ALBERT

Description: Abstract 2053 Poster Board II-30 Activating mutations in the receptor tyrosine kinase FLT3 are present in up to 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a potential target for kinase inhibitor therapy. AC220, a potent and selective FLT3 inhibitor, is currently in Phase II clinical trials in both FLT3-ITD positive and WT patients. Similar to imatinib therapy for CML, it is possible that targeted therapies will be delivered chronically. Therefore, we examined the efficacy of AC220 in a limited versus chronic dosing regimen in the homozygous FLT3-ITD-dependent MV4-11 disseminated mouse disease model. As AML is a heterogeneous disease, we also examined the in vivo efficacy of AC220 in the MOLM-14 disease model. This cell line is heterozygous for a FLT-3 ITD mutation in addition to carrying the MLL-AF9 fusion. In the MV4-11 model, a 30 day dosing regimen was compared to chronic dosing. In the vehicle control group, median survival time following inoculation was 47 days, with mortality preceded by clinical signs of disease and detection of MV4-11 cells in the blood. No specific clinical signs or body weight loss were attributed to the study drug. AC220 demonstrated dose-dependent efficacy from 0.1 mg/kg to 10 mg/kg orally once per day for 30 days. The 0.1 mg/kg group had a marginal (10%) increase in life span (ILS) relative to vehicle control, while a significant increase of survival was observed at the 1.0 mg/kg dose (55% ILS). The 10 mg/kg dose led to 80% survival at study termination, day 172 (〉250% ILS). Prolonged survival with AC220 correlated with delayed disease onset as measured by clinical signs and detection of circulating MV4-11 cells. Chronic dosing in the 1.0 mg/kg group further delayed disease onset and mortality with an ILS of 155% relative to vehicle, and 63% relative to 30-day dosing. Similar to the 30-day dosing group, chronic administration of AC220 at 10 mg/kg led to 80% survival at day 172. A separate study was conducted to examine the relationship between bone marrow engraftment, tumor burden in peripheral blood and disease onset. At day 20, engraftment was detected only in the bone marrow. At day 35, when clinical signs of disease are typically apparent, levels of tumor cells as high as 80% and 35% were detected in the bone marrow and peripheral blood, respectively. AC220 given for 28 days at 1.0mg/kg delayed median onset of disease by 24 days (63%), with tumor burden undetectable in the absence of clinical signs of disease. The 28 day dosing at 10.0mg/kg completely inhibited disease onset, with no detectable tumor burden in either blood or bone marrow through the end of the study (terminated on day 130, 〉200% ILS). In the MOLM-14 model, median survival time of untreated or vehicle treated animals was 22 days. In a 21 day dosing regimen, AC220 demonstrated dose dependent efficacy, providing 9%, 64% and 127% ILS at 0.1, 1.0 and 10 mg/kg respectively. Prolonged survival correlated with a delay in both disease onset and detection of circulating MOLM-14 cells. Similar to the MV4-11 model, temporal analysis confirmed bone marrow engraftment prior to detectable levels in the circulation. At 13 days post-transplant, tumor cells are only detectable in the bone marrow, while by day 19, when clinical signs are apparent, tumor burden in the bone marrow and periphery of vehicle treated animals are as high as 35% and 6%, respectively. Unlike the MV4-11 model, dosing at 10 mg/kg for 21 days did not prevent disease onset after cessation of dosing, indicating that tumor cells were not completely eliminated, although detectable levels are not present in bone marrow or circulation until days 47 and 52, respectively. These data suggest that AC220 is efficacious against both FLT3-ITD homozygous and heterozygous genotypes, and that a chronic dosing regimen may provide greater disease protection than a limited course of therapy. This study is consistent with observed efficacy in AML patients treated continuously with AC220. Disclosures: Brigham: Ambit Biosciences: Employment. Belli:Ambit Biosciences: Employment. Breider:Ambit Biosciences: Employment. Bhagwat:Ambit Biosciences: Consultancy. Wierenga:Ambit Biosciences: Employment. Armstrong:Ambit Biosciences: Employment.

Description: Abstract 1538 Acute myeloid leukemia patients harboring an activated FLT receptor have a poorer prognosis following standard chemotherapy treatment compared to patients with a wild type receptor. This suggests that the combination of FLT3 inhibitor with chemotherapy may improve clinical outcome in this patient population. AC220, a potent and selective FLT3 inhibitor, has a subnanomolar anti-proliferative EC50 against cells containing activated FLT3 receptor. Using the homozygous FLT3-ITD (internal tandem duplication) cell line MV4-11 we previously demonstrated that treatment of cells with AC220 concurrent with or the day following treatment with the individual chemotherapeutic agents generally provided additive to slightly synergistic effects in vitro, with no indications of drug antagonism or increased toxicity as measured by body weight changes and CBC analysis (ASH 2009). Because induction therapy in AML patients is typically comprised of Cytarabine (Ara-C) plus an anthracyline, we sought to more closely approximate this chemotherapeutic dosing regimen in a preclinical model. In the current study, we treated MV4-11 tumor bearing animals with two cycles of ‘5+3 therapy’; 5 days of Ara-C at 80mg/kg IP concurrent with 3 days of daunorubicin (DNR) at 1 mg/kg IV with cycles separated by 10 days. These doses were at or near the MTD for tumor bearing SCID mice. AC220 at 0.5 mg/kg PO was delivered either concurrently and continuously throughout the two cycles or was delivered episodically for 7 days immediately following the end of the DNR dosing period or the end of the Ara-C dosing period. Monotherapy with the chemotherapeutic agents alone or in a 5+3 combination dosing regimen provided measurable but statistically non-significant anti-tumor benefit with a tumor growth delay (TGD) of 7 days for the 5+3 cohort (endpoint =1500 mm3). AC220 alone delivered continuously at 0.5 mg/kg from the beginning of the dosing period did provide significant anti-tumor benefit, with a tumor growth delay (TGD) of 21 days and a tumor growth inhibition (TGI) of 17% when measured at the end of the first cycle (day 30). Continuous AC220 treatment initiated on day 1 in conjunction with 5+3 therapy led to a slightly increased TGD of 23 days and further reduced the TGI to 12%. AC220 alone delivered episodically for 7 days at 0.5 mg/kg starting on either day 4 or on day 6 (mimicking post-DNR or post-Ara-C treatment, respectively) provided a TGD of 11 days. Layering 7 day episodic dosing of AC220 immediately following treatment with DNR or Ara-C in a 5+3 regimen led to a TGD of 16 or 18 days, respectively. However, comparison of the TGI at the end of the first cycle of therapy suggests that episodic dosing of AC220 post-AraC is more efficacious (TGI 22%) than episodic dosing of AC220 post-DNR (TGI 35%). These results suggest that episodic dosing of AC220 post-5+3 therapy or continual dosing of AC220 plus 5+3 induction therapy may provide additional benefit over chemotherapy alone. Disclosures: Belli: Ambit Biosciences: Employment, Equity Ownership. Dao:Ambit Biosciences: Employment. Wierenga:Ambit Biosciences: Equity Ownership. Armstrong:Ambit Biosciences: Employment, Equity Ownership.

Description: Abstract 2052 Poster Board II-29 AC220, a potent and selective FLT3 inhibitor, has a sub-nanomolar anti-proliferative EC50 against cells containing activated FLT3 receptor. Acute myeloid leukemia patients harboring an activated FLT3 receptor have a poorer prognosis following standard chemotherapy compared to patients with a wild-type receptor. This suggests that the combination of FLT3 inhibitor with standard chemotherapeutic regimens may improve efficacy. Other investigators have reported potential antagonistic consequences when dosing the clinical compound lestaurtinib prior to chemotherapy agents utilizing both in vitro tumor cell lines (Levis, M. et al. Blood 2004;104:1145-1150) and primary patient samples (Brown, P. et al. Leukemia 2006;20:1368-1376). We therefore explored the effect of combining AC220 with cytarabine (ara-C), cladribine, etoposide and daunorubicin against the homozygous FLT3 ITD (internal tandem duplication) cell line MV4-11 in vitro. Data were analyzed utilizing the median effect method of Chou and Talalay (Chou TC, Talalay P. Adv. Enz. Reg. 1984;22:27-55). The results indicated that delivery of AC220 concurrent with or the day following treatment with the individual chemotherapeutic agents generally provided additive to slightly synergistic effects. Unlike the results with lestaurtinib, treating the cells with AC220 prior to the addition of the chemotherapeutic agent did not result in significant antagonism. To determine whether these in vitro results translated to increased in vivo efficacy, two experiments were performed. In the first experiment, mice bearing MV4-11 solid tumors were treated with decitabine (5-aza) at 1 or 3 mg/kg QD X 5 alone or in combination with AC220 at 0.5 mg/kg delivered QD either concurrent with 5-aza or immediately following treatment. Three cycles of treatment were delivered, and, in cycle two and three, AC220 dosing was continuously maintained. AC220 alone was tumor-static regardless of treatment initiation date. 5-aza alone at 3 mg/kg reduced tumor volume by 50%, whereas 5-aza at 1 mg/kg was ineffective. When the two drugs were delivered concurrently, or when a single cycle of 5-aza preceded AC220, the combination led to an additive antitumor effect. Importantly, there was no indication of antagonism on tumor volume, body weight and WBC counts with either of the tested schedules, indicating that the combination of AC220 with 5-aza is safe and efficacious in the MV4-11 solid tumor mouse flank model. In the second experiment, mice were treated with ara-C at 30 mg/kg on a 10 day on, 14 day off, 10 day on regimen, either alone or in combination with AC220 at 1.0 mg/kg QD. AC220 dosing was initiated concurrent with the first treatment of ara-C and maintained over the 34 day period, or immediately following the first ara-C cycle and given for 14 days. AC220 alone led to tumor regression and a 20% remission rate with no cures. Ara-C alone reduced tumor burden by approximately 50% following the first cycle of treatment. Ara-C further reduced tumor burden in mice treated with AC220, and AC220 was efficacious when dosed following 10 day treatment with ara-C. Episodic dosing of ara-C concurrent with continuous AC220 leads to a significant increase in TTE (time to endpoint, 1500mm3) relative to AC220 alone (105 vs. 80 days, respectively). In this cohort, remission rate was 30% and two mice achieved cures. As with 5-aza, there was no indication of drug specific antagonism with ara-C on either of the schedules tested. Together, these data indicate that the combination of AC220 with ara-C or 5-aza, when delivered concurrently or episodically, is safe and leads to increased efficacy in the MV4-11 solid tumor mouse flank model. This result merits further investigation in future clinical trials. Disclosures: Belli: Ambit Biosciences: Employment. Dao:Ambit Biosciences: Employment. Bhagwat:Ambit Biosciences: Consultancy. Wierenga:Ambit Biosciences: Employment. Armstrong:Ambit Biosciences: Employment.