Ambit Biosciences Research Articles

Crenolanib Is A Highly Potent, Selective, FLT3 TKI with Activity Against D835 Mutations

Abstract 1341Approximately 23% of acute myeloid leukemia (AML) patients have internal tandem duplications (ITD) of the type III receptor tyrosine kinase FLT3. FLT3/ITD mutations correlate with poor survival rates in AML patients. This has made FLT3 a promising target for treating AML, leading to the development of a series of small molecule FLT3 tyrosine kinase inhibitors (TKIs). Quizartinib is the most potent FLT3 TKI developed to date, and has achieved a high response rate as a monotherapy in relapsed/refractory FLT3/ITD AML patients. However, during clinical trials of quizartinib, multiple patients developed point mutations of aspartate 835 (D835) which conferred resistance to treatment with FLT3 TKIs. FLT3/D835 mutations can also occur at disease presentation with or without the presence of ITD mutations, though the prognostic significance in these cases is less well-defined. Therefore, a FLT3 TKI which also has activity against FLT3/D835 point mutations has significant potential to be clinically beneficial for a number of patients. Crenolanib is a next generation FLT3 TKI which is nearly as potent as quizartinib (IC50 in human plasma of 35 nM versus 18 nM for quizartinib). Furthermore, in clinical trials of quizartinib, some subjects experienced myelosuppression, which may have been associated with inhibition of the related type III receptor tyrosine kinase, c-Kit. Crenolanib is 33-fold less active against c-Kit as compared to FLT3. The functional significance of this finding was tested with colony formation assays of normal donor bone marrow (n=3). The abundance of erythroid colonies with 200 nM of drug compared to control was 23% and 75% when treated with quizartinib and crenolanib, respectively. These data suggest that clinically, crenolanib may not have the significant myelosuppressive effects as observed for quizartinib in clinical trials. Crenolanib was studied in phase II trials as a PDGFR inhibitor in glioma and GIST patients. Pharmacokinetic data from these trails show that patients are able to achieve drug levels well above the IC90 for FLT3 in plasma. Plasma samples were obtained from these patients and utilized for plasma inhibitory activity (PIA) assays. The results of these assays suggest that adequate FLT3 inhibitory concentrations of this drug can easily be achieved with safe and tolerable dosing schedules. The activity of crenolanib was tested in series of primary samples with a variety of FLT3 mutations. One patient sample containing a D835V mutation was treated with 20 nM of sorafenib, quizartinib, and crenolanib and Western blotted for pFLT3. Only crenolanib showed any activity against this sample, with an IC50 of 2 nM in culture medium. In another case, blasts were obtained from a patient treated with quizartinib who then relapsed. In a comparison of sorafenib, quizartinib, and crenolanib, only crenolanib had a significant cytotoxic effect. These data strongly suggest that crenolanib is an important potential new TKI for treating FLT3/D835 mutated AML. Accrual into the trial is ongoing (NCT01522469). Disclosures:Muralidhara:Arog Pharmaceuticals LLC: Employment. Ramachandran:Arog Pharmaceuticals LLC: Employment. Levis:Ambit Biosciences: Consultancy.

A Phase I Study of AC220 in Combination with Cytarabine and Etoposide in Relapsed/Refractory Childhood ALL and AML: A Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Study

AbstractAbstract 3605 Background:AC220 is a novel class III receptor tyrosine kinase (RTK) inhibitor that is potent and highly selective for mutant and wild type (WT) FLT3 and other class III RTK’s including KIT, CSF1R, RET and PDGFR. In childhood acute myeloid leukemia (AML), ∼18% of children have FLT3 internal tandem duplication mutations (FLT3-ITD), and ∼10% high WT FLT3 expression. FLT3-ITD is associated with poor prognosis. In childhood acute lymphoblastic leukemia (ALL), the highest levels of FLT3 mRNA expression occur in cases of infants (80%) and childhood ALL with MLL rearrangements (MLL-r) (5%), both conferring poor prognosis.1,2 Study Design:TACL 2009–004is a first-in-children study using AC220 in combination with cytarabine and etoposide. Children > 1 month and < 21 years of age with relapsed/refractory AML or MLL-rearranged ALL are eligible. A standard 3+3 dose escalation design is utilized. The three doses tested (25, 40 and 60 mg/m2/day) are significantly lower than those tested in adults. Dose escalation past 60 mg/m2 occurs only if adequate biologic activity as determined by a plasma inhibitory assay (PIA) is not achieved. Intravenous (IV) cytarabine (1 gm/m2/dose every 12 hours) and IV etoposide (150 mg/m2/dose daily) are given over 5 days. AC220 is administered once daily as an oral solution on days 7–28. Patients can receive up to 2 courses of therapy. PIA testing is performed at trough time points weekly during exposure to AC220 to determine biologic activity. Results:To date, 13 patients (pts) were enrolled and 12 are evaluable for toxicity and response. One pt died from infectious complications (not drug-related) after a single dose of AC220 and was replaced. Median age at study entry was 10.2 years (range 11 mo – 20 yrs), average number of prior regimens was 2.8 (range 1–5), and 5 pts had prior stem cell transplant. Nine pts had relapsed AML, 2 had relapsed MLL-r ALL, and 1 had secondary AML. Of patients with AML, 4 had FLT3-ITD mutations and one had a D835 mutation. Toxicities were consistent with intensive relapsed leukemia regimens. Across all dose levels, non-hematologic toxicities ≥ grade 3 attributed to AC220 included vomiting (n=1), elevated transaminases (n=1), anorexia (n=2), and infection (n=3). One pt experienced a dose-limiting toxicity (DLT) on dose level 2 (40 mg/m2/day) of recurrent grade 3 elevated lipase. Dose level 2 was expanded to 6 pts without additional DLTs. Of 3 pts treated at 60 mg/m2/day, there have been no DLTs. Near total (>99%) inhibition of FLT3 phosphorylation by PIA is seen in every patient across all dose levels. Of 12 pts evaluable for response to date, 1 patient achieved a complete response (CR), 3 achieved complete response with incomplete neutrophil and platelet recovery (CRi), 5 had stable disease (SD), and 3 had progressive disease (PD). Responses in the 4 FLT3-ITD pts include 1 CR, 2 CRi and 1 SD. The FLT3-ITD patient with SD had reduction in marrow blasts without peripheral blood count recovery. An additional 6 pts will be enrolled at 60 mg/m2/day to complete safety evaluation and confirm biologic activity. Conclusions:AC220 plus intensive chemotherapy is well tolerated at doses up to 60 mg/m2/day with near complete inhibition of FLT3 phosphorylation in all pts tested to date. Response rates to date in pre-treated children with relapsed FLT3-ITD AML are encouraging. Disclosures:Off Label Use: AC220 in relapsed/refractory pediatric acute leukemia. Gammon:Ambit Biosciences: Employment.

AC220, a Potent and Specific FLT3 Inhibitor, Enhances the Activity of Combined Cytarabine and Daunorubicin Chemotherapy in a FLT3-ITD Model of AML

Abstract 1538Acute 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.

Detection of Phosphorylated and Total Flt3 and STAT5 in Whole Blood: Modulation by AC220 From Phase I and II Trials in AML

Abstract 2607Fms-like tyrosine kinase 3 (Flt3) plays a critical role in the leukemogenesis of a significant percentage of acute myeloid leukemias (AML) and acute lymphoblastic leukemias making it an attractive target for therapeutic intervention with small molecule inhibitors. To evaluate the pharmacodynamic (PD) effects of AC220, a highly selective and potent Flt3 inhibitor, in phase I and II clinical trials, we developed Flt3 immunoassays using the Meso Scale Discovery (MSD) 96-well platform. Several antibodies were screened to identify capture and detection pairs for both total (t) and phospho (p) Flt3 that were optimized for selectivity, sensitivity, and reproducibility. To validate the assays, cell lysates from SEM-K2 cells that express constitutively phosphorylated Flt3, the MSD assays reproducibly detected tFlt3 with high signal to background ratios, and consistently tracked changes in Flt3 phosphorylation when cells were subjected to Flt3 inhibitors. The functionality of these assays was minimally affected in the context of a whole blood matrix, as p/tFlt3 levels were similarly detected when SEM-K2 cells were spiked into human blood before lysis.To determine the PD effects of AC220 in humans, we tested whole blood samples from a phase I trial in predominantly relapsed or refractory AML patients (pts). AC220 was dosed once daily with escalations from 12 to 450 mg/day. Blood samples were initially examined in 6 pts taken pre-drug administration, at 2, 9, and 24 hrs, and 8 and 15 days post-administration. AC220 markedly reduced pFlt3 levels in the blood lysates at the 2 hr time point and this reduction was maintained over the 5 subsequent time points. Interestingly, the tFlt3 levels increased in all 6 pts at 2 and 9 hrs after treatment, began decreasing by 24 hrs (albeit still elevated from pre-dose levels), and was severely reduced by day 8 and day 15, consistent with elimination of Flt3-expressing peripheral blasts. Expanding the analysis to 45 patients from this phase I study, statistically significant reductions were observed in pFlt3 levels at 2 hrs, 24 hrs, and 15 days after dosing. Furthermore, pts receiving >90 mg demonstrated significantly greater reductions in pFlt3 compared to those receiving <90 mg. There was also significantly higher inhibition of pFlt3 in pts with Flt3 harboring the internal tandem duplication (ITD) activating mutation compared to those lacking this mutation. Importantly, absolute levels, as well as reductions in tFlt3 from day 1 to day 15 showed a statistically significant (p<0.001) rank correlation with the absolute levels and reductions in peripheral blast counts over the same time period. These results demonstrate that the Flt3 levels detected by the MSD assay effectively parallels the peripheral disease burden in AML pts.More recently, the PD effects of AC220 were assessed for pts from the exploratory part of an ongoing phase II trial in relapsed/refractory Flt3 ITD+ AML pts. Blood was taken at day 1 just before the first dose, and 24 hrs and 8 days following the initial dose. Phosphorylated STAT5, a marker closely associated with the Flt3 ITD mutation, was also monitored using a commercially available MSD assay. Blood samples from 52 pts were tested in both MSD assays. For Flt3, 13 pts were excluded from the analysis due to signals below the level of quantitation. Of the 39 pts remaining, a median pFlt3 inhibition of 75% (mean 70%) was observed at 24 hrs following the first dose, consistent with a direct effect of AC220 on its intended target. Similar to what was observed in the phase I trial, 52% of the pts had an increase in tFlt3 levels at 24 hrs. Importantly, 47% of pts had >90% reduction in tFlt3 levels after one week on AC220, signifying a major inhibitory effect on peripheral blast counts. Consistent with the strong linkage between Flt3 ITD autoactivation and STAT5 phosphorylation, there was a significant (p<0.006) correlation between changes in pFlt3 and pSTAT5 levels 24 hrs after dosing. These newly developed assays allow for the rapid assessment of Flt3 autophosphorylation and correlation with downstream signaling and peripheral disease burden. Disclosures:Nepomuceno:Ambit Biosciences: Employment, Equity Ownership. Gunawardane:Ambit Biosciences: Employment, Equity Ownership. Rooks:Ambit Biosciences: Employment, Equity Ownership. Armstrong:Ambit Biosciences: Employment, Equity Ownership.

Validation of FLT3-ITD As a Therapeutic Target in Human Acute Myeloid Leukemia

Abstract 937 Background. Activating in-tandem duplication (ITD) mutations within the FLT3 juxtamembrane region are detected in ∼25% of adult acute myeloid leukemia (AML) cases and portend a poor prognosis. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity, as evidenced by a composite complete remission rate of 43% in 53 chemotherapy refractory/relapsed AML patients evaluable for an interim analysis of the ongoing phase II study in FLT3-ITD+ relapsed/refractory AML (Cortes et al, EHA 2011 abstract 1019). Many patients who initially respond ultimately suffer disease progression. We sought to utilize the clinical efficacy of AC220 to determine if FLT3-ITD is a valid therapeutic target in human AML. A saturation mutagenesis strategy identified amino acid substitutions at three amino acid residues (F691, D835, Y842) in FLT3-ITD that confer a high degree of resistance to AC220 in vitro. Of these, substitutions at F691 and D835 conferred the greatest degree of relative resistance. Results. To assess the validity of FLT3-ITD as a therapeutic target in AML, we analyzed paired pretreatment and relapse samples obtained from a cohort of 9 FLT3-ITD-positive AML patients enrolled in the exploratory part of the ongoing phase II study of AC220 in relapsed/refractory AML (clinicaltrials.gov NCT00989261) who relapsed after initially achieving morphologic clearance of bone marrow blasts to <5% on treatment. Through RT-PCR subcloning and sequencing of the FLT3 kinase domain, we observed evolution of one or more acquired kinase domain mutations on the FLT3-ITD containing allele in all 9 cases at relapse. Clinically detected mutations were restricted to the “gatekeeper” residue F691 and/or the activation loop residue D835 (F691L, n=3, D835Y, n=4; D835V, n=2, D835F, n=1). Each of these mutations successfully transformed Ba/F3 cells to IL-3 independence and conferred substantial resistance to AC220 in cell growth and cell-based biochemical assays. All of the AC220-resistant mutations conferred substantial in vitro cross-resistance to sorafenib, which has been reported to harbor clinical activity in a small number of FLT3-ITD-associated AML cases.In an expanded analysis of genomic DNA samples from 30 patients enrolled in the exploratory part of the Phase II study who discontinued study drug for any reason, we observed the occurrence of acquired mutations in the kinase domain (D835 and F691) in a total of 10 of 30 (33%) patients at the off study timepoint. One patient had a D835Y mutation prior to going on AC220.Molecular docking studies were undertaken to provide mechanistic insights into the structural basis of resistance conferred by AC220-resistant mutations. These studies revealed that AC220 likely binds strongly to the DFG-out inactive FLT3 kinase domain. AC220 directly interacts with the gatekeeper residue F691, explaining the drug-resistance associated with the F691L mutation. Mutations at D835 and Y842 may potentially de-stabilize the inactive conformation, and result in a more active, AC220 binding-deficient FLT3 kinase. Indeed, substitutions at these residues are known to activate FLT3 in the absence of an ITD mutation.To more precisely assess the frequency and identity of resistance-conferring mutations at relapse, we analyzed a subset of samples using single molecule real-time (SMRT™; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the kinase domain of FLT3-ITD alleles. With this assay, more than 350 reads of >1000 nucleotides were reliably obtained. Analyses of pretreatment and relapse samples from four patients confirmed the presence of resistance-conferring FLT3-ITD kinase domain mutations at F691 or D835 at relapse in 36–60% of FLT3-ITD sequence reads. Additionally, this method detected polyclonal resistance in two of the four samples assessed. Conclusions. Our studies validate FLT3-ITD as a therapeutic target in a proportion of AML cases, and demonstrate that the clinical activity of AC220 is mediated by FLT3-ITD inhibition. AC220-resistant FLT3-ITD gatekeeper and activation loop mutations identified in clinical samples from relapsing patients represent high-value therapeutic targets for next-generation FLT3 inhibitors. Disclosures:Off Label Use: AC220 is an investigational agent and has no approved drug indication in AML. Chin:Pacific Biosciences: Employment. Hunt:Ambit Biosciences: Employment. Levis:Ambit Biosciences, Inc: Consultancy. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.

Statin Treatment Prevents FLT3 Glycosylation and Overcomes Resistance to FLT3 Tyrosine Kinase Inhibitors

Abstract 1421FLT3 is a receptor tyrosine kinase that is expressed on hematopoietic stem and progenitor cells where it functions in cell differentiation, survival and proliferation. It is also one of the most frequently mutated genes in acute myeloid leukemia (AML), and has thus become a target for modulation by the use of FLT3 tyrosine kinase inhibitors (TKI). Unfortunately, clinical resistance emanating via several different mechanisms has limited the potential benefit of FLT3 TKI. Some of these include FLT3 mutations that reduce drug binding, elevated FLT3 ligand (FL) levels that shift the dose-response curve and activation of parallel signaling pathways. These all pose major challenges to TKI effectiveness that must be overcome to improve patient outcomes. Since FLT3 is dependent upon N-linked glycosylation for its maturation and surface localization, we sought to determine whether statins might disrupt FLT3 signaling. Statins inhibit the mevalonate pathway and reduce levels of all ensuing end-products including dolichol, which transfers preassembled oligosaccharides onto nascent polypeptides. Here, we demonstrate by Western blotting that statins can indeed prevent complex FLT3 glycosylation, thus leading to loss of surface receptor expression. Immunofluorescence microscopy confirms a reduction in surface localization and a concomitant increase in intracellular FLT3/ITD accumulation. Interestingly, this aberrant localization was associated with increased STAT5 activation while inhibiting both MAP kinase and AKT phosphorylation. We have extended our previous findings that statins are cytotoxic to mutant FLT3 expressing cell lines to examine whether they are also able to overcome the resistance mechanisms discussed above. We show that the following mechanisms of resistance could be circumvented by fluvastatin. First, stimulation of BaF3 FLT3/ITD cells with exogenous FL at physiologic concentrations induces a three-fold increase in the IC50 for inhibition of phosphorylated FLT3/ITD by the FLT3 TKI lestaurtinib. Pretreatment with fluvastatin for 24 h eliminated surface FLT3/ITD: FL interactions and restored the potency of lestaurtinib. Secondly, resistance to another clinical FLT3 TKI, sorafenib, caused by the FLT3/ITD N676K or D835Y mutations increased the IC50 from 5 nM for BaF3/ITD cells to 30 nM and >50 nM, respectively. In contrast, both of these mutants remained just as sensitive to treatment with fluvastatin as were the BaF3/ITD cells (approx. IC50 of 0.2 μM). A third mechanism of resistance that can be circumvented by fluvastatin is the over expression and/or activation of alternative pathways that can compensate for inhibited FLT3. A model of this is rescue of BaF3 FLT3/ITD cell lines from FLT3 TKI-mediated cytotoxicity by stimulation with IL-3. When this cell line is stimulated with IL-3, fluvastatin is able to inhibit activation of STAT5, AKT and MAP kinase caused by the IL-3 receptor, as well as FLT3/ITD signaling, leading to cell death. Fluvastatin was also effective in other cell lines expressing constitutively activated FLT3 (Molm-14, MV4;11, HB1119 and SEM-K2 cells) and in other cell lines in which glycosylated transmembrane receptors drive proliferation (mutant c-Kit in Kasumi cells) but not in cells that rely upon intracellular kinases (BCR-ABL in K562 cells). Importantly, fluvastatin also reduced FLT3 glycosylation in and was cytotoxic to primary AML patient samples harboring FLT3/ITD mutations at therapeutically achievable concentrations (1 μM). Finally, fluvastatin reduced engraftment of BaF3 FLT3/ITD cells transplanted in syngeneic Balb/c mice and prolonged their survival. These results demonstrate that statins, a class of drugs already FDA approved, might be useful, either alone or in combination with a FLT3 TKI, in the management of FLT3 AML cases including those resistant to FLT3 TKI. Disclosures:Levis:Ambit Biosciences, Inc: Consultancy.

The Response of FLT3/ITD AML to FLT3 Inhibition: Apoptosis of Peripheral Blood Blasts and Differentiation of Bone Marrow Blasts

Abstract 943FLT3/ITD mutations are present in approximately 23% of adult AML cases. FLT3/ITD therefore presents an attractive therapeutic target for tyrosine kinase inhibitors. To date, the clinical responses to FLT3 inhibitors have been primarily limited to clearance of peripheral blood blasts, while bone marrow blasts remain largely unaffected. AC220 (quizartinib), a novel FLT3 inhibitor with 10–50 times more potency in vivo than other FLT3 inhibitors, has had a high response rate in an exploratory subset of patients in an ongoing clinical trial for relapsed and refractory FLT3/ITD AML. A high proportion of FLT3/ITD AML patients treated with AC220 displayed the typical rapid clearance of peripheral blasts seen with other FLT3 inhibitors. Strikingly, however, (as reported at last year’s ASH meeting), the bone marrow blasts underwent terminal differentiation in response to AC220 treatment over the course of 4–8 weeks, as established by the presence of the same FLT3/ITD mutation in mature circulating neutrophils isolated after treatment with AC220. This differentiation was frequently accompanied by a syndrome of fever, skin nodules resembling Sweet’s syndrome, and pulmonary nodules. To study the mechanisms associated with this phenomenon, we developed an in vitro model of FLT3 inhibitor-induced differentiation. In order to more closely reproduce the bone marrow microenvironment in vitro, we prepared human stromal cell layers derived from healthy bone marrow donors and co-cultured them with Molm14 cells (FLT3/ITD cell line), as well as with primary AML blasts from FLT3/ITD AML patients in the presence and absence of AC220. In the absence of co-culture with stroma, both the Molm14 cell line and the primary samples underwent rapid apoptosis in the presence of increasing concentrations of AC220, similar to the response of peripheral blood blasts in AC220 -treated patients. However, when grown in co-culture with stroma, Molm14 cells underwent relatively rapid (24–48 hours) morphologic differentiation, as evidenced by the development of characteristic multi-lobed nuclei, and by reduction of nitroblue tetrazolium (NBT) after stimulation with endotoxin (respiratory burst activity). Similar data were observed in Molm14 cells treated with the FLT3 inhibitor sorafenib, indicating that this differentiation is likely a class effect of FLT3 inhibitors. We have also used our model system to study differentiation in primary AML patient blasts. Pre-treatment blasts were obtained from a patient enrolled on the AC220 trial (who exhibited terminal differentiation of marrow blasts in response to AC220), and differentiation was induced in vitro by co-culturing the blasts with stroma in the presence of AC220. After five days of drug exposure during stromal co-culture, the blasts remained viable (assessed by trypan blue exclusion), but morphologically began to resemble myelocytes, and demonstrated reduction of NBT in response to endotoxin. Flow cytometry analysis of these blasts after eight days of AC220 exposure demonstrated increased side scatter, expression of CD15, and loss of CD117 and CD34, all indicative of maturation. Blasts cultured in the absence of stroma displayed decreased P-FLT3, P-STAT5, and P-ERK in response to AC220. In contrast, blasts exposed to AC220 on stroma demonstrated continued activity of P-ERK despite loss of P-FLT3 and P-STAT5. CEBPalpha protein levels were significantly higher in blasts on stroma, but decreased rapidly over the first 48 hours of culture. These findings confirm that a FLT3/ITD mutation can be sufficient to induce a differentiation block, and, in the bone marrow microenvironment, FLT3 inhibition does not induce immediate apotosis, but rather differentiation. The continued activity of P-ERK (presumably by parallel signaling pathways activated by stromal cells) may be responsible for the survival of the blasts in the setting of FLT3 inhibition, with CEBPalpha inducing a differentiation program in this context. We conclude, therefore, that the response of a FLT3/ITD AML patient to potent FLT3 inhibition consists of rapid apoptosis of blasts in the peripheral blood, and, in most cases a slower process of terminal differentiation in bone marrow blasts. Furthermore, our in vitro model will allow us to explore the precise mechanisms whereby differentiation is blocked in FLT3/ITD AML, and how this can be overcome with FLT3 inhibition and other targeted therapies. Disclosures:Borowitz:BD Biosciences: Research Funding. Levis:Ambit Biosciences, Inc: Consultancy.

Phospho-Specific Flow Cytometry of Fixed Whole Blood During AML Clinical Trials to Monitor In Vivo FLT3 Inhibition in Leukemic Blasts,

Abstract 3508Although first-generation FLT3 inhibitors may have had limited anti-leukemic effects due to suboptimal target inhibition, newer drugs such as AC220 and KW-2449 have substantially greater in vitro potency and bioavailability. Ex vivo assays such as the plasma inhibition assay (PIA) are useful to estimate free drug bioavailability, but direct confirmation of biochemical FLT3 inhibition in leukemic blasts in vivo has proven more challenging to employ systematically for drug development. Here we report the development of a fixed whole blood intracellular flow cytometry platform to measure real-time signal inhibition during a clinical trial of the second-generation FLT3 inhibitor KW-2449. Methods:Anticoagulated blood samples were aliquoted into FACS tubes within four hours of collection; a subset was exposed to signaling inhibitors (KW-2449, rapamycin × 30 min.) or activators (phorbol ester/PMA or FLT3 ligand/FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed, red cells were lysed with the permeabilizing agent triton X-100, and specimens were stored at −20C in glycerol medium. Subjects’ samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single cocktail of antibodies. Blasts were identified by CD45 and side scatter (SSC) and confirmed by multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive gates for phospho-proteins were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls. Results:Despite adequate controls, flow demonstrated limited changes in FLT3-ITD+ blasts' pSTAT5 signal following either FL stimulation or ex vivo KW-2449 treatment of these peripheral blood primary samples. This contrasted with the FLT3-ITD+ cell line Molm14, in which FLT3 inhibition reduced pSTAT5. However, the PI3K/AKT/mTOR downstream target ribosomal protein S6 (S6) was consistently observed to be constitutively phosphorylated in both Molm14 cells and peripheral blood FLT3-ITD+ AML blasts. pS6 in all FLT3-ITD+ samples markedly augmented with ex vivo FL, and decreased following ex vivo KW-2449 treatment. We therefore serially monitored S6 phosphorylation during therapy on a phase 1/2 trial of KW-2449.In this clinical trial, subjects were treated with KW-2449 every 6–8 hours, due to the drug’s relatively short half life. 10 subjects (9 FLT3-ITD+, 1 FLT3-WT) provided serial blood samples for analysis. All FLT3-ITD+ subjects had blasts identifiable by morphology and immunophenotype. Samples with as few as 500 blasts/uL were informative for pS6. In all cases, blasts showed dynamic changes in pS6 in response to ex vivo FL. As previously described using intracellular flow cytometry, pS6 in primary AML samples was heterogeneous, and, at basal state, frequently only demonstrable in a subset of blasts. We observed constitutive S6 phosphorylation in 8/9 subjects’ leukemic cells. The mean percentage of blasts with constitutive pS6 was 21% (median 7%, range 5–70%). To directly quantify FLT3 kinase inhibition in vivo, we serially monitored pS6 in blasts by flow prior to and following their initial oral KW-2449 dose. In 8/8 patients with baseline constitutive S6 phosphorylation, blood obtained two hours following the initial dose showed marked reduction in the percentage of pS6+ blasts to a mean of 3.8% (median 1.3% range 0.1 to 20%). This reflected an 83% mean reduction in the percentage of pS6+ blasts. PIA was performed in 8/9 of FLT3-ITD+ subjects and confirmed that potent FLT3-inhibitory concentrations were present 2 hours after a single dose of KW-2449 (mean reduction from baseline of 79% for pFLT3 and 88% for pSTAT5). Two subjects’ samples were followed serially by flow cytometry throughout the dosing interval. One showed sustained inhibition (consistent with concurrent PIA), while in the other, pS6 returned to baseline within 4–6 hours of the initial dose (concurrent PIA not done). Summary:We confirm that PI3K/AKT/mTOR is a major downstream pathway of FLT3 signaling in primary AML samples. We further demonstrate the feasibility of intracellular flow cytometry for S6 phosphorylation to monitor the biochemical efficacy of FLT3 inhibitors in patients. Studies are underway to correlate biochemical FLT3 inhibition by flow cytometry with clinical response/resistance to KW-2449 and other FLT3 inhibitors. Disclosures:Sato:Kyowa Hakko Kirin Co., LTD: Employment. Akinaga:Kyowa Hakko Kirin Co., LTD: Employment. Rao:Kyowa Hakko Kirin Co., LTD: Employment. Levis:Kyowa Hakko Kirin Co., LTD: Research Funding; Ambit Biosciences: Consultancy. Carroll:Kyowa Hakko Kirin Co., LTD: Research Funding.

Persistent ERK Activation in Bone Marrow Blasts May Account for the Difference in Bone Marrow Versus Peripheral Blood Response to FLT3 Inhibition in FLT3/ITD AML

Abstract 736Given the poor prognosis of AML patients with FLT3/ITD mutations, a number of small molecule FLT3 inhibitors are being investigated in clinical trials for these patients. When administered as monotherapy, these agents induce clearance of peripheral blood blasts, but usually have only minimal effects on bone marrow blasts. For example, treatment with sorafenib, while effective in clearing the peripheral blood of blasts, has resulted in only modest marrow blast reductions (Borthakur et al, Haematologica 2010). In contrast, AC220 (quizartinib), a more recently developed FLT3 inhibitor, has induced a much higher rate of marrow responses in an ongoing clinical trial (Cortes et al, 2011 EHA, abstract #1019). We wished to investigate the difference in response to FLT3 inhibition between peripheral blood and bone marrow blasts, and to better understand the basis for the difference in apparent efficacy between sorafenib and AC220. In our approach, we used a FLT3/ITD cell line (Molm14) and primary blasts from FLT3/ITD AML patients treated with sorafenib and AC220 either in suspension or in co-culture with human bone marrow stromal cells. We found that both sorafenib and AC220 could successfully inhibit FLT3, STAT5, AKT, and ERK signaling in Molm14 cells in suspension at concentrations below 100nM, with apoptosis induced in a dose-dependent fashion. The IC50 for inducing apoptosis was 0.4 nM for AC220 and 3.3 nM for sorafenib. The co-culture of Molm14 with bone marrow stroma did not affect the ability of AC220 and sorafenib to inhibit FLT3 and STAT5. However, downstream signaling of AKT and ERK was augmented by co-culture with stroma, and, in comparison with suspension cultures, 3-fold higher concentrations of sorafenib and AC220 were required to inhibit them. The addition of exogenous FL (2.5 ng/mL) to Molm14 cells co-cultured with stroma further enhanced the phosphorylation of FLT3, Akt and ERK, but not of STAT5. Cell survival (as determined by an apoptosis assay) correlated tightly with Akt and ERK phosphorylation. By comparison, treatment with another cytokine, IL-3, had no effect on the response to FLT3 inhibitors on or off stroma. Thus, in the Molm14 model, the resistance to FLT3 inhibitors conferred by both FL and bone marrow stroma could be overcome by increasing the dosage of drugs. In Molm14 cells co-cultured with stroma and with FL, the IC50 for inhibition of ERK was 20nM and 100nM for AC220 and sorafenib, respectively. In primary FLT3/ITD AML blasts, STAT5 activation did not appear to be affected by FLT3 inhibition, either on or off stroma. Strikingly, ERK was strongly activated in these blasts when they were co-cultured with stroma, and this activation was unaffected by FL or by FLT3 inhibition. As reported previously, FLT3/ITD AML blasts cultured in suspension underwent apoptosis in response to FLT3 inhibition. However, when cultured for several days on stroma, FLT3 inhibition did not induce apoptosis. We conclude that bone marrow stroma provides survival signals to FLT3/ITD AML blasts that allow them to survive in the presence of even very potent FLT3 inhibition. We have previously presented data that FLT3 inhibition by AC220 induces terminal differentiation of bone marrow blasts (rather than apoptosis) in vivo (Levis et al, 2010 ASH, abstract #660). Our data suggests that FLT3 inhibition needs to be extremely potent in order to induce a meaningful affect on leukemia cells in the bone marrow, explaining the difference in responses between sorafenib and the more potent inhibitor AC220. The persistent activation of ERK in blasts on stroma irrespective of the activation of the FLT3 signaling pathway suggests that this represents an important survival signal conferred by the bone marrow stroma that may account for the difference in response to FLT3 inhibition observed between peripheral blood and bone marrow blasts. Disclosures:Levis:Ambit Biosciences, Inc: Consultancy.

Terminal Differentiation of FLT3/ITD AML Blasts In Patients Treated with the FLT3 Inhibitor AC220

AbstractAbstract 660AML is characterized by abnormal proliferation of myeloid cells that have a block in differentiation. FLT3/ITD mutations are relatively common in AML, and previous in vitro studies have demonstrated that signaling from ITD-mutated FLT3 blocks myeloid differentiation through repression of CEBP/a. As part of an ongoing phase 2 trial, we treated 6 patients with FLT3/ITD AML who were refractory to either primary induction therapy or salvage therapy after relapse with the highly potent and selective FLT3 inhibitor AC220. At the start of therapy, all 6 patients had circulating blasts (mean 9864 blasts/mm3; median 2970) and the median blast percentage in the bone marrow was 71.5%. Western blotting revealed a high level of sustained in vivo FLT3 inhibition in all patients. By Day 8, no patient had detectable blasts in the peripheral blood. After 14 days of treatment with AC220, all 6 patients displayed striking differentiation to the myelocyte stage within the bone marrow. By light microscopic evaluation of bone marrow aspirates, myelocytes (promyelocytes, myelocytes, and metamyelocytes) increased from a median of 10.5% pre-treatment to 52% after 2 weeks. Most patients were neutropenic on Day 1 of treatment (mean 574, median 560 neutrophils/mm3), but rose to a mean of 3275 neutrophils/mm3 after 4–8 weeks of treatment (median time to peak 34 days). By Day 28 of treatment, marrows were most often still hypercellular, but consisted primarily of fully differentiated neutrophils. Marrow blasts were markedly reduced or absent by Day 28 in all 6 cases (mean 2.3%, median 1.5%). In all 6 patients the FLT3/ITD mutation originally detected at the beginning of treatment was present in the marrow and peripheral blood despite the absence of circulating blasts after the first week of therapy. The FLT3 mutant allelic ratio did not change between pre-therapy and Day 28. Neutrophils were isolated to homogeneity (confirmed by cytospin) from peripheral blood by double ficoll density centrifugation. Using genomic DNA obtained from these purified neutrophils, we confirmed by PCR that the FLT3/ITD mutation was present, at a similar ratio as compared with the pre-treatment blasts. However, there was no detectable expression of FLT3 either by RNA (quantitative PCR) or protein (western blotting and flow cytometry) in these neutrophils. The isolated neutrophils morphologically resemble normal neutrophils by light microscopy, and by flow cytometry they express the differentiation antigen CD15 and CD11b, and have lost expression of immature markers such as cKIT and CD34. Stimulation of these neutrophils by endotoxin results in normal respiratory burst activity, as measured by reduction of nitroblue tetrazolium. They also express lactoferrin and MMP-9, proteins typically expressed in mature neutrophils. Clinically, lung nodules and fever occurred in 3 of the 6 patients within 14 days of the peak neutrophil count. They were not treated with steroids, but rather with antibiotics, and in all cases resolved. Other patients on this trial have developed Sweet's syndrome during the neutrophil surge. CEBPa transcript levels in Molm14 cells (an AML cell line with a FLT3/ITD mutation) rose 3–5-fold over baseline following treatment with AC220. This is consistent with our previously published findings, and suggests at least one mechanism for the observed release of the differentiation block observed in the AC220-treated patients. These clinical and correlative laboratory results suggest that effective, sustained in vivo FLT3 inhibition in AML patients with FLT3/ITD mutations induces terminal differentiation in blasts in many ways similar to that seen with all trans retinoic acid in acute promyelocytic leukemia. Furthermore, these findings demonstrate the direct link between the growth factor receptor pathway and control of differentiation, and provide new insight into mechanisms of leukemogenesis. Disclosures:Levis:Ambit Biosciences, Inc: Consultancy.

Patients with FLT3/ITD AML May Benefit From Allogeneic Transplant In First Remission: Outcomes From a Consecutive Series of Patients at a Single Institution

AbstractAbstract 2172AML patients with FLT3/ITD mutations have an inferior survival compared to AML patients with wild-type (WT) FLT3, primarily due to an increased relapse rate. Allogeneic transplant represents a post-remission therapy that is effective at reducing the risk of relapse for many cases of poor-risk AML. Whether or not allogeneic transplant in first complete remission (CR) can improve outcomes for patients with FLT3/ITD AML remains controversial. Our institution has adopted a policy of pursuing allogeneic transplant, including the use of alternate donors, for FLT3/ITD AML patients in remission.As part of an IRB-approved study, we performed a review of the clinical data from November 1, 2004 to October 31, 2008 on all adult patients under the age of 60 presenting in consecutive fashion to the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins with newly diagnosed non-M3 AML. We followed their outcomes through August 1, 2010.During the study period, 133 previously untreated AML patients between the ages of 20 and 59 were diagnosed and received induction and consolidation therapy at our institution. Of these 133 patients, 31 (23%) harbored a FLT3/ITD mutation at diagnosis. Fourteen of the total 133 patients were deemed unfit for intensive therapy, all WT patients. Of these, 6 (5%) received investigational agents on protocol while the other 8 (6%) were treated with combinations of supportive care and cytotoxic agents off protocol. Of the remaining patients, 119/133 (89%) received timed- sequential intensive induction therapy (TST) with infusional cytarabine, an anthracycline or anthracenedione, and a third drug. The majority, 95/133 (71%), received the three drug TST regimen of cytarabine, daunorubicin, and etoposide delivered in a timed sequential fashion, while 24/133 (18%) were treated with TST containing cytarabine, mitoxantrone, and the investigational agent flavopiridol on an institutional protocol. 72/119 (61%) achieved a CR with induction therapy (55 patients in AcDVP16 group and 17 patients in FLAM group). The OS (overall survival) from the time of diagnosis for the FLT3/ITD AML patients was compared to the OS of the entire cohort (n=133) (Figure 1). The median OS for the FLT3/ITD group was 19.3 (range 0.0–69.9) months and the median OS for the WT patients was 15.5 (range 0.7 to 64.6) months (p=0.56). Of the FLT3/ITD patients, 20/31 (65%) achieved a complete remission with intensive induction therapy. Of these 20 patients in CR1, 11 (55%) underwent an allogeneic transplant in first remission (4 HLA-matched myeloablative sibling, 5 HLA-matched myeloablative unrelated donor, and 2 nonmyeloablative haplo-identical related donor) compared to only 18/102 (17%) WT patients were transplanted in first CR, a statistically different proportion compared to the FLT3/ITD patients (p<0.0001). The 9 FLT3/ITD patients who were not transplanted in CR1 had issues related to lack of donor or overall comorbidities. There was one patient who went on to transplant after first relapse. The median relapse-free survival in the FLT3/ITD non- transplant group was 8.6 (range 5.3–43.3) months while the median relapse-free survival in the FLT3/ITD transplant group was 54.1 (range 6.4–69.9) months (p=0.02) (Figure 2).Historically, OS for FLT3/ITD AML patients is significantly worse than for AML patients lacking this mutation. However, the OS for the 31 FLT3/ITD patients reported here was comparable to the 102 patients with WT FLT3 over the same 4 year time period. One difference that might have contributed to the surprising outcomes for the FLT3/ITD group is our aggressive pursuit of allogeneic BMT in CR1 within this group (60% of FLT3/ITD vs 17% with WT). Our single institution study of consecutively treated AML patients supports the hypothesis that allogeneic transplant in early CR1 improves the long term outcomes for FLT3/ITD AML. [Display omitted] [Display omitted] Disclosures:Levis: Ambit Biosciences, Inc: Consultancy.

Statin Treatment Inhibits FLT3 Signaling by Preventing Glycosylation of the Mature Receptor and Leads to Cell Death In Mutant FLT3 Expressing Cells

AbstractAbstract 779FLT3 is a tyrosine kinase receptor expressed on the surface of hematopoietic stem cells/progenitors and contributes to leukemogenesis as evidenced by its frequent mutation in acute myeloid leukemia (AML). FLT3 normally undergoes N-linked glycosylation to produce an immature 130 Kd protein in the endoplasmic reticulum followed by further processing in the Golgi to form the final 160 Kd mature glycoprotein that translocates to the surface. At the surface, FLT3 binds to FLT3 ligand (FL) resulting in phosphorylation and activation of multiple signaling cascades. FLT3 activating mutations primarily occur as internal tandem duplications (ITD) within the juxtamembrane domain or as point mutations in the kinase domain and have become the target of numerous small molecule tyrosine kinase inhibitors (TKI), several of which have entered clinical trials for AML. However, clinical trials to date have shown limited success due to a variety of reasons including the emergence of resistance mediated by alternative pathway activation, the acquisition of point mutations, expression of increased FLT3 ligand levels and poor inhibition of FLT3 kinase activity for a number of reasons. Statins are a well-defined class of drugs that have been safely used for treatment of hypercholesterolemia. Here, we show for the first time that statins can inhibit constitutive activation of both kinase domain and ITD mutants. Western blotting demonstrates that fluvastatin inhibits FLT3 phosphorylation by preventing receptor glycosylation, initially leading to loss of the mature receptor followed by instability of the receptor and subsequent degradation of FLT3 protein and ultimately, cell death. MTT analysis indicates that BaF3 cells transfected with FLT3/ITD are killed in a dose-dependent fashion by fluvastatin with an IC50 of <1 uM, a level which has been achieved in clinical trials. Annexin V binding confirms that BaF3/ITD cells undergo apoptosis when treated with Fluvastatin. Fluvastatin treatment causes a reduction in surface FLT3 expression as measured by flow cytometry that parallels induction of apoptosis. This finding is consistent with results obtained by immunofluoresence microsopy showing increased intracellular retention of FLT3/ITD. Additionally, fluvastatin eliminates the 2–3 fold shift in the dose-response to TKI seen when FLT3/ITD expressing cells are stimulated by FL. Also, a number of mutations that confer high level resistance to TKI are overcome by fluvastatin. These results demonstrate that statins, a class of drugs already clinically approved, might be useful in the management of AML cases in which FLT3 is over expressed, mutated or resistant to other clinically available inhibitors, either alone or more likely in combination with FLT3 TKI. Disclosures:Levis:Ambit Biosciences, Inc: Consultancy.

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AC220, a Potent, Selective, Second Generation FLT3 Receptor Tyrosine Kinase (RTK) Inhibitor, in a First-in-Human (FIH) Phase 1 AML Study.

Abstract 636Activating mutations in the FLT3 RTK are present in ∼30% of AML patients (pts), who have a significantly worse prognosis than pts with wild type (WT) FLT3. AC220 is a novel 2nd generation RTK inhibitor with potent in vitro and in vivo activity in FLT3- and KIT-dependent tumor cell lines. It is highly selective for both WT and mutant FLT3 with significant activity against KIT. A first-in-human Phase 1 study investigating AC220 in predominantly relapsed or refractory AML pts, unselected for FLT3 mutations, was recently completed, using a standard 3+3 dose escalation with 50% dose increments. AC220 was administered once daily as an oral solution initially with an intermittent dosing (ID) regimen: 14 days on and 14 days off (1 cycle). Starting dose was escalated from 12 to 450 mg/day ID. Additional cohorts were investigated on a continuous dosing (CD) regimen: 200 and 300 mg/day for 28 days (1 cycle). A total of 76 pts (46 male, 30 female) were dosed with AC220. Median age was 60 yrs (23-86), median number of prior therapies was 4 (0-9), 12 pts had prior allogeneic transplant and 3 elderly pts (≥72 yrs) unfit for induction chemotherapy were previously untreated. Eighteen pts (24%) had FLT3 ITD mutations, 47 (62%) were WT, and 11 (14%) were undetermined. Pts were also evaluated for PK, phosphorylated (p) FLT3, pKIT, pSTAT5, and ex vivo plasma inhibitory activity. AC220 plasma exposure was sustained between dose intervals and continued to increase in a dose-proportional manner from 12 to 450 mg with a half-life of ∼1.5 days. An active metabolite, AC886, was detected that has similar potency and activity to AC220. Patient plasma at '12 mg potently inhibited pFLT3 in ex vivo FLT3-ITD cell lines and complete inhibition of pFLT3 in WT cell lines was observed at higher doses. Target inhibition was also observed, with suppression of pFLT3, pSTAT5 and pKIT in peripheral blasts. The most commonly reported possibly drug-related AEs were GI events, peripheral edema, and dysguesia, which were Grade ≤2. DLT was observed at 300 mg CD and 200 mg CD was declared as the MTD. Two pts at 300 mg CD had possibly study drug-related DLTs with grade 3 QTc prolongation, but had confounding factors including concomitant medications known to prolong QTc. Responses (IWG criteria) were observed in 23 (30%) pts. PR and CR were observed as low as the 18 and 40 mg cohorts, respectively. Most responses occurred within cycle 1. Overall, 9 (12%) pts had a complete response (CR) with 2 CR, 5 CRi, and 2 CRp. One of these pts also had complete resolution of leukemia cutis. In addition, 14 (18%) pts achieved PR. Overall median duration of response (MDOR) was 14 (4-62+) weeks and overall median survival (MS) was 14 (1-68+) weeks. In FLT3-ITD pts the MDOR was 12 (4-27+) weeks and the MS was 18 (3-42) weeks. In FLT3-WT pts the MDOR was 32 (8-62+) weeks and the MS was 11 (1-68+) weeks. 10 (56%) of 18 FLT3-ITD pts were responders (1 CR, 3 CRi, 6 PR), 9 (19%) of 47 FLT3-WT pts (1 CRi, 2 CRp, 6 PR), and 4 (36%) of 11 undetermined pts (1 CR, 1 CRi, 2 PR). At 200 mg CD (MTD expansion), 4 of 6 FLT3-ITD pts responded (1 CR, 1 CRp, 1 CRi, 1 PR). Of the 4 responders, 2 failed prior treatment with sorafenib and 2 previously refractory pts went onto transplant. The 2 FLT3-ITD non-responders had 6 and 8 prior lines of therapy, respectively. These encouraging efficacy results and an acceptable safety profile warrant continued evaluation of AC220 as monotherapy and in combination therapy for the treatment of AML. Phase 2 studies in FLT3-ITD positive and WT pts are in progress. Disclosures:Cortes:Ambit Biosciences: Research Funding. Foran:Ambit Biosciences: Research Funding. Ghirdaladze:Ambit Biosciences: Research Funding. DeVetten:Ambit Biosciences: Research Funding. Zodelava:Ambit Biosciences: Research Funding. Holman:Ambit Biosciences: Research Funding. Levis:Ambit Biosciences: Consultancy, Research Funding. James:Ambit Biosciences: Employment. Zarringkar:Ambit Biosciences: Employment. Gunawardane:Ambit Biosciences: Employment. Armstrong:Ambit Biosciences: Employment. Padre:Ambit Biosciences: Employment. Wierenga:Ambit Biosciences: Employment. Corringham:Ambit Biosciences: Employment. Trikha:Ambit Biosciences: Employment.

AC220, a FLT3 Inhibitor, Increases Survival in Two Genotypically Distinct FLT3-ITD Models of Acute Myeloid Leukemia and Provides Sustained Protection Following Chronic Administration.

Abstract 2053Poster Board II-30Activating 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.

AC220, a Potent and Specific FLT3 Inhibitor, Enhances the Cytotoxic Effects of Chemotherapeutic Agents in Cell Culture and in Mouse Tumor Xenografts.

Abstract 2052Poster Board II-29AC220, 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.

Comparison of Steady-State Imatinib (IM) Trough Levels, Clinical Response, and Safety Between Caucasian and Asian Patients with Chronic Lyeloid Leukemia in Chronic Phase (CML-CP) Treated with 400mg and 800mg Daily Doses of IM in the Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) Study.

Abstract Abstract 1127 Poster Board I-149 Background In the TOPS study, IM trough levels (Cmin) were collected from different race groups, mainly Caucasian and Asian, but also Black and others. Inter-ethnic differences in the PK of a drug are known to be important factors accounting for inter-individual variation in drug responsiveness. This analysis reports the comparison between Caucasian and Asian CML patients (pts) treated at doses of 400 mg QD and 400 mg bid (800 mg daily) of IM Cmin on Day 29 of initial treatment, clinical response, safety and tolerability. Methods Steady state IM Cmin on Day 29 and clinical response and safety data obtained during the first 12 months (mos) of treatment were obtained from pts randomized 2:1 to 800 mg or 400 mg daily IM. The steady-state Cmin was defined as predose concentration collected approximately within ±3 hours of the scheduled dosing time on Day 29. The associations of race with the rates of major molecular response (MMR) and complete cytogenetic response (CCyR) were evaluated. Correlation of IM exposure with clinical response (MMR and CCyR) was assessed by grouping pts into quartiles based on their measured IM Cmin levels at Day 29. The safety endpoint for each pt was the presence or absence of an adverse event (AE) of grade 3 or higher in the first 12 mos from the first dose. Results IM Cmin levels were available from 229 pts in TOPS including 54 Caucasians, 18 Asians, and 14 Black and others at 400 mg (total 86) and 103 Caucasians, 29 Asians, and 11 Black and others at 800 mg (total 143). For the first 12 mos, the means of the average dose intensities for Asian (mean [range], 362 [267-400] in 400 mg and 666 [226-800] in 800 mg) were not significantly different from Caucasian (386 [204-597] in 400 mg and 666 [289-800] in 800 mg) (P=0.070 and P=0.995 for the 400 mg and 800 mg arms, respectively). Mean (± SD) of IM Cmin levels (ng/mL) with respect to race are shown in Table 1. IM Cmin was slightly over-proportional to dose and showed large interpatient variability (CV=42-60%) for both dose groups regardless of the race group. In the lower quartile Cmin group (Cmin&lt;1290 ng/mL), the differences in CCyR and MMR rates between Asian and Caucasian pts were significant (P=0.031 and P=0.022 respectively), which was probably due to a higher rate of dose interruptions in the 1st 12 mos in Asian pts. A definitive conclusion cannot be drawn due to the small number of Asian pts. Occurrences of at least one grade 3 or 4 adverse event were found to be significantly higher in Asian pts (69% and 75% in the 1st 6 and 12 mos respectively) compared to Caucasian pts (53% and 57% in the 1st 6 and 12 mos respectively) (P=0.028 and P=0.008 respectively). Conclusion The results of this analysis from TOPS show that IM Cmin levels were similar between Caucasian and Asian CML pts in each treatment arm. There were no major differences in efficacy, as measured by MMR and CCyR rates by 12 mos, between Asian and Caucasian pts. There were no unexpected differences in patterns of AEs between Caucasian and Asian pts; however, occurrences of one or more grade 3 AEs were higher in Asian. Further analysis on a larger group of CML pts will be needed to evaluate the impact of AE rate differences between Caucasian and Asian pts. Disclosures Kim: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Wyeth: Research Funding. Granvil:Novartis: Employment. Demirhan:Novartis: Employment. Reynolds:Novartis: Employment. Jin:Novartis: Employment. Wang:Novartis: Employment, Equity Ownership. Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau. Cortes-Franco:Novartis: Honoraria, Research Funding, Speakers Bureau; Wyeth: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents &amp; Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents &amp; Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding. Guilhot:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria.

Steady-State Imatinib Trough Levels as Well as Dose Interruptions Are Associated with Clinical Response (CCyR and MMR) and Adverse Events (AEs) in Patients with Chronic Myeloid Leukemia (CML) Receiving IM as Frontline Therapy.

Abstract 2213Poster Board II-190 Background:Correlations between IM trough plasma levels (Cmin) and clinical response have been previously reported [Picard et al. Blood 2007; Larson et al. (IRIS) Blood 2008; Guilhot et al. (TOPS) ASH 2008]. This analysis correlates IM Cmin on Day 29 of initial treatment with complete cytogenetic response (CCyR) and major molecular responses (MMR) at 12 months and with cumulative Grade 3&4 toxicity over 12 months based on data pooled from 2 studies, IRIS (400 mg qd) and TOPS (400 mg bid (800 mg/daily) vs 400 mg qd), in newly diagnosed, previously untreated, Ph+ CML-CP. Methods:Steady-state Cmin was defined as predose blood level collected within ±3 hours of the scheduled dosing time on Day 29 without any dose interruptions within 5 days prior to PK sampling. The correlation between IM Cmin and CCyR and MMR at 12 months was studied by two approaches: 1) analysis of outcomes by quartile groups based on patients' IM Cmin levels; 2) logistic regression analysis with Cmin as a continuous variable plus Sokal risk scores and cumulative days with any dose interruptions during the initial 12 months. Safety parameters included Grade 3&4 AEs, as well as all frequently-occurring (>10%) AEs of any grade that occurred during the 12 months. Patients with missing covariates were excluded. Results:Steady-state IM Cmin trough levels were available in 526 patients: 319 in IRIS and 207 from TOPS. At the time of assessment most patients received either 400 mg or 800 mg; 8 patients received reduced doses (6 at 300 mg; 2 at 600 mg). The median IM Cmin [25-75% quartiles] for 400 mg in the pooled dataset was 943 ng/mL [688-1280 ng/mL], and that for 800 mg was 2910 ng/mL [2333-3900 ng/mL]. IM Cmin showed large inter-patient variability for both 400 mg and 800 mg dose groups (52.7% and 39.9%, respectively). Both CCyR and MMR rates at 12 months were significantly correlated with IM Cmin on Day 29. Besides Cmin on Day 29, Sokal risk scores and cumulative dose interruptions (due either to treatment-related toxicities or non-adherence) were significant covariates for 12 month CCyR and MMR. Patients with high Sokal scores (H) had lower CCyR and MMR rates than those with low Sokal scores (L), 64% (H), 69% (intermediate (I)), and 83% (L), respectively, for CCyR, and 37%, 48%, and 59%, respectively, for MMR. Response rates at 12 months were significantly lower for patients with cumulative dose interruptions > 28 days (in the first 12 months): 45% vs 76% for CCyR, and 27% vs 48% for MMR. Modeling predicts that at a Cmin level of 1000 ng/mL and assuming no or minimal dose interruptions, the CCyR at 12 months would be 85%, 78%, and 68% for L, I, and H Sokal risk patients, respectively, and for MMR 55%, 45% and 36%, respectively. If the Cmin were 2000 ng/mL, the CCyR at 12 months would be 93%, 89%, and 83% for L, I, and H Sokal risk patients, respectively, and for MMR 65%, 55% and 44%, respectively. The predicted CCyR and MMR would be lower if there were dose interruptions. Patients who had Grade 3&4 AEs over first 12 months period (n=136) had a higher IM Cmin on Day 29 (median [25-75% quartiles], 1985 [982-2943] ng/mL vs 1010 [728-1468] ng/mL, P<0.001), than those without (n=390) as well as longer cumulative dose interruptions (20 [8-41] days vs 0 [0-2] days, P<0.001), lower CCyR rate (66%; 77/117 vs 75%; 277/369, P=0.05), and lower MMR rate (37%; 49/131 vs 48%; 155/323, P=0.006). Most Grade 3&4 AEs were treatment-related hematologic AEs with median times to onset between 50-63 days. Regression analysis showed the correlation between hematologic Grade 3&4 AEs and IM Cmin level for the population (Figure). Among all-grade non-hematologic AEs, rash and vomiting were associated with higher IM Cmin levels. [Display omitted] Conclusion:IRIS+TOPS pooled data confirmed earlier findings that higher steady-state IM levels correlate with better CCyR and MMR responses but also with more Grade 3&4 treatment-related toxicities. Dose interruptions compromise CCyR and MMR rates at 12 months. IM Cmin levels provide additional information together with clinical response and tolerability to inform dose changes for individual patients. Disclosures:Larson:Novartis: Consultancy, Honoraria, Research Funding. Chia:Novartis: Employment. Granvil:Novartis: Employment. Guilhot:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria. Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. O'Brien:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Wyeth: Research Funding. Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding. Nedelman:Novartis: Employment, Equity Ownership. Wang:Novartis: Employment, Equity Ownership.

The Oncogenic Role of Tumor Suppressor Protein p27 in Ph+ Chronic Myeloid Leukemia.

Abstract 3276Poster Board III-1 Background:Several studies have indicated that BCR-ABL causes cell cycle defects by interfering with the cell cycle regulatory functions of p27, a Cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. Studies in BCR-ABL positive cell lines have shown that BCR-ABL promotes proteasomal degradation of p27 in a pathway that involves the SCFSKP2 ubiquitin ligase, while cytoplasmic mislocalization has been described in primary CML cells. It has been suggested that the principal effect of this cytoplasmic mislocalization is to remove p27 from the nucleus, thereby relieving Cdks from p27 inhibition. However, recent studies have shown that a p27 mutant (p27CK-), that cannot bind to Cdks or Cyclins, actively contributes to oncogenesis. This raises the question as to whether cytoplasmic mislocalization of p27 in CML cells may in fact promote leukemogenesis rather than merely compromise Cdk inhibition. We therefore hypothesized that the net contribution of p27 in CML is to promote leukemogenesis due to the oncogenic activity of cytoplasmic p27. Experimental approach and results:We determined p27 localization in BCR-ABL positive cell lines and CD34+ progenitor cells from newly diagnosed chronic phase CML patients (N=7) and from CML patients in blast crisis (N=2) by immunoblotting of nuclear and cytoplasmic cellular fractions. We found that p27 is predominantly cytoplasmic in most CML cell lines and in CD34+ cells from 8/9 (89%) patient samples, including patients in blastic phase. Cytoplasmic localization of p27 in CD34+ cells from CML patients was also confirmed by immunofluorescence analysis. Further, we observed that inhibition of BCR-ABL kinase by imatinib, an Abl kinase inhibitor increased nuclear p27 in all cell lines tested and in 4/9 patient samples (3/7 chronic phase and 1/2 blastic phase samples). However, we did not observe a substantial change in the cytoplasmic p27 levels. Similar results were obtained in Ba/F3 and 32D murine hematopoietic cell lines expressing BCR-ABL when compared with the respective parental cells. Further, SKP2 was up-regulated in CD34+ cell from CML patients as compared to the normal patients consistent withSKP2 mediated down-regulation of nuclear p27. These data suggest that nuclear but not cytoplasmic p27 levels are predominantly regulated by BCR-ABL kinase activity. To test whether p27 is crucial for BCR-ABL-driven leukemia, we compared leukemogenesis between recipients of BCR-ABL transduced p27+/+ and p27-/- bone marrow. Mice transplanted with BCR-ABL infected p27-/- marrow had significantly longer median survival (70 days, range 48-150 days) compared to recipients of p27+/+ marrow (37 days, range 14-56 days) (p=0.0123). To exclude that this difference was related to the differences in homing and engraftment capabilities of p27+/+ and p27-/- bone marrow cells, we compared short term homing and long term engraftment of p27+/+ and p27-/- bone marrow cells transplanted into wild-type recipients and found no differences. These data suggest that the net contribution of p27 to BCR-ABL-mediated leukemogenesis is positive. Further, to investigate the contribution of nuclear p27 to leukemogenesis, we utilized marrow from p27S10A mice in the murine CML model. In p27S10A mice, p27 is nuclear to to abrogation of the phosphorylation site implicated in nuclear export. We injected BCR-ABL transduced bone marrow cells of p27S10A and p27+/+ mice into wild-type recipients and compared the disease progression. We observed that mice transplanted with BCR-ABL infected p27S10A marrow had significantly longer median survival (28 days, range 23-79 days) compared to the recipients of p27+/+ marrow (23 days, range 21-38 days) (p=0.0139). This data is consistent with nuclear tumor suppressor function of p27. Combined with the data above, this suggests that cytoplasmic p27 promotes BCR-ABL mediated leukemogenesis. Conclusions:Our data suggest that though nuclear p27 functions as a tumor suppressor, the net contribution of p27 in CML might be oncogenic due to an oncogenic role of the increased cytoplasmic p27. Restoring nuclear p27 or reducing cytoplasmic p27 may be therapeutically useful in malignancies with low nuclear and high cytoplasmic p27 expression. Disclosures:Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Deininger:Genzyme: Research Funding; BMS: Consultancy; Novartis: Consultancy, Honoraria; Ariad : Research Funding.

Development of a Small-Molecule Inhibitor Screen to Rapidly Identify Key Signaling Pathways in Leukemogenesis.

Abstract 708Aberrantly activated tyrosine kinases and their associated signaling pathways are critical to leukemogenesis and primary acute myeloid leukemia (AML) cell viability. While aberrant kinase activation has been confirmed in a significant percentage of AML, constitutive phosphorylation of STAT5, a marker of tyrosine kinase activation, is present in the majority of AML samples indicating that as yet unidentified tyrosine kinases can be aberrantly activated and contribute to leukemogenesis. Efforts to identify activating tyrosine kinase mutations using high-throughput sequencing have identified low frequency mutations of uncertain functional significance. Because these studies failed to detect additional high-frequency kinase mutations, the identity and mechanism of tyrosine kinase activation may be unique in many AMLs. To avoid the imitations of high-throughput sequencing, we have developed a functional assay that can rapidly and simultaneously identify therapeutic targets while providing therapeutic options. Methods:To rapidly identify activated kinase pathways in individual, primary AML samples, we have developed a small-molecule inhibitor array which includes 90 small-molecule, cell-permeable inhibitor compounds including a core of 36 tyrosine kinase inhibitors that covers the majority of the tyrosine kinome. Many of the inhibitors are available for clinical use or are in clinical development. In this assay, inhibitors were placed in 96-well plates at four serial dilutions to allow IC50 calculations. Three days after adding primary AML cells to each well, we performed an MTS cell viability assay to evaluate the effect of each inhibitor on cell viability. Because most inhibitors affect multiple kinases, we compared target specificities of compounds that decrease primary AML cell viability with those that have no effect to identify potential targets. Results:In preliminary proof-of-principal experiments, we tested leukemia cell lines with known activating tyrosine kinase mutations and Ba/F3 cell lines expressing activated tyrosine kinases. Appropriate inhibitor sensitivity profiles were obtained in CMK cells which depend on a JAK3 A572V mutation for viability, MKPL-1 cells with an activating CSF1R translocation, and in a Ba/F3 line expressing JAK2 V617F. In addition to the primary target, downstream targets were frequently identified; MKPL-1 cells also showed sensitivity to phosphoinositol 3-kinase and NFKB inhibitors. Thus, not only primary targets but the downstream signaling pathways critical to leukemic cell viability can be highlighted using this assay. To date, we have analyzed approximately 150 primary leukemia and lymphoma samples. In some cases, targets could be identified by comparison of overlapping kinase specificities for compounds that decreased leukemic cell viability and subtraction of possible kinase targets inhibited by compounds that had no effect on viability. However, many cases exhibited complex, often unique, inhibitor sensitivity profiles that complicated target identification. Comparison with sensitivity profiles for known aberrantly activated kinases was useful when available. Accordingly, additional leukemia cell lines and Ba/F3 lines that depend on a single aberrantly activated tyrosine kinase for viability are being evaluated. Automated scripts that correlate the leukemic cell inhibitor sensitivity with the inhibitor target specificity are also in preparation. Conclusions:These preliminary data demonstrate that the small-molecule inhibitor functional assays can rapidly identify disease causing genes, provide insights into their mechanism of action, and suggest therapeutic options. The distinct patterns of tyrosine kinase sensitivity in these samples support the hypothesis that tyrosine kinases and related pathways contributing to leukemogenesis in each patient may be different and that targeted therapy will be most effective when administered on an individualized basis. Disclosures:Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding.