List, Alan F.

In this phase 1 trial, inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was associated with clinically meaningful responses in 5 of 15 patients with relapsed or refractory chronic myelomonocytic leukemia (CMML). Preliminary data suggest that this approach may be tractable in CMML bearing activating NRAS mutations.

Background: Only a small subset of Lower risk (LR) MDS patients benefit from treatment with rhu-Erythropoietin (Epo). We previously reported that lenalidomide (LEN) restores sensitivity to Epo in MDS progenitors by inducing the formation of lipid rafts that are enriched for signaling competent, JAK2/Epo-receptor complexes (McGraw K, et. al. PLoS One 2014; Basiorka A, et. al. Cancer Res 2016). In the MDS-002 and MDS-005 trials, treatment with LEN monotherapy gave rise to RBC transfusion-independence (TI) in 26% of azanucleoside-naïve, transfusion-dependent (TD) LR, non-del(5q) MDS patients for a median of 10.2 and 7.75 months, respectively. In a pilot study of Epo-refractory LR-MDS patients, the addition of epoetin alfa (EA) to LEN treatment yielded erythroid responses in 28% of patients who were unresponsive to LEN alone, suggesting that LEN may overcome clinical resistance to augment response to rhEpo (Komrokji R, et. al. Blood 2012). To test this hypothesis, we performed a randomized phase III trial comparing treatment with LEN to LEN+EA in LR non-del(5q) MDS patients who were refractory to, or not candidates for treatment with rhEpo. Methods: Patients with Low or Intermediate-1 IPSS risk MDS with hemoglobin <9.5 g/dL who were unresponsive to rhEpo treatment or were TD (>2 units/month) with serum Epo >500mU/mL were eligible. Patients were stratified by serum Epo level and prior rhEpo (EA vs. darbepoetin vs. none) then randomized to treatment with LEN 10 mg/d x21d q4wk (Arm A) or LEN + EA 60,000U SC/wk (Arm B). The primary endpoint was major erythroid response (MER) at week 16 which was defined according to transfusion status at baseline: (1) achievement of RBC-TI for ≥ 8 consecutive weeks AND a sustained ≥1 g/dL hemoglobin rise compared to mean pre-transfusion baseline value in TD patients; and (2) a >2 g/dL rise in hemoglobin without transfusion for ≥ 8 consecutive weeks in non-TD patients (<4U RBC/8 wks). Arm A non-responders were offered cross-over to combined therapy. Results: Among 205 patients randomized, 14 were excluded from the primary analysis due to a 4 month interruption in drug supply. Among the 195 evaluable patients, 96 were assigned to Arm A and 99 to Arm B. Baseline characteristics were well balanced between arms with 85% of patients heavily TD, receiving a median of 4 units/8 weeks. Overall, 93% of patients received prior treatment with Epo and 18% azanucleosides. In an intent to treat analysis, 28/99 patients (28.3%) in Arm B achieved MER compared to 11/96 (11.5%) in Arm A (P=0.004). Among 136 patients who completed 16 weeks of study treatment, 28/72 (38.9%) and 10/64 (15.6%) achieved MER, respectively (P=0.004). Forty-four Arm A non-responders crossed over to combination-therapy with 11 patients (25%) experiencing a MER. Multivariable logistic regression analysis identified only treatment with LEN + EA as an independent covariate for erythroid response (P=0.01). Responses were durable with MER median duration of 23.8 months in Arm B compared to 13 months in Arm A. There was no significant difference in the frequency or distribution of >Grade 3, non-hematologic adverse events between treatment arms. Two patients progressed to AML while on study (Arm A), and no thromboembolic events were reported. Conclusions: LEN restores sensitivity to rhEpo in otherwise refractory, LR-non-del(5q) MDS patients to yield a significantly higher frequency of durable major erythroid responses compared to LEN alone. The addition of LEN to EA treatment is an effective strategy for the management of Epo-refractory patients with a potential duration of benefit extending to years. Disclosures List: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Verma:Janssen: Research Funding; BMS: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Komrokji:JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; celgene: Consultancy; pfizer: Consultancy. Luger:Onconova: Research Funding; Pfizer: Honoraria; Seattle Genetics: Research Funding; Cyslacel: Research Funding; Biosight: Research Funding; Ariad: Research Funding; Agios: Honoraria; Genetech: Research Funding; Jazz: Honoraria; Daichi Sankyo: Honoraria; Kura: Research Funding; Celgene: Research Funding. Mattison:Pfizer: Membership on an entity's Board of Directors or advisory committees. Altman:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; France Foundation: Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; prIME Oncology: Speakers Bureau; PeerView: Speakers Bureau; Cancer Expert Now: Consultancy; Novartis: Consultancy; Biosight: Other: US Lead. Claxton:Astellas Pharma: Other: Pharma support of clinical studies; Merck Sharp & Dohme Corp.: Other: Pharma support of clinical studies; Cyclacel Pharmaceuticals, Inc.: Other: Pharma support of clinical studies; Medimmune Inc.: Other: Pharma support of clinical studies; Novartis Pharmaceuticals: Other: Pharma support of clinical studies; Celgene Corporation: Other: Pharma support of clinical studies; Incyte Corporation: Other: Cyclacel Pharmaceuticals, Inc; Daiichi Sankyo Co. and Ambit Biosciences Corp: Other: Pharma support of clinical studies. Artz:Miltenyi: Research Funding. Tallman:Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Lenalidomide used for treatment non-del 5q myelodysplastic syndromes.

BACKGROUND: CMML is a myelodysplastic/myeloproliferative neoplasm with a median survival of 32 months and no therapies that improve its natural history. We have previously demonstrated that CMML bone marrow mononuclear cells (BMNCs) are hypersensitive to GM-CSF and that the GM-CSF axis is a viable therapeutic target (Padron et al., Blood 2013). Lenzilumab is a novel, humaneered IgG1κ monoclonal antibody, with high affinity for human GM-CSF that has activity in preclinical models of CMML. We report a Phase 1 clinical trial testing the safety and preliminary efficacy of this agent in CMML. METHODS: The study was approved by scientific and ethical review boards. This was a multicenter Phase 1 study designed to evaluate the safety and determine the recommended phase 2 dose of lenzilumab in subjects with CMML. Dose escalation proceeded using a standard 3+3 study design to determine the maximum tolerated dose (MTD). Three dose cohorts included 200 mg, 400 mg, and 600 mg, were given IV on day 1 and 15 of cycle 1 and then only on day 1 of subsequent 28-day cycles. Key inclusion criteria included a WHO-defined diagnosis of CMML and a platelet count greater than 20 x103 cells/dL. Response was evaluated utilizing the MDS/MPN International Working Group Criteria (Savona Blood 2015). Pharmacokinetic analysis and pharmacodynamics were evaluated by pSTAT5 by flow cytometry. RESULTS: Between July 2016 and June 2018, a total of 15 patients were enrolled. The median age at study entry was 74 years (range 52-85) and 80% were male. Nine patients were classified as CMML-0, 3 as CMML-1, and 3 as CMML-2. Seventy three percent of patients had normal cytogenetics or -Y. The most commonly mutated genes at screening included TET2 60%, ASXL1 53%, SRSF2 47%, and RAS pathway (i.e. NRAS or CBL) mutations 40%. Nine patients were previously treated with hypomethylating agents and/or experimental therapies, 3 were treated with hydroxyurea only, and 3 were untreated. The mean Hgb was 9.7g/dL (7.6-14g/dL), the mean platelet count was 147 x103 cells/dL (16-942 x103 cells/dL), and 66% of cases were MPN-CMML by the French-American-British classification at study entry. Three patients were enrolled at each dose level and an additional 6 patients were enrolled at 600mg as planned. Consistent with prior studies of lenzilumab, no dose limiting toxicities were identified and no treatment emergent grade 3 or 4 toxicities were reported. The mean duration on therapy was 221.8 days (14-787 days) and the majority of patients discontinued study drug because of disease progression or lack of response (69%). Five of 15 (33%) patients enrolled achieved clinical benefit by MDS/MPN IWG criteria with 3 platelet responses, 1 neutrophil response, and 1 spleen response. An additional patient had bone marrow myeloblast reduction from 6% to 1% which allowed that patient to undergo allogeneic stem cell transplantation. Clinical response was not statistically associated with somatic mutations or changes in pSTAT5 between screening and cycle 3. However, 3 of 4 patients with NRAS mutation achieved clinical benefit or had clinical meaningful bone marrow myeloblast reductions. CONCLUSION: Lenzilumab is well tolerated in patients with CMML, with no grade 3 or 4 treatment emergent adverse events or DLTs reported. Durable clinical benefit was achieved in 33% of patients and one patient was bridged to allogenic transplant, providing proof of concept that GM-CSF inhibition has activity in CMML. The favorable safety and activity profile of lenzilumab warrants future evaluation as part of a combination regimen targeted to specific subtypes more likely to respond, including patients with NRAS mutations. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Sallman:Celgene: Research Funding, Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Research Funding. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Komrokji:celgene: Consultancy; Agios: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; pfizer: Consultancy; DSI: Consultancy; Incyte: Consultancy. Lo:Humanigen: Employment. Durrant:Humanigen: Employment. Chappell:Humanigen: Employment. Ahmed:Humanigen: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. 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. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.

Abstract CONTEXT AND OBJECTIVE: Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) is a provisional entity included in the MDS/MPN overlap syndromes, with features of refractory anemia with ring sideroblasts (RARS) and thrombocytosis (platelet count ³ 450 x 10/L), recently renamed MDS/MPN with ring sideroblasts and thrombocytosis. Management of RARS-T is extrapolated from MDS and MPN treatment options namely for alleviating symptoms of anemia and, occasionally, for thrombocytosis. Individual case reports have suggested that lenalidomide may be effective in treating RARS-T. In non-del 5q, low-risk MDS, 25% of patients (pts) became red blood cell transfusion independent (RBC-TI). In the phase II study of lenalidomide in non-del 5q lower risk MDS, 35% of pts with RARS treated with lenalidomide achieved RBC-TI. The primary objective of this study is to evaluate a series of pts with RARS-T and compare hematological improvement (HI) responses among different treatment options. METHODS: This is a retrospective, single-center study. Patients with RARS-T were identified from the MDS Database at Moffitt Cancer Center. Samples for somatic mutation testing were obtained at time of referral. Descriptive statistics were used to describe baseline characteristics and response rates to treatment. The primary endpoint was HI using international working group criteria (IWG) 2006. The median overall survival (OS) was calculated from time of diagnosis. RESULTS: We identified 33 pts with RARS-T. The mean age was 67 years (38-80), 15 pts were male (45.5%), and majority were Caucasians (n=27, 82%). According to international prognostic scoring system (IPSS), 24 patients (73%) were low risk and 9 pts (27%) were int-1 risk. By revised IPSS, 11 pts (33%), 18 (55%), 3 (9%), and 1 (3%) were very low, low, intermediate, and high risk, respectively. The mean WBC was 7.7 (2.8-25.7), the mean Hemoglobin was 9.9 g/dl (7.3-12.4), and the mean platelet count was 682 (372-1644). Among 20 pts who had Next generation sequencing for somatic mutations at time of referral, 19 pts (95%) had SF3B1 mutation and 4 pts (20%) had JAK-2 V617 F mutation. The median OS was 90 mo (95% CI 47-134). For pts with JAK-2 V617F mutations, the median OS was 110 mo versus 74 mo for those with wild type (p=0.24). The rate of AML transformation was 3% (one patient only). Among the 33 pts, 8 pts had no treatment (observation only), 22 pts (67%) received erythroid stimulating agent (ESA) prior to any active therapy, 2 patients received lenalidomide with no prior ESA, and 1 pt ruxolitinib with no prior ESA. Twelve pts (36%) received lenalidomide (7 as first line active therapy after ESA and 2 without prior ESA exposure) and 13 pts (33%) received azacitidine (6 as first line active therapy after ESA). The HI rate for ESA was 36% (12/22) with a median duration of ESA treatment of 33mo. The HI rate for lenalidomide was 50% (6/12) with a median duration of lenalidomide treatment of 10.3mo. Among the 9 pts who received lenalidomide as first line active therapy (2 pts without prior ESA exposure) HI rate was 56%. The HI rate for azacitidine was 15% (2/13) with a median duration of azacitidine treatment of 6.3mo. CONCLUSIONS: Pts with RARS-T had favorable outcomes, with long overall survival and a low rate of AML transformation. Rates of hematological improvement with the use of azacitidine were lower than expected. Lenalidomide is an effective treatment option and should be considered when treating patients with RARS-T. Figure. Figure. Disclosures Sallman: Celgene: Research Funding, Speakers Bureau. List:Celgene: Research Funding. Komrokji:Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau.

Abstract Since the identification of the JAK family of intracellular tyrosine kinases and their downstream signaling partners, the STATs (Signal Transducers and Activators of Transcription), evidence has accumulated that activation of JAK signaling pathways can contribute to proliferation and survival of cancer cells. A direct link to oncogenesis was made when an activated form of JAK2 (JAK2 V617F) was identified in the majority of patients with myeloproliferative disorders (MPDs) including approximately half of patients with myelofibrosis (MF). Introduction of this JAK2 mutation into the bone marrow of mice induced erythrocytosis [Wernig et al. (2006) Blood107:4274] supporting JAK2 as a relevant target for treatment of myeloproliferative disorders. We have identified a novel small molecule inhibitor of JAK2, XL019, which may prove to have utility in treating MPDs. XL019 is a potent and reversible inhibitor of the JAK2 enzyme, with a Ki of 2 nM, and shows excellent selectivity (minimum of 50-fold selectivity against >120 protein kinases tested including other JAK family members). XL019 downregulates STAT signaling in cell lines expressing both wild type and activated forms of JAK2. IC50s for inhibition of STAT5 phosphorylation by XL019 ranged from 623 nM (HEL92.1.7) to 3398 nM (KG-1) in tumor cell lines. XL019 showed increased potency in primary human erythroid cells in vitro, where the IC50 for inhibition of EPO-stimulated phospho-STAT5 was 64 nM. XL019 inhibits proliferation in cell lines harboring activated or overexpressed JAK2, including certain lines derived from patients with Hodgkin’s Lymphoma (L-1236, 928 nM IC50), AML (MV4-11, 992 nM IC50), essential thrombocythemia (SET-2, 386 nM IC50), and erythroleukemia (HEL92.1.7, 6777 nM IC50). Preclinical single-dose pharmacodynamic studies demonstrate a potent effect of XL019 on JAK-STAT signaling in HEL92.1.7, CFPAC-1 and DU 145 xenograft tumors. Twice daily dosing (bid) led to substantial tumor growth inhibition in the DU145 and HEL models (maximum tumor growth inhibition of 86% and 60%, respectively), accompanied by increases in tumor cell apoptosis (4 – 4.4 fold increase) and decreases in tumor microvasculature (44% reduction in DU 145 xenograft tumors). Based on these encouraging data, XL019 is being evaluated in subjects with primary or post PV/ET MF in a Phase I dose escalation study, which has completed enrollment of the first cohort. The primary objectives of this study are to determine the safety and tolerability of XL019 when administered orally once daily for 21 days in 28 day cycles. Secondary objectives include determination of the pharmacokinetics and pharmacodynamics of XL019, and to evaluate response using the International Working Group for MF consensus response criteria. We have developed a flow-based mechanistic assay for JAK2 activity, which measures GM-CSF-stimulated phospho-STAT5 in peripheral granulocytes in whole blood samples. Additional pharmacodynamic assessments for this study include JAK2 V617F allele burden, erythropoietin-independent colony formation, cytokine levels, and changes in bone marrow histology. Our initial findings regarding the pharmacokinetics, pharmacodynamics, tolerability and efficacy of XL019 in MF patients will be presented.

Abstract MDS with chromosome 5q deletion [del(5q)] is uniquely sensitive to clonal suppression by lenalidomide (LEN), yielding high rates of cytogenetic and erythroid response. Although haploinsufficiency for a candidate tumor suppressor gene within the common deleted segment (CDS) has been implicated in the pathogenesis of del(5q) MDS, no specific gene products account for the karyotype-dependent LEN sensitivity. Using primary AML cells, we showed that LEN is selectively cytotoxic to del(5q) cells, inducing cell cycle arrest at the G2 > G1 junctures and apoptosis in a concentration-dependent fashion. Now we show that the CDS-encoded Cdc25C and PP2A phosphatases (PPTs) serve as molecular targets for LEN. Cdc25C and PP2A are dual specificity PPTs that regulate cell cycle. The genes encoding the catalytic subunit alpha (PP2ACa), the dominant catalytic isoform, and the regulatory subunit B (beta isoform PR 52) of the PP2A holoenzyme reside within the del(5q) CDS. Real-time RT-PCR showed that Cdc25C and PP2ACa gene expression is significantly reduced in primary del(5q) AML and MDS BM-MNC compared to non-del5q and normal controls. A Cdc25C FISH probe confirmed presence of only a single allele in del(5q) specimens [n=5] vs. normal karyotype [n=5] with no overlap with the EGR1 locus. We found that LEN inhibited rHu-Cdc25C activity with IC90 inhibitory concentrations ≤10nM. Similarly, PP2A PPT activity was inhibited by LEN in enzyme immunoprecipitates from Ramos cells at sub-micromolar concentrations. In U937 cells, we confirmed intracellular Cdc25C and PP2A inhibition, showing concentration-dependent retention of the inhibitory p-Tyr15 -Cdc2 substrates for Cdc25C with corresponding phosphorylation of the PP2A substrate Ser216 Cdc25C, findings reproduced by treatment with the PP2A selective antagonist, fostreicin. Cdc25C immunoprecipitation showed that phosphorylation of Ser216 Cdc25C increased binding to the 14-3-3 protein with consequent cytoplasmic sequestration confirmed by fluorescence microscopy. Phosphorylation of histone H2A.X Ser139 was unchanged after LEN treatment, indicating that phosphorylation of the inhibitory Cdc25C serine does not arise from upstream DNA damage activated kinases. Importantly, siRNA suppression of Cdc25C gene expression in U937 cells promoted LEN-specific apoptosis in 45% of cells, which increased to 68% after fostreicin inhibition of PP2A. These data indicate that haplodeficiency for the Cdc25C and PP2ACa genes promotes selective sensitivity of del(5q) cells to LEN-induced apoptosis. Haplodeficiency of critical drug-able targets may represent a novel strategy for tumor selective therapy.

Abstract Background: The few therapies available to treat higher-risk MDS and CMML have limited impact on outcome. We previously reported initial results of S1117, which compared overall response rates (ORRs) of azacitidine (AZA) monotherapy to AZA combined with the histone deacetylase inhibitor vorinostat (VOR), or the immunomodulator lenalidomide (LEN)( ASH 2014 LBA-5). We now report updated response data and overall survival (OS), subgroup analyses, impact of cytogenetics, and effect of treatment center volume/centers of excellence on outcome. Methods: This randomized, Phase II study (ClinTrials.gov # NCT01522976) enrolled higher-risk MDS (International Prognostic Scoring System (IPSS) Int-2 or High and/or bone marrow blasts ≥5%) and CMML adult patients (pts) with <20% blasts from 3/12-6/14 to receive AZA (75 mg/m2/d on d1-7 of a 28d cycle), AZA + LEN (10 mg/d on d1-21), or AZA + VOR (300 mg BID on d3-9). Pts continued treatment until treatment failure, defined as disease progression, relapse, significant or unresolved toxicity, or lack of response. Dose reductions occurred for grade ≥3 adverse events (per NCI CTCAE) or delayed count recovery. Cytogenetic risk groups were defined per IPSS-R. The primary endpoint was improvement in ORR, by intention to treat and reviewed centrally, of one of the combination arms vs. AZA per 2006 International Working Group MDS response criteria (complete response (CR) + partial response (PR) + hematologic improvement (HI)). OS was from study entry. MDS Centers of Excellence (MCE) were defined per MDS Foundation; center volume was defined as low (1-4 pts enrolled) or high (5-17 pts). Results: Of 277 pts, 92 received AZA, 93 AZA+LEN, and 92 AZA+VOR. Baseline characteristics, previously reported, were similar across arms. Pts received a median of 22 weeks of therapy and were followed for a median of 10 months (range: 0-30). Non-protocol defined dose modification and protocol discontinuation due to toxicity occurred more frequently in combination arms vs. AZA (p=.0014 and p=.018, respectively). Responses are now assessable in all pts (Table 1). ORR was statistically similar for combination arms vs. AZA, with a trend for longer response duration (p=.083) for combinations. Within HI, AZA+LEN pts had higher HI-n than AZA pts (16% vs. 5%, p=.031). ORR for CMML pts was significantly higher for LEN+AZA vs. AZA (63% vs. 29%, p=.04), with a trend for longer response duration for combinations (p=.06); no differences in ORR were seen for therapy-related MDS, IPSS subgroups, or transfusion-dependent pts. Allogeneic transplantation rates were similar. Median OS (Figure) for AZA:AZA+LEN:AZA+VOR was 15:18 (p=.38):17 (p=.17) months; p=.19 for combination arms vs. AZA. Median OS after failure was 7:9 (p=.6):9 (p=.05) months; p=.15 for combination arms after failure vs. AZA. For pts on therapy >6 months, there was a trend (p=.08) for higher ORR for AZA+LEN vs. AZA, though response duration was similar; median OS was 18:21 (p=.44 vs. AZA):21 months (p=.45 vs. AZA). Cytogenetic risk category distribution and ORR was similar across arms. OS (compared to Very Good/Good) was worse for Poor (HR 2.07, p=.022) and Very poor (HR 4.41, p<.001), without significant modification by treatment arm (Table 2). Compared to pts without identified cytogenetic abnormalities (abn), ORR across arms was better for pts with Chr 5 abn (OR 2.38, p=.004); OS was better for normal (HR .42, p<.001) and worse for Chr 5 abn (HR 3.1, p<.001), -7 (HR 2.69, p<.001), and 17p (HR 2.61, p<.001). While small numbers prevented definitive conclusions for treatment arm effect, combinations trended towards improving OS in Normal and Chr 5 abn only. The outcome of all pts and pts on discrete study arms treated at MCE (n=75) or high volume (n=138) sites were similar to non-MCE or low-volume sites for ORR, non-protocol defined dose modifications, dose adjustment in first 4 cycles, time to off-protocol (HR 1.2, p=.21 and HR .94, p=.64), and OS (HR .81, p=.3 and HR .77, p=.12). Conclusions: In higher-risk MDS pts, ORR and OS was similar for AZA monotherapy compared to combination arms, while for CMML pts, ORR was significantly higher with AZA+LEN. For cytogenetic subgroups, OS was worse for Chr 5 abn, -7, and 17p, and may be improved by combinations in normal or Chr 5 abn. MCE or treatment at a high volume site did not impact these effects or outcomes. Figure 1. Responses Figure 1. Responses Figure 2. Cytogenetics Figure 2. Cytogenetics Figure 3. Figure 3. Disclosures Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. List:Celgene Corporation: Honoraria, Research Funding. Odenike:Sunesis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stone:Merck: Consultancy; Celgene: Consultancy. Gore:Celgene: Consultancy, Honoraria, Research Funding. Buckstein:Celgene: Honoraria, Research Funding. Fang:Affymetrix: Research Funding. Attar:Agios Pharmaceuticals: Employment. Erba:Ariad: Consultancy; Daiichi Sankyo: Consultancy; Sunesis: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Incyte: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jannsen (J&J): Other: Data Safety and Monitoring Committees ; Ariad: Consultancy; Celgene: Consultancy, Speakers Bureau; GlycoMimetics: Other: Data Safety and Monitoring Committees; Seattle Genetics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Millennium/Takeda: Research Funding; Celator: Research Funding; Celator: Research Funding; Astellas: Research Funding; Astellas: Research Funding; Sunesis: Consultancy; Daiichi Sankyo: Consultancy; GlycoMimetics: Other: Data Safety and Monitoring Committees; Jannsen (J&J): Other: Data Safety and Monitoring Committees.

Abstract Introduction: CMML is a clinically heterogeneous myeloid neoplasm hallmarked by the coexistence of dysplastic and proliferative patho-clinical features, which can include cytopenias, constitutional symptoms, splenomegaly, and leukocytosis. However, according to the FAB classification schema, CMML may be differentiated in dysplastic and proliferative subgroups only by the presence of leukocytosis in the latter (WBC ≥13 x 109/L). We hypothesize that incorporation of other clinically discriminating features may yield a more informative CMML stratification system. To address this, we propose three distinct CMML categories and explore their clinical relevance leveraging our existing international CMML database (Padron E et al. Blood Cancer J. 20151). Method: 1622 WHO-defined CMML cases diagnosed between 1973 and 2014 were collected from eight large cancer centres that include up to 80 discrete data elements as previously described1. Cases were placed into three clinically distinct groups and the Pearson Chi-Square test and the Kruskal-Wallis test were applied respectively to compare categorical and continuous characteristics. The Kaplan-Meier (KM) method was used to estimate median OS and the log rank test was used to compare survival curves.Cox models whereapplied to obtain univariate and adjusted hazard ratios. Identification of optimal cut-off values for continuous variable was supported by graphical inspection of martingale residuals from the null Cox model. Statistical analyses were done in SPSS v23 and R v.3.3.0. Results: We propose three categories to delineate clinically distinct CMML subtypes: (1) Myelodysplastic (MD)-CMML: WBC≤10 x 109/L, PB-immature myeloid cells (IMC) = 0%, no splenomegaly (2) MD/MP-CMML: WBC 10-20 x 109/L or WBC ≤10 x 109/L but PB-IMC>0% and/or splenomegaly (3) Myeloproliferative (MP)-CMML: WBC >20 x 109/L. A recursive partitioning approach was used to identify the WBC cut points, with splenomegaly and IMC added to more accurately depict the MPN aspect of CMML. Numbers of patients included in the MD-, MD/MP-, and MP-CMML subcategories were 319 (19.7%), 789 (48.6%) and 514 (31.7%), respectively (Table 1). According to the FAB criteria, the MD/MP group included 521 (66%) MD- (WBC ≤13 x 109/L) and 268 (34%) MP-CMML (WBC>13 x 109/L) patients suggesting that the proposed classification clinically reclassifies FAB-defined CMML. Within the MD/MP group, 344 patients (21.2%) had a WBC <10 x 109/L but with IMC>0% and/or splenomegaly. Comparison of overall survival (OS) among proposed groups demonstrated that this classification schema was capable of discriminating the CMML natural history (Figure 1). In comparison to MD/MP-CMML, the unadjusted OS Hazard Ratio (HR) was 0.60 (95% CI 0.49-0.73) for MD-CMML and 1.57 (95% CI 1.36-1.81) for MP-CMML (p<0.001 for both). This difference was retained after adjusting for BM-Blasts (< vs ≥5%), IPSS or CPSS cytogenetics (High-risk vsInterm/low-risk), and elevated levels of LDH (HR was 0.73 with 95% CI 0.57-0.93, p=0.011 for MD-CMML, and 1.33 with 95% CI 1.12-1.58, p=0.001 for MP-CMML, respectively). We next explored whether each proposed group may have distinct variables that uniquely govern its prognosis. Peripheral blood blasts >5%, RBC- and PLT-transfusion-dependence predicted poor OS only in the MD and MD/MP-subcategories (p<0.005), but had no impact in the MP-CMML. Male gender and elevated LDH were only significantly associated with prognosis in the MD/MP group (p<0.05 and p<0.005, respectively) while absolute lymphocyte count >2.5 x 109/L and absolute monocyte count >10 x109/L were uniquely predictive for shorter survival in the MP-CMML subcategory (p<0.005). Notably, we additionally identified gene mutations that uniquely predicted prognosis in each proposed group (Table 2). Conclusions: We demonstrate that our proposed 3-group clinical classification schema is capable of independently stratifying prognosis. Further, our analysis identified clinical and genetic variables that uniquely govern each group's prognosis, suggesting independent clinical behaviour. Further investigations are warranted to validate these groups. Disclosures Jabbour: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Fenaux:Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau.

Abstract Background: MDSs are clonal hematopoietic stem cell malignancies characterized by cytopenias resulting from ineffective hematopoiesis. A phase 2 study of lenalidomide (MDS-002) demonstrated hematological improvement in patients with MDS without an associated deletion 5q cytogenetic abnormality (List AF, et al. Haematologica2006;91[suppl1]:379[abst 1032]). We now report multicenter results after 〉2 yrs follow-up. Methods: A total of 214 pts with transfusion-dependent anemia were categorized into the following IPSS MDS risk groups after central review of bone marrow morphology, karyotype, and peripheral blood findings: Low/Int-1 (168 [78%]); Int-2/High (8 [4%]); and unclassified (38 [18%]). Patients were unclassified for missing or inadequate marrow studies for central review (morphology [29] or cytogenetic [7]) or other diagnoses (AML [1], atypical CML [1]). Patients were evaluated for frequency and duration of red blood cell transfusion independence (RBC-TI) (IWG criteria), hemoglobin (Hgb) response, and safety. The starting dose of lenalidomide was 10 mg (daily or daily × 3 wks q28d cycle). Results: Overall, 56 (26%) of 214 enrolled patients achieved RBC-TI. Median time to RBC-TI was 5 wks, median duration of RBC-TI response was 41.0 wks (range, 8.0–136.4), and median Hgb increase achieved during RBC-TI was 3.2 g/dL (range, 1.0–9.8). An additional 36 patients experienced a ≥ 50% reduction in RBC transfusions (overall hematological improvement in 92 [43%] patients). Overall, 47 (22%) patients had an abnormal karyotype at baseline and 9 (19%) patients achieved a cytogenetic response (4 complete). The most common drug-related grade 3/4 adverse events (AEs) were neutropenia and thrombocytopenia (25% and 20%, respectively). Deep vein thrombosis occurred in only 2 (1%) patients. The duration of RBC-TI was ≥ 52 weeks in 21 (38%) patients. Among these 21 patients, 14 (67%) did not require a lenalidomide dose reduction during the first 52 weeks of treatment. Analyses of response variables will be presented. Dose-limiting neutropenia/thrombocytopenia was not reported after 52 weeks. The most commonly reported AEs after 52 weeks were mild-moderate diarrhea and fatigue (38% and 29%, respectively). Conclusion: Lenalidomide is an active, well-tolerated treatment in MDS patients with transfusion-dependent anemia that is not associated with a deletion 5q abnormality. The rate of erythroid hematologic improvement and duration of RBC-TI is encouraging and offers a possible alternative to cytokine therapy.