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Abstract Introduction Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders characterized by dysplasia in one or more hematopoietic cell lines, peripheral cytopenia(s), and distinct cytogenetic abnormalities. The presence of certain cytogenetic abnormalities [e.g. 7/del(7q), -5/del(5q), 13/del(13q), i(17p)], in the setting of unexplained persistent cytopenia, has been considered presumptive evidence of MDS, despite definitive morphologic features of MDS. Conversely, chromosomal abnormalities such as isolated 20q- are not considered definitive evidence for MDS, in the absence of dysplasia. Based on our previous findings, patients with isolated 20q- without morphologic features of a myeloid neoplasm have varied clinical outcomes (Jawad MD et al. Am J Hematol.2016). In this study, we investigate the frequency of recurrent somatic gene mutations in these patients, and their relationship to progression to a myeloid neoplasm. Methods We retrospectively reviewed the bone marrow pathology reports of patients evaluated at our institution from 01/2005 to 09/2015 who had isolated 20q- in at least 2 of 20 metaphases. Patients with isolated 20q- and without myeloid neoplasms were included in the study, along with medical record review. A 35-gene next generation sequencing (NGS) panel (OncoHeme) targeting myeloid neoplasms was performed on available archived bone marrow aspirate pellets. Statistical analyses were performed using JMP Pro software version 13. Parametric (t-test) or nonparametric tests such as the Fisher-exact test, chi-square test, or Wilcoxon rank-sum test, were utilized as appropriate for test variables. Statistical significance was based on a 2-sided significance level of .05. Kaplan-Meier analysis was used for analyzing time to progression to myeloid neoplasms and progression-free survival (i.e. time to progression to myeloid neoplasms or death from any cause). Results Fifty-six (42 males, 14 females) patients with isolated 20q- were identified. The average percentage of 20q- was 38.5 % (±29.3). The median age was 68 years (range: 44-90) and the median hemoglobin (Hb), absolute neutrophil count (ANC) and platelet count (Plt) were 11.7 g/dL (range: 8.3-15.1), 2.7 x 10(9)/L (range: 0.6-9.4) and 138 x 10(9)/L (range: 16-392), respectively. The indications for bone marrow biopsies ranged from cytopenia (21), multiple myeloma (20), non-Hodgkin lymphoma (10), amyloidosis (2), CLL (2) and transplant donor evaluation (1). Twenty-three patients harbored pathogenic mutations (15 had one mutation, 1 had two mutations and 7 had three mutations). The identified pathogenic mutations included ASXL1 (8), TET2 (8), SRSF2(3),PHF6 (2), SF3B1 (2), CBL (2), U2AF1 (2), IDH1 (1), IDH2 (1), BCOR (1), JAK2 (1), PTPN11(1), TP53 (1) and RUNX1 (1). Nine patients (16.1%) progressed to myeloid neoplasms (5 MDS, 2 acute myeloid leukemia, 2 chronic myeloid neoplasm-not otherwise specified) at a median follow-up of 36.7 months (range: 1.9-57.4). Of these 9 patients, 5 had one mutation (BCOR, TET2, DNM3TA, CBL, IDH2), 1 had two mutations (SF3B1/CBL) and 3 had three mutations (ASXL1/IDH1/SRSF2,DNM3TA/PTPN11/TET2, PHF6/TET2/TET2). In contrast, only 29.8% of non-progressed cases showed at least 1 mutation. Kaplan-Meier analysis demonstrated the presence of genetic mutations in patients with isolated 20q- carried significantly higher chance of progression to myeloid neoplasms (p<.05, Figure 1A and 1B). However, the number of genetic mutations was not predictive of progression in our relatively small study cohort (p=.66). There was no significant difference between those who did and did not progress with respect to age, gender, Hb, ANC, Plt, 20q- or mutational allele burden. Conclusions Most of the patients with isolated 20q- and without morphologic features of a myeloid neoplasm did not harbor myeloid neoplasm associated gene mutations and further did not develop a myeloid neoplasm during the course of our study. Among patients with pathogenic mutations, nearly one-third showed progression to a myeloid neoplasm. Of the clinical and laboratory variables, the presence of genetic mutation is a significant risk factor for progression to a myeloid neoplasm. This study revealed the underlying molecular heterogeneity of isolated 20q- in the absence of morphologic features of a myeloid neoplasm, and underscored the prognostication value of genetic mutation analysis in these cases. Disclosures No relevant conflicts of interest to declare.

Abstract The current view is that treatment failures of AML patients are due to persistence of leukemia stem cells (LSCs). The presence of FMS-like tyrosine kinase-3 (FLT3) Internal tandem duplication (ITD) is associated with poor prognosis. But, FLT3 tyrosine kinase inhibitors (TKI) demonstrate transient clinical activity in FLT3-ITD+ AML patients. Persistent FLT3-ITD+ AML LSC represent a source of relapse. There is a pressing need to target LSC and improve outcomes for FLT3-ITD+ AML patients. PRMT1, the predominant arginine methyltransferase, has been implicated in pathogenesis of AML rare subtype (i.e., acute megakaryoblastic leukemia). Herein, we show that PRMT1 protein expression is significantly increased in LSC-enriched AML CD34+CD38- cells relative to the counterparts of normal peripheral blood stem cells (PBSC) (AML n=9, normal n=8, p=0.0004,). Following PRMT1-knockdown (KD), AML CD34+ cells (n=15) demonstrated varying degrees of apoptosis while the survival of normal cells were not affected. Interestingly, we observed significant apoptosis-induction in a subset of samples bearing FLT3-ITD mutation (6 out of total 15) upon PRMT1-KD (ShCtrl 13.9±3.6%, ShPRMT1 32.5±4.6%, p<0.001). PRMT1-KD induced apoptosis was also more evident in cord blood (CB) CD34+ cells expressing FLT3-ITD (ShCtrl 19.4±1.3%, ShPRMT1 45.2±2.5%, p<0.001) relative to that of FLT3-WT (ShCtrl 27.47±1.8%, ShPRMT1 36.9±1.6%, p=0.0167). In 293T cells ectopically overexpressing FLT3-WT or FLT3-ITD, co-immunoprecipitation (co-IP) indicated greater interaction between PRMT1 and FLT3-ITD. Through RNA-Seq profiling two AML lines (MV4-11, OCI-AML3) plus one FLT3-ITD transduced CB CD34+ cells with PRMT1-KD, we obtained a differentially-regulated gene set as a "PRMT1 signature". This signature was enriched in FLT3-ITD+ AML relative to FLT3-WT AML according to ssGSEA analysis using two AML datasets (GSE14468, GSE10358), indicating PRMT1 may cooperate with FLT3-ITD regulating AML maintenance. Given that PRMT1 directly interacts with FLT3-ITD, we next asked whether PRMT1 catalyzes FLT3-ITD protein methylation. Through mass-spectrometry analysis of a FLT3-ITD+ AML specimen and in vitro methylation assay, we identified that PRMT1 catalyzes FLT3-ITD arginine (R) methylation (Me) at two conserved residues, 972 and 973. Using in-house R972/973 Me antibody, we validated the expression of FLT3 R-Me in FLT3-ITD AML speciemens (7 out of 7). To test R-Me function, we transduced MLL-AF9 (MA9) overexpressing murine c-Kit+ cells with methylation-deficient FLT3-ITD (R972/973K, arginine [R] to lysine [K]) construct, and found that MA9 cells expressing R972/973K underwent more apoptosis than that of WT FLT3-ITD (WT FLT3-ITD 9.7±1.1%, R972/973K 23.7±2.1%, p=0.003). The double transformed cells were further transplanted into recipients for leukemia development. Mice receiving MA9 cells expressing R972/973K exhibited longer survival (median survival: WT FLT3-ITD 36 days, R972/973K 50 days, p=0.002, n=6). Mechanistically, expression of R972/973K did not affect the total tyrosine phosphorylation level of FLT3-ITD. Additionally, FLT3-ITD R-Me expression persisted after a TKI (AC220) treatment. These facts indicated that FLT3-ITD R-Me function is independent of FLT3-ITD kinase activity. We then used a FLT3-ITD+ patient derived xenograft (PDX) model to assess the effects of TKI and PRMT1 inhibition. Following engraftment >1% in peripheral blood, we divided mice (n=24) into 4 groups and treated each with vehicle, MS023 (a type I PRMT inhibitor, ACS Chem Biol. 2016;11:772-781) (160 mg/kg/i.p), AC220 (10 mg/kg/i.g) or combination for 4 weeks. MS023 treatment downregulating FLT3 R-Me levels enhanced elimination of FLT3-ITD AML cells by AC220 treatment (AC220 24.6±13.4% vs combination 7.6±6.5%, p=0.02, n=6). At 16 weeks post-secondary BMT, significant AML burden in single drug treated transplants was observed, but less AML cells were detected in combination-treated transplants (AC220 52.3%, vs combination 25.4%, p<0.001, n=6). MS023 had little effect on long-term in vivo engraftment of CD34+ from a human CB specimen (vehicle 76.3±5.9%, MS023 72.2±3.4%, p=0.21, n=5). In summary, our study demonstrated PRMT1 overexpression contributes to AML stem/progenitor cell survival possibly through FLT3-ITD methylation, supporting further exploration into how PRMT1-mediated FLT3 methylation governs LSC survival. Disclosures Khaled: Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy; Juno: Other: Travel Funding.