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PURPOSE Approximately 20% of patients with TP53-mutant myelodysplastic syndromes (MDS) achieve complete remission (CR) with hypomethylating agents. Eprenetapopt (APR-246) is a novel, first-in-class, small molecule that restores wild-type p53 functions in TP53-mutant cells. METHODS This was a phase Ib/II study to determine the safety, recommended phase II dose, and efficacy of eprenetapopt administered in combination with azacitidine in patients with TP53-mutant MDS or acute myeloid leukemia (AML) with 20%-30% marrow blasts (ClinicalTrials.gov identifier: NCT03072043 ). RESULTS Fifty-five patients (40 MDS, 11 AML, and four MDS/myeloproliferative neoplasms) with at least one TP53 mutation were treated. The overall response rate was 71% with 44% achieving CR. Of patients with MDS, 73% (n = 29) responded with 50% (n = 20) achieving CR and 58% (23/40) a cytogenetic response. The overall response rate and CR rate for patients with AML was 64% (n = 7) and 36% (n = 4), respectively. Patients with only TP53 mutations by next-generation sequencing had higher rates of CR (69% v 25%; P = .006). Responding patients had significant reductions in TP53 variant allele frequency and p53 expression by immunohistochemistry, with 21 (38%) achieving complete molecular remission (variant allele frequency < 5%). Median overall survival was 10.8 months with significant improvement in responding versus nonresponding patients by landmark analysis (14.6 v 7.5 months; P = .0005). Overall, 19/55 (35%) patients underwent allogeneic hematopoietic stem-cell transplant, with a median overall survival of 14.7 months. Adverse events were similar to those reported for azacitidine or eprenetapopt monotherapy, with the most common grade ≥ 3 adverse events being febrile neutropenia (33%), leukopenia (29%), and neutropenia (29%). CONCLUSION Combination treatment with eprenetapopt and azacitidine is well-tolerated yielding high rates of clinical response and molecular remissions in patients with TP53-mutant MDS and oligoblastic AML.

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.

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.

PURPOSE Impaired response to erythropoietin underlies ineffective erythropoiesis and anemia in myelodysplastic syndromes (MDS). We investigated whether treatment with lenalidomide (LEN), which augments erythropoietin receptor signaling in vitro, can restore and improve hemoglobin response to epoetin (EPO) alfa in patients with lower-risk, non-del(5q) MDS who have anemia that is refractory to or have low probability of benefit from treatment with recombinant erythropoietin. METHODS In a phase III, US intergroup trial, we randomly assigned patients to receive either LEN and EPO alfa or LEN alone following stratification by serum erythropoietin concentration and prior erythropoietin treatment. RESULTS A total of 195 evaluable patients were randomly assigned: 99 patients to the LEN-EPO alfa cohort and 96 to LEN alone. After four cycles of treatment, the primary end point of major erythroid response (MER) was significantly higher (28.3%) with the combination compared with LEN alone (11.5%) ( P = .004). Among 136 patients who completed 16 weeks of study treatment, 38.9% and 15.6% achieved MER, respectively ( P = .004). Additionally, minor erythroid response was achieved in 18.2% and 20.8% of patients, for an overall erythroid response rate of 46.5% versus 32.3%. Among LEN nonresponders, 38 crossed over to the addition of EPO alfa with 10 patients (26.3%) achieving a MER. Responses to the combined treatment were highly durable with a median MER duration of 23.8 months compared with 13 months with LEN alone. CONCLUSION LEN restores sensitivity to recombinant erythropoietin in growth factor–insensitive, lower-risk, non-del(5q) MDS, to yield a significantly higher rate and duration of MER compared with LEN alone (funded by the National Cancer Institute; E2905 ClinicalTrials.gov identifier: NCT02048813 ).

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.

7044 Background: Myelodysplastic syndromes (MDS) are characterized by aberrant maturation, ineffective hematopoiesis, cytopenia, and progression to acute myeloid leukemia. MDS pathogenesis is multifactorial and potentially linked to constitutive innate immune stimulation converging upon the NLRP3 inflammasome to induce pyroptosis, a caspase-1 dependent cell death. Inflammasome assembly is initiated by both cell-extrinsic stimuli including S100A9, the TLR4 and CD33 ligand, and cell-intrinsic danger signals licensing caspase-1 which activates IL1b and beta-catenin resulting in cell death and cellular proliferation leading to maturation and differentiation blocks. Further, EYA2 has been suggested to be an inflammasome activator, whereas cPLA2 has been suggested to be an inhibitor. The purpose of this study is to determine whether immunohistochemistry (IHC) may be utilized to assess expression of inflammasome components. Methods: An IRB protocol was approved prior to initiating this study. We retrospectively identified 43 low risk MDS patients. A tissue microarray (TMA) was constructed utilizing MDS bone marrow biopsy samples (2-3 representative cores per sample). IL-1, S100A9, EYA2, cPLA2, beta-catenin, and TLR4 expression were assessed by IHC after validation of each antibody. IHC expression was scored independently by two hematopathologists by calculating scores (product of staining intensity x percent expression). IHC expression was compared using Spearman correlation estimate. Demographic and clinical data were collected and correlated with IHC expression using Kruskal-Wallis test, Spearman correlation, and Logrank test. Results: Patients were median 72 years of age, 67% men and included 47% MDS-MLD, 35% MDS-RS, 14% MDS-SLD, 2% MDS del5q and 2% MDS-U. IL-1 expression correlated with beta-catenin expression, r = 0.42, 95% CI 0.115 to 0.658 (p = 0.007). There was a trend towards significance between IL-1 and cPLA2, r = 0.30 (p = 0.067); S100A9 and cPLA2, r = 0.31 (p = 0.052); and S100A9 and EYA2, r = 0.31 (p = 0.057). Percentage EYA2 expression correlated with blast count, r = 0.425 (p = 0.008). The IHC expression of these antigens did not correlate with WHO MDS subclassification, IPSS, R-IPSS, disease progression, or survival (p > 0.05). Conclusions: IHC staining of inflammasome activators using TMA may allow better characterization of molecular pathways contributing the MDS pathogenesis. A correlation was seen between expression of antigens known to be increased downstream of NLRP3 inflammasome activation. Furthermore, increased expression of EYA2 correlated with blast count. A future study will compare expression patterns between normal, low risk MDS and high risk MDS samples and correlate these findings with clinical outcome data to further elucidate the pathogenesis of MDS and identify potential targetable markers for novel therapeutic strategies.

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.

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.

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.

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.

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).

Altres ajuts: National Cancer Institute/National Institute of Health (5 R01CA131076-04); Molecular Genomics Facility (P30-CA076292); Sociedad Española Hematología y Hemoterapia (2014 SGR225); Fundació Internacional Josep Carreras i Celgene Spain. RTICC/RD12/0036/0044 Abstract:P53 is a key regulator of many cellular processes and is negatively regulated by the human homolog of murine double minute-2 (MDM2) E3 ubiquitin ligase. Single nucleotide polymorphisms (SNPs) of either gene alone, and in combination, are linked to cancer susceptibility, disease progression, and therapy response. We analyzed the interaction of TP53 R72P and MDM2 SNP309 SNPs in relationship to outcome in patients with myelodysplastic syndromes (MDS). Sanger sequencing was performed on DNA isolated from 208 MDS cases. Utilizing a novel functional SNP scoring system ranging from +2 to -2 based on predicted p53 activity, we found statistically significant differences in overall survival (OS) (p = 0.02) and progression-free survival (PFS) (p = 0.02) in non-del(5q) MDS patients with low functional scores. In univariate analysis, only IPSS and the functional SNP score predicted OS and PFS in non-del(5q) patients. In multivariate analysis, the functional SNP score was independent of IPSS for OS and PFS. These data underscore the importance of TP53 R72P and MDM2 SNP309 SNPs in MDS, and provide a novel scoring system independent of IPSS that is predictive for disease outcome.

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.

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.

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.

Stabilization of p53 in erythroid precursors in response to nucleosomal stress underlies the hypoplastic anemia in myelodysplastic syndromes (MDS) with chromosome 5q deletion [del(5q)]. We investigated whether cenersen, a clinically active 20-mer antisense oligonucleotide complementary to TP53 exon10, could suppress p53 expression and restore erythropoiesis in del(5q) MDS. Cenersen treatment of ribosomal protein S-14-deficient erythroblasts significantly reduced cellular p53 and p53-up-regulated modulator of apoptosis expression compared with controls, accompanied by a significant reduction in apoptosis and increased cell proliferation. In a two-stage erythroid differentiation assay, cenersen significantly suppressed nuclear p53 in bone marrow CD34+ cells isolated from patients with del(5q) MDS, whereas erythroid burst recovery increased proportionally to the magnitude of p53 suppression without evidence of del(5q) clonal suppression (r = −0.6; P = 0.005). To explore the effect of p53 suppression on erythropoiesis in vivo, dexamethasone, a glucocorticoid receptor-dependent p53 antagonist, was added to lenalidomide treatment in eight lower-risk, transfusion-dependent, del(5q) MDS patients with acquired drug resistance. Transfusion independence was restored in five patients accompanied by expansion of erythroid precursors and decreased cellular p53 expression. We conclude that targeted suppression of p53 could support effective erythropoiesis in lenalidomide-resistant del(5q) MDS.

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.

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.

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in -7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

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.

Introduction: TP53 mutant (mTP53) MDS and AML represent a distinct molecular cohort with poor outcomes. Hypomethylating agents (HMA) have emerged as preferred treatment for these patients with CR rate of 20-30% and median OS of 6-12 months. APR-246 is a mutant p53 activator with single agent activity in mTP53 AML. We report initial phase 1b results of APR-246 + AZA in mTP53 MDS/AML. Methods: Eligible pts included HMA naïve mTP53 MDS and oligoblastic AML (≤ 30% blasts) ≥ 18 years of age. Pts received APR-246 in a 3+3 dose escalation design (50, 75, 100 mg/kg lean body weight) 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. Primary objective was safety with AEs graded by CTCAE v4.03 and DLT assessment over 6 weeks. Secondary endpoints 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. Results: As of Jan 1, 2017, 9 pts (33% male; median age 65 years (39-73)) have enrolled with 3 pts per cohort. Three pts had AML-MRC and 6 had MDS; all pts had poor risk cytogenetics (11% poor, 89% very poor) and higher risk disease by IPSS-R (22% high, 78% very high). Median BM blasts were 18% (4-30). Seven pts (78%) remain on study: 2 pts in the 50mg/kg cohort discontinued treatment (Tx), 1 pt due to infection during C2 who later died of sepsis unrelated to Tx, and 1 pt electively discontinued in durable marrow CR (mCR) after 5 cycles of therapy. Median time on study is 106 days (14-221). Tx related AEs during the lead-in phase (all G1) included ataxia (n=1), dizziness (n=1), and facial numbness (n=1). AEs occurring in > 1 pt included dizziness (n=3), nausea (n=3), neutropenia (n=3), thrombocytopenia (n=3), infection (n=3), headache (n=2), pain (n=2), weakness (n=2), falls (n=2), facial numbness (n=2), and ataxia (n=2); all G1/G2 except neutropenia/thrombocytopenia (G4). No Tx-related SAEs or DLTs have occurred to date. Five of six pts were response evaluable with 1 pt discontinuing tx prior to 1st disease assessment. ORR by IWG was 100% with 4 CR (80%, 3/3 in DL2) and 1 mCR. All CR patients achieved complete cytogenetic response. One CR patient achieved a 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 (55-70%) which normalized on serial assessment ( Conclusions: APR-246 + AZA combination is well tolerated in mTP53 MDS/AML. Responses have been achieved in all pts (80% CR) accompanied by deep molecular remissions. The maximum tolerated dose has not been reached and dose escalation is ongoing. Citation Format: David A. Sallman, Amy DeZern, Kendra Sweet, David P. Steensma, Thomas Cluzeau, Mikkael Sekeres, Guillermo Garcia-Manero, Gail Roboz, Amy McLemore, Kathy McGraw, John Puskas, Ling Zhang, Chirag Bhagat, Armin Graber, Najla H. Al Ali, Eric Padron, Roger Tell, Jeffrey E. Lancet, Pierre Fenaux, Alan F. List, Rami S. Komrokji. Phase Ib/II combination study of APR-246 and azacitidine (AZA) in patients with TP53 mutant myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT068.

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.

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.

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.

Background: The myelodysplastic syndromes (MDS) are senescence-dependent stem cell malignancies. Although germ line mutations in RUNX1, CEBPA, ETV6, or GATA2 may underlie familial cases, inherited genetic polymorphisms influencing MDS susceptibility has not been evaluated. We performed the first genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) linked to MDS predisposition. Methods: DNA from 1361 MDS patients from 9 international centers was genotyped on the Affymetrix Genome-Wide Human SNP Array 6.0; all patients signed informed consent. Data on 4597 healthy controls genotyped on the same platform were obtained from the Database of Genotypes and Phenotypes (dbGaP). Applying standard quality control metrics and limiting to individuals of European ancestry, we analysed 999 MDS patients and 4,309 controls at 545,924 markers. We used logistic regression to determine associations with MDS after appropriate adjustment assuming an additive genetic model. Results: We identified 15 SNPs at 9 loci associated with MDS, including rs6780298 (3q13; p = 5.0×10-6) and rs1206819 (20q13; p = 1.54×10-6) that mapped to the intragenic regions of MORC1 and EYA2, respectively; and rs2473784 (1p31; p = 3.32×10-7) that mapped downstream of DEPDC1. Other hits (p≤5×10-6) at 4q28, 5p14, 9p22, 6p22, 10q21 are in or near SLC7A11, GUSBP1, SH3GL2, MOG and TRIM27, respectively. Using publicly available gene expression profiling data, we confirmed all of these genes are expressed in the hematopoietic compartment and MORC1, EYA2, DEPDC1, and SH3GL2 are increased in leukemic samples suggesting that variation in these genes may be functionally relevant in myeloid malignancies. Notably, EYA2 (20q13) flanks the commonly deleted region in del(20q) MDS and has oncogenic properties in solid tumors. SH3GL2 is very highly upregulated in leukemic samples and interacts with Dynamin-1, a GTP-binding protein involved in cellular trafficking and is similarly associated with solid tumorigenesis. To validate our observed associations, we analysed the 15 SNP markers in an independent set of 12,385 individuals of whom 117 developed hematologic malignancy (including MDS) during follow up. The markers at 1p31 including rs2473784 and 4 others in strong linkage disequilibrium were associated with individuals who developed hematologic malignancy (p Conclusions: These data provide the first genome-wide identification of germline variants associated with MDS. Current work is underway to replicate these findings in an independent sample set of MDS patients and healthy controls. If confirmed, these SNPs may serve as biomarkers to identify individuals at risk for MDS and potentially support new prevention strategies. Citation Format: Kathy McGraw, Chia-Ho Cheng, Ann Chen, Giulio Genovese, Thomas Cluzeau, Hui-Yi Lin, Bartlomiej Przychodzen, Mar Mallo, Leonor Arenillas, Azim Mohamedali, Lionel Ades, Ashley Basiorka, Brittany Irvine, David Sallman, Eric Padron, Lubomir Sokol, Andrea Pellagatti, Chimene Brest, Sophie Raynaud, Bjorn Nilsson, Jacqueline Boultwood, Benjamin Ebert, Francesc Sole, Pierre Fenaux, Ghulam Mufti, Jaroslaw Maciejewski, Peter Kanetsky, Alan List. Identification of genetic polymorphisms associated with myelodysplastic syndromes by genome-wide association study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2570.

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.

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.

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.

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.

Approximately 30-40% of patients with myelodysplastic syndromes (MDS) develop acute myeloid leukemia (AML). Several recurrent mutations have been identified in MDS using next-generation sequencing (NGS) technology and recent studies have greatly illuminated the molecular landscape of this disorder. However, the molecular events driving MDS progression to AML remain poorly understood. In order to investigate the genetic basis of leukemic transformation in MDS during disease progression, we evaluated the frequency and chronology of the acquisition of mutations using a targeted NGS myeloid gene panel on serial (paired) samples from 41 MDS patients before (pre-progression) and after disease progression (post-progression) to a more advanced subtype (n=7) or to AML (n=34). The mutational profile was characterized using a TruSeq Custom Amplicon panel (Illumina) targeting the hotspots of 31 recurrently (>1%) mutated genes in myeloid malignancies. Samples were run on an Illumina MiSeq and variants were annotated and filtered using Illumina VariantStudio v2.1.36, and interpreted according to the ACMG recommendations. The proportion of sequencing reads reporting a given mutation (variant allele frequency, VAF) was used to estimate the fraction of tumor cells carrying that mutation, and to determine whether mutations are clonal (in all tumor cells) or subclonal (in a fraction of tumor cells). A total of 100 and 123 mutations across 23 genes were identified in pre- and post-progression samples, respectively. The number of mutations was generally higher in the post-progression samples: the number of cases with one or two mutations was 24 in pre-progression samples and 16 in post-progression samples, while the number of cases with three or four mutations was 12 in pre-progression samples and 18 in post-progression samples. Several cases showed ≥5 mutations. The most frequently mutated genes (in >15% of samples) were ASXL1, TET2, SRSF2, U2AF1, RUNX1 and TP53; ASXL1 was the top ranking mutated gene with a frequency of 44% in pre-progression samples and 46% in post-progression samples. SF3B1, the most frequently mutated gene in MDS, was mutated in only two cases in our cohort. This finding is consistent with the strong association of SF3B1 mutation with the low-risk MDS subtype RARS, and its status as a good prognostic marker. Our patient cohort is highly selected, comprised only of patients whose disease has progressed and our data thus indicate that ASXL1 mutations are strongly associated with MDS cases that show disease progression and conversely that SF3B1 mutations are rarely associated with MDS disease progression. Mutations of genes involved in splicing (U2AF1, SRSF2), chromatin modification (EZH2, ASXL1) and DNA methylation (TET2, IDH1/2) were present in the pre-progression samples for almost all cases with mutations of these genes; these mutations often occur in founding clones (VAF >40%) and may play a role in disease initiation. Mutations of genes involved in transcriptional regulation (RUNX1, ETV6, PHF6) and signal transduction (NRAS, KRAS) were found in many cases in the post-progression sample only, suggesting that these are often late events that may co-operate with early events to drive disease progression. Interestingly, co-occurrence of NRAS and ASXL1 mutations was a frequent event in post-progression samples (n=5) in our cohort. It has been reported that NRas mutation and Asxl1 loss co-operate to drive myeloid proliferation and myeloid leukemia in mice, and our data support this observation. The average VAF of some mutations changed markedly during disease progression, with NRAS showing the largest VAF fold increase among genes mutated in >5 cases. The majority of subclones containing mutations in RUNX1 or ASXL1 expanded with disease progression. EZH2 mutations were mainly in the founding clone, while TP53 mutations were mainly in a subclone. For six cases we sequenced an additional serial sample, thus giving more precise information on the mutational profile evolution during disease progression. This is the first study to investigate the mutation status of a large group of MDS patients showing disease progression by the study of serial samples using a NGS myeloid gene panel. This study informs the timing of mutation acquisition during disease initiation and progression in MDS and closely related conditions, illuminating the genetic basis of leukemic transformation in these disorders. Disclosures No relevant conflicts of interest to declare.