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Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/CAT1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a post-translational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors--by inhibition of deoxyhypusine synthase--abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This impacted pathway is critical for eIF5A-regulated erythropoiesis as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins were especially sensitive to the loss of hypusine and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in del(5q) myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPC, synchronizing mitochondrial metabolism with erythroid differentiation.

Individuals with age-related clonal hematopoiesis (CH) are at greater risk for hematologic malignancies and cardiovascular diseases. However, predictive preclinical animal models to recapitulate the spectrum of human CH are lacking. Through error-corrected sequencing of 56 human CH/myeloid malignancy genes, we identified natural CH driver mutations in aged rhesus macaques matching genes somatically mutated in human CH, with DNMT3A mutations being the most frequent. A CH model in young adult macaques was generated via autologous transplantation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9–mediated gene-edited hematopoietic stem and progenitor cells (HSPCs), targeting the top human CH genes with loss-of-function (LOF) mutations. Long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared with minimal expansion of clones bearing other mutations. Although the blood counts of these CH macaques were normal, their bone marrows were hypercellular and myeloid-predominant. TET2-disrupted myeloid colony-forming units isolated from these animals showed a distinct hyperinflammatory gene expression profile compared with wild type. In addition, mature macrophages purified from the CH macaques showed elevated NLRP3 inflammasome activity and increased interleukin-1β (IL-1β) and IL-6 production. The model was used to test the impact of IL-6 blockage by tocilizumab, documenting a slowing of TET2-mutated expansion, suggesting that interruption of the IL-6 axis may remove the selective advantage of mutant HSPCs. These findings provide a model for examining the pathophysiology of CH and give insights into potential therapeutic interventions.

Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53 mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. In this study, we characterized the immunological features of the malignant clone and alterations in the immune microenvironment in patients with TP53-mutant and wild-type MDS or sAML. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of patients with TP53 mutations, which is associated with MYC upregulation and marked downregulation of MYC’s negative regulator miR-34a, a p53 transcription target. Notably, patients with TP53 mutations display significantly reduced numbers of bone marrow–infiltrating OX40+ cytotoxic T cells and helper T cells, as well as decreased ICOS+ and 4-1BB+ natural killer cells. Further, highly immunosuppressive regulatory T cells (Tregs) (ie, ICOShigh/PD-1−) and myeloid-derived suppressor cells (PD-1low) are expanded in cases with TP53 mutations. Finally, a higher proportion of bone marrow–infiltrating ICOShigh/PD-1− Treg cells is a highly significant independent predictor of overall survival. We conclude that the microenvironment of TP53 mutant MDS and sAML has an immune-privileged, evasive phenotype that may be a primary driver of poor outcomes and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly defined subpopulation.

Studies comparing the prognostic role of RUNX1 mutations (RUNX1mut) in acute myeloid leukemia (AML) and acute myeloid leukemia-with myelodysplasia-related changes (AML-MRC) are limited. Our study e...

Hypomethylating agent (HMA) failure myelodysplastic syndrome (MDS) patients have poor outcomes and urgent need for novel therapies. Hedgehog pathway signaling upregulation plays a central role in myeloid neoplasm pathogenesis and leukemia stem cell survival. We evaluated the efficacy and safety of the smoothened inhibitor glasdegib in HMA-failure MDS (n = 35, median age 73 years). According to the International Prognostic Scoring System and the MD Anderson Global Risk Model, 54% and 77% had higher risk disease, respectively. Overall response was 6% (n = 2), and best response was marrow complete remission with hematologic improvement in both patients. Median OS and median follow-up were 10.4 and 42.8 months, respectively. Drug response/stable disease (SD) resulted in better OS than treatment failure (20.6 [95% CI, 10.4-] vs 3.9 months [95% CI, 0.7–9.1]; P < .0001). Response/SD was confirmed to be an independent covariate for improved OS (P < .0001). Grade 3 or higher infections occurred in 11% of patients (n = 4); non-hematologic toxicities were rare. Early mortality (< 30 days) occurred in 11% of patients (n = 4). Glasdegib was well tolerated among HMA-failure MDS patients, although single-agent activity was limited. SD or better resulted in notably superior OS. These results support further investigation of glasdegib, potentially in novel drug combinations, in MDS patients.

Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell malignancies that can phenotypically resemble other hematologic disorders. Thus, tools that may add to current diagnostic practices could aid in disease discrimination. Constitutive innate immune activation is a pathogenetic driver of ineffective hematopoiesis in MDS through Nod-like receptor protein 3 (NLRP3)–inflammasome-induced pyroptotic cell death. Oxidized mitochondrial DNA (ox-mtDNA) is released upon cytolysis, acts as a danger signal, and triggers inflammasome oligomerization via DNA sensors. By using immortalized bone marrow cells from murine models of common MDS somatic gene mutations and MDS primary samples, we demonstrate that ox-mtDNA is released upon pyroptosis. ox-mtDNA was significantly increased in MDS peripheral blood (PB) plasma compared with the plasma of healthy donors, and it was significantly higher in lower-risk MDS vs higher-risk MDS, consistent with the greater pyroptotic cell fraction in lower-risk patients. Furthermore, ox-mtDNA was significantly higher in MDS PB plasma compared with all other hematologic malignancies studied, with the exception of chronic lymphocytic leukemia (CLL). Receiver operating characteristic/area under the curve (ROC/AUC) analysis demonstrated that ox-mtDNA is a sensitive and specific biomarker for patients with MDS compared with healthy donors (AUC, 0.964), other hematologic malignancies excluding CLL (AUC, 0.893), and reactive conditions (AUC, 0.940). ox-mtDNA positively and significantly correlated with levels of known alarmins S100A9, S100A8, and apoptosis-associated speck-like protein containing caspase recruitment domain (CARD) specks, which provide an index of medullary pyroptosis. Collectively, these data indicate that quantifiable ox-mtDNA released into the extracellular space upon inflammasome activation serves as a biomarker for MDS and the magnitude of pyroptotic cell death.

Accumulating evidence implicates innate immune activation in the pathobiology of myelodysplastic syndromes. A key myeloid-related inflammatory protein, S100A9, serves as a Toll-like receptor ligand regulating tumor necrosis factor-α and interleukin-1β production. The role of myelodysplastic syndrome-related inflammatory proteins in endogenous erythropoietin regulation and response to erythroid-stimulating agents or lenalidomide has not been investigated. The HepG2 hepatoma cell line was used to investigate in vitro erythropoietin elaboration. Serum samples collected from 311 patients with myelodysplastic syndrome were investigated (125 prior to treatment with erythroid-stimulating agents and 186 prior to lenalidomide therapy). Serum concentrations of S100A9, S100A8, tumor necrosis factor-α, interleukin-1β and erythropoietin were analyzed by enzyme-linked immunosorbent assay. Using erythropoietin-producing HepG2 cells, we show that S100A9, tumor necrosis factor-α and interleukin-1β suppress transcription and cellular elaboration of erythropoietin. Pre-incubation with lenalidomide significantly diminished suppression of erythropoietin production by S100A9 or tumor necrosis factor-α. Moreover, in peripheral blood mononuclear cells from patients with myelodysplastic syndromes, lenalidomide significantly reduced steady-state S100A9 generation (P=0.01) and lipopolysaccharide-induced tumor necrosis factor-α elaboration (P=0.002). Enzyme-linked immunosorbent assays of serum from 316 patients with non-del(5q) myelodysplastic syndromes demonstrated a significant inverse correlation between tumor necrosis factor-α and erythropoietin concentrations (P=0.006), and between S100A9 and erythropoietin (P=0.01). Moreover, baseline serum tumor necrosis factor-α concentration was significantly higher in responders to erythroid-stimulating agents (P=0.03), whereas lenalidomide responders had significantly lower tumor necrosis factor-α and higher S100A9 serum concentrations (P=0.03). These findings suggest that S100A9 and its nuclear factor-κB transcriptional target, tumor necrosis factor-α, directly suppress erythropoietin elaboration in myelodysplastic syndromes. These cytokines may serve as rational biomarkers of response to lenalidomide and erythroid-stimulating agent treatments. Therapeutic strategies that either neutralize or suppress S100A9 may improve erythropoiesis in patients with myelodysplastic syndromes.

MYC gene amplification and somatic mutations are frequent in both adult and pediatric AML although how MYC drives and contributes to the development and maintenance of AML has not been resolved. Transcription factor EB (TFEB) is a master regulator of genes that control autophagy and lysosome biogenesis, a central catabolic recycling pathway that regulates cell survival. Given the oncogenic effects of MYC in AML and that the induction of autophagy compromises AML cell growth and survival, we tested if the oncogenic effect of MYC depends on its suppression of TFEB transcription programs in AML. In support of this hypothesis, inducible MYC expression in K562 and THP-1 leukemia cells was sufficient to suppress expression of TFEB and its target genes. Further and conversely, MYC knockdown in NB4 AML cells provoked increased expression of TFEB mRNA and protein, as well as increased expression of TFEB target genes. Notably, dose response studies demonstrated that expression of TFEBS211A, constitutively nuclear form of TFEB that is refractory to control by mTORC1 signaling, dramatically impairs proliferation of HL60, OCI-AML2 and OCI-AML3 AML cells. In addition, induction of TFEBS211A provoked rampant apoptosis. Of important, overexpression of TFEBS211A in HL-60 and OCI-AML3 cells was also sufficient to promote monocytic and granulocytic differentiation, as judged by morphological changes and the acquisition of mature monocytic and granulocytic markers including CD11b, Gr1, and CD15. To identify TFEB targets that might contribute to myeloid/granulocytic differentiation, we performed RNA-seq analysis of HL60 leukemia cells engineered to inducibly express the TFEBS211A transgene. Using a cut-off of fold change>4 with q Surprisingly, among genes induced by TFEB in HL60 cells is IDH1, which catalyzes the production of α-ketoglutarate (α-KG), a required substrate of the TET family of dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In particular, TFEBS211A expression provoked increases in levels of α-KG and significant increases in global levels of 5hmC in genomic DNA of HL60 leukemia cells both ex vivo and in vivo. Furthermore, TFEB-mediated induction of IDH1/2 mRNA and protein, and of IDH1 promoter activity, was antagonized by inducible expression of MYC. To assess the global effects of TFEB on 5mC/5hmC landscapes, we performed paired reduced representation bisulfite (BS)- and oxidation bisulfite (oxBS)-sequencing. As predicted, TFEBS211A induced both loss and gains of 5mC, but there were more losses (n = 722) than gains (n = 459) across all 22 chromosomes. Quite remarkably, and consistent with TFEB-provoked increases of 5hmC signals, TFEBS211A exclusively induced 5hmC gains (in a total of 863 genes), and 37% and 36% of these 5hmC gains occurred in promoter regions and CpG islands, and across all 22 chromosomes. Comparison of BS- and oxBS-seq versus RNA-seq analyses of HL60 cells expressing TFEB revealed significant changes in mRNA levels and concomitant differential changes in 5mC and 5hmC marks in KLF4, KLF6, STAT3, TP73, andFOXO1 that have pivotal roles in controlling myeloid cell differentiation and death. Collectively, these findings demonstrate that MYC suppresses TFEB expression and function in AML cells, and that TFEB functions as a tumor suppressor that provokes AML cell differentiation and death. Strikingly, these responses rely on epigenetic control, where TFEB directly induces the transcription of IDH1 and IDH2 to provoke global hydroxylation of 5-methylcytosine and the expression of genes that drive terminal differentiation and apoptosis. Thus, a MYC-TFEB-IDH1/2-TET2 circuit controls AML cell fate. Disclosures Murphy: Merck: Research Funding; Puma Biotech: Research Funding. Ballabio:CASMA Therapeutics: Other: Co-Founder. Kaufmann:Takeda Pharmaceuticals: Research Funding.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancies. Known predisposing factors to adult MDS include rare germline mutations, cytotoxic therapy, age-related clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. To date, no published studies characterizing MDS-associated germline susceptibility polymorphisms exist. We performed a genome-wide association study of 2 sample sets (555 MDS cases vs 2964 control subjects; 352 MDS cases vs 2640 control subjects) in non-del(5q) MDS cases of European genomic ancestry. Meta-analysis identified 8 MDS-associated loci at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2) that approached genome-wide significance. Gene expression for 5 loci that mapped within or near genes was significantly upregulated in MDS bone marrow cells compared with those of control subjects (P < .01). Higher PLA2G4A expression and lower EYA2 expression were associated with poorer overall survival (P = .039 and P = .037, respectively). Higher PLA2G4A expression is associated with mutations in NRAS (P < .001), RUNX1 (P = .012), ASXL1 (P = .007), and EZH2 (P = .038), all of which are known to contribute to MDS development. EYA2 expression was an independently favorable risk factor irrespective of age, sex, and Revised International Scoring System score (relative risk, 0.67; P = .048). Notably, these genes have regulatory roles in innate immunity, a critical driver of MDS pathogenesis. EYA2 overexpression induced innate immune activation, whereas EYA2 inhibition restored colony-forming potential in primary MDS cells indicative of hematopoietic restoration and possible clinical relevance. In conclusion, among 8 suggestive MDS-associated loci, 5 map to genes upregulated in MDS with functional roles in innate immunity and potential biological relevance to MDS.

Despite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1β (IL-1β) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and β-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear β-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention.

Molecular predictors of response in patients with myeloid neoplasms treated with lenalidomide

Myelodysplastic syndromes (MDS) are genetically diverse malignancies with peripheral cytopenias, dysplastic hematopoiesis, and increased risk for acute myeloid leukemia (AML) transformation. Recent investigations indicate that somatic, myeloid-specific gene mutations refine clinical staging to alter estimates of overall survival (OS), and should be included in current risk stratification models.1 Hence, the identification of these mutations and corresponding protein expression levels has increasing clinical utility. TP53 mutations are found in 5–10% of MDS patients, are enriched in patients with isolated del(5q), complex cytogenetics, or MDS with fibrosis (MDS-F), and are associated with an overall worse prognosis.1–5 Next-generation sequencing (NGS) is a valuable ancillary tool, however, the technology may not be economically feasible for routine community use. Alternatively, immunohistochemistry (IHC) is fast, reproducible, and cost effective for routine laboratory use. In this study, we explore the relationship between p53 expression and TP53 gene mutation in MDS and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC). Additionally, we investigate correlations between p53 expression and clinical characteristics, including TP53 mutation variant allele frequency (VAF), myeloblast percentage, cytogenetic characteristics and outcome.

Summary Background NLRP3 inflammasome-directed pyroptotic cell death drives ineffective haemopoiesis in myelodysplastic syndromes. During inflammasome assembly, the apoptosis-associated speck-like protein containing a CARD (PYCARD, commonly known as ASC) adaptor protein polymerises into large, filamentous clusters termed ASC specks that are released upon cytolysis. Specks are resistant to proteolytic degradation because of their prion-like structure, and therefore might serve as a biomarker for pyroptotic cell death in myelodysplastic syndromes. Methods This observational cohort study was done at the H Lee Moffitt Cancer Center (Tampa, FL, USA). Patients with myelodysplastic syndromes, healthy controls, and patients with non-myelodysplastic syndrome haematological cancers or type 2 diabetes were recruited. We used confocal and electron microscopy to visualise, and flow cytometry to quantify, ASC specks in peripheral blood and bone marrow plasma samples. Speck percentages were compared by t test or ANOVA, correlations were assessed by Spearman's rank correlation coefficient, and biomarker efficiency was assessed by receiver operating characteristics and area under the curve (AUC) analysis. Findings Between Jan 1, 2005, and Jan 12, 2017, we obtained samples from 177 patients with myelodysplastic syndromes and 29 healthy controls for the discovery cohort, and 113 patients with myelodysplastic syndromes and 31 healthy controls for the validation cohort. We also obtained samples from 22 patients with del(5q) myelodysplastic syndromes, 230 patients with non-myelodysplastic syndrome haematological cancers and 23 patients with type 2 diabetes. After adjustment for glucose concentration, the log10-transformed mean percentage of peripheral blood plasma-derived ASC specks was significantly higher in the 177 patients with myelodysplastic syndromes versus the 29 age-matched, healthy donors (−0·41 [SD 0·49] vs −0·67 [0·59], p=0·034). The percentages of ASC specks in samples from patients with myelodysplastic syndromes were significantly greater than those in samples from individuals with every other haematological cancer studied (all p Interpretation Our results underscore the pathobiological relevance of ASC specks and suggest that ASC specks are a sensitive and specific candidate plasma biomarker that provides an index of medullary pyroptotic cell death and ineffective haemopoiesis in patients with myelodysplastic syndromes. Funding T32 Training Grant (NIH/NCI 5T32 CA115308–08), Edward P Evans Foundation, The Taub Foundation Grants Program, the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the H Lee Moffitt Cancer Center and Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (P30-CA076292).

Introduction: TP53 gene mutations (mTP53), found in up to 20% of MDS or AML pts and 30-40% of therapy-related (TR) MDS/AML cases, represent a distinct molecular cohort with poor outcomes. Hypomethylating agents (HMA) are the standard of care with CR rates of ~20% and median OS of 7-8 months. APR-246 is a novel, first-in-class small molecule that selectively induces apoptosis in mTP53 cancer cells via thermodynamic stabilization of the p53 protein and shifting equilibrium toward the wild-type conformation. We previously reported the Phase 1b results of APR-246+AZA with no DLTs, transcriptional activation of p53 targets and high response rates, identifying a Phase 2 (P2) dose of 4500mg days 1-4 (Sallman et al., ASH 2018). We report herein the planned, completed phase 2 results. Methods: This is a multicenter Phase 1b/2 trial of APR-246+AZA in HMA-naïve mTP53 higher risk MDS, MDS/MPN and oligoblastic AML (≤ 30% blasts) pts (NCT03072043). P2 pts received APR-246 4500mg IV (days 1-4) + AZA 75 mg/m2 SC/IV x 7 days (days 4-10 or 4-5 and 8-12) in 28 day cycles. Primary objective was CR rate by International Working Group (IWG) 2006 criteria. Secondary objectives included ORR, OS, outcome following allogeneic hematopoietic stem cell transplant (allo-HSCT), and both next generation sequencing (NGS) and p53 immunohistochemistry (IHC) to monitor clonal suppression and remission depth as prognostic covariates. For minimal residual disease (MRD) analysis, a custom target-capture NGS assay was developed using unique molecular Identifiers for error correction with a 0.1% limit of detection. Results: As of July 15, 2019, 55 pts were enrolled (6 P1; 49 P2) with a median age 66 years (34-85; 47% male). By WHO, 40 pts had MDS, 11 AML-MRC and 4 CMML/MDS-MPN; 85% had complex cytogenetics and 33% TR-MDS/AML. All pts had higher risk disease by IPSS-R (7% Intermediate, 24% High, 69% Very High). Fifty pts (91%) had a TP53 missense mutation in the DNA binding domain with multiple mutations in 18 (33%), and median variant allele frequency (VAF) of 25%. In 34 pts (62%), TP53 was the sole mutation. Median time on treatment is 154 days (11-392) with 8 pts ongoing. Eighteen pts (33%; 40% of evaluable pts) discontinued study treatment to proceed to allo-HSCT. Treatment (Tx)-related AEs in ≥ 20% of pts included nausea/vomiting (58%), dizziness (31%), constipation (24%), neuropathy (22%), leukopenia (22%) and thrombocytopenia (20%; all G1/G2 except cytopenias (G3/G4). Tx-related febrile neutropenia and anemia occurred in 9% and 5% of pts with no other G3/G4 event in >1 pt. Thirty and 60 day mortality was 2% (n=1) and 6% (n=3), respectively. At data cutoff, 45pts were response evaluable with a median follow up of 10.5 months (Fig 1A). ORR by IWG was 87% (39/45) with 24 CR (53%), 8 marrow CR (mCR)+HI (18%), 3 HI alone (7%), and 4 with mCR (9%). Of 6 non-responders, 4 had stable disease and 2 pts had progressive disease. Median time to response was 2.1 months (0.1-5.4) and median duration of response of 6.5 months. CR rate for MDS was 61% (20/33), 50% for AML (4/8) and 0% for MDS/MPN (0/4) with an 88% ORR rate for MDS/AML and 75% for MDS/MPN. An isolated mTP53 was predictive for a higher CR rate (69% vs 25%; P=.006) with a trend for higher ORR (93% vs 75%; P=.17). Additionally, pts with >10% p53 IHC+ BM-MNC was a covariate associated with higher CR rate (66% vs 13%; P=.01). Complete and partial cytogenetic response occurred in 41% (n=18) and 18% (n=8) of pts, respectively. On serial TP53 NGS using a VAF cutoff of 5%, 39% (n=21) of patients achieved NGS negativity, which was associated with improved OS (12.8 vs 9.2 months; P=.02). In NGS- pts, the median MRD VAF at maximum clearance was 0.63% (0.0%-5%) with 5 pts (11%) MRD negative. By intention-to-treat analysis, median OS was 11.6 months (95% CI 9.2-14) with significantly longer OS in responding pts (12.8 vs 3.9 months; P Conclusions: APR-246+AZA is a well-tolerated combination with high response rates in mTP53 MDS/AML. Response durations are promising accompanied by a high fraction of cytogenetic and deep molecular remissions leading to encouraging outcomes post-HSCT. These data support the ongoing, randomized phase 3 study of APR-246+AZA versus AZA alone in mTP53 MDS (NCT03745716). Disclosures Sallman: Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Research Funding; Incyte: Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding, Speakers Bureau. DeZern:Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Steensma:Stemline: Consultancy; Pfizer: Consultancy; Aprea: Research Funding; H3 Biosciences: Other: Research funding to institution, not investigator.; Astex: Consultancy; Arrowhead: Equity Ownership; Onconova: Consultancy; Summer Road: Consultancy. Roboz:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Cluzeau:Jazz Pharma: Consultancy; Abbvie: Consultancy; Menarini: Consultancy. Sweet:Incyte: Research Funding; Celgene: Speakers Bureau; Pfizer: Consultancy; Agios: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Stemline: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Korbel:Aprea Therapeutics: Employment. Attar:Aprea Therapeutics: Employment. Kantarjian:Astex: Research Funding; Amgen: Honoraria, Research Funding; Ariad: Research Funding; BMS: Research Funding; AbbVie: Honoraria, Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Immunogen: Research Funding; Jazz Pharma: Research Funding; Takeda: Honoraria; Pfizer: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Agios: Honoraria, Research Funding; Cyclacel: Research Funding. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Fenaux:Aprea: Research Funding; Astex: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Jazz: Honoraria, Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Komrokji:JAZZ: Consultancy; Novartis: Speakers Bureau; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy; JAZZ: Speakers Bureau; Agios: Consultancy.

Improvements in antisense technology have now enabled clinically relevant therapeutic credentialing of the noncoding genome. MALAT1 is a long non-coding RNA that, among other functions, is thought to serve as a nuclear scaffold for splicing and transcription factors. MALAT1 expression is associated with inferior prognosis across solid tumors and its depletion impairs proliferation and metastasis in preclinical solid tumor models. We found that elevated MALAT1 levels are independently associated with inferior overall survival in patients with CMML. Further, RNA-sequencing of primary CMML monocytes identified MALAT1 as the fourth most over-expressed transcript compared to controls. Therefore, we explored the biologic relevance and therapeutic candidacy of MALAT1 across several human and murine models of CMML. First, we crossed NRASQ61R/+Mx1-cre driven mice, which display a CMML-like phenotype, to MALAT1KO/KOmice. Although MALAT1KO/KOmice did not have abnormalities in complete blood counts, immunophenotyping of the hematopoietic stem cell (HSC) compartment identified statistically significantly lower numbers of HSC compared to wild type (WT) controls and a non-significant decrease in NRASQ61R/+/MALAT1KO/KOcompared to NRASQ61R/+alone. This decrease in HSC was not a result of impaired self-renewal as no differences were observed in these models after in vivo competitive transplant experiments. Therefore, we reasoned that MALAT1 expression may be controlling HSC differentiation. To test this, we transformed bone marrow cells from these models with an estrogen-regulated (ER) Hoxb8 construct enabling cells to maintain an HSC state until ER is withdrawn and myeloid differentiation is induced. ER-Hoxb8NRASQ61R/+/MALAT1KO/KOcells had increased basal levels of Gr-1 compared to ER-Hoxb8 transformed NRASQ61R/+alone that was dramatically enhanced upon ER withdrawal suggesting that MALAT1 depletion regulates myeloid differentiation. These findings were validated by assessment of morphology, transcriptome, and in vivo immunophenotyping of bone marrow and spleen cells. Further, moribund NRASQ61R/+/MALAT1KO/KOmice displayed a reduction in organomegaly typically associated with leukemic burden. We validated this in human monocytic leukemia by generating MALAT1 depleted THP-1 isogeneic cell lines using the CRISPR/Cas9 system. MALAT1 depleted THP-1 cells (MKO) had greater terminal differentiation according to immunophenotypic markers and morphology that was greatly enhanced when treated with phorbol myristate acetate. Last, MKO orthotopic xenografts demonstrated inferior human leukemia engraftment and decreased spleen and liver weights, and heterotopic xenografts exhibited reduced tumor volume, collectively suggesting diminished leukemic burden. Because ATRA has been clinically tested in CMML with minimal effects, we next explored whether MALAT1 depletion could potentiate ATRA differentiation in CMML. First, we treated MKO cells with ATRA and observed a large induction of myeloid differentiation by marker expression and morphologic assessment compared to isogenic controls. This was validated by NRASQ61R/+/MALAT1KO/KOmice demonstrating that ATRA more robustly induced differentiation compared to vehicle which was not seen in NRASQ61R/+/MALAT1+/+mice. Next, we tested MALAT1 antisense oligonucleotides (ASOs) currently under clinical development in THP-1 cells +/- ATRA and demonstrated both an increase in myeloid differentiation and apoptosis compared to ATRA alone. To test this therapeutic strategy in primary CMML specimens, we generated CMML patient-derived xenografts (n=30 mice) and treated each with ASO, ATRA, the combination, or controls and identified a more robust reduction in human HSC engraftment with the combination. To explore the mechanistic basis for these findings, we performed RNA-sequencing of MALAT1-depleted or control cells and identified that CREB target genes were differentially expressed. Basal protein levels of p-CREB were also decreased in MKO cells and were further reduced in the nucleus of MKO by western and microscopy. Lastly, overexpression of WT or constitutively active CREB but not its dominant negative rescued the differentiation effect seen in ATRA treated MKO cells. Taken together, MALAT1 is a novel, CREB-dependent regulator of myeloid differentiation and its depletion potentiates ATRA therapy. Disclosures Cluzeau: Menarini: Consultancy; Jazz Pharma: Consultancy; Abbvie: Consultancy. Komrokji:celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy. MacLeod:Ionis Pharmaceuticals: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Epling-Burnette:Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Incyte Corporation: Research Funding.

Background: Only a small subset of Lower risk (LR) MDS patients benefit from treatment with rhu-Erythropoietin (Epo). We previously reported that lenalidomide (LEN) restores sensitivity to Epo in MDS progenitors by inducing the formation of lipid rafts that are enriched for signaling competent, JAK2/Epo-receptor complexes (McGraw K, et. al. PLoS One 2014; Basiorka A, et. al. Cancer Res 2016). In the MDS-002 and MDS-005 trials, treatment with LEN monotherapy gave rise to RBC transfusion-independence (TI) in 26% of azanucleoside-naïve, transfusion-dependent (TD) LR, non-del(5q) MDS patients for a median of 10.2 and 7.75 months, respectively. In a pilot study of Epo-refractory LR-MDS patients, the addition of epoetin alfa (EA) to LEN treatment yielded erythroid responses in 28% of patients who were unresponsive to LEN alone, suggesting that LEN may overcome clinical resistance to augment response to rhEpo (Komrokji R, et. al. Blood 2012). To test this hypothesis, we performed a randomized phase III trial comparing treatment with LEN to LEN+EA in LR non-del(5q) MDS patients who were refractory to, or not candidates for treatment with rhEpo. Methods: Patients with Low or Intermediate-1 IPSS risk MDS with hemoglobin 2 units/month) with serum Epo >500mU/mL were eligible. Patients were stratified by serum Epo level and prior rhEpo (EA vs. darbepoetin vs. none) then randomized to treatment with LEN 10 mg/d x21d q4wk (Arm A) or LEN + EA 60,000U SC/wk (Arm B). The primary endpoint was major erythroid response (MER) at week 16 which was defined according to transfusion status at baseline: (1) achievement of RBC-TI for ≥ 8 consecutive weeks AND a sustained ≥1 g/dL hemoglobin rise compared to mean pre-transfusion baseline value in TD patients; and (2) a >2 g/dL rise in hemoglobin without transfusion for ≥ 8 consecutive weeks in non-TD patients ( Results: Among 205 patients randomized, 14 were excluded from the primary analysis due to a 4 month interruption in drug supply. Among the 195 evaluable patients, 96 were assigned to Arm A and 99 to Arm B. Baseline characteristics were well balanced between arms with 85% of patients heavily TD, receiving a median of 4 units/8 weeks. Overall, 93% of patients received prior treatment with Epo and 18% azanucleosides. In an intent to treat analysis, 28/99 patients (28.3%) in Arm B achieved MER compared to 11/96 (11.5%) in Arm A (P=0.004). Among 136 patients who completed 16 weeks of study treatment, 28/72 (38.9%) and 10/64 (15.6%) achieved MER, respectively (P=0.004). Forty-four Arm A non-responders crossed over to combination-therapy with 11 patients (25%) experiencing a MER. Multivariable logistic regression analysis identified only treatment with LEN + EA as an independent covariate for erythroid response (P=0.01). Responses were durable with MER median duration of 23.8 months in Arm B compared to 13 months in Arm A. There was no significant difference in the frequency or distribution of >Grade 3, non-hematologic adverse events between treatment arms. Two patients progressed to AML while on study (Arm A), and no thromboembolic events were reported. Conclusions: LEN restores sensitivity to rhEpo in otherwise refractory, LR-non-del(5q) MDS patients to yield a significantly higher frequency of durable major erythroid responses compared to LEN alone. The addition of LEN to EA treatment is an effective strategy for the management of Epo-refractory patients with a potential duration of benefit extending to years. Disclosures List: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Verma:Janssen: Research Funding; BMS: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Komrokji:JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; celgene: Consultancy; pfizer: Consultancy. Luger:Onconova: Research Funding; Pfizer: Honoraria; Seattle Genetics: Research Funding; Cyslacel: Research Funding; Biosight: Research Funding; Ariad: Research Funding; Agios: Honoraria; Genetech: Research Funding; Jazz: Honoraria; Daichi Sankyo: Honoraria; Kura: Research Funding; Celgene: Research Funding. Mattison:Pfizer: Membership on an entity's Board of Directors or advisory committees. Altman:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; France Foundation: Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; prIME Oncology: Speakers Bureau; PeerView: Speakers Bureau; Cancer Expert Now: Consultancy; Novartis: Consultancy; Biosight: Other: US Lead. Claxton:Astellas Pharma: Other: Pharma support of clinical studies; Merck Sharp & Dohme Corp.: Other: Pharma support of clinical studies; Cyclacel Pharmaceuticals, Inc.: Other: Pharma support of clinical studies; Medimmune Inc.: Other: Pharma support of clinical studies; Novartis Pharmaceuticals: Other: Pharma support of clinical studies; Celgene Corporation: Other: Pharma support of clinical studies; Incyte Corporation: Other: Cyclacel Pharmaceuticals, Inc; Daiichi Sankyo Co. and Ambit Biosciences Corp: Other: Pharma support of clinical studies. Artz:Miltenyi: Research Funding. Tallman:Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Lenalidomide used for treatment non-del 5q myelodysplastic syndromes.

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

TP53 gene mutations occurring in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are associated with high-risk karyotypes including 17p abnormalities, monosomal and complex cytogenetics. TP53 mutations in these disorders portend rapid disease progression and resistance to conventional therapeutics. Notably, the size of the TP53 mutant clone as measured by mutation allele burden is directly linked to overall survival (OS) confirming the importance of p53 as a negative prognostic variable. In nucleolar stress-induced ribosomopathies, such as del(5q) MDS, disassociation of MDM2 and p53 results in p53 accumulation in erythroid precursors manifested as erythroid hypoplasia. P53 antagonism by lenalidomide or other therapeutics such as antisense oligonucleotides, repopulates erythroid precursors and enhances effective erythropoiesis. These findings demonstrate that p53 is an intriguing therapeutic target that is currently under investigation in MDS and AML. This study reviews molecular advances in understanding the role of p53 in MDS and AML, and explores potential therapeutic strategies in this era of personalized medicine.

Loss of heterozygosity affecting chromosome 7q is common in acute myeloid leukemia and myelodysplastic syndromes, pointing toward the essential role of this region in disease phenotype and clonal evolution. The higher resolution offered by recently developed genomic platforms may be used to establish more precise clinical correlations and identify specific target genes. We analyzed a series of patients with myeloid disorders using recent genomic technologies (1458 by single-nucleotide polymorphism arrays [SNP-A], 226 by next-generation sequencing, and 183 by expression microarrays). Using SNP-A, we identified chromosome 7q loss of heterozygosity segments in 161 of 1458 patients (11%); 26% of chronic myelomonocytic leukemia patients harbored 7q uniparental disomy, of which 41% had a homozygous EZH2 mutation. In addition, we describe an SNP-A–isolated deletion 7 hypocellular myelodysplastic syndrome subset, with a high rate of progression. Using direct and parallel sequencing, we found no recurrent mutations in typically large deletion 7q and monosomy 7 patients. In contrast, we detected a markedly decreased expression of genes included in our SNP-A defined minimally deleted regions. Although a 2-hit model is present in most patients with 7q uniparental disomy and a myeloproliferative phenotype, haplodeficient expression of defined regions of 7q may underlie pathogenesis in patients with deletions and predominant dysplastic features.

Telomeres are specialized structures providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis. Accelerated telomere attrition in patients with myelodysplastic syndrome (MDS) occurs by an undefined mechanism. Although the MDS clone originates within the myeloid compartment, T-lymphocytes display repertoire contraction and loss of naive T-cells. The replicative lifespan of T-cells is stringently regulated by telomerase activity. In MDS cases, we show that purified CD3+ T-cells have significantly shorter telomere length and reduced proliferative capacity upon stimulation compared with controls. To understand the mechanism, telomerase enzymatic activity and telomerase reverse transcriptase (hTERT), gene expression were compared in MDS cases (n=35) and healthy controls (n=42) within different T-cell compartments. Telomerase activity is greatest in naive T-cells illustrating the importance of telomere repair in homeostatic repertoire regulation. Compared with healthy controls, MDS cases had lower telomerase induction (P

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self-renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down-regulation of members of the leukaemia-associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate-1) and higher risk (Intermediate-2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.

Background: The pathogenesis of Myelodysplastic Syndromes (MDS) is linked to constitutive innate immune stimulation that converges upon the NLRP3 inflammasome to induce pyroptosis, a caspase-1 dependent cell death. We have shown that inflammasome assembly is initiated by both cell-extrinsic stimuli such as S100A9 elaborated by Myeloid-Derived Suppressor Cells (MDSC), as well as cell-intrinsic somatic gene mutations (SGM) (Basiorka A, et. al. Blood 2016). SGM of varied classes evoke replication stress caused by transcriptional pauses that can expose genomic DNA to cytosolic sensors through unresolved R-loops or micronuclei formation. The cGMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) is a cell-intrinsic DNA surveillance pathway recognizing both cytosolic pathogenic and autologous DNA, leading to interferon stimulated gene (ISG) transcription and NLRP3 inflammasome activation, key biological features of MDS (Pellagatti A, et. al. Blood 2006; 108:337.). Here, we investigate the contribution of genomic cytosolic DNA engagement by cGAS-STING to NLRP3 inflammasome activation in MDS. Methods: MDS patient and healthy donor bone marrow mononuclear cells (BMMC) were isolated by Ficoll®-Hipaque method from consented participants at the Moffitt Cancer Center or the National Taiwan University Hospital (NTUH). Immortalized murine C57BL/6 Tet2-/- and MX1Cre/SRSF2P95H as well as respective wild type (WT) control BMMCs were used as MDS SGM models. Results: We first assessed cGAS-STING activation in MDS BMMC by measuring ISG response by microarray, demonstrating profoundly increased expression of ISG15, CXCL10, Samd9l, and Ifi27l2 in MDS BMMC (n=213) compared to healthy control BMMC (n=20) (p Conclusion: These data indicate that cGAS-STING engages redundant sources of cytosolic genomic-DNA in MDS to initiate a Type I interferon response and NLRP3 inflammasome activation. Inhibition of the cGAS-STING axis may represent a novel therapeutic strategy for investigation in MDS. Disclosures List: Celgene: Research Funding.

Introduction: TP53 mutant (mTP53) MDS and AML, accounting for 5-10% of de novo MDS and 25-30% of therapy related MDS (t-MDS), represent a distinct molecular cohort with inferior outcomes. Hypomethylating agents (HMA) are preferred treatments for patients (pts) with these mutations, although with CR rates of only 20-30% and median OS of 6-12 months. APR-246 is a novel, first-in-class small molecule that selectively induces apoptosis in mTP53 cancer cells through mutant p53 protein re-activation by restoring the wild-type conformation, with single agent activity in mTP53 AML. We report the planned, completed Phase 1b results of APR-246+ azacitidine (AZA) in mTP53 MDS/AML. Methods: This is a multicenter Phase 1b/2 trial of APR-246+AZA in HMA naïve mTP53 MDS and oligoblastic AML (≤ 30% blasts) pts ≥ 18 years of age. Pts received APR-246 in a 3+3 dose escalation design (50, 75, 100 mg/kg lean body weight (equivalent to 4500mg fixed dose based on PK studies)) IV daily over 4 days in a lead-in phase (days -14 to -10) followed by the same dose of APR-246 (days 1-4) + AZA 75 mg/m2 SC/IV over 7 days (days 4-10 or 4-5 and 8-12) in 28 day cycles. The primary objective was to define safety and the recommended Phase 2 dose (RP2D), with AEs graded by CTCAE v4.03 and DLT assessment over 6 weeks. Secondary objectives included response by IWG 2006 criteria as well as serial next generation sequencing (NGS) and p53 IHC for evaluation of clonal suppression and remission depth as predictors of outcomes. For minimal residual disease (MRD) analysis, a custom target-capture NGS assay was developed using unique molecular Identifiers for error correction with a limit of detection of 0.1% with results validated by pt specific digital droplet PCR (ddPCR). Nanostring nCounter RNA expression analysis was conducted on a panel of 770 genes after the lead-in phase to assess transcriptional effects induced by APR-246. Results: As of July 30, 2018, 12 pts (42% male; median age 66 years (39-73)) were enrolled. Three pts had AML-MRC and 9 had MDS; all pts had poor risk cytogenetics (17% poor, 83% very poor) and higher risk disease by IPSS-R (25% high, 75% very high). T-MDS occurred in 5 pts (42%) and 7 pts (58%) were transfusion-dependent at baseline. Median BM blasts were 9% (4-30). Eleven of 12 pts (92%) had a TP53 missense mutation in the DNA binding domain with multiple mutations in 4/12 pts (33%). For 9/12 pts (75%), TP53 was the sole mutation. Median time on study is 176 days (41-298) with 7 pts ongoing. Treatment (Tx) related AEs during the lead-in phase (all G1) included nausea (n=5), neuropathy (n=5), decreased appetite (n=2), and dizziness (n=2) which were all transient. Tx related AEs occurring in > 1 pt in the combination phase included nausea/vomiting (n=6), dizziness (n=3), headache (n=3), neuropathy (n=3), fall (n=2), pruritus (n=2), thrombocytopenia (n=6), neutropenia (n=5), and leukopenia (n=4); all G1/G2 except cytopenias (G3/G4). No DLTs have occurred to date. Eleven of twelve pts were response evaluable with 1 pt discontinuing tx prior to 1st disease assessment (Fig 1A). ORR by IWG was 100% (11/11) with 9 CR (82%) and 2 marrow CR (mCR; 18%). Median time to first response was 70 days (4-91) and one CR patient achieved mCR and partial cytogenetic response after APR-246 lead-in prior to combination therapy. All CR pts had high p53 positivity by IHC at baseline (25-80%) which normalized on serial assessment with the 2mCR pts having Conclusions: APR-246+AZA combination is well tolerated in mTP53 MDS/AML. Responses have been achieved in all evaluable pts (82% CR) accompanied by deep molecular and durable remissions. The RP2D of APR-246 is a fixed dose of 4500mg days 1-4 in combination with AZA and phase 2 accrual has begun. Disclosures Sallman: Celgene: Research Funding, Speakers Bureau. Sweet:Agios: Consultancy; Phizer: Consultancy; Agios: Consultancy; BMS: Honoraria; Celgene: Honoraria, Speakers Bureau; Jazz: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; BMS: Honoraria; Phizer: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy; Jazz: Speakers Bureau; Astellas: Consultancy. Cluzeau:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; Sanofi: Speakers Bureau; Menarini: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Roboz:Orsenix: Consultancy; Cellectis: Research Funding; Eisai: Consultancy; Astex Pharmaceuticals: Consultancy; Argenx: Consultancy; Pfizer: Consultancy; Bayer: Consultancy; Eisai: Consultancy; Sandoz: Consultancy; Jazz Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Otsuka: Consultancy; Bayer: Consultancy; Aphivena Therapeutics: Consultancy; Celltrion: Consultancy; Argenx: Consultancy; Celgene Corporation: Consultancy; Daiichi Sankyo: Consultancy; Celltrion: Consultancy; Sandoz: Consultancy; Astex Pharmaceuticals: Consultancy; Aphivena Therapeutics: Consultancy; Orsenix: Consultancy; Otsuka: Consultancy; Roche/Genentech: Consultancy; Roche/Genentech: Consultancy; Janssen Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; AbbVie: Consultancy; Janssen Pharmaceuticals: Consultancy; Celgene Corporation: Consultancy; AbbVie: Consultancy; Cellectis: Research Funding. Bhagat:Genoptix: Employment. Tell:Aprea Therapeutics: Employment. Fenaux:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Roche: Honoraria. List:Celgene: Research Funding. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Constitutive innate immune activation is a pathogenetic driver of Myelodysplastic Syndromes (MDS) that directs ineffective hematopoiesis by NLRP3 inflammasome (IFM) assembly and pyroptotic cell death. IFM activation involves recruitment of caspase-1 (casp1) through the adapter protein, ASC, to facilitate autocatalytic cleavage of the zymogen to its active form that is responsible for interleukin (IL)-1β maturation, membrane pore formation and pyroptosis. Oxidized mitochondrial DNA (ox-mtDNA) has been proposed to serve as an alarmin that can activate the IFM by interaction directly with NLRP3 or engagement by DNA sensors, Toll-like receptor 9 (TLR9) and Cyclic GMP-AMP synthase (cGAS). Upon cytolysis, ox-mtDNA is released, permitting interaction with pattern recognition receptors on neighboring cells (Grishman, Pediatric Research, 2012, Shimada, 2012, Immunity. Vollmer, 2004, Immunology). Here, we investigate ox-mtDNA as an IFM-activator and pyroptotic biomarker in MDS. Incubation of TLR9 expressing cell lines, SKM1 (high expresser) and U937 (moderate expresser) with 50ng/mL ox-mtDNA (ND1 gene, amplified with oxidized guanosine) induced IFM activation evidenced by increased p-NFkβ, casp1 and IL-1β cleavage, ASC oligomerization and liberation of ASC specks. Direct interaction of ox-mtDNA with NLRP3 was confirmed by NLRP3 immunoprecipitation followed by probing for mtDNA using ND1 and CYTB specific primers and GAPDH primers as negative genomic control; mtDNA oxidation status was confirmed by dot blot. Furthermore, significantly increased expression of interferon stimulated genes (ISG) was seen in MDS bone marrow (BM) specimens (p≤0.01) compared to normal donors indicating TLR9 and/or cGAS activation. Ox-mtDNA engagement of TLR9 and cGAS was confirmed in MDS specimens by IF colocalization with corresponding IFM activation, as well as in MDS somatic gene mutation murine models (Tet2, SRSF2, U2AF) vs. Wt controls. Evaluation of surface TLR9 by flow cytometry showed significantly increased membrane expression in MDS CD34+ BMMC (n=4) vs. healthy donors (n=13) (p Collectively, these data indicate that ox-mtDNA both directly engages NLRP3 and the DNA sensors TLR9/cGAS to induce IFM activation and pyroptosis, creating a feed forward inflammatory cascade that extends to neighboring cells. Ox-mtDNA may serve as a biomarker and companion diagnostic for pyroptosis execution in MDS. Disclosures List: Celgene: Research Funding.

Background: Myelodysplastic Syndromes (MDS) are genetically and hematologically diverse stem cell malignancies pathogenetically linked to constitutive innate immune activation. Other than rare germline mutations and age, the only known predispositions to adult MDS include prior cytotoxic therapy, clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. Few studies investigating the genetic susceptibility to MDS have been performed owing to the limitations of SNP-array sample size. Here, we report the results from the first unbiased genome wide analysis of germline polymorphisms associated with non-del(5q) MDS using a multinational curated data set. Methods: Association analyses were performed on 2 sample sets (set 1: 555 cases, 2,964 controls; set 2: 352 cases, 2,640 controls) and combined by meta-analysis. Standard SNP- and sample-level QC was applied. Haplotype reference consortium (HRC) imputation was done by the Michigan imputation server (Rsq>0.4) providing 23,278,269 markers for analysis. Gene expression sequencing was performed on an independent sample set from the National Taiwan University Hospital (213 MDS cases, 20 healthy donors; HumanHT-12 v4 Expression BeadChip; Chuang et al. Leukemia 2015). Functional analyses were performed as described. Results: Eight MDS associated loci were identified with lead variants, rs6683416, rs34539210, rs341274, rs1634783, rs7099032, rs2947170, rs4404050, and rs1206818, at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2), respctively. Odds ratio (OR) and p-values of each are listed in Table 1. Using gene expression profiling in an independent MDS sample set, we found expression of these five candidate genes was significantly increased in MDS vs controls (p Conclusion: We describe here the first MDS susceptibility loci ever identified the majority of which have a direct relationship to innate immune activation, a driver of MDS pathogenesis. Expression of genes housing these loci is increased in MDS with demonstrable prognostic and biological relevance. Further, functional studies implicate EYA2 as a novel, biologically rational target for MDS treatment. The direct functions of each polymorphism as well as the potential relationship between these predisposition loci to age-related clonal hematopoiesis merits further investigation. Disclosures Cluzeau: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Menarini: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau. Sallman:Celgene: Research Funding, Speakers Bureau. Sokol:Spectrum Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Mallinckrodt Pharmaceuticals: Consultancy. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. List:Celgene: Research Funding.

Granulocyte-macrophage–colony-stimulating factor (GM-CSF) hypersensitivity is a hallmark of juvenile myelomonocytic leukemia (JMML) but has not been systematically shown in the related human disease chronic myelomonocytic leukemia (CMML). We find that primary CMML samples demonstrate GM-CSF–dependent hypersensitivity by hematopoietic colony formation assays and phospho-STAT5 (pSTAT5) flow cytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signaling-associated mutations. When compared with IL-3 and G-CSF, GM-CSF hypersensitivity was cytokine specific and thus a possible target for intervention in CMML. To explore this possibility, we treated primary CMML cells with KB003, a novel monoclonal anti–GM-CSF antibody, and JAK2 inhibitors. We found that an elevated proportion of immature GM-CSF receptor-α(R) subunit-expressing cells were present in the bone marrow myeloid compartment of CMML. In survival assays, we found that myeloid and monocytic progenitors were sensitive to GM-CSF signal inhibition. Our data indicate that a committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML, consistent with results in JMML. These preclinical data indicate that GM-CSF signaling inhibitors merit further investigation in CMML and that GM-CSFR expression on myeloid progenitors may be a biomarker for this therapy.

Introduction: MDS encompasses a heterogeneous group of clonal neoplastic hematopoietic stem cell diseases characterized by ineffective hematopoiesis and frequent progression to acute myeloid leukemia (AML). While the advent of hypomethylating agent (HMA) therapy has modestly improved survival in higher-risk MDS, outcomes following HMA failure are dismal. The hedgehog (HH) signaling pathway is an important regulator of hematopoietic cell survival and differentiation, with upregulation of HH target genes and signaling proteins a frequent occurrence in MDS and AML. PF-04449913 (PF-04) is an orally bioavailable inhibitor of Smoothened (SMO), which demonstrated antileukemic activity in a prior phase 1 study. We performed a phase 2 study of PF-004449913 in patients with MDS following HMA failure. Objectives: 1) primary: to estimate overall IWG-2006 response rate (CR + PR + HI + marrow CR) to PF-04449913 in patients with relapsed/refractory MDS and CMML; 2) secondary: to assess safety , overall survival, time to AML transformation, and effect of PF-04 on HH target gene expression. Methods: This was a single center, open-label phase 2 study. Key eligibility criteria included: 1) patients age ≥ 18 with MDS, CMML, or AML (20-29% blasts) following failure of HMA; 2) ECOG 0-2; 3) Adequate kidney and hepatic function. Treatment consisted of PF-04 at 100 mg/day in 4-week cycles for 4 cycles, with continuation allowed for achievement of stable disease or better. Dose increase to 200 mg/day was permitted after cycle 2 for patients with stable disease. Bone marrow biopsy for response assessment occurred after cycle 2, cycle 4, and every 4th cycle thereafter. For correlative studies, total cellular RNA was isolated from ficolled mononuclear cells in baseline marrow samples, and qPCR performed for expression of GLI1, PTCH1, and SMO). Results: Thirty five patients were enrolled, 24M and 11F. Median age was 75 years (range 55-88). A majority of patients had higher-risk (54%) vs lower-risk (43%) MDS by IPSS at time of enrollment. A total of thirty four patients (97%) had received prior azacitidine for a median of 9 cycles (range 1-88), while 6 patients had received decitabine for a median of 4 cycles (5 of whom had also received azacitidine). The median number of cycles of PF-04 received was 3 (range 1-11). All 35 patients were evaluable for response. Two of 35 patients (6%) achieved response per MDS IWG 2006 criteria, including one patient with HI-N/HI-P and another with marrow CR and HI-N. Nineteen additional patients (54%) had stable disease following cycle 2 (8 weeks). With a median follow-up of 10.1 months, the median survival for all treated patients was 10.2 months (95% CI 6.8-13.6 mo)(Figure 1). Median survival for patients with low/INT-1 risk MDS was longer than for INT-2/high risk patients (22.4 mo vs 7.5 mo, p=0.013). Six patients (17%) died while on-treatment, but none of the deaths were considered related to PF-04. The most common treatment-emergent adverse events included myalgia/muscle cramps (65%), dysgeusia (60%), nausea (31%), anorexia (31%), oral mucositis (25%), and AST elevation (22%). The vast majority of these events were grade 1/2. Except for infection (11%), there were no treatment-emergent grade 3/4 events in > 10% of patients. Only 5 patients (14%) required dose reduction to 50 mg/day due to intolerance. In the pharmacodynamics analysis, responding patients had higher baseline expression of SMO, PTC ,and GLI1 than non-responders, while non-responders had higher post-treatment increases in these transcripts compared with responders. Conclusions: PF-04 was well tolerated in patients with advanced, HMA refractory MDS, but had limited efficacy in this unselected, heavily pre-treated patient population. The pharmacodynamic analysis suggests predictive potential of HH pathway gene expression for response warranting further investigation. Figure 1 Figure 1. Disclosures Komrokji: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy. Sweet:Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Pfizer: Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding.

Background: Treatment with rhu-Epo ameliorates anemia in a subset of LR-MDS patients, however, effective salvage therapy is limited. LEN promotes erythroid lineage competence and expansion of primitive erythroid precursors in vitro. In the MDS-002 and MDS-005 trials, treatment with LEN improved erythropoiesis, yielding RBC transfusion-independence in 26% of azanucleoside-naïve, transfusion-dependent (TD) LR, non-del(5q) MDS patients for a median of 10.2 and 7.75 months, respectively. We previously reported that LEN restores Epo-responsiveness in MDS progenitors by inducing formation of lipid rafts enriched for signaling competent JAK2/Epo-receptor complexes and excluding large isoforms of the JAK2/lyn kinase-phosphatase CD45 (McGraw K, et. al. PLoS One 2014; Basiorka A, et. al. Cancer Res 2016). In a pilot study of Epo-refractory MDS patients, addition of EA yielded erythroid responses in 28% of patients who were unresponsive to LEN alone, suggesting that LEN may overcome resistance and augment response to rhEpo (Komrokji R, et. al. Blood 2012). To test this hypothesis, we performed a randomized phase III trial comparing treatment with LEN to LEN+EA in LR non-del(5q) MDS patients who were refractory to, or not candidates for treatment with rhEpo. Methods: Patients with Low or Intermediate-1 (Int-1) risk IPSS MDS with hemoglobin 2 units/mo) with serum Epo >500mU/mL were eligible for study. Patients were stratified by serum Epo level and prior rhEpo (EA vs. darbepoetin vs. none) then randomized to treatment with LEN 10 mg/d x21d q4wk (Arm A) or LEN + EA 60,000U SC/wk (Arm B). Primary endpoint was IWG 2006 major erythroid response (MER) rate after 4 cycles. Arm A non-responders were offered cross-over to combined therapy. Secondary endpoints included analysis of response biomarkers. Results: Between April 2009 and May 2016, 248 patients were enrolled and 195 were randomized and will be included in the primary analysis. Interim analysis of 163 patients (Arm A, 81; B, 82) accrued before July 2015 showed that the study met predefined stopping criteria. Baseline characteristics were balanced between arms. Median age was 74 years (range, 47-89) receiving a median of 2 RBC units/mo (0-8). Overall, 64 (39%) patients had Low IPSS risk and 90 (55%) Int-1 risk. Among these, 150 received prior rhuEpo (92%) and 27, azanucleosides (17%). In an ITT analysis, MER rate was significantly higher with combination therapy, Arm B 25.6% (n=21) vs. Arm A 9.9% (n=8) (P=0.015). Among 116 patients evaluable at week 16, 33.3% (20/60) and 14.3% (8/56) achieved MER, respectively (P=0.018), with a median response duration of 25.4 months vs. not reached in Arm A responders. Response to combined treatment was associated with baseline CD45-isoform distribution in erythroid precursors. Patients achieving MER had a significantly lower CD45 RA+RB:RO ratio (median, 1.51) compared to non-responders (median, 4.21; P=0.04), favoring homo-dimerization of the short CD45-RO isoform and inhibition of phosphatase activity. MER rate in Arm B patients with a low isoform ratio (< median) was 72.7% vs. 18.2% in the high ratio group (P=0.03). Thirty-four Arm A non-responders crossed over to combination-therapy with only 1 MER. There was no difference in the frequency or distribution of >Grade 3, non-hematologic AEs. Conclusions: LEN restores sensitivity to rhEpo in Epo-refractory LR-non-del(5q) MDS patients to yield durable and significantly higher rates of erythroid response to combination treatment without added toxicity. Erythroid CD45 isoform profile may serve as a response biomarker for selection of candidates for combination therapy. Disclosures Bennett: Celgne: Membership on an entity's Board of Directors or advisory committees. Altman:Syros: Honoraria; Janssen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau. Schiffer:Teva: Other: DSMB member; BMS: Research Funding; Ariad: Research Funding; Pfizer: Other: DSMB member.

Background: The EZH2 gene (Enhancer of Zester homolog 2), located on chromosome (chr) 7q, is mutated in ~10% of patients with myelodysplastic syndromes (MDS) or MDS/myeloproliferative neoplasm (MDS/MPN). EZH2 gene mutations are associated with negative clinical outcomes; however, the role of EZH2 protein in MDS or MDS/MPN is largely unknown. In contrast to lymphomas or solid tumors in which expression is upregulated, decreased EZH2 mRNA expression was recently reported in 47% of MDS patients compared to normal controls and was more prevalent in patients with chr7 abnormalities. Furthermore, patients with decreased EZH2 expression lacking chr7 abnormalities had better overall survival (OS) (Cabrero M et al 2016). This study aims to explore EZH2 protein expression by immunohistochemistry (IHC) in MDS and MDS/MPN and the relationship to gene mutation and clinical outcome. Methods: Retrospective analysis (Institutional Review Board approved) of MDS or MDS/MPN cases seen at Moffitt Cancer Center between 7/2013-5/2015 with myeloid targeted, next generation sequencing (NGS) panel was performed. Bone marrow biopsies corresponding to NGS date were used for IHC. Nuclear EZH2 expression was assessed semi-quantitatively using an IHC score calculated by multiplying signal strength (0-3+) with percent positivity in hematopoietic cells. OS was considered duration between date of diagnosis and date of death. P-values were determined using Chi-squared, student t-test, ANOVA, or log-rank analysis with p Results: EZH2 mutations were identified in 69/620 MDS and MDS/MPN patients (11.13%). Of these, IHC was performed on 44 patients with EZH2 mutations including 33 MDS and 11 MDS/MPN [median age 74 years (y), male to female (M: F) ratio 2:1], and on 25 EZH2 wild type (WT) patients (median age 70.5 y, M:F ratio 2.1:1, including 22 MDS and 3 MDS/MPN). While mean EZH2 IHC score was reduced in all MDS cases compared to hematologically normal, WT controls (n=5), more controls are required to reach statistical significance (MDS IHC score 77.60±8.09 control score 102.50±14.00, p=0.167). However, there was significantly decreased expression in EZH2 mutant compared to EZH2 WT cases (IHC score, 0.5716±0.09 vs 1.113±0.11, p Conclusion: EZH2 mutation is associated with reduced protein expression, chr7 abnormalities and inferior OS in MDS and MDS/MPN. Analysis of a larger cohort is warranted to explore the role of EZH2 protein in the pathogenesis and natural disease history of MDS and MDS/MPN. Figure OS of patients with MDS and MDS/MPN with or without EZH2 mutation Figure. OS of patients with MDS and MDS/MPN with or without EZH2 mutation Disclosures Komrokji: Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: We reported that ineffective hematopoiesis in MDS results from caspase-1-dependent, pyroptotic cell death. Pyroptosis, which culminates in cytolysis, is initiated by the NOD-like receptor NLRP3, a redox-sensitive, cytosolic sensor of danger signals. Upon activation, NLRP3 recruits the ASC (apoptosis-associated speck-like protein containing a CARD (caspase activation and recruitment domain)) adapter protein that polymerizes to create large cytoplasmic filaments. Exposure of the ASCCARDdomain on the filament surface fosters docking of pro-caspase-1 that initiates processing of pro-IL1β and -IL18. Large cytoplasmic aggregates formed by filament clusters are known as ASC specks. Upon cytolysis, ASC specks are released into the extracellular space where they retain catalytic activity and propagate inflammation. Given the magnitude of pyroptosis in the bone marrow (BM) and maturing cells, we hypothesized that detection of ASC specks in peripheral blood (PB) plasma may serve as a biologically-rational MDS biomarker. Methods: We optimized a flow cytometry assay for detection of extracellular ASC specks averaging 1µm in size. Following protein quantitation, 300µg was aliquoted from each donor and stained with rabbit-anti-ASC 1o-Ab at a 1:1500 dilution for 60' at 37°C. 1:1500 dilution 2o-Ab was added and incubated for 30' at 37°C. Sample acquisitions were made on a BD FACSCalibur flow cytometer. PB plasma samples were obtained from normal donors (ND, n=40) and patients with MDS (n=220), de novo AML (n=16), 2o AML (n=26), CMML (n=20), CLL (n=50), CML (n=52), ALL (n=7), ET (n=20), PV (n=20), myeloma (n=20) and patients with Type-2 diabetes (T2D, n=25) on IRB-approved protocols, providing >90% power to detect significant differences between diseases. The diagnostic biomarker is defined as % of PB plasma ASC specks adjusted for glucose. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were used to evaluate classification accuracy of the biomarker at varied cutoff points. Results: The % PB-ASC specks was significantly increased in lower-risk (LR) MDS vs. ND (n=196, 27.7±1.5 vs. 7.2±0.7, respectively; p=2.8x10-32). No significant difference was observed between ND and higher-risk (HR) MDS (12.4±3.0), however, % of PB-ASC specks was significantly increased in LR (n=196) vs. HR (n=16) MDS (p= 2.0x10-5). Because hyperglycemia stimulates NLRP3 inflammasome assembly and activation, we measured plasma glucose by colorimetric assay to adjust for this potentially confounding variable. % PB-ASC specks were normalized to plasma glucose concentration and corrected for volume. Glucose-adjusted speck % in LR-MDS remained significantly higher vs. ND (1.2±0.2 and 0.02±3.0x10-3, respectively; p=3.7x10-6). Notably, the corrected % PB specks was not significantly greater in HR-MDS (0.6±0.5) vs. ND or between LR-MDS vs. HR-MDS, although the HR-MDS sample size is small. Importantly, LR-MDS samples (1.2±0.2) displayed significantly greater corrected % ASC specks compared to de novo AML (0.3±0.2, p=2.3x10-3), 2o-AML (0.16±0.05, p=5.5x10-5), CMML (0.03±0.01; p=4.4x10-6), CLL (0.03±4.4x10-3, p=4.4x10-6), CML (0.04±0.01; p=5.2x10-6), ALL (0.2±0.09, p=9.9x10-5), ET (0.17±0.07, p=8.7x10-5), PV (0.12±0.06, p=3.8x10-5), myeloma (0.14±0.04, p=3.8x10-5) and PB from non-cancer patients with T2D (p=4.8x10-6), suggesting specificity for MDS (see Figure). A cut off of 0.053 was selected to minimize total misclassification error (false positive + false negative). With this cutoff, the biomarker achieves 99% sensitivity and 81% specificity in classifying MDS from ND. The corresponding PPV and NPV are both 95%. In a preliminary cohort of lenalidomide-treated LR-MDS patients, PB-ASC specks decreased a mean of 48% (range, 3-69%) at week 16, suggesting that specks may serve as a biomarker index of ineffective hematopoiesis. Conclusion: ASC specks are readily quantified by flow cytometry and profoundly increased in PB plasma of MDS patients compared to ND and other hematologic malignancies. ASC specks are a sensitive and specific diagnostic biomarker for MDS that may also serve as an index of disease activity and emerging treatment response. Disclosures Pinilla-Ibarz: Novartis: Consultancy; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau. Komrokji:Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

Background While lenalidomide (LEN) shows high efficacy in myelodysplastic syndromes (MDS) with del[5q], responses can be also seen in patients presenting without del[5q]. We hypothesized that improved detection of chromosomal abnormalities with new karyotyping tools may better predict response to LEN. Design and methods We have studied clinical, molecular and cytogenetic features of 42 patients with MDS, myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes and secondary acute myeloid leukemia (sAML) without del[5q] by metaphase cytogenetics (MC) who underwent therapy with LEN. Results Fluorescence in situ hybridization (FISH) or single nucleotide polymorphism array (SNP-A)-based karyotyping marginally increased the diagnostic yield over MC, detecting 2/42 (4.8%) additional cases with del[5q], one of whom were responded to LEN. Responses were more often observed in patients with a normal karyotype by MC (60% vs abnormal MC; 17%, p = .08) and those with gain of chromosome 8 material by either of all 3 karyotyping methods (83% vs all other chromosomal abnormalities; 44% p = .11). However, 5 out of those 6 patients received combined LEN/AZA therapy and it may also suggest those with gain of chromosome 8 material respond well to AZA. The addition of FISH or SNP-A did not improve the predictive value of normal cytogenetics by MC. Mutational analysis of TET2, UTX, CBL, EZH2, ASXL1, TP53, RAS, IDH1/2, and DNMT-3A was performed on 21 of 41 patients, and revealed 13 mutations in 11 patients, but did not show any molecular markers of responsiveness to LEN. Conclusions Normal karyotype and gain of chromosome 8 material was predictive of response to LEN in non-del[5q] patients with myeloid malignancies.

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3 +/- 1.4(SE) vs. 25.6 +/- 3.2 aggregates/cell; p 3-fold increase in cluster size (p

Introduction: Accumulating evidence implicates innate immune activation in the pathobiology of myelodysplastic syndromes (MDS) and its inflammatory bone marrow microenvironment. Excess elaboration of S100A9 accompanied by secondary induction of TNFα and IL-1β, characterize the inflammatory milieu found in lower risk MDS. Erythroid stimulating agents (ESA) and lenalidomide (LEN) are erythropoietic promoters with known anti-inflammatory properties. To date, the role of such inflammation parameters in endogenous erythropoietin (Epo) regulation and response to such treatments has not been investigated. Herein, we investigated the role of these inflammatory cytokines on Epo elaboration and response to ESA and LEN in MDS. Materials and Methods: The HepG2 hepatoma cell line was used to investigate Epo elaboration in vitro. Serum collected from 311 MDS patients from 3 centers (AOU Careggi, University of Firenze; Saint Louis hospital in Paris; H. Lee Moffitt Cancer Center in Tampa) were investigated. 125 were obtained prior to ESA treatment and 186 prior to LEN or LEN+EPO treatment. ELISAs for S100A9, S100A8, TNFα, IL1β and EPO were performed. Clinical data were analyzed from all patients. Spearman's correlation, Mann-Withney and Jonckheere-Terpstra tests were used for analysis of continuous variables. Chi-square test was used for analysis of non-continuous variables. Results: Median age of the patients was 76 years [41-94]. IPSS was low, intermediate-1, intermediate-2 and high risk in 47%, 49%, 3% and 1% of patients, respectively; whereas 28%, 42%, 22%, 7% and 1% of patients were very low, low, intermediate, high and very high risk according to IPSS-R. Serum S100A9 concentration was significantly higher in patients with lower risk versus higher risk MDS (17,798 pg/ml vs 15,291 pg/ml, p=0.01). No significant difference was observed for TNFα and IL1 β according to IPSS or IPSS-R. ELISA quantitation following 24h in vitro stimulation of HepG2 cells with S100A9 (10 to 20 µg/ml), TNFα (10 to 100 ng/ml) or IL1β (10 to 100ng/ml) showed that each cytokine significantly suppressed Epo elaboration in a concentration-dependent manner. To validate these findings, we assessed the relationship between serum concentration of these inflammatory cytokines and Epo level in MDS patients. We observed a significant negative correlation between TNFα and Epo concentration (r=-0.158, p=0.01), and a corresponding significant negative correlation between S100A9 and Epo (r=-0.143, p=0.01). We also found a significant positive correlation between S100A9 and S100A8 (r=0.757, p Conclusion: These findings demonstrate that S100A9 and its NFκ-B transcriptional target, TNFα, directly suppress Epo elaboration and endocrine response to anemia in MDS. These inflammatory proteins may serve as rational biomarkers for response to lenalidomide and/or ESA treatment and merit prospective validation. Therapeutic strategies that either neutralize S100A9 or suppress elaboration of this alarmin may improve erythropoiesis in MDS by restoring Epo response and suppressing innate immune effectors. Disclosures Fenaux: Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

Autoimmune disorders (AIDs) are commonly observed in up to 30% of myelodysplastic syndromes (MDS) patients. Clinical manifestations such as acute neutrophilic febrile dermatosis, rheumatoid arthritis, inflammatory bowel disease, pulmonary infiltrates and peripheral polyneuropathy, precede or accompany the diagnosis of MDS. In fact, large-scale epidemiologic studies have suggested that patients with AIDs have an elevated risk of developing MDS. To gain more insight into the association of MDS and AIDs, bone marrow mononuclear cells available from 202 patients were sequenced for 'inflammsome' pathway genes NLRP3 and MYD88, which have been shown to carry mutations in various AIDs. According to WHO classification, the patient cohort included 49% RA/RARS/RCMD, 24% RAEB, 6% 5q syndrome, 10% AML, 6% MPD/MDS and 5% tMDS. Median age of the cohort was 62 years (IQR 54.8-62.3). Autoimmune data was available on 129 patients and 49/129 patients had evidence of AID. 31 patients were diagnosed with MDS-associated 'Sweets Syndrome' AID. Myeloid neoplasm-related 22 gene panel mutation data was available for 171 patients. Initially all protein coding exons of NLRP3 and MYD88 genes were sequenced in 31 cases diagnosed with MDS-Sweets syndrome. Targeted sequencing (Miseq, Illumina) revealed NLRP3 variants in 6/31 cases. NLRP3 heterozygous variants detected were V200M (n=1/31, 3%), Q705K (n=4/31, 13%) and T954M (n=1/31, 3%). Two of these variants (V200M and Q705K) have been previously described as pathogenic and linked with various autoimmune disorders1,2. Variant T954M is reported in COSMIC v73 database as a confirmed somatic mutation. The frequency of these variants in general European population (NHLBI ESP 2015) varies in-between 0.6% to 5% (V200, rs121908147 MAF= 0.7%; Q705, rs35829419 MAF= 5% and T954, MAF= NLRP3 heterozygous variants V200M and Q705K were observed in 2.3% (n=4/171) and 12% (n= 20/171), of the patients screened, respectively. T954M variant was not detected in the follow-up cohort. Sequencing of the paired constitutional DNA (CD3+ lymphocytes/ Skin biopsy), where available (n=10), confirmed that V200M and Q705K are inherited SNPs. The frequency of these NLRP3 variants observed among our study population is marginally higher than previously described in the general European population (Pearson chi-square test, P >0.05) In order to detect the effects of the NLRP3 variant on the inflammasome pathway, we quantified the serum cytokine (IL1β, IL18, TNFα and INFγ) levels using ProcartaPlex Human Cytokine & Chemokine Panel 1A (34 plex, eBioscience) kit in patients with NLRP3 variants (MDS/sweets syndrome, n =3) vs NLRP3 wildtype (MDS-Sweets syndrome, n=5; MDS/non-sweets syndrome, n=2). No difference was observed in the serum cytokine levels between two patient groups. Next, we investigated the mRNA levels of the NLRP3 and other genes that lie downstream of the NLRP3 inflammasome pathway (IL1β, IL18, TNFα, CASPASE-1 and INGγ). 34 patients (NLRP3 variant, n= 22 and NLRP3 Wildtype, n= 12) were included in this analysis. An increased trend in the IL18 mRNA levels was observed in patients with NLRP3 variant vs NLRP3 wildtype. In conclusion, our study shows that the NLRP3 variants are enriched in MDS patients with AIDs compared to historical data on the general population. Patients with NLRP3 variants have an increased levels of IL18 mRNA levels. A larger study to explore the inflammasome pathway is needed to define the molecular pathogenesis of the autoimmunity associated with MDS. Identifying the casual genomic variants of AIDs in MDS may provide relevance to development of novel therapeutic strategies. References 1. Verma D, Sarndahl E, Andersson H, et al. The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1beta and IL-18 production. PLoS One. 2012;7(4):e34977. 2 . Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat Genet. 2001;29(3):301-305. Disclosures Kulasekararaj: Alexion: Consultancy. List:Celgene Corporation: Honoraria, Research Funding.

Recent studies suggest that aging-associated inflammation, or "inflammaging", contributes to genetic instability and MDS predisposition. Although innumerable somatic genetic events have been annotated in recent years, including many that are not unique to MDS, they are not sufficient for disease initiation. The precise underlying mechanisms conducive to the emergence of these genetic events also remain to be delineated. We reported that bone marrow (BM) myeloid derived suppressor cells (MDSC) activated by the damage associated molecular pattern (DAMP) protein S100A9, promote ineffective hematopoiesis and the development of MDS. Inflammaging associated alterations in metabolism have been implicated in predisposition to cancer development with age. Here we report that S100A9 and ROS-induced inflammaging are associated with insulin resistance and hyperglycemia in the BM microenvironment that triggers activation of adaptive oncogenic pathways and genomic instability in HSPC. Glucose concentrations were markedly elevated in MDS BM plasma vs. age-matched controls, and directly correlated with S100A9 concentration (r=0.513, P=0.003, n=41). The magnitude of BM-glucose elevation significantly exceeded that in the peripheral blood and negatively correlated with the proportion of HSPC while directly correlating with BM MDSC percentage. S100A9 transgenic (Tg) mice displayed age-dependent elevation of glucose in peripheral blood and BM when compared to wild type mice accompanied by accumulation of somatic mutations (SM) by sequencing in aged S100A9-Tg compared to younger counterparts. NGS of 38 primary MDS BM specimens showed that SM common to MDS, such as those involving ASXL1, U2AF1 and DNMT3A, we re present only in high glucose stratified MDS-BM specimens (glucose > 110 ug/ml). Furthermore, there was a strong correlation between the cellular ROS/nuclear-β-catenin to DNA damage (γH2AX+ cells) linking S100A9-induced ROS accumulation to genetic instability. Elevation in BM plasma glucose was specifically associated with upregulation of the fat mass and obesity associated (FTO) transcript and protein in both MDS BM and S100A9Tg mice. FTO is a risk factor for type 2 diabetes, and encodes an α-ketoglutarate-dependent dioxygenase that functions as an RNA demethylase specific for N 6-methyladenosine (m6A) residues, targeted by splicing factors. Both human and murine MDS specimens displayed decreased m6A mRNA methylation compared to controls. FTO knockdown with CRISPR increased mRNA m6A methylation in MDS primary specimens, whereas overexpression of FTO led to a corresponding decrease that was enhanced by S100A9 stimulation. Moreover, S100A9 treatment induced FTO and demethylation of m6A mRNA in human and murine BM cells. These effects were accompanied by disruption of spliceosomes in the nucleus, as demonstrated by delocalization of SRSF2 from nuclear speckles into the cytoplasm where they colocalize with FTO in MDS patients. Interestingly, from the S100A9Tg mouse sequencing studies, we discovered that the mutation hotspots were in locations specific for histone H3K27 acetylation which have been previously linked to genomic instability, as well as splicing regulation, and regulated by histone deacetylase 1 (HDAC1). Reduced HDAC1 levels are known to make cells hypersensitive to DNA-damaging insults, such as those inducing ROS, similar to what we observed with treatment of S100A9 in healthy human bone marrow, MDS patient samples and S100A9Tg mice. Furthermore, this correlated with increased levels of DNA damage in our patient samples and in our murine S100A9Tg model as measured by phosphorylated γH2AX histones. Our studies provide a biological rationale for the initiation of DNA-genetic damage under inflammatory senescence conditions in MDS. These findings demonstrate that S100A9-induced inflammation activates a signaling cascade that enhances development of splicing variants and somatic gene mutations critical to MDS pathogenesis. Disclosures List: Celgene Corporation: Honoraria, Research Funding.

Background: TP53 mutations have been reported in 5-10% of patients with myelodysplastic syndromes (MDS) with a higher frequency in those who harbor del(5q) and complex cytogenetics. Next generation sequencing (NGS) data in large clinical cohorts has revealed TP53 mutation is a strong independent prognostic factor. Limited studies have shown an association of p53 protein overexpression with TP53 mutation as well as other clinical characteristics, such as blast count, cytogenetics, and mutant TP53 variant allele frequency (VAF). The study aims to validate the association of p53 overexpression by immunohistochemical (IHC) staining with TP53 mutation, and to explore its relationship to clinical outcome and mutation burden in MDS. Methods: We retrospectively analyzed MDS patients with or without transformation to acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) diagnosed between 7/2013 and 5/2015. Only those cases with available bone marrow (BM) biopsies with good quality (>1.0 cm in length) corresponding to the NGS testing date were included. Five normal BMs were used as controls. IHC was performed according to our institutional standard protocol and modified in accordance with the manufacturer. Nuclear expression of p53 was assessed semi-quantitatively using an IHC score calculated by multiplying IHC stain intensity with percent positivity in hematopoietic cells. Paired t-test was used for numerical parameters. Hazard ratios were generated using the standard cox proportional harzard model. Survival was analyzed by the Kaplan-Meier method and overall survival (OS) was defined as the duration between date of diagnosis and date of death. Results: Of 201 patients with myeloid malignancies, 29 (14%) harbored clinically significant mutations by NGS testing. Twenty two of these (9 AML-MRC,13 MDS) had available BM biopsies. An additional 32 patients (27 MDS, 5 AML) with wild type (WT) TP53 were included. Clinicopathologic differences of these groups are summarized in Figure 1. IHC analysis showed significantly (p=1.21x10-6) elevated nuclear p53 expression in TP53 mutated patients (mean score, 1.11± 0.164) compared to WT (0.037 ±0.014). A higher p53 IHC score in mutated patients correlated with a complex karyotype (n=14) compared to those without (n=6) (1.235 ± 0.206, and 0.580 ± 0.257, respectively, p=0.054). A significant positive correlation between IHC score and cytogenetic risk group according to revised international prognostic scoring system ( R-IPSS) existed in our cohort (p1.0 [16 month (95% CI, 8-23)] (p= 0.007). Similarly, with a p53 IHC score cutoff of 0.5, median OS was again not reached in those patients with 0.5 (p=0.015). HR for IHC score > 0.5 was 3 (95% CI, 1.2-8), p=0.02. HR for IHC score >1.0 was 3.5 [(95% CI, 1.3-9), p=0.01]. Conclusion: p53 overexpression in MDS patients correlates with mutated TP53 and mutation burden. A higher IHC score was associated with poorer clinical features and outcomes. A larger cohort is warranted to define its diagnostic or prognostic utility. Figure 1. Clinicopathologic Characteristics in Our MDS/AML-MRC Cohort Figure 1. Clinicopathologic Characteristics in Our MDS/AML-MRC Cohort Disclosures Komrokji: Celgene: Consultancy, Research Funding; Pharmacylics: Speakers Bureau; Incyte: Consultancy; Novartis: Research Funding, Speakers Bureau. Padron:Incyte: Research Funding; Novartis: Speakers Bureau. Lancet:Celgene: Consultancy, Research Funding; Amgen: Consultancy; Pfizer: Consultancy; Boehringer-Ingelheim: Consultancy; Kalo-Bios: Consultancy; Seattle Genetics: Consultancy. List:Celgene Corporation: Honoraria, Research Funding.

Myelodysplastic syndrome (MDS) is one of the major types of acquired bone marrow failure characterized by impaired peripheral blood cell production (anemia and cytopenias). Although much has been learned regarding the molecular genetic events involved in the pathogenesis of the MDS hematopoietic stem/progenitor cell compartment (HSC/HPC), investigations of the environmental pressures underlying disease initiation have been limited. Prior investigations have shown that inflammation in the bone marrow microenvironment contributes to hematopoietic impairment, with inflammatory molecules providing regulatory cues driving the proliferation and apoptotic death of HSC/HPC. We have reported that, the accumulation of myeloid-derived suppressor cells (MDSCs) in the local inflammatory bone marrow microenvironment plays a major role in the direct pathogenesis of MDS. MDSCs function by producing mediators and inflammatory cytokines capable of suppressing hematopoiesis and are defined by the lack of all lineage markers and only one key surface receptor, CD33 (CD33+HLA-DR−Lin−). We recently uncovered that this receptor is greatly expressed in MDSC isolated from patients with MDS and plays an important role in MDSC-mediated hematopoietic suppressive function. We also found that this ITIM (immune-receptor tyrosine-based inhibitor motif) containing molecule could induce suppressive cytokines after its engagement with its newly identified ligand S100A9. Therefore, we tested the hypothesis that with a fully human IgG1 monoclonal antibody against CD33 (mAb33.1) we could prevent the engagement of this ligand/receptor pair as well as induce ADCC of pathogenic MDSC through the reduction in the accumulation of CD33+ MDSC, the immune suppression and the restoration of hematopoiesis in MDS bone marrow specimens. After testing primary specimens with mAb33.1 we saw a significant increase in ADCC activity via NK as evidenced by significant reduction in the proportion of MDSC in culture as compared to isotype control treated cells. This decrease correlated with an increase in CD107a granule mobilization as well as an increased cytotoxicity in a killing assay without changing the proportion of NK cells in the culture. Functionally, the concentration of secreted IL-10, TGFb and VEGF were decreased, as was the gene expression of these suppressive cytokines after treatment with mAb33.1 but was restored when the antibody was cross-linked with an anti-human IgG antibody demonstrating the blocking ability of the antibody in preventing CD33 downstream signaling. Importantly, all of these observations correlated with the restoration of hematopoiesis, as there was a significant increase in the formation of CFU-GM and BFU-E colonies on a methylcellulose assay (n=9). In addition mAb33.1 also displayed a protective effect on HSC by blocking ROS production and reducing DNA damage, as demonstrated by comet and H2AX assays. This work provides the ground for the development of a novel group of therapies directly aimed at the suppressive MDSC and blockage of their signaling, rather than directly targeting the malignant clone, with the long term goal of improving the local microenvironment. This strategy will provide the background to assess its clinical potential to serve as a therapeutic target in MDS and AML. Disclosures No relevant conflicts of interest to declare.

Erythropoietin receptor (EpoR) signaling is impaired in patients with Myelodysplastic Syndromes (MDS) despite appropriate growth factor production and cellular receptor display. We previously reported that EpoR signaling is dependent upon receptor localization within membrane lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity (McGraw KL, et al. PLoS One 2012). Here, we show that MDS erythroid progenitors display markedly diminished raft assembly (p=0.005) and smaller raft aggregates (p=0.023) compared to normal controls. Because lenalidomide triggers raft coalescence in T-lymphocytes to promote immune synapse formation, we assessed the effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lipid rafts were isolated from UT7 cells using ultracentrifugation and identified by GM-1 dot blot and Lyn kinase western blot. Lenalidomide rapidly induced lipid raft formation in UT7 cells which was confirmed by confocal microscopy visualization of GM-1 fluorescence. Lenalidomide also significantly induced lipid raft formation in pooled MDS erythroid progenitors (CD71+, cKit+) from 11 patients [mean raft size, control (n=569) vs. lenalidomide treatment (n=659), p Disclosures List: Celgene: Consultancy.

Abstract 920 Background: Stabilization of p53 as a result of ribosomal stress is a key effector of the hypoplastic anemia in del(5q)MDS. We recently reported that lenalidomide (LEN) stabilizes MDM2 to promote p53 degradation, fostering cell cycle transition and arrest of del(5q) progenitors in G2/M (Wei S, et. al. Oncogene 2012). P53 expression in erythroid precursors (EP) decreased in responders, but was up-regulated upon emergence of resistance to LEN (Wei S, et. al. PNAS 2010). We hypothesized that targeted suppression of p53 may be an effective strategy to restore erythropoiesis in del(5q)MDS EP after LEN-treatment failure. Methods: To test this hypothesis, we first investigated the in vitro effects of cenersen (Aezea®, Eleos Inc. Plymouth, MN), a 20mer phosphorothioate antisense oligonucleotide complementary to the exon 10 coding sequence of the TP53 gene transcript, on cellular p53 expression and colony forming capacity (CFC) in EP. Cenersen, through RNase H-mediated site specific mRNA cleavage, down regulates wild type & mutant TP53 mRNA and protein expression. Using a 2-stage EP differentiation assay, CD34+ cells from MDS patient specimens (del5q, n=11; non-del5q, n=4; normal donor, n=5) were cultured in StemSpan® SFEM expansion medium (StemCell Technologies, Vancouver, Canada) supplemented with SCF, EPO, and dexamethasone for 3–10 days, then transfected with 40nM cenersen or scrambled oligodeoxynucleotides using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). Cells were harvested after 3 days for analysis of p53 expression by fluorescence microscopy, FISH and methylcellulose cultures. CFC was assessed after 7–14 days incubation. Results: Del(5q) EP displayed greater nuclear p53 fluorescence intensity (FI) than non-del(5q)MDS or controls [del(5q) mean =82.12±11.4, non-del(5q)=41.55±13.05, controls=63.58±10.14, P=0.07; del(5q) vs. controls, P=0.33]. Treatment with cenersen significantly reduced nuclear (> 50%) and cytoplasmic p53 expression (> 59%) in EP in 9 of 11 del(5q) specimens, (mean nuclear FI, cenersen=41.75±9.02 vs. non-sense=86.84±13.12, P=0.01; mean cytoplasmic FI, cenersen= 23.38±3.14 vs. non-sense= 41.34±6.21, P=0.02). Similar but non-significant trends in nuclear p53 were observed in control (P=0.19) and non-del5qMDS (P=0.29) EP, whereas cytoplasmic p53 was significantly reduced only in controls (>52%, P=0.03; non-del5q, P=0.35). Cenersen treatment significantly increased recovery of BFU-E and CFU-E (n=15, P=0.01; n=13,P=0.0001, respectively) in MDS specimens, whereas there were no consistent changes in granulocyte-macrophage or mixed lineage colony yields. Similar but less robust improvements in CFC were observed in control samples with cenersen treatment (CFU-E, n=4, P=1.0; BFU-E, n=5, P=0.43). The magnitude of improvement in BFU-E in MDS specimens highly correlated with the extent of p53 suppression (nuclear Spearman r= -0.64, P=0.009; cytoplasmic r= -0.65, P=0.008). Changes in CFU-E showed no significant correlation with changes in nuclear (P=0.65) or cytoplasmic (P= 0.89) p53 expression. FISH analysis of cytospin preparations following oligonucleotide treatment of del(5q) EP using the EGR1 5q31/5p15.31 deletion probe (Cytocell, Cambridge UK) showed no significant differences between cenersen and non-sense treated cells (P=0.55, n=10). To determine if in vivo p53 suppression can restore transfusion independence (TI) after secondary LEN treatment failure, we treated 8 LEN-resistant, transfusion-dependent del(5q) MDS patients with dexamethasone (Dex) 20 mg weekly and continued LEN 5–10 mg/day. The glucocorticoid receptor acts as a ligand-dependent transcription factor and mutual antagonist of p53. All patients had Conclusions: Targeted suppression of p53 restores effective erythropoiesis in del(5q) MDS in the absence of clonal suppression. A clinical trial testing treatment with cenersen +Dex in LEN-resistant patients is currently in development. Disclosures: Smith: Smith Holdings LLC: Equity Ownership, Smith Holdings owns rights to the drug involved in this study. Patents & Royalties; Eleos, Inc: Eleos has licensed rights to the drug from Smith Holdings. Patents & Royalties. Daugherty:Eleos, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Wells:Alexion: Honoraria; Janssen: Honoraria; Celgene : Honoraria; Novartis: Honoraria, Research Funding. List:Celgene: Consultancy. Off Label Use: lenalidomide combined with Dexamethasone in lenalidomide refractory patients .

Abstract 3793 Introduction: Chronic Myelomonocytic Leukemia (CMML) is a genetically diverse hematologic malignancy characterized by cytopenias with or without leukocytosis, marrow dysplasia, monocytosis, splenomegaly, and a propensity to transform to Acute Myeloid Leukemia (AML). It is among the most aggressive chronic myeloid malignancies with a three year overall survival approximating twenty percent. The current treatment armamentarium is generally ineffective and mostly derived from studies that focused on Myelodysplastic Syndromes (MDS). As has been well defined in Juvenile Myelomonocytic Leukemia (JMML), we previously reported that primary CMML bone marrow mononuclear cells exhibit GM-CSF-dependent pSTAT5 hypersensitivity. To determine whether this signaling pathway is a viable therapeutic target, we present the preclinical characterization of KB003 activity on primary CMML bone marrow mononuclear cells. Methods: KB003 was engineered and provided by KaloBios Pharmaceuticals. The ability of KB003 to neutralize GM-CSF was tested in two ways using doses that ranged from .01 to 10,000 ng/ml of KB003. First, 10 ng/ml of GM-CSF was used to induce IL-8 production by the U937 cell line as measured by ELISA. Second, the GM-CSF-dependent cell line, MO7e, was cultured in the absence and in the presence of 0.1, 1, and 10 ng/ml of GM-CSF with or without KB003. Apoptosis and viability was assessed by annexin and 4′,6-diamidino-2-phenylindole (DAPI) staining by flow cytometry. To determine anti-GM-CSF sensitivity in CMML, primary cryoperserved bone marrow samples from 10 patients were reconstituted in prewarmed StemSpan H3000 with 10% FBS and microcultured in a 96 well plate with increasing doses of GM-CSF and KB003. After a 48-hour incubation period, cells were stained with myeloid-specific antibodies (CD34, CD38, CD14, CD33) and measured by multi-color flow cytometry. DAPI was used to measure cell viability. MethoCult hematopoietic progenitor assays were used to quantify the expansion and differentiation of CMML mononuclear cells in the presence of GM-CSF and escalating concentrations of KB003 in those samples with sufficient cell number for analysis (n=7). Results: KB003 effectively neutralized GM-CSF-induced IL-8 secretion in U937 cells with an IC50 of 48.2 ng/ml, as shown in Figure 1a. A dose of 0.1, 1, and 10 ng/ml of GM-CSF was used to protect MO7e cells from apoptosis and a survival benefit was achieved at each dose tested (Fig 1b). In this assay, KB003 neutralized 0.1 ng/ml of GM-CSF, but was unable to neutralize GM-CSF at higher doses (10 ng/ml). Using CMML samples, doses ranging from 10 to 100 μg/ml of KB003 were tested in apoptosis and in colony formation assays. Using the gating strategy shown in Figure 2a, the percentage of mature (CD33+/CD14+) and immature (CD33+/CD14-) myeloid subpopulations within the viable gate after KB003 treatment was decreased (Figure 2b). As expected, KB003 had no effect on CD3+ T-cells in the mixed culture populations. In addition to myeloid subpopulations identified by CD14, the CD33+/CD38+ cells were also more sensitive than CD33+/CD38- or CD38+/CD34+ inhibition by KB003 (Figure 2c, p Conclusion: GM-CSF neutralization using KB003 suppresses CMML progenitor survival in vitro. These preclinical data suggest that KB003 and other GM-CSF signaling axis inhibitors merit further investigation in CMML. A more committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML. This is consistent with the results showing CD38 to be a marker of GM-CSF-dependent pSTAT5 sensitivity in JMML and may prove to be predictive biomarker for anti-GM-CSF therapy in CMML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3455 Background: Lenalidomide (LEN) and its analogue, pomalidomide, promote erythroid lineage competence and in vitro colony-forming capacity. In patients with non-del(5q) myelodysplastic syndrome (MDS), LEN restores erythropoiesis in a subset of patients (List et al. N Eng J Med 2005;352:549). Investigations by Ebert et al. showed that such responders to LEN treatment display repression of erythroid specific genes and that LEN restored transcriptional response to erythropoietin (Epo) (Ebert BL et al. PLoS Medicine 2008;5(2):e35), suggesting that LEN enhances Epo receptor (R) signal fidelity. We previously reported that LEN induces cellular expression of JAK2 associated EpoR in a concentration-dependent manner, however, the mechanism of regulation is unclear (Basiorka et al. Blood 2011,118: 2382a). Recent investigations implicated inhibition of the cereblon RING (really interesting new gene) finger domain containing E3-ubiquitin ligase complex as a key target of the immunomodulatory drugs (IMiDs) responsible for the teratogenic effects of thalidomide and the cytotoxic effects of LEN in multiple myeloma (Ito T et al. Science 2010; 327:1345–50; Zhu YW et al. Blood 2011;118:4771–9). We recently showed that LEN also interacts with the RING finger E3 ubiquitin ligase, murine double minute 2 (MDM2) to inhibit ligase ubiquitination and stabilize the protein (Wei et al. Oncogene, MS#ONC-2011-01840R, 2012). Because EpoR turnover is regulated by ubiquitination and proteasomal degradation, we evaluated the effects of LEN on the E3-ubiquitin ligase, RNF41, which regulates steady state or ligand independent, Janus kinase (JAK2) associated Type I receptor internalization (Wauman et al. J Cell Science. 2011;124:921–932). We hypothesized that LEN upregulates JAK2/EpoR expression through inhibition of RNF41 function, thereby increasing EpoR expression and enhancing JAK2 competent receptor signaling. Methods and Results: Treatment of the UT-7 erythroid progenitor cell line with cycloheximide ±1μM LEN showed that LEN stabilized cellular EpoR (T1/2, LEN >72h vs. 56h). To determine if the effects of LEN on receptor turnover are restricted to Type 1 cytokine receptors, we examined the effects of LEN on cellular expression of IL3-R (Type 1) and c-Kit (Type 2). LEN up-regulated IL3-R expression in a concentration-dependent fashion, whereas c-Kit expression was unchanged, confirming Type 1 receptor specificity. To determine if LEN alters EpoR/RNF41 interaction, we assessed protein association after LEN treatment. Immunoprecipitation (IP) of either EpoR or RNF41 followed by immunoblot (IB) for the binding partner showed that LEN promoted EpoR/RNF41 association in a concentration dependent manner. To investigate the effects of LEN on RNF41 function, we assessed protein specific ubiquitination after proteasomal inhibition with bortezomib followed by LEN treatment. IP of RNF41 and EpoR followed by ubiquitin IB showed that LEN inhibited RNF41 auto-ubiquitination in a concentration-dependent fashion accompanied by a corresponding decrease in EpoR ubiquitination, suggesting that LEN inhibits RNF41 ubiquitination to increase EpoR accumulation. To confirm that RNF41 is the principal target of LEN responsible for EpoR stabilization, we transfected HEK293T cells with EpoR and/or RNF41 expression vectors using the calcium phosphate method. Steady state EpoR expression was lower in EpoR/RNF41 cells compared with cells transfected with EpoR alone. Moreover, EpoR upregulation by LEN was abrogated in EpoR/RNF41 cells indicating that cellular RNF41 is a critical determinant of EpoR upregulation by LEN. Immunohistochemical staining of 16 bone marrow biopsies from non-del(5q) LEN-treated MDS patients are in progress to determine the relationship between cellular RNF41 level in erythroid precursors and clinical response. Conclusion: Our findings suggest that LEN acts as a broad RING finger E3-ubiquitin ligase inhibitor, whose targets extend to the Type 1 cytokine receptor specific, RNF41. RNF41 inhibition by LEN promotes accumulation of signaling competent JAK2/EpoR complexes that may augment Epo responsiveness. Further investigation is warranted to determine if erythroid expression level of RNF41 may serve as a biomarker for response to LEN in patients with non-del(5q) MDS. Disclosures: List: Celgene: Consultancy.

Abstract 788 Understanding the pathophysiology of myelodysplastic syndrome (MDS) is limited by a complex molecular mechanism and lack of an adequate animal model that recapitulates the role of inflammation in the abnormal hematopoiesis. We recently showed that patients with MDS have expansion of inflammation-related hematopoietic suppressive cells called immature myeloid-derived suppressor cells (MDSC) that display direct cytotoxic and suppressive effects on autologous hematopoietic progenitor cells (HPCs). Expansion of bone marrow (BM) MDSCs contributed to the production of inflammatory cytokines and reduced HPC survival underlying BM failure in lower risk patients. Here we provide evidence that MDSC activation, expansion and development is driven by overexpression of inflammatory-related signaling molecules, myeloid-related protein 8 (MRP8, encoded by S100A8) and MRP14 (encoded by S100A9). Both MRP proteins serve as the native endogenous ligands for Toll-like receptor 4 (TLR4), which is an important damage-associated molecular pattern (DAMP) mediating inflammatory response. We found higher expression of MRP8 and MRP14 in BM mononuclear cells from MDS patients compared to healthy donors, in whom these proteins were not detectable. High surface expression of both TLR2 and TLR4 in MDS MDSCs compared to healthy donor MDSCs confirmed that this signaling pathway is activated in MDS. Inhibition of MRP8/MRP14 proteins in MDSCs using specific shRNAs dramatically attenuated IL-10 and TGF-β production and rescued BFU-E and CFU-GM colony formation of autologous bone marrow progenitors. These data show that inflammation-associated MRP8/MRP14 expression plays a critical role in the suppressive activities of MDS MDSCs. We therefore generated S100A9 transgenic mice (S100A9Tg) overexpressing the murine MRP14 homologue and investigated the role of this protein in bone marrow failure. Significant MDSC accumulation was evident in the BM of S100A9Tg mice by 6 weeks, but not in S100 knockout (KO) or wild type (WT) mice. Similar to human MDS, MDSCs from S100A9Tg mice, but not S100KO or WT mice, significantly inhibited BFU-E colony formation. Depletion of MDSCs in vitro rescued BM colony formation in the S100A9Tg mice indicating that the BM suppression is mediated by MDSC cells. TGF-β and IL-10 secretion was significantly increased in S100A9Tg mice, substantiating the role of S100A9 as an essential inflammatory factor that regulating MDSC suppressive activity. Analogous to human MDS, 6-month old S100A9Tg mice developed ineffective hematopoiesis with severe anemia, leukopenia, and thrombocytopenia accompanied by MDS-like morphological features. BM aspirates and core biopsies from S100A9Tg mice were hypercellular with trilineage cytological dysplasia characteristic of MDS. Treatment with ATRA, which induced the differentiation of MDSCs rescued hematopoiesis in S100A9Tg mice. Our findings indicate that primary BM expansion of MDSC is sufficient to perturb hematopoiesis and result in the development of MDS, supporting the notion of microenvironment-conducive oncogenesis. S100A9Tg transgenic mice provide a novel in vivo model of human MDS for target discovery and testing of novel therapeutics. Disclosures: No relevant conflicts of interest to declare.