To see the other types of publications on this topic, follow the link: CRISPR/Cas9, flow cytometry, order of event.
Journal articles on the topic 'CRISPR/Cas9, flow cytometry, order of event'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'CRISPR/Cas9, flow cytometry, order of event.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Adriaanse, Fabienne R. S., Sadie M. Sakurada, Shondra M. Pruett-Miller, Ronald W. Stam, Michel C. Zwaan, and Tanja A. Gruber. "Non-Coding HOX Fusions in Pediatric Non-Down Syndrome Acute Megakaryoblastic Leukemia." Blood 134, Supplement_1 (2019): 533. http://dx.doi.org/10.1182/blood-2019-127014.
Full textAbstract:
The homeobox (HOX) genes are a highly conserved family of transcription factors involved in embryonic patterning as well as adult hematopoiesis. Dysregulation of HOX genes, in particular upregulation of HOXA cluster genes, is a frequent event in Acute Myelogenous Leukemia (AML). Recently, we performed a detailed genomic analysis on pediatric non-Down Syndrome Acute Megakaryoblastic Leukemia (non-DS-AMKL) and identified novel fusions involving a HOX cluster gene in 14.9% of the cases. While most fusions were predicted to lead to an in-frame functional protein, several fusions included a non-coding HOX antisense gene (PLEK-HOXA11-AS, C8orf76-HOXA11-AS, HOXA10-AS-CD164) that were predicted to result in a loss of function of these regulatory transcripts. The functional consequence of these events, however, remain unknown. HOXA11-AS (human) and Hoxa11os (mouse) have been previously shown to have mutually exclusive expression with the Hoxa11 transcript throughout development. We therefore hypothesized that loss of function of non-coding HOX antisense genes as a result of these structural variations would cause upregulation of nearby coding HOXA genes that in turn promote leukemogenesis. To test this hypothesis, using CRISPR-Cas9 technology, we genome edited the human AMKL cell line CMK to carry the PLEK-HOXA11-AS translocation. qRT-PCR of HOXA11-AS and HOXA9-11 transcripts in this cell line recapitulated the pattern seen in patient specimens. Specifically, HOXA11-AS expression was significantly diminished while HOXA10 and HOXA11 transcripts were upregulated 1.8-2.5-fold when compared to parental CMK cells (p=0.0385 and p=0.006 respectively). To further investigate the loss of HOXA11-ASin vivo a CRISPR-Cas9 Hoxa11os knockout mouse model was established. qRT-PCR on bone marrow confirmed the loss of Hoxa11os transcripts in heterozygous (Hoxa11os1+/-) and homozygous (Hoxa11os-/-) mice of both genders (p=<0.0001-0.0012). Consistent with Hoxa11os knockdown, Hoxa11 transcripts were upregulated in male (1.8-fold p=0.0023 Hoxa11os+/-, and 2-fold p=0.0052 Hoxa11os-/-)and female (1.3-fold p=0.0074 Hoxa11os+/- and 2.2-fold p=0.0226 Hoxa11os-/-) bone marrow compared to wild type gender matched littermates. Interestingly, flow cytometry analysis of progenitor subsets revealed gender specific findings. We found a significant increase in the frequency of the lineage negative, Sca-1 and c-Kit positive (LSK) population in males (0.13% of total bone marrow Hoxa11os+/+, 0.19% p=0.0214 Hoxa11os+/-, and 0.25% p=0.0001 Hoxa11os-/-) compared to wild type male littermates but not in female mice at 8 weeks of age. In contrast an increase in the megakaryocyte-erythroid (MEP) population was seen only in the female setting (0.07% Hoxa11os+/+, 0.15% p=0.0055 Hoxa11os+/-, and 0.165% p<0.0001 Hoxa11os-/-). Limiting dilution colony forming assay confirmed the higher LSK frequency with a 2-fold increase in the number of colonies for male knockout marrow compared to wild type marrow in contrast to the female setting where no significant differences were seen. As hormonal signals have been shown to regulate expression of HOX genes and differences in clonogenicity of male and female stem cells has been previously demonstrated, we reasoned this phenomenon could be secondary to extrinsic stimuli in vivo. The relatively uniform Hoxa11 levels in male and female knockout mice, however, suggested that cell intrinsic factors may also play a role. We therefore overexpressed HOXA11 into male and female wild type bone marrow ex vivo for colony forming assays to determine if elevated levels of the HOXA11 protein led to functional differences. This assay demonstrated a clear enhancement of self-renewal in male but not female bone marrow in contrast to HOXA9 overexpression which serially replated in both genders. Combined these data demonstrate that loss of function alterations in Hoxa11os transcripts lead to upregulation of Hoxa11 and gender specific hematopoietic progenitor cell perturbations. Ongoing efforts include competitive transplant studies as well as RNA and ChIP sequencing to identify gender specific downstream targets of Hoxa11 in the hematopoietic compartment in order to understand the selective expansion of progenitor subsets and male specific self-renewal capacity of this protein. These data will contribute to our understanding on how HOXA11-AS translocations promote oncogenesis. Disclosures Zwaan: Daiichi Sankyo: Consultancy; Sanofi: Consultancy; Roche: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Incyte: Consultancy; Celgene: Consultancy, Research Funding; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Janssen: Consultancy. Gruber:Bristol-Myers Squibb: Consultancy.
2
Ivy, Kathryn S., Candace H. Cote, and Paul Brent Ferrell. "IDH2 Mutations Induce Altered STAT Signaling and Cytokine Responses Which Are Restored By Enasidenib." Blood 132, Supplement 1 (2018): 1468. http://dx.doi.org/10.1182/blood-2018-99-117783.
Full textAbstract:
Abstract Acute myeloid leukemia (AML) is a heterogeneous myeloid malignancy characterized by mutational and clonal heterogeneity. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are common, occurring in approximately 15-20% of patients, and actionable, with recently approved inhibitors for both mutations. These inhibitors lead to leukemia cell differentiation in vitro, in vivo and in patients. Healthy myeloid differentiation is governed by precise regulation of intracellular signaling, but this regulation is disrupted in AML. Given that signal transducer and activator of transcription (STAT) proteins are involved in both leukemogenesis and myeloid differentiation, we sought to determine the role of phosphorylated STATs and other signaling proteins in mIDH AML and inhibitor-induced differentiation of mIDH2 AML. To simultaneously dissect single cell signaling, differentiation and epigenetic changes, we employed fluorescence flow cytometry and mass cytometry to study an in vitro model of mIDH2R140Q leukemia, consisting of a parental TF-1 leukemia cell line and a CRISPR/Cas9 gene-edited mIDH2R140Q-mutated cell line. In order to generate provoked signaling profiles, we stimulated both the wild-type IDH2 and mutant IDH2 cells with nine cytokines. Additionally, we treated both cell lines and primary mIDH2 AML samples ex vivo with enasidenib and measured changes in high-dimensional single cell phenotype and phospho-protein expression via mass cytometry. mIDH2 leukemia cells displayed elevated basal pSTAT1, pSTAT3 and pNFkB-s529 expression, and concomitant low basal IkBa levels when compared to parental cells. Further, mIDH2 cells had increased PMA induced pS6 and IL1β induced pNFkB-s529 than the parental cell line, while wtIDH2 cells had higher levels of cytokine-induced pSTAT1, pSTAT3 and pSTAT5. After prolonged enasidenib treatment (28 days), model mIDH2 cells expressed lower basal pSTAT1, pSTAT3 and pNFkB-s529 than vehicle-treated mIDH2 cells. Further, when stimulated with GM-CSF, enasidenib-treated mIDH2 leukemia cells showed increased response at pSTAT3 and pSTAT5 as compared to vehicle-treated leukemia cells. We dissected enasidenib-induced differentiation using 18 cell surface markers and visualized results using t-distributed stochastic neighbor embedding (tSNE). Presence of mIDH2 leads to baseline expression differences including higher CD90 and CD71. Following treatment with enasidenib, mIDH2 leukemia cells had increased CD45 and CD11b expression as compared to vehicle-treated controls. Additionally, prolonged treatment with enasidenib increased proliferation as shown by increased Ki67 and decreased histone hypermethylation at suppressive histone marks, H3K27 and H3K9, while 7 days of enasidenib did not result in changes to these marks. We further explored the impact of mIDH2 in primary leukemia samples. First, we developed an ex vivo stromal co-culture system that allowed for treatment and expansion of four primary mIDH2 AML samples for 16 days. While the basal levels of signaling markers were sample dependent, consistently across all samples enasidenib-treated primary AML was more sensitive to IL-6 and GM-CSF-induced pSTAT1, pSTAT3 and pSTAT5 signaling. The enasidenib-treated AML also showed increased expression of mature myeloid markers, including CD33 and CD11c. Here we have shown the presence of mIDH2 mutations leads to decreased STAT signaling in response to cytokine stimulation as compared to wtIDH2 AML cells. We have also demonstrated increased response of pNFkB-s529 and pS6 to cytokine stimulation in mIDH2 AML cells as compared to wtIDH2 AML cells. Moreover, in both mIDH2 AML cell line and primary samples, IDH2 inhibitor-induced differentiation restored sensitivity to cytokine responses and reduced histone hypermethylation. Future work exploring these aberrant signaling events may reveal precise connections between mutated IDH, associated epigenetic changes, and intracellular signaling, potentially uncovering synergistic therapeutic strategies for mIDH AML. Disclosures Ferrell: Incyte: Research Funding.
3
Pabst, Gabriel, Johannes Foßelteder, Angelika Schlacher, et al. "Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells." Blood 138, Supplement 1 (2021): 2160. http://dx.doi.org/10.1182/blood-2021-149591.
Full textAbstract:
Abstract Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays. Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition. Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed "Acute Myeloid Leukemia" among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells. Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. Figure 1 Figure 1. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.
4
Xiong, Kun. "Feasibility assessment of using cRISPR-Cas9 to improve the infiltration of CAR-T cells in solid tumors." Theoretical and Natural Science 60, no. 1 (2024): 46–51. http://dx.doi.org/10.54254/2753-8818/60/20241395.
Full textAbstract:
Abstract. Chimeric antigen receptor T-cell immunotherapy (CAR-T) has been developing for decades, CAR-T is playing an increasingly important role in tumor treatment. However, because fibroblasts (CAFs) in solid tumors secrete proteins and glycans to form ECM, CAR-T is faced with the challenge of improving tumor invasion. To this end, a new scheme was put forth to alter CAR T cells such that they release heparinase (HPSE) to break down heparan sulfate proteoglycan (HSPG), which is covered on the outermost layer of the cancerous cells by CAF and released. In order to solve the above problems, CRISPR-Cas9 was proposed to modify CAR T to enhance the secretion of HPSE. Although this method has the advantage of broad spectrum, it still has certain defects. The matrix characteristics of different solid tumors and the subpopulations and regulatory mechanisms of HPSE need to be further studied. The off-target effects of CRISPR-Cas9 gene editing technology and the high cost and time-consuming problems of flow cytometry also limit its application. It is anticipated to enhance cell infiltration in solid tumors, boost CAR-T therapy's effectiveness in treating solid tumors, and advance the field of CAR-T therapy.
5
Lanjewar, S. N., and K. R. Bondioli. "205 Optimization of Transfection Efficiency for CRISPR/Cas9-Induced Genomic Editing in Porcine Fibroblast Cells." Reproduction, Fertility and Development 30, no. 1 (2018): 243. http://dx.doi.org/10.1071/rdv30n1ab205.
Full textAbstract:
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system creates DNA double-stranded breaks (DSB) at specific sequences and allows efficient genomic modification, even in species previously resistant to gene editing. The DSB can be repaired using non-homologous end joining (creating insertions/deletions) or by homology directed repair (HDR) using a donor DNA with small changes at the cut site, giving rise to precise targeted modifications. Despite growing interest in genome editing using RNA-guided endonucleases, the efficiency of HDR is only 0.5 to 20%. The objective of this study was to optimize transfection conditions in order to increase efficiency of HDR for CRISPR/Cas9 targeted genomic editing of porcine cells. We utilised the Swedish mutation of the porcine APP gene causing early-onset Alzheimer’s disease. We first tested co-transfection of 2 plasmids, one containing our guide RNA (gRNA) and another containing the Cas9 nuclease, using square-wave electroporation. Upon analysis via T7 endonuclease assay I, this method failed to produce a DNA DSB at the target site. Next, we tested transfection of a single vector containing both the gRNA and Cas9 nuclease. Three gRNAs targeting exon 17 of the porcine APP gene were constructed and inserted into CRISPR/Cas9 pGuide-it plasmids expressing green fluorescent protein (GFP). Plasmid DNA was transfected into cultured porcine fibroblast cells by 3 methods: Lipofectamine 2000, square-wave electroporation, and exponential-wave electroporation. To determine which method yielded the highest transfection rates, cells were analysed using flow cytometry to detect GFP expression. The transfection efficiency, percentage of cells expressing GFP, was analysed by one-way ANOVA and individual pair wise comparisons. Twelve microliters of Lipofectamine 2000 per well of a 6-well plate with 200 ng of plasmid DNA per μL of Lipofectamine was used to optimize transfection rates, as suggested by the manufacturer. Removal of transfection media after 48 h yielded higher transfection rates than removal after 24 h (6.9% ± 0.7 v. 2.2% ± 0.1; P = 0.02). For electroporation, 12.5 and 25 μg of plasmid DNA per mL was added to 0.2- and 0.4-mm gap cuvettes, respectively, each containing cell suspensions of 1 × 106 cells mL−1. Square-wave electroporation was performed at 300 V for three 1-ms pulses in 0.2-mm cuvettes. Exponential-wave electroporation was performed at 350 V and 500 μFD in both 0.2-mm and 0.4-mm cuvettes. Exponential-wave electroporation containing 25 μg of plasmid DNA/mL of cell suspension yielded the highest average transfection efficiency, 22.8% (P < 0.00001), compared with square-wave electroporation and transfection using optimized Lipofectamine 2000 conditions (9.1 and 1%, respectively). All 3 gRNAs resulted in similar transfection rates. In conclusion, efficiency of transfection of the CRISPR/Cas9 system into porcine cells is optimized using exponential-wave electroporation of a single plasmid CRISPR system.
6
Haase-Kohn, Cathleen, Markus Laube, Cornelius K. Donat, Birgit Belter, and Jens Pietzsch. "CRISPR/Cas9 Mediated Knockout of Cyclooxygenase-2 Gene Inhibits Invasiveness in A2058 Melanoma Cells." Cells 11, no. 4 (2022): 749. http://dx.doi.org/10.3390/cells11040749.
Full textAbstract:
The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.
7
Beigl, Tobias B., Ine Kjosås, Emilie Seljeseth, Nina Glomnes, and Henriette Aksnes. "Efficient and crucial quality control of HAP1 cell ploidy status." Biology Open 9, no. 11 (2020): bio057174. http://dx.doi.org/10.1242/bio.057174.
Full textAbstract:
ABSTRACTThe near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.
8
Foßelteder, Johannes, Angelika Schlacher, Gabriel Pabst, et al. "Introduction and Genetic Correction of Calreticulin Mutations in Human Hematopoietic Stem and Progenitor Cells Sheds Light on MPN Pathogenesis." Blood 138, Supplement 1 (2021): 2541. http://dx.doi.org/10.1182/blood-2021-147919.
Full textAbstract:
Abstract Introduction: Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype. Methods: We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential. Results: Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody. Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p&lt;0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation. When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors. To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF. Conclusion: Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. Disclosures Greinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.
9
Castro, Jesus, Mariana Gaelzer, and Scott Welford. "Abstract 209: Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice." Cancer Research 82, no. 12_Supplement (2022): 209. http://dx.doi.org/10.1158/1538-7445.am2022-209.
Full textAbstract:
Abstract Therapeutic options for glioblastoma multiforme (GBM), the most common and fatal brain tumor, have been limited over the course of recent decades, leaving radiation as one of the few effective therapies. While advancements in radiation therapy have improved life expectancy and patient outcome, exposure to radiotherapy causes normal tissue toxicity and damage to the central nervous system, resulting in cognitive impairment and negative patient side effects. In the hippocampus, the memory processing center of the brain, radiotherapy-induced elevated glutamate levels lead to over-excitation of N-methyl-D-aspartate receptors (NMDAR) and thus cell death. To investigate the NMDAR, we repurposed a NMDA subtype specific inhibitor (Ifenprodil) and treated non-tumor mice before radiation. We found that Ifenprodil rescues cell death and cognition by inhibiting the NR2B subunit of NMDA receptors and preventing over-excitement-induced excitotoxicity, despite presence of high glutamate levels. In order to relieve glioma patients of the negative side effects of radiotherapies, we aim to further investigate the critical role of NR2B inhibition in non-tumor and tumor-bearing mice to reduce excitotoxicity and prevent damage to the central nervous system, opening avenues for therapeutic targeting and drug development. To accomplish this, we designed promotor-specific plasmids, only activated when present in their designed neuronal cell types, to initiate CRISPR/Cas9 genome editing and knock out the NR2B subunit of NMDAR. With NR2B knocked out across 5 different cell types, we exposed hippocampal organotypic slice cultures, derived from non-tumor mice, to 2 Gy radiation and ran flow cytometry to reveal which neuronal NR2B subunits play critical roles in excitotoxic phenotypes. To further these findings, we utilized a recently developed in-utero-electroporation mouse model that produces glioblastomas in immunocompetent mice by inactivating select tumor-suppressor genes via CRISPR/Cas9 genome editing at embryonic day 14.5. By applying different CRISPR/Cas9 gRNAs into our tumor-bearing mice, we can not only explore the robust role of NR2B inhibition in tumor brains with varying expressions, but also shed light onto how NR2B inhibition protects normal brain while leaving tumors radiosensitive. In conclusion, this work seeks to further develop our understanding of radiotherapy-induced cytotoxicity in order to prevent cognitive impairment and negative side effects in patients, improving their quality of life. Ultimately, the results from our findings will lead to the advancement of targeted drug development and therapeutic options. Citation Format: Jesus Castro, Mariana Gaelzer, Scott Welford. Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 209.
10
Soerensen, Johannes Frasez, Carina Agerbo Rosenberg, Katharina Wolter, et al. "Assessing Lipid Nanoparticle RNA Delivery Including CRISPR-Cas9 Based Therapy to Bone Marrow Cells with Emphasis on Leukemic Blasts - a Proof-of-Principle Study." Blood 144, Supplement 1 (2024): 7451. https://doi.org/10.1182/blood-2024-199992.
Full textAbstract:
Acute myeloid leukemia (AML) with the translocation t(8;21)(q22;q22.1) resulting in the fusion oncogene RUNX1::RUNX1T1 is a well-described subtype of AML. Generally perceived as associated with a favorable prognosis, the main cause of mortality in these patients remain relapse, occurring in an estimated 40% of patients leading to increased mortality. Gene editing technology CRISPR-Cas9, has in previous research been demonstrated to be able to disrupt the RUNX1::RUNX1T1 fusion gene. The disruption leads to inhibited leukemic cell growth and proliferation, suggesting its potential as a future therapeutic in the treatment of AML (Neldeborg et al. PMID: 37464068). One of the main challenges in translating RNA-based technologies, such as CRISPR-Cas9, to clinical testing remains in vivo delivery. Lipid nanoparticles (LNPs) are emerging non-viral vectors capable of delivering RNA-based therapies, such as CRISPR, in vivo. In this study, we investigated LNPs as a potential vector for delivery of RNA, including CRISPR components, to leukemic cell lines as well as to patient peripheral blood (PB) and bone marrow (BM) cells. LNPs were synthesized from SM-102 or D-lin-MC3-DMA, DSPC, Cholesterol and DMG-PEG2000 in molar ratio 50:10:39:1. The LNPs were loaded with: (i) dual guide RNAs (gRNA) targeting introns in RUNX1::RUNX1T1, (ii) Cas9mRNA, or (iii) GFPmRNA. Human AML cell line Kasumi-1, RUNX1::RUNX1T1 positive, was used for in vitro experiments. Evaluation of CRISPR cleavage and subsequent non-homologous end-joining, when treated with our dual-gRNA and Cas9-mRNA approach, was evaluated by PCR as well as with digital PCR to assess disruption efficiency. Paired primary patient PB and BM samples from 11 patients and BM from 3 patients evaluated for hematological disease or treatment response were used to assess LNP transfection when treated with LNPs loaded with GFPmRNA. A multiparameter flow cytometry panel was utilized to quantify GFP expression in different hematopoietic cell subsets. LNP transfection efficiency in Kasumi-1 cells was detected with high GFP expression (&gt;90%). Treatment of Kasumi-1 cells with dual LNP-gRNA and LNP-Cas9mRNA resulted in consistent disruption of RUNX1::RUNX1T1 albeit at low absolute efficiency as compared to electroporation of the ribonucleoprotein complex. The transfection potential of LNPs was interrogated in an ex vivo analysis of primary patient PB and BM cells. In the BM compartment, high GFP expression was detected in the immature myeloid- and leukemic blast subset (median 50.2% (95% CI 42.4% - 72.2%)), T-cells (median 29.1% (95% CI 19.9% - 37.4%)) and the monocytes (median 58.7% (95% CI 35.2% - 79.0%)). There was no apparent GFP expression detected in the B-cells nor granulocytes. A similar tendency was observed in the PB. Comparing the two LNP constructs, the LNPs synthesized using ionizable lipid SM-102 showed higher efficiency in the immature myeloid- and leukemic blast subset (p=.008) and the T-cells (p=.001) as compared to the LNPs synthesized using D-Lin-MC3-DMA. Our data demonstrates that LNPs can deliver RNA cargo to several types of primary patient cells in PB and BM. Importantly, at high efficiency in the leukemic blast subset. Furthermore, our data indicates that different LNP constructs may change the transfection efficiency. Lastly, we have demonstrated that LNPs can deliver dual-gRNA and Cas9-mRNA to Kasumi-1 cells and disrupt the RUNX1::RUNX1T1 fusion gene. Collectively, our results suggest a future role of LNPs as vectors of RNA-based therapies in hematology, including CRISPR components aimed at disrupting the RUNX1::RUNX1T1 fusion oncogene. Future research efforts will aim to optimize the CRISPR-mediated disruption efficiency of the RUNX1::RUNX1T1 fusion gene when delivered in LNPs in order to elucidate its therapeutic potential.
11
Kaur, Surinder, Stephen C. Alley, Matt Szapacs, et al. "2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A – Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine)." Bioanalysis 14, no. 9 (2022): 505–80. http://dx.doi.org/10.4155/bio-2022-0078.
Full textAbstract:
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “Context of Use – COU”); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.
12
Putino, Rossana, Alessia Boaretto, Gennaro Bruno та ін. "Abstract B110: β3-adrenergic receptor as a new molecular target in neuroblastoma treatment". Molecular Cancer Therapeutics 22, № 12_Supplement (2023): B110. http://dx.doi.org/10.1158/1535-7163.targ-23-b110.
Full textAbstract:
Abstract Purpose of study: β3-Adrenergic receptor correlation with tumor growth and progression in preclinical and clinical studies in neuroblastoma. Experimental procedures: Knock-out of β3-Adrenergic Receptor (β3-AR) in Neuroblastoma (NB) cell lines using CRISPR-Cas9 technology. In vivo experiment using β3-AR knock-out cells in order to evaluate the tumor growth in mice. RNA Sequencing of tumor masses derived from mice to investigate β3-AR Dependent tumor signaling pathways. Flow Cytometry analysis to observe the expression level of β3- Adrenergic Receptor on circulating tumor cells (CTCs) in peripheral blood and on disseminated tumor cells (DTCs) in bone marrow from NB patients with low-risk and high-risk prognosis. Summary of data: CRISPR/Cas9 technology was performed to obtain ADRB3-/- clones from human and murine NB cell lines. ADRB3-/- knocked-out cells were inoculated in murine models NU-Foxn1nu and A/J mice, showing an absence of tumor growth compared to wild type NB cells. Subsequently, RNA sequencing on tumor masses was performed to investigate β3-AR-dependent tumor signaling pathways. RNA-sequencing analysis showed many pathways were altered. Notably, downregulation of genes involved in epithelial–mesenchymal transition (EMT) pathway and genes involved in cell adhesion were underlined in Knock-out vs control, correlated with the absence of tumor growth. Cytofluorimetric analysis of β3-AR level expression were conducted on circulating tumor cells (CTCs) in peripheral blood and on disseminated tumor cells (DTCs) in bone marrow from NB patients with low-risk and high-risk prognosis to evaluating the expression levels of β3-AR. As expected, most of the high-risk NB patients showed high percentage of β3-AR+ cells among CTCs and DTCs, while the majority of low-risk patients showed lower percentage. Statement of the conclusions: Ours data showed that β3-AR could be a potential target therapy for neuroblastoma treatment. In fact, his deletion results in an absence of tumor growth and progression as demonstrate in mice in vivo experiments. In addition, NB patients with lower β3-AR level, showed a better prognosis . Furter studies should be focused on the pathways related to β3-AR. Moreover, new drugs will be tested in order to normalize the level of β3-AR in NB patients. Citation Format: Rossana Putino, Alessia Boaretto, Gennaro Bruno, Gianluca Mattei, Annalisa Tondo, Claudio Favre, Maura Calvani. β3-adrenergic receptor as a new molecular target in neuroblastoma treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B110.
13
Eken, Janneke, Fleur R. M. Havenaar, Edwin Quinten, et al. "Oncogenic CARD11 Mutations and Autonomous BCR Signaling Act As Functionally Equivalent Alternative Drivers in ABC-DLBCL." Blood 144, Supplement 1 (2024): 1619. https://doi.org/10.1182/blood-2024-204704.
Full textAbstract:
Diffuse large B-cell lymphoma of activated B-cell type (ABC-DLBCL) is characterized by chronic signaling of the B-cell receptor (BCR) pathway with constitutive NF-kB activation. We have recently identified autonomous, i.e. antigen-independent, signaling originating from individual BCR complexes as the cause of BCR pathway activation in the majority of ABC-DLBCL cases (Eken et al., J Exp Med 2024). As a pure immunological driver, autonomous BCR signaling cannot be recognized by sequence analysis nor be readily added to proposed genetic DLBCL subclassifications. Besides autonomous BCR signaling, recurrent activating mutations of various members of the BCR signaling cascade, in particular CD79 and CARD11, and in the TLR pathway member MYD88 contribute to the ABC phenotype. Intriguingly, lack of autonomous BCR signaling is associated with a homozygous CARD11 L251P mutation in the OCI-Ly3 ABC-DLBCL cell line. We therefore investigated the function of recurrent CARD11 mutations in the CARD11 wild-type ABC-DLBCL cell line TMD8. TMD8 cells express a BCR with intermediate-high autonomous signaling and are highly sensitive to the BTK inhibitor acalabrutinib (IC50 = 0.005 nM) by the MTS viability assay. TMD8 cells were gene edited by CRISPR/cas9 homology-directed repair to express the presumed oncogenic CARD11 coiled-coil region mutations L251P, D230N, K215N, and R337Q, cloned, and analyzed by sanger sequencing. Introduction of a hemizygous CARD11L251P/- allele converted TMD8 cells to full acalabrutinib resistance (IC50 &gt;62 μM). Hemizygous and homozygous R337Q mutations had negligible effects (CARD11R337Q/-: IC50 = 0.003 nM; CARD11R337Q/R337Q: 0.007 nM). Homozygous K215N and D230N mutations induced partial resistance to acalabrutinib (CARD11K215N/K251N clones: IC50 = 0.25-0.42 nM; CARD11D230N/D230N: IC50 = 0.127 nM). Monoallelic mutations had variable but less pronounced effects (CARD11K215N/WT clones: IC50 = 0.007-0.184 nM; CARD11D230N/WT: IC50 = 0.009 nM). In accordance with the observed sensitivities of CARD11-edited TMD8 clones to BTK inhibition, only CARD11L251P/- clones were able to survive knock-out of their autonomously signaling BCR by CRISPR/cas9-mediated non-homologous end-joining. After BCR knock-out, an IgM-negative population of app. 20% remained detectable in CARD11L251P/- TMD8 cells by flow cytometry for at least 4 weeks of cell culture. For all other CARD11-mutant clones, IgM-negative cells consistently amounted to &lt;1%, even after successful retroviral transduction with the non-signaling OCI-Ly3 IgG BCR. Furthermore, competitive co-culture of three independent TMD8 CARD11L251P/- clones with mock gene-edited control clones failed to showed any growth advantages. In a reciprocal driver-targeting approach, OCI-Ly3 were transduced with the TMD8 BCR and a CRISPR/cas9-resistant but otherwise wild-type CARD11 allele. CRISPR/cas9-mediated knock-out of the CARD11L251P alleles increased acalabrutinib sensitivity from an IC50 &gt;96 μM in CARD11L251P/L251P/tgCARD11WT OCI-Ly3 cells to 0.002-7.2 μM in CARD11ko/ko/tgCARD11WT cells. All tested gene-modified TMD8 and OCI-Ly3 clones remained equally sensitive to downstream BCR signaling inhibition with the MALT1 inhibitor MI2. In conclusion, we have developed an in vitro system to quantify the effects of defined mechanisms of BCR pathway activation in ABC-DLBCL. Our reciprocally controlled results demonstrate that the strongly activating CARD11 L251P mutation and a BCR with sufficiently strong autonomous signalling provide functionally equivalent but alternative oncogenic growth and survival signals in experimental systems of ABC-DLBCL. Other recurrent mutations of the coiled-coil CARD11 region, even with a CADD-PHRED score of up to 5, have weaker or virtually undetectable effects. In the presence of these mutations, a BCR without autonomous signaling activity fails to rescue knock-out of the autonomous BCR signaling activity, indicating insufficiency of tonic BCR signaling. The low prevalence of the CARD11 L251P mutation emphasizes the importance of antigen-independent autonomous BCR signaling as a frequent non-genetic immunological driver mechanism and the need for detailed dissection of the relative functional contribution of genetic and immunological drivers, including the zygosity of driver mutations, in order to develop optimal individualized targeted therapy for DLBCL patients.
14
Qazi, Areebah, Sophia Agrusa, Swornalata Pukhrambam, et al. "Abstract 1455: Impact of ZFHX3 loss on endometrial cancer progression." Cancer Research 85, no. 8_Supplement_1 (2025): 1455. https://doi.org/10.1158/1538-7445.am2025-1455.
Full textAbstract:
Abstract Endometrial cancer (EC) is the sixth most common cancer among women and ranks as the leading type of gynecological cancer. Although EC is often diagnosed early, current treatment plans significantly limit fertility-sparing options, posing challenges for younger patients. This research focuses on the role of the gene ZFHX3 (ATBF1) to understand its effects on the advancement of endometrial cancer. In order to investigate this, we first transfected 12Z-ESR1 epithelial endometrial cells with ZFHX3 siRNA to create transient gene knockdowns. Following cell collection, western blotting was performed to measure ZFHX3 protein expression levels. Furthermore, we used CRISPR Cas9 plasmid guides tagged with GFP to establish more stable knockdowns. After bacterial transformation, we utilized flow cytometry to sort GFP-expressing cells and confirm successful gene editing. Subsequent western blots were conducted to validate which guide were successful in creating an efficient knockdown of ZFHX3. In vivo studies complemented these cellular experiments. We dissected the uteri of mice with ZFHX3 homozygous and ZFHX3/Pten heterozygous knockdowns. Analysis through H&E staining allowed us to quantify the number of endometrial glands. Our findings revealed that ZFHX3 loss disrupts progesterone signaling, a critical regulator of endometrial function, and leads to increased cell proliferation, which may drive tumor growth. These results highlight ZFHX3 as a potential target for therapeutic strategies aimed at balancing fertility preservation and inhibiting the progression of cancer. Citation Format: Areebah Qazi, Sophia Agrusa, Swornalata Pukhrambam, Juveria Ali, Sanjeev Ganesh, Jessica Back, Mike R. Wilson. Impact of ZFHX3 loss on endometrial cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1455.
15
Ranjan, Nikhil, Michael Cole, Gloria F. Gerber, et al. "Genetic or Epigenetic CR1 Deficiency Defines Catastrophic Antiphospholipid Syndrome (CAPS) and Response to Complement Inhibition." Blood 144, Supplement 1 (2024): 138. https://doi.org/10.1182/blood-2024-207793.
Full textAbstract:
Introduction: CAPS is a life-threatening manifestation of the antiphospholipid syndrome (APS) characterized by widespread thrombosis and multi-organ failure. Despite standard-of-care therapies, CAPS is associated with &gt;30% mortality. Complement is implicated in the pathophysiology of thrombosis in CAPS, and complement inhibition shows efficacy as salvage therapy. We previously used the modified HAM (mHam) assay to demonstrate complement activation in &gt;85% of CAPS sera (Chaturvedi, Blood, 2020). Further, 48% (9/19) of CAPS patients had rare germline variants in complement regulatory genes, a frequency similar to atypical hemolytic uremic syndrome. Of these, 33% (3/9) harbored rare variants in Complement Receptor 1 (CR1), which have not been reported in aHUS. CR1 is expressed predominantly on erythrocytes, and essential for the clearance of immune complexes and complement regulation. We investigated the functional significance of the CR1 variants and molecular mechanisms regulating associated CR1 expression in CAPS. Methods: We performed CRISPR/Cas9 genome editing of TF-1 (erythroleukemia) cells to generate CR1 “knock-out (KO)” (positive control) and “knock-in (KI)” lines with patient specific CR1 variants. CR1 mRNA and protein expression were quantified by real-time quantitative PCR (RT-qPCR), immunoblots, and flow cytometry. To assess complement activity directed against these cell lines, we measured complement-mediated cell killing using the mHam assay. Briefly, cells were incubated with 20% normal human serum and cell killing was measured using a WST-1 dye. Next whole blood was collected from patients with history of CAPS or anticoagulant refractory thrombotic APS. We quantified surface CR1 expression on erythrocytes by flow cytometry. To assess the role of methylation in CR1 expression, we performed reduced-representation bisulfite sequencing (RRBS) of the CR1 promoter region of healthy controls and CAPS cohort. Results: We previously identified a germline CR1 V2125L variant (rs202148801) in a woman with triple positive APS and pregnancy-associated CAPS. Pedigree analysis confirmed that this variant was inherited from her mother. We generated a TF-1V2125L cell line and found that the variant leads to significantly reduced expression of CR1 at the transcriptional and translational levels. Another CR1 variant G2109S, identified in a woman with preeclampsia but no thrombosis did not lead to diminished CR1 expression and therefore, served as a control. Functionally, the mHam showed that V2125L led to increased complement-mediated cell death induced by NHS (23% cell kill in TF-1CR1-/- vs 21% in TF-1V2125Lvs 7% in TF-1WT cells). Lastly, to confirm this variant leads to reduced CR1 expression in the patient, we performed flow cytometry of the patient's RBCs and found reduced surface CR1 expression. This observation led us to analyze erythrocyte CR1 expression by flow cytometry in 3 additional triple-positive APS patients with a history of CAPS or recurrent thrombosis refractory to anticoagulation. While these patients did not possess germline variants in CR1, we found a significant reduction in surface CR1 expression (n=4, 13.22%±3.77) as compared to healthy controls (n=22; 78.89%±2.98) and APS patients (n=6; 82.43%±6.34). To investigate the molecular mechanisms behind reduced CR1 expression, we performed RRBS of the CR1 promoter region, which demonstrated hypermethylation in these patients but not in the patient with the CR1 V2125L variant. This explains CR1 downregulation in CAPS patients without germline mutations. Lastly, these 3 patients were initiated on C5 inhibition (C5i) at the discretion of the treating clinician (duration of therapy 2 mo-9 y). Thrombosis rates reduced from 54.3 events per 100-patient-years to 10.2 events/100 patient years after starting C5i. Notably the one thrombotic event on C5i occurred in the postoperative period in the setting of delayed C5i administration (1 week late). Conclusion: CR1 deficiency, due to genetic or epigenetic down-regulation identifies a subset of CAPS that is particularly responsive to C5i.
16
Dong, Mengmeng, Enfan Zhang, Haimeng Yan, et al. "Macrophages Promote DNA Repair of Double Strand Break in Multiple Myeloma Cells By Non-Homologous End Joining(NHEJ), Nevertheless Decrease Its Accuracy." Blood 134, Supplement_1 (2019): 3087. http://dx.doi.org/10.1182/blood-2019-126753.
Full textAbstract:
Multiple myeloma (MM) is a hematological malignancy of B cells, characterized by clonal proliferation of malignant plasma cells. DNA damage and genomic instability play an important role in the pathogenesis of MM. Based on the characteristics of high heterogeneity and genomic instability of MM, and the protective effect of MΦs on MM cells (MMCs), our study intended to further clarify whether MΦs affect MMCs DNA damage response (DDR) and DNA repair, and the relationship between MΦs and genomic instability of MMCs. We found that the content of MΦs in bone marrow biopsy of MM patients was related to the results of cytogenetics (tested by FISH). The higher the content of MΦs, the more complicated the cytogenetic abnormalities of patients, especially in the IgH translocation, D13S319 locus deletion and RB1 deletion. In our study, MΦs were harvested from peripheral blood monocytes (PBMCs) , which were incubated for 7 days with M-CSF. Flow cytometry was used to detect M-CSF induced macrophages (MΦs) in vitro, and CD163 and CD206 were highly expressed in MΦs which implied that MΦs tended to be M2 type. The incubated MΦs were used in the following experiments. Our study showed that MΦs reduced the baseline γH2AX of MMCs, and contributed to MMCs surviving in the case of genomic instability detected by Western blot and immunofluorescence. We also confirmed that MΦs contribute to repairing the DNA damage in myeloma cells with the methods of comet assay. In the case of severe injury of MMCs' DNA, MΦs promoted the DDR and DNA damage repair. We examined the effects of macrophages on HR and NHEJ using U2OS cells. HR repair was measured in U2OS-HR cells loaded with SCR reporter (HR reporter) while NHEJ repair was measured in U2OS-NHEJ cells loaded with vGEJ reporter (NHEJ Reporter). We found that macrophages increased NHEJ but had no sense on total HR. In order to detect NHEJ level in endogenous genes, we adopted paired gRNA-CRISPR/Cas9 system. AAVS1 and HBB were used as detection genes, and the sequence of about 250 bp near the NHEJ interface was sequenced by NGS. The results proclaimed that the MΦs co-culture group significantly increased the efficiency of NHEJ, and decreased proportion of accurate NHEJ repair. In addition, analysis of the length of base sequence loss showed that the probability of base loss >3 bp in the MΦs co-culture group was higher than that MMCs in the group cultured alone. In the HBB site, MΦs also prolonged the average length of base loss in NHEJ. Furthermore, we used gRNA-CRISPR/Cas9 technology to cause fixed-point cleavage in AAVS1 and HBB respectively, and detected translocation by PCR and NGS with comparing the translocation reads between different groups. The results showed that MΦs promoted the probability of chromosomal translocation, which was of great importance in MM's occurrence and progression. Disclosures No relevant conflicts of interest to declare.
17
Corat, Marcus A. F., Jean-Yves Metais, and Cynthia E. Dunbar. "Progress Towards Creation of a Rhesus Macaque Animal Model for PNH Disease Via Crispr/Cas9 Technology to Knock out the PIG-a Gene." Blood 124, no. 21 (2014): 4389. http://dx.doi.org/10.1182/blood.v124.21.4389.4389.
Full textAbstract:
Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem/progenitor cell (HSPC) disease characterized by severe intravascular hemolysis, bone marrow failure, and propensity to thrombosis, causing early death in untreated patients. PNH has been linked to acquired somatic loss-of-function mutations in the X-linked PIG-A gene in HSPC, with resultant disruption of the first step of the biosynthesis of GPI anchors and loss of cell-surface expression of GPI-linked proteins such as CD55 or CD59. PNH red cells are sensitive to complement-mediated lysis due to loss of these GPI-linked proteins. Even though the molecular mechanism of PNH has been well-characterized, the apparent clonal dominance of PIG-A mutant HSPC over residual unmutated HSPC is still poorly understood. Murine models for PNH generated via conditional knockout of PigA do not recapitulate the hemolysis, thrombotic phenotype, or clonal dominance. We took advantage of the newly-described CRISPR-Cas9 gene editing technology to disrupt the PIG-A gene via non-homologous end joining DNA repair and generate a relevant animal model for PNH, in order to investigate these important pathophysiologic questions in rhesus macaques, a species phylogenetically closely related to humans. We constructed a lentiviral vector expressing GFP, Cas9, and one of a series of five guide RNAs manually designed to target the rhesus macaque PIG-A gene at several sites within exon 2, where most human missense mutations are clustered. Two of these five gRNAs also 100% matched homologous sequences in the human PIG-A gene, while three of the gRNAs had 1-2 mismatches with the human sequence. We characterized the efficiency of each guide at the genotypic level using the SURVEYOR assay for locus disruption and direct sequencing, and at the phenotypic level by flow cytometry for GPI-linked proteins. We used human K562 or rhesus macaque FRhK-4 cell lines for proof of concept in vitro studies. The majority of human K562 cells transduced with vectors expressing guide RNAs targeting human PIG-A sequences in exon 2 gradually lost expression of cell surface CD55 and CD59, approximately 70% of double negative for CD55 and CD59 markers by 3 weeks following transduction, in contrast to K562 cells transduced with control vectors not expressing gRNA. It is notable that of the 5 guides tested in K562 cells, the 2 guides resulting in efficient knockdown of CD55 and CD59 were targeted to regions with 100% homology between the rhesus and the human sequence of PIG-A. The other 3 guides with only 1 or 2 mismatches to the human sequence were very inefficient at gene disruption in human cells, suggesting high fidelity and limited off-target effects The SURVEYOR assay confirmed disruption of the K562 PIG-A gene by these two gRNAs (see Figure). Upon sequencing, we demonstrated a variety of indels consisting of insertions or deletions of 1 to 40 nucleotides at the target site in the PIG-A gene. Analysis for off-target indels is ongoing. Similar experiments were carried out in the FRhK4 rhesus cell line, with the SURVEYOR assay demonstrating gene disruption with all five gRNA targeting sequences, all 100% homologous to the rhesus PIG-A gene targets (see Figure) . In FRhK-4 rhesus cells all the PIG-A gRNAs, knocked down CD55 and CD59 expression. gRNAs #4.1, #6.2 and #9.1 showed higher efficiency than the others, with approximately 78% of double negative cells for CD55 and CD59 markers 3 weeks following transduction. We have validated the lentiCRISPR-/Cas9 approach for use in rhesus cells for PIG-A knockout in vitro, and the guide-RNAs have shown effectiveness and specificity. We are in the process of implementing the technique in primary rhesus macaque CD34+ HSPC prior to proceeding to transplantation of these cells in an autologous rhesus model, allowing investigation of the mechanisms of clonal dominance and thrombosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
18
Chen, Manling, Yihan Mei, Xiaoyu Liu, et al. "Pre-Leukemic Transformation of the Bone Marrow Microenvironment Induced By AML1-ETO Fusion Protein." Blood 142, Supplement 1 (2023): 5613. http://dx.doi.org/10.1182/blood-2023-179881.
Full textAbstract:
Objective: The bone marrow (BM) microenvironment, especially stromal cells (SCs) and cytokines, plays a critical role in supporting normal hematopoiesis. However, in cases of hematological malignancies such as acute myeloid leukemia (AML), leukemia cells can reshape the BM microenvironment to promote their survival. Despite this, there is still limited understanding of the changes in AML1-ETO fusion protein induced pre-leukemia microenvironment niche. In this study, the alterations of the BM microenvironment in the Aml1 Eto/+; Mx1-Cre (AE KI) mouse model are thoroughly explored and analyzed. This investigation will encompass the study of SCs, cytokines, their sources, and their potential impacts on the occurrence and progression of AML induced by AML1-ETO. Methods: After 4 weeks of PIPC induction, proportion changes of endothelial cells (ECs), osteoblasts, mesenchymal stem cells (MSCs) in the BM of Aml1 Eto/+; Mx1-Cre mice (experimental group) and Aml1 Eto/+; w/o Mx1-Cre mice (control group) were analyzed by flow cytometry. Bulk RNA sequencing (RNAseq) and single cell RNA sequencing (scRNAseq) techniques were applied for analysis of abnormal hematopoietic Lin -Sca-1 +c-Kit + (LSK) stem cells. Cytokine levels were detected using ELISA and Luminex bead assay. Additionally, CRISPR-Cas9 technology was used to knock out the target genes and their functions were validated. Results: Following 4 weeks of PIPC induction, the experimental group exhibited a considerable reduction in the proportion and absolute number of ECs, osteoblasts and MSCs, with ECs being the most significantly reduced. The Luminex assay revealed a substantial increase in cytokine release in the BM of the experimental group, including IL4, IL10, TNFa, Cxcl10, Cxcl11, Ccl1, Ccl2, Ccl4, Ccl7, etc. Bulk RNAseq studies of LSK cells showed that the inflammatory pathway in the experimental group was significantly upregulated. To further explore the release sources of cytokines in the BM and heterogeneity in LSK cell cytokine secretion, scRNAseq analysis of LSK cells was conducted in the AE KI group, which were classified into 9 subgroups. Subgroups 1 and 5 exhibited a significant upregulation of Ccl2, Ccl4, Cxcl2, and IL15. Subgroups 4 and 8 showed upregulation of Ccl2 and IL4. Subgroups 0 and 2 exhibited upregulation of Ccl4, while subgroups 6, 7, and 9 showed upregulation of Cxcl2. Notably, subgroups 1 and 5 also displayed increased expression of Ccr5, which is the receptor for Ccl2. To further validate the origins of cytokines, LSK cells were sorted from experimental group and the control group for in vitro culture, respectively. Subsequently, the cell culture supernatants were collected for Luminex assay, revealing a notable upregulation of Ccl2 and Ccl7 in the experimental group. Furthermore, when comparing m-cherry + LSK cells (AML1-ETO positive) with m-cherry - LSK cells (AML1-ETO negative) from the same mouse, a significant increase in the Ccl2 levels in the m-cherry + LSK cell culture supernatant was observed. Flow cytometry analysis of LSK cells further revealed high expression of Ccl2 and its receptor Ccr5 in Ccl2 + LSK cells. In order to gain a deeper understanding of the functions of Ccl2 and Ccl7, CRISPR-Cas9 technology was used to knockout Ccl2 and Ccl7 in c-kit + cells, which were then transplanted into mice for further investigation, respectively. After 3 months, partial restoration of ECs was observed compared to that of the scramble control group. Conclusion: Our findings suggested that significant changes occurred in the BM microenvironment in AML1-ETO-induced pre-leukemia stage. These changes were characterized by a decrease in SCs and an increase in pro-inflammatory cytokine levels. Moreover, our study identified the heterogeneity in cytokine transcriptional profiles among AE KI LSK cells. The secretion of Ccl2 and Ccl7 by AE KI LSK cells with a blockage in hematopoietic differentiation seemed to be involved in the reduction of stromal cell population. These results provided valuable insights for further exploration of the intricate interactions between abnormal hematopoietic stem cells and the BM microenvironment in AML1-ETO-induced pre-leukemic and leukemic states. Understanding these interactions is crucial for advancing our knowledge of the underlying mechanisms involved in the development and progression of AML.
19
Wakabayashi, Aoi, Maryanne Kihiu, Malini Sharma, et al. "Interrogating Post-Transcriptional Mechanisms of Fetal Hemoglobin Regulation." Blood 138, Supplement 1 (2021): 3079. http://dx.doi.org/10.1182/blood-2021-151488.
Full textAbstract:
Abstract Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion. Depletion of RBM12 protein in HUDEP2 cells and human CD34 + hematopoietic stem and progenitor cells (HSPC) via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by HbF flow cytometry, HBG1/2 mRNA, and protein (γ-globin). Cell viability and maturation of RBM12 perturbed cells were largely intact. Additionally, RBM12 depletion in CD34 + HSPCs derived from SCD patients resulted in reduced percentage of sickled cells under hypoxic conditions. Unexpectedly, reduction of RBM12 had minimal effect on BCL11A and LRF expression suggesting that RBM12 may regulate HbF through a pathway that is indirectly related or independent of these transcription factors. RBM12 is an RBP that is widely expressed across diverse cell types and contains multiple RNA recognition motifs (RRM). While it has been implicated in various cancers and neurological disorders, its functions are not well studied. As an RBP, RBM12 can carry out several roles of post-transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. As these activities are executed in different cellular compartments, we set out to narrow down RBM12 function by assessing its subcellular localization. Immunofluorescence staining revealed strong nuclear presence of RBM12, suggesting that it functions via mRNA biogenesis and/or processing. RNASeq and LC-MS/MS analysis of RBM12 KO CD34 + HSPCs revealed modest changes in the transcriptome and proteome. In order to gain mechanistic insight into RBM12 in the context of HbF regulation, we performed cDNA rescue experiments in RBM12-deficient HUDEP2 clones. Overexpression of full length RBM12 restored HbF repression. Notably, four out of the five RRMs were dispensable for HbF silencing, but RRM1 was essential for this activity. Interestingly, an extended form of RRM1 was also sufficient for HbF silencing. Mechanistic studies of this RRM1 module are underway and will be discussed. In sum, the identification of RBM12 as a regulator of HbF production represents a previously undescribed post-transcriptional layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this understudied RBP in the context of HbF regulation which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. Disclosures Blobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy.
20
Barta, Stefan K., Marco Ruella, Stephen J. Schuster, et al. "CD5-Deleted Chimeric Antigen Receptor Cells (Senza5™ CART5) to Enhance Immunotherapy Against T-Cell Non-Hodgkin Lymphoma: A First-in-Human Phase I Clinical Trial." Blood 144, Supplement 1 (2024): 7221. https://doi.org/10.1182/blood-2024-201063.
Full textAbstract:
Background and Significance: Outcomes for relapsed or refractory (RR) T-cell lymphomas (TCL) are poor with standard therapies and cure is elusive. While in B-cell malignancies CAR T-cell (CART) therapy has changed the treatment paradigm, its development in TCL faces many challenges, mainly related to the fact that normal and malignant T cells share similar antigens. This can result in fratricide during CART manufacture and T-cell aplasia after CART treatment. To overcome these obstacles, we used CRISPR-Cas9 CD5 short-guide RNA to delete CD5 (Senza5™) in T cells and created a 4-1BB costimulated anti-CD5 CAR product (CART5). This product (Senza™ CART5) features two T-cell populations: CD5 knocked out (CD5KO) CAR5+ T cells that will exert the anti-tumor effect and CD5KO CAR-neg T cells which are expected to provide anti-infective (e.g., EBV, CMV) immunity in case of T-cell toxicity. Preclinically, Senza™ CART5 demonstrated increased CART5 expansion and enhanced antitumor efficacy in TCL xenograft models compared to wild-type (WT) CART5. Study Design and Methods: In this single-center first-in human phase I trial, we will determine the recommended phase 2 dose (RP2D) of Senza5™ CART5 cells in participants with relapsed or refractory CD5 positive T cell NHL based on their safety, tolerability, pharmacokinetics. Participants will be treated on one of 5 dose levels (DL) of Senza5™ CART5 cells. The dose levels will be determined using a Bayesian Optimal Interval (BOIN) design. Participants will receive 3x106 (DL -1) to 1.25x108 (DL4) CART5+ cells based on the assigned DL, preceded by lymphodepletion with fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 (Flu/Cy) IV daily on days -5 to -3 (or bendamustine 90mg/m2 IV daily on days -5 and -4 if contraindications to or unavailability of Flu/Cy). For participants with a WBC &lt;1000/μL lymphodepletion may be omitted. We will assign cohorts of up to 3 participants sequentially to a dose level based on the number of dose-limiting toxicities (DLTs) observed at the current dose level under a pre-specified scheme assuming a targeted DLT rate of ≤0.3. The RP2D will be determined based on feasibility, safety, and efficacy data. Patients 18 years or older with confirmed RR CD5-positive non-leukemic T-cell lymphoma with ≥50% expression of CD5 on malignant cells and no circulating malignant CD5+ cells as determined by peripheral blood flow cytometry will be eligible if they have an adequate performance status (ECOG PS 0-2) and organ function. Participants must have a sufficient cell dose for an autologous hematopoietic cell transplant (HCT) available or be human leukocyte antigen-typed and have an identified stem cell donor that could provide immune function backup in the event of T-cell aplasia. Patients with prior allogeneic HCT are currently excluded. The study will continue to enroll and treat participants until a maximum of 9 participants are infused and evaluable for DLT assessments at a given dose level, or a maximum of 30 DLT-evaluable participants from all dose levels are infused. The primary objective is to determine the RP2D of Senza5™ CART5 cells. Secondary objectives include safety as determined by frequency and severity of treatment-related adverse events, as well as efficacy by assessing overall and complete response rates, duration of response, progression-free and overall survival. Manufacturing feasibility will be determined by the frequency of product release failures and occurrence of dose failures (inability to meet targeted dose). Exploratory objectives will evaluate the persistence and trafficking of Senza5™ CART5 cells in blood and tumor by characterizing the kinetics of the infused cells by flow cytometry and qPCR. We will perform gene expression profiling of the tumor and tumor microenvironment and measure systemic soluble cytokines before and after treatment. We will also assess the impact of treatment on normal T cells, and the persistence of CD5KO untransduced T cells that are infused as part of the Senza5™ CART5 product by multicolor flow cytometry, qPCR, and single-cell RNA/TCR sequencing. The trial is registered at clinicaltrials.gov as NCT06420089 and open to accrual. Enrollment is ongoing.
21
Berrien-Elliott, Melissa M., Wong Pamela, Carly Neal, et al. "Primary Human NK Cell Gene-Editing Reveals a Critical Role for NKG2A in Cytokine-Induced Memory-like NK Cell Responses." Blood 134, Supplement_1 (2019): 3237. http://dx.doi.org/10.1182/blood-2019-129162.
Full textAbstract:
Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell anti-leukemia potential is unclear. Paradigm-shifting reports have shown that NK cells exhibit "memory-like" properties following hapten exposure, virus infection, or combined cytokine pre-activation. Human cytokine-induced memory-like (ML) NK cells display enhanced re-stimulation responses to numerous activating stimuli, including tumor target cells. This has been translated in clinical trials as cellular therapy for rel/ref AML patients (NCT#), and the dose escalation of a phase 1/2 study has been completed (PMID). Donor memory-like NK cells expanded in patients' blood and bone marrow and retained enhanced functionality ex vivo, with 7 of 11 patients achieving CR/CRi. Since NK cell recognition depend on signals from multiple activating and inhibitory receptors, we developed mass cytometry panels to immunophenotype and track the diversity and effector functions of these human in vivo-differentiated memory-like NK cells. Previous work showed that that in vivo-differentiated memory-like (ML) NK cells were distinct from baseline (BL) NK cells from the same donor, as well as NK cells from normal donor PBMC. Multidimensional analyses revealed a memory-like phenotype: CD56hi CD11blo CD62L+ NKG2Ahi NKp30hi Ki-67+. Furthermore, Citrus analyses revealed that higher NKG2A expression was significantly correlated with treatment failure. NKG2A is a C-type lectin receptor with two immunoreceptor tyrosine-based inhibitory motifs. Signaling through NKG2A is achieved when it engages its ligand, HLA-E. HLA-E is a non-classical major histocompatibility complex class I molecule that is expressed abundantly on many normal tissue types as well as tumors, including AML. Based on these findings that NKG2A is upregulated on memory-like NK cells and the intensity of NKG2A on memory-like NK cells correlated with patient responses, we hypothesized that NKG2A/HLA-E interactions represent a major barrier to memory-like NK cell responses. CRISPR based gene editing of primary human NK cells has been technically challenging. In order to interrogate the role NKG2A may play in limiting ML NK cell responses, we optimized the MaxCyte GT electroporation system to introduce Cas9 and guide RNA into freshely isolated, purified human NK cells. As proof of principle, we introduced Cas9 and gRNA targeting CD56 into NK cells and assessed CD56 expression a week later. We observed a 96.5% ± 0.8% (SD) reduction in median CD56 expression as determined by flow cytometry, with little impact on cell viability (90.3% ± 2.7% live v 87.0% ± 3.1% live ΔCD56) after electroporation. Next we introduced Cas9 and gRNA against NKG2A into freshly isolated, purified human NK cells. After electroporation, cells were briefly incubated with IL-12/IL-15/IL-18, overnight. The cytokines are washed away and the cells incubated for 4 days in low-dose IL-15, which was required for their survival. NKG2A frequency was decreased 64.72% (ΔNKG2A v Control; 42.3-85.7% range, ± 13.18% SD) by 4 days post-electroporation. We compared the ability of these cells to respond to HLA-E+ K562 leukemia targets and observed a significantly enhanced ML NK cell response by ΔNKG2A ML NK cells compared to control ML NK cells (19.04 ± 5.9% IFN-γ+ v 34.9 ± 8.8% ΔNKG2A IFNγ+; Mean ± S.D.). Finally, we infused ΔNKG2A ML NK or control ML NK cells into NSG recipient mice and assessed the spleen at D7 and D14 for persistence and NKG2A expression. We were able to detect ΔNKG2A ML NK and control ML NK cells at both time points and the ΔNKG2A ML NK cells remain NKG2A-negative, post-transfer. Using gene-editing approaches, the data reveal an important inhibitory role for NKG2A on ML NK cell responses against HLA-E+ targets. Primary human NK cells are notoriously difficult to modify by virus or electroporation. Indeed, most reports utilize expanded NK cells or cord-blood differentiated NK cells which were edited in the stem cell stage. This report demonstrates that mature NK cells can be modified with little ex vivo manipulation with high efficiency and viability. This method has broad potential to expand our understanding of human NK cell biology using genetic loss or gain of function techniques, as exemplified by identification of NKG2A as a critical ML NK cell checkpoint. Figure Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).
22
Zhang, Yanlei, Hechun Ma, Boang Han, et al. "Abstract 2666: Genetic ablation of B2M leads to resistance to PD-1/PD-L1 blockade in vivo." Cancer Research 84, no. 6_Supplement (2024): 2666. http://dx.doi.org/10.1158/1538-7445.am2024-2666.
Full textAbstract:
Abstract Immune checkpoint inhibitors (ICIs) has revolutionized the treatment landscape of various cancers by reinvigorating the exhausted T cells in patients. However, the therapeutic efficacy is largely confined due to the primary or acquired resistance to anti-PD-1 (L1) therapy in many patients. Recently, the mechanisms of resistance to ICIs has been extensively elucidated from different aspects and emerging sequencing data from clinical samples has pointed to IFN-γ signaling defects and antigen presentation loss in patients who are resistant to PD-1(L1) blockade. The loss-of-function mutations in JAK1 and JAK2 results in lack of response to IFN-γ signaling and incapacity to upregulate PD-L1 and MHC-I, which subsequently leads to noninflammatory TMEs and resistance to anti-PD-1/PD-L1. The loss-of-function of B2M is likely a common resistance mechanism that the intratumoral cytotoxic CD8+ T cells are failed to be boosted owing to antigen presentation loss. In order to obtain higher clinical benefits in various cancers, it’s appealing and urgent to develop mechanism-based strategies to overcome resistance to ICI-based immunotherapy. To this end, we developed a genetically acquired resistant tumor model with B2M mutations in syngenic MC38 tumor cells using CRISPR/Cas9 technology, which is ‘hot’ tumor to PD-1/PD-L1 blockade. The expression of B2M was detected by western blotting and flow cytometry. Mouse B2M-knockout MC38 tumors became resistant to PD-1/PD-L1 blockade in vivo. Notably, we found a remarkable increase of CD8+ T cell infiltration in B2m-KO MC38 tumors in comparison to parental MC38 tumors upon anti-PD-1 treatment, which further explains that antigen presentation loss results in inactivation of cytotoxic CD8+ T cells within the tumor microenvironments (TMEs), subsequently leading to resistance and poor survival to ICI therapy.In summary, our data consistently indicated the importance of MHC-I expression in T cell activation in the TMEs and the possibility to dig out the pathway involved in MHC-I expression independent of IFN-γ signaling by CRISPR-based screening to overcome MHC-I deficiency-induced resistance in the future. In addition, it’s possible to leverage the cytotoxic NK cells and CD4+ T cells in antitumor immunity against MHC-II+ tumors even with the loss of MHC-I molecules. For example, high dose of IL-2RB-biased IL-2 agonist preferentially binding to the dimeric IL-2R can stimulate Teff and NK cells independent of immune checkpoint expression. Likewise, cytokine modified anti-PD-1 fusion formulation like anti-PD-1-IL-2 mutein and anti-PD-1-IL-15 mutein are promising to overcome the resistance. Furthermore, it has been suggested that the resistance due to genetic mutations of MHC-I could be overcome by combining NF-B targeted therapies. Citation Format: Yanlei Zhang, Hechun Ma, Boang Han, Yi Li, Ping Yang, Zhen Li, Dongxiao Feng, Lei Ci, Ruilin Sun, Daniel X. He. Genetic ablation of B2M leads to resistance to PD-1/PD-L1 blockade in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2666.
23
Pilunov, Artem, Dmitrii Romaniuk, Savely Sheetikov, et al. "Development of T-Cell Therapy Targeting Hematopoietic Minor Histocompatibility Antigen HA-1." Blood 134, Supplement_1 (2019): 5749. http://dx.doi.org/10.1182/blood-2019-130552.
Full textAbstract:
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative therapy for hematological malignancies yet in nearly one-third of patients, it is followed by a relapse of the disease contributing to high mortality. In fully HLA-matched allo-HSCT graft versus leukemia reaction is driven by the recognition of the minor histocompatibility antigens (MiHAs) - endogenous polymorphic peptides presented by MHC. Particularly, HA-1 MiHA is a promising target for immunotherapy. HA-1 is presented by frequent among Caucasians HLA allele - A*02:01. The single nucleotide variation in ARGHAP45 gene which generates the MiHA has the optimal allelic distribution, thus immunogenic mismatch occurs in 30% of allo-HSCT. Also, ARGHAP45 is overexpressed in certain types of leukemia. Here we aim to develop HA-1-specific T-cells for post-transplant relapse therapy. To obtain the sequences of HA-1-specific T-cell receptors (TCRs), naive CD8+ T-cells from 3 HLA-A*02:01 positive and HA-1 negative donors were expanded in vitro using autologous dendritic cells pulsed with HA-1 peptide. Antigen-specific cells were enriched by CD137 marker expression or by HLA-tetramer staining, RNA from positive and negative fractions was isolated for cDNA library preparation. The α and β TCR-repertoires were sequenced using the Illumina MiSeq system. The representative enrichment plot is shown in Figure 1 (A - α chains, B - β chains). Each circle represents a unique TCR. The vertical axis shows the normalized frequency in enriched fraction, the horizontal axis shows the normalized frequency in tetramer or CD137 negative flowthrough. HA-1-specific TCRs are denoted by green filled circles.TCRs were considered to be HA-1-specific if they were significantly enriched in CD137+ or tetramer+ fraction (exact Fisher test, p=0.05). In total, 49 α and 80 β chains were described. To determine the degree of similarity between HA-1-specific TCRs Levenstein distance was calculated between amino acid sequences of complementarity-determining region 3 for both chains. Sequences of previously published HA-1-specific TCRs were also included in the analysis (Verdijk et.al., Haematologica, 2002; Bleakley et.al., 2017, WO2018058002A1). α chains demonstrated low degree of mutual similarity, the majority of sequences did not belong to any cluster (Figure 2A, sequences with the Levenstein distance <3 are connected). In contrast, a significant proportion of β chains were organized in a few clusters containing sequences from all 3 donors and previously published data (Figure 2B). We selected 14 α and 12 β HA-1-specific TCR chains (marked by the black dots in Figure 2). Clones were picked to represent separate clusters of similarity to Levenstein metrics, and unique sequences. Selected α and β-chains were cloned for subsequent functional screening in different combinations. Besides, we developed the modular lentiviral backbone for manufacturing HA-1 specific transgenic CD8+ T-cells. Our approach utilizes Golden Gate Cloning, which allows rapid assembly of lentiviral backbone carrying any combination of TCR α and β chains fused with the selective marker for sorting via p2A peptides. We used truncated CD34 as a transduced cell surface marker for the rapid separation of transduced cells by clinical-grade antibodies and subsequent expansion. In order to prevent the mispairing of transgenic TCR with endogenous one, CRISPR/Cas9 knockout strategy of endogenous TCR chains was developed. We used guide RNAs specific to TRAC,TRBC1 and TRBC2 genes and recombinant Cas9. The efficiency was demonstrated on Jurkat E6-1 cell line, the knockout was confirmed both by flow cytometry and genotyping of the modified cells using fragment analysis. Constant regions of the transgenic TCRs were modified to prevent cleavage by Cas9, the resistance was confirmed by in vitro Cas9 digestion assay. Moreover additional cysteines were introduced in the constant regions of transgenic TCRs for increased transgenic TCR stability. Cytotoxic activity of modified cells will be confirmed on lymphoblastoid cell lines and patient leukemia samples, cytokine secretion of modified cells will be detected using ELISPOT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. Disclosures No relevant conflicts of interest to declare.
24
Shen, Yandong, Kyle R. Crassini, Michael E. O'Dwyer, et al. "The Dual PI3/PIM-Kinase Inhibitor, Ibl-202, Is Highly Synergistic with Venetoclax Against CLL Cells, and TP53-Knock-out Cells, and Under Conditions That Mimic the Tumor Microenvironment." Blood 132, Supplement 1 (2018): 1870. http://dx.doi.org/10.1182/blood-2018-99-115574.
Full textAbstract:
Abstract Background The B-cell receptor (BCR) signaling pathway and the pro-survival Bcl-2-family of proteins play crucial roles in the pathogenesis of chronic lymphocytic leukemia (CLL). Constitutive activity of the BCR signaling pathway and overexpression of Bcl-2 promote CLL-cell survival, proliferation and drug resistance. BCR-targeted therapies, most notably ibrutinib and idelalisib and the Bcl-2 inhibitor Venetoclax, demonstrate the potential of targeting these pathways. However, there is no evidence that these novel agents are curative in the event of relapse. Treatment options remain limited for patients treated with these agents, in particular those with TP53 lesions. In our recent study (Crassini et al., BJH 2018) we demonstrated efficacy of the PI3/PIM kinase inhibitor, IBL-202 (Inflection Biosciences, Ltd), against CLL cells. In the current study we investigated the effects of combining IBL-202 with Venetoclax on primary CLL cells and both wild-type and TP53 deficient OSU-CLL cells. Methods Primary CLL cells were co-cultured with CD40L-expressing fibroblasts. We established a TP53 knock-out OSU-CLL cell line (OSU-TP53ko) using the CRISPr-Cas9 system. Cell viability was assessed using the mitochondrial dye DilC1(5), propidium iodide and flow cytometry. Synergy between IBL-202 and Venetoclax was evaluated by determining combination indices (CI) using the Compusyn software. The effects of the drugs on cell cycle and proliferation of the OSU-CLL cell lines were assessed using propidium iodide or carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. The effects of the drugs on the migratory capacity of CLL cells were assessed by determining changes in CXCR4 expression and CLL-cell migration along an SDF-1a gradient. The mechanisms of action of the drugs were investigated by immunoblotting. Results IBL-202 and Venetoclax were highly synergistic against primary CLL cells co-cultured with CD40L-fibroblasts, with a CI of 0.4 at a fractional effect of 0.9 (Figure A and B). Synergy between the drugs was consistent with a significant (P < 0.05) reduction in the IC50 for both drugs. Synergy was also observed against wild-type (WT) and TP53ko OSU-CLL cells, with CI values of < 0.5 at fractional effects of 0.5 and 0.9. Synergy was consistent with significantly greater cytotoxic effects of the drugs in combination (Figure C. WT : P = 0.002 and TP53ko : P = 0.002). IBL-202 and Venetoclax in combination induced cell cycle arrest and slowed the proliferation of both cell lines. Immunoblotting of primary CLL cells showed IBL-202, alone and in combination with Venetoclax, inhibited AKT phosphorylation and reduced the expression of Mcl-1 and Bcl-xL. A greater than additive effect of IBL-202 and Venetoclax was observed on the migratory capacity of CLL cells, reducing the number of cells migrating towards SDF1-a. The effects of the drugs on cell migration were consistent with reduced expression of CXCR4. Conclusions The synergy we observed between IBL-202 and venetoclax against primary CLL cells cultured under conditions that mimic the tumor microenvironment suggests this drug combination may be effective against CLL cells within the lymph nodes and bone marrow. Furthermore, the efficacy of the combination against the TP53ko OSU-CLL cell line suggests the combination may be a highly effective treatment strategy for poor risk CLL disease. Figure A and B - Synergy of IBL-202 and Venetoclax against primary CLL cells. Figure C - Cytotoxic effects of IBL-202 in combination with Venetoclax against OSU and OSU-TP53ko CLL cells Disclosures O'Dwyer: Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees.
25
Gupta, Dipti, Yannis Hara, Samuel Lessard, et al. "Catalytic Activity of Heme-Regulated eIF2 Alpha Kinase (HRI) Regulates Fetal Hemoglobin." Blood 136, Supplement 1 (2020): 7. http://dx.doi.org/10.1182/blood-2020-139991.
Full textAbstract:
Sickle cell disease (SCD) is a hereditary disorder occurring due to a mutation in the β- globin gene resulting in hemoglobin polymerization and sickling of red blood cells that drives an array of severe pathophysiologies. SCD patients with hereditary persistence of fetal hemoglobin mutations show amelioration of disease symptoms. HRI is a heme sensing eIF2α kinase belonging to the integrated stress response pathway, primarily regulating the hemoglobin synthesis in RBCs. Under low levels of heme, HRI undergoes auto-phosphorylation and subsequently phosphorylates its substrate eIF2α. This impedes the global protein translation and selectively activates ATF4 translation thereby initiating a transcriptional stress response. HRI protein deletion has been shown to induce fetal hemoglobin (HbF) in vitro by reducing BCL11A levels (Grevet et al., 2018). It has also been shown that ATF4 binds to enhancer region of BCL11A promoting its expression, revealing a direct regulation of HRI from eIF2α to ATF4 to BCL11A to γ-globin (Huang P, 2020). As it is well-established that protein kinases can exert dual kinase and scaffold functions, we explored whether the regulation of HRI on HbF is driven by its catalytic or non-catalytic activity. We generated HRI kinase dead (KD) mutant HUDEP-2 cells, with a mutation (K196R) in HRI ATP binding site that is predicted to result in loss of its auto-phosphorylation (Bauer B. N., 2001). HRI KD mutant HUDEP-2 was created using CRISPR Cas9 and homologous recombination approach, followed by clonal selection that yielded two homozygous clones, referred as KD clones. To further compare the scaffold and kinase function of HRI, we created HRI knockout (KO) in HUDEP-2 cell line using CRISPR Cas9 approach. We isolated clonal populations with 3 clones each with heterozygous and homozygous editing. Western analysis of KD clones showed that HRI protein levels remain unchanged. About 50% and a 100% loss in HRI protein level was observed in heterozygous and homozygous KO clones respectively. To confirm the loss in kinase activity of HRI, we differentiated KD clones for 7 days in vitro and subjected them to iron depleted conditions. As expected, HRI WT cells under iron depleted conditions showed robust activation in the p-eIF2α levels indicative of increase in HRI activity. KD clones lacked increase in p-eIF2α levels under these conditions confirming the loss of kinase activity of HRI in KD clones. HRI KD and KO clones were characterized to evaluate the HbF regulation by differentiating in vitro for 7-8 days. KD clones showed 4-5-fold induction in HbF levels measured by flow cytometry. Heterozygous and homozygous KO clones showed an equally strong activation in HbF levels. KD and KO clones showed downregulation in BCL11A and ATF4 protein levels. These results indicate that HRI regulates HbF as a function of its catalytic activity. We further employed RNA-seq to study the global transcriptomics in HRI WT and KD mutant clones at day 0 and day 8, under steady state and iron depleted conditions. RNA-seq confirmed the strong activation of HBG1/2 levels and robust silencing of BCL11A levels in KD clones at day 8. A weaker induction in HBG1/2 levels and minor reduction on BCL11A levels were observed at day 0. These results indicate that HRI may play a role in HbF silencing in a stage specific manner. HRI WT cells under iron depleted condition showed activation of ATF4 pathway, indicative of activation of HRI stress pathway. As expected, HRI KD population showed no regulation of ATF4 pathway under iron depleted conditions. Interestingly, we did not observe any changes in the BCL11A gene expression in WT cells under iron depleted conditions. Further evaluation needs to be carried out in order to understand the regulation of BCL11A by ATF4 under low iron mediated stress. Fewer gene changes were observed in KD populations under iron depleted conditions indicative of HRI playing a vital role as a heme sensor. Overall, our results provide evidence to support that HRI regulates HbF though its catalytic activity and demonstrates its role as heme sensor in HUDEP-2 cells. Disclosures Gupta: Sanofi: Current Employment. Hara:Sanofi: Current Employment. Lessard:Sanofi: Current Employment. Sturtevant:Sanofi: Current Employment. Krishnamoorthy:Sanofi: Current Employment. Demers:Sanofi: Current Employment. Hicks:Sanofi: Current Employment.
26
Iyer, Prajish, Brian Jiang, Girish Venkataraman, et al. "Glycerolipid Metabolism Via the MGA-MYC-NME1-PGP Axis As a Key Regulator of Oxidative Phosphorylation in Richter's Transformation." Blood 142, Supplement 1 (2023): 79. http://dx.doi.org/10.1182/blood-2023-179427.
Full textAbstract:
Despite advances in targeted therapies for chronic lymphocytic leukemia (CLL), Richter's transformation (RT) remains a clinical challenge. RT is an aggressive shift from CLL to lymphoma, associated with metabolic changes and aberrations in the MYC transcriptional network. Genomic analysis of paired CLL and RT cases revealed recurrent mutations or deletions in the MYC suppressor Max-gene-associated (MGA) increase from 5% (CLL) to 30% (RT) cases, highlighting its critical yet unexplored role as a driver event. We recently developed a murine RT model using B cell-restricted in vitro editing of LSK progenitors from Cd19Cre-Cas9-del(13q)-Sf3b1 ( Sf3b1-K700E) with Mga-targeting sgRNAs. This murine model demonstrated upregulation of Mga targets Myc and Nme1 (nucleoside diphosphate kinase), a distinct OXPHOS-based transcription signature, increased oxygen consumption rate, elevated ROS levels, suggesting a significant link between the Mga-Myc-Nme1 axis and OXPHOS dysregulation in RT. Given the crucial role of metabolic reprogramming in cancer, we seek to understand the molecular mechanisms of metabolites driving OXPHOS and accelerating CLL transformation. Unraveling these processes could revolutionize therapeutic approaches for managing RT in clinical settings. To identify metabolites driving dysregulated OXPHOS in RT, we first conducted paired RNA-seq and unlabeled metabolomic profiling on normal and RT mouse splenic B cells (n=3/group). To translate our discovery from the murine RT model to human disease, we performed metabolomic profiling using MEC1 and Nalm6 cell lines with MGA deletion. Our metabolomic analysis of murine RT cells revealed 44 upregulated metabolites, including TCA cycle substrates (glutamine fold change (FC)=9.6, p=0.001;asparagine FC= 4.0, p=0.006), fatty-acid intermediates (glycerol-3P FC=11.7, p=0.003; carnitine FC=4.3, p=0.02; palmitate FC= 4.1, p= 0.08), and glycolysis intermediates (3-phosphoglycerate FC= 4.0, p=0.03; UDP-glucose FC=12.7, p=0.02; glycine FC=4.7, p=0.03). Moreover, integrated metabolite analysis (MetaboAnalyst v5.0) identified 63 common upregulated metabolites in MGA KO (knockout) human cell lines and murine RT, enriching glycerolipid, and fatty acid oxidation, associated pathways, indicating a potential role for MGA deletion in driving dysregulated fatty acid metabolism. Pathway enrichment analysis using integrated RNA-seq and metabolite analyses showed that the glycerolipid pathway was one of the most enriched pathways among mouse RT and B-cell lines (Nalm6 and MEC1) with MGA KO. Additionally, two key fatty-acid enzymes, fatty acid synthetase (FASN) and phosphoglycerate phosphatase (PGP), displayed significant mRNA and protein changes, as validated by qPCR and immunoblotting. Furthermore, elevated lipid accumulation and increased free glycerol were confirmed based on lipi-green flow cytometry and glycerol-bioluminescent assays, suggesting an important role of glycerolipid metabolism in MGA KO-associated OXPHOS. PGP, a glycerol-3-phosphatase, regulates glycerol-3-P levels, a key intermediate in glucose, lipid, and energy metabolism. Overexpression of MYC and NME1 increases PGP levels in B-cell lines (MEC1, Nalm6, and Jeko), confirming PGP as a direct target of the MGA-MYC-NME1 axis. To determine the role of PGP in MGA KO (knock-out) cells, we used CRISPR/Cas9 to KO PGP in MEC1, Nalm6, and JeKo cell lines. KO of PGP decreased cell growth and reduced OXPHOS compared to controls based on CCK8 absorbance and Seahorse Mitostress assays respectively. Moreover, PGP KO downregulated the mTOR (p4E-BP1) pathway, emphasizing the significance of dysregulated glycerol and fatty acid metabolism in CLL to RT-driven OXPHOS. Our study reveals key metabolites driving RT and highlights the crucial role of the MGA-MYC-NME1-PGP axis in regulating OXPHOS in Mga KO RT cells. Our results thus suggest targeting glycerolipid metabolism and related pathways may offer effective strategies to conquer RT
27
Wenthe, Sophia, Kelsie Becklin, Brett Napiwocki, Emma Kozurek, Branden Moriarity, and Jong Hyuk Kim. "Abstract 198: Unveiling chromatin accessibility landscape and convergent oncogenic pathway in angiosarcoma models using induced pluripotent stem cells." Cancer Research 82, no. 12_Supplement (2022): 198. http://dx.doi.org/10.1158/1538-7445.am2022-198.
Full textAbstract:
Abstract Angiosarcoma is a rare soft tissue sarcoma that forms malignant vessels. Angiosarcomas are aggressive and highly metastatic, resulting in a poor prognosis. Recurrent somatic mutations in TP53 and genes involved in PI3K/AKT/mTOR pathway such as PIK3CA and PIK3R1 are identified in angiosarcomas. However, angiosarcomas are genomically complex, and the oncogenic mechanisms are virtually unknown. Due to its rarity, establishment of experimental tumor models is an unmet need for angiosarcoma research. In this study, we used human induced pluripotent stem cells (iPSCs) to develop a novel, reliable model for angiosarcoma recapitulating the genomic complexity and the tumor immune landscape. Specifically, we induced TP53 mutations in human iPSCs using CRISPR/Cas9 with validation of p53 deficiency by gene sequencing and Western blotting. We then established protocols to differentiate genetically engineered iPSCs to mesoderm and subsequently bi-potential hemangioblasts, which are considered the putative cell-of-origin of angiosarcoma. We found that isogenic wild-type (WT) and TP53 mutant iPSCs were capable of generating hemangioblasts, with no significant differences in morphology or growth patterns observed between WT and mutant cells. Putative iPSC-derived hemangioblasts were CD34+ by flow cytometry and contained primitive endothelial cells with the capacity to form tube-like structures in Matrigel. RNA-seq data libraries were generated to profile global gene expression in non-differentiated cells, mesodermal precursors, hemangioblasts, and endothelial cells derived from WT and p53 mutant iPSCs during differentiation over time (day 0, 2, 5, 8). Principal component analysis revealed that gene expression patterns were altered between WT and p53 mutant cells during differentiation, representing distinct gene signatures unique to each cell type. Intriguingly, transcriptomic alteration of p53 mutant iPSC-derived cells was more variable than that of WT. Our data also showed that p53 mutation induced dysregulation of genes associated with chromosome maintenance, extracellular matrix organization, hemostasis, and receptor tyrosine kinase signaling in iPSC-derived hemangioblasts when compared isogenic WT controls. Our data highlights the role that mutant p53 plays in the induction of genomic instability and transcriptional programs that regulate hemogenic and endothelial function during differentiation. Additionally, ATAC-seq data were generated from WT and p53 mutant iPSC-derived cells to determine chromatin accessibility dynamics and identify key transcription factors that activate convergent vascular tumorigenic pathways. We are currently generating iPSC harboring co-mutations in TP53 and PIK3CA in order to determine the phenotype and tumorigenic capacity of engineered iPSC-derived hemangioblasts in xenograft models. Citation Format: Sophia Wenthe, Kelsie Becklin, Brett Napiwocki, Emma Kozurek, Branden Moriarity, Jong Hyuk Kim. Unveiling chromatin accessibility landscape and convergent oncogenic pathway in angiosarcoma models using induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 198.
28
Shi, Yuanfei, Yanchun Zhao, Yi Xu, et al. "Effects and Molecular Mechanism of Inhibiting p53 Signaling Pathway By NSUN6 on the Resistance to BCL-2 Inhibitor for Diffuse Large B-Cell Lymphoma." Blood 144, Supplement 1 (2024): 6238. https://doi.org/10.1182/blood-2024-202205.
Full textAbstract:
Background: Most DLBCL patients with recurrent and refractory DLBCL have high BCL-2 expression, but some patients are resistant to BCL-2 inhibitors for unknown reasons, the mechanism of which is unknown yet. We constructed a DLBCL cell line resistant to venetoclax in the early stage to explore its molecular mechanism of resistance. We found that m5C methyltransferase NSUN6 was highly expressed in DLBCL resistant cell lines, and also highly expressed in lymph nodes of relapsed and refractory DLBCL patients. Those with high expression had poor prognosis. Knocking down NSUN6 can significantly reduce the tumor burden and prolong the survival cycle of experimental mice. Therefore, we speculate that NSUN6 may be an important target for venetoclax resistance in relapsed and refractory DLBCL patients. Molecular complex detection (MCODE) and RNA-seq analysis revealed that NSUN6 mainly exerts its effect by inhibiting the p53 signaling pathway, and the specific mechanism remains to be explored. This project aims to elucidate the roles of NSUN6 and p53 signaling pathways in venetoclax resistance at the molecular, cellular, and animal levels based on previous research. Exploring how m5C methylation modification mediates venetoclax resistance and regulates the p53 pathway, providing a theoretical basis for addressing ABT-199 resistance in relapsed and refractory DLBCL patients. Methods: This study successfully constructed the venetoclax resistant strain OCI-Ly1 in DLBCL cell lines, established a cell line-derived xenograft (CDX) model in DLBCL mice, and identified the venetoclax resistant gene NSUN6 through RNA seq sequencing and MCODE method. NSUN6 knockdown and overexpression cell lines were successfully constructed using CRISPR-Cas9 technology and lentivirus methods. Using methods such as fluorescence quantitative PCR, Western blot, immunohistochemistry, co immunoprecipitation (Co-IP), flow cytometry, and cell proliferation experiments to explore the molecular mechanisms of venetoclax resistance at the cellular, animal, and primary patient levels, in order to systematically and comprehensively reveal the important role of NSUN6 in venetoclax resistance. Results: We successfully constructed venetoclax-R in DLBCL and elucidated that NSUN6 is the main resistance gene of venetoclax, revealing that NSUN6 is an important adverse factor affecting patient prognosis. Elucidating that knocking down NSUN6 in OCI-Ly1-R can reverse the development of venetoclax resistance. We proving that the p53 pathway is an important pathway for NSUN6 to function. Search for target genes regulated by NSUN6 and investigate the effect of knocking out NSUN6 on the m5C modification level of target genes. Conclusion: Our research takes DLBCL cell lines, primary cell samples, and BALB/C nude mice as research objects. Based on existing research, the biological effects of NSUN6 and the feasibility of reversing venetoclax resistance are verified in vitro and in vivo, and the specific mechanism of NSUN6 mediated venetoclax resistance is elucidated.
29
McCONVILLE, Michael, Toby Thomas, Catherine Valadez, et al. "Familial Thrombocytopenia-Associated Germline ETV6 P214L Mutation Results in XPO1-Mediated Nuclear Export." Blood 142, Supplement 1 (2023): 1298. http://dx.doi.org/10.1182/blood-2023-182269.
Full textAbstract:
In recent years, a number of heterozygous germline mutations in ETV6 have been associated with familial thrombocytopenia and predisposition to hematologic malignancies. While most of these missense mutations occur in the DNA-binding-domain of ETV6, the recurrent P214L mutation is unique in that it occurs in the central disordered region of the protein. The ETV6 P214L mutation has previously been shown to disrupt the nuclear localization of ETV6 and the ability of ETV6 to act as a transcriptional repressor. Unexpectedly, we have found that the P214L missense mutation creates a recognition motif for the nuclear export protein XPO1. We have demonstrated in cellular and protein reconstitution assays that XPO1 directly engages the P214L but not the wild-type ETV6 construct. Consistent with this mechanism, we are able to fully rescue the nuclear localization of ETV6 P214L in mammalian cells with small molecule XPO1 inhibitors. We have also found that once localized to the nucleus, the P214L mutant construct exhibits functional consequences indistinguishable from wild-type ETV6 in Ba/F3 transformation assays. When looking at the evolutionary conservation of the ETV6 amino acid sequence involved in the XPO1 recognition motif, we observed that the synonymous P216L mutation in the mouse ETV6 ortholog does not create a nuclear export signal. In order to study XPO1-mediated export of ETV6 P214L and its impact on hematopoiesis, we have generated a mouse model in which two point mutations (P216L and S211I) have been introduced into a single endogenous ETV6 allele, which precisely matches the sequence of this region in human P214L carriers. Using CRISPR-Cas9, we generated 3 transgenic mouse lines: Etv6 P216L, Etv6 S211I, and Etv6 S211IP216L. Immunofluorescence of fixed bone marrow shows that ETV6 is only mislocalized in megakaryocytes from ETV6 S211I P216L mice. Complete blood counts revealed a significant decrease in platelet counts in ETV6 S211I P216L heterozygotes (485 x 10 3/µL) compared to ETV6 S211I heterozygotes (1140 x 10 3/µL, p&lt;0.0001), ETV6 P216L heterozygotes (1065 x 10 3/µL, p&lt;0.0006), and wild-type littermates (1254 x 10 3/µL, p&lt;0.0001). Flow cytometry phenotyping of bone marrow from the three transgenic lines and wild-type littermates shows a significant decrease in the absolute numbers of hematopoietic stem cells (HSCs) and megakaryocyte precursors (MkPs) in ETV6 S211I P216L mice. In summary, we have determined that XPO1-mediated nuclear export is the mechanistic basis for ETV6 P214L mislocalization. We developed a mouse model that recapitulates this export signal in the endogenous ETV6 allele and found that this model demonstrates a thrombocytopenic phenotype as seen in human patients. Reductions in HSCs and MkPs suggest both a decreased fitness of HSCs, as well as dysregulated megakaryopoiesis in this model.
30
Liu, Jile, Xiaomei Zhang, Rui Sun, et al. "Oncolytic Herpes Simplex Virus-Loaded CD19 CAR-T Cells Improves the Therapeutic Effect of Diffuse Large B-Cell Lymphoma." Blood 144, Supplement 1 (2024): 4810. https://doi.org/10.1182/blood-2024-205138.
Full textAbstract:
Background In the clinical practice of treating patients with diffuse large B-cell lymphoma, CD19 CAR-T cell therapy has failed to achieve the desired therapeutic effect and is highly prone to recurrence. In the field of solid tumors, the combined therapy of CAR-T cell therapy and oncolytic viruses has made breakthrough progress. Therefore, we introduce this combined therapeutic idea into the treatment of diffuse large B-cell lymphoma. In order to deal with the characteristic that diffuse large B-cell lymphoma is highly prone to systemic metastasis and to avoid the problem that oncolytic viruses are neutralized by antibodies in the blood,we abandon the two common administration methods of oncolytic viruses, intravenous infusion and intratumoral injection, and chose to load the oncolytic viruses onto CAR-T cells for combined treatment. MethodWe used the CRISPR Cas9 technology to knockout the cytotoxic genes ICP34.5 and ICP47 on Herpes Simplex Virus (HSV), and added the GFP expression cassette to obtain the oncolytic herpes simplex virus-GFP（ oHSV-GFP）. By co-culturing oHSV-GFP (MOI = 100) with CD19 CAR-T cells in a 37°C, 5% CO2 incubator for 24 hours, CD19 CAR-ToHSV cells were obtained, and evaluated using flow cytometry and qPCR techniques. We evaluated its anti-tumor effect and safety using the human diffuse large B-cell lymphoma cell line (U2932) and the human diffuse large B-cell lymphoma animal model. ResultFlow cytometry analysis demonstrated that this method could successfully prepare CD19 CAR-ToHSV cells, and the proportion of CD19 CAR-T cells expressing fluorescence accounted for approximately 10% of the total. It was verified again by qPCR technology. Using CCK8 to evaluate the proliferation situation, we found that this preparation method had no effect on the proliferation of CAR-T cells. CD19 CAR-T cells and CD19 CAR-ToHSV cells were co-cultured with diffuse large B-cell lymphoma cell lines respectively. We found that the anti-tumor effect of CD19 CAR-ToHSV cells was significantly better than that of CD19 CAR-T cells. In the evaluation of cytokines, the secretion of IFN-γ by CD19 CAR-ToHSV cells was significantly higher than that of CD19 CAR-T cells, which might be one of the reasons for its high tumor effect. In the animal model of diffuse large B-cell lymphoma, we found that the tumor volume of mice treated with CD19 CAR-ToHSV cells was significantly smaller than that of the control group and the mice treated with CD19 CAR-T cells. Meanwhile, immunohistochemistry showed that the infiltration of CAR-T cells in the CD19 CAR-ToHSV cell treatment group was significantly better than that in the CD19 CAR-T cell group. This proved the superior inhibitory effect on tumor growth and tumor infiltration ability of CD19 CAR-ToHSV cell therapy. Through qPCR analysis of the main organs and tumors of mice, we found that only the tumor showed viral replication. Evaluation of HE staining sections of the liver, spleen and kidney of mice did not reveal obvious tissue damage. This preliminarily proved the safety of CD19 CAR-ToHSV cell therapy. ConclusionOncolytic herpes simplex virus-loaded CD19 CAR-T cells is a relatively safe and effective method to enhance the therapeutic effect of diffuse large B-cell lymphoma.
31
Walker, Janek S., Kerstin Wenzl, Joseph P. Novak, et al. "Truncating SPEN Mutations Highlight BN2-Subtype DLBCL with Aggressive Biology and Features of Immune Evasion." Blood 142, Supplement 1 (2023): 4140. http://dx.doi.org/10.1182/blood-2023-181974.
Full textAbstract:
Introduction: Molecular classifications have been developed to characterize the heterogeneity of diffuse large B cell lymphoma (DLBCL), with activation of BCL6 and Notch signaling pathways acting as driving pathogenic features in C1/BN2 subtype tumors. Among C1/BN2 defining variants are truncating/inactivating SPEN mutations. SPEN regulates the Notch signaling pathway by initiating formation of a repressive complex that facilitates transcriptional repression of Notch target genes. Our group has identified SPEN mutations enriched in DLBCL cases failing to achieve event-free status at 24 months (EFS24). This contrasted with generally favorable outcomes in C1/BN2 tumors, suggesting a potential high-risk molecular feature with unexplored significance. Presenting an opportunity to expand underlying mechanisms and risk stratification in C1/BN2 tumors, we aimed to use a functional genomic approach, combined with clinical data, to characterize the alternative biology stemming from SPEN truncating mutations. Methods: FFPE tumor biopsies from newly diagnosed DLBCL cases (N=444) enrolled in the Mayo Clinic/Iowa Lymphoma Molecular Epidemiology Resource (MER) program were analyzed by WES (n=404) and RNA-seq (n=321). Biologic and clinical variables were annotated for all cases. CRISPR/Cas9 introduced truncating mutations to the endogenous SPEN locus in OCI-LY3 & SU-DHL-2 ABC-type human cell lines to establish models of SPEN depletion. Functional in vitro analysis utilized standard methods for RNA-seq, cell proliferation, western blot, and flow cytometry. Results: WES identified 29/404 ndDLBCL (7.2%) harboring SPEN mutations; n=17 missense and n=12 nonsense or frameshift insertions/deletions (truncating variants - [trunc]), the later considered as the focal point of our study. Against all cases, SPEN trunc cases displayed enrichment for high-risk factors including non-GCB COO (p=0.06, OR=3.7, 95% CI [0.9, 22.2]) and primary refractory disease (PTR; p=0.02, OR=5.0, 95% CI [1.05, 19.9]), and demonstrated inferior OS (p=0.06, HR=2.2), where a majority of clinical progression events occurred within 12 months of diagnosis. Leveraging RNA-seq, we observed 11 of 11 SPEN trunc cases exhibiting a double-hit negative gene expression signature, 9/11 cases with inflammatory or depleted microenvironment (LME), and 8/11 with a signature associating with intermediate/high risk of EFS24 failure. Applying the LymphGen algorithm, 5/12 SPEN trunc cases received the BN2 classification, observing co-occurring mutations with CD70 (p&lt;0.01, OR=9.68), BRCA2 (p=0.03, OR=5.84), TET2 (p=0.02, OR=4.91), IRF4 (p=0.02, OR=7.27), and PTPRD (p&lt;0.01, OR=8.31) genes, and were mutually exclusive with ARID1A, BCL2, EZH2, FAS, HLA-B, IRF8, and TMEM30A mutations ( Figure 1A). Isolating SPEN trunc against BN2 cases without SPEN variants (n=14), despite similar clustering of non-GCB COO (p=0.7), PTR was exclusively observed in SPEN trunc cases, accompanied again by inferior OS (p=0.07, HR=4.2; Figure 1B). In the OCI-LY3 SPEN depletion model, GSEA analysis from RNA-seq revealed activation of E2F (NES=2.7; p=9.4E-20) and MYC target genes (NES=2.3; p=4.1E-6), while both OCI-LY3 and SU-DHL-2 models featured depletion of antigen presentation genes (NES= -1.8, p=5.7E-4; NES= -2.2, p=3.8E-7, respectively). A decrease of MHC class II proteins (HLA-DR, HLA-DP) was confirmed by flow cytometry in both models (p&lt;0.01). Hypothesizing loss of SPEN enhances reliance on the BCL6-HDAC3 axis featured in BN2 tumors, we treated both SPEN depletion models with a class-I HDAC inhibitor highly selective for HDAC3, BRD-3308, and indeed observed a 1.4 and 2.4-fold increase in sensitivity compared to WT cells (p=0.07, p&lt;0.01, respectively). Significance: We find SPEN trunc mutations in poor acting DLBCL cases with clinical and biologic characteristics that diverge from C1/BN2 biology. The enhanced rate of clinical progression highlights urgency to better characterize these tumors. Our data suggests loss of SPEN regulatory mechanisms may act cooperatively with C1/BN2 biology to facilitate tumor immune evasion and drive progression through Notch-mediated pathways. Importantly, in vitro evidence shows SPEN deficient lymphoma cells are susceptible to targeted therapeutics against dependent pathways, providing rationale for advancing individualized care strategies for affected patients.
32
Skuli, Sarah, A'Ishah Bakayoko, Gerald Wertheim, et al. "The Mevalonate Pathway Is a Therapeutic Target in TP53 Mutant Acute Myeloid Leukemia." Blood 142, Supplement 1 (2023): 408. http://dx.doi.org/10.1182/blood-2023-185059.
Full textAbstract:
Introduction: Acute myeloid leukemia (AML) with mutations in the TP53 tumor suppressor gene is the most fatal of AMLs with a median overall survival of only six months due to chemotherapy resistance. To understand biologic differences in TP53 mutant (MT) AML, as compared to wildtype (WT) AML, we performed transcriptomic analysis on sorted patient samples. Gene set enrichment analysis demonstrated significant upregulation of the cholesterol biosynthesis or mevalonate pathway in TP53 MT AML. In TP53 MT solid tumor models, the mevalonate pathway plays a key role in tumorigenesis and metastasis. We hypothesized that the mevalonate pathway is essential for chemotherapy resistance of TP53 MT AML and represents a novel therapeutic target. Methods: We developed chemoresistant, isogenic TP53 MT AML cell lines using CRISPR/Cas9 technology in the MOLM14 AML cell line. We used primary TP53 MT AML patient samples to perform colony forming unit (CFU) assays, in vitro assays with a serum substitute and cytokines, and a patient derived xenograft (PDX) model of TP53 MT AML. Results: We first sought to determine if TP53 MT AML exhibits upregulation of mevalonate pathway activity in response to cytarabine (AraC), the backbone of AML therapy. We treated our isogenic TP53 MT AML cell lines for 24 hours (h) with AraC and assessed mevalonate pathway gene expression and metabolites using qRT-PCR and liquid chromatography high resolution mass spectrometry (LC-HRMS), respectively. Only TP53 MT cell lines respond to AraC with a significant upregulation of key mevalonate pathway genes and metabolites. We then determined if TP53 MT AML is sensitized to AraC by inhibition of the mevalonate pathway with a statin. We pretreated our isogenic cell lines with 24h rosuvastatin before 24h AraC and assessed cell viability with flow cytometry with AnnexinV/7AAD staining and XTT assays. We also performed CFU assays in TP53 MT AML patient samples treated with the two drugs alone or in combination and assessed CFUs after 14 days. These studies revealed a synergistic reduction in cell viability and CFUs in TP53 MT AML by rosuvastatin in combination with AraC. We next addressed the mechanism by which a statin sensitizes TP53 MT AML to AraC. Our group and others have demonstrated enhanced mitochondrial activity is associated with AML chemoresistance. We hypothesized that mevalonate pathway byproducts, known to be crucial for mitochondrial functions, contribute to the mitochondrial response to AraC. We treated TP53 MT AML cell lines as above with rosuvastatin and AraC and used seahorse technology to measure oxidative phosphorylation (OXPHOS). We also used electron microscopy, flow cytometry of mitochondrial protein, TOM20, and qRT-PCR for mitochondrial DNA content to quantify and characterize mitochondria. These data demonstrate that TP53 MT AML cell lines, compared to WT, exhibit a significant increase in OXPHOS after 24h that is due to an increase in total mitochondrial. These effects are abrogated by pretreatment with a statin. We also validated these findings in primary TP53 MT AML patient samples in vitro. Importantly, co-treatment of the isogenic TP53 MT AML cell lines with a soluble form of mevalonate or a downstream byproduct, geranylgeranyl pyrophosphate (GGPP), recovered OXPHOS and subsequent chemoresistance. Overall, this data supports a model where TP53 MT AML cells dynamically upregulate the mevalonate pathway to regulate OXPHOS and avoid DNA damage-induced cell death (Figure 1). Finally, we studied a PDX model of TP53 MT AML. Engrafted mice were treated with AraC for five days alone or in combination with high dose rosuvastatin. Mice were harvested on day eight with assessment of leukemic burden by flow, OXPHOS by seahorse and mevalonate byproducts by LC-HRMS. TP53 MT AML leukemic burden was only significantly reduced by the combination therapy. Consistent with in vitro findings, mice sacrificed three days after completing AraC continue to demonstrate enhanced OXPHOS and increased mevalonate byproducts. This response is blunted by rosuvastatin. Conclusions: These results demonstrate that TP53 MT AML requires the mevalonate pathway for chemotherapy resistance and targeting of this pathway in combination with chemotherapy may improve clinical responses. Analysis is ongoing to determine the role of GGPP in regulating the mitochondrial response to DNA damage in TP53 MT cells in order to elucidate novel therapeutic approaches.
33
Coutinho de Oliveira, Beatriz, Saaurav Bari, Jessica Kerr, et al. "Abstract 3807: Validation of a functional genomics screen based on lentiviral integration site analysis reveals single-gene disruptions enhancing CAR19 T-cell effectiveness in preclinical models." Cancer Research 85, no. 8_Supplement_1 (2025): 3807. https://doi.org/10.1158/1538-7445.am2025-3807.
Full textAbstract:
Abstract Anti-CD19 CAR (CAR19) T-cell therapy has revolutionized the treatment for relapsed and refractory B-cell malignancies, showing long-term efficacy and potential cures. However, while many patients, including those with Chronic Lymphocytic Leukemia (CLL), exhibit initial signs of response, nearly two-thirds will eventually relapse. This highlights the critical need to understand treatment failure mechanisms and to identify targets to improve therapies. Our team previously reported an exceptional response in a CLL patient treated with CAR19 T-cells, which coincided with significant expansion of a CD8+ CAR T-cell clone harboring a disruption of the TET2 gene from an integration event of the lentiviral construct. Building on this finding, we conducted a large study analyzing lentiviral vector integration sites (LVIS) in 40 CAR19-treated Acute Lymphoblastic Leukemia (ALL) and CLL patients. Consistent with other studies, our data revealed a correlation between LVIS and therapeutic outcomes. To test whether these vector-mediated gene disruptions influence CAR T-cell growth kinetics and anti-tumor efficacy, we designed a CRISPR library targeting 180 LVIS-associated genes using 4-8 guide RNAs (gRNAs) per gene, including positive and negative controls. Our custom vector was engineered to drive the expression of both the anti-CD19 CAR and gRNAs under the promoters EF1a and human U6, respectively. To generate these modified CAR T-cells, we first transduced healthy donor T-cells at low multiplicity of infection, ensuring single-gene edits per cell, purified for CAR positivity three days post-transduction, and expanded cells for additional 10 days. The impact of each gene edit on CAR T-cell fitness was assessed in our well-established in vitro repeat tumor challenge and an in vivo xenogeneic model using the aggressive leukemia cell line NALM6. In both scenarios, we identified several genes with increased frequency (Log2FC ≥ 0.25, FDR ≤ 0.1) upon Robust Rank Aggregation (RRA) analysis using the MAGeCKFlute package in R, suggesting a potential fitness advantage. To validate the top candidates from our screen, we utilized Cas9-ribonucleoprotein (RNP) complexes with 2 independent gRNAs compared to AAVS1 (safe-targeting) and wild type controls. The targeted disruptions of the genes PCNX1 and PDCD10, among others, exhibited superior proliferative capacity upon our in vitro assay compared to controls. Ongoing studies include in vivo validation, phenotypic analyses of all candidates through spectral flow cytometry, and RNA sequencing to elucidate the biological roles of these genes in T-cell function. This preclinical package has the potential to advance into a Phase 1 clinical trial, where our optimized CAR T-cell product will be tested in CLL and potentially other refractory/relapsed B-cell malignancies. Citation Format: Beatriz Coutinho de Oliveira, Saaurav Bari, Jessica Kerr, Victoria Putzbach, Yee Peng Phoon, Joseph A. Fraietta, Wei-Ting Hwang, Frederic D. Bushman, Jan Joseph Melenhorst. Validation of a functional genomics screen based on lentiviral integration site analysis reveals single-gene disruptions enhancing CAR19 T-cell effectiveness in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3807.
34
Isabelle, Colleen, Amy Boles, Kathleen McConnell, et al. "Pathobiology and Targeting of CD38 in Cutaneous T-Cell Lymphoma." Blood 142, Supplement 1 (2023): 1650. http://dx.doi.org/10.1182/blood-2023-189556.
Full textAbstract:
Introduction: Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature CD4+ T-cells that primarily affects the skin. Despite a wide variety of topical and systemic therapies for patients, CTCL remains difficult to treat. Because of its high likelihood of relapse and resistance, new therapeutic approaches are needed. We recently showed strong CD38 expression on neoplastic T-cells from patients with mature T-cell neoplasms, including CTCL. In this study, we evaluated i) the role of CD38 in CTCL pathogenesis ii) strategies to enhance CD38 expression in CTCL and iii) tested efficacy of combination therapy in vivo. Methods: Skin and peripheral blood samples from patients and healthy donors were evaluated for CD38 expression by immunohistochemistry (N=7), microarray (N=82), single-cell RNA-sequencing (N=11), and flow cytometry (N=14). To investigate the role of CD38 in CTCL, we generated CD38 knockout cell lines (H9, HH, and HuT-78) using CRISPR/Cas9-mediated genomic deletion. The CD38 wild-type (CD38 WT) and knockout (CD38 KO) cells were purified, cultured, and used for downstream functional analysis. We evaluated the lines for growth, metabolic capacities (XFe24 Seahorse assay), and engraftment potential in vivo. For in vivo therapeutic studies, we transduced firefly-luciferase gene into H9 CD38 WT and CD38 KO cells, and engrafted them intravenously into immunodeficient NOD Rag -/-γc -/- mice. Mice were treated with anti-CD38 antibody daratumumab or IgG control and disease progression was monitored over time using an in vivo imaging system. For combination studies, we evaluated CD38 expression by flow cytometry in H9 cells treated with increasing doses of HDAC inhibitor, panobinostat (5nM, 10nM, 25nM) for 24, 48, and 72 hours. Next, we tested co-treatment with panobinostat and daratumumab in vivo in mice engrafted with H9 CD38 WT cells with four treatment groups (vehicle/IgG, panobinostat/IgG, vehicle/daratumumab, and panobinostat/daratumumab). Results: Patient skin biopsies showed increased CD38 expression at both protein and RNA levels (log fold change 4.8; p&lt;0.0001 by Mann-Whitney test). Across nine days of tracking under standard cell culture conditions, CD38 KO CTCL cells showed minimal growth differences compared to CD38 WT (p=0.22 by linear regression). However, CD38 KO CTCL cells showed significantly increased basal and maximum oxygen consumption rates (OCR) via seahorse assay when compared to CD38 WT cells (basal OCR 2.26 fold increase, p&lt;0.0001; max OCR 1.83 fold increase, p&lt;0.0001 by Mann-Whitney test). Using IVIS luminescent imaging, we found significantly increased in vivo growth of CD38 KO CTCL cells compared to CD38 WT cells (CD38 WT=2.2e8 photons/sec average total flux, N=7; CD38 KO=1e9 photons/sec, N=5; p=0.003 by Mann-Whitney test). These data suggest that the loss of CD38 in CTCL cells can accelerate the development of tumors in mice. In order to study the effectiveness of a targeted drug against CD38 in CTCL, we used anti-CD38 antibody daratumumab in our H9 xenograft model. Daratumumab treatment had a significant therapeutic effect on CD38 WT tumors compared to isotype control, reducing tumor burden by 84% (daratumumab average total flux=1.4e7 photons/sec, N=4; IgG average total flux=9.0e7 photons/sec, N=3; p=0.0002). Additionally, we explored methods to increase CD38 expression on CTCL cells. We found that panobinostat significantly increased CD38 expression on H9 CTCL cells in a dose dependent manner across multiple time points (max 85% increase in CD38 expression with 25nM panobinostat vs. DMSO at 72 hours; p&lt;0.0001 by 2way ANOVA). In vivo testing demonstrated a statistically significant improved survival benefit in mice treated with the combination of panobinostat and daratumumab compared to mice that received daratumumab alone (vehicle median survival 23 days, N=4; daratumumab alone median survival 32 days N=4; panobinostat alone median survival 27.5 days, N=4; combination median survival 39 days, N=4; p=0.01 by log-rank test). The prolonged average survival benefit amounted to approximately one quarter of their entire overall lifespan. Conclusion: Our studies demonstrate strong evidence for further study into how CD38 regulates the growth and survival of CTCL cells. Our data also provide preliminary evidence for the clinical usefulness of combination therapies that increase CD38 expression to enhance tumor cell immunotherapeutic targeting.
35
Aruljothi, Charlesantony, Subin S. George, Patrick Somers, et al. "Regulation of Ribosomal RNA Synthesis in Myeloid Progenitors By Cell Type Specific Transcription Factors." Blood 136, Supplement 1 (2020): 11–12. http://dx.doi.org/10.1182/blood-2020-138776.
Full textAbstract:
Haematopoiesis relies on the ability of hematopoietic stem cells to progress through a systematic hierarchy to produce lineage-restricted progenitors that terminally differentiate into phenotypically distinct types of mature hematopoietic cells. This process is precisely coordinated by the combinatorial activity of lineage-specifying transcription factors (TFs). Indeed, the critical transcriptional program of every hematopoietic cell type, and indeed of all cell types throughout the body, requires a set of core TFs for its proper execution. A frequently-overlooked component of the cellular transcriptional program is the transcription of ribosomal RNA (rRNA), the major component of ribosomes. Ribosomal RNA comprises 90% of total cellular RNA, and its transcription from hundreds of rDNA genes by RNA polymerase I is one of the most intense transcriptional processes in the cell. Different progenitor and mature cell types in the hematopoietic tree have different sizes, ribosome abundances, and rates of protein synthesis. Tight control of ribosome abundance is essential for a normal cellular proteome, and different cell types within the hematopoietic tree have varied rRNA transcription rates. However, there has been limited study of the molecular basis of lineage-specific regulation of rDNA transcription in hematopoiesis. We explored the binding to rDNA of over twenty key hematopoietic TFs that were determined by the Broad Institute DepMap database to be crucial for survival of hematopoietic cell lines. Using a customized bioinformatics pipeline, we mapped over three hundred ChIP-Seq datasets for these factors (generated by us as well as publicly available from ENCODE and GEO) to mouse and human rDNA assemblies, and found that several essential hematopoietic TFs such as MYC, MYB, RUNX1, PU.1, CEBPA and others bind to rDNA at conserved sites and motifs (Fig A). MYC is well-known as a master regulator of rDNA, and RUNX1 was recently reported to bind rDNA, but most of the others have never been linked to rDNA, and their functional roles in regulating rDNA transcription have not been explored. We picked for further study CEBPA, a crucial TF required for specification of granulocyte-monocyte progenitors (GMPs). For our experiments, we used the mouse HOXA9-ER cell line, which mimics GMPs. We used CRISPR/Cas9 and homologous recombination to fuse FKBPV degron into bi-allelic endogenous loci of the Cebpa gene in HOXA9-ER cells, and, upon addition of dTAG-13 (the ligand for FKBPV), the CEBPA-FKBPV fusion protein could be rapidly degraded within 2 hours (Fig B, C), providing us an experimental system to study the immediate consequences of CEBPA loss. In order to quantify the rate of rRNA transcription, we devised an assay titled "47S FISH-Flow" that combined fluorescent in-situ hybridization (FISH) using probes against nascent 47S rRNA with flow cytometry (Fig D, E). This assay not only allows us to quantify the rate of rRNA transcription on a per-cell basis in millions of cells, but also allows us to separately gate and quantify rRNA transcription in different stages of the cell cycle, eliminating a major confounder in bulk cell studies - cell cycle distribution. Using 47S FISH-Flow, we observed that degradation of CEBPA in the HOXA9-ER mouse GMP cell line led to decrease in synthesis of 47S rRNA within hours (Fig F) before any change in cell cycle or growth kinetics, and was followed by growth arrest in 24 hours. In summary, we show that several critical hematopoietic TFs show abundant, conserved binding to rDNA, and the depletion of CEBPA rapidly reduces nascent rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by a wide range of lineage-specific hematopoietic TFs. Figure Disclosures No relevant conflicts of interest to declare.
36
Barwe, Sonali P., Meredith Tavenner, Patrick Van Berkel, et al. "Identification of DLK1 As a Novel Therapeutic Target in Down Syndrome Myeloid Leukemia." Blood 144, Supplement 1 (2024): 209. https://doi.org/10.1182/blood-2024-211364.
Full textAbstract:
The current standard-of-care for myeloid leukemia associated with Down syndrome (ML-DS) relies on cytotoxic chemotherapy. Although primary ML-DS has favorable prognosis, the 10-20% of patients who develop relapsed leukemia or have refractory disease show a 3-year event-free survival less than 21% (Raghuram et al., 2023). Novel treatment options are needed to improve outcome in these patients with relapsed or refractory disease. The genomic landscape of ML-DS has been characterized by multiple studies (Labuhn et al., 2019; Nikolaev et al., 2013; Sato et al., 2024; Yoshida et al., 2013). In addition to trisomy 21, ML-DS samples possess mutations in the major hematopoietic transcription factor GATA1 and co-operating mutations in three major classes of proteins - cohesin complex components, epigenetic modulators and signaling molecules. However, there are no studies on differential gene expression analysis in ML-DS samples compared to normal bone marrow cells. We have generated ML-DS patient-derived xenograft (PDX) models using primary bone marrow samples from patients with ML-DS (Barwe et al., 2019). We conducted transcriptome analysis using these PDX models (n=4) supplemented with an ML-DS cell line (CMK) to identify differentially expressed genes in ML-DS compared to CD34+ cells isolated from normal bone marrow specimens (n=4). Filtering the differentially expressed genes to shortlist cell surface resident proteins, DLK1 was revealed as one of the top ten targets overexpressed in ML-DS. Analysis of a larger dataset also showed that DLK1 is overexpressed in ML-DS. In this dataset, the mean DLK1 TPM scaled counts in ML-DS bone marrow samples (257.20±73.81, n=77) were significantly greater than the normal bone marrow specimens (2.23±0.24, n=68, p&lt;0.0001). DLK1 expression on the cell surface of ML-DS cell and PDX lines was confirmed by flow cytometry. To determine the role of DLK1 in ML-DS, we generated CMK cells with CRISPR/Cas9 mediated DLK1 knockout. The percentage of EdU positive cells determined by flow cytometry was significantly reduced by 67% and 65% in two distinct knockout clones (P&lt;0.01), indicating that DLK1 indcues ML-DS cell proliferation. When injected in NSG-SGM3 mice, the CMK cells with DLK1 knockout showed minimal engraftment with less than 0.1% human cells in bone marrow, while the mice injected with wild-type CMK cells showed 63% human cell population 5 weeks post cell injection (P&lt;0.001, n=3 each). Consistent with low bone marrow load, the median survival of mice engrafted with DLK1 knockout cells was significantly longer than those with CMK wild-type cells (P&lt;0.05, n=5 each). Thus, our results demonstrate that DLK1 knockout in ML-DS cells suppressed cell proliferation and delayed in vivoengraftment in the bone marrow. We used DLK1-targeting antibody drug conjugate (ADCT-701, referred to as DLK1-ADC) with a DNA intercalating agent pyrrolbenzodiazepine. Isotype control antibody (Iso-ADC) with the same payload was used as control. While DLK1-ADC failed to induce cell death in DLK1 knockout cells, the IC50 for CMK wild-type cells was 0.027 nM. CMK cells with shRNA mediated DLK1 knockdown showed increased IC50 (0.172 nM). Iso-ADC did not have a significant effect on cell viability in any of these lines within the tested concentration range. These results highlight the target-dependent specificity and efficacy of DLK1-ADC in inducing ML-DS cell death. DLK1-ADC also suppressed the viability and colony forming ability of CPCT-0010, a PDX model generated from a patient with refractory ML-DS. In this PDX model in vivo, DLK-1ADC showed a dose-dependent improvement in median survival by 17, 22.5 and 24 days respectively when treated with 0.25, 0.5 or 1 mg/Kg DLK1-ADC i.v. in comparison with Iso-ADC at the same dose (P&lt;0.05). DLK1-ADC dosed twice at 1 mg/Kg also prolonged survival in three distinct PDX models generated using primary cells from patients with refractory ML-DS. Finally, DLK1-ADC, but not Iso-ADC cured 2 of 3 mice injected with a refractory ML-DS PDX model while the third mouse showed an 84-day prolongation of survival compared to that of mice treated with Iso-ADC. Thus, taken together, we have identified DLK1 as a novel therapeutic target for refractory ML-DS and show that targeting DLK1 may be a novel treatment option for patients with refractory ML-DS.
37
Dahlgren, Anna R., Francesca Careddu, Raymond Adili, et al. "Identification of the Role of SEL1L in Platelet Function with Implications for Atypical Equine Thrombasthenia in Thoroughbred Horses." Blood 142, Supplement 1 (2023): 3944. http://dx.doi.org/10.1182/blood-2023-187146.
Full textAbstract:
Atypical Equine Thrombasthenia (AET) is a recessive heritable platelet disorder in horses due to aberrant platelet signaling after thrombin stimulation, preventing platelets from fully activating or efficiently binding to fibrinogen, leading to prolonged bleeding. To date, AET has only been identified in the Thoroughbred horse breed. A prevalence study performed at one breeding farm found that one in every 150 Thoroughbreds was affected. Despite the negative effects on horse health and racing performance, the underlying etiology of this unique platelet disorder is unknown. Here we show that a variant in SEL1L, a gene which encodes a protein known to play a pivotal role in endoplasmic reticulum-associated protein degradation (ERAD) and not previously known to be involved in platelet function, leads to AET. In order to identify the underlying genetic mechanism, we performed whole genome sequencing on five affected, one equivocal, and eleven control Thoroughbred horses. A whole-genome association study was conducted, and associated variants were filtered by determining if they were in or near genes known to be expressed in platelets. This evaluation identified three putative loci: a missense variant in SEL1L (p.Ile604Val), a 1.6kb deletion in a long non-coding RNA ( AL355838.1) and a 73bp intronic deletion in VIPAS39. The long non-coding RNA AL355838.1 did not show expression in horse platelets, and there was no differential expression of SEL1L or VIPAS39 mRNA identified between affected and unaffected individuals. However, a significant difference in SEL1L protein expression was observed in AET-affected horses' platelets. Flow cytometry and immunofluorescence studies demonstrated that SEL1L is located intracellularly in equine platelets, but not in the α-granule, and moves to the surface of the platelet upon activation with thrombin. Although the template bleeding time was normal, platelets from horses that were homozygous for the SEL1L variant showed defective spreading on collagen. To support SEL1L as the causative gene and dissect the underlying mechanisms, we undertook studies in three additional models. Differentiation of human megakaryocytes revealed the presence of two SEL1L protein isoforms, p100 and p38, and both showed increased expression during megakaryopoiesis, although only p100 was delivered to mature platelets. We perfused whole blood from Mx1-Cre +; Sel1l fl/fl conditional knockouts and controls through a collagen-coated microfluidic chamber. We found that significantly fewer platelets from knockout mice bound to the collagen as compared to control siblings. Finally, a knockout zebrafish line was developed by deleting exons 3-20 using CRISPR/Cas9 mediated genome editing. Similar to the mouse, laser-mediated arterial endothelial injury of 5-day old mutant larvae resulted in significantly fewer thrombocytes (the fish platelet equivalent) adhering to the injured vessel wall as compared to wild-type siblings. Overall, these data support the conclusion that the SEL1L missense variant leads to AET, and that SEL1L has a previously undescribed and conserved role in platelet function, particularly the ability of platelets/thrombocytes to adhere to exposed collagen at sites of endothelial injury. These results will allow the Thoroughbred industry to easily diagnose AET-affected horses and make appropriate decisions for their racing and breeding careers. These data also provide an excellent introduction to further investigation into the understudied and potential role of ERAD proteins in human platelet disorders.
38
Meyer, Tatjana, Nikolaus Jahn, Anna Dolnik, et al. "BRCA1/2 Containing Complex 3 (BRCC36) Is Recurrently Mutated in AML with t(8;21) and Associated with Increased Sensitivity to Chemotherapy through Impairment of the DNA Damage Repair Pathway." Blood 132, Supplement 1 (2018): 1487. http://dx.doi.org/10.1182/blood-2018-99-114143.
Full textAbstract:
Abstract Introduction BRCA1/BRCA2-containing complex 3 (BRCC36) is a Lys63-specific deubiquitinating enzyme (DUB) involved in DNA damage repair. Mutations in BRCC36 have been identified in 2-3% of patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). The role of BRCC36 mutations in de novo AML and their impact on DNA damage-inducing cytotoxic chemotherapy sensitivity is not clear. Aim We aimed to determine the incidence of BRCC36 mutations in AML and their impact on outcome and drug sensitivity in vitro. Methods We analyzed the entire coding region of BRCC36 for mutations in 191 AML cases with t(8;21) (q22;q22.1) and 95 cases with inv(16) (p13.1q22) using a customized targeted sequencing panel. Data for de novo AML was derived from The Cancer Genome Atlas Research Network (TCGA) data set (NEJM 2013). Lentiviral CRISPR/Cas9 was used to inactivate BRCC36 in t(8;21)-positive AML cell lines - Kasumi-1 and SKNO-1 - and murine hematopoietic stem and progenitor cells (LSKs). Knockout was confirmed by a cleavage assay as well as Western blot. AML1-ETO-9a was expressed by a retroviral vector. Cell lines and LSK cells were treated with different concentrations of doxorubicin or cytarabine and their viability was assessed seven days post treatment. DNA damage was assessed through phospho-γH2AX staining using flow-cytometry. Results BRCC36 mutations were identified in 7 out of 191 patients (3.7%) with t(8;21) AML and none of 95 patients with inv(16). In the TCGA data set one out of 200 patients (0.5%) with de novo AML had a BRCC36 mutation. This patient had a complex karyotype and would be considered as secondary AML with myelodysplastic-associated changes according to the 2016 WHO classification. Six of the 7 mutations were missense or nonsense mutations that were predicted to be deleterious to BRCC36 function. One mutation affected a splice site at exon 6, resulting in an impaired splicing capability. With intensive standard chemotherapy all patients with BRCC36 mutations achieved a complete remission and had an estimated relapse-free and overall survival of 100% after a median follow up of 4.2 years. Given its role in DNA damage repair, we hypothesized that BRCC36 inactivation sensitizes AML cells to DNA-damage inducing drugs. In order to test this, we generated BRCC36 knockout Kasumi-1 and SKNO-1 cell lines using CRISPR-Cas9. BRCC36 inactivation had no impact on cell growth on either of the cell lines. However, we found that BRCC36 knockout cells were significantly more sensitive to doxorubicin as compared to the parental cells with normal BRCC36. This was accompanied by a significant increase in DNA damage as assessed by phospho-γH2AX in BRCC36 knockout vs control cells after doxorubicin treatment. In contrast, BRCC36 inactivation had no impact on cytarabine sensitivity. We next assessed drug sensitivity in primary murine leukemic cells expressing AML1-ETO-9a. Again, inactivation of BRCC36 resulted in a significant higher sensitivity to doxorubicin but not cytarabine. Conclusion We found BRCC36 to be recurrently mutated in t(8;21)-positive AML Inactivation of BRCC36 was associated with impairment of the DNA damage repair pathway and thus higher sensitivity to DNA damage-inducing chemotherapy. This might be also reflected by the favorable clinical outcome of patients with BRCC36 mutated t(8;21)-positive AML, a finding which has to be confirmed in a large patient cohort. Disclosures Paschka: Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Otsuka: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Other: Travel support; Janssen: Other: Travel support; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Astex: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Bullinger:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding.
39
Ghobadi, Armin, Ibrahim Aldoss, Shannon L. Maude, et al. "WU-CART-007 (WT-7), an Allogeneic CAR T-Cell Targeting CD7 in Relapsed/Refractory (R/R) T-Cell Acute Lymphoblastic Leukemia/Lymphoma (T-ALL/LBL): Phase 2 Results." Blood 144, Supplement 1 (2024): 3450. https://doi.org/10.1182/blood-2024-202005.
Full textAbstract:
R/R T-ALL/LBL are challenging malignancies to treat with few treatment options and high rates of relapse and mortality. WT-7 is a CD7-targeted CAR T-cell product with CRISPR/Cas9 deletion of CD7 and T-cell receptor alpha constant (TRAC), to prevent fratricide and enable the use of healthy donor allogeneic T-cells (Leedom, et al. ASH 2021). We report time-to-event (TTE) update from the Phase 2 portion of WU-CART-007 1001 (NCT04984356), a Phase 1/2 study of WU-CART-007 in patients (pts) with R/R T-ALL/LBL, and a focus on subgroup analysis of RP2D. In the Phase 2 portion, pts received a single infusion of 900 million WT-7 cells on Day 1 following enhanced lymphodepleting (eLD) chemotherapy (fludarabine 30 mg/m2/day x 4 days and cyclophosphamide 1000 mg/m2/day x 3 days). Disease response was assessed by a Day 28 bone marrow (BM) assessment and a CT/PET, if applicable. Composite complete remission (CRc) was defined as combination of CR (BM blasts &lt;5% with absolute neutrophil count ≥1,000/μL and platelet count ≥100,000/μL) and CRi (CR with incomplete hematologic recovery), and SUV uptake &lt; than liver/mediastinum for those with extramedullary disease (EMD). Objective response rate (ORR) was defined as CRc plus partial response (PR; reduced uptake compared to baseline in EMD). Duration of response (DOR) is measured from initial response to relapse or death, follow up time is calculated from first dose to death or last follow date, and TTE endpoints are calculated using Kaplan-Meier statistics. Pharmacokinetics were measured by ddPCR andimmunophenotyping. MRD was measured centrally by flow cytometry, threshold &gt;0.01%. As of 24 Jul 2024, 13 pts were dosed, median age was 23 years (range 14 - 47). Pts were heavily pretreated with a median of 5 lines of therapy (range 1 - 9); 46% (6/13) had prior allogeneic hematopoietic stem cell transplant (allo-HSCT). Disease burden at baseline consisted of EMD in 38.4% (5/13), and a median BM blast percentage of 82.5% (range 5-95%) in pts with BM disease (8/13). Treatment-related adverse events of ≥ Grade (G) 3 were observed in 10/13 (77%) pts. Cytokine Release Syndrome (CRS) occurred in 13/13 (100%) pts; most (69%; 9/13) had G1-2 CRS events; four (31%) pts had G≥3 CRS, which was managed with steroids (75%), tocilizumab (100%), anakinra (25%). A single event of immune effector cell-associated neurotoxicity (G1) was reported in one pt (8%). Severe (≥ G3) infections were observed in 46% (6/13), including sepsis 31% (4/13), opportunistic viral infections, otitis externa, fungal pneumonia in 1 patient (8%) each. One case of G2 graft vs. host disease was reported. Two G5 events occurred: sepsis due to fungal infection on day 13, and an event of multi-organ failure occurred in the setting of G3 CRS and fulminant disease progression on Day 7 post infusion. ORR and CRc amongst evaluable pts were 91% (10/11) and 73% (8/11; 6 CR, 2 CRi), respectively. Minimum Residual Disease (MRD) data were available for 6 pts who achieved CRc, with 83% (5/6) achieved MRDneg CR. Five patients received consolidating allo-HSCT. With a follow-up time of up to 9.9 months (m), median DOR has not been reached (95% CI: 0.5, NE; range 0.5-9.1 m); 4 pts remain in continuous CR at 9.1, 8.2, 6.7, and 6.7 m. In 5 pts with EMD disease, a relatively more challenging population, the ORR was 80% (4/5; 2CR/2PR). For pts that achieved PR the decrease in total disease burden ranged from 78.5-95.7% (median 87.7%) by Lugano Criteria. Expansion of WT-7 cells peaked on Day 10 in the peripheral blood (median 237,827 copies/μg DNA), persisting out to Day 90. No pt tested developed novel anti-HLA or anti-drug antibodies against the CAR construct. A further analysis exploring the impact of MRD response was performed on all treated patients irrespective of dose level. A time-to-event Kaplan-Meier analysis showed median duration of response for pts with MRDneg CR/CRi (n=7) was 6.6 m (95%CI: 1.8, NE) vs. 3.7 m (95%CI: 0.5, NE) for those with CR/CRi MRDpos (n=3). Similar findings were identified for overall survival (OS), for pts that achieved MRDneg OS was 11.5 m (95%CI: 2.7, NE) vs. 6.1 m (95%CI: 1.4, NE) for MRDpos pts. WT-7 has demonstrated evidence of anti-leukemic activity with an acceptable safety profile in heavily pre-treated R/R T-ALL/LBL pts. A follow up study, NCT06514794, in R/R T-ALL/LBL including pediatric pts 1 year of age and older will begin enrolling in late 2024; exploratory MRDpos cohort may be initiated after safety is confirmed in the R/R setting.
40
Stopka, Tomas, Jarmila Vargova, Karina Vargova, et al. "Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Is a New Biomarker for Mantle Cell Lymphoma: Expression, Localization, and Phosphorylation Study." Blood 128, no. 22 (2016): 1767. http://dx.doi.org/10.1182/blood.v128.22.1767.1767.
Full textAbstract:
Abstract Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases. In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (NMCL=6, NCLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells. In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker. Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604. Disclosures No relevant conflicts of interest to declare.
41
Barrio, Santiago, Larissa Haertle, Umair Munawar, et al. "Clonal Competition Models to Understand Progression and Resistance in Myeloma." Blood 134, Supplement_1 (2019): 1807. http://dx.doi.org/10.1182/blood-2019-130812.
Full textAbstract:
Background: Progression and relapse in Multiple Myeloma (MM) is induced by changes in the clonal tumor composition. In order to better understand the mechanisms underlying these dynamics, we developed clonal competition models based on the co-culture of fluorescent labelled isogenic MM cells, with or without the alteration under study. Methods: To understand the effect of mono- and bi-allelic TP53 lesions, we use the AMO1 cell line, one of few myeloma cell lines harbouring wild type TP53 (WT). After modification with CRISPR / CAS9, we selected subclones with mono- and/or bi-allelic deletion of TP53. For the characterization of alterations in RAS, we selected OPM2 cells, one of the few lines with the RAS pathway intact. Furthermore, we generated the KRAS WT, G12A and A146T sublines by stable transfection with Sleeping Beauty vectors. To study mutations related to resistance to IMIDs and PIs, we introduced mutations in the target genes IKZF1 (WT, A152T, Q170D or R439H), CUL4B (KO), and PSMB5 (WT or A20T ) in AMO1 and L363, cell lines sensitive to IMiD or PI treatment. In addition, all WT and mutant sublines were also stably transformed with E-GFP or LSS-mkate2-RFP for flow cytometry analysis. Results: We recently demonstrated that lesions in TP53, both mono- and bi-allelic, induce a growth advantage to the affected cells. In the current study, we also observed an increased fitness in KRAS mutated cells (G12A or A146T vs WT) independent of treatment. We co-cultivated KRAS mutant with WT cells at a ratio 1:3 in two independent experiments, with the color labelling switched (red/green wt/mutant and vice-versa). KRAS G12A clone significantly expanded and reached 50% of the cells at day 40. Likewise, A146T clone outcompeted WT cells, but the time required to represent the majority of cells in the coculture was longer. We next explored the effects of resistance mutations and drug exposure. Both the IKZF1 A152T and CUL4B KO mutants outcompete WT cells in the presence of Lenalidomide (LEN). The same effect was observed for the PSMB5 A20T mutant exposed to Bortezomib (BOR). This selection ("Survival Fitness") did not occur without the presence of the drug. Thus, resistance related mutations seem only to provide a fitness advantage under drug exposition. In addition, both the CUL4B KO and PSMB5 A20T mutants were overcome by WT cells when the drug was removed from co-culture, suggesting that these lesions provide a survival disadvantage without the selective pressure of IMiD or PI. This may provide an explanation for the low mutation rate in this gene in recent sequencing publications, as usually samples are not obtained under selective pressure but in treatment free intervals. IKZF1 mutations outside the IMiDs / CRBN binding area (Q170D and R439H) provided no advantage to the cells. Conclusions: Our clonal competition assays provide novel insights on the impact of point mutations on the fitness of affected myeloma subclones, either with or without the selective pressure of therapy. Figure Disclosures Martinez-Lopez: Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; F. Hoffmann-La Roche Ltd: Honoraria; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; BMS: Honoraria, Other: Advisory boards; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria.
42
Schimpf, Marlise L. Guerrero, Courtney C. Sparger, Hsuan-Ting Huang, Dean Wade, and Maria E. Figueroa. "Abstract A37: PDZD2 is essential for steady-state hematopoiesis and its 37-kDa secreted product, sPDZD2, functions as a soluble tumor suppressor in AML." Blood Cancer Discovery 4, no. 3_Supplement (2023): A37. http://dx.doi.org/10.1158/2643-3249.aml23-a37.
Full textAbstract:
Abstract PDZ domain-containing protein 2 (PDZD2) is almost universally silenced through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein that it is cleaved into a 37kDa protein that is secreted to the tissue microenvironment (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using ELISA. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected lower levels in AMLs. In addition, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition, upregulation of differentiation markers (CD15/CD11b) and cell cycle arrest in G0/G1. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot. In vitro treatment with r-sPDZD2 led to inhibition of CREB(S133) phosphorylation, as well as increased GSK3b(S9), RSK1/2(S221/S227) and p53(S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a and CD7. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts and hemoglobin levels, an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML. Citation Format: Marlise L Guerrero Schimpf, Courtney C Sparger, Hsuan-Ting Huang, Dean Wade, Maria E. Figueroa. PDZD2 is essential for steady-state hematopoiesis and its 37-kDa secreted product, sPDZD2, functions as a soluble tumor suppressor in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A37.
43
Mishima, Yuji, Jiantao Shi, Michele Moschetta, et al. "In Vivo Analysis of Clonal Evolution of Multiple Myeloma." Blood 128, no. 22 (2016): 799. http://dx.doi.org/10.1182/blood.v128.22.799.799.
Full textAbstract:
Abstract Introduction According to the clonal evolution model, tumor progression proceeds in a branching rather than in a linear manner, leading to substantial clonal diversity and coexistence of genetically heterogeneous sets of subclones. Unlike many cancers, in which the evolutionary history can only be inferred from the established disease, Multiple Myeloma (MM) has well defined precursor states, which offer a unique opportunity to study the sequential evolution of the disease. In MM, multiple subclones can co-exist because they are of similar fitness, potentially interact with each other or with the surrounding microenvironment and further disseminate to spatially separate areas of the bone marrow (BM). Therefore, in order to accurately predict the course of the disease, we require methods to estimate clone-specific growth rates within the BM and define clones that have the propensity of dissemination. Methods We developed a MM metastatic xenograft model by performing tumor-bearing bone chip implantation to SCID-beige mice (SCID-murine model) and examining tumor clones that are present in the implanted bone chips (primary sites) compared to those present in the distant BM sites (metastatic sites). To obtain a perspective of clonal heterogeneity in vivo, we used the "rainbow" system by which fluorescent proteins were infected into cells using lentiviruses to label the cells with 15 distinctive fluorescence profiles (rainbow MM cells). Rainbow MM cells with equal proportion of all 15 colors were injected into donorfemurs and implanted into recipient mice. After paralysis, the mice were sacrificed and tumor cells were analyzed using flow cytometry and confocal microscopy. To further investigate the dynamics of heterogeneity at the single cell level, similar experiments were performed using a DNA-barcode library. For genomic and transcriptomic characterization, primary and metastatic tumor clones were purified by sorting and underwent whole exome and RNA sequencing. To identify key regulators of the metastatic process, we conducted in vivo CRISPR library screening of the most critical targets identified. Briefly, the MM cell library was prepared by transduction of sgRNAs targeted for 20 genes and control sgRNAs to MM.1S cells stably expressing Cas9. The cell library was used in SCID-murine model and the fractions of each sgRNA were calculated in the primary and metastatic sites to identify genes that facilitate tumor metastasis. Results We found that the 15 rainbow subpopulations were present with equal distribution in the primary sites but not at the metastatic sites. Specific subclones (winner clones) had a greater advantage of growing in the metastatic site. Interestingly, the winner clones were similar between the bilateral femurs of most of the mice, suggesting the existence of potential metastatic subclones. Experiments using DNA-barcoding further demonstrated that single clones could become disproportionately present in the metastatic sites, even though they account for a smaller fraction of the primary tumors. Confocal imaging showed the difference in cluster structures between primary and metastatic tumors. Most of the clusters in the metastatic sites consisted of cells of single colors. RNA sequencing analysis of two human MM cell lines derived from these mouse models demonstrated a distinct gene expression profile of the metastatic tumors compared to the primary sites. By intersecting differentially expressed genes, we identified 110 shared up-regulated genes and 238 shared down-regulated genes, which we designated as the "metastatic signature". Gene Set enrichment analysis of the metastatic signaturein publicly available MM patient datasets (GSE6477 and GSE2658) demonstrated that this signature significantly correlated with overall survival and with clinical progression from MGUS/smoldering MM to overt myeloma and relapsed disease. Finally, the CRISPR in vivo screening prioritized two transcription factors as the key regulatory molecules, namely EGR3 and ATF3. Conclusions Here, we demonstrate that in vivo clonal evolution can be characterized using an in vivo model of MM. The data defines specific subclones that have a higher metastatic potential and are likely driver clones for tumor metastasis in MM. On the molecular level, a metastatic gene signature was found and two genes were identified as potential regulator of MM metastasis. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Hatake:Chugai: Research Funding; Meiji-Seika: Consultancy; Kyowa Kirin: Honoraria, Research Funding; Otsuka: Consultancy. Scadden:Dr. Reddy's: Consultancy; Bone Therapeutics: Consultancy; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlaxoSmithKline: Research Funding. Ghobrial:Amgen: Honoraria; BMS: Honoraria, Research Funding; Noxxon: Honoraria; Takeda: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria.
44
Cai, Shuyang, Honghu Li, Ruxiu Tie, Qian Luo, He Huang, and Jiahui Lu. "Clec16a-Mediated Mitophagy Modulates Zebrafish Definitive Hematopoiesis." Blood 144, Supplement 1 (2024): 30. https://doi.org/10.1182/blood-2024-198955.
Full textAbstract:
Hematopoietic stem and progenitor cells (HSPCs) generate all blood cell lineages throughout the lifespan of vertebrates. The emergence of HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process, which is finely regulated by a variety of signaling pathways. Previous studies have highlighted the essential roles of pattern-recognition receptors such as Toll-like receptors, RIG-I-like receptors, and NOD-like receptors in EHT. However, whether members of the C-type Lectin Receptors (CLRs) family participate in vertebrate embryonic hematopoiesis remains unclear. To explore the potential pathways involved in hematopoietic ontogeny and differentiation during this stage, we screened the dynamic expression of CLR family genes and other inflammation-related genes in published scRNA-seq data from vertebrate embryos, including zebrafish, mouse, and human, during HSPC development. To verify the expression of CLR family genes in vitro, we used the HSC-like cell differentiation system of mouse and human embryonic stem cells (ESCs). These results suggest that Clec16a is highly expressed during embryonic HSPC development both in vivo and in vitro and may play an important role in the emergence of HSPCs in vertebrates. In order to validate this hypothesis, we utilized the well-established zebrafish model for studying embryonic hematopoiesis to conduct in vivo functional experiments. First, we synthesized probes from the full-length mRNA of clec16a and utilized them to observe the in-situ expression of clec16a in zebrafish at different developmental stages. The whole-mount in situ hybridization (WISH) experiment demonstrated that clec16a was expressed from the 1-cell stage and, importantly, showed specific expression in the AGM region at the 24-28hpf, which coincides with the onset and generation of HSPCs. Then, morpholinos were utilized to knockdown clec16a in transgenic embryos Tg(runx1/kdrl), Tg(cmyb/kdrl), and Tg(CD41). Then mutants generated by CRISPR-Cas9 were used to perform WISH and qPCR to detect specific markers such as runx1, cmyb (HSPCs), rag1 (lymphocytes), gata1a (erythrocytes), and l-plastin (myelocytes). The results showed that clec16a was required for HSPC emergence, expansion, and differentiation. Mechanistically, we sorted EGFP+ cells in Tg (fli1a:EGFP) zebrafish embryos at 26 hpf to perform RNA-Seq, which was the stage where EHT and these EGFP+ cells contained HEs and HSPCs. After bioinformatics analysis of sequencing data and considering published reports, we found that mitophagy in HEs and HSPCs was inhibited in clec16a-deficient zebrafish embryos. Transmission electron microscopy showed that clec16a knockdown led to a reduction in mitochondria in HEs, with disrupted outer membranes, damaged cristae structures, and enlarged intermembrane spaces. Furthermore, immunofluorescence and flow cytometry revealed elevated levels of mitochondrial reactive oxygen species (ROS), and treatment with mitochondrial antioxidants could reduce ROS levels while restoring hematopoiesis. These findings suggest that clec16a regulates EHT and HSPC generation and differentiation through mitophagy. Furthermore, combined with transcriptomic sequencing and proteomics, we discovering that the key mitophagy gene prkn might play a crucial role. Rescue experiments showed that overexpression of prkn could ameliorate the phenotypes caused by inhibited mitophagy, reduce ROS levels, and restore the hematopoietic defects in clec16a-deficient embryos. Therefore, we propose the existence of a clec16a-prkn-ROS regulatory axis that is essential for the development and differentiation of HSPCs during zebrafish embryogenesis. In summary, our work is the first to identify the role of the CLRs family gene clec16a in zebrafish embryonic hematopoiesis. We elucidate the mechanism by which clec16a-mediated mitophagy regulates embryonic hematopoiesis through ROS, highlighting the critical role of the mitophagy protein prkn in this process. This study not only provides new theoretical insights into the complex biological process of HSPC emergence, offers a novel perspective on the regulatory network between the innate immune system and embryonic hematopoiesis, but also proposes new approaches for the in vitro cultivation and expansion of HSCs.
45
Couch, Tyler A., Zachary C. Murphy, Michael Getman, Ryo Kurita, Yukio Nakamura, and Laurie A. Steiner. "Human Erythroblasts with c-Kit Activating Mutations Have Reduced Cell Culture Costs and Remain Capable of Terminal Maturation and Enucleation." Blood 132, Supplement 1 (2018): 2315. http://dx.doi.org/10.1182/blood-2018-99-117157.
Full textAbstract:
Abstract There is a constant need for red blood cells for transfusion therapy in the treatment of anemias and acute injury. As all blood products for transfusion come from donors, there are concerns over shortages and safety. Furthermore, many patients with transfusion-dependent anemias risk alloiumminization. The in vitro production of red blood cells would address these problems, especially as they can be genetically engineered to prevent alloimmunization. Numerous erythroid culture systems now exist for the in vitro production of red blood cells. Hematopoietic stem and progenitor cells (HSPCs) obtained from umbilical cord or peripheral blood can be differentiated into erythrocytes, however, they are limited in expansion. While umbilical cord HSPCs have greater expandability than peripheral blood, the resulting erythrocytes contain fetal globins. Pluripotent stem cells can also be used as a starting source, however only a small percentage of the cells can be differentiated into erythroblasts which also suffer from low enucleation rates. Presently, the cost of in vitro production of a unit of red cells is greater than an order of magnitude higher than obtaining it from a donor largely due to the medium and cytokine costs (Timmins & Nielsen, Trends Biotechnol, 2009). A relatively new approach of immortalizing early erythroblasts allowing unlimited expansion as well as terminal maturation and enucleation shows great therapeutic promise (Kurita et al., PLoS One, 2013; Huang et al., Mol Ther, 2014; Trakarnsanga et al., Nat Commun, 2017). However, these immortalized erythroblasts are still reliant on two costly cytokines: stem cell factor (SCF) and erythropoietin (Epo). Mutations in exon 17 of the receptor tyrosine kinase gene KIT are frequently seen in acute myeloid leukemias, gastrointestinal stromal tumors, and mast cells leading to mastocytosis. These mutations cause the c-Kit protein to spontaneously activate and transduce signal in the absence of SCF (Kit-ligand). To generate an SCF-independent HUDEP-2 cell line (Kurita et al., PLoS One, 2013), we used CRISPR/Cas9 to introduce missense and frameshifting mutations within the vicinity of Asp816 in exon 17 of the KIT gene. The resulting monoclonal cell lines were selected for by removing SCF from the expansion medium and were subsequently named KIT-CAT (KIT with Constitutively Activating Transformation). To better understand what KIT mutations allowed or impaired terminal maturation, monoclonal cell lines were genotyped by Sanger sequencing. Three cell lines with unique genotypes were chosen for further analysis. All three KIT-CAT lines had a shorter doubling time compared to HUDEP-2 cells (16.7 vs 18.9 hrs, p=0.020) and were no longer dependent on SCF or Epo. However, two of the three KIT-CAT lines showed more robust proliferation with Epo in the expansion medium. The addition of SCF to the medium caused no increase in c-Kit activation by Western blotting for phosphorylation at Tyr703. Furthermore, the low molecular weight and immature form of c-Kit is also phosphorylated in KIT-CAT cells, but not HUDEP-2 cells, indicating c-Kit activation occurs before trafficking to the cell membrane where SCF would bind (Tabone-Eglinger et al., Clin Cancer Res, 2008). Key features of erythroblast maturation are the decrease in cell and nuclear size which can be measured using imaging flow cytometry (McGrath et al., Methods, 2017). While in expansion phase, all 3 cell lines were larger in cell and nuclear area compared to the parental HUDEP-2 line. By day 6 of maturation, all three cell lines had statistically significant decreases in cell and nuclear size indicating maturation. By day 13 of culture, Wright-Giemsa staining showed that the majority of the cells were orthochromatic erythroblasts or enucleate reticulocytes. Reducing cell culture costs is needed for in vitro manufacturing of red blood cells to be economically feasible. These results show that a c-Kit activating mutations in human erythroblasts removes the cost of SCF and reduces the cost of Epo while still allowing for terminal maturation and enucleation. Disclosures No relevant conflicts of interest to declare.
46
El Hoss, Sara, Sylvie Cochet, Auria Godard, et al. "Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease." Blood 136, Supplement 1 (2020): 14–15. http://dx.doi.org/10.1182/blood-2020-137477.
Full textAbstract:
Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.
47
Guruprasad, Puneeth, Alberto Carturan, Yunlin Zhang, et al. "Modulation of the Btla-HVEM Axis to Enhance CAR T Cell Immunotherapy Against Cancer." Blood 142, Supplement 1 (2023): 768. http://dx.doi.org/10.1182/blood-2023-189633.
Full textAbstract:
Introduction: The efficacy of adoptive T cell immunotherapies against cancer, such as chimeric antigen receptor (CAR) T cells, is severely blunted by the immunosuppressive tumor microenvironment (TME). We sought to investigate the role of the TME in cancer resistance to effector T cells in order to define actionable targets to enhance CAR T cell immunotherapies. We initially used Hodgkin lymphoma (HL) as an ideal tumor model since it is characterized by a TME that is profoundly infiltrated by immunosuppressive cells, and then we expanded our findings to multiple cancer models. We first sought to identify the dominant interactions of immunosuppressive cellular compartments and effector T cells by analyzing single-cell RNA sequencing data on a total of 26 (4 exploratory + 22 validation) HL patient tumor biopsies. Using the CellPhoneDB algorithm, we inferred that the ligand B- and T-lymphocyte attenuator (BTLA) on effector T cells and the receptor Herpesvirus entry mediator (HVEM, TNFRSF14) on immunosuppressive cells (e.g., regulatory T cells, monocytes) strongly interact in the TME and promote T cell dysfunction ( Fig 1a). Akin to the canonical checkpoint PD1, BTLA recruits two potent tyrosine phosphatases, SHP-1 and SHP-2, to disable early T cell activation. Thus, we rationalized that BTLA expression on T cells might reduce their anti-tumor function. We hypothesized that deleting BTLA in CAR T cells would abolish BTLA-HVEM trans interactions at the immunological synapse and unleash the cytotoxic potential of CAR T cells. Methods and Results: We first generated BTLA KO anti-CD30 CAR T (CART30) cells against HL. To test their function in vivo, 15x10 6 HDLM-2 (CD30+HVEM+ HL) cells were subcutaneously implanted into NSG mice on day 0, and on day 62, 3x10 5 CAR30+ T cells were infused intravenously. Our results demonstrated that BTLA KO significantly enhances the function of anti-CD30 CAR T cells in HVEM+ HL ( Fig 1b), as assessed via tumor size (caliper) and CART30 expansion in the peripheral blood (flow cytometry). Additionally, we generated BTLA KO 4-1BBζ CART19 cells, which showed greater in vivo anti-tumor function in a subcutaneous tumor model of DLBCL (CD19+ HVEM+ OCI-Ly18). Serum collected from OCI-Ly18-bearing NSG mice infused with BTLA KO CART19 was enriched in effector cytokines (e.g., TNF, IFNγ, IL-2) as measured by Luminex. We then extended these findings into HVEM+ solid tumor models. BTLA KO improved tumor control in vitro in short-term killing experiments (Incucyte SX5) for both CAR and TCR T cells, respectively directed against prostate cancer (HER2+ PC-3) and melanoma (GP-100+ DM-6). Importantly, we demonstrated that BTLA KO in primary BALB/c-derived murine CAR T cells enhances tumor control in A20 murine lymphoma. In this model, BTLA KO tumor-infiltrating CAR T cells showed substantially reduced exhaustion in the TME relative to wild-type CAR T cells. Mechanistically, we showed that targeted mutations in BTLA intracellular tyrosine motifs effectively reduce the recruitment of SHP-1/2 and preserve the docking domain of the pro-stimulatory Grb2, thus maintaining high NFAT signaling and increased persistence in vivo. These effects were dependent on the expression of HVEM on tumor cells. Finally, we found that high BTLA RNA expression in tisagenlecleucel infusion products correlates with poor response to treatment in patients with DLBCL and FL (NCT02030834). Conclusion: Our results reveal a critical role of the BTLA-HVEM axis in inhibiting CAR T cell function, and demonstrate that CRISPR-Cas9 deletion of BTLA leads to enhanced anti-tumor efficacy in multiple models of cancer. The key mechanism is the reduction of SHP-1/2 recruitment and the consequent increase in CAR T cell activation. The results of this study will be translated into a first in human clinical trial of BTLA-deficient CAR T cells for relapsed or refractory cancer.
48
Cai, Shuyang, Honghu Li, Ruxiu Tie, et al. "Nlrc3 Signaling Is Indispensable for Hematopoietic Stem Cell Emergence Via Notch Signaling in Vertebrates." Blood 142, Supplement 1 (2023): 2675. http://dx.doi.org/10.1182/blood-2023-186693.
Full textAbstract:
Hematopoietic stem and progenitor cells (HSPCs) generate all the lineages of blood cells throughout the lifespan of vertebrates. The emergence of HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process, which is finely tuned by a variety of signaling pathways. Previous studies have emerged the essential roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in EHT. However, whether the nucleotide-binding domain leucine-rich repeat (NLR) containing family members participate in vertebrate embryonic hematopoiesis remains unclear. To explore the potential pathways involved in hematopoietic ontogeny and differentiation in this stage, we screened the dynamic expression of NLR family genes and other genes related to inflammation in the published scRNA-seq data in vertebrates' embryos including zebrafish, mouse and human during HSPC development. And to verify the expression of NLR family genes in vitro by using HSC-like cells differentiation system of mouse and human embryonic stem cells (ESCs), these results suggest that NLRC3 was highly expressed during embryonic HSPC development in vivo and vitro and might play an important role in HSPC emergence in vertebrates. In order to validate this hypothesis, we synthesized probes from the full-length mRNA of nlrc3 and utilized them to observe the in-situ expression of nlrc3 in zebrafish at different developmental stages. The WISH experiment demonstrated that nlrc3 was expressed from the 1-cell stage and importantly, showed specific expression in the AGM region at the 24-28hpf and CHT region at 72hpf, which coincides with the onset and generation of HSPC generation. Then morpholino were utilized to knockdown nlrc3 in transgenic embryos Tg( runx1:EGFP/ kdrl:mCherry), Tg( cmyb:EGFP/ kdrl:mCherry) and Tg( CD41:GFP), and mutants generated by CRISPR-Cas9 were used to perform whole-mount in situ hybridization (WISH) and qPCR to detect specific markers such as runx1, cmyb(HSPCs), rag1(lymphocytes), gata1a(erythrocytes) and l-plastin(myelocytes), etc. The results showed that nlrc3 was required for HSPCs emergence, expansion and differentiation. In primitive hematopoietic wave, nlrc3 signaling may not impact primitive erythropoiesis but required for primitive neutrophils and macrophages. Mechanistically, we sorted EGFP + cells in Tg ( fli1a:EGFP) zebrafish embryos at 28 hpf to perform RNA-Seq, which was the stage where EHT and these EGFP + cells contained hemogenic endothelial cells. After bioinformatics analysis of sequencing data, we found that the effect of nlrc3 in zebrafish hematopoiesis is not based on affecting the development of arterial vessels or causing apoptosis. By using targeted inhibitor such as DAPT and JSH23, conditional rescue model like Tg ( hsp70:GAL4/ UAS:NICD), overexpression expression experiment in Tg ( tp1:EGFP/ kdrl:mCherry) and nlrc3 -/- mutants, etc, through Imaging systems and qPCR we found that NF-kB- nlrc3-Notch- hey1 axis triggers HSPCs emergence and differentiation in zebrafish. To further extend our findings in mammals, we utilized Nlrc3 knockout mice to explore whether Nlrc3 signaling was evolutionarily conserved in HSPCs emergence. Hematogenic endothelial cells (CD31 + CD41 - CD45 - TER119 -) from the embryonic AGM tissues at E10.5 were sorted and cultured in OP9 for 4 days and flow cytometry was performed to assess that c-Kit +CD45 + hematopoietic cells and CD31 +Sca-1 +CD201 + hematopoietic stem cells were notably decreased in these Nlrc3-/- embryos. Colony-forming unit-cell (CFU-C) assay and BM transplantation experiments were used to observe that fetal liver HSCs from E14.5 Nlrc3-/- embryos was decrease in the number and impaired in function in vitro and in vivo. Finally qPCR using mRNA from FL-derived LSK ( Lin-c-Kit+Sca-1+) cells further demonstrated that the NF-kB- Nlrc3-Notch- Hey1 axis orchestrated development of embryonic HSCs was highly conserved in vertebrates. Overall, by summarizing the results of the physiological and developmental roles in vivo and in vitro, we uncover an indispensable role of Nlrc3 signaling in vertebrates' HSPCs emergence, our findings provide new insights into inflammation-related hematopoietic ontogeny and Nlrc3 signal is of major importance since this investigation might be taken into consideration when recapitulation definitive hematopoiesis in vitro.
49
Heatley, Susan L., Elyse C. Page, Laura N. Eadie, et al. "Modeling Relapsed, Refractory Acute Lymphoblastic Leukemia from a Child with Neurofibromatosis." Blood 138, Supplement 1 (2021): 1317. http://dx.doi.org/10.1182/blood-2021-150086.
Full textAbstract:
Abstract Neurofibromatosis type 1 (NF-1) is an autosomal dominant disorder affecting approximately 1:3000 individuals globally. While approximately 50% are familial, with over 3000 causative germline variants in the neurofibromatosis (NF1) gene identified, the remainder occur sporadically. These mutations lead to haploinsufficiency of NF1 and neurofibromin, a tumor suppressor and important negative regulator of RAS signaling. Children with NF-1 have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop acute lymphoblastic leukemia (ALL). A 9-year-old male presented in 2015 with persistent migratory subcutaneous swellings and multiple bony aches with lytic lesions on bone imaging. He had a high white cell count with eosinophilia (WCC 43.4 x 10 9/L, eosinophils 23.87 x 10 9/L) with no circulating blasts, 10% marrow blasts (CD10+/CD19+/CD34+) and was CNS negative. Although previously undiagnosed, NF-1 was clinically suspected due to typical skin changes. He was diagnosed with iAMP21 ALL and NF-1 was confirmed with the identification of a germline NF1 donor splice site mutation (c.1845G&gt;A:p.L615=). Bone marrow cells were sorted by flow cytometry on CD19 positivity and underwent transcriptomic sequencing. This revealed a P2RY8-CRLF2 gene fusion, with no other clinically relevant variants, while a custom Taqman low density array indicated high-risk B-ALL subtype Ph-like ALL. Multiplex ligation-dependent probe amplification (MLPA) confirmed iAMP21 and also identified IKZF1 exon 2-3 and BTG1 deletions. Treatment followed the high-risk B-ALL arm of the AEIOP-BFM ALL2009 protocol due to persistent end-consolidation MRD in addition to iAMP21 and the Ph-like phenotype. He relapsed three years later off treatment and was refractory to both salvage chemotherapy and blinatumomab. The iAMP21, P2RY8-CRLF2 gene fusion, IKZF1 exon 2-3 and BTG1 deletions remained detectable. Whole exome sequencing of CD19 positive samples from diagnosis, relapse and mesenchymal stem cells (germline control) was performed, identifying a NF1 c.7400dupT:p.L2467 frameshift (fs) mutation only at relapse. To understand the implications of NF1 p.L2467fs, the P2RY8-CRLF2 gene fusion was first transduced into the interleukin 3 (IL3) dependent murine pro-B cell line Ba/F3. P2RY8-CRLF2 alone is not transforming and is thought to be a secondary event in iAMP21 ALL, providing an ideal model to study the cumulative effect of the NF1fs. The NF1fs was then introduced to the P2RY8-CRLF2 cells by CRISPR/Cas9. A proliferation assay was performed without IL3 and demonstrated the P2RY8-CRLF2+NF1fs cell line was IL3 independent, indicative of leukemic transformation, whereas all other lines were not (vs Ba/F3, p = 0.001). Neurofibromin can be constitutively phosphorylated at the c-terminus, negatively regulating NF1-GAP activity, suppressing RAS signaling and inducing cell cycle arrest. Therefore, to demonstrate loss of function due to the c-terminus NF1 p.L2467fs and increased RAS signaling, western blotting for pERK was performed. Significant upregulation of pERK was confirmed in P2RY8-CRLF2+NF1fs in comparison to Ba/F3 control cells (p=0.007) (Figure 1). The MEK inhibitors trametinib and mirdametinib are in clinical trials for NF-1 patients and have shown efficacy in ALL models with RAS mutations. In a 3-day cell death assay, only P2RY8-CRLF2+NF1fs demonstrated sensitivity to trametinib (LD 50 P2RY8-CRLF2 = &gt;6.4 µM, NF1fs = &gt;6.4 µM, P2RY8-CRLF2+NF1fs =1.7µM; p &lt; 0.001) and mirdametinib (LD 50 P2RY8-CRLF2 = &gt;16 µM, NF1fs = &gt;16 µM, P2RY8-CRLF2+NF1fs = 8.3 µM; p &lt; 0.0001) (Figure 2). Here, we have demonstrated a LOF NF1fs mutation using an in-vitro model of ALL. Germline NF1 haploinsufficiency and a second hit NF1 mutation in ALL is limited to one report of monozygotic twins with neurofibromatosis. We propose that NF1 p.L2467fs caused bi-allelic LOF and therefore contributed to relapse in this patient. An understanding of the genomic complexities that lead to relapse may also inform personalized treatment strategies. While this patient subsequently achieved remission with inotuzomab and underwent successful stem cell transplantation, the sensitivity to MEK inhibitors is an exciting development for neurofibromatosis patients with ALL. Figure 1 Figure 1. Disclosures Yeung: Amgen: Honoraria; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. White: BMS: Honoraria, Research Funding; Novartis: Research Funding.
50
Ghobadi, Armin, Ibrahim Aldoss, Shannon L. Maude, et al. "Phase 1/2 Dose-Escalation/Dose-Expansion Study of Anti-CD7 Allogeneic CAR-T Cells (WU-CART-007) in Relapsed or Refractory (R/R) T-Cell Acute Lymphoblastic Leukemia/ Lymphoblastic Lymphoma (T-ALL/LBL)." Blood 142, Supplement 1 (2023): 770. http://dx.doi.org/10.1182/blood-2023-178723.
Full textAbstract:
T-ALL/LBL are challenging hematologic cancers with high rates of relapse and mortality in both children and adults. Despite success in B-cell malignancies, development of CAR-T cell therapy for T cell malignancies has been complicated by induction of fratricide and risk of malignant cell contamination of the drug product in the autologous setting. WU-CART-007 is a CD7-targeted CAR-T cell products with CRISPR/Cas9 deletion of CD7 and T-cell receptor alpha constant (TRAC), to prevent fratricide and enable the use of healthy donor allogeneic T-cells, respectively (Leedom et al. ASH 2021). This off-the-shelf allogeneic CAR-T cell product is being developed for the treatment of CD7 + malignancies. WU-CART-007 1001 (NCT04984356) is a global first-in-human, Phase 1/2 single-agent study of WU-CART-007 in patients (pts) with R/R T-ALL/LBL. The recommended Phase 2 dose (RP2D) of WU-CART-007 is 900 million (M) cells administered on day 1 following lymphodepleting chemotherapy (LDC). Two different LDC regimens have been tested: standard LDC (fludarabine 30 mg/m 2/day x 3 days and cyclophosphamide 500 mg/m 2/day x 3 days), and enhanced LDC (eLDC; fludarabine 30 mg/m2/day x 4 days and cyclophosphamide 1000 mg/m2/day x 3 days).Disease response is assessed by Day 28 bone marrow (BM) aspirate/biopsy, and CT/PET, if applicable, per NCCN Guidelines Version 2.2022; Pharmacokinetics (PK) are measured by ddPCR; samples are collected for immunophenotyping by flow cytometry (FACS). As of July 21, 2023, 18 pts have been dosed with WU-CART-007(n = 11 T-ALL, n = 7 T-LBL); 3 with 100M (DL1), 3 with 300M (DL2), 6 with 600M (DL3), and 6 with 900M (DL4/RP2D) cells in a single infusion. A total of 15 pts received standard LDC while 3 received eLDC at the DL4/RP2D. Median age is 33.5 years (range 20-68). Pts were heavily pretreated with a median of 4 prior lines of therapy (range 2 - 7), 28% (5/18) relapsed following an allogeneic HSCT. Disease burden at baseline consisted of extramedullary disease (EMD) in 28% (5/18) of pts, and a median BM blast count of 60% (range 5-98%) in pts with BM disease (13/18). Overall WU-CART-007 demonstrated manageable safety profile; treatment-related adverse events of ≥ G3 were observed in 8/18 (44%) pts. Cytokine release syndrome (CRS) was observed in 14/18 (78%) pts. Most (72%; 13/18) pts had G1-2 CRS events; a single G3 CRS event was reported which resolved within 72 hours after receiving tocilizumab, dexamethasone, and low-dose vasopressors. Grade 1 ICANS was reported in one patient at DL3, which resolved spontaneously. No GvHD, prolonged T-cell aplasia, or prolonged pancytopenia in the absence of disease were observed. One unrelated DLT lead to cohort expansion at DL3. The majority of deaths were due to disease progression. There were two Grade 5 events not attributed to WU-CART-007, both due to fungal infection. WU-CART-007 showed dose-dependent anti-tumor activity with no responses seen at DL1. In evaluable patients at DL≥ 2 (n=12) Composite Complete Remission Rate (CRc; CR + CRi + CRh): 58% (86% MRD neg), median duration of response 12.3 weeks (1.1 - 29.4); two patients successfully received a consolidating allogeneic-HSCT. At the RP2D, the CRc rate was 60% (3/5; 2 CR, 1CRi). Molecular expansions of CAR-T cells peaked on day 10 in peripheral blood (mean 66,069 copies/ug DNA) and persisted out to day 56. Of all pts tested (n=15), none developed novel anti-HLA antibodies against the donor, and no anti-drug antibodies against the CAR-construct were detected. Phenotypic analysis revealed in vivo WU-CART-007 cells expressed activation markers (KI67, CD38, HLA-DR) and were largely an effector memory CD45RA + (EMRA) CM phenotype (CD45RA +, CD197 +). WU-CART-007 has demonstrated an acceptable safety profile and preliminary evidence of anti-leukemic activity. This program advances CAR-T cell therapy in heavily pre-treated patients with R/R T-ALL/LBL. Enrollment is ongoing.