acute leukemia or diffuse large B-cell lymphoma. In 3 cases a solid cancer was observed. Conclusion: BR combination showed to be as effective as more intensive salvage regimens in pretreated WM patients. Treatment showed to be well tolerated even in elderly patients with limited episodes of myelosuppression and infections when compared to purine analogues including regimens. Disclosures No relevant conflicts of interest to declare.
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Adekola, Kehinde U. A., Vivian Liu, Shuo Ma, Steven T. Rosen, and Mala Shanmugam. "Targeting Glucose Metabolism and Compensatory Metabolic Resistance in Chronic Lymphocytic Leukemia." Blood 120, no. 21 (November 16, 2012): 1801. http://dx.doi.org/10.1182/blood.v120.21.1801.1801.
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Abstract Abstract 1801 Chronic lymphocytic leukemia (CLL) is the most common leukemia seen in Western countries, primarily in the elderly, with a median age of diagnosis of 72 years. CLL is characterized by the aggressive accumulation of monoclonal peripheral (mature) CD5+ B cells in primary and secondary lymphoid tissues. Several classes of drugs currently exist to treat CLL and these include - nitrogen mustard alkylating agents, purine analogs, monoclonal antibodies, cyclin dependent kinase inhibitors, BTK and PI3K inhibitors. CLL can become resistant to existing therapies necessitating the need for identification of new targets and therapeutic strategies. Unique metabolic dependencies of cancer cells have been identified, further investigation of which could provide tumor cell specific targeting modalities. A myriad of tumor cells exhibit increased glucose uptake and metabolism of glucose via the in-efficient glycolytic pathway, a phenomenon first described by Warburg in the 1900's. Restriction of glucose utilization and metabolism has been shown to chemosensitize and/or elicit toxicity in a wide range of cancers. CLL metabolism is a relatively unexplored area. We sought to determine the dependency of CLL on glucose metabolism. As there are currently no CLL cell lines we used primary patient samples for these studies. CLL cells harvested from PBMCs were cultured in both glucose free and glucose containing media. Our results show that when CLL cells are cultured in these media, there is variation in sensitivity to glucose deprivation ranging from very sensitive to highly resistant (Figure 1). We have investigated possible resistance mechanisms and alternate sources of energy in CLL that could be responsible for maintaining viability even during glucose-withdrawal. We first investigated a role for glutamine. CLL cells sensitive to glucose withdrawal and cultured in the absence of glutamine, did not exhibit enhanced toxicity. These results suggest that cells resistant to glucose withdrawal were not maintaining viability via glutamine metabolism. The role of the mitochondrial metabolism was also investigated. We observed that when the CLL cells are cultured in the absence of glucose and are substituted with galactose, there is a rescue effect, with cell viability being restored. This rescue effect is also observed (although not to the same extent as with galactose) when the CLL cells in the absence of glucose are treated with 2-methylpyruvate (2MP). 2MP feeds directly into the mitochondria and can bypass the glycolytic pathway. CLL cells were also treated with metformin, which is complex-1 inhibitor, and we observed enhanced cell death. The results with galactose, 2MP and metformin all suggest that mitochondrial metabolism is an integral part of CLL metabolism, potentially playing a compensatory role upon glucose-withdrawal. The role of autophagy was also investigated and, using chloroquine we observed that autophagy was pro-survival in CLL patient samples. In summary we have observed varying sensitivity of CLL patient samples to glucose deprivation, and identified resistance mechanisms and alternative sources of energy in CLL cells. The reduction of extracellular glucose has been shown to induce resistance of normal cells to DNA damaging therapeutics, and enhancement of sensitivity in cancer cells, suggesting that glycolysis inhibition may expand the therapeutic window of currently used therapeutics. Targeting these unique metabolic dependencies in CLL could provide strategies to chemo-sensitize and target CLL more effectively with less toxicity. Figure 1: Variable sensitivity of CLL patient samples to 48-hour glucose free culture Figure 1:. Variable sensitivity of CLL patient samples to 48-hour glucose free culture Disclosures: No relevant conflicts of interest to declare.
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Spurgeon, Stephen, Talia Pindyck, Marc M. Loriaux, Craig Okada, Kamal Abbi, Yiyi Chen, Zunqui Chen, Elana Mater, and Elliott Epner. "Cladribine Plus Rituximab Is An Effective Therapy for Newly Diagnosed Mantle Cell Lymphoma." Blood 116, no. 21 (November 19, 2010): 4910. http://dx.doi.org/10.1182/blood.v116.21.4910.4910.
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Abstract Abstract 4910 Background: Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma that is incurable with standard chemotherapy and remains a therapeutic challenge. Despite improved outcomes in MCL there is no consensus on the best initial treatment. Options vary from aggressive treatment strategies that incorporate multi-agent induction chemotherapy and consolidative transplant to less intensive treatment strategies that utilize alkylators, purine nucleoside analogues, and the monoclonal antibody rituximab. Although, higher response rates have been seen with aggressive approaches, their impact on overall survival is not yet fully appreciated and many patients are not candidates for such approaches. Thus, finding less intensive induction regimens is imperative. The combination of rituximab plus cladribine has shown activity across a number of B-cell malignancies and the NCCN treatment guidelines currently include this regimen for the initial treatment of MCL; noting that there are few data available to substantiate this recommendation. The largest prospective experience (n=29) with R-cladribine for the initial treatment of MCL comes from the North Central Cancer Group. They reported an overall response rate (ORR) of 66% with a 52% complete remission (CR) rate and a 2 year progression free survival (PFS) of 43%. Given its therapeutic potential and increasing popularity, more data are needed to verify the benefits of the R-cladribine regimen. Therefore, to explore the role of R-cladribine in the treatment of newly diagnosed MCL, we performed a retrospective chart review of patients with newly diagnosed MCL treated with R-cladribine. Methods: We reviewed the charts of 31 patients with newly diagnosed MCL initially seen at two university hospitals and at an associated VA that were treated with R-cladribine. One patient had been previously treated with 2 cycles of R-CHOP;, all other patients were untreated. All patients had measurable disease and follow up imaging (CT and/or PET/CT scans) before and at the completion of therapy. Post treatment bone marrow biopsies were not available for all patients. Chemotherapy included: cladribine 5mg/m2 given over two hours on days 1–5; and rituximab given on days 1, 8, 15, and 22 with the first cycle and then on day 1 with subsequent cycles. Each cycle was 28 days for up to a total of 6 cycles. Patients with an initial response received maintenance rituximab. Results: The median age of our cohort was 67 years (48-86) with 42% of patients ≥ 65 years. All patients had advanced stage disease (stage ≥ 3) and the majority of patients had poor risk disease. For example, 20/31 (65%) of patients had high FLIPI (≥ 3) and11/31 (37%) had high MIPI (≥ 6). Of the 24 patients in whom beta2-microglobulin was available, 11 (46%) had levels ≥ 3.5 mg/L. The overall response rate (ORR) was 87% with 19/31(61%) of patients achieving a complete remission (CR/CRu). At a median follow up of 21.5 months (2-85 months) the 2 year PFS rate is 65% and the OS rate is 74%. For those subjects achieving a CR/CRu with a median follow up of 23 months, 1/19 (5.3%) has relapsed. No significant trends were seen regarding response rate and pre-treatment disease defining parameters including Ki67, beta2-microglobulin, FLIPI, or MIPI. However, CR was associated with improved survival (p = <.0001) while high MIPI was associated with worse survival (p=0.0317). There was one toxic death (neutropenic sepsis) related to treatment. Conclusion: The combination of rituximab plus cladribine appears to be an effective initial therapy in MCL. The higher response rates seen in this series may be the result of patient selection and/or increased rituximab exposure. Rituximab maintenance may also be an important component of ongoing disease control in responding patients. These data support the ongoing evaluation of rituximab plus cladribine in combination with novel agents. Prospective single arm studies incorporating R-cladribine with other novel agents such as vorinostat, bortezomib, or temsirolimus are ongoing. Disclosures: No relevant conflicts of interest to declare.
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Ray, Arghya, Leutz Buon, Yan Song, Dharminder Chauhan, and Kenneth C. Anderson. "Interaction of Plasmacytoid Dendritic Cells and Myeloma Cells Trigger Tumor Promoting Transcriptional Changes in Multiple Myeloma Cells." Blood 134, Supplement_1 (November 13, 2019): 510. http://dx.doi.org/10.1182/blood-2019-123333.
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Introduction We have previously shown that increased numbers of plasmacytoid dendritic cells (pDCs) in bone marrow (BM) of multiple myeloma (MM) patients promote tumor cell growth, survival, and drug resistance; as well as suppress T and NK cell mediated anti-MM immunity (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2015, 29:1441-1444). Here, we analyzed genetic changes in MM cells triggered by coculture with pDCs using next generation sequencing (NGS). Functional validation of NGS data was performed in our coculture models of pDC-T-MM cells to assess impact on tumor cell surface phenotype, growth, survival, and drug resistance; as well as cytotoxic T lymphocyte (CTL) activity against MM. We identified and validated the metabolic ectoenzyme CD73/NT5E, implicated in cancer metabolism and immunosuppression via nucleotide degradation pathway, as a novel therapeutic target in MM. Methods Purified MM patient pDCs were cocultured with autologous MM cells or allogeneic MM cell lines (1pDC/5MM) for 48h, followed by separation of MM cells from pDCs using flow cytometry. Total RNA from MM cells was subjected to RNAseq analysis using Illumina Next Generation Sequencing (NGS). Raw sequence data were analyzed using VIPER workflow generating differential expression (DEseq2) and KEGG pathway. Statistical significance: log2FC (fold change) values in coculture vs control, with an FDR (False Discovery Rate) value of <0.05, was considered significant (CI > 95). Linear model for RNAseq analysis (Limma) and its GUI (Glimma) were also utilized for the visualization of data. Results RNA-seq data was analyzed using negative binomial distribution (DEseq2) and linear (Limma) models. Results showed 9200 and 9250 genes were differentially expressed (p < 0.05). MM cells cultured with or without pDCs clustered into two distinct groups, based upon pDC-MM contact-dependent transcriptional changes. Pathway enrichment analysis showed that pDCs interaction with MM cells regulates multiple physiological processes in MM cells including DNA replication/repair, purine/pyrimidine metabolism, and cell cycle. Hierarchical clustering showed increased expression of genes in MM cells after coculture with pDCs (log2FC range: ± 6.0): TLR7/9 (0.5; p=0.02), HDAC6 (0.65; p=0.00002), CD274 (0.6; p=0.02), or IL3Rα/CD123(0.1; p<0.05). On the other hand, pDCs reduces expression of CASP3 (-1.049; p= 1.1e-7), BAK1 (-0.5; p=0.000043), ADAM33 (-1.36; p=0.004), and BAD (-0.14; p = 0.0048) in MM cells. We validated the functional significance of pDC-induced gene alterations in MM cells using coculture model of patient MM-pDCs and autologous tumor cells. For example CD73 levels further increases in MM cells after coculture with pDCs (MFI: 1.2-fold vs MM; p = 0.008; CD73+ cells: 1.15-fold vs MM; n = 5; p = 0.005); and anti-CD73 Ab (1.0 µg/ml) treatment of autologous pDC-T cell cocultures (1pDC/10T cells) induces MM-specific CD8+ CTL activity against both autologous and allogeneic tumor cells. Furthermore, combining anti-CD73 Ab and TLR7 agonist triggers more robust MM-specific CD8+ CTL activity than either agent alone (% MM lysis: anti-CD73 Ab plus TLR7 agonist: 60-70%; TLR7 agonist: 40%; and anti-CD73 Ab: 30%; p = 0.009; n = 5). Conclusions Our RNA-seq and NGS analysis of pDCs-triggered transcriptome changes in MM cells identifies genes and pathways mediating tumor growth and immunosuppression, which therefore represent targets for novel therapeutics to improve patient outcome. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.
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Ray, Arghya, Leutz Buon, Yan Song, Dharminder Chauhan, and Kenneth C. Anderson. "Plasmacytoid Dendritic Cells Trigger Contact-Dependent Tumor Promoting Genetic Alterations in Multiple Myeloma Cells." Blood 132, Supplement 1 (November 29, 2018): 244. http://dx.doi.org/10.1182/blood-2018-99-117887.
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Abstract Introduction We identified an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in multiple myeloma (MM) pathogenesis. Specifically, we found increased numbers pDCs in MM BM vs normal BM. pDCs protect tumor cells from therapy-induced cytotoxicity; promote tumor growth and survival, as well as suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323). Aberrant pDC function is evidenced in their interactions not only with MM cells, but also with immune effector T cells and NK cells in the MM BM milieu (Ray et al, Leukemia 2015, 29:1441-1444). Directly targeting pDC interactions with MM and immune effector cells in the MM BM milieu will be required to enhance both anti-tumor immunity and cytotoxicity. To further characterize the role of pDCs in MM pathogenesis, we utilized our pDC/MM co-culture model and analyzed genetic changes in MM cells after coculture with pDCs using next generation sequencing (NGS). NGS data was confirmed using flow cytometry analysis, and functionally validated in cocultures of pDC-MM-T cells, as well as cytotoxic T lymphocyte assays. Based on our NGS study, we have identified the metabolic ectoenzyme NT5E/CD73 as a promising novel therapeutic target in MM; primarily for its role in cancer metabolism and immunosuppression via nucleotide degradation pathway. Methods. Patient BM/PB pDC and MM cells were evaluated by flow cytometry following immunomagnetic separation (purity> 95%). pDCs were cocultured with autologous MM cells or allogeneic MM cell lines (pDC/MM; 1:5) for 48h, followed by separation of MM cells from pDCs using FACS. Total RNA from MM cells was subjected to RNAseq analysis using Illumina Next Generation Sequencing (NGS). Raw sequence data were subjected to VIPER workflow generating differential expression (DEseq2) and KEGG pathway analyses. For RNAseq analysis, log2FC (fold change) values in coculture vs single control, with an FDR (False Discovery Rate) value of <0.05, was considered significant (CI > 95). Linear model for RNAseq analysis (Limma) and its GUI (Glimma) were used for the visualization of data. Results A total of 19,569 and 25,436 transcripts were analyzed through negative binomial (DEseq2) and linear (Limma) models, respectively. We found that 9200 (47.01%) and 9250 (36.36%) genes were differentially expressed (p < 0.05). The sample-sample correlation analysis showed that MM cells cultured with or without pDCs fall into 2 different groups with distinct clustering properties (n=3), suggesting that pDCs trigger genetic alterations in MM cells. Furthermore, pathway analysis showed that pDCs interaction with MM cells modulates various physiological processes in MM cells including Cell cycle, DNA replication/repair (Fanconi anemia) and Purine/Pyrimidine metabolism. Hierarchical cluster analysis demonstrated upregulation or downregulation of several genes in MM after coculture with pDCs (log2FC range: ± 6.0). For example, pDCs trigger upregulation of CD274 (0.6; p=0.02), IL3Rα/CD123(0.1; p<0.05), TLR7/9 (0.5; p=0.02), and HDAC6 (0.65; p=0.00002) in MM cells. Conversely, pDCs decreased the expression of BAK1 (-0.5; p=0.000043), BAD (-0.14; p = 0.0048), ADAM33 (-1.36; p=0.004), and CASP3 (-1.049; p= 1.1e-7) in MM cells. The functional significance of these findings is exemplified in our studies for NT5E/CD73 (log2FC: 0.43; p=0.0002). Specifically, we found that MM patient cells or cell lines express CD73, which further increases upon coculture with MM-patient derived pDCs (MFI: 1.2 fold vs MM; p = 0.008; CD73+ cells: 1.15 fold vs MM; n=5; p = 0.005). Treatment of autologous pDC-T cell cocultures (pDC:/T; 1:10 ratio) with anti-CD73 Ab (1.0 µg/ml) triggers a robust MM-specific CD8+ CTL activity against both autologous and allogeneic MM cells. A more robust MM-specific CD8+ CTL activity was noted when anti-CD73 Ab was combined with TLR7 agonists (%MM lysis: anti-CD73 Ab + TLR7 agonist: 60-70%; anti-CD73 Ab: 30%; TLR7 agonist: 40%; p = 0.009; n= 5). Conclusions Collectively, our RNAseq NGS analysis of pDCs-induced gene changes in MM cells validates our previously published findings (Ray et al Leukemia 2014; 28(8): 1716-1724; Leukemia 2015; Leukemia 2017; 31:2652-2660; Leukemia 2018,32:843-846) that these accessory cells induce growth, survival and drug resistance in tumor cells. Importantly, these studies delineate novel genes and pathways mediating these sequelae which represent targets for future therapies. Disclosures Anderson: Gilead: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Celgene: Consultancy; Oncopep: Equity Ownership.
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Bhingarkar, Aboli, Hima V. Vangapandu, Sanjay Rathod, Keito Hoshitsuki, and Christian A. Fernandez. "Amino Acid Metabolic Vulnerabilities in Acute and Chronic Myeloid Leukemias." Frontiers in Oncology 11 (July 1, 2021). http://dx.doi.org/10.3389/fonc.2021.694526.
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Amino acid (AA) metabolism plays an important role in many cellular processes including energy production, immune function, and purine and pyrimidine synthesis. Cancer cells therefore require increased AA uptake and undergo metabolic reprogramming to satisfy the energy demand associated with their rapid proliferation. Like many other cancers, myeloid leukemias are vulnerable to specific therapeutic strategies targeting metabolic dependencies. Herein, our review provides a comprehensive overview and TCGA data analysis of biosynthetic enzymes required for non-essential AA synthesis and their dysregulation in myeloid leukemias. Furthermore, we discuss the role of the general control nonderepressible 2 (GCN2) and-mammalian target of rapamycin (mTOR) pathways of AA sensing on metabolic vulnerability and drug resistance.
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Yu, Yajie, Jane Ding, Shunqin Zhu, Ahmet Alptekin, Zheng Dong, Chunhong Yan, Yunhong Zha, and Han-Fei Ding. "Therapeutic targeting of both dihydroorotate dehydrogenase and nucleoside transport in MYCN-amplified neuroblastoma." Cell Death & Disease 12, no. 9 (August 30, 2021). http://dx.doi.org/10.1038/s41419-021-04120-w.
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AbstractMetabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics.
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Cheung, Chantal Hoi Yin, Chia-Lang Hsu, Chao-Yin Tsuei, Tzu-Ting Kuo, Chen-Tsung Huang, Wen-Ming Hsu, Yun-Hsien Chung, et al. "Combinatorial targeting of MTHFD2 and PAICS in purine synthesis as a novel therapeutic strategy." Cell Death & Disease 10, no. 11 (October 17, 2019). http://dx.doi.org/10.1038/s41419-019-2033-z.
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Abstract MYCN-amplified (MNA) neuroblastoma is an aggressive neural crest-derived pediatric cancer. However, MYCN is indispensable for development and transcriptionally regulates extensive network of genes. Integrating anti-MYCN ChIP-seq and gene expression profiles of neuroblastoma patients revealed the metabolic enzymes, MTHFD2 and PAICS, required for one-carbon metabolism and purine biosynthesis were concomitantly upregulated, which were more susceptible to metastatic neuroblastoma. Moreover, we found that MYCN mediated the folate cycle via MTHFD2, which contributed one-carbon unit to enhance purine synthesis, and further regulated nucleotide production by PAICS in response to cancer progression. Dual knockdown of the MYCN-targeted gene pair, MTHFD2 and PAICS, in MNA neuroblastoma cells synergically reduced cell proliferation, colony formation, migration ability, and DNA synthesis. By systematically screening the compound perturbagens, the gene expression levels of MTHFD2 and PAICS were specifically suppressed by anisomycin and apicidin across cell lines, and our co-treatment results also displayed synergistic inhibition of MNA neuroblastoma cell proliferation. Collectively, targeting a combination of MYCN-targeted genes that interrupts the interconnection of metabolic pathways may overcome drug toxicity and improve the efficacy of current therapeutic agents in MNA neuroblastoma.
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Zhu, Ying, Tingting Li, Suzane Ramos da Silva, Jae-Jin Lee, Chun Lu, Hyungjin Eoh, Jae U. Jung, and Shou-Jiang Gao. "A Critical Role of Glutamine and Asparagine γ-Nitrogen in Nucleotide Biosynthesis in Cancer Cells Hijacked by an Oncogenic Virus." mBio 8, no. 4 (August 15, 2017). http://dx.doi.org/10.1128/mbio.01179-17.
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ABSTRACT While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway. IMPORTANCE We have previously found that Kaposi’s sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.
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Ortiz, Raúl, Gloria Perazzoli, Laura Cabeza, Cristina Jiménez-Luna, Raquel Luque, Jose Prados, and Consolación Melguizo. "Temozolomide: An Updated Overview of Resistance Mechanisms, Nanotechnology Advances and Clinical Applications." Current Neuropharmacology 18 (June 26, 2020). http://dx.doi.org/10.2174/1570159x18666200626204005.
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: Temozolomide (TMZ), an oral alkylating prodrug which delivers a methyl group to purine bases of DNA (O6- guanine; N7-guanine and N3-adenine), is frequently used together with radiotherapy as part of the first-line treatment of high-grade gliomas. The main advantages are its high oral bioavailability (almost 100% although the concentration found in the cerebrospinal fluid was approximately 20% of the plasma concentration of TMZ), its lipophilic properties, and small size that confer the ability to cross the blood-brain barrier. Furthermore, this agent has demonstrated activity not only in brain tumors but also in a variety of solid tumors. However, conventional therapy using surgery, radiation, and TMZ in glioblastoma results in a median patient survival of 14.6 months. Treatment failure has been associated with the tumor drug resistance. This phenomenon has been linked to the expression of O6-methylguanine-DNA methyltransferase, but the mismatch repair system and the presence of cancer stem-like cells in tumors have also been related to TMZ resistance. The understanding of these mechanisms is essential for the development of new therapeutic strategies in the clinical use of TMZ, including the use of nanomaterial delivery systems and the association with other chemotherapy agents. The aim of this review is to summarize the resistance mechanisms of TMZ and the current advances to improve its clinical use.