Tibes, Raoul

Abstract Background: Large case-control studies have identified common epidemiologic exposures associated with risk of AML development, including obesity [BMI ≥30 kg/m2] associated with 2-fold increase risk. However it is not known whether individual risk factors are associated with unique AML genetic lesions. We have recently identified a significant association of some common exposures including obesity with unique disease cytogenetic subgroups in a cohort of n=295 consecutive AML patients diagnosed and treated at Mayo Clinic [Cancer Epidemiol 39:1084, 2015]. Aberrant DNA methylation is a hallmark of cancer and has been reported in AML, and we therefore performed an analysis of global DNA methylation in patients in this cohort to determine whether specific epidemiologic exposures are associated with a unique pattern of DNA methylation. Methods: Patients in the initial cohort were previously reported. An available baseline AML bone marrow cytogenetic pellet from diagnosis was identified for n=148 patients (50.2%). Following IRB approval and using previously reported techniques [Leukemia 24:1283, 2010], leukemia DNA was isolated from the remnant diagnostic cytogenetic samples and processed on the HumanMethylation450 BeadChip array (Illumina). Individual CpG dinucleotides were assigned a Beta value (i.e. methylation value) ranging from 0 (unmethylated) to 1 (methylated). We excluded those with minor methylation changes (<0.2 between any two samples), resulting in 383,524 CpG's. For the purposes of this first analysis we have focused only on CpG's with an inter-quartile range (IQR, i.e. difference between 75th and 25th percentiles) of greater than 0.25, comprising a total of 39,657 CpG's. An unsupervised cluster analysis of differential methylation was generated ("Heatmap", Figure). Univariate and multivariable analyses were performed adjusting for age as a continuous variable to determine the association of individual exposures with unique methylation clusters (primary endpoint), and we also evaluated the association of clinical characteristics and clinical outcomes after therapy (secondary endpoint). The median age of patients was 69 yrs (range 19-89), male 65%, and Caucasian 89%. The median BMI was 27.2 (range 16.9-59.9), and n=47 patients (32%) had BMI ≥30. Standard cytogenetic risk groups were applied [favorable (n=8), normal (n=61), intermediate abnormal (n=32) and poor (n=47)]. Results: We identified 4 distinct methylation clusters (Figure: Cluster #1, Black horizontal bar; Cluster #2, Green; Cluster #3, Blue; and Cluster #4, Red). Multivariate analysis of select variables is shown in Table. Patients in cluster #1 were significantly more likely to have a BMI ≥30 [Odds Ratio (OR) 2.49, p=0.017] and more likely to have favorable risk cytogenetics although this was not statistically significant [OR 6.71, p=0.088], while those in cluster #2 were significantly more likely to be have a history of regular NSAID use [OR 3.26, p=0.03] and less likely to have normal [OR 0.40, p=0.044] and possibly intermediate abnormal cytogenetics [OR 0.34, p=0.051]. Those in cluster #3 were significantly younger [median age 48 yrs, p<0.001] and were also more likely to have prior history MDS although this was not statistically significant [OR 3.7, p=0.082]. Cluster #4 was not correlated with any distinct exposure apart from possible decreased alcohol use [OR 0.3, p=0.079]. History of past or current tobacco use, regular acetaminophen use, documented myelotoxin exposure, family history, and secondary AML did not correlate with a unique methylation cluster. There was no independent association of methylation cluster with overall survival (p=0.46), although among patients receiving intensive therapy cluster #2 was associated with lower complete remission rate [OR 0.28 (95% CI 0.09-0.86), p=0.026]. Conclusion: This first analysis of AML molecular epidemiology and clinical outcome suggests that specific disease risk factors (esp. obesity and age) are associated with unique leukemia DNA methylation clusters. In addition, aberrant methylation is associated with some clinical features and with outcome after therapy, suggesting that epidemiologic risk factors may have a prognostic impact. This provides novel insight into leukemogenesis, and will guide prospective studies of AML epidemiology and outcome. Figure. Figure. Disclosures Foran: medscape: Honoraria; pfizer: Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; novartis: Honoraria; karyopharm: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding.

Abstract Background Myeloid neoplasms (MN) occurring secondary to treatment of non-malignant disease, especially autoimmune diseases (AID), and the overlap between them, is an increasing clinically important observation. Confounding variables for myeloid transformation include the nature of autoimmune disease as an incessant, chronically activated inflammatory cascade that has been associated with an increased risk of myeloid malignancy, and disease modifying therapies, often with cytotoxic and immunosuppressive drugs. Thus, the addition of therapeutic agents for treatment of AID theoretically amplifies the opportunity for MN to develop in genetically susceptible individuals. To our knowledge, there is no large study evaluating a comprehensive primary AID population that developed MDS or AML with detailed assessment of therapeutic interventions for AID. The presented study queries the association of cytotoxic, anti-inflammatory, and immune-modulating agents to treat AID as risk factors for developing myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). Methods In a 10-year period from 2004-2014, a total of 40,011 patients with an ICD-9 coded diagnosis of AID were seen at Mayo Clinic Arizona and Florida campuses; 311 patients from this population had a concomitant coded diagnosis of MDS and/or AML. In an IRB approved, retrospective chart review, we confirmed 86 cases meeting strict inclusion criteria for AID and confirmed MDS or AML. We performed a case-control match from the same primary autoimmune population for age, gender, and autoimmune specific diagnosis at a 2:1 ratio querying for therapeutic interventions. Results Myeloid neoplasm specifics in our case cohort of 86 patients were 55 MDS, 21 de novo AML, and 10 AML with history of MDS. Average age was 72 years with a slight male predominance (57%). Rheumatoid arthritis (26.4%), psoriasis (20.7%), systemic lupus (13.8%), was the most common autoimmune profiles. Median onset of autoimmune disease to diagnosis of myeloid neoplasm was 6 years (range 1-54 years). Up to 26% controls documented no systemic agent for treatment of autoimmune disease and similarly 12.8% of the case cohort (p=0.142). Case and control populations treated with systemic therapies had similar exposures by category for immune-modulating agents (33.7% and 37.8% respectively) and cytotoxic therapies (47.7 and 44.8% respectively). A total of 57/86 cases (66.3%) received either a cytotoxic or immune-modulating agent. In comparison, 105/172 (61.1%) controls received either agent; p=0.495. Azathioprine was the only statistically significant agent exposure observed more frequently in the study cohort than controls with an OR of 7.05 (p= < 0.001). There was no increased incidence of MDS or AML in TNF-antagonist treated patients. No significant correlation for duration of agent exposure by drug category was observed. Discussion In a large primary AID population, azathioprine exposure was associated with a 7 fold risk of MDS or AML (p=< 0.001). Notable, but not statistically significant case cohort trends among cytotoxic agents was exposure to cyclophosphamide (OR 3.58) followed by mitoxantrone (OR 2.73). Methotrexate, mercaptopurine, and mycophenolate had favorable odd ratios, but these were not statistically significant. No association was found for TNF-inhibitory agents. Finally, there was no association of drug exposure time to the incidence in development of MDS or AML. Disclosures Dueck: Bayer: Honoraria. Mesa:Gilead: Research Funding; Novartis: Consultancy; Celgene: Research Funding; Incyte Corporation: Research Funding; Galena: Consultancy; Ariad: Consultancy; CTI: Research Funding; Promedior: Research Funding. Al-Kali:Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding. Foran:Millennium Pharmaceuticals, Inc.: Research Funding; novartis: Honoraria; karyopharm: Honoraria; medscape: Honoraria; pfizer: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding.

Abstract Introduction: Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) result in recruitment of another HMT, DOT1L, to a multi-protein complex. This leads to abnormal methylation of Histone H3 lysine 79 (H3K79) at MLL target genes and enhanced expression of leukemogenic genes such as HOXA9 and MEIS1 ( Krivstov, 2007). Pinometostat is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs. Pinometostat treatment of MLL-rearranged cells and xenografts reduced histone H3K79 methylation, decreased MLL target gene expression, and induced selective leukemia cell kill (Daigle, 2013). We report the safety, activity, pharmacokinetics (PK) and pharmacodynamics (PD) in the phase 1 trial of pinometostat in adult patients (pts) with relapsed/refractory (R/R) leukemia. Methods: This open label dose escalation/expansion study of pinometostat enrolled pts >18 years (yrs) with R/R leukemia (NCT01684150). In the dose-escalation phase, pts with AML, ALL, mixed lineage leukemia (MLL), myelodysplastic syndrome, myeloproliferative neoplasm or chronic myeloid leukemia were eligible. Eligibility in the two expansion cohorts: 90 mg/m2 (n = 17) and 54 mg/m2 (n = 6), was restricted to pts with MLL-r or MLL-partial tandem duplication (MLL-PTD). Pinometostat was given via continuous intravenous infusion (CIV) for 21 of 28 day cycles in the dose escalation phase and CIV for 28 of 28 day cycles in the expansion phases, until disease progression or unacceptable toxicity. All pts underwent serial collection of PK and peripheral blood mononuclear cells (PBMC) for PD. Leukemic blasts were isolated from PBMCs using flow cytometry and quantified for dimethylation of H3K79 (H3K79-me2) by ChIP-Seq. Results: As of 28-June-2015, 49 pts have enrolled in the dose escalation and expansion phases. Pts receiving 21/28 day infusions: 12 (n=1), 24 (n=5), 36 (n=4), 54 (n=6) and 80 mg/m2/day (n=3). Pts receiving 28/28 day infusions: 54 (n=6) and 90 mg/m2/day (n=24). Table 1. Patient Characteristics n (%) Median age, yrs (range) 51 (19 - 81) Sex (M / F) 27/22 Diagnosis MLL-r* 29 (59) AML MLL-PTD** 5 (9) MLL-wt 7 (14) ALL MLL-r* 5 (10) MLL-wt 1 (2) MLL MLL-r 1 (2) CMML MLL-r 1 (2) # of prior therapeutic regimens 1 - 2 29 (59) 3 - 4 18 (36) >4 2 (4) Prior allogeneic hematopoietic cell transplant 20 (41) * centrally confirmed by karyotype/FISH ** centrally confirmed by NGS (next generation sequencing) Adverse events (AEs) reported in >15% of pts regardless of attribution were: nausea, constipation, vomiting, abdominal pain, diarrhea, hypocalcemia, hypokalemia, hypomagnesemia, fatigue, fever, peripheral edema, mucositis, febrile neutropenia, leukocytosis, anemia, cough, dyspnea, and pneumonia. Grade ≥3 non-hematologic related toxicities include: hypophosphatemia (n=1), decreased ejection fraction (n=3), or elevated transaminases (n=1). Nine patients had leukocytosis (absolute monocyte and neutrophil 50% above baseline and above upper limit of normal) or differentiation. The median days of pinometostat treatment was 35 days (range 3-189 days). To date, objective responses observed are morphologic CR (1 pt), cytogenetic CR (MLL negative by FISH) (1 pt), PR (1 pt) and resolution of leukemia cutis (3 pts). Dose proportional PK was observed with rapid attainment of steady-state plasma concentrations (Css) on Day 1 of treatment. Plasma Css correlated with inhibition of global H3K79-me2 in PBMCs. H3K79-me2 ChIP-Seq demonstrated pinometostat induced reductions in methylation at MLL -r target genes HOXA9 and MEIS1 (median inhibition = 61%: range = 13-91%) in all 9 pts analyzed to date from the 90 mg/m2 expansion cohort. Inhibition of H3K79-me2 in leukemic blasts is consistent with DOT1L suppression. PK-PD relationships in both expansion cohorts using both free and total plasma Css are being explored. Conclusions: Pinometostat administered as a CIV to adults with R/R leukemia has an acceptable safety profile. Clinical activity as demonstrated by both marrow responses and resolution of leukemia cutis were observed. In addition, analysis of H3K79-me2 by ChIP-Seq demonstrated PD reductions in the methylation of MLL-r target genes following pinometostat exposure, as expected from DOT1L inhibition. Relationships between PK exposure, reductions in pinometostat induced H3K79-me2 levels and clinical response are being interrogated. Disclosures Stein: Seattle Genetics: Consultancy; Agios Pharmaceuticals: Consultancy. Rizzieri:Teva: Other: ad board, Speakers Bureau; Celgene: Other: ad board, Speakers Bureau. Berdeja:Novartis: Research Funding; Takeda: Research Funding; BMS: Research Funding; Curis: Research Funding; MEI: Research Funding; Abbvie: Research Funding; Onyx: Research Funding; Array: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Acetylon: Research Funding. Altman:Novartis: Other: Advisory board; Spectrum: Other: Advisory board; Ariad: Other: Advisory board; Seattle Genetics: Other: Advisory board; BMS: Other: Advisory board; Astellas: Other: Participation in an advisory board December 2013. Thomson:Epizyme, Inc: Employment. Blakemore:Epizyme: Employment. Daigle:Epizyme, Inc: Employment. Fine:Epizyme: Employment. Waters:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy. Ho:Epizyme, Inc: Employment.

Abstract Background: Cytosine arabinoside (ara-C) remains the backbone of most regimens for the treatment of AML. The incorporation of ara-C into DNA activates checkpoint kinase 1 (Chk1) causing replication fork slowing, the accumulation of cells in S phase, and diminished ara-C cytotoxicity. The selective Chk1 inhibitor MK-8776 abrogates ara-C-induced S-phase arrest, thereby enhancing ara-C-induced apoptosis in vitro.1 A phase I trial demonstrated the safety of MK-8876 in combination with infusional ara-C and correlative studies demonstrated increased ara-C-induced H2Ax phosphorylation in AML cells, consistent with unrepaired DNA damage.2 Herein, we report data from a phase II trial comparing ara-C plus MK8876 with ara-C alone. Methods: Patients with relapsed or refractory AML were randomized to receive either ara-C 2 gm/m2 via continuous infusion (CIV) over 72 hours on days 1 and 10 with MK-8776 100 mg flat dose infused over 30 minutes on days 2, 3, 11, and 12 (combo); or ara-C alone on the same schedule (control). The primary end point was response rate (CR+CRi). Adverse events (AEs) for all patients were reported according to the NCI-CTCAE version 4.0. Correlative studies were performed on blood and marrow samples collected prior to treatment on days 1, 2 and 3 and at one hour after MK-8776 infusion (PB only). Marrow and circulating blasts were analyzed using semi-quantitative flow cytometric determinations of blast cell gH2AX expression, a marker of DNA damage. Results: Thirty-two patients were treated at 4 sites with 14 assigned to the combo arm and 18 to the control arm. The study was stopped early due to lack of drug availability. The median age was 60 (range 29-72) and 17 (53%) were male. Nine (28%) had refractory AML, 16 (50%) were in first relapse, and 7 (22%) were in second relapse. Eleven (34%) had undergone a prior bone marrow transplant (BMT). Risk stratification classified 1 (3%) patient as favorable risk disease, 11 (34%) as intermediate-1, 8 (25%) as intermediate-2, and 11 (34%) as adverse risk. There were 5 (36%) responses (CR/CRi) and 1 (7%) partial response (PR) in the combo arm, and 8 (44%) responses and 1 (6%) PR in the control arm. Median survival did not differ significantly between the two groups (5.9 months in the combo arm vs. 4.2 months in the control arm). Sub-group analysis showed increased responses in the combo arm for the patients in first relapse (50% vs. 40%) and those with a prior BMT (50% vs. 29%). Most non-hematologic grade 3/4 treatment-related AEs were similar in the two groups including febrile neutropenia (64% vs. 61%), infectious complications (50% vs. 50%), and sepsis (21% vs. 17%). Patients in the combo arm experienced more QT prolongation (21% vs. 0%), hypertension (21% vs. 0%), and GI toxicities (21% vs. 0%); while those in the control arm had increased renal (0% vs. 11%) and liver toxicity (7% vs. 17%). Two deaths were attributed to treatment, and both were in the control arm. Eight combo patients had serial PB samples and 2 had serial marrow samples studied by flow cytometry. There was an increase in the percentage of gH2AX+ CD34+ cells on Day 2 directly after MK-8876 infusion (16.9% prior and 36.4% at one hour), which occurred to a lesser extent on Day 3 after MK-8876 infusion (12.7% prior and 15.6% at one hour). Three of eight patients demonstrated a sustained increase in gH2AX+ CD34+ from Day 2 to Day 3, and all three achieved either a PR or CR. Conclusion: This is the first phase II study building on prior work suggesting that inhibition of Chk1 would increase response to ara-C in patients with relapsed or refractory AML. The study confirmed that treatment with the combination is feasible with similar toxicities between the treatment and control groups. The response rates were similar between the two groups with improved responses in patients receiving the combo in first relapse and following BMT. Correlative studies support the finding that responding patients show a sustained increase in DNA damage and this may serve as a biomarker in future studies. Chk1 remains a potential target to improve treatment outcomes with ara-C. References: 1. Schenk EL et al. Clin Cancer Res. 2012 Oct 1; 18(19):5364-73. 2. Karp JE et al. Clin Cancer Res. 2012 Dec 15; 18(24):6723-31. Disclosures No relevant conflicts of interest to declare.

Abstract Background: International Working Group (IWG) has set criteria in 2003 for response assessment in patients with AML; at which time intensive chemotherapy was the main therapy (Cheson, JCO 2003). Hypomethylating agents (HMA) were approved shortly after for the treatment of elderly AML (Fenaux, JCO 2009; Dombret et al Blood 2015). Although few patients achieve a deep response, the majority will have stable disease (SD), some of which could last several months while patients continue to receive HMA and gain clinical benefit. We hereby describe outcomes for patients who achieve SD with HMA for the treatment of elderly unfit AML patients. Methods: A total of 56 AML patients' data from Feb 2007 to June 2015 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our institution. Patients were considered for our study if they received HMA as frontline therapy for their AML. Patients who received HMAs after induction chemotherapy failure or for their MDS were excluded. Response was identified based on 2003 IWG AML response criteria. Patients with SD were categorized into short-lasting (<6 cycles of HMA, cohort 1) or long-lasting (≥6 cycles of HMA, cohort 2) SD.Prognostic factors were analyzed by univariate and multivariate analyses. Survival estimates were calculated using Kaplan-Meier curves and univariate and multivariate analyses was based on log-rank testing using JMP software version 10. Results: i) Characteristics: Fifty six AML patients were treated with HMA as frontline therapy with median age of 76 years (range, 59-91), 68% of which were males. Therapy related AML (T-AML) was seen in 2% of the cases. Cytogenetics were favorable risk in 2%, intermediate in 62%, and poor in 36%. Complete remission (CR) was found in 10 (18%) patients, CR with incomplete count recovery in 3 (5%), partial remission (PR) in 3 (5%) patients. Forty patients were non-responders (SD). ii) Characteristics of HMA SD cohorts: Upon comparison of cohort 1 versus cohort 2 there was no statistical significance in median age, white blood count, absolute neutrophil count, platelets, peripheral blood and bone marrow blasts, bone marrow cellularity and sex. Median number of cycles was expectedly higher in cohort 2 vs 1 (9 vs 2, p<0.0001). Intermediate cytogenetic risk was more common in cohort 2 vs 1 (90% vs 43%, p=0.006), while poor risk was more common in cohort 1 vs 2 (57% vs 10%, p=0.006). Only 10% had complex karyotype in cohort 2 compared to 50% in cohort 1 (p=0.017). NPM1 mutation was absent in all cases, while FLT3 mutation was present 30% in cohort 1 and 25% in cohort 2 (p = 0.9). Decitabine was more commonly given in cohort 1 vs cohort 2 (80% vs 50%, p =0.07). iii) Clinical outcome of HMA SD cohorts : Median overall survival was higher in long-lasting SD patients (cohort 2) vs short-lasting SD (18 vs 4 months, p = 0.0002). Upon multivariate analysis, both age (p <0.0001) and long-lasting SD (p =0.01) were statistically significant for overall survival but not cytogenetics risk grouping nor type of HMA administered. Conclusions: Standard response criteria for AML by IWG were developed for intensive chemotherapy and do not apply to newer agents, specifically HMA. Stable disease (SD) could be meaningful to some elderly AML patients when treated with HMA. Intermediate cytogenetics and azacitidine use were more frequent in patients with long-lasting SD. Long-lasting SD did confer a survival benefit in elderly AML patients. Newer response criteria for assessing novel agents' efficacy are needed in this population. Figure Figure. Disclosures Tibes: Italfarmaco S.p.A.: Consultancy, Honoraria. Foran:pfizer: Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; medscape: Honoraria; karyopharm: Honoraria; novartis: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. Al-Kali:Novartis: Research Funding; Celgene: Research Funding.

Background: Although 5-azacitidine (5-AZA) is the standard of care for patients with higher-risk patients with Myelodysplastic Syndrome (MDS) almost all patients will fail 5-AZA therapy with the majority of patients progressing within 2 years. Two-year survival for MDS patients refractory to 5-AZA is only 15%. There is a need for more effective therapies for these patients with higher-risk MDS. The Inhibitors of Apoptosis (IAP) Proteins are a family of molecules that suppress apoptotic cell death. Expression of IAPs is dysregulated in MDS, supporting potential as therapeutic targets. SMAC (second mitochondrial activator of caspases) is an IAP antagonist, resulting in caspase activation, inhibition of the pro-survival transcription factor NF-kB and increased apoptosis leading to tumor cell death. Birinapant is a potent bivalent SMAC mimetic in clinical development for the treatment of several cancers. Pre-clinical data support its investigation in myeloid malignancies, specifically MDS. Methods: This Phase 1b study was designed to determine the MTD and recommended Phase 2 dose and schedule of birinapant in combination with 5-AZA in patients with higher-risk MDS who are naïve to or have relapsed or are refractory to 5-AZA. Secondary objectives included determining the clinical activity of the combination of birinapant plus 5-AZA, and assessing the pharmacokinetics (PK) and exploratory biomarkers. Results: Twenty-one patients were enrolled, of whom 15 had high/very high risk MDS, with median IPSS score of 3 (range, 1.5-8). Median age (range) was 73 years (48-84 years). Eight patients were therapy-naïve. All patients received 5-AZA, administered either intravenous (IV; 13 patients) or subcutaneous (SC; 8 of whom 4 later switched to IV), at 75 mg/m2 for 7 days of a 28-day cycle in combination with birinapant. Birinapant was administered IV twice weekly (Days 1 and 4) at 13 mg/m2 for either 3 of 4 weeks (15 patients; Cohort 1 and Expansion Cohort) or 4 of 4 weeks (6 patients; Cohort 2) in 28-day cycles. No cycle 1 dose-limiting toxicities were observed. Adverse events ≥Grade 3 observed in two or more patients were thrombocytopenia (9 patients); pneumonia (including fungal pneumonia; 8); anemia (8); leukopenia (7); febrile neutropenia (7) neutropenia (5); fatigue (4); respiratory failure (3); cellulitis or soft tissue necrosis (3); and nausea, vomiting, or hypoxia (2 patients each). SAEs seen in more than one patient included pneumonia (6 patients, including fungal pneumonia); febrile neutropenia (4); sepsis (3) and cellulitis or soft tissue necrosis (3). Grade 3 abdominal soft tissue necrosis occurred in the area of subcutaneous injections of 5-AZA, and two events of Grade 3 cellulitis were judged related to treatment. These local injection site reactions were more severe than expected with SC 5-AZA, and were not observed with subsequent IV administration. These events were not considered DLTs. Treatment-related suppression of NF-k B activity was evident in circulating blast cells from patients in Cohort 1 suggesting pharmacodynamic activity of birinapant at this dose and schedule. Based on this, the Phase 2 dosing regimen has been recommended as birinapant administered at 13 mg/m2 IV twice weekly for 3 weeks of a 28-day cycle in combination with 5-AZA at 75 mg/m2 IV daily for 7 days of a 28-day cycle. Although not a primary endpoint, evidence of clinical activity, as demonstrated by significant reduction in marrow blast percentages, was seen, including 3 patients considered relapsed or refractory to prior therapy. Nine patients of 20 evaluable (45%) demonstrated either a ≥50% decrease in marrow blast count, or a blast count of ≤5% at the end of Cycle 1 or 2. Two additional patients (10%) demonstrated hematological improvement in one or more peripheral cell lineages without transfusions. In addition, 2 patients, one of whom had previously received 5-AZA, could be bridged to stem cell transplant subsequent to response with birinapant plus 5-AZA. Several patients remain in active follow-up. Conclusion: This Phase 1b trial confirms the acceptable safety profile of the combination of birinapant administered with IV 5-AZA. Evidence of clinical activity in both 5-AZA naïve and refractory patients provide the rationale for an ongoing global randomized, blinded Phase 2 study comparing birinapant in combination with 5-AZA vs. 5-AZA alone in patients with higher-risk MDS in the front-line setting. Disclosures Wang: Immunogen: Research Funding. Rakkar:TetraLogic Pharmaceuticals: Research Funding. Minderman:TetraLogic Pharmaceuticals: Research Funding. Burns:TetraLogic Pharmaceuticals: Employment. Tibes:TetraLogic Pharmaceuticals: Research Funding.

Abstract Background and purpose of the study: Several reports have documented that histone deacetylase inhibitors induce neoplastic cells to undergo growth arrest, differentiation and/or apoptotic cell death. Among these agents, Givinostat inhibits proliferation of cells bearing the JAK2V617F mutation and has shown clinical significant activity with good tolerability in patients with chronic myeloproliferative neoplasms, including polycythemia vera (PV). In two phase II studies [Rambaldi et al. Br J Haematol 2010;150:446, Finazzi et al. Br J Haematol 2013;161:688] the maximum administered dose (150 mg/day) was generally well tolerated. Assuming a linear relationship between dose and efficacy, greater clinical efficacy could be expected with increased doses of Givinostat. Methods: This is a two-part, multicenter, open label, non-randomized, phase Ib/II study to assess the safety and tolerability, maximum tolerated dose (MTD) and preliminary efficacy of Givinostat in patients with JAK2V617F positive PV. Part A (i.e. phase Ib) is the dose escalation portion of the study, while Part B assesses the preliminary clinical efficacy of Givinostat at the MTD defined in Part A. Eligible patients for this study include JAK2V617F positive PV patients, who have active and/or uncontrolled disease, defined as: (i) Hematocrit (HCT) ≥ 45% or HCT <45% normalized by phlebotomy, and (ii) PLT > 400 x109/L, and (iii) WBC > 10 x109/L. Study therapy is administered in 28 day cycles (cycle is defined as 4 weeks of treatment). Disease response has been evaluated according to the clinico-haematological European LeukemiaNet (ELN) criteria [Barosi et al. Blood 2009;113:4829] at Cycles 3 and 6. Disease-related symptoms have been reported directly by the patients who completed the MPN-SAF QoL form. The study lasts up to a maximum of 24 weeks of treatment. However, after completion of the trial, all patients achieving clinical benefit are allowed to continue treatment with Givinostat in a long-term study (ClinicalTrials.gov ID: NCT01761968). In Part A, the Cycle 1 has been used to define the MTD of Givinostat. Only dose limiting toxicities (DLTs) experienced during Cycle 1 were considered for dose escalation decisions. Part B is the cohort expansion part of the study to assess the preliminary clinical efficacy of Givinostat at the MTD in evaluable patients Results: In Part A, 12 patients with active and/or uncontrolled disease, were treated with Givinostat, 6 patients at DL1 (100 mg b.i.d.), 3 patient at DL6 (150 mg/die) and 3 patients at DL0 (50 mg b.i.d.). Only one DLT .(grade 3 dyspepsia) was observed in the 6 patients treated at DL1 during the Cycle 1. Givinostat treatment was generally well tolerated: one patient developed grade 4 and two patients grade 3 thrombocytopenias at DL1 after the Cycle 1. Complete/partial response was observed in 100% of evaluable patients at Cycle 3 (Figure). At that time, an important reduction of WBC and PLT count (about 50% in both cases), an HCT control without phlebotomy, a net decrease of MPN-SAF total score and JAK2V617F allele burden (about 50% and 30%, respectively) have been noted and generally maintained at Cycle 6. One out of 3 (33%) patients with splenomegaly at baseline normalized the spleen at Cycle 6. PK/PD evaluations are currently under analysis. Part B started on December 2015 and is currently recruiting patients. At the time of ASH Meeting, a presentation of the preliminary results of the interim analysis (IA) is planned. The objective of this IA is to evaluate the response rate of the first 12 treated and evaluable patients. Conclusions: Taking into account the continuous schedule of the drug as prescribed in this current study, the daily dosage of 200 mg has been identified as the MTD of Givinostat in PV patients. Givinostat treatment is generally well tolerated and shows preliminary clinical efficacy in PV patients with an active/uncontrolled disease. Response rate of patients in Part A C3D28 = Cycle 3 Day 28, or three months of treatment. C6D28 = Cycle 6 Day 28, or six months of treatment. Two patients dropped-out the study Part A, due to Informed Consent Form withdrawal, while 1 patient left the study for AE (i.e. grade 3 dyspepsia, DLT). At C3D28, 1 Complete Response (CR) and 8 Partial Response (PR) have been observed. At C6D28, 1 Complete Response (CR), 7 Partial Response (PR) and 1 No Response (NR) have been observed Figure Figure. Disclosures McMullin: Novartis: Honoraria, Speakers Bureau. Vannucchi:Novartis: Honoraria, Speakers Bureau. Mesa:Ariad: Consultancy; CTI: Research Funding; Galena: Consultancy; Celgene: Research Funding; Promedior: Research Funding; Novartis: Consultancy; Incyte Corporation: Research Funding; Gilead: Research Funding. Tibes:Italfarmaco S.p.A.: Consultancy, Honoraria. Bettica:Italfarmaco S.p.A.: Employment. DI Tollo:Italfarmaco S.p.A.: Employment.

Abstract Cytarabine (AraC) resistance is a fundamental feature of refractory/relapsed AML. RNA interference (RNAi) screens conducted in our laboratory recently identified WEE1 kinase (WEE1) as one of the top candidate genes and target in leukemias in combination with AraC. WEE1 is a tyrosine kinase belonging to the Ser/Thr family of protein kinases and acts as a negative regulator of mitotic entry by controlling DNA damage (DDR) and cell cycle checkpoint responses. The WEE1 inhibitor MK1775 potently synergizes with AraC ex vivo and in vitro and clinical trials are in preparation. However, the mechanism of action for the anti-leukemic activity of MK1775 with AraC remains unknown. To elucidate genes mediating activity of the combination, we first performed siRNA rescue screens silencing a custom set of 44 genes involved in WEE1 regulation under combined AraC + MK1775 to identify sensitizers and markers of resistance. The MRN (MRE11, Rad51, NBS1) complex and particularly NBS1 were potent modifiers of AraC and MK1775. Focusing on NBS1 since it is proposed to centrally regulate the defense capacity of leukemic cells, we identified that NBS1 phosphorylation at Ser343 (the ATM regulation site) is significantly altered both in cell lines and primary AML samples under combined AraC+MK1775 treatment as compared to single agent MK1775. In parallel, lower phosphorylation of ATMS1981(an autophosphorylation site in response to DNA strand breaks), was observed indicating that the ATM-CHEK1 pathway is not activated under co-treatment. Further Homologous recombination (HR)-mediated repair was compromised by AraC+MK1775 shown by DR-GFP expression vector to measure intracellular HR capacity: post-transfection of the I-SceI nuclease which cleaves non-functioning GFP tandem repeats to form a functional GFP unit, the HR was reduced with the combination. Consistently other HR markers decreased as well. Delayed accumulation of Cyclin A (indicative of S-phase progression) and greater inhibition of phospho-Cdk2Y15in synchronized cells treated with AraC + MK1775 in comparison to controls was observed. In addition the cell cycle was globally dysregulated by slower S-phase kinetics (progression), a completely abrogated G2/M checkpoint/phase as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high single and double strand breaks (ɣH2AX) were observed with an increase in phospho-histone H3 in AraC + MK1775 treated cells compared to untreated cells or MK1775 single agent, confirming faster mitotic entry. Changes were followed by massive induction of apoptosis. Since WEE1 is implicated in leukemic stem cell maintenance we examined the long term effects of the combination in colony forming assays. AraC + MK1775 treated leukemic cells obtained from patients with AML were re-plated on Methocult after drug washout and colonies counted after 14 days. While MK1775 as a single agent could reduce colony formation by 4 fold compared to controls and lower dose AraC, co-treatment with low to moderate doses of AraC and MK1775 reduced colony formation by more than 7 fold and to almost zero in some primary specimens. Taken together, these results suggest that leukemia cells co-treated with AraC + MK1775 lost their ability to activate DNA damage and repair pathways mainly by compromising the MRN complex via NBS1 with subsequently reduced HR. The combination (as opposed to single agents) almost complete dysregulated the cell cycle and its checkpoints lead to DNA damage, genomic instability and rapid exit from the cell cycle with cell death via apoptosis. Thus we have molecularly characterized the detailed mechanisms underlying the potent AraC+WEE1 inhibition in AML and describe for the first time a therapeutic combination that has the potential to abrogate the MRN and NBS1 repair capacity which is central for drug resistance in AML. A key implication of our work is to provide a clinical rationale, mechanistic understanding and suggestions for biomarkers to clinically evaluate AraC + MK1775 in patients with AML. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2617 Introduction: ABT-348 is a potent, novel, adenosine triphospate competitive inhibitor of Aurora A, B and C kinases. ABT-348 is also a potent inhibitor of all members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinase (RTK) families. ABT-348 has demonstrated strong antitumor activity in a variety of preclinical cancer cell line models, in vitro and in vivo, including leukemias as monotherapy and in combination with the DNA methyltransferase inhibitor, azacitidine. Objectives: The primary objectives of this study are to determine the safety and pharmacokinetics (PK) of orally and IV administered ABT-348 as monotherapy or in combination with azacitidine in patients (pts) with advanced hematologic malignancies. Secondary objectives include determination of the maximum tolerated dose (MTD) and exploration of biomarkers associated with ABT-348 activity. Methods: This phase 1 dose-escalation study of ABT-348 utilizes a modified continual reassessment method in pts with advanced hematologic malignancies: acute myelogenous leukemia (AML), acute lymphoblastic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia and myelodysplasia (MDS). Adult pts with histological or cytological confirmed disease, ECOG status 0–2, adequate hematological, renal and hepatic function and without significant hypertension or proteinuria are eligible. This study has four arms consisting of ABT-348 administered on a 28-day (D) cycle. Pts receive ABT-348 monotherapy once daily (QD, Arm A) or twice daily (BID; Arm B) on Days (D) 1, 8, and 15 of a 28-day cycle under fasting conditions. In Arm C, ABT-348 is given on the same schedule as Arm A in combination with azacitidine (75 mg/m2) administered IV or SC on Days 1 – 7 of each 28-day cycle. In Arm D, pts receive ABT-348 monotherapy on the same schedule as Arm A, but via IV administration. Samples are collected for PK and biomarker analyses. Pts are treated until progressive disease (PD) or unacceptable toxicity. Adverse event (AE) severity is graded using NCI-CTCAE v4.0. Results: 39 pts (median age, 66 y [45–86]) have enrolled (Arm A, n=32; Arm B, n=4; Arm C, n=3). Of these pts, 27 had AML, 11 had MDS and 1 had CML. Pts received a median of 4 prior therapies (range, 1 – 8). Best response to last prior therapy was: complete or partial response in 3 pts, stable disease in 11 pts, PD in 24, and not determined in 1. Dose escalation is shown in the table. The first dose-limiting toxicity (DLT) of grade 3 pancreatitis was observed at 640 mg in Arm A. Due to this DLT, along with a second case of grade 3 pancreatitis, further enrollment in Arm A was held and Arms B and C were opened. A DLT of grade 4 acute kidney injury was seen at 440 mg in Arm C. ABT-348-related AEs in greater than 2 pts were proteinuria (23%), nausea (21%), diarrhea (18%), hypertension (13%), vomiting (10%) and fatigue (8%). Grade 3/4 ABT-348-related AEs were hypertension (13%), pancreatitis (5%), acute kidney injury (3%) and diarrhea (3%). One patient in Arm A achieved a CRi (640 mg). Five of 32 pts in Arm A were treated for ≥4 cycles, including 2 of 7 pts at ABT-348 doses ≥ 640 mg. Based on preliminary PK analyses, Cmax and AUC of ABT-348 were approximately dose proportional in the dose range studied (10–690 mg); co-administration of azacitidine had no apparent effect on ABT-348 PK. Evidence of dual aurora/VEGF receptor kinase inhibition has been demonstrated by induction of polyploidy and PlGF, respectively. 33 pts have discontinued and 6 remain on study. Conclusions: ABT-348 has demonstrated on-target biomarker and clinical activity in pts with advanced hematologic magnancies, and was well tolerated at doses below 640 mg in Arm A. PK appears to be dose-proportional. Dose escalation is ongoing in Arms B and C (with azacitidine); an IV formulation of ABT-348 will also be evaluated. Disclosures: Off Label Use: Clofarabine in AML. Tibes:Abbott: Research Funding. Chiu:Abbott: Employment, Owns stocks Other. Xiong:Abbott: Employment, own Abbott stock Other. Qin:Abbott: Employment. Ansell:Abbott: Employment, own Abbott stock Other. Albert:Abbott: Employment, own Abbott stock Other. Tse:Abbott: Employment, own Abbott stock Other. Oliver:Abbott: Employment, own Abbott stock Other. Sajwani:Abbott: Employment, own Abbott stocks and participated in study design, data collection, analysis, interpretation and reviewing approving study documents related to ABT-348 programs. Other. McKee:Abbott: Employment, own Abbott stocks Other. Ricker:Abbott: Employment, own Abbott stocks Other.

Abstract Background: Pegylated interferon alpha-2a (Peg INF2a) has been demonstrated to be active therapy for high-risk essential thrombocythemia (ET), polycythemia vera (PV), and early myelofibrosis (MF). We retrospectively analyzed the outcomes of Peg INF2a therapy in myeloproliferative (MPN) patients treated outside the constraints of a clinical trial. Methods: Charts were analyzed for demographic and clinical data. Toxicity to therapy was assessed per CTCAE 3.0 criteria. Therapeutic responses for ET and PV were calculated by the revised ELN/IWG-MRT criteria including complete remission (CR), partial resmission (PR), no response (NR), or progressive disease (PD). Responses in MF were calculated by EUNMET: complete response (CR), major response (MR), moderate response (MoR), minor response (MiR) and NRand the revised IWG-MRT/ELN criteria: CR, PR, clinical improvement (CI), stable disease (SD) or PD. Results: Patients: 75 patients were identified overall. There were 36 PV patients (48%), 20 ET patients (26.7%), and 19 MF patients (25.6%). Thirteen MF patients were post-PV/ET MF. The median age at diagnosis was 51.5 yrs (range 28.8-75.1). JAK2 V617 mutation was present in 53 patients (70.7%). Median baseline hemoglobin (g/dL), leukocyte (x10 9), and platelet count (x109) for PV was 13.6, 8.6, 369, for ET was 12.5, 6.8, 517, and for MF was 11.4, 8.0, and 420, respectively. DIPSS risk category for the 19 MF patients: Low in 6 (31.5%) patients, Intermediate-1 in 3 (15.7%), Intermediate-2 in 8 (42%), and High in 2 (10.5%) patients. The majority of patients (82.2%) had received at least one prior cytoreductive therapy for their disease. Therapy: Median starting dose of Peg INF2a was 45 micrograms/week (range 45-90). The median peak dose was 90 micrograms/week (range 45-270). The median tolerated dose was 60 micrograms/week (range 5.6-180). The median duration of treatment was 24 months (range 3.6-85). Toxicity: Overall the Peg INF2a was well tolerated. Hematological toxicity included: leukopenia at grade 1 in 13 patients (17.3%), grade 2 in 5 patients (6.7%), and grade 3 in 1 patient (1.3%), anemia at grade 1 in 10 patients (13.3%) and grade 2 in 1 patient (1.3%), thrombocytopenia at grade 1 in 13 patients (17.3%) and grade 2 in 1 patient (1.3%). The most common non-hematologic toxicity included: fatigue at grade 1 in 14 patients (18.7%) and grade 2 in 4 patients (5.3%), transaminitis at grade 1 in 6 patients (8%) and grade 2 in 3 patients (4%), myalgias at grade 1 in 4 patients (5.3%). Response: See Table #1 PV/ET: 56 patients were evaluated by ELN/IWG-MRT criteria overall: A complete remission (CR) was seen in 8 patients (14.3%), a partial remission (PR) in 18 patients (32.1%), either a CR or PR in 18 patients (32.1%) when histologic remissions were unable to be documented due to lack of restaging bone marrow examination, no response in 11 patients (19.6%), and progressive disease in 1 patient (1.8%). Of the 12 patients receiving at least1 phlebotomy per month, 10 patients (83.3%) became phlebotomy independent with therapy. Of the 20 ET patients, 12 patients (60%) had platelet normalization (<400 x 109). MF: 19 patients were evaluated by IWG-ELN criteria: a PR was seen in 2 patients (10.5%), CI in 4 patients (21.1%), SD in 12 patients (63.2%), and PD in 1 patient (5.3%). Utilizing EUNMET critieria: 1 patient (5.3%) with a CR, 5 patients (26.3%) with a MR, 3 patients (15.8%) with MoR, 5 patients (26.3%) with MiR, and 5 patients (26.3%) with NR. Table 1. Peg INF2a Patient Responses RESPONSE Disease IWG-ELN response criteria: (Patient #, %) (Barosi, Blood 2013, Tefferi, Blood 2013) EUMET response criteria: (Patient #, %) (Barosi, Blood 2005) PV: N=36 CR: 3 pts (8.3%) PR: 14 pts (38.8%) CR/PR: 14 pts (38.8%) NR: 5 pts (13.8%) PD: 0 NA ET: N=20 CR: 5 pts (25%) PR: 4 pts (20%) CR/PR: 4 pts (20%) NR: 6 pts (30%) PD: 1 (5%) NA MF: N=19 IWG-ELN: PR: 2 pts (10.5%) CI: 4 pts (21.1%) SD: 12 pts (63.2%) PD: 1 pt (5.3%) EUNMET: CR: 1 pt (5.3%) MR: 5 pts (26.3%) MoR: 3 pts (15.8%) MiR: 5 pts (26.3%) NR: 5 pts (26.3%) NA: Not applicable Conclusions: Peg INF2a is active and well-tolerated when administered outside of the support of a clinical trial. Given the majority of patients had previously failed cytoreductive therapy these results substantiate prior reports of efficacy of Peg INF2a in MPNs. Disclosures Mesa: Pfizer: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding; NS Pharma: Research Funding; Genentech: Research Funding.