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Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis and high rates of relapse despite aggressive treatments including high dose chemotherapy. To understand the clonal dynamics and genetic evolution of relapsed AML, we analyzed a cohort of 142 previously published genotyped and paired diagnosis-relapse AML samples. 40% of queried cases exhibited no major changes in somatic mutations upon relapse, and genetically stable clonal structure correlated with increased relapse probability. Thus, we hypothesized epigenetic reprogramming plays a role in these cases and AML relapse in general. Here, we examine the epigenetic landscape of relapsed AML and characterize the cis and trans regulatory elements that correlate with AML relapse in the presence or absence of genetic evolution. Methods: We identified 27 viable cryopreserved paired diagnosis and relapse samples from the same patient treated at Stanford with high-dose chemotherapy. Leukemic blasts and (when possible) leukemia stem cell (LSC) enriched populations were FACS purified and prepared for genotyping with a myeloid malignancy targeted sequencing panel as well as ATAC-seq for chromatin accessibility profiling. Single-cell ATAC-seq was further performed on select samples to investigate regulatory reprogramming in cell subpopulations. We then performed integrative analysis to uncover the interplay between genetic lesions, epigenetic regulatory programs, and gene accessibility in relapsed AML. Results: Genotyping analysis of these AML specimens revealed that 40% of samples exhibited no changes in AML-related genetic alterations upon relapse (hereby referred to as "stable" samples). Chromatin accessibility analysis revealed these stable samples had a distinct epigenetic signature, modulating similar gene accessibility programs and sharing enhancer loci that become accessible across all stable relapse samples. These sequences included genes involved in chromatin organization and compaction, as well as those involved in transcriptional control of hematopoietic differentiation and myeloid cell maturation. We also observed several regulatory signatures present at relapse specific for AML subtypes, including NPM1/FLT3 double mutant AML and those with mutations in transcription factors such as CEBPA or RUNX1. We then performed single-cell ATAC-seq on genetically stable samples to further characterize the sub-clonal epigenetic dynamics between the diagnosis and relapse cells. Several samples exhibited regulatory heterogeneity in multiple cell subpopulations that changed significantly at relapse. One subpopulation of interest was characterized by increased GATA and RUNX family transcription factor motif accessibility at relapse, indicating a shift toward a less differentiated progenitor cell phenotype. In addition, we identified a subpopulation of cells at diagnosis that were epigenetically similar to the major epigenetic states present at relapse, indicating that selection for specific epigenetic subclones may occur in AML patients during therapy in the absence of additional genetic lesions. Finally, given the critical role of LSCs in AML pathogenesis and their possible role as a reservoir for AML relapse, we analyzed LSC-enriched subpopulations in a subset of our cohort. ATAC-seq analysis indicated these cells shared several LSC-specific epigenetic features between samples, are distinguished from leukemia blasts by distinct regulatory programs, and undergo epigenetic remodeling between initial diagnosis and relapse. Gene accessibility analysis also revealed a shared LSC gene expression signature that also shifted at relapse. These data indicate a specific, distinct epigenomic signature for LSC enriched cell populations, and that epigenetic evolution at relapse occurs intracellularly, rather than reflecting heterogeneity in cellular subpopulations upon AML relapse. Conclusion: This study reveals that epigenetic remodeling in the absence of genetic evolution is a mechanism through which AML relapse occurs. We show that chromatin reorganization of genes and regulatory sequences occurs in these AML cells, leading to a permissive cell state that might be resistant to conventional treatment. Ongoing work includes dissecting the subclonal structure of these AML cells and identifying the relationship between gene regulatory networks that contribute to relapse. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. 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..

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.

Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis even with aggressive treatments including high dose chemotherapy. While most patients enter clinical remission, these remissions are often short-lived leading to chemotherapy-resistant relapsed disease that accounts for the majority of deaths. We undertook a meta-analysis of published datasets consisting of 142 genotyped paired diagnosis-relapse AML samples to understand the genetic evolution of AML between the two disease states. This analysis determined that a plurality of cases exhibited the same mutations at diagnosis and relapse, and that genetically stable clones were associated with an increased probability of relapse. The finding that many cases exhibited no clonal genetic evolution upon relapse, yet exhibited chemotherapy resistance, lead us to hypothesize that epigenetic evolution plays a significant role in AML relapse. Here, our objective was to investigate the epigenetic evolution and cis and trans regulatory elements that correlate with AML relapse. Methods: We identified 27 paired diagnosis and relapse specimens from patients treated at Stanford with high dose chemotherapy regimens. Leukemic blasts, and in some cases leukemia stem cell (LSC)-enriched fractions, were purified by FACS. Cells were then analyzed through a multi-omic platform including genotyping with a myeloid malignancy targeted panel, RNA-seq, and ATAC-seq to obtain a molecular and chromatin accessibility profile of each sample. The resulting data set was analyzed to investigate epigenetic evolution in relapsed AML. Results: Genotyping analysis of banked AML specimens identified a similar pattern of genetic evolution as our meta-analysis, with several samples exhibiting the same mutations at diagnosis and relapse. We used an epigenetic matrix of chromatin accessibility data obtained from purified cell populations within the hematopoietic hierarchy and implemented this with the CIBERSORT algorithm to map the regulatory programs active in diagnosis and relapsed AML blasts. This analysis revealed a general trend of epigenetic states associated with more primitive cells (such as hematopoietic stem and progenitor cells) active at relapse, as opposed to more differentiated myeloid cell programs active at diagnosis. Focusing further on samples with no genetic changes between the two disease states, we observed several samples with substantial epigenetic evolution at relapse, with AML blasts shifting from a more differentiated myeloid cell profile to that of stem and progenitor cells. These changes were associated with a loss of accessibility in PU.1 and CEBPα transcription factor motifs, with a corresponding increase in GATA and RUNX motifs, suggesting epigenetic remodeling contributes to relapse even in the absence of genetic changes. We have additionally identified various categories of relapse samples in our cohort that share similar epigenetic profiles relating to genotype; NPM1 and FLT3 double mutant samples, for example, shared active chromatin accessibility features. Given the key importance of LSCs in AML pathogenesis and their potential role in chemotherapy resistance, we further undertook an analysis of cellular subpopulations enriched for these cells in a subset of our sample cohort. ATAC-seq analysis of CD34+CD38- cell fractions revealed these cells share many epigenetic features between samples, yet also have distinct regulatory programs from those active in leukemia non-stem cells and exhibit similar epigenetic reprogramming between diagnosis and relapse. This analysis further indicates that epigenetic evolution at relapse occurs at the single cell level, rather than reflecting selection of cellular subpopulations at relapse. Ongoing work involves identifying the specific regulatory programs upregulated in relapse samples, and LSCs specifically, to understand how these programs contribute to relapse at the gene regulatory level. Conclusion: Our results indicate a substantial role for epigenetic evolution in AML, with the activation of more primitive stem and progenitor programs upon relapse. We have also identified epigenetic classifications for several relapse samples that correspond to genotype and characterized the regulatory programs associated with relapse. We hope this work will permit a deeper understanding of the evolutionary factors that guide AML relapse. Disclosures Majeti: Zenshine Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Coherus BioSciences: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Research Funding; BeyondSpring Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties: pending patent application on CD93 CAR ; Forty-Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences, Inc.: Patents & Royalties: inventor on patents related to CD47 cancer immunotherapy.

Introduction: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in the United States, accounting for more than 11,000 deaths each year and with a 5-year overall survival rate of less than 30%. With the exception of Gemtuzumab ozogamycin, an anti-CD33 antibody drug conjugate, the landmark success of immunotherapy in other hematologic malignancies has not translated to AML. Chimeric antigen receptor (CAR) T cell therapy, in which T cells are engineered with redirected tumor specificity, holds promise for the treatment of AML, but optimal antigens for CAR targeting of AML remain to be defined. We identified CD93 as a novel target for AML CAR therapy given high expression on many AML samples and an important role in leukemia stem cell (LSC) biology. Here, we describe anti-leukemic efficacy of CD93 CAR T cells both in vitro and in murine xenograft models. Consistent with predictions based on CD93 expression within the hematopoietic compartment, we demonstrate minimal CAR T cell toxicity to hematopoietic progenitors. However, we identify endothelial cell toxicity as a significant on-target, off-tumor toxicity. We also analyze endothelial expression of other common AML targets including CD123 and CD38 at baseline and in an inflammatory microenvironment and propose a strategy to incorporate endothelial expression considerations into rational design of combinatorial CAR T cells for AML. Results/Methods: CD93 was expressed to some degree on 24/25 (96%) of primary AML specimens, and was homogeneously expressed in 17/25 (68%). T cells were engineered to express second generation CD93 CARs based on the scFv of a humanized monocloncal CD93 antibody (F11) linked to CD28-CD3ζ or 4-1BB-CD3ζ intracellular domains (CD93-28z and CD93-BBz, respectively). CD93-28z and CD93-BBz CAR T cells incubated in vitro with target cells demonstrated AML-specific cytokine production measured by ELISA and cytotoxicity measured by IncucyteTM assay. CD93 CAR T cell treatment of NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML line THP-1 resulted in improved leukemic control in comparison to mock-treated mice. In a patient derived xenograft model of primary AML, CD93 CAR T cell treatment resulted in significantly improved leukemic clearance, T cell expansion, and prolonged survival compared to mock-treated mice. CD93 CAR T cells were incubated with cord-blood derived CD34+ cells to evaluate CD93 CAR recognition of hematopoietic stem cells (HSCs) and other hematopoietic progenitors. In an ELISA, CD93 CAR T cells did not produce cytokines against the bulk CD34+ population, in contrast to a positive control of AML. Additionally, after a 24h co-culture, CD93 CAR T cells did not kill any hematopoietic progenitor cells as assessed by flow cytometry. Furthermore, a methycellulose based colony forming assay confirmed that CD93 CAR T cells do not impact hematopoietic progenitor multipotent functional ability. We analyzed CD93 expression by immunohistochemistry of a tissue microarray of normal tissues. H-scores of all tissues analyzed were Hematopoietic cells and endothelial cells have a common developmental origin, and other AML CAR targets have been described as expressed on endothelial cells either at baseline or in the presence of inflammatory cytokines. A targeted analysis of CD123, CD38, and CD33 revealed that CD123 and CD38 are also expressed on endothelial cells, especially when cells are pretreated with IFN𝛾 and TNF⍺. Conclusion: Progress in generating effective CAR T cells for acute myeloid leukemia (AML) has been hampered the paucity of AML cell surface antigens that are not expressed on vital tissues. Combinatorial antigen targeting will likely play a major role in advancing CAR T cell therapy for AML. Our data support that endothelial expression at baseline and in an inflammatory microenvironment should be considered as a factor in any rational combinatorial targeting strategy, and that creative CAR engineering will be necessary for certain AML targets, including CD93, to circumvent endothelial toxicity. Disclosures Richards: Stanford University: Patents & Royalties: pending patent application for CD93 CAR. Sotillo:Lyell Immunopharma: Consultancy, Other: Consultancy. Hong:Genentech, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Majzner:Zai Lab: Consultancy; Xyphos Biopharma: Consultancy; Aprotum Group: Consultancy; GammaDelta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Illumina Radiopharmaceuticals: Consultancy; Lyell Immunopharma: Consultancy. Majeti:CircBio Inc.: Research Funding; Gilead Sciences, Inc.: Patents & Royalties: inventor on patents related to CD47 cancer immunotherapy; Stanford University: Patents & Royalties: pending patent application on CD93 CAR ; BeyondSpring Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Forty-Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Coherus BioSciences: Membership on an entity's Board of Directors or advisory committees; Zenshine Pharmaceuticals: 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. Mackall:Lyell Immunopharma: Consultancy, Current equity holder in private company; BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy.

Introduction: AML is an aggressive cancer that develops from the sequential accumulation and clonal expansion of somatic mutations in hematopoietic stem and progenitor cells. Recent next-generation sequencing (NGS) studies of AML have correlated mutations with clinical outcomes and response to targeted therapies. Additionally, emerging reports have suggested that increased clonal heterogeneity and mutation burden tend to correlate with worse survival outcomes. However, due to previous cohort sizes, the architecture of clonal evolution and variant allele frequency (VAF) of recurrent mutations have yet to be robustly correlated with response to therapy or with more granular risk stratification. To address previous limitations, we combined available datasets of sequenced AML to model features of clonality and determine their correlations with clinical outcomes and drug sensitivity. Methods: A systematic literature review was performed to identify cohorts of clinically annotated and genetically profiled adult AML. Studies were included if: (i) their sequencing panel targeted at least 30 of the most commonly mutated genes, (ii) censored overall survival data was reported, and (iii) data were publicly available. An additional cohort of patients profiled at Stanford was also included. Leveraging statistical learning methods and robust clonal modeling algorithms (PyClone and ClonEvol), we performed a meta-analysis of the clonal architecture of mutations, their temporal relationships, sensitivity to drugs, and correlation with outcomes in AML. Results: A total of 12 studies were aggregated into a uniformly annotated database comprising 2,987 AML patient samples profiled with an array of DNA sequencing modalities (2,884 with VAFs) and ex vivo drug screening results (nsamples = 562; ndrugs = 122); survival outcomes were available for 2,606 patients. To investigate broad features of leukemia evolution, we used clonal modeling algorithms to infer clonal architecture. Interestingly, patients exhibiting linear evolution (sequential mutations in the same clone) displayed worse outcomes compared to those with branched architecture (distinct subclonal populations). Additionally, mutational burden and clonal heterogeneity only stratified patients with branched structure. These results motivated us to understand how the temporal acquisition of mutations might further stratify outcomes. Using dynamic VAF thresholds, we identified novel high-risk patient populations for 15 recurrently mutated genes. Greater VAF was associated with statistically significant improved survival in genotypes such as GATA2mut and WT1mut and with worse outcomes for patients with NRAS and NF1 mutations. Next, we leveraged VAFs to infer the temporal ordering of individual mutations and functional mutation categories. Patients where NRAS mutations occurred before GATA2 mutations showed a significant correlation with worse outcomes. We also observed that patients in which (i) DNA methylation mutations occurred before those in tumor suppressors and (ii) splicing factor mutations occurred before RTK/RAS signaling components showed significantly shortened overall survival. These results indicate that patients with the same genotype can be stratified by the timing of mutations in the clonal evolution of their leukemia. Finally, we used linear regression between drug sensitivity and VAF to identify several mutations which predict drug sensitivity exclusively in a VAF-dependent manner. Increased WT1 VAF correlated with sensitivity to ABT-737 and elevated FLT3-TKD VAF predicted sensitivity to cabozantinib, among other clinically notable drug-gene relationships. These results suggest potential biomarkers for clinical response to emerging targeted agents. Conclusions: We show that VAF can identify novel high-risk patient populations at the individual mutation level (e.g. BCOR and NF1) and can also be leveraged to stratify outcomes based on inferring the temporal ordering of mutations (e.g. NRAS and GATA2). Our observation that patients with leukemias exhibiting branched evolution showed improved survival compared to linear evolution was also striking and warrants further experimental and clinical validation. Incorporating these results with our findings of drug sensitivity validate the clinical utility of integrating clonal analysis into the molecular evaluation and treatment of AML. Disclosures Majeti: Zenshine Pharmaceuticals: 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; Stanford University: Patents & Royalties: pending patent application on CD93 CAR ; Coherus BioSciences: Membership on an entity's Board of Directors or advisory committees; BeyondSpring Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Forty-Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences, Inc.: Patents & Royalties: inventor on patents related to CD47 cancer immunotherapy; CircBio Inc.: Research Funding.

Introduction: Mutations in additional sex combs like 1 (ASXL1) are recurrently found in myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but can also be found in pre-malignant states such as clonal hematopoiesis of indeterminate potential (CHIP). However, the mechanisms by which these mutations contribute to disease initiation and progression remain unresolved. Initial observations using murine models suggested a loss of function of the ASXL1 protein as relevant to disease. However more recently, several studies have suggested that recurrent mutations of ASXL1 result in a change-of function caused by a truncated ASXL1 protein. Most of these studies have been performed in a murine system and have used over-expression of truncated ASXL1. Methods: To interrogate the functional relevance of recurrent mutations in ASXL1 in human HSPCs, we generated a novel CRISPR/Cas9-mediated model that allows for the introduction of truncations or complete knockout of ASXL1 into CD34+ cord blood cells. Importantly, this is the first model which efficiently introduces these mutations into the native ASXL1 locus in human cord-blood and bone-marrow derived HSPCs while simultaneously introducing a fluorescent marker corresponding to the genotype, so individual cells can be tracked in vitro and in vivo. Results: Using this system, we demonstrate that truncated, but not deleted, ASXL1 shows a proliferative advantage, decreased ability to differentiate along the megakaryocyte and erythroid lineages, as well as increased serial replating in vitro. Interestingly, truncation of ASXL1 alone did not result in a myeloid differentiation block in vitro, in line with observations of mutant HSPCs in CHIP. In vivo, CD34+ cells harboring truncation of ASXL1 exhibited myeloid skewing and a proliferative advantage in competitive engraftment studies using a xenograft NSGS mouse model (both p Conclusion: In summary, our work demonstrates the utilization of a novel CRISPR/Cas9 model of human ASXL1-mutant HSPCs that closely resembles the phenotype seen in individuals with ASXL1 mutant CHIP and patients with myeloid malignancies carrying mutant ASXL1. Additionally, our model allows us to directly compare the deletion of ASXL1 with the truncation, adding to the evidence that truncated ASXL1 phenocopies pre-malignant and malignant hematopoiesis with recurrent ASXL1 mutations. Finally, we provide the first model for the spontaneous progression of pre-malignant human CHIP to myeloid malignancy, which should enable further studies of leukemogenesis and potentially disease prevention. Disclosures Majeti: FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy.

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.

BCL2 is an antiapoptotic protein commonly expressed in hematologic malignancies. Overexpression of BCL-2 is a poor prognostic factor in acute myeloid leukemia (AML). Venetoclax (ABT-199) is a highly selective BCL2 inhibitor that can induce cell death in multiple leukemia cell lines. Recently, venetoclax received an FDA breakthrough therapy designation for use in combination with hypomethylating agents in treatment-naïve patients with AML who are unfit for intensive chemotherapy. However, venetoclax was only modestly effective as monotherapy in relapsed/refractory AML (19% CR/CRi). The aim of the current study is to integrate genomic and functional screen data to identify biomarkers to predict venetoclax sensitivity and resistance in AML, and to identify potential venetoclax combination treatment strategies. In this study, we investigated approximately 200 AML patient samples and correlated clinical parameters, whole exome sequence data, and RNAseq gene expression data with in vitro drug screening data (drug area under the curve (AUC)) to identify subsets of AML samples with sensitivity or resistance to venetoclax alone and in combinations with 10 small molecular inhibitors (Array-382, dasatinib, JQ-1, idelalisib, quizartinib, palbociclib, panobinostat, ruxolitinib, sorafenib, and trametinib). For gene expression, we observed that venetoclax correlated with 3 gene expression clusters (coefficient frequency: 0.94, 0.80 and 0.71 respectively) among 21 gene expression clusters in AML, associated with innate immune system, neutrophil degranulation, and interleukin-10 signaling. Among the BCL2 gene family, venetoclax AUC positively correlated with BCL2A1 (r=0.59, p In summary, we have identified that CD14 and/or CLEC7A could be used as biomarkers to predict venetoclax sensitivity in AML, and we propose to combine venetoclax and palbociclib to treat patients with a venetoclax resistant profile (high CD14/CLEC7A expression or high BCL2A1 expression, or presence of KRAS mutations). Disclosures Druker: Beta Cat: Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Bristol-Meyers Squibb: Research Funding; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Novartis Pharmaceuticals: Research Funding; Millipore: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Patient True Talk: Consultancy; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Celgene: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Majeti:BioMarin: Consultancy; Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Incyte: Research Funding; AstraZeneca: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Leap Oncology: Equity Ownership; Seattle Genetics: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Syros: Research Funding; Aptose: Research Funding; Agios: Research Funding.

Introduction: Mutations substituting arginine 132 of isocitrate dehydrogenase 1 (IDH1) are recurrent in acute myeloid leukemia (AML) and several other cancers, resulting in the aberrant production of the onco-metabolite, R-2-hydroxyglutarate (2-HG), as well as an inability to convert cytoplasmic alpha-ketoglutarate to isocitrate via reductive carboxylation. Currently, small molecules that effectively inhibit the neomorphic enzyme and abrogate the production of 2-HG, such as AG-120, are in clinical trials with promising results. However, these inhibitors have not proven to be curative in most AML cases, indicating a need for additional targeted therapies. We have previously investigated synthetic lethal vulnerabilities in IDH1-mutated AML and identified an interaction with BCL2 leading to increased susceptibility to ABT-199 (Chan et al, 2015). Synthetic lethal approaches targeting 2-HG independent metabolic vulnerabilities conferred by mutant IDH1 may complement IDH1 mutant inhibitors. Using a novel computational method (MiSL) based on Boolean implication (if-then rules) mining of pan-cancer data, we identified acetyl CoA carboxylase (ACACA) as a potential druggable target in IDH1-mutated AML. ACACA is the rate-limiting step in the de novo synthesis of fatty acids, and mutant IDH1 leads to a reduction in malonyl-CoA, a key building block for fatty acids, in a 2-HG independent manner. This finding led us to investigate a potential synthetic lethal interaction between mutant IDH1 and ACACA based on the hypothesis that the combination causes marked inhibition of fatty acid synthesis required for cell growth. Methods: Boolean implications (MiSL) were used to identify candidate synthetic lethal interactions with mutant IDH1 by isolating genes deleted only in the absence of the mutation and with differential gene expression within pan-cancer TCGA data. Validation was performed using THP-1 cells transduced with doxycycline-inducible wildtype and R132H mutant IDH1 lentiviral vectors, and primary patient IDH1-mutant and wildtype AML samples, using both shRNAs and a targeted pharmacologic inhibitor of ACACA. Metabolomics was performed using semi-targeted mass spectrometry and liquid chromatography. Finally, primary AML samples and IDH1-mutant and wildtype cancer cell lines (HT-1080, U118, U87) were transduced with validated shRNA and engrafted into NSG mice. Results: Our computational method found that IDH1 mutation and ACACA deletions were mutually exclusive in pan-cancer TCGA data, ACACA deletions resulted in lowered expression of ACACA, and ACACA was differentially over-expressed in IDH1-mutant AML compared to IDH1-wildtypeAML. Pharmacologic inhibition of ACACA with 2 uM TOFA caused a marked reduction in cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox; p = 0.0001). Similarly, knockdown of ACACA with independent shRNAs caused a defect in viable cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox) or with scrambled shRNA (p=0.009, shRNA #1 vs. scrambled; p=0.01, shRNA #2 vs. scrambled). Primary IDH1 R132 mutated purified AML blasts were selectively sensitive to TOFA treatment compared to IDH1 wildtype normal karyotype blasts (IC50 0.6 uM vs 6 uM, p=0.009) in viable growth assays. Furthermore, when transduced with lentivirus encoding shRNA to ACACA, primary IDH1-mutant AML cells exhibited markedly reduced engraftment of RFP-positive human CD45+CD33+ leukemic cells compared to scrambled non-targeting shRNA (p < 0.05, Mann-Whitney U). As predicted, IDH1-mutant AML blasts pre-treated with 10uM AG-120 (sufficient to inhibit production of detectable 2-HG) remained susceptible to ACACA inhibition in vitro. Strikingly, in vivo models of IDH1 R132C mutated, but not wildtype, sarcoma cell lines exhibited a dramatic decrease in cell growth after ACACA inhibition that was not reversible by treatment with AG-120. Finally, metabolomic profiling revealed a major perturbation in multiple phospholipid fatty acid species and decreased malonyl-CoA conferred by IDH1 R132H, consistent with our proposed mechanism. Conclusion: We have identified de novo lipogenesis through ACACA as a critical metabolic vulnerability linked to IDH1 mutation in AML and provide evidence that therapeutic inhibition of ACACA with small molecules may be beneficial in AML, as well as in other cancers with IDH1 mutations. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Understanding the contribution of abnormal genetic and epigenetic programs to acute myeloid leukemia (AML) is necessary for the integrated design of genetic and epigenetic targeted therapies. While epigenetic therapies have been used to treat AML, so far efficacy has been limited. To this point, the reversibility of epigenetic modifications in AML is poorly understood, as are the relative contributions of the leukemic genetic and epigenetic programs to disease pathogenesis. To investigate these questions, we sought to reprogram primary AML leukemic blasts into induced pluripotent stem cells (iPSCs) and assess the effect of epigenetic reprogramming on leukemic behavior. We generated iPSCs from several AML patients with 11q23/MLL translocations using classic iPSC reprogramming factors (Sox-2, Klf4, Oct4, and Myc). Reprogrammed AML cells generated iPSCs as indicated by classic pluripotent features including embryonic stem cell morphology, expression of pluripotent markers, and formation of teratomas in vivo. In addition, AML iPSCs retained all the karyotypic and genetic abnormalities of the original AML patient. Despite presence of these mutations, AML iPSCs were able to differentiate into multiple cell types with normal function. Thus, human AML cells can be epigenetically reprogrammed into a pluripotent state despite the presence of leukemic driver mutations. Surprisingly, when AML iPSCs were differentiated in CD43+45+ hematopoietic cells, a leukemia phenotype re-emerged. Differentiated hematopoietic cells from AML iPSCs exhibited exclusively granulocytic-monocytic differentiation in colony forming assays, and serial replating potential in contrast to control iPSCs. When transplanted into immunodeficient mice, hematopoietic cells from AML iPSCs formed aggressive myeloid leukemias as evidenced by peripheral blood and bone marrow CD33+ myeloid engraftment, enlarged spleens, secondary transplant potential, and death from fulminant leukemia. These data indicate that epigenetic reprogramming alone was insufficient to eliminate leukemic behavior. To further investigate these epigenetic changes during reprogramming, we performed DNA methylation and gene expression analysis using 450K BeadChip arrays and RNAseq, respectively. Unsupervised hierarchical clustering demonstrated clustering of undifferentiated AML iPSCs with control iPSCs. In contrast, hematopoietic cells differentiated from AML iPSCs clustered exclusively with primary AML cells. Through gene ontology enrichment analysis, hematopoietic differentiated AML iPSCs were enriched for hypomethylation and gene activation of hematopoietic and leukemogenesis gene sets, including MLL gene targets, as compared to undifferentiated AML iPSCs. These data demonstrate that DNA methylation and gene expression profiles are reset upon epigenetic reprogramming of AML cells, but re-emerge upon hematopoietic differentiation, coinciding with re-emergence of the leukemic phenotype. Lastly, we demonstrated that our AML iPSC model can be used to physically separate and functionally profile genetic subclones within an AML patient. In one patient, we identified distinct AML iPSC genetic subclones (KRAS wildtype and G13D mutant) that could be prospectively separated and demonstrated differential growth properties and therapeutic susceptibilities. To determine the clinical relevance of these subclones, we sequenced this patient at relapse. Strikingly, the KRAS mutant clone, which was dominant at diagnosis, was absent at disease relapse implying relative chemotherapy resistance and competitive outgrowth of the KRAS wildtype subclone. Indeed, in our AML iPSC model, the KRAS wildtype clone demonstrated increased resistance to cytarabine in colony assays. We then investigated in a cohort of AML patients whether cytarabine sensitivity differed between KRAS wildtype and mutant patients. Strikingly, KRAS AML patients were more resistant to cytarabine in vitro compared to KRAS G13D patients. Thus, our AML iPSC model predicted clonal relapse in this patient and identified differential subclonal sensitivity to chemotherapy as a mechanism for relapse. In summary, pluripotent reprogramming of AML resets leukemic methylation/gene expression patterns, facilitates clonal targeting, and predicts subclonal relapse. Disclosures Chao: Forty Seven Inc.: Employment, Equity Ownership, Patents & Royalties. Majeti:Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

Recurrent mutations in the components of the cohesin complex (RAD21, SMC1A, SMC3, and STAG2) have been identified in human AML and other myeloid malignancies, and have been shown to occur as pre-leukemic mutations in HSC. Cohesin functions to hold chromatin strands within a ring-like structure composed of the four core components, and although its best-established role is to maintain the polarity of sister chromatids during mitosis, cohesin is also involved in double-stranded DNA damage repair and regulation of transcription. As little is known about their contributions to leukemogenesis, we sought to investigate the effects of cohesin mutants on human hematopoiesis, particularly hematopoietic stem and progenitor cells (HSPC). Introduction of mutant cohesin into AML cell lines and primary human HSPC resulted in a differentiation block with an increased frequency of CD34+ progenitor cells. A similar phenotype was observed with knockdown of core component RAD21 both in vitro and in vivo, indicating that mutant cohesin can act either through haploinsufficiency or dominant-negative mechanisms. Mutant cohesin increased the serial replating ability of HSPC in vitro and showed enrichment for HSC and leukemia stem cell gene expression programs, indicating an effect to enforce stem cell functions. Furthermore, we observed a skewing toward the myeloid lineage in cohesin mutant colonies cultured in methylcellulose, which was recapitulated by a strong myeloid skewing of human engrafted cells in vivo. Thus, mutant cohesin enforces stem cell programs and impairs human hematopoietic progenitor differentiation. Since cohesin complex mutations were identified in pre-leukemic HSC in many of the cases we investigated, we hypothesized that they may impart their phenotype in a cell context-dependent manner. To investigate this hypothesis, six human HSPC subpopulations (HSC, MPP, LMPP, CMP, GMP, and MEP) were isolated from cord blood, and these cells were transduced with cohesin WT, cohesin mutant, RAD21 shRNA, or control lentivirus. Transduced cells were then cultured in either myeloid differentiation or erythroid differentiation-promoting conditions. Strikingly, a strong myeloid differentiation block was only observed with cohesin mutant-transduced HSC and MPP, but not GMP. Similarly, a strong erythroid differentiation block was also observed in HSC and MPP, but not MEP. These results indicate that the effect of mutant cohesin is context dependent and restricted to the most immature HSPC. We next sought to elucidate the mechanism by which cohesin mutants exert their effects on human HSPC. Since the cohesin complex functions to establish and maintain DNA accessibility, and knockdown of cohesin can led to a decrease in chromatin accessibility at transcription factor (TF) clustered regions (Yan et al., 2013), we hypothesized that cohesin mutants impart their phenotypic effects through modulation of chromatin accessibility. To investigate this hypothesis, we used a newly developed method known as ATAC-Seq (Buenrostro et al., 2013) to assess genome-wide accessibility in cohesin WT and mutant HSPC. As expected, we found that cohesin mutants exhibited globally reduced chromatin accessibility at transcriptional regulatory elements. However, we detected increased chromatin accessibility at motifs for transcription factors known to be highly expressed in and critical regulators of HSPC including ERG, GATA2 and RUNX1. Further footprinting analysis, a proxy for ChIP-Seq experiments, showed a strong enrichment of binding of these factors in the mutant cells compared to WT cells. Based on these results, we developed a model in which the functional effects of mutant cohesin on human HSPC are mediated by transcription factors exhibiting increased chromatin accessibility such as ERG, GATA2 and RUNX1. From this model, we hypothesized that knockdown of these transcription factors would prevent the enforcement of stem cell programs and increase in CD34-expressing cells observed with cohesin mutants. As predicted, knockdown of ERG, GATA2, or RUNX1, but not GATA1 or PU.1, in the presence of cohesin mutants completely prevented the increase in CD34-expressing cells. These results strongly support our proposed model that mutant cohesin impairs hematopoietic differentiation and enforces stem cell programs through the modulation of ERG, GATA2, and RUNX1 chromatin accessibility, expression, and activity. Disclosures Majeti: Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Introduction: Somatic mutations in cancer can directly or indirectly perturb signalling and metabolic pathways that can render a cancer cell susceptible to synthetic lethality. We have developed a novel computational method to accelerate identification of synthetic lethal partners for recurrent mutations in acute myeloid leukemia. Our method is based on the hypothesis that, across multiple cancers, synthetic lethal partners of a mutation will be amplified more frequently or deleted less frequently, with concordant changes in expression, in primary tumor samples harboring the mutation of interest.It uses Boolean implication (if-then rules) mining (Sinha et al, Blood 2015) to efficiently identify candidate synthetic lethal partners of a given mutation. The method is distinct from existing work in that it is not reliant on data collected from cell-lines, which are not biologically equivalent to primary tissue and do not always share the composition of mutations found in vivo, but instead utilizes large pan-cancer primary patient datasets. Pan-cancer analysis discovers robust relationships that are more likely to be independent of cancer subtypes, as well as increases statistical power. Methods: We utilized TCGA data of 12 non-AML cancer data-sets (TCGA Research Network et al, Nat. Gen. 2013) for which recurrent AML mutations were present with a frequency of at least 2.5%. These mutations include Cohesin, IDH1, WT1, KRAS, and RUNX1. Boolean implications (FDR < 0.05) were used to identify genes that have more copies in the presence of a mutation as determined by (i) preferred amplification in the presence of the mutation - if gene B is amplified, then mutation A is present, (ii) deletion only in the absence of the mutation - if mutation A is present, then gene B is not deleted. Next, we remove genes that are passengers in large chromosomal alterations using gene expression filtering. Finally, the resulting gene set is filtered by differential gene expression in AML to yield the set of candidate synthetic lethal (SL) partners for a given mutation in AML. Results: To validate our novel method, we compared our putative SL partners to an independent shRNA library screen (DECIPHER) performed in our laboratory for the IDH1 R132 mutation (mut) expressed in THP-1 cells using a doxycycline-inducible promoter (Chan et al, Nat. Med. 2015). We found 6 out of 29 predicted genes showed synthetic lethality when knocked down in the presence of the mutation (Fisher's exact test, p=0.002) indicating our method could find experimentally confirmed interactions. Interestingly, our method predicted Bcl-w to be a SL partner of IDH1 mut, consistent with the SL interaction we previously described between Bcl-2 family members and IDH1 mut in primary AML. Importantly, we found that acetyl-CoA carboxylase alpha (ACACA), the rate-limiting enzyme that controls lipid biosynthesis, was predicted to be a strong SL partner for IDH1 mut. Selective inhibition of ACACA with independently validated shRNA or the small molecule inhibitors, 5-(tetradecyloxy)-2-furoic acid (TOFA) and Soraphen A, prevented cell proliferation in the presence of IDH1 mut but not with IDH1 wildtype. (R)-2-hydroxyglutarate inhibited oxidative phosphorylation and sensitised cells to ACACA inhibitors suggesting the interaction was mediated through the oncometabolite. Gene expression profiling of IDH1 mut cells indicated upregulation of lipid biogenesis pathways (PHOSPHOLIPID METABOLISM, p=0.001). Furthermore, gene expression of ACACA is higher in primary IDH1 mut samples compared to IDH1 wildtype (p=0.008, fold change = 1.2), and cultured primary IDH1mut blasts show selective sensitisation to ACACA inhibition in vitro (n=5/6 IDH1mut/IDH1 wt, p=0.04). Conclusion: We have developed a computational tool that can predict SL interactions for recurrent mutations in AML, with applicability to other cancers. Our method identified de novo lipogenesis as a critical metabolic pathway linked to a specific mutation and suggests therapeutic inhibition of ACACA with small molecules may be beneficial in IDH1 mut AML. This is consistent with recent understanding of the Warburg effect, which postulates that certain oncogenic mutations may indirectly stimulate macromolecule biosynthesis pathways to promote unrestrained cell growth. Our results indicate that a function of the IDH1 mutation is to inhibit oxidative phosphorylation and stimulate de-novo lipid synthesis. Disclosures Majeti: Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Human acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by rare self-renewing leukemia stem cells (LSC), which must be eliminated in order to cure the patient. We originally identified increased expression of CD47 on human AML LSC compared to their normal counterparts. CD47 is a widely expressed cell surface protein that functions as a regulator of phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, SIRP-alpha, which in turn delivers an inhibitory signal for phagocytosis. In this way, CD47 essentially functions as a “don't eat me” signal. We previously found increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, and demonstrated that blocking monoclonal antibodies directed against CD47 enabled the phagocytosis and elimination of AML, non-Hodgkin's lymphoma (NHL), and many solid tumors in xenograft models. These results established CD47 as a promising clinical therapeutic antibody target for human AML and other cancers. Towards this goal, we have now developed a novel humanized anti-CD47 antibody with potent efficacy and favorable toxicokinetic properties as a candidate human therapeutic. A novel monoclonal anti-human CD47 antibody, 5F9, was generated, and antibody humanization was carried out by grafting its complementarity determining regions (CDRs) onto a human IgG4 format. The resulting humanized 5F9 antibody (Hu5F9-G4) bound monomeric human CD47 with an 8 nM affinity. Hu5F9-G4 induced potent macrophage-mediated phagocytosis of primary human AML cells in vitro and completely eradicated human AML in vivo, leading to long-term disease-free survival of patient-derived xenografts. Moreover, Hu5F9-G4 synergized with rituximab to eliminate NHL engraftment and cure xenografted mice. Finally, toxicokinetic studies in non-human primates showed that Hu5F9-G4 could be safely administered intravenously at doses able to achieve potentially therapeutic serum levels. Thus, Hu5F9-G4 is actively being developed for clinical trials in human cancers including AML. Citation Format: Ravi Majeti. Preclinical development of a humanized anti-CD47 antibody targeting AML stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA13.

The cohesin complex is a multiprotein complex involved in a number of cellular processes including sister chromatid cohesion in mitosis, replication fork organization, and regulation of chromatin accessibility for gene expression. Mutations in genes encoding the members of the cohesin complex (SMC1A, SMC3, STAG2, and RAD21) occur in about 10-15% of de novo acute myeloid leukemia (AML) patients. Apart from AML, cohesin mutations have been found in many human cancers indicating a central role for this complex in oncogenesis. In AML, our prior studies have demonstrated that cohesin mutations occur in pre-leukemic hematopoietic stem and progenitor cells (HSPC) that retain normal differentiation potential. Thus, these mutations are likely key initiating events in leukemia pathogenesis. Due to their importance in AML evolution, we sought to determine the effect of these mutations on human hematopoiesis. Cohesin mutations typically occur as heterozygous mutations throughout the genes suggesting either a haploinsufficiency or dominant negative effect. Co-immunoprecipitation experiments in primary human AML samples showed marked decrease in binding between RAD21 and SMC1A in RAD21/SMC1A-mutant AML. These results suggest a dominant negative effect of cohesin mutants on complex formation. In an effort to characterize the phenotype of cohesin complex mutations in AML, we generated human AML cell lines engineered to express wildtype (WT) or mutant cohesin components under the control of a doxycycline-inducible promoter. We chose the TF-1 erythroleukemia cell line due to its ability to differentiate down the erythroid lineage in response to erythropoietin (EPO). We found that cohesin mutant cell lines showed a significant decrease in erythroid differentiation upon exposure to EPO as determined by surface expression of glycophorin A (GPA) and RNA expression of fetal hemoglobin and KLF-1, a key erythroid transcription factor, suggesting that cohesin mutations act in a dominant negative manner to impair differentiation. We next investigated the impact of cohesin complex mutations on normal HSPCs from primary human cord blood. We transduced CD34+ cord blood cells with lentivirus encoding constitutive expression of either WT or mutant cohesin components. Transduced cells were isolated and cultured under several conditions. First, cells were cultured with cytokines designed to promote retention of HSPCs, and cord blood cells expressing mutant cohesin showed significant retention of CD34+ expression as compared to WT or control cells. Second, cells were cultured under conditions designed to promote granulocytic/monocytic differentiation, and cohesin mutant-expressing cells showed a significant decrease in CD14+ expression compared to controls. Third, cells were cultured under conditions designed to promote erythroid differentiation, and cohesin mutant cells showed a significant decrease in CD71 and GPA-double positive erythroid cells. Together, this data suggests that cohesin complex mutations impart a differentiation block on primary human HSPCs. Finally, we investigated whether cohesin mutations affected the serial colony replating ability of human HSPCs in vitro. Primary human cord blood HSPCs were transduced with cohesin mutant-encoding lentivirus, sorted, and cultured in methylcellulose for 14 days. No differences were observed in the colony number or type in the primary plating. However, cohesin-mutant cells exhibited increased serial replating potential beyond the 3rd replating, with essentially no control or WT colonies after the 2ndreplating. In summary, our results indicate that cohesin complex mutations impair HSPC differentiation and increase in vitro replating of primary human cells. The mechanisms by which this occurs are currently being investigated, but preliminary data suggests that mutations in cohesin affect global chromatin accessibility. These results are consistent with a model of mutational acquisition in AML that we have proposed, in which pre-leukemic mutations occur in genes involved in global regulation of gene expression through epigenetic mechanisms that impair differentiation and/or affect self-renewal (such as IDH1/2, TET2, DNMT3A, and cohesin), whereas late mutations occur in genes that generally lead to an increase in activated signaling and proliferation (such as FLT3 and RAS). Disclosures No relevant conflicts of interest to declare.

Xenotransplantation into immunocompromised mice is a powerful tool for studying human normal and malignant hematopoiesis. However, xenotransplant models do not necessarily recapitulate human physiology since environmental factors provided by bone marrow (BM) niche cells may differ between mice and humans. Although existing mouse models allow for propagation of hematopoietic stem and progenitor cells (HSPCs) and aggressive acute leukemias, engraftment of more committed normal progenitor cells and many other hematopoietic malignancies is still lacking. Here, we report the development of a novel mouse model bearing a subcutaneously accessible human bone ossicle formed by in situ differentiation of BM-derived mesenchymal stromal cells (MSCs). These human ossicles contain a functional humanized BM niche that facilitates robust engraftment of normal human HSPC and exhibits superior engraftment and expansion of primary AML. Interestingly, this model allows robust engraftment of PML-RARA positive APL and JAK2 V617F+ PMF, both of which fail to engraft in conventional xenotransplantation models. Subcutaneous injection of human BM-derived MSCs resulted in the formation of human ossicles through endochondral ossification resulting in a bone marrow cavity harboring normal mouse hematopoietic elements. After conditioning irradiation, direct intraossicle injection of cord blood CD34+ HSPC (n=5) resulted in robust multi-lineage human engraftment including B cells, T cells, NK cells, mature neutrophils, eosinophils, monocytes, as well as red blood cell precursors and platelets, as assessed by flow cytometric analysis of aspirates taken directly from the ossicles. The self-renewal potential of engrafted HSPCs was demonstrated by re-establishing multi-lineage hematopoiesis in secondary recipients. Unlike conventional NSG mice, this xenograft model allowed engraftment of phenotypically defined L-MPP (n=7) and GMP (n=7). Engraftment analysis 2 weeks post transplantation showed human engraftment with a majority of CD33+ myeloid cells (LMPP: 77.3±14.4%; GMP: 97.3±1.8%) and intermediate (LMPP: 20.8±14.4%) to low (GMP: 1.8±1.8%) numbers of B lymphoid cells. In the investigation of AML, direct intraossicle transplantation of blasts (n=5) resulted in faster (as early as 6 weeks) and significantly higher leukemic engraftment in humanized niches compared to mouse BM. Furthermore, engraftment was also significantly higher in humanized niches after intravenous transplantation and demonstrated a paratrabecular engraftment pattern, suggesting preferential homing and expansion of AML cells in a human BM microenvironment. Limiting dilution analysis revealed a 10-250 fold higher leukemia-initiating cell frequency using direct intraossicle transplantation compared to intrafemoral injection into mouse BM. Intraossicle transplantation also allowed for engraftment and propagation of PML-RARA-positive APL blasts (n=3) resulting in almost 100% leukemic chimerism and granulocytic sarcoma formation 10-15 weeks post-transplantation. Finally, transplantation of PMF patient-derived CD34+ cells (n=5) into the humanized niche led to robust engraftment with up to 50% human chimerism, and further subpopulation studies demonstrated that PMF-engraftment was exclusively initiated from the HSC (CD34+/CD38-/CD90+) population. Engrafted cells showed marked myeloid skewing with JAK2 V617F-positive cells, and detection of only minimal JAK2 wild type lymphoid cells. In summary, we report the development of a novel xenotransplantation model using human bone ossicles formed by in situ differentiation of BM-derived MSCs that substantially improves the engraftment of normal and malignant hematopoietic populations, leading to the formal demonstration that PMF-initiating cells reside in the immunophenotypic HSC compartment. Disclosures No relevant conflicts of interest to declare.

Inhibition of glutaminase (GLS), the principal enzyme in the glutamine utilization pathway that coverts glutamine (Gln) to glutamate (Glu), is an attractive therapeutic approach in many cancers. Gln plays a unique role in the metabolism of proliferating cancer cells, providing building blocks to sustain cell proliferation and regulating redox homeostasis and signal transduction pathways. Recent findings indicate that leukemic cells depend on Gln as a major carbon source for growth and survival [Willems et al., Blood, 2013]. We previously reported that a subset of acute myeloid leukemia (AML) cell lines are sensitive to Gln deprivation as well as inhibition of glutaminase by the small molecule BPTES [Matre et al. ASH 2013 #606]. Here we report the efficacy of CB-839, a novel, potent, orally bioavailable GLS inhibitor currently under clinical investigation, in the Gln-dependent subset of AML. First, expression of GLS gene splice variants glutaminase C (GAC) and kidney glutaminase (KGA) and of the GLS2 gene was determined through analysis of RNA sequencing data from 173 newly diagnosed AML patients in the TCGA dataset. Of the GLS gene splice variants, the expression levels of GAC were much higher than those of KGA; GLS2 was expressed at low levels. Levels of both GAC and KGA mRNA were significantly higher (two-sample Wilcoxon test) in AML patients with complex cytogenetics and monosomal karyotype (n=31) than in those with diploid AML (n=88, p=0.019 and p=0.01); GAC levels were higher in core-binding factor AML (n=14) than in diploid AML (p=0.018). These findings indicate high expression of the GLSGAC splice variant in specific AML subsets. Analysis of a panel of AML cell lines showed that, in a subset of leukemia cells, CB-839 treatment decreased viable cell number and induced apoptosis. In sensitive cell lines (Molm14, OCI-AML3, MV4;11), CB-839 decreased viable cell number with IC50s between 10nM and 100nM and induced significant apoptosis. HL60, MOLM13, KG1α, and OCI-AML2 cells were less sensitive (IC50 100-1000nM) and responded with minor induction of cell death. CB-839 decreased viability by >40% in blasts from 9 of 20 (45%) primary AML samples. GC- or LC-MS metabolic profiling of OCI-AML3 and THP1 cell lines as well as primary patient samples revealed that GLS inhibition by BPTES or CB-839 was accompanied by concomitant decrease in concentration of downstream GLS metabolites such as glutamate, α-ketoglutarate (a-KG), aspartate, fumarate, and malate. Investigation of the effects of CB-839 on mitochondrial OXPHOS by the Seahorse Bioscience XF96 Analyzer showed that CB-839 exposure for 16 h caused a dose-dependent decrease in maximal respiratory capacity in OCI-AML3 cells, indicating reduced availability of the substrates for OXPHOS. Similar results were obtained upon treatment with BPTES and in AML cells stably transduced with GLS shRNA. Gln, through Glu, is a precursor for cellular α-KG, which can undergo further metabolism through the Krebs cycle or be further metabolized to 2-hydroxyglutarate (2-HG) by mutant isocitrate dehydrogenase (IDH). In THP1 cell lines stably transduced with doxycycline-inducible mutant IDH1-R132H or IDH2-R140Q construct, CB-839 exposure for 4 days reduced intracellular 2-HG oncometabolite levels by >50%. This was associated with induction of differentiation marker CD11b and morphological signs of differentiation in CB-839–treated IDH2-R140Q cells [30%±2% increase in CD11b mean fluorescent intensity (p In summary, these results indicate that GLS is a relevant therapeutic target in AML, warranting future inclusion of GLS inhibitors in the armamentarium of multi-agent therapeutic approaches. In particular, reduction of production of the oncometabolite 2-HG in conjunction with therapeutic blockade of Gln metabolism may serve as a tailored therapeutic strategy in IDH-mutated AML cells. Disclosures Konopleva: Calithera Biosciences: Research Funding.

CD47 is an anti-phagocytic cell surface protein mediating cancer cell evasion of phagocytosis by the innate immune system. Preclinical data suggest that CD47 is appears to be an indispensable means by which many cancer cells, including cancer stem cells, overcome intrinsic expression of their pro-phagocytic “eat me” signals. Blockade of CD47 with mAbs enables phagocytosis of cancer cells in vitro and in vivo. We have developed a novel humanized mAb (huCD47-Ab) that specifically binds CD47 and blocks it from interacting with its ligand, signal regulatory protein–α; (SIRPα), on phagocytic cells, resulting in the phagocytosis and elimination of cancer cells through their “eat me” signals. Normal cells generally do not express eat me signals, and are unaffected by CD47 blocking mAb. We observed expression of CD47 on aggressive pediatric tumors, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), Ewing's sarcoma, neuroblastoma, and several cancers of the central nervous system (CNS), including atypical tetroid rhabdoid tumor (ATRT), primitive neuroectodermal tumor (PNET), medulloblastoma (MB), and pediatric high-grade glioma (pHGG). In-vitro phagocytosis assays, of primary patient samples shows significant engulfment by human peripheral blood derived macrophages upon incubation with the HuCD47-Ab antibody as compared to control. Furthermore we observe potent anti-tumor efficacy and survival benefit in MB, pHGG, ATRT and PNET orthotopic xenografts models establishing the ability of the huCD47 antibody to across the blood brain barrier by the CD47 blocking antibody. Using a novel human neural stem cell/brain tumor co-engraftment orthotopic xenograft model we show specificity of the HuCD47 antibody to only tumor cells and not normal human cells. We further observe potent anti-tumor efficacy in non-CNS tumor (ALL, AML and Osteosarcoma) xenograft models as well. Furthermore we demonstrate that HuCD47-Ab can be administered safely to non-human primates at therapeutic serum levels. Pre-IND and IMPD meetings with the FDA in the US and the MHRA in the UK, have been held, and the clinical trials for adult patients with advanced stage malignancies will start in early 2014. After determining safety in these phase I trials, we initiate protocols for adult CNS tumors as part of expansion cohorts, and subsequently extend the trials to pediatric cancer patients, given manageable toxicity and signs of efficacy in the adult patients. This abstract is also presented as Poster A86. Citation Format: Siddhartha S. Mitra, Sharareh Gholamin, Jens-Peter Volkmer, Abdullah Feroze, Jie Liu, Achal Achrol, Lijuan Wang, Leanne Sayles, Michael Zhang, Kathleen Sakamoto, Michelle Monje-Deisseroth, Yoon-Jae Cho, Alejandro Sweet-Cordero, Ravi Majeti, Samuel Cheshier, Irving Weissman. Overcoming immune evasion in pediatric hematologic and solid tumor malignancies: A preclinical study using a humanized anti-CD47 antibody. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR12.

PI3Kinase/Akt signaling is frequently activated in acute myelogenous leukemia (AML) and inhibitors of this pathway have shown some promise in both pre-clinical and early clinical studies. In particular, much attention has been focused on components of the mTOR complex as downstream effectors of Akt activation that promote cell proliferation and survival in AML and other malignancies. We have discovered a direct effect of activated and phosphorylated Akt in increasing ribosomal RNA (rRNA) synthesis in both leukemic cell lines and primary AML cells by a novel pathway that is independent of mTOR. We have defined TIF-90 as a splice variant of TIF-1A, a molecule that tethers RNA Polymerase I to the ribosomal DNA promoter and is required for rRNA synthesis. TIF-90 is localized in the nucleolus and directly co-locates with Pol I in imaging, pull down, and rDNA promoter occupancy studies. Activation and phosphorylation of Akt correlates with high levels of TIF-90 expression and increased TIF-90 occupancy of the rDNA promoter in primary AML cells, while overexpression of constitutively activated Akt (Akt-Myr) increases rRNA synthesis in a TIF-90-dependent fashion in cell lines. Activation of Akt increases the expression of TIF-90 by inhibiting its ubiquitination and proteasomal degradation by the p53 ubiquitin ligase, Mdm2. Furthermore, activated Akt directly inhibits the cleavage of the actin-binding protein, Filamin A, into a 90 kDa fragment that interacts with TIF-90 and prevents it from occupying the rDNA promoter. Expression of the 90 kDa Filamin A cleavage fragment alone is sufficient to inhibit rRNA synthesis by binding to TIF-90. Thus, inhibition of Filamin A cleavage by Akt is associated with increased rRNA synthesis. The Akt inhibitor AZD8055, but not the mTORC1 inhibitor Rapamycin, promotes the cleavage of filamin A, decreasing both TIF-90 occupancy of the rDNA promoter and rRNA synthesis. Examination of 21 primary AML samples reveals an inverse correlation between the phosphorylation of Akt and the cleavage of Filamin A; conversely, AZD8055 inhibits both Akt phosphorylation and rRNA synthesis in these samples while promoting the cleavage of Filamin A. These data demonstrate a novel pathway by which Akt directly enhances rRNA synthesis. This pathway is active in primary AML cells and in cell lines that have high levels of phosphorylated Akt. Inhibition of rRNA synthesis has been shown to result in nucleolar disruption, cell cycle arrest, and ultimately cell death. Our data support an mTOR-independent effect of Akt activation on cell proliferation that would require direct targeting of PI3Kinase or activated Akt to have a major therapeutic effect in AML. Disclosures: No relevant conflicts of interest to declare.

Background Despite the addition of tyrosine kinase inhibitors to treatment regimens for Philadelphia chromosome-positive (Ph+) precursor B cell acute lymphoblastic leukemia (B-ALL), the prognosis for adults with this disease remains poor, highlighting the need for better treatments. Ph+ B-ALL is characterized by frequent deletions in transcription factors required for normal B cell development. While these mutations are not obviously “druggable” by conventional means, these findings indicate that B cell maturation arrest is crucial to disease pathogenesis. This suggests that strategies to induce differentiation of B-ALL blasts might provide a novel avenue for therapy, but, to date, no such therapies have been developed. Previous studies have demonstrated that normal B cell progenitors retain the potential to transdifferentiate into cells of the myeloid lineage, and we hypothesized that B-ALL cells might possess this latent myeloid potential as well. Methods and Results To investigate the transdifferentiation potential of B-ALL cells, we FACS-purified B-ALL blasts from 12 patients with Ph+ disease and exposed them to myeloid differentiation-promoting cytokines in vitro for 12 days. In 6 of 12 cases, culturing leukemic blasts under these conditions led to their transdifferentiation into cells that morphologically resembled normal human macrophages. These cells expressed typical monocyte/macrophage markers including CD14, CD11b, and CD11c and downregulated the B cell marker CD19. In addition, the transdifferentiated cells were able to perform phagocytosis and generate an oxidative burst, functions that are typical of macrophages. Using gene expression profiling and gene set enrichment analysis (GSEA), we found that the transdifferentiated B-ALL blasts possess a gene expression profile that most closely resembles macrophage/monocyte gene expression signatures. Furthermore, using fluorescence in situ hybridization for BCR-ABL and immunoglobulin heavy chain clonality assays, we demonstrated that these macrophages were clonally related to the B-ALL blasts initially isolated from the patients. The CCAAT/enhancer-binding protein alpha (CEBPα) has been shown to induce transdifferentiation of normal precursor B cells to the macrophage linage, and we sought to determine whether this effect would be seen in primary B-ALL blasts as well. Similar to the effect seen using myeloid cytokines, we found that ectopic expression of CEBPα in B-ALL blasts led to upregulation of CD14 and downregulation of CD19 in these primary human B-ALL cells. To determine if transdifferentiation impairs leukemogenicity, we transplanted macrophages derived from primary B-ALL samples into immunodeficient NOD/SCID/IL2R-gamma null (NSG) mice. The transdifferentiated cells failed to engraft whereas primary Ph+ B-ALL blasts showed robust engraftment, demonstrating a detrimental effect of myeloid transdifferentiation on leukemogenicity. B-ALL blasts that remained CD19hi/CD14lo after culture were still capable of engrafting NSG mice and causing disease, indicating that ex vivo culturing alone did not eliminate leukemogenicity. Conclusions Here, we demonstrate that primary Ph+ B-ALL blasts can transdifferentiate to the myeloid lineage by either exposure to myeloid differentiation-promoting cytokines or ectopic expression of CEBPα. The resulting cells resemble normal human macrophages in morphology, immunophenotype, gene expression, and function. Most importantly, we show that transdifferentiated B-ALL blasts failed to engraft immunocompromised mice and cause leukemia. Our findings suggest that the induction of transdifferentiation represents a novel therapeutic strategy for the treatment of Ph+ B-ALL. Disclosures: No relevant conflicts of interest to declare.

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in about 15% of acute myeloid leukemia (AML) patients. Studies of paired samples at diagnosis and relapse have demonstrated that IDH mutations, in contrast to FLT3 or RAS mutations, are stable during disease evolution. This finding indicates that a small population of cells harboring IDH mutations can persist in remission and eventually contribute to relapse. Therapeutic strategies that eradicate this IDH-mutated population have the potential to result in long-term remission. Small molecule inhibitors specific for the mutant IDH enzymes have recently been developed (Wang et al Science 2013), but it is not known if they are effective in targeting primary AML cells including the leukemic stem cell (LSC) compartment in vivo. We sought an alternative approach to target IDH-mutated cells based on the concept of non-oncogene addiction which refers to the increased dependence on a subset of non-mutated genes for survival in response to activation of a specific oncogene. This dependency can be exploited therapeutically by inhibiting the activity of these non-oncogenes resulting in selective elimination of malignant cells, a phenomenon known as synthetic lethality (SL). In an effort to identity SL targets against IDH mutations, we performed a pooled lentiviral RNA interference (RNAi) screen to search for genes that, when inhibited, led to the selective elimination of mutant IDH1 expressing cells. Our lentiviral short hairpin RNA (shRNA) library consisted of 27,500 unique shRNAs targeting 5,043 human genes of relevance to cancer biology. Each shRNA was tagged with a unique barcode sequence which permitted downstream identification by sequencing. The lentiviral shRNA library was transduced into a human AML cell line engineered to express mutant IDH1 (R132H) under the control of a doxycycline-inducible promoter. Following transduction, the cells were cultured in doxycycline to deplete shRNAs that were synthetic lethal to mutant IDH1. The relative abundance of each shRNA was subsequently determined by high-throughput sequencing of the barcode. Using a stringent algorithm designed to minimize false positive hits, the prosurvival gene BCL-2 was identified as one of the top SL hits. We confirmed that RNAi-mediated knockdown of BCL-2 expression was selectively lethal to mutant IDH expressing AML cell lines and further demonstrated that exposure to a cell-permeable form of (R)-2-hydroxyglutarate, the oncometabolite produced by mutant IDH, was sufficient to induce BCL-2 dependence. Similarly, pharmacologic inhibition of BCL-2 with ABT-199, a novel orally bioavailable and highly specific inhibitor of BCL-2 (Souers et al Nature Medicine 2013), was significantly more toxic to mutant IDH expressing AML cell lines than isogenic cell lines with wildtype (WT) IDH. We next investigated the impact of IDH mutation status on ABT-199 sensitivity of primary AML cells and found that FACS-purified blasts with IDH mutations were 10-fold more sensitive to ABT-199 than blasts with WT IDH in ex vivo culture conditions. Normal cord blood hematopoietic stem and progenitor cells were highly resistant to ABT-199 treatment ex vivo suggestive of a wide therapeutic index. To demonstrate in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with primary human IDH-mutated leukemic cells with either ABT-199 at a dose of 100 mg/kg/day or vehicle control for 7 consecutive days by oral administration. Bone marrow engraftment analysis before and after treatment showed a 10 to 20-fold reduction in leukemic burden in ABT-199 treated mice, whereas no difference was seen in vehicle-treated mice. Importantly, bone marrow cells collected from ABT-199 treated mice failed to engraft in secondary transplant recipients indicative of a loss of LSC activity. In separate experiments, lentiviral transduction of BCL-2 shRNA vectors into IDH-mutated primary AML cells to knockdown BCL-2 expression impaired their engraftment in NSG mice, further validating the detrimental effect of BCL-2 inhibition on IDH-mutated LSCs. In summary, our results indicate that IDH mutations increase BCL-2 dependence in leukemic cells including LSCs and identify a subgroup of patients that is likely to respond to pharmacologic BCL-2 inhibition. Our data provide the preclinical rationale for investigating the use ABT-199 in this patient subgroup in clinical trials. Disclosures: No relevant conflicts of interest to declare.

Abstract 210 The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. Our data indicate that HSCs (Lin-CD34+CD38-CD90+CD45RA-) from MDS patients exhibit widespread gene dysregulation (3,258 differentially expressed genes, FDR We used flow cytometry to assess the expression of 25 candidate disease markers on HSCs in bone marrow (BM) samples from patients with therapy related MDS (n=26). Nine surface markers were upregulated on MDS HSCs as compared with cord blood HSCs in at least 50% of samples. The most frequently upregulated surface marker was CD99 (85%). CD99 was also upregulated on HSCs in 27/37 (73%) de novo MDS cases. To determine whether CD99 upregulation was specific to MDS, we analyzed 78 paired diagnosis/relapse AML specimens encompassing all non-M3 subtypes of AML and a variety of cytogenetic subgroups. CD99 was found to be frequently overexpressed both at diagnosis (81%) and at relapse (83%). Notably, the mean intensity of CD99 expression was significantly higher at relapse (p=0.007). This suggests that CD99 is selected for during relapse and may represent a high risk AML marker. To investigate whether CD99 is upregulated in other myeloid disorders, we measured CD99 in CML samples (n=12). CD99 was decreased in chronic phase CML HSCs (7/10) but increased in blast crisis CML HSCs (2/2), suggesting that it may be a marker of leukemic transformation. Nevertheless, its overexpression in low-risk MDS suggests a distinct role in MDS biology. In CD34+ AMLs, CD99 expression was significantly higher in the stem cell enriched CD34+CD38- fraction compared with bulk blasts (p=0.003). In most samples (91%), CD99 high cells within the CD34+CD38- fraction exhibited a CD90-CD45RA+ “LMPP-like” immunophenotype, previously shown to be enriched for leukemia-initiating cell activity, whereas CD99 low cells exhibited a CD45RA- phenotype resembling normal HSCs and MPPs. Thus, CD99 may be preferentially expressed at higher levels on leukemic stem cells (LSC). We therefore used CD99 expression levels to prospectively purify potential LSCs (high CD99) from residual normal HSCs (low CD99) by sorting CD34+CD38- cells from three primary AML specimens into complete methylcellulose. CD99 low cells generated more myeloid colonies than CD99 high cells (mean 33 and 12, respectively, p=0.02). PCR-based assays revealed that molecular abnormalities associated with the prospectively sorted leukemias were present in CD99 high derived colonies, but absent in CD99 low derived colonies (two Flt3-ITD+ and one AML1-ETO+ samples tested). Transplantation of CD34+CD38-CD99 low cells from a Flt3-ITD+ AML sample into sublethally irradiated NOD/SCID/IL2-R null mice (NSG) (n=2) led to lympho-myeloid engraftment at 12 weeks (2/2 transplanted mice), with Flt3-ITD absent in the engrafted cells. Together, these data indicate that CD99 expression can distinguish LSCs from residual normal HSCs. To study the function of CD99 in AML, we measured CD99 expression on 14 AML cell lines, identifying 12 with high levels, including HL60 and MOLM13. Using a lentiviral vector to introduce a CD99 shRNA into HL60 cells, we achieved an 8.2-fold reduction in CD99 surface expression. Cells expressing the CD99 shRNA exhibited decreased growth in vitro compared with vector control (p=0.05). When these cells were transplanted into NSG mice, animals transplanted with CD99 shRNA expressing cells demonstrated a significant improvement in survival compared with vector controls (median OS 26d vs. 22d, p=0.04, log-rank). Conversely, NSG mice transplanted with MOLM13 cells overexpressing CD99 (4.7-fold increased surface expression) demonstrated a significantly impaired survival as compared with vector controls (median OS 11d vs. 28d, p=0.05, log-rank). In summary, our results establish CD99 as a cell surface protein that is highly expressed in MDS HSCs, AML blasts, and LSCs. Using CD99 expression to prospectively isolate residual normal HSCs from LSCs may provide a useful tool for the performance of purged autologous stem cell transplants. Furthermore, CD99 expression appears to promote leukemic cell growth, particularly in vivo. Further studies are needed to fully establish the role of CD99 in the biology of MDS and AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 1231 Background: Current methods for defining and isolating human hematopoietic stem and progenitor cells using surface markers enrich for unique functional properties of these populations. However, significant functional heterogeneity in these compartments remains with important implications for understanding normal and altered hematopoiesis. Using flow sorting to enrich >10,000 cells as progenitor subpopulations, we previously characterized the gene expression signature of normal human HSC (Majetiet al 2009 PNAS 106(9):3396–3401). We hypothesized that interrogation of the transcriptomes of single cells from this compartment could resolve remaining heterogeneity and help identify and better define features of progenitor cells and hematopoietic stem cells (HSCs). Methods: Using normal human bone marrow aspirates and a FACS Aria II instrument equipped with a specialized single-cell sorting apparatus, we sorted cells enriched for HSCs based on expression of Lin-CD34+CD38-CD90+CD45RA− into 1-cell, 10-cell, 100-cell, and 40000-cell (bulk) representations. We used at least 5 replicates per group and verified single cell deposition by direct visualization. We amplified cDNA from these corresponding inputs using an exponential whole transcriptome amplification (WTA) scheme (Miltenyi SuperAmp), and evaluated gene expression profiles by two microarray platforms (Agilent/GE Healthcare 60K, and Affymetrix U133 plus 2.0), and by RNA-Seq (Illumina). We used gene expression correlation between replicates within and between microarrays as means of assessing methodological reproducibility and estimating population heterogeneity. Results: Whole transcriptome amplification yielded cDNA ranging from 0.2–1 kb for 10 and 100 cells, with significantly lower size distribution of amplified cDNA observed for single cells. Gene expression profiles had significantly better replicate reproducibility and array coverage with the Agilent microarray platform when compared with the Affymetrix U133 Plus 2.0 platform (gene coverage of 84 % for 100 cells, 73 % for 10 cells and 50% for 1 cell for Agilent vs 24 % for 100 cells, 11 % for 10 cells and 5.7% for 1 cell for Affymetrix). RNA-Seq profiling of the same populations is ongoing with major technical optimizations focused on reducing amplification of non-human templates while maintaining library complexity and representation. Using biological replicates for each input size, we observed high inter-replicate correlation levels for expression profiles obtained for bulk sorted HSCs from 8 healthy donors (∼40000-cells, average r=0.97) and for 100-cell and 10-cell inputs from a single donor (r=0.96–0.99, respectively). While intra-array concordance of replicate measurements (n=14642) was high (r>0.91) within each of 5 single cells from a single donor, comparison of 5-single cells from the same donor identified significant heterogeneity, when compared to the 10-cell and 100-cell sub-clusters (Figure 1). Individual genes characteristically expressed by these heterogeneous single cell populations are currently being investigated by FACS and Fluidigm arrays. A larger experiment characterizing 192 single progenitor cells, employing Agilent microarrays and RNA-Seq is currently in progress. Conclusions: Single cell transcriptome profiling is feasible, with best performance on 60-mer microarrays. Single cell transcriptomes exhibit lower, but reasonable levels of reproducibility (r>0.7) and precision as compared with higher cell numbers. Gene expression profiles of single cells capture gene expression heterogeneity in HSCs. Disclosures: No relevant conflicts of interest to declare.