Tibes, Raoul

Abstract Background: Gilteritinib (ASP2215) is a novel, highly selective, potent oral FLT3/AXL inhibitor with preclinical activity against FLT3-ITD activating and FLT3-D835 resistance mutations. The objectives of this phase 1/2 study were to assess gilteritinib safety/tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) profiles after single- and multiple-day dosing, and antileukemic effects in patients with R/R AML. Methods: This open-label study (NCT02014558) enrolled patients (≥18 yr) into 1 of 7 dose-escalation cohorts (20-450 mg once daily [QD]) or concomitant dose-expansion cohorts. While confirmed FLT3 mutation was not an inclusion criterion, each expanded dose level enrolled ≥10 patients with FLT3 mutations (FLT3mut+); 120 and 200 mg dose levels were further expanded with ≥40 FLT3mut+ patients. The choice to expand these dose cohorts was based upon FLT3 inhibition in correlative assays and clinical activity seen during dose escalation. Safety and tolerability were primary endpoints; blood samples were drawn from patients in the dose-escalation cohorts to evaluate gilteritinib PK parameters and PD effects. Antileukemic response rates (eg, complete remission [CR], CR with incomplete platelet recovery [CRp], CR with incomplete hematological recovery [CRi], overall response rate [ORR]) were secondary endpoints. Results: Patients (N=252; 129M:123F, median age 62 yr [range: 21-90]) enrolled between October 2013 and August 2015 received ≥1 dose of gilteritinib. The study population was heavily pretreated: 70% (n=177) had ≥2 prior AML therapies, 29% (n=73) had a prior stem cell transplant, and 25% (n=63) had prior TKI treatment with sorafenib most commonly used. Across the study, 194 patients had a locally confirmed FLT3 mutation (ITD, n=159; D835, n=13; ITD-D835, n=16; other, n=6). For all enrolled patients, progressive disease (n=75), lack of efficacy (n=44), adverse events (n=34), and death (n=29) were the most common reasons for treatment discontinuation. Seven deaths were considered possibly/probably related to treatment: pulmonary embolism, respiratory failure, hemoptysis, intracranial bleed, ventricular fibrillation, septic shock, and neutropenia (all n=1). Maximum tolerated dose was determined to be 300 mg when 2 of 3 patients in the 450 mg cohort experienced diarrhea and/or hepatic transaminase elevation as dose-limiting toxicities. Diarrhea (16%) and fatigue (15%) were the most commonly reported treatment-related adverse events of any grade. Less than 5% of patients (11/252) had a maximum post-baseline QTcF interval >500 msec. Gilteritinib concentrations were generally dose proportional and showed both a long-elimination half-life (45-159 h) and substantial accumulation (3.2-10 fold) by day 15. An exposure-related increase in the inhibition of FLT3 phosphorylation with increasing doses of gilteritinib was also observed. Gilteritinib showed strong antileukemic activity in FLT3mut+ patients (ORR=49%); response was observed less frequently in patients with wild-type FLT3 (ORR=12%). While CR, CRi, and CRp occurred at all doses, responses were enriched among FLT3mut+ patients with gilteritinib steady-state trough concentrations ≥100 ng/mL, which correlated with potent FLT3 inhibition in PD assays and corresponded to doses ≥80 mg. The ORR in 169 FLT3mut+ patients receiving ≥80 mg was 52% (Table); median overall survival in this patient population was ~31 wk (range: 1.7-61; Figure) and median duration of response was 20 wk (range: 1.1-55). Clinical responses occurred in FLT3mut+ patients with -ITD, -D835, and both mutations (ORR: 55%, 17%, and 62%, respectively) as well as in FLT3mut+ patients with or without prior TKI treatment (ORR: 42% vs 56%, respectively). Conclusions: This PD-driven, first-in-human study shows that gilteritinib was well tolerated and generated frequent, prolonged, clinically important responses in FLT3mut+ patients with R/R AML. Antileukemic responses were enriched in FLT3mut+ patients treated at doses that consistently and potently inhibited FLT3 phosphorylation. The survival of these patients appears better than expected for this patient population when treated with standard therapy. Our data suggest that FLT3 inhibition may improve survival in patients with FLT3mut+R/R AML; as such, phase 3 testing of oral gilteritinib 120 mg QD in patients with FLT3mut+R/R AML after first-line therapy is underway (NCT02421939). Disclosures Perl: Astellas US Pharma Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Asana Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arog Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Altman:Janssen: Other: advisory board; BMS: Membership on an entity's Board of Directors or advisory committees; Spectrum: Other: advisory board; Ariad: Other: advisory board; Seattle Genetics: Other: advisory board; Syros: Other: advisory board. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Smith:Astellas: Research Funding. Erba:Jannsen: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding; Agios: Research Funding; Juno: Research Funding; Incyte: Consultancy, DSMB, Speakers Bureau; Daiichi Sankyo: Consultancy; Ariad: Consultancy; Amgen: Consultancy, Research Funding; Astellas: Research Funding; Gylcomimetics: Other: DSMB; Seattle Genetics: Consultancy, Research Funding; Sunesis: Consultancy; Novartis: Consultancy, Speakers Bureau; Celator: Research Funding; Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy. Gill:Astellas: Employment. Goldberg:Bristol Myers Squibb, Novartis: Speakers Bureau; Novartis: Consultancy; COTA Inc: Employment; Pfizer: Honoraria; Neostem: Equity Ownership. Jurcic:Astellas: Research Funding. Larson:Astellas: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy. Liu:Astellas: Employment. Ritchie:Celgene: Consultancy, Other: Travel, Accomodations, Expenses, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Ariad: Speakers Bureau; Pfizer: Consultancy, Research Funding; Astellas Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; NS Pharma: Research Funding. Schiller:Incyte Corporation: Research Funding. Strickland:Celator: Research Funding; Cyclacel: Research Funding; Karyopharm Therapeutica: Research Funding; GlaxoSmithKline: Research Funding; Baxalta: Consultancy; Boehringer Ingelheim: Consultancy, Research Funding; Ambit: Consultancy; Alexion Pharmaceuticals: Consultancy; Astellas Pharma: Research Funding; CTI Biopharma: Consultancy; Daiichi Sankyo: Consultancy; Sunesis Pharmaceuticals: Consultancy, Research Funding; Abbvie: Research Funding; Sanofi: Research Funding. Wang:Incyte: Speakers Bureau; Immunogen: Research Funding. Stuart:Sunesis: Consultancy, Honoraria, Other: Travel, Accomodations, Expenses, Research Funding; Agios: Research Funding; Incyte: Research Funding; Bayer: Research Funding; Celator: Research Funding; Astellas: Research Funding. Martinelli:Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Roche: Consultancy, Speakers Bureau; MSD: Consultancy; Genentech: Consultancy; Novartis: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Bahceci:Astellas: Employment. Levis:Millennium: Consultancy, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

Abstract Background: Acute myeloid leukemia (AML) is an aggressive form of blood cancers with a wide range of response and relapse rates using standard chemotherapy regimen (commonly known as 7+3). Several genes (FLT3, IDH1, RAS) have been targeted using small molecule tyrosine kinase inhibitors with encouraging results. The stem cell receptor tyrosine kinase KIT is expressed more than 10% in the blasts in 95% of relapsed AML cases and mediates leukemic proliferation and has anti-apoptotic effects (Domen and Weissman 2000). AML with high KIT expression is associated with poorer outcome (Del Poeta, Venditti et al 2003). Goals: To study the efficacy and safety of combination 7+3 and nilotinib in patients (pts) with AML and KIT expression. Primary goal is to determine the complete response (CR) rate; while secondary goals include 2-year overall survival (OS) and disease free survival (DFS) in addition to safety of DATA regimen. Methods: A single arm, Phase II study, enrolled pts at Mayo Clinic (MN and AZ). Appropriate IRB was obtained and study was registered (NCT 01806571). Pts were enrolled if they were newly diagnosed with AML with KIT (CD117) expression of 20% or higher on myeloblasts by flow cytometry. KIT mutations were allowed if present. Nilotinib 300 mg twice daily was given on days 4-14 of induction and consolidation; and continuous daily maintenance therapy for up to 2 years. Cytarabine 100 mg/m2/day continuous IV x7 days plus daunorubicin 60 mg/m2 IV daily x3 days were used for induction , while consolidation used standard cytarabine 3 gm/m2 twice daily days 1, 3, 5 for a total of 4 cycles. This is a Simon 1-stage design with a safety analysis after enrolling 12 pts, and an interim analysis after enrolling 18 out of 43 pts. If 11 or fewer pts achieve complete remission, the regimen will be considered ineffective. Results: Eighteen pts have been enrolled between July 2013 and July 2015, 17 of which have baseline data. Median age was 58 years (range 24-65) with 76.5% being male. Median laboratory findings include hemoglobin of 9.3 gm/dL, platelets of 52 x109, and white blood count of 7.0 (0.5-125). Cytogenetics were normal in 41% of the pts. Favorable cytogenetics were seen in 2 pts (inv 16). FLT3 testing was done on 15 pts and was positive in 27%. KIT gene sequencing (exon 8, 9, 10, 11, 17) revealed no pathogenic mutation. Median number of cycles was 3 (range 1-7). Six pts had treatment delays, with 2/13 (15%) delays being due to non-hematologic toxicities. No delays or missed doses affected cytarabine and daunorubicin administration. Out of 15 pts evaluable for response, 12 (80%) achieved CR (or CR with incomplete platelet recovery). Six of the 12 pts (50%) who achieved remission needed a second induction on protocol. Two out of 15 did not respond (did not get re-induction on protocol). One pt died before disease assessment due to liver failure (G5, had only one dose of nilotinib and toxicity was attributed to daunorubicin). Of the 14 pts having at least 1 bone marrow biopsy, the overall CR rate was 86%. Five (33%) pts proceeded to allogeneic stem cell transplant, all are alive and none were able to initiate protocol nilotinib maintenance therapy. Only 2 out of 12 (17%) pts relapsed after achieving CR, one was secondary AML from myelodysplastic syndrome with complex karyotype and the other was therapy-related AML with t(9;11). Only 2 (11%) pts died at time of report. Thirteen pts were evaluated for adverse events (AE). Six pts had G4 non-hematological AEs. Five of 12 G4 AEs were related to infection, 2 were electrolyte imbalances, and 2 were heart failures. Most common G3 non-hematological AEs were febrile neutropenia (53%), hypophosphatemia (23%), hyperglycemia (23%), and hypertension (23%). In agreement with the favorable outcomes, the molecular target validation revealed that nilotinib treatment down-regulates the expression of KIT, Sp1, DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b. Conclusion: Combination daunorubicin and cytarabine with nilotinib (DATA) appears to be safe and effective. Interim results show an acceptable safety profile in the first 12 evaluable pts with most common AE being infection as expected. Thirty day mortality is acceptable (7%). DATA regimen has encouraging CR rates of 80% (intent to treat) and 86% in assessed pts, with half of the pts who achieved remission requiring 2 cycles of induction. Relapse rate seems to be low at 17%. We will continue accrual until all pts accrued for final results. Disclosures Al-Kali: Celgene: Research Funding. Off Label Use: This is a Phase II study of combination nilotinib to standard chemotherapy in patients diagnosed with AML.. Tibes:TetraLogic Pharmaceuticals: Research Funding.

Abstract Background: Histone deacetylase inhibitors (HDIs) have shown clinical activity in myeloid malignancies, albeit insufficient to justify single-agent therapy. Thus, we have sought targets/therapies that synergistically enhance the activity of HDIs for combination therapy. We conducted RNA-interference (RNAi) drug modifier screens with the HDI SAHA, and have previously reported convergence of multiple screen hits on different points of the p38-SAPK/JNK signaling pathway. However, in subsequent pharmacological studies, well-characterized p38α/β inhibitors SB2012190 and LY2228820, and JNK inhibitor SP600125 did not modulate SAHA anti-leukemic activity. Nonetheless, a putative MK2 substrate-selective p38 inhibitor known as CMPD1 (Biochemistry 2004 Sep 21; 43(37):11658-71) was found to potently synergize with SAHA in all AML cell lines tested (N=8) in a dose-dependent manner. Further, using ex vivo cultures of primary myeloid malignancies (N=14), CMPD1 showed strong, selective synergy with SAHA in malignant CD34+ isolated cells, as compared to little or no synergy in CD34-depleted cells. Furthermore, CMPD1 specifically synergized with SAHA and a similar HDI panobinostat, but was found to interact antagonistically with cytarabine, and only additively with azacitidine. Herein, we present additional data from our investigation to better understand the mechanism of CMPD1 synergy with HDIs for future clinical translation. Results: Concurrent cell cycle and apoptosis measurements by flow cytometry show that single-agent CMPD1 results in a potent G2/M arrest that is resolved over time without a significant induction of apoptosis, whereas the combination of SAHA + CMPD1 results in a similar or increased level of G2/M arrest, which conversely culminates in a significant induction of apoptosis. The synergistic dose of single-agent SAHA used in these flow cytometry studies did not cause any significant cell cycle changes, and did not induce significant apoptosis alone in the AML cell lines studied. CMPD1 is known to be a MK2 substrate-selective p38 inhibitor; however, we find that CMPD1 potently increases the phosphorylation of MK2 at doses strongly synergistic with SAHA. This is not a universal activity of p38 inhibitors in our in vitro AML models, as the p38 inhibitor SB202190 does not increase MK2 phosphorylation alone or in combination with SAHA. We further hypothesized that CMPD1 synergizes with SAHA through a transcription-based mechanism, thus we measured mRNA expression by next-generation sequencing after SAHA and CMPD1 treatment alone, and in combination, in an AML cell line TF-1. We find that SAHA + CMPD1 treatment induces a synergistic increase in transcript levels of: i) several chemokines such as IL-8, CXCL1, CXCL2, and CXCL3, ii) inhibitor of DNA binding proteins ID2 and ID3, and iii) cell cycle regulator p21. Transcript levels of the Hippo pathway transcription factor YAP1 were also found to be increased by CMPD1 treatment in TF-1. We confirm an increase of total YAP1 protein levels by western blot. We also find an increase of YAP1 phosphorylation at serine 127 in CMPD1 or CMPD1 + SAHA treated samples in several AML cell lines, yet we find that Ser-127 phosphorylated YAP1 is located in the nucleus. Consistent with YAP1 Ser-127 phosphorylation, we find that upstream signaling kinase LATS1 is activated/phosphorylated at serine 909 in AML cell lines after treatment with CMPD1 or SAHA + CMPD1. We also confirm a synergistic increase in p21 protein levels upon SAHA + CMPD1 treatment by western blot in several AML cell lines. Changes in the protein levels of ID2 and ID3 were confirmed in TF-1 by western blot; however, ID protein levels appear to be uniquely affected by CMPD1 or the combination in a cell line-dependent manner. Conclusion: CMPD1 selectively synergizes with histone deacetylases inhibitors in myeloid malignancies. The synergistic activity of this combination highlights the potential for selectively targeting malignant CD34+ cells. The phosphorylation of LATS1 observed is consistent with i) the known roles for LATS1 in cell cycle regulation, ii) the G2/M arrest observed with CMPD1, and iii) the downstream phosphorylation of YAP1 observed. These findings linking CMPD1 with LATS1/YAP1 await further study and characterization. Although CMPD1 is not a clinically-relevant compound, CMPD1 may be used as a scaffold or tool compound to enable future development and research. Disclosures Mesa: Pfizer: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Genentech: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Gilead: Research Funding; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding.

Abstract Background: A diagnosis of cancer leads to significant information requirements to facilitate health care decision making, understand management options, and health care system navigation. Many patients demonstrate a knowledge deficit that can lead to decreased quality of life and poor health care compliance. This study sought to evaluate the impact of a patient centered cancer symposium on knowledge level, symptom burden, and desired information in a broad population of cancer patients. Methods: Surveys were distributed to attendees of the Mayo Clinic "Living with and Surviving Cancer" patient symposium in January 2015. A follow up survey was sent to participants 3 months after the symposium. Surveys included demographic data, questionnaire evaluating disease comprehension, symptom burden, desired information and quality of life. Results: Demographics: 113 patients completed the pre-intervention survey. Average age of participants was 64.7 years. Disease types represented included hematologic malignancies (N=50), specifically multiple myeloma (N=24), lymphoma (N=17), leukemia (N=6), MPN/MDS (n=3), as well as solid tumor malignancies (N=77). Highest knowledge topics were disease (80%), screening tests (74%), and monitoring tools (72%). Lowest knowledge topics were financial management (13%), legal issues (13%), and pain management (35%). Pre and Post Analysis: Of the 113 initial participants, 79 patients completed the pre- and post-symposium surveys. In the post-symposium setting significant improvement was noted in understanding of disease (pre 80% vs post 92%), treatment options (pre 60% vs post 76%), nutrition (pre 68% vs post 84%) and legal issues (pre 15% vs post 32%). Non-significant knowledge improvement was noted in areas of risk factors and symptoms, screening/monitoring tools, disease side effects, managing stress/fatigue, end of life decision making and health care navigation. Most patients desired increased understanding regarding disease, screening tests, nutrition, and stress and fatigue management. The level of patient reported desire for information of these topics decreased in the post-symposium setting, statistically significant decrease noted in 4 of 5 topics assessed. In hematology patients who completed pre and post surveys (N=36), a higher level of baseline knowledge was reported regarding disease understanding (89%), tools monitoring response (83%) and recurrence (75%), symptoms (83%), relapse symptoms (64%) and treatment options (72%). In this group the decrease in desire for information was less prominent in the post-symposium setting. Discussion: This data demonstrates improved patient reported understanding on a variety of topics with durable response at 3 months. A majority of patients reported adequate baseline knowledge of many topics prior to the symposium but also a desire of increased cancer specific information. Knowledge deficit in cancer care includes disease specific topics, social stressors and health care navigation. The percentage of participants reporting a high desire for more information remained high but did decrease, suggesting decreased knowledge deficit in the post-symposium setting. Patient education should strive to be accessible, all-inclusive and reactive to patient needs. Table 1.Reported "quite a bit" or "very much" understanding and desired information regarding cancer carePrePostP-valueN=79(%)N=79(%)I understand…How to manage financial considerations of my disease/treatment39(51)41(53)0.85How to manage my pain24(34)23(32)0.81Risk factors that can lead to my disease37 (49)42 (56)0.56How to navigate the health care system43(55)47(60)0.56How to manage fatigue38(50)43(56)0.54Process of making end of life decisions33(47)41(53)0.52Screening tests60 (76)63 (81)0.5Symptoms associated with relapse35(45)42(54)0.27Disease symptoms54(70)60(76)0.2Tools monitoring recurrence49(62)56 (72)0.19Tools monitoring response56(71)63(81)0.15How to manage stress46(60)56(71)0.12Legal issues of my disease11(15)24(32)<0.05Treatment options47(60)60(76)<0.05What to eat49(68)66(84)<0.05My disease62(80)71(92)<0.05I desire increased understanding…Of my disease59(77)53(70)0.4Of managing stress50(65)35(51)<0.05Regarding nutrition64(84)54(70)<0.05Of the screening tests62(79)43(57)<0.05How to manage fatigue54(72)42(54)<0.05 Disclosures Tibes: TetraLogic Pharmaceuticals: Research Funding. Mesa:Novartis. Research- incyte, Gilead, cti, Genentech, promedior, NS Pharma: Consultancy.

Abstract Introduction: ASP2215 is a highly selective inhibitor of AXL and FMS-like tyrosine kinase-3 (FLT3) receptors. ASP2215 is active against both FLT3 internal tandem duplication [ITD] and D835 mutations. Prior analyses of an open-label, dose-escalation/dose-expansion study in subjects with relapsed or refractory acute myeloid leukemia (R/R AML) show ASP2215 was well tolerated from 20-300 mg and associated with antileukemic activity in FLT3 mutation-positive (FLT3+) patients at ≥80 mg with minimal activity observed in wild-type FLT3 subjects. Here we describe the tolerability and potent activity of ASP2215 in a large cohort of FLT3+ patients. Methods: Patients (≥18 years) with R/R AML were assigned to treatment in dose-escalation cohorts or were randomized to an open dose level in the dose-expansion cohorts. Although FLT3 mutation was not an inclusion criterion, each expanded dose level enrolled ≥10 FLT3+ subjects; 120 mg and 200 mg dose levels were further expanded with ≥40 FLT3+ subjects. Tolerability was assessed by adverse event (AE) monitoring. Response assessment was based on modified Cheson criteria and duration of response and overall survival were calculated using Kaplan-Meier estimates. Results: As of June 19, 2015,215 patients with a median age of 61 yr (range: 21-90) had received ≥1 dose of ASP2215 (safety population). Across the safety population, 65% of subjects received ≥2 prior lines of AML therapy, 29% had a hematopoietic stem cell transplant prior to ASP2215 treatment, and 23% had prior tyrosine kinase inhibitor (TKI) treatment. Approximately 73% of patients were FLT3+, of which 137 had FLT3-ITD mutation, 7 were FLT3-D835+, and 9 had both FLT3-ITD and D835. Treatment-related AEs of all Grades, reported in ≥10% of the safety population were diarrhea (16%), fatigue (13%), and increased AST (11%); <2% of subjects reported a Grade ≥3 QTc prolongation. Activity was assessed in the 133 FLT3+ patients treated with ASP2215 ≥80 mg. Overall response rate (ORR; composite complete [CRc] plus partial remission [PR]) for all FLT3+ subjects was 55% (Table). Median overall survival for FLT3+ patients receiving ASP2215 ≥80 mg was ~29 weeks (95% CI: 22-32) and was similar for patients who achieved CRc or PR (Figure). Treatment with ≥80 mg ASP2215 was associated with an ORR of 60% in FLT3-ITD subjects; ORR for the other FLT3 populations was 29% (Table). No difference was observed in median ORR of ASP2215 (≥80 mg) in TKI-naïve patients (55%) and patients with prior TKI treatment (55%). Conclusions: ASP2215, a novel AXL/FLT3 TKI, was well tolerated in subjects with R/R AML and demonstrated a strong antileukemic activity in FLT3+ subjects. Importantly, the ASP2215 response rate in these FLT3+ patients was independent of prior TKI treatment. Even in this heavily pretreated population, the survival of R/R FLT3+ AML patients who received ≥80 mg ASP2215 was longer than prior reports of cytotoxic chemotherapy or other FLT3 inhibitors. Table. ASP2215 Response Assessment 80 mg 120 mg 200 mg 300 mg 450 mg Total All FLT3+ Subjects Population, n 12 52 57 10 2 133 CRc, n (%) 5 (42) 25 (48) 28 (49) 3 (30) 0 61 (46) PR, n (%) 3 (25) 3 (6) 3 (5) 3 (30) 0 12 (9) ORR, n (%) 8 (67) 28 (54) 31 (54) 6 (60) 0 73 (55) Subjects with FLT3-ITD Only Population, n 10 46 50 8 0 114 CRc, n (%) 4 (40) 23 (50) 26 (52) 3 (38) 0 56 (49) PR, n (%) 3 (30) 3 (7) 3 (6) 3 (38) 0 12 (11) ORR, n (%) 7 (70) 26 (57) 29 (58) 6 (75) 0 68 (60) Subjects with FLT3-D835 and Subjects with FLT3-ITD and FLT3-D835 Population, n 2 5 5 1 1 14 CRc, n (%) 1 (50) 1 (20) 2 (40) 0 0 4 (29) PR, n (%) 0 0 0 0 0 0 ORR, n (%) 1 (50) 1 (20) 2 (40) 0 0 4 (29) CRc, composite complete remission (complete remission + complete remission with incomplete platelet recovery + complete remission with incomplete hematologic recovery); ORR, overall response rate; PR, partial response. NR, no response. Subjects with non-evaluable data (N=8) were not included in this curve. Figure 1. Overall Survival by Best Overall Response Achieved with ASP2215 ≥80 mg Across All FLT3+ Subjects Figure 1. Overall Survival by Best Overall Response Achieved with ASP2215 ≥80 mg Across All FLT3+ Subjects Disclosures Altman: BMS: Other: Advisory board; Novartis: Other: Advisory board; Spectrum: Other: Advisory board; Ariad: Other: Advisory board; Seattle Genetics: Other: Advisory board; Astellas: Other: Participation in an advisory board December 2013. Off Label Use: ASP2215 is currently under investigation for the treatment of AML and is not yet approved.. Perl:Arog Pharmaceuticals: Consultancy; Asana Biosciences: Consultancy; Actinium Pharmaceuticals: Consultancy; Ambit/Daichi Sankyo: Consultancy; Astellas US Pharma Inc.: Consultancy. Cortes:Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Novartis: Consultancy, Research Funding; Teva: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Levis:Arog: Research Funding; Ambit: Research Funding; Takeda: Research Funding; Astellas: Consultancy. Smith:Plexxikon: Research Funding; Astellas: Research Funding. Claxton:NCI: Research Funding; Medimmune, Inc: Research Funding; Ambit Biosciences Corp: Research Funding; Incyte Corporation: Research Funding; Merck Sharp & Dohme Corp: Research Funding; Astellas Pharma: Research Funding. Erba:Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Ariad: Consultancy; Celgene: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; GlycoMimetics: Other: Data Safety and Monitoring Committees ; Jannsen (J&J): Other: Data Safety and Monitoring Committees ; Seattle Genetics: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Amgen: Consultancy, Research Funding; Celator: Research Funding; Millennium/Takeda: Research Funding; Astellas: Research Funding; Sunesis: Consultancy; Celator: Research Funding; Pfizer: Consultancy; Astellas: Research Funding; Daiichi Sankyo: Consultancy; Sunesis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; GlycoMimetics: Other: Data Safety and Monitoring Committees ; Jannsen (J&J): Other: Data Safety and Monitoring Committees ; Daiichi Sankyo: Consultancy. Gill:Astellas Pharma US, Inc: Employment. Goldberg:Cyclacel: Research Funding; Celetor: Research Funding; Pfizer: Research Funding; Ambit: Research Funding; Astellas: Research Funding. Jurcic:Astellas Pharma: Research Funding. Larson:Astellas: Consultancy, Research Funding. Lui:Astellas Pharma US, Inc: Employment. Ritchie:Incyte: Speakers Bureau; Novartis: Speakers Bureau; Ariad: Other: Advisory Board; Celgene: Speakers Bureau; Onyx: Speakers Bureau. Sargent:Astellas Pharma US, Inc: Employment. Schiller:Sunesis: Honoraria, Research Funding. Strickland:Sunesis Pharmaceuticals: Other: Steering Committee and Advisory Board Participation; Alexion Pharmaceuticals: Other: Advisory Board Particpation; Amgen: Other: Advisory Board Particpation; Daiichi-Sankyo: Other: Advisory Board Particpation; Boehringer-Ingelheim: Other: Advisory Board Particpation. Wang:Immunogen: Research Funding. Stuart:Sunesis: Honoraria, Other: Advisory Board, Research Funding; Astellas Pharma, Inc: Research Funding. Baldus:Novartis: Research Funding. Martinelli:MSD: Consultancy; ARIAD: Consultancy; BMS: Speakers Bureau; Pfizer: Consultancy; Novartis: Speakers Bureau; Roche: Consultancy. Bahceci:Astellas Pharma Global Development: Employment.

Abstract BACKGROUND: Azacitidine and decitabine are indicated for the first line treatment of intermediate and high risk MDS. About 40% to 47% of patients respond to 1st line hypomethylating agents. The median duration of response is 13 months. There are no effective therapies for patients who fail to respond to or progress on hypomethylating agents. A phase II trial of high dose lenalidomide (50mg/day) induction therapy followed by low dose (10mg/day) maintenance therapy in elderly patients with AML had demonstrated a high CR/CRi of 30%. CR was restricted to patients with presenting WBC counts <10000/µL, peripheral blast counts <1000/ µL and < 30%, many of whom could have been classified as having MDS rather than AML by FAB criteria. With the rationale that MDS and AML represent a continuum of disease, and the promising results in AML we aimed to assess the clinical activity of high dose lenalidomide by conducting a clinical trial of high dose Lenalidomide induction followed by low dose maintenance in patients with hypomethylating agent refractory MDS. METHODS: We conducted an open label, single arm, multi-center phase II trial of high dose lenalidomide induction (50mg/day for two 28 day cycles) followed by low dose lenalidomide maintenance therapy (10mg/day for 12 cycles) for patients with MDS (as defined by FAB criteria) refractory to hypomethylating agents. Patients who had failed to show CR, PR or HI (hematological improvement) after at least 6 cycles of azacytidine or decitabine or who had progressed on treatment were considered eligible. Patients were enrolled in a Simon's optimal two-stage design with an intended sample size of 43. The primary endpoint was proportion of confirmed responses (CR, PR or HI), as defined by international working group criteria, during the 14 cycles of treatment. RESULTS: 24 patients were enrolled in the first stage of the study. 16 patients completed at least one cycle of therapy and were considered evaluable for efficacy. Efficacy: Characteristics and outcomes of patients are detailed in Table 1. There were no complete or partial remissions. Marrow complete remission (MCR) was seen in 8/16 patients (50%). One of the 8 patients with a MCR had 5q- karyotype; 3/8 had trisomy 8 and one patient had a complex karyotype with monosomy 7. Median duration of MCR was 70 days (range 28-336 days). MCR lasted beyond the high dose induction period in 2 patients, one of whom had a 5q interstitial deletion (112 days) and one patient with trisomy 8 (336 days). Hematologic improvement (HI) was seen in 2/16 patients (12.5%) (Refer to table below). Toxicity: ≥grade 3 toxicities included pneumonia (12/24, 50%), sepsis (4/24, 17%), febrile neutropenia (8/24, 33%), rash (2/24, 8%), thromboembolic event (1/24), MI (1/24), new cancer -mammary analogue salivary carcinoma- (1/24). TABLE 1 Pt No: Age IPSS Prior cycles of 5-aza/ decitabine Cycles of Lenalidomide Reason off trial Best Response 1 69 1.5 2 4 Pancytopenia MCR 2 78 3 8 1 Death-Unrelated SD 3 73 2 7 1 Consent withdrawn SD 4 87 2 14 12 Progression MCR, HI-p&n 6 72 2 13 2 Thrombocytopenia MCR 7 69 2 13 1 PE SD 8 75 1 7 2 New Cancer MCR 9 74 1 4 3 Progression SD 10 75 2 0 2 Progression SD 11 72 1 13 3 Thrombocytopenia MCR 12 75 3 6 1 Progression PD 13 78 1 9 3 Gr3 Fatigue MCR 14 70 0 19 2 Progression MCR, HI-p 15 72 1 23 1 Progression PD 16 73 1 8 2 Gr 3 fatigue MCR CONCLUSIONS: High dose lenalidomide followed by standard dose maintenance in hypomethylating agent refractory MDS resulted in a marrow complete remission rate of 50% and hematological improvement rate of 12.5%. Future protocols should explore whether an extended schedule of intermittent dosing of high dose lenalidomide would allow for improved efficacy with acceptable toxicity. Disclosures Off Label Use: Lenalidomide is used in this study in patients with MDS both with and without deletion 5q.Use in patients without deletion 5q is off label use.. Tibes:Tetralogic Pharmaceuticals: Research Funding. Stockerl-Goldstein:Celgene: Speakers Bureau. Jacoby:Norvo Nordisk: Consultancy. Cashen:Celgene: Speakers Bureau. Vij:Onyx: Honoraria, Research Funding; Sanofi: Honoraria; Jannsen: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Celgene: Honoraria, Research Funding.

Abstract Abstract 420 Primary central nervous system lymphoma (PCNSL) is a rare and aggressive non-Hodgkin lymphoma that is confined to the CNS because of a poorly understood neurotropism. Most of PCNSL (90%) are part of the immune-privileged site-associated DLBCL (IPDLBCL). IPDLBCL consist in late–germinal center or post–germinal center lymphoid cells but that show very distinct characteristics that separate them from systemic DLBCL. It is still a matter of debate whether the PCNSL differ from nodal DLBCL with respect to molecular features and pathogenesis and also if there is a genomic signature specific of PCNSL. Only few genetic studies have been performed in PCNSL, partly due to the rarity of the tumors and the limited amount of available tissue. To gain insight into the genomic basis of PCNSL, we performed an integrated, high-throughput, genomic analysis in 17 immunocompetent, EBV- and HIV- cases. B-cell differentiation status was characterized by immunostains for CD10, MUM-1, and BCL-6. Either frozen samples or formalin fixed embedded paraffin sections from 17 PCNSL were studied by array-based comparative genomic hybridization (aCGH) using Sureprint G3 (1 million probes) array. Massively parallel whole-exome sequencing was performed in 4 of these cases. Additionally, 2 cases were analyzed by mate-pair whole genome sequencing searching for chromosomal breakpoints. Sanger DNA sequencing was used for validation. All cases were characterized by complex genomic aberrations with a median of 21 copy-number abnormalities (CNA, range 10–49), 4 structural abnormalities, 6 frameshift indels and 99 nonsynonymous exonic mutations. Focal deletion affecting CDKN2A (9p21) was the most common CNA, found in 14 of 17 cases (82%); with 6 of these cases (35%) having homozygous deletion. The second most frequent CNA involved the HLA genes (6p21), found in 11 of 17 (65%) cases; 4 of them (23%) with homozygous deletions. We identified recurrent CNA and mutations in several genes previously found in systemic DLBCL. Thus, PRDM1 (BLIMP1) was deleted or mutated in 47% of cases and the translocation IgH-BCL6 was found in 30% of PCNSL. Furthermore, recurrent mutations were found in NF-kB genes CD79B (75%, 3 of 4 cases analyzed), MYD88 (70%, 7 of 10), TNFAIP3 (50%, 2 of 4) and CARD11 (50%, 2 of 4). Additionally, recurrent abnormalities were found in B2M, BCL7A, CD58, CIITA, ETV6, GNA13, PAX5, TMEM30A and TP53. Nevertheless, we identified several recurrent genetic alterations not described in systemic DLBCL. TOX (a regulator of T-cell development) and TBL1XR1 (a negative regulator of the NF-kB and Wnt pathways) were either deleted or mutated in 30% of PNCSL, something not previously described in systemic DLBCL. Additionally, chromosomal breakpoints either in DLGAP1 or DLGAP2 (play a role in neuronal cell signaling) were found in 18% of PCNSL but not in systemic DLBCL. Moreover, mutations in ATM (master controller of cell cycle checkpoint), BAI3 (inhibitor of brain angiogenesis), BTG2 (cell cycle arrest), KDM6B (histone demethylase), PKC family members PRKCD/PRKCDE, genes from the histone cluster, the protocadherin family and the WD repeat domain were found in 10% to 50% of PCNSL. Pathway analysis including the most commonly affected genes in PCNSL showed an enrichment of networks associated with immune response, NF-kB pathway, proliferation, regulation of the apoptosis and lymphocyte differentiation and activation. In summary, we show evidence of a highly complex genome and identified a subset of genes with potential relevance in PCNSL pathogenesis. The genomic profile described here reinforces the existence of a specific molecular signature in PCNSL, thus helping to genetically differentiate this entity from the nodal DLBCL and related lymphomas. Disclosures: Stewart: Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy; Binding site: Consultancy; Millenium: Consultancy; AMGEN: Consultancy; Celgene : Research Funding; Onyx: Research Funding; prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Abstract Background: Histone deacetylase (HDAC) inhibitors (HDIs) vorinostat (SAHA) and romidepsin are approved for the treatment of cutaneous T-cell lymphoma, while belinostat has recently been approved for the treatment of peripheral T-cell lymphoma. SAHA exhibits clinical activity in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), although insufficient to justify single-agent therapy for these indications. The precise clinical mechanism of SAHA and other HDIs remains incompletely characterized, impeding the design and implementation of rational HDI-based therapeutic combinations for myeloid malignancies. To circumvent this lack of mechanistic understanding, we conducted RNA-interference (RNAi) modifier screens of the kinome and phosphatome to identify signaling pathways that modulate SAHA (HDI) anti-leukemic activity in AML cell lines TF-1, HEL and THP-1 for rapid translation of findings from ex vivo validation to clinical application. Results: Multiple screen hits (both kinases and phosphatases) converge on distinct points of the p38-SAPK/JNK signaling pathways, suggesting involvement of these stress-activated pathways in SAHA anti-leukemic activity. However, the most well characterized p38α/β inhibitors SB202190 and LY2228820 do not augment SAHA anti-leukemic activity at any dose across the broad dose ranges tested. Similarly, the JNK inhibitor SP600125 does not modulate SAHA activity at doses that inhibit JNK. However, the putative MK2a substrate-selective p38 inhibitor CMPD1 (Boehringer-Ingelheim; Davidson, W., et. al., Biochemistry 2004 Sep 21;43(37):11658-71) selectively potentiates the activity of SAHA in all AML cell lines tested (N=8), as well as in ex vivo cultures of primary myeloid malignancies (N=14), including polycythemia vera, chronic myelomonocytic leukemia, MDS/myeloproliferative neoplasm overlap syndrome, MDS transformed to AML, and de novo AML. The observed dose-dependent synergy occurs at nanomolar doses consistent with the reported apparent inhibitory constant for CMPD1 inhibition of p38-dependent phosphorylation of MK2a. Further, SAHA + CMPD1 synergy is significantly greater in CD34+-selected as compared to CD34+-depleted cell populations ex vivo. Furthermore, CMPD1 is similarly synergistic with the HDI panobinostat in vitro, but interacts antagonistically with cytarabine and additively with 5-azacitidine. This suggests that CMPD1 interacts specifically with SAHA and panobinostat in AML. The synergistic interaction between CMPD1 and SAHA is independent of ROS induction, as pretreatment with N-acetylcysteine does not abrogate synergy, despite reducing single-agent SAHA activity. While CMPD1 is reported to be an MK2a substrate-selective p38 inhibitor, CMPD1 does not affect the regulation of several canonical MK2 targets at doses that are potently synergistic in AML cell lines, including the phosphorylation of HSP27 or AATF. To begin to probe the mechanism of CMPD1 and SAHA synergy more extensively, mRNA expression was measured by next-generation sequencing after SAHA and CMPD1 treatment alone and in combination in the AML cell line TF-1. Sequencing data is currently being analyzed and findings will be presented. Conclusion: RNAi knockdown of several p38-SAPK/JNK pathway components modulates SAHA activity in malignant myeloid cells; however, common pharmacological sensitizing targets remain elusive. We hypothesize that MK2 and/or a yet unrecognized pathway modulated by CMPD1 can be targeted for HDI combination therapy in malignant myeloid cells. Current and ongoing results from this study will provide validated targets for rational HDI combination in myeloid malignancies. Disclosures Off Label Use: LDE225/Sonidegib as investigational agent. Mesa:Incyte, CTI, NS pharma, Gilead, Celgene: Research Funding.

Abstract Abstract 1851 Background: In previous work we performed RNAi sensitizer screening of epigenetic modulation with 5-Azacytidine and the human kinome. Few kinase hits were identified. Therefore we asked if phosphatases might be involved in modulating epigenetic therapies. Thus in order to identify potentiators of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) we performed high-throughput RNA-interference (RNAi) lethality screening using the myeloid cell lines HEL, THP-1 and TF-1 in combination with SAHA. Methods: A small-interfering RNA (siRNA) library targeting 206 phosphatases with 4 sequences per target gene was used for this study. siRNA were transfected in a single-siRNA-per-well format in 384-well plates using commercial lipid-based transfection reagents. Non-silencing siRNA were included as negative controls to access non-specific siRNA toxicity and universal lethal siRNA were included as positive controls to indirectly assess functional transfection efficiency. After a 96 hour incubation period, cell viability/proliferation was measured with Cell Titer Glo. For analysis of screen data, log2 values of (SAHA + siRNA treatment/siRNA treatment only) were calculated. Potentiation was defined as two standard deviations from the median log2 ratio of all siRNA (excluding positive controls) on a plate-by-plate basis. Hits were defined as ≥ 2/4 siRNA sequences meeting potentiation criteria. Results: Transfection efficiency was 95, 90 and 70% for HEL, THP-1 and TF-1, respectively. All three cell lines were similarly sensitive to SAHA with IC50 values of 0.3, 0.4 and 0.6 μM for HEL, THP-1 and TF-1, respectively. Resulting SAHA concentrations for screens were between the IC5 to IC30. For THP-1, 5 phosphatases qualified according to the hit selection criteria applied indicating strong potentiation to SAHA. Of these 5 hits, 3 did not exhibit strong reductions in viability with siRNA alone, suggesting a SAHA-dependent relationship. For HEL and TF-1, although no phosphatases met the strict selection cutoff, there were several phosphatases exhibiting potentiation, suggesting biological relevance. The primary screening data and preliminary analysis will be validated with rigorous secondary experimentation and top hits for all cell lines will be investigated and advanced. Conclusions: While there are numerous kinase inhibitors in the clinic and in preclinical development, phosphatases are much less explored as therapeutic targets. Validated potentiating targets from this RNAi screen can facilitate the development of rational SAHA combination therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 5061 Background: Ruxolitinib was approved for commercial use for the therapy of intermediate- and high-risk myelofibrosis (MF) in the USA in November 2011. We sought to assess the initial clinical experience on the use of ruxolitinib outside of the clinical trial setting at our high volume MPN program. Methods: Twenty-eight patients with intermediate- or high-risk myelofibrosis (including PMF, post-PV and post-ET myelofibrosis) prescribed ruxolitinib at our center within the first seven months after FDA approval were included in this analysis. Baseline disease information, treatment history, dynamics of ruxolitinib dosing, impact on cytopenias, splenic response, and symptoms were assessed. Results: A total of 28 patients with post-ET MF (n=7), post-PV MF (n=6) and primary MF (n=15) began ruxolitinib between November, 2011 and May, 2012. Twenty-seven patients had been treated with previous therapies. The JAK2V617F mutation was present in 79% of patients. The mean number of weeks from diagnosis of MF to treatment with ruxolitinib was 218 (8–929). The average spleen size at the start of therapy was 11. 5cm (0–36) below the left costal margin. DIPSS Plus scores classified patients as Intermediate-1 (53%), Intermediate-2 (29%) and High risk (18%). Symptoms at the start of therapy included fatigue (78%), early satiety (25%), night-sweats (68%), abdominal pain (61%), pruritis (32%), weight-loss (57%), bone pain (21%), and fevers (11%). The average hemoglobin concentration, WBC count and platelet counts were 10. 9 g/dL (7. 6–13. 8), 13. 5 × 10(9)/L (3. 1–53. 8) and 295 × 10(9)/L (32–844) respectively. The median duration to follow-up was 72 days (4–144 days). The majority of patients began therapy at 20 mg BID (68%). Most patients seen in follow-up experienced reduction in splenic size (64%), 36% of which had complete resolution of palpable splenomegaly within a mean follow-up of 78 days (10–142). Significant improvements were noted in symptoms including fatigue (68%), early satiety (71%), night sweats (47%), abdominal pain (71%), pruritis (55%), weight loss (69%), bone pain (33%) and fevers (33%). Dosage reductions were required for four patients with anemia (n=3) and thrombocytopenia (n=1). Two patients required the addition of anagrelide/hydroxyurea for platelet counts >1, 000 × 10(9)/L. Excluding patients with preexisting thrombocytosis >600 × 10(9)/L, the percentage reduction of platelets from baseline by weeks 4, 9 and 15 were 34%, 13%, and 12% respectively. Eight patients required red blood cell transfusions for anemia, three of which who had begun therapy with hemoglobin concentrations under 9 g/dL. Excluding the 8 transfusion-dependent patients, the percentage reduction in hemoglobin concentration from baseline by weeks 4, 9, and 15 was 7%, 6%, and 14% respectively. Non-hematological toxicities requiring dosage adjustments included hyperkalemia (n=1) and fatigue/dizziness/headaches (n=1). Five patients underwent dose increases for lack splenic (n=3) and symptomatic (n=2) response. Six patients discontinued the drug for disease progression requiring stem cell transplant (n=2), lack of constitutional symptom improvement (n=1), diarrhea (n=1), thrombocytopenia (n=1) and anemia (n=2). Five patients had platelet counts below 100 × 10(9)/L (19–94 × 10(9)/L) and began therapy at reduced starting doses. All patients seen in follow-up reported significant improvements in symptoms and splenomegly was reduced >50% in two patients seen in follow-up within 4 weeks of initiating therapy. Two patients underwent dose increases after 5 months of therapy to enhance response. No bleeding events were reported. By week 15, no decrease was noted in platelet levels from baseline in the 3 patients seen in follow-up. Two patients required danzol (n=1) or platelet transfusions (n=1) for thrombocytopenia. Conclusion: Randomized controlled trials have proven ruxolitinib's efficacy in reducing constitutional symptoms, cytopenias and splenomegaly in myelofibrosis patients. This study represents a confirmation of the efficacy this drug provides in reducing splenomegaly and symptoms observed in clinical trials when utilized in a standard practice setting. Toxicities are minimal and have been generally well controlled with dose reductions, even in patients with baseline thrombocytopenia. In conclusion, ruoxlitinib appears to be an efficacious and well-tolerated therapy with limited adverse effects. Disclosures: Mesa: Incyte: Research Funding; Lilly: Research Funding; Sanofi: Research Funding; NS Pharma: Research Funding; YM Bioscience: Research Funding.