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Background: The normal function of isocitrate dehydrogenase 1 (IDH1) is to catalyze the conversion of cytoplasmic isocitrate to α-ketoglutarate (aKG) in the Krebs cycle. In hematologic malignancies a hotspot mutation at arginine 132 within the conserved active site of IDH1 leads to loss of the expected Krebs Cycle reaction as well as production of the oncometabolite 2- hydroxyglutarate (2-HG). In acute myeloid leukemia (AML) increased cellular 2-HG levels has been shown to impact the tumor microenvironment and promotes tumorigenesis via paracrine stimulation as well repression of the tumor associated immune system. Ivosidenib (AG-120) is a small molecule inhibitor of mIDH1 and is FDA approved for the treatment of patients (pts) with relapsed refractory IDH1 mutant AML. Ivosidenib is known to lower 2-HG levels, independent of response. By reducing 2-HG levels that promote immune escape of IDH-mutant tumors with ivosidenib, we hope to harness the known programmed death receptor pathway dysregulation in AML and high risk myelodysplastic syndrome (MDS). Study Design and Methods: IVO-NIVO is a single arm, multi-institutional phase II investigator initiated clinical trial with a safety cohort to confirm safety and tolerability of combination AG-120 and nivolumab. To be included pts must have MDS or AML with a documented IDH1 mutation in blood or bone marrow within 30 days of inclusion based on mutational testing by PCR or sequencing in a CLIA certified laboratory and be willing to undergo a bone marrow biopsy. Pts will initially start therapy with AG-120, 500mg by mouth daily for 1 cycle. A cycle is defined as 28 days of therapy. On cycle 2 day 1 enrolled pts will receive nivolumab 480 mg that will subsequently be infused every four weeks. Disease response to treatment will be serially assessed through the evaluation of blood (at least monthly) and bone marrow biopsies and aspirates. A complete response assessment will be performed after 6 cycles of therapy. Transplant eligible pts may proceed to allogeneic stem cell transplant (ASCT) if complete response (CR) or morphologic complete remission with incomplete count recovery (CRi) is achieved. Pts that are not eligible for ASCT will continue to receive AG-120- nivolumab combination therapy until documented progression or unacceptable toxicity (figure 1). Safety Cohort: We will have an interim safety analysis after the first 6 pts finishing the first cycle of combination therapy to ensure dosing is safe and tolerable. Pts will undergo clinical assessment with each cycle of therapy, with particular attention to known toxicities like differentiation syndrome that may be life threatening. If 0 to 1 of the first 6 subjects experienced unacceptable toxicities the study will continue as planned. If 2 pts experienced unacceptable toxicities, accrual will be temporarily suspended in order to conduct a review of safety data. If after review, a safety issue is identified and can be corrected, the protocol will be amended accordingly, and enrollment will restart. If an additional 2 pts (4 of 12) or more experience an unacceptable toxicity the patient will be taken off trial and the trial will be terminated. Statistical plan: It is estimated that 45 pts will be enrolled on trial. Using optimal Simon Stage 2 minimax design, if less than 7 of the first 16 pts respond the treatment will be study will be stopped for futility (type 1 error 0.05, type 2 error 0.2). Primary endpoint: We aim to improve the cumulative response rate after 6 cycles of therapy from 40% to 60% (Two-sided alpha risk of 0.05, 1-Beta of 0.8 = 38 pts + 7 additional pts for potential drop-off observed in prior studies) in pts with mIDH1 relapsed/ refractory AML and MDS. Based on published data, it is known that overall response rate is approximately 40% to single agent AG-120 in the relapsed/ refractory IDH1 mutant AML population. Therefore, we expect a 20% ORR improvement compared to historical control after 6 cycles of treatment, based on MDS International Working Group 2006 criteria and AML MDS International Working group MDS/AML criteria. Secondary endpoints: Progression-free survival (the interval between the time of initiation of olaparib to the time of documentation of olaparib failure or last follow-up), overall survival (the interval between the time of initiation of olaparib to the time of death or last follow-up) and percentage of pts that can be bridged to ASCT. Disclosures Prebet: novartis: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Consultancy; novartis: Honoraria; pfizer: Honoraria; novartis: Honoraria; Boehringer Ingelheim: Research Funding; novartis: Honoraria; pfizer: Honoraria; pfizer: Honoraria; Tetraphase: Consultancy; Agios: Consultancy, Research Funding; pfizer: Honoraria; novartis: Honoraria; Boehringer Ingelheim: Research Funding. OffLabel Disclosure: We will use Ivosidenib and nivolumab in combination as a part of a clinical trial to improve care of patients with relapsed/ refractory AML and MDS

Background: Recurring mutations have been identified in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) which translate to therapeutic targets. Isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations occur in ~20% of AML, and up to 12% of patients with MDS. Three conserved mutational hotspots in the IDH enzymes alter their function and lead to the production of (R)-2-hydroxyglutarate (2HG), an oncometabolite with numerous downstream effects, including impaired DNA damage repair. Specifically, homologous recombination (HR) is impaired by inhibiting the function of histone demethylases that are critical for HR and recruitment of the HR machinery to sites of DNA damage. In HR deficient tumors poly-ADP ribose polymerase (PARP) enzymes mediate a key salvage pathway. PARP inhibition in HR deficient tumors leads to synthetic lethality via simultaneous inhibition of HR and SSB mediated DNA repair. Our group previously demonstrated synthetic lethality with PARP inhibition in IDH mutant cells lines, and other IDH mutant models including primary patient-derived cell lines and genetically-matched tumor xenografts. Study Design and Methods: The PRIME trial (NCI10264) is a proof of concept, biomarker-driven, multi-institution, phase II open label clinical trial to assess the overall response of IDH1/2 mutant relapsed/refractory AML and MDS to PARP inhibitor monotherapy with olaparib. The clinical trial is executed by the Experimental Therapeutics Clinical Trials Network of the NCI. The Cancer Therapy Evaluation Program will provide olaparib. Eligibility criteria include documented IDH1 or IDH2 mutation in blood or bone marrow within 30 days of enrollment based on mutational testing by PCR or sequencing in a CLIA certified laboratory and willingness to undergo a bone marrow biopsy. Patients will be treated with olaparib 300 mg q12hrs each day of a 28-day cycle, using a tablet formulation, until disease progression, unacceptable toxicity, withdrawal of consent or death. Blood and bone marrow samples for 2-HG analysis will be collected prior to starting therapy and after 1 cycle (28 days), cycle 2, 3, 6, 9, 12 or when there is concern for disease progression (Figure 1). A Simon two-stage optimal design will be used to test the null (ORR=10%) versus the alternative hypothesis (ORR=40%) in each arm. In the first stage, 9 patients will be accrued in each arm. If one or fewer responses are observed in these 9 patients, that arm will be stopped early for futility. Otherwise, 11 additional patients will be accrued for a total of 18 in each arm. We reject the null hypothesis if at least 5 responses are observed in these 20 patients. In each arm, we have approximately 90% power to detect a 30% increase in ORR at a one-sided type I error rate of 0.05. Primary endpoint: Overall response rate (ORR) of 40%, i.e., a 30% ORR improvement (40% vs. historical control ORR = 10%) based on MDS International Working Group 2006 criteria and AML MDS International Working Group 2003 criteria after 6 cycles of treatment. Cumulative ORR will include complete remission, complete remission with incomplete blood count recovery, partial response, and bone marrow complete remission. Secondary endpoints: Progression-free survival (the interval between the time of initiation of olaparib to the time of documentation of olaparib failure or last follow-up) and overall survival (the interval between the time of initiation of olaparib to the time of death or last follow-up) for the trial. Exploratory studies: The PRIME trial will also test the utility of 2-HG and DNA damage markers such as γ-H2AX as potential biomarkers of response to olaparib. Using multiple viability assays on leukemia cell lines and bone marrow cultures we will assess synergistic therapeutic combinations to further improve outcomes in this patient population. To confirm efficacy in vivo without undue toxicity, promising combination therapies will be confirmed in cytokine-humanized immunodeficient "MISTRG" mice. We will also examine the impact of PARP inhibitors on the genomic, proteomic, metabolomic and immunologic landscape of IDH 1/2-mutant hematologic malignancies using DNA whole exome sequencing (WES), RNA-Seq, and liquid chromatography-mass spectrometry assessment of oncometabolites. Disclosures Bindra: Cybrexa: Consultancy, Equity Ownership. Prebet:pfizer: Honoraria; pfizer: Honoraria; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; novartis: Honoraria; Genentech: Consultancy; Boehringer Ingelheim: Research Funding; novartis: Honoraria; Boehringer Ingelheim: Research Funding; Agios: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria. OffLabel Disclosure: We will be using PARP inhibitors as a novel therapy for patients with relapsed or refractory AML and high risk MDS based on preclinical data.

Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects. To address this issue, we profiled the transcriptome of HEL erythroleukemic cell lines expressing wild-type (WT) and mutant U2AF1. U2AF1 S34F and Q157R mutants induced widespread alterations in splicing patterns of 3250 and 1791 genes respectively, with an overlap of 23.8% genes. On the other hand, we observed only minor alterations in gene expression levels. Meta-analysis and comparison with published RNA sequencing datasets on U2AF1 mutants revealed both conserved and unique splicing changes, with a strong enrichment for genes involved in cell cycle (P=6.7E-15) and DNA repair (P=2.6E-5). Confirming previous literature, U2AF1 S34F preferentially leads to the exclusion of exons preceded by 3' splice sites bearing an intronic UAG motif, while U2AF1 Q157R preferentially excludes exons starting with the AGA motif (Figure 1A). Collectively, the S34F mutation has a stronger effect on splicing, ultimately decreasing the global translation state of cells. To understand how mutations eventually result in the observed splicing alterations, we also profiled with unprecedented resolution the RNA interactome of the physiological and pathological U2AF heterodimer. We first performed enhanced crosslinking immunoprecipitation (eCLIP) on U2AF1 WT, U2AF1 mutants and U2AF2. Comparison of U2AF1 and U2AF2 binding profiles revealed a high degree of similarity, suggesting that they mostly bind to RNA as a tight dimer. Only by performing fractionated eCLIP on U2AF1 we were able to isolate, at the molecular level, the individual contributions of the U2AF components in the recognition of the 3' splice site. In particular, we deconvolved the U2AF2 signal, insisting on the polypyrimidine region, and the U2AF1 signal, peaking on the AG dinucleotide at the intronic end (Figure 1B). Importantly, the S34F mutant displays an aberrant binding profile, with a specific peak on the nucleotide in position -3, matching the sequence specificity previously observed in aberrant splicing events (Figure 1A-B). Systematic analysis of bound junctions suggests a complex model where the S34F mutation does not simply abrogate the ability of U2AF1 to bind splicing junctions ending with the UAG sequence, but rather alters the conformation of the U2AF complex bound to RNA, resulting in a differential ability to effectively recruit the U2 complex. To confirm this model, we identified and validated a set of gain-of-function splice junctions in genes contributing to hemopoiesis and cell cycle, characterized by increased binding of U2AF1 S34F mutant and parallel decreased binding of U2AF2. In summary, we identified novel RNA sequence and structure determinants of U2AF complex conformation, uncovered by the binding alterations induced by the U2AF1 S34F mutation. Our data further dissect the complexities of post-transcriptional regulation and provide the basis for development of U2AF directed cancer therapies. Disclosures Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.

Acute myeloid leukemia (AML) remains a disease with high unmet medical need. While most patients respond to initial therapy, few are cured, relapse rates are high, and most patients eventually develop life-threatening complications. FLT3-mutant disease is a particularly aggressive subtype. Recent approval of drugs targeting FLT3-mutant disease have improved short-term outcomes but not all patients respond, and duration of response is limited by secondary mutations that impede FLT3 inhibitor (FLT3i) binding (intrinsic factors) and by secreted stromal factors that activate alternative pro-survival pathways (extrinsic factors). Heat-shock protein 90 (HSP90) is a chaperone protein involved in many cellular processes and inhibition of HSP90 can have pleiotropic effects in targeting cancer cells such as degradation of oncoproteins that drive survival and proteins that mediate protective signaling. Here, we describe the nonclinical activity of LAM-003, an orally bioavailable HSP90 inhibitor (HSP90i) under clinical development for AML. To assess the anti-leukemic activity of LAM-003, we tested a panel of AML cell lines and primary AML samples. LAM-003 inhibited proliferation of both FLT3-mutant and wild-type cell lines, with preferential activity against cells harboring FLT3-ITD (geometric mean FLT3-ITD EC50 = 670 nM [n=8] vs FLT3 WT EC50 = 1400 nM [n=16]). Additionally, we observed that LAM-003 was potent in a subset of the FLT3 WT cells. To explore whether LAM-003 was effective against tumors driven by oncoproteins that are client proteins of HSP90, we focused on AML cells harboring FLT3-ITD. We confirmed that LAM-003 reduced cell surface FLT3-ITD expression and downstream signaling in MV-4-11 and MOLM-13 cells, consistent with HSP90i-mediated degradation of FLT3-ITD. In BA/F3 cells expressing FLT3-ITD with various secondary resistance mutations, we observed that LAM-003 elicited a dose-dependent reduction of FLT3 mutant cell surface expression. Moreover, BA/F3 cells expressing FLT3-ITD and the F691L mutation exhibited the expected resistance to crenolanib, yet LAM-003 retained anti-proliferative activity. Additionally, MOLM-13 cells harboring a FLT3 D835Y mutation demonstrated expected resistance to the FLT3i sorafenib and tandutinib yet remained sensitive to LAM-003. Finally, primary AML blasts harboring a D835 mutation displayed sensitivity to LAM-003 when tested ex vivo. To evaluate the potential of LAM-003 to overcome bone-marrow-stroma-derived resistance, FLT3-ITD AML cell lines (MV-4-11, MOLM-13, MOLM-14) were assayed in unconditioned or stromal-cell-conditioned medium. Conditioned medium dramatically reduced the potency of FLT3i but LAM-003 demonstrated equal potency under both conditions. We also showed that stromal cell co-culture induced FLT3i resistance in MOLM-13 cells whereas LAM-003 retained potent activity. Recognizing that the inherent genetic heterogeneity of AML blasts limits the curative potential of a single drug, we performed in vitro studies to identify drugs that synergize with LAM-003 in 3 FLT3-ITD AML cell lines. Synergy was demonstrated with FLT3i, daunorubicin, azacitidine or cytarabine, with the most robust synergy being observed with venetoclax. Extending the evaluation to AML cells wild type for FLT3 and cell lines from other hematologic indications (multiple myeloma, diffuse large B-cell lymphoma and mantle cell lymphoma), we found that the synergy was not limited to cells harboring FLT3-ITD, but rather correlated with BCL-2 abundance, suggesting a fundamental mechanism of action that depends on BCL-2 family-mediated survival. Mechanistic studies demonstrated that the combination of LAM-003 and venetoclax inhibited AKT-mediated regulation of GSK3B, resulting in MCL-1 degradation. In vivo studies using a MOLM-13 systemic model of FLT3-ITD AML demonstrated that LAM-003 monotherapy significantly improved animal survival and that the combination of LAM-003 and venetoclax significantly prolonged animal survival compared with each single agent. These nonclinical studies demonstrate that LAM-003 exhibits antileukemic activity, overcomes mechanisms of FLT3i resistance and potently synergizes with existing AML drugs. As such, our data provide strong rationale for evaluation of LAM-003 in an ongoing clinical trial in patients with AML (NCT03426605). Disclosures Beeharry: AI Therapeutics: Employment, Equity Ownership. Landrette:AI Therapeutics: Employment. Grotzke:AI Therapeutics: Employment. Gayle:AI Therapeutics: Equity Ownership. Young:AI Therapeutics: Employment, Equity Ownership. Miller:Incuron, Inc.: Consultancy; Cleveland Biolabs, Inc: Employment, Equity Ownership; Calistoga Pharmaceuticals, Inc.: Equity Ownership; AI Therapeutics: Consultancy, Equity Ownership; VelosBio Inc.: Employment, Equity Ownership; Acerta Pharma, Inc.: Equity Ownership. Xu:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rothberg:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lichenstein:AI Therapeutics: Employment, Equity Ownership.

Hematopoietic stem cell (HSC) self-renewal and lineage output are orchestrated by multiple regulatory layers, including RNA modifications. N6-methyladenosine (m6A) is an abundant modification found in RNAs which affects the translation and stability of modified transcripts. The effects of m6A are determined by m6A writers (install m6A), erasers (remove m6A) and readers (recognize m6A). In embryonic stem cells, deletion of the m6A writer METTL3 enforces a naïve pluripotent state. This raises the question of whether m6A RNA methylation analogously regulates stem cell self-renewal and differentiation in somatic stem cells such as HSCs. Using a Vav-Cre/Mettl3 (VCM3) hematopoietic-specific knockout mouse model, we show that loss of the RNA m6A writer METTL3 in fetal HSCs results in hematopoietic failure and perinatal lethality. At E14.5 (FL) hematopoiesis loss of Mettl3/m6A results in hematopoietic failure with expansion of Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) that are defective in the production of progenitors and mature blood cells, as evidenced by failure to rescue lethally irradiated congenic recipient mice in transplant experiments. The relative defect I hematopoiesis was further demonstrated by competitive transplant experiments, in which transplanted KO FL were consistently out-competed by WT FL. Interestingly, BrdU/7AAD labeling reveals a significant proliferative defect with reduced BrdU uptake in VCM3 KO FL cells, and specifically in Lin-c-Kit+Sca-1- (LK) progenitor cells. RNA-seq analysis of FL LSK cells reveals that loss of m6A results in upregulation of multiple 2'-5'-oligoadenylate synthetase (OAS) family genes. Interestingly, the majority of OAS family genes are not m6A modified in several m6A sequencing data sets. We therefore hypothesized that the OAS genes might be regulated at the transcriptional level. We performed cleavage under targets and release using nuclease (CUT&RUN) analysis and found that OAS family genes are transcriptionally activated, as evidenced by significant increase in H3K4 trimethylation (H3K4me3) at their respective promoter regions. The OAS family genes are activated by the presence of double stranded RNA (dsRNA), which can arise either endogenously or as a pathogen-associated trigger of the innate immune system in the context of viral infection. The dsRNA response includes three major response mechanisms. First, OAS genes facilitate RNase L dimerization, which mediates cleavage of cellular tRNA and rRNA, resulting in translation and proliferation arrest. Second, activation of the PKR/eIF2a pathway also results in translation arrest. Lastly, activation of the MDA-5/RIG-I/MAVS response induces interferon signaling, which further limits cellular proliferation. Staining of VCM3 WT & KO FL cells with the dsRNA-specific J2 antibody directly demonstrated significant accumulation of dsRNA in KO FL cells. We further demonstrated activation of the OAS/RNaseL axis in KO cells by quantification of tRNA-His-36 cleavage, which acts as a sensitive marker of RNase L activity. Phosphorylation of both PKR and eIF2a in KO cells demonstrated enhanced activity of this pathway. Lastly, we found a significant upregulation of interferon pathway genes in KO cells, and demonstrated that CRISPR mediated knockout of Mavs in VCM3 KO FL cells diminishes the activity of interferon response genes and rescues the Mettl3 KO phenotype in colony forming unit (CFU) assays. These results suggest that in the absence of m6A, endogenous dsRNA formation is enhanced, resulting in an associated inflammatory response which partially accounts for the hematopoietic defect observed in Mettl3 KO mice. In conclusion, our study suggests a novel, protective role for the m6A RNA modification in preventing endogenous dsRNA formation and aberrant activation of a detrimental innate immune response during mammalian hematopoietic development. Disclosures Fan: IsoPlexis: Other: served on the scientific discovery board, Patents & Royalties; Singleron Biotechnologies: Other: served on the Scientific Advisory Board; BioTechne: Other: served on the Scientific Advisory Board. Flavell:Rheos Biomedicines: Equity Ownership; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; SMOC: Equity Ownership; Zai labs: Consultancy.

Introduction: While the use of immune checkpoint inhibitors (ICI) is now an established standard of care in the management of many solid tumors, the efficacy of this approach in myeloid neoplasms (MN) is still being explored. Preliminary data regarding efficacy presented to date are modest at best. Since many leukemia physicians lack extensive experience using ICI, and MN patients experience deep thrombocytopenia and bleeding risk precluding lung or colon biopsies to assess for immune-related adverse events (irAEs), MN presents a particularly difficult space for ICI development. Particular concern may center around distinguishing progressive infections (especially fungal) from immune pneumonitis and the appropriateness of steroids in such settings We report a single-center experience on the use of ICI in MN. Methods: We retrospectively analyzed data from patients (pts) with acute myeloid leukemia (AML) or higher-risk (International Prognostic Scoring System [IPSS] INT-2 or high-risk) myelodysplastic syndromes (MDS) treated with ICI therapy from April 16th, 2015 to June 3rd, 2019. We collected data on patient age, sex, race, Performance Status (ECOG PS), WBC, hemoglobin (Hgb), platelet count, serum metabolic and coagulation parameters, AML disease risk (cytogenetic and molecular) or MDS risk by IPSS score, prior lines of therapy (for refractory/relapsed [R/R] pts), ICI used (classified by targeted immune checkpoint to maintain trial confidentiality) as well as concurrent leukemia-directed therapy, irAEs, transfusion requirements, response to therapy and overall survival (OS). Results: Twenty-seven (13 AML and 14 MDS) pts received ICI in the context of clinical trials. Median age was 73 years, 63% were male, and 95% had an ECOG PS of 0 or 1 (Table 1). Nineteen pts (59%) had R/R disease, of whom 14 previously-received hypomethylating agent (HMA) therapy while 5 received intensive induction therapy. Median number of prior therapies was 1 (range [R], 1-3). Median follow-up was 11.3 months (R, 0.5-46.7). 54% of AML pts had cytogenetically or molecularly-defined poor risk disease; 86% of MDS pts had IPSS high-risk disease. Complex karyotype and TP53 mutation was noted in 35% and 35% of pts, respectively. (Table 1). Fifteen pts (56%) received a PDL1 inhibitor in combination with a HMA, while 4 (19%) pts received either a PD1 inhibitor in combination with a histone deacetylase inhibitor or CTLA4 inhibitor monotherapy. Two pts (7%) received dual ICI with both a PDL1 inhibitor and CTLA4 inhibitor (Table 1). The median number of ICI cycles administered was 4 with the main reason for discontinuation being lack of response (39%). Notably 27% discontinued because of intolerance to therapy. Almost half (n=12, 44%) of pts experienced an irAE; 4 pts had a grade 3 irAE. There were no grade 4 irAEs, but one patient with MDS developed multi-organ grade 3 irAEs and despite appropriate steroid therapy died due to irAEs with necrotizing myocarditis (with multifocal lymphocytic inflammatory infiltrate) and pericarditis confirmed on post-mortem examination. Of pts experiencing an irAE, 69% (including all pts with grade 3 irAEs) received high-dose corticosteroid therapy with a median time to initiation of 3 days (R, 1-14). No biopsies were performed due to bleeding risk in setting of cytopenia. Two of six newly-diagnosed AML pts achieved CR in response to ICI+HMA; one of 2 newly-diagnosed MDS pts developed CR following ICI+HMA. No R/R AML or MDS pts responded. Half of pts proceeded to an additional line of therapy after ICI. Two pts (7%) proceeded to allogeneic stem cell transplant (alloSCT), both receiving anti-CTLA4 monotherapy x 4 cycles with last cycle 62 and 400 days prior to alloSCT, respectively. One pt developed stage 1 acute graft versus host disease (GVHD) of gut with complete response to steroids and another with moderate, classic chronic GVHD without unusual GVHD. No AML pts in this study remain alive. Median OS for newly-diagnosed AML pts treated with ICI + HMA was longer than those with R/R disease (11.6 vs. 5.0 months) at a median follow-up of 11.6 months (Table 2). Conclusions: Patients with AML and MDS treated with ICI have a significant risk of irAEs requiring provider diligence and possibly quick and life-saving intervention. Larger prospective studies of ICI in AML and MDS patients are needed to better define the role of ICB in management of these cancers. Disclosures Podoltsev: Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AI Therapeutics: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Research Funding; Arog Pharmaceuticals: Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Sunesis Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; Samus Therapeutics: Research Funding; Kartos Therapeutics: Research Funding. Prebet:Genentech: Consultancy; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; novartis: Honoraria; Boehringer Ingelheim: Research Funding; novartis: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; pfizer: Honoraria; novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Agios: Consultancy, Research Funding; novartis: Honoraria; pfizer: Honoraria; pfizer: Honoraria. Gore:Celgene Corporation: Consultancy, Research Funding. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:Seattle Genetics: Consultancy, Other: fees for non-CME/CE services ; miRagen: Consultancy; Mallinckrodt: Consultancy; Spectrum: Other: fees for non-CME/CE services ; Acrotech: Consultancy; Eisai: Consultancy. Huntington:Celgene: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; AbbVie: Consultancy; Pharmacyclics: Honoraria; Genentech: Consultancy; DTRM Biopharm: Research Funding. Neparidze:Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees; Janssen Scientific Affairs, LLC: Research Funding. Zeidan:Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Seattle Genetics: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; BeyondSpring: Honoraria; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding. OffLabel Disclosure: Immune checkpoint inhibitor therapy is not approved for use in patients with acute myeloid leukemia or myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are heterogeneous bone marrow failure neoplasms marked by cytopenias, reduced quality of life and predilection to transform into AML. While several treatments for AML have recently been approved, the available treatments for MDS are lacking, and adaptation of AML therapy to MDS is complicated. This is due, in part, to the heterogeneity of MDS. Despite this heterogeneity, most clonal cells in MDS have an imbalance of mitochondrial-controlled BCL2 family proteins resulting in dysregulated apoptosis. These anti- (or pro-) apoptotic proteins compete for ligand to block (or promote) apoptosis, providing an opportunity to selectively target anti-apoptotic proteins and advance therapy for MDS. Venetoclax (VEN), a newly FDA-approved therapy that specifically inhibits the anti-apoptotic protein BCL2, has yielded response rates of up to 50-70% in elderly AML including impressive responses in transformed MDS which previously failed DNMTi (DiNardo et al, 2019, Wei et al, 2019). Upregulation of the anti-apoptotic protein, induced myeloid cell leukemia-1 (MCL1), is a known resistance mechanism in AML resistant or refractory to BCL2 inhibition (Pan et al, 2014), and MCL1 increases when some MDS samples are treated with BCL2 inhibitors (Jilg et al, 2016). Therefore, strategic inhibition of BCL2 and/or MCL1 is a logical therapeutic approach in MDS. We have shown efficacy of MCL1 inhibitors in the laboratory against AML patient samples that are dependent on MCL1 protein or resistant to BCL2 inhibition, including AML cells that arose from MDS (Ramsey et al, 2018). Here, our goal was to determine the sensitivity of MDS cells to inhibition of specific anti-apoptotic proteins, elucidate the characteristic determinants of response, and investigate synergy with combined BCL2 and MCL1 inhibition. We cultured MDS patient samples and determined the in vitro sensitivity of 35 MDS patient samples to selective BCL2, BCL-XL and MCL1 inhibitors using CellTiter-Glo to determine the relative cell viability concentrations (GI50) for each selective inhibitor after 48 hours of exposure. While there was little sensitivity to BCL-XL inhibition across all samples, we detected a gradient of low to high response to the BCL2 inhibitor with low to higher blast count MDS subtypes; higher blast count MDS (EB1 and EB2) were more sensitive than low blast count subtypes (RS-SLD/MLD and MLD). Interestingly, nearly all MDS subtypes were sensitive to the selective MCL1 inhibitor, S63845. To determine if there were any correlations between sensitivity to specific inhibitors and mutational status, a targeted NGS panel of 37 commonly mutated genes in myeloid disease was conducted on all samples. As expected, we observed an increased number of SF3B1 mutations in the lower blast count MDS-RS patient samples. Likewise, though there were only two samples in this cohort containing mutations in PTPN11, one was completely resistant to BCL2 inhibition.(Stevens et al, ASH 2018) Otherwise, we did not observe any correlation between specific mutations and BH3 dependence. Since upregulation of MCL1 is seen in VEN treated MDS cells and is a known resistance mechanism for VEN treatment in AML, we treated these same patient samples with VEN+S63845 to determine any synergistic benefit of combining these drugs. While there were differential responses to VEN monotherapy between subtypes, all MDS subtypes exhibited response benefit to the addition S63845 to VEN. Drug synergy was confirmed with the Zero Interaction Potency model. These results were further corroborated by increased annexin-V staining and reduced colony formation in methylcellulose. Toxicity experiments in the MISTRG immunocompromised animal model indicate that the combination of selective inhibitors of MCL1 and BCL2 are tolerated, and treatment of MDS within these xenografts is underway and will be presented. Overall, our data suggests that higher blast count MDS subtypes (EB1 and EB2) are more likely to respond to VEN monotherapy than low blast count subtypes, while all MDS subtypes may respond to MCL1 inhibition. Moreover, drug synergy can be obtained across all subtypes of MDS by combining BCL2 and MCL1 inhibitors. BCL2 inhibition is changing the standard of care in AML, thus, refining the design of clinical trials testing BCL2 and MCL1 inhibitors in MDS and the precision of patient selection for therapy is a great priority. Disclosures Savona: Boehringer Ingelheim: Patents & Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are heterogeneous clonal disorders. Isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations are detected in ~20% of AML and ~5% of MDS, in which they confer gain of a neomorphic function that leads to the production of (R)-2-hydroxyglutarate (2HG). Targeted inhibition of mutant IDH1/2 has resulted in significant responses in IDH1/2 mutant MDS and AML but is not curative and patients relapse (Stein et al. Blood 2016, DiNardo et al. N Engl J Med 2018). 2HG accumulation inhibits the function of histone demethylases (KDM4A and KDM4B) that are critical for the homologous recombination (HR) DNA repair pathway and consequently for the repair of DNA double strand breaks (DSBs) (Mallette et al. EMBO J 2012, Sulkowski et al. Sci Transl Med 2017). In HR deficient tumors, Poly-ADP-Ribose Polymerase (PARP) is essential for DNA single strand break (SSB) repair. In IDH mutant tumors PARP inhibitors induce synthetic lethality by suppressing the repair of SSBs, which eventually get converted into DSBs (Javle and Curtin Br J Cancer 2011). We previously demonstrated that in AML, IDH1/2 mutations impair DNA damage response by inducing a defect in HR, and that this renders leukemia cells susceptible to PARP inhibitors in vitro. We hypothesized that this vulnerability would also exist in IDH mutant MDS and more importantly, that this vulnerability would persist in MDS/AML resistant to IDH1/2 inhibitors. To determine whether PARP inhibition targets IDH mutant MDS/AML in vivo, we took advantage of 2 syngeneic mouse models of MDS and AML relying on co-mutation of SRSF2/IDH2 and FLT3/IDH2, respectively. Olaparib (PARP inhibitor) effectively targeted IDH2 mutant but not IDH2 wild type MDS/AML (Fig. 1A). We next sought to determine whether PARP inhibition mediated synthetic lethality persists in MDS/AML resistant to targeted IDH inhibition. We transduced IDH2 mutant murine cells with IDH2 WT or IDH2 MUT lentiviral vectors carrying one of two published resistance mutations. While these resistance mutations conferred resistance to the targeted IDH2 inhibitor Enasidenib, cells remained sensitive to Olaparib (Fig. 1B). Patient MDS/AML is heterogeneous and in general carries additional genetic mutations and epigenetic alterations. We therefore engrafted IDH1/2 WT and MUT MDS/AML patient samples in cytokine humanized immunodeficient mice and treated with vehicle of Olaparib. Engrafted mice were assigned to vehicle or Olaparib 8 weeks after transplantation based on equal engraftment levels determined by BM aspiration. Mice were treated with vehicle or Olaparib via IP injection for 21 days. Human engraftment levels and plasma 2-HG levels were significantly reduced in Olaparib treated animals when compared to pre-treatment and vehicle-treated mice (Fig. 1C). Of note, when equal numbers of huCD34+ cells from vehicle or Olaparib treated mice were transplanted into next generation mice, engraftment was significantly higher for recipients of human cells from vehicle than Olaparib treated mice, suggesting that Olaparib is toxic to leukemia initiating cells. In contrast, IDH WT MDS/AML was insensitive to Olaparib treatment (Fig. 1C). In conclusion, PARP inhibition is effective in vivo against IDH mutant MDS/AML and can overcome targeted IDH inhibitor resistance. Disclosures Flavell: Zai labs: Consultancy; SMOC: Equity Ownership; Troy: Equity Ownership; Artizan Biosciences: Equity Ownership; GSK: Consultancy; Rheos Biomedicines: Equity Ownership. Prebet:Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; novartis: Honoraria; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; Agios: Consultancy, Research Funding; novartis: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; Genentech: Consultancy; pfizer: Honoraria; novartis: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria; pfizer: Honoraria. Bindra:Cybrexa: Consultancy, Equity Ownership.

Hematopoietic stem cells (HSC) mainly engage glycolysis while leukemia stem cells (LSC), such as in acute myeloid leukemia (AML), heavily rely on mitochondrial (Mt) respiration (i.e. oxidative phosphorylation, OxPhos) to fuel energy. Growing evidence suggests this metabolic reprogramming confers therapeutic vulnerabilities in AML. BCL2 is overexpressed in LSC from AML patients, while BCL2 inhibitors, such as venetoclax (VEN), have been shown to suppress OxPhos in LSC and to eradicate LSC. In clinical practice, half of the patients fail to respond to VEN. VEN is a costly medication and patients who are resistant to VEN forgo alternative treatment at that time. Prediction of the response to VEN and strategies to circumvent resistance are urgently needed. BCL2 is shown to increase Mt Ca2+ levels ([Ca2+]m), which enhance OxPhos through activation of the Ca2+-sensitive dehydrogenases within the tricarboxylic acid (TCA) cycle. G protein-coupled receptor 68 (GPR68) is a proton sensor, activating phospholipase C that leads to releasing of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and elevation of cytosolic Ca2+ levels ([Ca2+]c). This prompted us to examine the cooperative effect of GPR68 and BCL2 on the Ca2+/OxPhos pathway in AML, and particularly in LSC. Expression of leukemic oncogenes (i.e. MLL-AF9 and HRASG12D) in mouse hematopoietic stem and progenitor cells, such as Lineage-Sca-1+cKit+ (LSK) cells or granulocyte-monocyte progenitor cells, promote leukemogenesis as evidenced by serial colony formation in vitro and leukemia development in vivo. We found significantly reduced colonies in oncogene-expressing LSK cells from Gpr68 knockout mice compared to wild type mice. Deletion of Gpr68 reduced [Ca2+]c in oncogene-expressing LSK cells. We next examined the function of GPR68 in human AML cell lines. Knockdown of GPR68 with shRNA reduced cell growth and colony formation, and induced apoptosis in AML cells. Knockdown of GPR68 also reduced [Ca2+]c and Mt membrane potential (Δψm) in AML cells, indicating reduced Mt OxPhos. These results suggest that GPR68 regulates the Ca2+/OxPhos pathway in AML cells. We next examined the cooperative effect of GPR68 and BCL2 by jointly inhibiting their activities with pharmacological agents (i.e. GPR68 antibody and VEN, respectively) in AML cell lines and AML patient-derived xenograft models. Of note, the expression of GPR68 was positively correlated with the sensitivity to VEN in AML cells. For AML cells that were resistant to VEN, GPR68 antibody but not an unrelated antibody increased the sensitivity to VEN by enhancing apoptosis, indicating that GPR68 and BCL2 co-regulate AML cell survival. We next examined the mechanism of this synthetic lethality by measuring cellular respiration. Single treatment with VEN reduced Δψm, ATP production and O2 consumption in AML cells. Cotreatment with VEN and GPR68 antibody further reduced Δψm, ATP production and O2 consumption in AML cells, indicating that GPR68 and BCL2 co-regulate Mt OxPhos. Given that GPR68 releases Ca2+ from ER to cytosol, while BCL2 maintains [Ca2+]m by inhibiting its extrusion from Mt, we hypothesize that the GPR68/BCL2 axis relocates Ca2+ from ER to Mt. As expected, treatment with GPR68 antibody reduced [Ca2+]c. Cotreatment with VEN and GPR68 antibody increased [Ca2+]c, indicating enhanced extrusion of Ca2+ from Mt to cytosol by VEN. Consistently, cotreatment with VEN and GPR68 antibody reduced the activity of isocitrate dehydrogenase, the rate limiting enzyme in the TCA cycle, in AML cells. These results indicate that GPR68 and BCL2 co-regulate the Ca2+/OxPhos pathway in AML cells and that co-inhibition of GPR68 and BCL2 may enhance lethality and overcome VEN resistance. In summary, our study suggests that the GPR68/BCL2 axis co-regulates AML cell survival by relocating Ca2+ from ER to Mt thus enhancing Mt OxPhos, and that disruption of the GPR68/BCL2 axis provides a novel therapeutic strategy to overcome resistance to VEN. Disclosures No relevant conflicts of interest to declare.

The murine host has remained a readily available and ethically acceptable model for the study of human diseases and therapeutic testing. Immunodeficient mouse models support engraftment of human hematopoietic stem cells (HSC) but with limitation in efficiency and mature lineage representation. Combined knock-in of several non-crossreactive human cytokines (M-CSF, IL3/GM-CSF, and Thrombopoietin) into the corresponding murine loci in the SRG strain (in short termed "MISTRG") has enhanced engraftment and maintenance of human HSCs with multi-lineage differentiation (Rongvaux et al. Annu Rev Immunol 2013, Deng et al. Nature 2015, Saito et al. Blood 2016, Theocharides et al. Haematologica 2016). Despite robust HSC engraftment and myelo- and erythropoiesis in bone marrow (BM), all humanized immunodeficient mouse models lack of mature human red blood cells (RBC), platelets, and myeloid cells in peripheral blood (PB) (Rahmig et al. Stem Cell Reports 2016, Yurino et al. Stem Cell Reports 2016, Song et al. Nat Commun 2019). Yet, full maturation and representation of all myeloid lineages in PB is essential to study diseases of the HSC, such as MDS, and of the RBC, such as sickle cell anemia or malaria. With universal absence of a murine adaptive immune system the culprit is likely the murine host's innate immune system. Previous studies have shown that treatment of engrafted mice with liposomal clodronate that abrogates murine (and human) macrophages, with or without cobra venom factor that eliminates complement, can increase mature human circulating RBC, but only transiently and with significant toxicity. We first sought to determine the site of huRBC sequestration and destruction. Intravital imaging after injection of CFSE labelled huRBC identified the murine liver as the major site of RBC destruction. While muRBC rapidly circulate through the liver circulation, huRBC have greatly increased transit times and are sequestered in liver vessels. We hypothesized that humanization of the murine host's liver could potentially alleviate huRBC sequestration and significantly increase circulating huRBC. In previous studies deletion of fumarylacetoacetate hydrolase (Fah) in the Rag-/-Il2rg-/- background has allowed humanization of the liver and served to study diseases such as malaria (Vaughan et al. J Clin Invest 2012). The liver is the site of synthesis of numerous proteins, some of which directly impact hematopoiesis and blood cells, such as complement. We deleted the Fah gene via CRISPR/Cas9 in MISTRG mice and crossed MISTRG-Fah-/- mice to homozygosity (MISTRGFah). MISTRGFah are viable, fertile, and healthy when maintained on drinking water supplemented with 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC), that blocks tyrosine metabolism upstream of Fah and prevents buildup of hepatotoxic metabolites. At 8 weeks of age we implanted MISTRGFah mice with commercially available, adult human hepatocytes (HuHep) via direct injection into the splenic vein, followed by gradual withdrawal of NTBC water. Regulated buildup of intracellular fumarylacetoacetate results in death of murine Fah-/- hepatocytes and regeneration with HuHep with up to 90% repopulation by HuHep (Azuma et al. Nature biotechnology 2007). When plasma human albumin levels reached 2mg/dL, indicative of significant (~80%) HuHep repopulation, we sublethally (80cGy) irradiated HuHepMISTRGFah mice and engrafted each mouse with 105 fetal liver (FL) derived CD34+ cells. 10 weeks post transplantation, mice were analyzed for engraftment and specifically erythroid maturation in PB. HuHepMISTRGFah mice had significantly higher levels of BM and interestingly spleen erythropoiesis and circulating huRBC in PB (Fig.1 a). CD235a+ huRBC in HuHepMISTRGFah mice are enucleated (Hoechst neg) and mature as evident by loss of CD49d (ITGA4) and gain of Band3 staining (Hu et al. Blood 2013) (Fig.1 b). Interestingly, human erythroid cells in MISTRG but not HuHepMISTRGFah mice are coated with murine complement C3 (muC3) (Fig.1 c) suggesting that liver humanization results in loss of muC3 expression. In conclusion, we have generated the first humanized mouse model with fully mature, circulating huRBC when engrafted with human CD34+ stem and progenitor cells. Ongoing studies are testing the applicability of this model to MDS and sickle cell disease. Disclosures Flavell: SMOC: Equity Ownership; Zai labs: Consultancy; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; Rheos Biomedicines: Equity Ownership.

Whole-exome sequencing identifies a novel somatic mutation in MMP8 associated with a t(1;22)-acute megakaryoblastic leukemia

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Autoimmune haemolytic anaemia (AIHA) is a disease characterised by the production of pathological antibodies that attach to the surface of a patient9s own red blood cells, resulting in haemolysis. It can present in either an acute or a chronic manner. In addition to the obvious consequence of anaemia, there are other potentially deadly complications that can arise from AIHA, such as venous thromboembolism (VTE) and pulmonary hypertension. We report a case of a 52-year-old woman who developed a pulmonary embolism (PE) soon after being diagnosed with AIHA. Despite having a very small pulmonary venous clot burden, she developed profound haemodynamic compromise with severe right ventricular dysfunction, which quickly reversed with inhaled nitric oxide treatment. This case makes an interesting observation of cell-free haemoglobin-associated nitric oxide scavenging as a mechanism of pulmonary hypertension and highlights the possible benefit of nitric oxide in treatment.

NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1+/− mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1+/− cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1+/− cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1+/− cells contributes to the MDS phenotype associated with NPM1 deficiency.

Inherited deficiency of acid β-glucosidase (GCase) due to biallelic mutations in the GBA (glucosidase, β, acid) gene causes the classic manifestations of Gaucher disease (GD) involving the viscera, the skeleton, and the lungs. Clinical observations point to immune defects in GD beyond the accumulation of activated macrophages engorged with lysosomal glucosylceramide. Here, we show a plethora of immune cell aberrations in mice in which the GBA gene is deleted conditionally in hematopoietic stem cells (HSCs). The thymus exhibited the earliest and most striking alterations reminiscent of impaired T-cell maturation, aberrant B-cell recruitment, enhanced antigen presentation, and impaired egress of mature thymocytes. These changes correlated strongly with disease severity. In contrast to the profound defects in the thymus, there were only limited cellular defects in peripheral lymphoid organs, mainly restricted to mice with severe disease. The cellular changes in GCase deficiency were accompanied by elevated T-helper (Th)1 and Th2 cytokines that also tracked with disease severity. Finally, the proliferation of GCase-deficient HSCs was inhibited significantly by both GL1 and Lyso-GL1, suggesting that the “supply” of early thymic progenitors from bone marrow may, in fact, be reduced in GBA deficiency. The results not only point to a fundamental role for GBA in immune regulation but also suggest that GBA mutations in GD may cause widespread immune dysregulation through the accumulation of substrates.

Little is known about how hypomethylating agents (HMAs) have been adopted into the treatment of myelodysplastic syndromes (MDS). We conducted a population-based study to assess the use of HMAs among 4,416 MDS patients (age ≥66 years) who were diagnosed during 2001–2005 and followed up through the end of 2007. Multivariate logistic regression models were utilized to evaluate the role of various patient characteristics. 475 (10.8%) patients had received HMAs by 2007, with the proportion increasing over time. Patients who were white (odds ratio (OR)= 0.66, 95% confidence interval (CI): 0.46–0.95), male (OR= 1.47, 95% CI: 1.19–1.82), young (Ptrend

Introduction: Venous thromboembolism (VTE), defined as deep venous thrombosis (DVT) and pulmonary embolism (PE), is a cause of significant morbidity and mortality worldwide, with an overall incidence of about 10,000,000 cases per year. The majority of VTEs are believed to be attributable to genetic factors. Yet, the five established heritable thrombophilias of factor V Leiden (FVL), prothrombin (PT) gene mutation, antithrombin (AT) deficiency, protein C (PC) deficiency, and protein S (PS) deficiency comprise only a small portion of VTEs, suggesting that further genetic factors contributing to VTE risk are unrecognized. In our previously published study, we performed whole exome sequencing (WES) in 64 patients with VTE and developed a 55-gene extended thrombophilia panel which identified 40 pathogenic or likely pathogenic variants or variants of uncertain significance (VUS) involving 22 different genes. Here, we present updated data from an expanded patient cohort. Methods: Blood was obtained from 101 patients with VTE. Genomic DNA was extracted and WES performed; mean coverage of the exome was 100x with 98% of the exome covered >/= 20 times. Variants were filtered for an allele frequency of 7% in the GnomAD database. A targeted analysis of the 55 genes in the extended thrombophilia panel was then performed. Variants in these genes were classified according to ACGM criteria as pathogenic, likely pathogenic, VUS, benign or likely benign. The number of patients with pathogenic or likely pathogenic variants and VUS in VTE patients was compared to a control population of 237 patients who had WES performed for reason other than VTE. The results of WES were also compared to those of traditional laboratory-based thrombophilia testing. Further, 17 VUS underwent in silico protein modeling to evaluate structural modifications. Results: Of the 101-patient study population, 46 were men and 55 women; 62% were Caucasian, 22% African American and 5% Hispanic; 15% had unprovoked VTE, 71% had provoked, 12% had both; and 55% had first degree family members with VTE. WES and extended thrombophilia testing identified a pathogenic or likely pathogenic variant or VUS in 69/101 (68%) VTE patients compared to 6/237 (2.5%) controls, a statistically significant difference (p-value A total of 72 genetic variants (26 previously published and 46 novel) were identified in 30 genes in the extended thrombophilia panel. 18 variants were categorized as pathogenic or likely pathogenic and 54 as VUS. 32 patients had pathogenic or likely pathogenic variants in one or more of the major thrombophilia genes, with FVL being the most common, and AT and PS deficiency being more common than PT gene mutation (FVL, n=15; PT gene mutation, n=3; AT deficiency, n=5; PC deficiency, n=3; PS deficiency, n=6). Of pathogenic variants in major thrombophilia genes, 15 were previously reported in literature, while 13 were novel. Of the 52 VUS, 17 were subjected to in silico analysis, including protein modeling, which suggested impaired protein folding in 14 variants. Conclusions: WES and extended thrombophilia testing reveal a high frequency of pathogenic or likely pathogenic variants or VUS in VTE patients compared to controls. These variants demonstrate good concordance with traditional laboratory-based thrombophilia testing. Several novel pathogenic or likely pathogenic variants were identified. In silico studies and protein modeling suggest that many VUS identified by this approach are likely to be deleterious to protein function. The results may underlie a strong genetic component to VTE. Next steps include further characterization of VUS using protein modeling, biochemical analysis, and epidemiological and familial studies to understand potential roles these variants may play in thrombosis. Table. Table. Disclosures Camire: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy.

The mechanism underlying thrombocythemia in patients with iron deficiency anemia remains unknown. Iron metabolism is highly conserved in humans and mice. We used Tmprss6-/- knockout (KO) mice as a genetic model of chronic iron deficiency anemia to study the effects of iron deficiency on lineage commitment. Tmprss6 is expressed in the liver, but not in hematopoietic cells, and is required for iron uptake from the diet. We observed that Tmprss6-/- mice have elevated platelet counts compared to littermate controls (average x1000/uL ± SD in wild type (WT): 477.9 ± 53.68, Tmprss6-/-: 915.9 ± 57; p CFU assays of single MEPs showed a striking increase in CFU-Mk (unilineage committed Mk progenitors) within the MEP gate in Tmprss6-/- at the expense of the bipotent CFU-Mk/E compared to WT (p To test the effect of altered iron content in vivo, we transplanted WT or KO BM into sublethally irradiated WT or Tmprss6-/- recipients. Tmprss6-/- recipients (of WT or KO BM) exhibited microcytic anemia and hypoferremia when compared to WT recipients (of WT and KO BM). Interestingly, the Mk-bias observed in non-transplanted Tmprss6-/- MEPs was recapitulated in Tmprss6-/-recipients (regardless of transplanted BM genotype) 6 months post-transplant (p < 0.009, n= 3 per group). We show using index single cell sorting followed by CFU assays that in WT mice, surface expression of transferrin receptor 1 (CD71) is a predictor of cell fate; cells from the MEP gate that grow as BFU-E have a higher CD71 mean fluorescence intensity (MFI) (179.9 ± 38.9) than cells that grow bipotent CFU-Mk/E colonies (65.9 ± 13.9) or Mk-only (CFU-Mk) colonies (24.9 ± 7.06; p RNAseq of WT and KO MEPs showed that 137 genes are differentially expressed (fold change: 1.5; p We then asked if the iron regulation of the MEP commitment observed in iron deficient mice was also true for human MEP. In order to disrupt the iron sensing pathways in primary human MEP, we performed shRNA-based knockdown of Transferrin Receptor 2 (TFR2) using two different sequence targets and found a similar decrease in proliferation (p=0.03) and skewing toward Mk commitment (p Regulation of erythroid differentiation by iron is thought to prevent inappropriate iron utilization when body stores are limited. To function in a protective manner, this checkpoint must act at stages prior to erythropoietin-mediated expansion of erythroid progenitors. Here we propose a model where iron acts on the bipotent progenitor, one possible source of Mk and E cells, to bias lineage commitment. These data support that low iron in the environment affects MEP metabolism, leading to lower ERK phosphorylation, slower proliferation and increased Mk commitment. Disclosures No relevant conflicts of interest to declare.

Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML. Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function. In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway. In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. Disclosures No relevant conflicts of interest to declare.

N6-methyladenosine (m6A) is the most abundant RNA modification, with key roles in RNA metabolism and regulation of gene expression. Recent studies have elucidated a role for m6A in normal hematopoiesis and myeloid malignancies. Constitutive deletion of the m6A methyltransferase Mettl3 in zebrafish enforces endothelial identity during the endothelial-to-hematopoietic transition, thus precluding normal developmental emergence of HSPCs. Studies conducted in human CD34+ cells, as well as human and murine AML cell lines have suggested that loss of METTL3 results in resolution of differentiation blockade, and impaired engraftment in murine transplantation assays. However, the effects of METTL3 deletion on hematopoietic stem cells in the context of an intact hematopoietic hierarchy in vivo have not yet been extensively characterized. To study the effects of Mettl3 deletion on the hematopoietic system in vivo, we generated Vav-Cre METTL3-/- (VCM3-/-) mice. Deletion of Mettl3 resulted in embryonic lethality, evidenced by skewing of Mendelian ratios at birth. Occasional stillborn VCM3-/- pups were smaller than wildtype littermates, and exhibited pallor, pancytopenia, and dramatically reduced marrow cellularity. To study the effects of Mettl3 deletion on embryonic hematopoiesis, we isolated fetal liver (FL) at embryonic day 14.5 for analysis. At E14.5, expected Mendelian ratios were preserved. Western blot and qPCR confirmed loss of Mettl3 expression in VCM3-/- mice. Reduction of total m6A levels in VCM3-/- mice was confirmed by ELISA. Flow cytometry for hematopoietic markers demonstrated a significant increase in the total number and frequency of Lin-Sca+c-Kit+ (LSK) cells in E14.5 VCM3-/- FL, with an increase in the frequency of HPC-1 (CD48+CD150-) and HPC-2 (CD48+CD150+) cells. To determine the function of VCM3-/- FL cells, we performed colony forming and transplantation assays. VCM3-/- FL cells demonstrated reduced colony forming ability in methylcellulose culture, and colonies that did arise were morphologically abnormal. VCM3-/- FL cells were also deficient in hematopoietic rescue assays, with all lethally irradiated recipient mice dying by day 14 post-transplant. Transplantation of CFSE labeled cells confirmed that absence of Mettl3 -/- hematopoiesis was not attributable to a homing defect. Competitive transplantation of VCM3-/- FL with Pep3b bone marrow similarly resulted in almost total loss of peripheral blood and bone marrow VCM3-/- engraftment, whereas mice transplanted with VCM3+/+ FL maintained chimerism at 8 weeks. Interestingly, VCM3-/- FL cells and FL LSK cells displayed no differences in apoptotic rate or cell cycle. To determine the mechanism underlying the observed phenotypes, we first performed RNA sequencing of VCM3+/+ and VCM3-/- FL LSK. Mettl3 deletion resulted in the increased expression of 701 transcripts, and reduced expression of 1395 transcripts. Gene ontology (GO) analysis revealed that upregulated genes were enriched for mitochondrial function, ribosome and ribonucleoprotein complex proteins and downregulated genes for cell adhesion and developmental processes. M6A RNA modification affects mRNA stability and translation. To determine the effect of m6A depletion on the hematopoieitic stem and progenitor cell proteome we are in the process of validating changes in protein levels of select genes essential in hematopoiesis. Previously, studies have demonstrated that deletion of METTL3 in human CD34+ hematopoietic cells and AML cell lines promote myeloid differentiation. Interestingly, we see a similar depletion of myeloid progenitors in VCM3-/- FL, with an increased percentage of mature myeloid CD11b+ cells. However, these results also coincide with an increased fraction of LSK HSPCs at E14.5. Interestingly, this resembles the METTL3 knockout phenotype in embryonic stem cells, which results in a reinforced naïve pluripotent state with impaired differentiation. Our ongoing studies seek to determine the role of m6A in FL HSC maintenance and differentiation. Disclosures No relevant conflicts of interest to declare.

Neutrophils play important roles in host innate immunity and various inflammation-related diseases. In addition, neutrophils represent an excellent system for studying directional cell migration. However, neutrophils are terminally differentiated cells that are short lived and refractory to transfection; thus, they are not amenable for existing gene silencing techniques. Here we describe the development of a method to silence gene expression efficiently in primary mouse neutrophils. A mouse stem cell virus-based retroviral vector was modified to express short hairpin RNAs and fluorescent marker protein at high levels in hematopoietic cells and used to infect mouse bone marrow cells prior to reconstitution of the hematopoietic system in lethally irradiated mice. This method was used successfully to silence the expression of Gbeta(1) and/or Gbeta(2) in mouse neutrophils. Knockdown of Gbeta(2) appeared to affect primarily the directionality of neutrophil chemotaxis rather than motility, whereas knockdown of Gbeta(1) had no significant effect. However, knockdown of both Gbeta(1) and Gbeta(2) led to significant reduction in motility and responsiveness. In addition, knockdown of Gbeta(1) but not Gbeta(2) inhibited the ability of neutrophils to kill ingested bacteria, and only double knockdown resulted in significant reduction in bacterial phagocytosis. Therefore, we have developed a short hairpin RNA-based method to effectively silence gene expression in mouse neutrophils for the first time, which allowed us to uncover divergent roles of Gbeta(1) and Gbeta(2) in the regulation of neutrophil functions.

Nat. Biotechnol. 32, 364–372 (2014); published online 16 March 2014; corrected after print 8 November 2017 In the version of this article initially published, the cells labeled Me290 were Me275 cells. Both Me275 and Me290 are human metastatic HLA-A201+ melanoma cell lines, and both of them were obtained from the Ludwig Cancer Institute.

Comorbid conditions have rarely been systematically studied among patients with myelodysplastic syndromes (MDS). We conducted a large population-based study to assess the role of comorbidity in the survival of newly diagnosed MDS patients. This study included 1708 MDS patients (age > or =66 years) diagnosed in the US during 2001-2002, with follow-up through the end of 2004. Hazard ratios (HRs) were estimated using multivariate Cox proportional hazard models. The median survival time was approximately 18 months. Fifty one percent of MDS patients had comorbid conditions. Patients with comorbid conditions had significantly greater risk of death than those without comorbidities. The HR was 1.19 (95% confidence interval (CI): 1.05-1.36) and 1.77 (95% CI: 1.50-2.08) for those with a Charlson index of 1-2 and > or = 3, respectively. The risk of death increases with Charlson index. MDS patients who have congestive heart failure or chronic obstructive pulmonary disease had significantly shorter survival than patients without those conditions, whereas diabetes did not appear to have an impact on survival. This study confirms comorbidity as a significant and independent determinant of MDS survival, and the findings underscore the importance to take comorbid conditions into account when assessing the prognosis of MDS.

Human cytokine knock-in mice are improved in vivo models for multilineage engraftment of mobilized PB CD34+ cells.Humanized mouse models might open new avenues for personalized studies of human pathophysiology of the hematopoietic and immune system.

SummaryMutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2’s normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.

In this issue of Blood, Xiao et al provide a direct link between the OTT protein and regulation of splicing of the RNA for c-MPL, which encodes the thrombopoietin receptor.1

Background A 73-year-old woman presented with acute lower back pain, fever, chills and arthralgias. She had previously had a positive protein derivative test with a negative chest X-ray; her medical history was also remarkable for a mitral valve prolapse. Initial symptoms resolved spontaneously without therapy, but fever recurred with associated arthralgias, myalgias, diffuse and worsening lymphadenopathy, splenomegaly, and bilateral pulmonary infiltrates. Investigations Physical examination, blood and urine cultures, MRI of the spine, echocardiogram, extensive serologies, serum and urine protein electrophoresis, immunofixation electrophoresis, bone-marrow aspiration and biopsy with flow cytometry, cytogenetics, and gene rearrangement studies, CT scan of the chest, abdomen and pelvis, whole-body PET, and lymph-node biopsy for histological examination, immunohistochemistry, and gene rearrangement studies. Diagnosis Angioimmunoblastic T-cell lymphoma. Management Steroids (prednisone, methylprednisolone), levofloxacin, isoniazid with pyridoxine, ciclosporin A, methotrexate, alemtuzumab, broad-spectrum antibiotics, Pneumocystis carinii prophylaxis, vancomycin, and clindamycin.

2-Hydroxyglutarate (2HG) exists as two enantiomers, R-2HG and S-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, while the latter is produced under pathologic process such as hypoxia. Recurring IDH1/2 mutations were first identified gliomas and acute myeloid leukemia (AML), and subsequently they were found in multiple other tumor types. Many IDH1/2-mutant tumors are known to be chemo- and radiosensitive, although the mechanisms underlying this enhanced sensitivity have been elusive. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect which renders tumor cells exquisitely sensitive to Poly (ADP-Ribose) polymerase (PARP) inhibitors. Remarkably, this “BRCAness” phenotype can be completely reversed by small molecule mutant IDH1/2 inhibitors, and it can be entirely recapitulated by treatment with either 2HG enantiomer in cells with intact IDH1/2. We performed a comprehensive series of studies directly implicate two αKG-dependent dioxygenases, KDM4A and KDM4B, as key mediators of the observed phenotype. In addition, we demonstrate that 2HG-induced HR suppression cannot be explained by mutant IDH1/2-associated alterations in NAD+ levels. We have demonstrated IDH1/2-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells and AML bone marrow cultures in vitro, as well as genetically-matched tumor xenografts in vivo. Finally, we have extended these findings to several structurally related and clinically relevant oncometabolites. We demonstrate profound synthetic lethality with PARP inhibitors in tumors which produce these other oncometabolites, and our data suggest a similar mechanism of action via which HR is suppressed. Small molecule inhibition of oncogenic kinases is a pillar of precision medicine in modern oncology, and this approach has been extrapolated to treat IDH1/2-mutant and other oncometabolite-producing cancers with small molecule inhibitors which block the neomorphic activity of the mutant proteins. The findings present here directly challenge this therapeutic strategy, and they instead provide a novel approach to treat these tumors oncometabolite-producing tumors with DNA repair inhibitors. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair and genetic instability. We previously reported that hypoxia suppresses HR, driving genetic instability and conferring a BRCAness phenotype in hypoxic tumor cells. It is tempting to speculate that the findings reported here provide a novel commonality between hypoxia and IDH1/2 mutations as mediating a “hit-and-run” mechanism for genetic instability and tumor progression through 2HG, but at the same time bestowing a vulnerability to PARP inhibition that can be therapeutically exploited. Based on these findings, we are planning a multi-center Phase II trial testing the efficacy of olaparib for the treatment of recurrent IDH1/2-mutant tumors, and we anticipate this trial will be open for enrollment later this year. Citation Format: Parker Sulkowski, Christopher Corso, Nathaniel Robinson, Susan Scanlon, Karin Purshouse, Hanwen Bai, Yanfeng Liu, Ranjini Sundaram, Denise Hegan, Nathan Fons, Gregory Breuer, Yuanbin Song, Ketu Mishra, Henk De Feyter, Robin de Graaf, Yulia Surovtseva, Maureen Kachman, Stephanie Halene, Murat Gunel, Peter Glazer, Ranjit S. Bindra. Oncometabolites induce a BRCAness state that can be exploited by PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-290. doi:10.1158/1538-7445.AM2017-LB-290

Purpose of review Neutrophils rapidly migrate to sites of injury and infection. Egress of neutrophils from the circulation into tissues is a highly regulated process involving several distinct steps. Cell-cell interactions mediated by selectins and integrins and reorganization of the actin cytoskeleton are key mechanisms facilitating appropriate neutrophil recruitment. Neutrophil function is impaired in inherited and acquired disorders, such as leukocyte adhesion deficiency and myelodysplasia. Since the discovery that deletion of all or part of chromosome 5 is the most common genetic aberration in myelodysplasia, the roles of several of the deleted genes have been investigated in hematopoiesis. Several genes encoding proteins of the serum response factor (SRF) pathway are located on 5q. This review focuses, in particular, on the role of SRF in myeloid maturation and neutrophil function. Recent findings SRF and its pathway fulfill multiple complex roles in the regulation of the innate and adaptive immune system. Loss of SRF leads to defects in B-cell and T-cell development. SRF-deficient macrophages fail to spread, transmigrate, and phagocytose bacteria, and SRF-deficient neutrophils show defective chemotaxis in vitro and in vivo with failure of inside-out activation and trafficking of the Mac1 integrin complex. Loss of the formin mammalian Diaphanous 1, a regulator of linear actin polymerization and mediator of Ras homolog family member A signaling to SRF, results in aberrant myeloid differentiation and hyperactivity of the immune system. Summary SRF is an essential transcription factor in hematopoiesis and mature myeloid cell function. SRF regulates neutrophil migration, integrin activation, and trafficking. Disruption of the SRF pathway results in myelodysplasia and immune dysfunction.

The actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization and stability. To determine the function of Tmod3 in platelet biogenesis, we studied Tmod3(-/-) embryos, which are embryonic lethal by E18.5. Tmod3(-/-) embryos often show hemorrhaging at E14.5 with fewer and larger platelets, indicating impaired platelet biogenesis. MK numbers are moderately increased in Tmod3(-/-) fetal livers, with only a slight increase in the 8N population, suggesting that MK differentiation is not significantly affected. However, Tmod3(-/-) MKs fail to develop a normal demarcation membrane system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured Tmod3(-/-) MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud sizes, including abnormally large proplatelet buds containing incorrect numbers of von Willebrand factor-positive granules. Tmod3(-/-) MKs exhibit F-actin disturbances, and Tmod3(-/-) MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod3(-/-) MKs show increased F-actin and redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing.

The overall goal of this project is to develop a model for the study of myelodysplasia (MDS), an acquired bone marrow failure syndrome in the aging population and in cancer survivors. MDS is inherently difficult to study. MDS stem cells cannot be grown in culture and in vivo models are thus the gold standard. However, MDS stem cells are diseased and fail to efficiently engraft in current immunodeficient mouse models. We have optimized engraftment of normal adult HSPC into MISTRG. We have determined optimal cell preparation, cell number, pre-engraftment irradiation dose, transplantation route and recipient age. We have optimized the analysis of engrafted humanized mice. We have successfully transplanted primary MDS bone marrow cells into MISTRG mice with successful engraftment and replication of the donor disease status. We have successfully performed secondary transplantation of MDS BM cells to propagate human MDS in MISTRG mice for therapeutic testing.

Tropomodulins (Tmod) bind tropomyosin and cap the pointed ends of actin filaments. Tmod3 is the only Tmod isoform detected in human and mouse platelets. To study its function in platelets we have e...

Mutations in key factors of the spliceosome occur in over 50% of patients with myelodysplastic syndromes (MDS) and thus splicing represents the most frequently affected pathway in MDS. The vast majority of mutations cluster in four genes: SF3B1, SRSF2, U2AF1, and ZRSR2. Mutations occur in a mutually exclusive manner suggesting a common mechanism by which they cause disease, and yet they segregate to specific subtypes of myeloid malignancies suggesting unique, mutation-specific functions. With the exclusion of ZRSR2, mutations occur at highly restricted residues and are strictly heterozygous, suggestive of gain of function mutations. We and others have shown that mutations affect RNA binding affinity and specificity, and can lead to a variety of outcomes including exon inclusion or skipping, alternative splice site selection, intron retention, and most recently alternative polyadenylation. Overall, these splice changes are subtle: only a small subset of all splice events is affected by these mutations and changes in isoform usage are < 10% for most alternative splice events identified. Furthermore, a direct connection between alternatively spliced transcripts and differentially expressed genes is often not seen. However, these alternative splice events affect genes critical to cellular function and hematopoiesis. Examples include EZH2, ABCB7, macroH2A1, and ATG7. Mutations in SRSF2 result in inclusion of a poison exon in EZH2 resulting in reduced protein levels of this critical epigenetic regulator. Mutations in SF3B1 cause alternative splicing of ABCB7 slating its RNA for non-sense mediated decay. Intriguingly, mutations in U2AF1 promote usage of a distal polyadenylation site in an essential autophagy factor, ATG7, resulting in inefficient translation and decreased protein levels. As a consequence, autophagy is impaired, which can promote cellular transformation, providing a possible explanation for the clonal advantage of cells with splicing factor mutations. Additional research is critical to further determine how alternative splicing contributes to the MDS phenotype with impaired hematopoietic differentiation and cellular function and eventual malignant transformation. Latest findings will be presented here with a focus on mechanistic, functional, and biologic effects of splicing factor mutations. Disclosures No relevant conflicts of interest to declare.

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer,negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.

Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo . MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro . To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2°CFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2°CFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2°CFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2°CFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0–10% of the vector containing 2°CFU-S from 8 recipients and expression in >50% of the vector-containing 2°CFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo .

Deregulation of microRNA (miRNA) expression can lead to cancer initiation and progression. However, limited information exists on the function of miRNAs in cancer maintenance. We examined these issues in the case of myeloproliferative diseases and neoplasms (MPN), a collection of hematopoietic neoplasms regarded as preleukemic, thereby representing early neoplastic states. We report here that microRNA-125a (miR-125a)–induced MPN display a complex manner of oncogene dependence. Following a gain-of-function genomics screen, we overexpressed candidate miR-125a in vivo, which led to phenotypes consistent with an atypical MPN characterized by leukocytosis, monocytosis, splenomegaly, and progressive anemia. The diseased MPN state could be recapitulated in a doxycycline-inducible mouse model. Upon doxycycline withdrawal, the primary MPN phenotypes rapidly resolved after the discontinuation of miR-125a overexpression. However, reinduction of miR-125a led to complex phenotypes, with some animals rapidly developing lethal anemia with extensive damages in the spleen. Forced expression of miR-125a resulted in elevated cellular tyrosine phosphorylation and hypersensitivity toward hematopoietic cytokines. Furthermore, we demonstrate that miR-125a targets multiple protein phosphatases. Our data demonstrate that miR-125a–induced MPN is addicted to its sustained overexpression, and highlight the complex nature of oncogenic miRNA dependence in an early neoplastic state.

Serum response factor and its transcriptional cofactor MKL1 are critical for megakaryocyte maturation and platelet formation. We show that MKL2, a homologue of MKL1, is expressed in megakaryocytes and plays a role in megakaryocyte maturation. Using a megakaryocyte-specific Mkl2 knockout (KO) mouse on the conventional Mkl1 KO background to produce double KO (DKO) megakaryocytes and platelets, a critical role for MKL2 is revealed. The decrease in megakaryocyte ploidy and platelet counts of DKO mice is more severe than in Mkl1 KO mice. Platelet dysfunction in DKO mice is revealed by prolonged bleeding times and ineffective platelet activation in vitro in response to adenosine 5′-diphosphate. Electron microscopy and immunofluorescence of DKO megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. Surprisingly, the DKO mice have a more extreme thrombocytopenia than mice lacking serum response factor (SRF) expression in the megakaryocyte compartment. Comparison of gene expression reveals approximately 4400 genes whose expression is differentially affected in DKO compared with megakaryocytes deficient in SRF, strongly suggesting that MKL1 and MKL2 have both SRF-dependent and SRF-independent activity in megakaryocytopoiesis.

Introduction: Venous thromboembolism (VTE) occurs with an incidence of 1-2 per 1000 individuals per year. Approximately 10-20% of patients with VTE have a heritable thrombophilia involving one of five known genes: Factor V, prothrombin (PT), antithrombin (AT), protein C (PC), and protein S (PS). Significant variability of laboratory functional assays as well as fluctuating plasma levels of AT, PC and PS lead not only to delay in initial thrombophilia screening but also to multiple rounds of costly testing. Whole exome sequencing (WES) is a potentially useful diagnostic tool for inherited thrombophilias as it avoids dependence on laboratory and situational variation in protein levels. We compiled a panel of 33 genes involved in thrombosis with a goal of investigating the diagnostic yield and cost of WES in comparison to traditional thrombophilia testing. Methods: Since January of 2014, we have been performing WES in patients with a personal and family history of VTE seen at Yale New Haven Hospital. Thus far, 18 such patients have had a complete WES analysis. Data regarding patient demographics, number and type of VTE events, family/surgical history, medical co-morbidities, and traditional laboratory testing for inherited thrombophilias was recorded. Costs of each test were determined based on the amount billed to insurance. WES focusing on 33 genes involved in thrombosis was performed and analyzed by the DNA Diagnostic Lab at the Yale School of Medicine. Positive WES testing was defined as identification of a pathogenic variant in a gene known to be associated with thrombophilia, found at a frequency consistent with frequency of the disease, and with evidence that the variant predisposes to thrombosis. Positive laboratory testing was defined as any test that led to an unequivocal diagnosis of Factor V Leiden, PT mutation, homozygous MTHFR mutation with hyperhomocysteinemia, or a deficiency in AT, PC, or PS. Results: All 18 patients (7 male, 11 female) were included in the final analysis. Median age at first VTE was 35.5 years (range, 14-78 years); median number of independent VTE events was 2 (range, 1-9). WES with our 33-gene thrombophilia panel was positive in 11 of 18 (61.1%) patients, while traditional laboratory testing was positive in only 2 of 16 (12.5%) cases (Table 1). There were no statistically significant differences in clinical characteristics between those patients with positive findings on WES versus those without. Identified variants included those in genes with well known roles in thrombosis (SERPINC1, PROS1, F5), and in genes with emerging data regarding thrombosis (HABP2, SERPINA10, SERPIND1). Two patients identified on WES as having PS deficiency, one with AT deficiency, and one with a non-Leiden Factor V mutation had laboratory testing that was either normal or uninterpretable. The total cost of WES at our institution was $1935.00; by comparison, among 16 patients who underwent laboratory testing, median cost of laboratory testing was $2892.70 (range, $406.90-$11419.80), and the cost of laboratory testing exceeded WES in 13 patients. Conclusions: WES using our 33-gene thrombophilia panel has higher diagnostic yield and is more cost effective than traditional thrombophilia testing. With increasing availability and declining cost, this thrombophilia gene panel has the potential to truly transform thrombophilia testing. Further investigation of the diagnostic power and phenotypic correlation of identified mutations is in progress. | Gender | Age (yrs) | Lab testing result | WES result | | ------ | --------- | ------------------------- | --------------------------------------------------------- | | M | 40 | Negative | SERPINA10 (Q384R) heterozygous | | M | 67 | Negative | SERPINC1 (S426W) heterozygous | | M | 30 | Negative | PROS1 (Y234C) heterozygous | | F | 34 | Negative | HABP2 (G508E) heterozygous | | F | 56 | APC resistance | F5 (R506Q) heterozygous | | F | 27 | F2 20210 G>A heterozygous | F2 20210G>A heterozygous; vWF P2063S heterozygous | | F | 37 | Negative | SERPIND1 (R468C) heterozygous | | F | 48 | Negative | F5 (T915S) heterozygous | | M | 20 | Negative | PROS1 (P76L) heterozygous | | M | 55 | Negative | SERPINA10 (21_23delCCT ) heterozygous | | F | 78 | Negative | HABP2 (G534E) heterozygous | | F | 49 | Negative | Negative | | F | 41 | Negative | Negative | | M | 32 | Negative | Negative | | F | 64 | Negative | Negative | | M | 42 | Negative | Negative | | F | 51 | Negative | Negative | | F | 28 | Negative | Negative | Table 1. Mutations detected by the 33 gene thrombophilia panel Disclosures No relevant conflicts of interest to declare.

Specific splicing Factor (SF) mutations are recurrent and mutually exclusive in hematopoietic diseases.Mutations in the splicing factor SRSF2 occur in nearly 40% of patients with CMML, 14% of MDS, and 19% of secondary AML, and portend a poor prognosis. SRSF2 binds to exonic splicing enhancers (ESEs), thereby affecting exon inclusion or exclusion. We have recently shown that SRSF2 mutations result in altered RNA binding affinity and specificity via in vitro structure-function studies, specifically isothermal titration calorimetry and nuclear magnetic resonance (NMR) modeling. While wildtype (WT) SRSF2 binds the consensus RNAs 5'-SSNG-3' equally well (S=G/C; N=A/T/C/G), mutant (MUT) SRSF2 shows increased affinity specifically for 5'-CCNG-3' and 5'-GCNG-3', due to structural changes in the N- and C-termini of the RRM (Kim et al, Cancer Cell, 2015). To determine how mutations in SRSF2 alter its function in vivo, we generated hematopoietic cell lines with inducible expression of Flag-tagged SRSF2WT or SRSF2MUT. We performed "High-Throughput Sequencing after UV-Cross-linking and RNA Immunoprecipitation" (HITS-CLIP) to identify specific RNAs bound by WT versus MUT SRSF2 and performed RNA deep sequencing (RNA-Seq) on the same cells to correlate RNA binding and splice events. HITS-CLIP reads were highly enriched in protein-coding RNAs (83%) and specifically exonic RNA sequences (>90%), consistent with the known function of SRSF2 as an ESE-binding protein and emphasizing the specificity of HITS-CLIP in detecting in vivo SRSF2-mRNA interactions. To determine whether the differences in in vitro binding affinities determined by isothermal calorimetry held true in vivo, we selected those RNA reads differentially bound by WT and MUT SRSF2 and analyzed the distribution of RNA consensus motifs within these regions. The 5'-CCNG-3' motif was significantly enriched over the 5'-GGNG-3' motif in SRSF2MUT bound RNAs, whereas the 5'-GGNG-3' motif was significantly enriched in SRSFWTbound reads. Crosslinking followed by reverse transcription results in single nucleotide deletions at the protein-RNA crosslink site, allowing determination of the exact protein binding site and resolution of the SRSF2-mRNA interaction at the single-nucleotide level. To compute the RNA binding consensus in an unbiased fashion we therefore performed MEME (Multiple Em for Motif Elicitation) analysis of only those reads with nucleotide deletions. We observed enrichment for CCNG in MUT-bound motifs, whereas WT-bound motifs were GA-rich, as has been previously shown (Pandit et al, Mol Cell, 2013). In addition, first analyses utilizing deletions to discern whether SRSF2 ESEs are located within specific regions of the exon suggest that while WT SRSF2binding maps to all exon regions (5', middle, 3'), more MUT SRSF2 binding occurs in the 5' region of exons. We next sought to identify RNA targets that are both differentially bound and alternative spliced in SRSF2 WT versus MUT cells. Interestingly, gene enrichment analysis within bound and alternatively spliced transcripts using "ToppGene" (https://toppgene.cchmc.org/enrichment.jsp), revealed significant enrichment for genes involved in mRNA binding, RNA processing, and RNA splicing in both our HITS-CLIP and transcriptome data sets. Strikingly, similar enrichment is found via RNA sequencing in primary patient samples with WT vs MUT SRSF2. To validate the correlation between RNA-binding and splicing, we verified several alternative splice events predicted to have differential transcript abundance in the WT vs MUT HITS-CLIP data set via exon specific RT-PCR. These splice events result in either altered protein function via in-frame inclusion/exclusion of an alternative exon, or altered protein abundance via frame-shift splice changes that lead to the introduction of a premature stop codon, slating transcripts for degration by non sense mediated decay. In summary, changes in consensus motif preference, emphasize the complex gain and loss of function nature of SRSF2 mutations, further underlining the highly context-specific SRSF2MUT-mRNA interactions. Additionally, alternative splicing of transcripts encoding other splicing factors may be the link between some of the mutually exclusive splicing factor mutations in MDS and provide a common pathway in the pathology and treatment of splicing factor mutant myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

De novo generation of HSCs has been described as a "holy grail" of stem cell biology, however the factors required for converting human pluripotent stem cells (PSCs) to true hematopoietic stem cells (HSCs) capable of robust long-term engraftment have yet to be fully characterized. Two groups have shown that injection of PSCs into immunodeficient mice leads to teratomas containing niches producing hematopoietic progenitors capable of long-term engraftment. Once these hematopoietic progenitors and their microenvironments are better characterized, this system could be used as a model to help direct in vitro differentiation of PSCs to HSCs. Toward this end, we have injected human PSCs into immunodeficient mice expressing human rather than mouse M-CSF, IL-3, GM-CSF, and thrombopoietin, as well as both human and mouse versions of the "don't eat me signal" Sirpa (collectively termed MISTRG mice). These cytokines are known to support different aspects of hematopoiesis, and thrombopoietin in particular has been shown to support HSC maintenance, suggesting these mice may provide a better environment for human PSC-derived HSCs than the more traditional mice used for human HSC engraftment. The majority of teratomas developed so far in MISTRG contain human hematopoietic cells, and the CD34+ population isolated from over half of the teratomas contained hematopoietic colony forming cells by colony forming assay. These findings further corroborate this approach as a viable method for studying human PSC to HSC differentiation. Disclosures No relevant conflicts of interest to declare.

Acute Megakaryoblastic Leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) that morphologically resembles abnormal megakaryoblasts. While extremely rare in adults, pediatric cases comprise 4-15% of newly diagnosed AML patients. Clinical outcomes for Down syndrome (DS) patients with AMKL are uniformly excellent, whereas studies on non-DS patients (non-DS-AMKL) are more variable with the majority reporting inferior survival rates compared to other AML subtypes. Furthermore, the recommendation for stem cell transplant (SCT) in first remission for non-DS-AMKL patients is not uniform among pediatric cooperative groups. Previous efforts have identified chimeric oncogenes in non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, MLL gene rearrangements and NUP98-KDM5A. The etiology of 30-40% of cases, however, remains unknown. To better understand the genomic landscape of non-DS-AMKL and its contribution to clinical outcomes, we performed RNA and exome sequencing on specimens from 115 patients compiled from eight institutions and three cooperative groups including 90 pediatric and 25 adult cases. Of the 104 patients for whom RNA was available, 27.8% (5/18) adult and 72% (62/86) pediatric cases carried a high confidence fusion event by RNAseq. The most frequent fusions in the pediatric cohort when combining RNAseq data, cytogenetics and RT-PCR include CBFA2T3-GLIS2 (17/90), MLL r (13/90), NUP98-KDM5A (9/90), and RBM15-MKL1 (9/90). Previously described low frequency fusions identified in this expanded cohort, include a case of NIPBL-HOXB9 and a novel but functionally analogous NIPBL-HOXA9 fusion. Similarly, a case carrying GATA2-HOXA10 was identified, which is functionally equivalent to the GATA2-HOXA9 fusion that has been reported in a single case. Chimeric transcripts not previously described include several fusions involving genes within the HOX cluster (HOTAIRM1-HOXA3, HOXA_AS3-HOXA9, EWSR1-HOXB8, PLEK-HOXA11-AS, and BMP2K-HOXD10 each in a single case). Collectively, fusions involving a HOX cluster gene (HOX r) occurred in 11% of the pediatric cohort. Single Nucleotide Variation (SNV) analysis of exome and RNAseq data on the cohort revealed the presence of truncating GATA1 mutations in one adult and 10 pediatric specimens lacking fusion genes. Patients carrying GATA1 mutations did not have stigmata of DS or evidence of mutant reads in germline DNA, suggesting they are not mosaics. To determine if these fusion events contribute significantly to gene expression patterns, samples with greater than 60% purity were subjected to unsupervised clustering. Confirming the strength of the fusions in altering gene expression signatures, samples clustered according to fusion subtype and were distinct from those carrying GATA1 mutations. Specifically MLL r, HOX r, NUP98-KDM5A, and CBFA2T3-GLIS2 cases formed distinct clusters. When analyzing differentially upregulated genes within these subgroups, HOX r cases demonstrated upregulation of a HOX gene signature. Combined with MLL r and NUP98-KDM5A, chimeric oncogenes also known to upregulate HOX cluster genes, roughly one-third of pediatric non-DS-AMKL patients carry a HOX gene expression program. These cases were distinct from those carrying the CBFA2T3-GLIS2 inversion. HOX genes play a significant role in normal hematopoietic development and data suggests that deregulated expression has a central role in the etiology of several subtypes of acute leukemia, in part through the acquisition of enhanced self-renewal. We evaluated our identified HOXr for their ability to serially replate in murine colony formation assays as a surrogate marker of this characteristic. Confirming their pathogenicity, chimeric transcripts conferred an enhanced ability to replate. We conclude that chimeric transcripts involving HOX cluster genes comprise a distinct subset of pediatric AMKL. Clinical outcome analyses between genomic subgroups of this heterogeneous malignancy may allow us to more effectively risk stratify these patients and determine those that may benefit from SCT in first remission. JdR and CB contributed equally FL, DR, MH-E, MF, CMZ, and TAG co-corresponding authors on behalf of AIEOP, BFM, DCOG, and SJCRH study groups Disclosures Shih: Novartis: Research Funding.

Myelodysplastic Syndromes (MDS) are a heterogeneous disorder of the hematopoietic stem cell caused by a large number of genetic and epigenetic alterations. With the development of novel therapeutics a reliable model to test the drugs' efficacy in correlation with genetic and epigenetic alterations and disease phenotype is essential. Recent advances in the field of MDS xenotransplantation have been achieved by transgenic expression of human cytokines in the murine host as well as by co-transplantation of primary patient derived mesenchymal stromal cells (MSCs) concurrent with MDS stem cells. However, neither model to date affords efficient transplantation of MDS at a scale that allows in vivo mechanistic studies or provides a platform to develop and test novel therapeutics. We sought to establish a MDS xenotransplantation model in humanized immunodeficient mice amenable to mechanistic in vivo studies and therapeutic testing. Several murine cytokines essential for hematopoiesis are non-crossreactive with their human counterpart. "MISTRG" mice express several human, non-crossreactive cytokines, namely M -CSF, I L-3, GM-CSF, and T hrombopoietin from the respective murine loci, as well as human macrophage receptor signal regulatory protein-alpha (S IRPα) to prevent murine macrophage-mediated immune rejection in the Rag2-/- IL2rγ-/- background (Rongvaux et al. Nature Biotech 32(4): 364 - 372, 2014). To establish a reliable, efficient MDS xenotransplantation model we optimized the host irradiation dose, transplantation route, CD34+ cell number and cell preparation. Mice were allowed to engraft for >10 weeks. Peripheral blood (PB), bone marrow (BM), and spleen were analyzed for engraftment by flow cytometry. BM, spleen, and liver were also fixed and sectioned for histologic analysis. Human CD45+ cells were sorted from engrafted MISTRG bone marrow and genomic testing was performed by cytogenetics, FISH, and/or targeted exome sequencing. MISTRG mice consistently supported higher engraftment in peripheral blood and bone marrow than NSG mice for the majority of MDS samples assessed. Out of 25 different patient's BM samples, including 6 RCMD, 5 RAEB I, 12 RAEB II, and 2 CMML patient samples, 23 samples engrafted in MISTRG mice, while 19 of the samples were transplanted concurrently into NSG mice (6 RCMD, 3 RAEB I, and 11 RAEB II patient samples) out of which 12 samples engrafted. Mice were classified as engrafted when huCD45+ cells accounted for over 1% of all nucleated cells in BM. On average, huCD45 engraftment was 7.3-fold higher in MISTRG than in NSG mice (17.78% vs. 2.45%). 56.1% of all MISTRG mice compared with 26.7% of NSG mice transplanted with MDS were engrafted. The number of engrafted MISTRG mice per sample ranged from 2-10 mice could be further improved with optimal bone marrow sample collection. We verified engraftment of the MDS clone via cytogenetics, FISH, and/or targeted exome sequencing, also revealing preserved clonal distribution and mutant allele frequencies in engrafted mice. Flow cytometric analysis of lineage differentiation revealed robust myeloid engraftment in MISTRG mice as opposed to NSG mice. In addition terminal differentiation of myeloid cells was markedly improved in MISTRG over NSG mice, with immunophenotypic concordance between engrafted MISTRG mice and the patient's primary bone marrow. Histologic analysis showed striking similarities between engrafted MISTRG bone marrow and the concurrent patient's bone marrow, such as marked dysplasia and clustering of human CD61 positive megakarocytes with resultant myelofibrosis as evident by reticulin staining. MISTRG mice lack the DNA repair defect inherent to NSG mice and are thus tolerant of chemotherapeutic agents. Studies testing hypomethylating drugs and targeted agents are now underway to establish MISTRG as promising "co-clinical" model for MDS. In conclusion, physiologic expression of essential non-crossreactive human cytokines greatly facilitates long-term engraftment of MDS patient derived CD34+ HSPCs in the murine immunodeficient host. MISTRG mice engraft lower and higher grade MDS with replication of the disease genotypes and phenotypes, supporting higher engraftment in bone marrow and blood than NSG mice for all MDS samples assessed. MISTRG mice may provide a xenotransplantation model for MDS allowing us to study the biology of the disease and to test therapeutics in vivo. Disclosures No relevant conflicts of interest to declare.