ALBERT

Description: 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

Description: 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.

Description: 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.

Description: CD47 is an anti-phagocytic signal and macrophage checkpoint that acute myeloid leukemia (AML) and other cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with its macrophage receptor SIRPα, thereby promoting phagocytosis of cancer cells. We have found in numerous preclinical studies that anti-CD47 Abs synergize with targeted Abs (such as rituximab and cetuximab) by promoting phagocytosis, and also enable antigen cross-presentation and activation of cytotoxic T cells. These preclinical findings are being translated into clinical results as we have established in several clinical trials promising preliminary evidence of 5F9's therapeutic potential. In this study, we hypothesized that combining 5F9 with azacytidine (AZA) would enhance therapeutic efficacy against AML. AZA (Vidaza®) is a hypomethylating and chemotherapeutic agent indicated for AML. AZA's anti-cancer mechanism of action is believed to be twofold, the first being induction of DNA demethylation and the second being its anti-metabolite activity. Interestingly, it has also been found that AZA can increase the expression of the anti-phagocytic signal, CD47, and the pro-phagocytic signal, calreticulin, in myeloid malignancies. Based on these previous findings, we hypothesized that AML cells may be more efficaciously eliminated using a combination of AZA and 5F9 through enhancement of AML cell phagocytosis. We first tested this hypothesis using an in vitro phagocytosis assay. AML cells (i.e. GFP-expressing HL60 cells) were incubated for 24 hours with 3µM AZA and afterwards, the HL60-GFP cells were co-cultured for 2 hours with either human macrophages plus IgG4 control or 5F9 (10µg/ml). Phagocytosis of HL60 AML cells was calculated as a percentage of GFP-positive macrophages (i.e. the amount of macrophages that engulfed GFP-positive HL60 cells), compared to total number of macrophages. Results were normalized to a condition that produced the maximum amount of phagocytosis (100%). We found that the combination of AZA with 5F9 enhanced the human macrophage-mediated phagocytic elimination of HL60-GFP cells compared to either agent alone (Fig. 1). Next, we asked whether we could confirm our in vitro findings in vivo utilizing an aggressive AML xenograft mouse model. HL60-GFP cells (500,000 cells/per mouse) expressing luciferase were engrafted by intravenous injection into 6 - 8 week old immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Three days post engraftment (PE), bioluminescence imaging was performed to assess AML engraftment based on total flux (photons/sec). Animals were randomized based on these values into 6 treatment cohorts with 8 animals per group. Treatment was performed as follows: (1) control (PBS) was initiated on day 4 PE and continued for 14 consecutive daily doses; (2) AZA (7.5 mg/kg) was initiated on day 4 PE and continued for 5 consecutive daily doses; (3) two cohorts of 5F9 (10mg/kg) were initiated at day 4 or day 7 PE and continued for 14 consecutive daily doses; and (4) two combination cohorts of AZA with 5F9 were initiated according to the 5F9 monotherapy dosing regimens. Routine bioluminescence imaging was performed during treatment and for several months after to assess AML burden and reoccurrence. Both combination cohorts inhibited AML growth as early as day 10 PE, and maintained elimination of growth and overall survival up to 255 days PE. In contrast, the AZA and 5F9 monotherapies initiated at day 7 PE (D7), decreased AML growth at day 10 PE, but failed to produce a durable response. Notably, as the AML expanded, all animals from the AZA cohort died by 46 days PE, and all animals from the 5F9 cohort died by 61 days PE. Of the 8 animals from the 5F9 cohort that received treatment on day 4 PE, only two animals demonstrated progressive disease and did not survive. The remaining animals from this cohort had no detectable AML cancer cells (Fig 2). In summary, the combination of 5F9 with AZA significantly enhanced the phagocytic elimination of AML cells by human macrophages in vitro, enhanced clearance of AML in vivo, and prolonged survival compared to single agent treatment with AZA or 5F9. These results support the rationale for investigating a combinatorial treatment of 5F9 and AZA in patients with AML. A clinical trial with this combination in patients with AML is currently ongoing (NCT03248479). Disclosures Feng: Forty Seven Inc: Employment, Equity Ownership. Gip:Forty Seven Inc: Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Zhao:Forty Seven Inc: Consultancy. Mata:Forty Seven Inc: Employment, Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Majeti:BioMarin: Consultancy; Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chen:Forty Seven Inc: Consultancy, Equity Ownership. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

Description: 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

Description: 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.

Description: Misregulation of genes that play an important role in transcription and chromatin biology is a salient feature of many hematological cancers, including acute myeloid leukemia (AML). Numerous genome-wide analyses in AML have provided evidence linking recurring genetic mutations to epigenomic alterations and disease-specific gene expression programs but the prognostic value and clinical utility of these findings remain unclear. Recently, a novel class of densely clustered cis-regulatory elements termed super-enhancers have emerged as key effectors initiating and maintaining cell type-specific gene expression in a variety of physiological and disease settings, including cancer. Tumor-specific super-enhancers regulate key oncogenes and other cancer-essential genes, providing a novel target discovery strategy for detecting both known and unrecognized cancer dependencies of high diagnostic and therapeutic value. Here we describe the discovery and characterization of super-enhancer domains across a cohort of nearly 50 AML patients and relevant normal hematopoietic stem and progenitor cell controls. We identified unique, clone-specific cancer targets encoding protein kinases, chromatin regulators, and lineage-specific transcription factors, including key drivers of AML such as FLT3, CDK6, and MYB. In addition, we have identified clusters of AML cases with shared super-enhancer domains suggesting convergence on common key drivers of AML. We provide the biological and disease relevance of super-enhancer-associated genes in the context of tumor cell state and drug-target discovery and establish a molecular rationale for developing therapies based on these new insights. Disclosures Eaton: Syros Pharmaceuticals: Employment, Equity Ownership. Lopez:Syros Pharmaceuticals: Employment, Equity Ownership. Ke:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Olson:Syros Pharmaceuticals: Employment, Equity Ownership. Loven:Syros Pharmaceuticals: Employment, Equity Ownership.

Description: 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.

Description: Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Recent genome-scale sequencing efforts have determined that on average, an individual AML case is associated with 5 somatic mutations in recurrently mutated genes. This finding raises the important question of how AML develops from normal hematopoietic stem and progenitor cells. Given that AML is characterized by the sequential acquisition of genetic lesions in a single lineage of cells, and that all cells in the myeloid lineage, apart from HSC, are short-lived, we proposed a model in which serial acquisition of mutations occurs in self-renewing HSC. We investigated this model and the nature of founder mutations through the genomic analysis of de novo AML and patient-matched residual HSC. Using exome sequencing, we defined mutations present in individual AML genomes from 19 cases, and screened for these mutations in the residual HSC. We identified multiple mutations present in residual HSC retaining normal multilineage differentiation in vivo, including mutations in IDH1/2, TET2, DNMT3A, and genes encoding the subunits of the cohesin complex. Through single cell analysis, we determined that as we hypothesized, a clonal progression of multiple mutations occurs in HSC. From these studies, we identified patterns of mutation acquisition in human AML. Our findings support a model in which mutations in "landscaping" genes, involved in global chromatin changes such as DNA methylation, histone modification, and chromatin looping, occur early in the evolution of AML, while mutations in "proliferative" genes such as FLT3 and KRAS occur late. Using this approach, we identified pre-leukemic HSC in a larger cohort of AML patients, and determined that their frequency within the stem cell compartment at the time of diagnosis varied widely from undetectable to nearly 100% of the cells. Stratifying these patients into two groups with either high or low frequencies of pre-leukemic HSC demonstrated that patients in the high group had much worse overall and relapse-free survival than those in the low group, indicating that the presence of pre-leukemic HSC may be critical for eventual clinical outcomes. To further investigate the response of pre-leukemic HSC to treatment, we analyzed the persistence of pre-leukemic mutations in patients in remission and found CD34+ progenitor cells and various mature cells that harbor pre-leukemic mutations. These findings indicate that pre-leukemic HSC can survive induction chemotherapy, identifying these cells as a potential reservoir for the re-evolution of relapsed disease. Finally, through the study of several cases of relapsed AML, we demonstrate various evolutionary patterns for the generation of relapsed disease, and show that some of these patterns are consistent with involvement of pre-leukemic HSC. Thus, our studies of pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations, suggest a potential mechanism contributing to relapse, and constitute a cellular reservoir that may need to be targeted for more durable remissions. Disclosures Majeti: Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: 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.