FLT3-mutated AML Research Articles

Administration of the Dual E-Selectin/CXCR4 Antagonist, GMI-1359, Results in a Unique Profile of Tumor Mobilization from the Bone Marrow and Facilitation of Chemotherapy in a Murine Model of FLT3 ITD AML

FLT3 mutated AML confers poor prognosis despite treatment with standard chemotherapy, attributable to bone marrow (BM) induced tumor cell resistance in the microenvironment via molecules such as CXCR4 and E-selectin. We recently reported that simultaneous targeting of CXCR4 and E-selectin with GMI-1359, a novel small molecule dual antagonist, mobilized leukemic cells into the circulation; further, combination with AraC and DNR resulted in prolonged survival in a FLT3-ITD AML murine xenograft model. Here we report a novel profile of leukemic cell mobilization induced by GMI-1359, its impact on survival when combined with chemotherapy, and toxicity profile supporting entry to the clinic.A comparison of tumor mobilization by GMI-1359 to E-selectin or CXCR4 antagonists was determined in NCR mice with established BM infiltrative MV4-11 FLT3 ITD human AML tumor (modified with mCherry and luciferase). Mice (n=18/group) were injected with GMI-1359 (40 mg/kg), a potent E-selectin antagonist (40 mg/kg), or plerixafor (5 mg/kg) and blood samples were assessed by flow cytometry at multiple times for human AML cells and murine WBC. Appearance of human AML cells in peripheral blood increased ~16-fold compared to untreated mice 8 h post treatment with GMI-1359, and remained elevated for the duration of the study (72 h). Tumor cell mobilization following treatment with the E-selectin antagonist was gradual, reaching a 9.7-fold increase 48 h post injection and trending down at 72 h. Administration of plerixafor induced a rapid 11-fold mobilization of tumor cells by 1 h returning to baseline at 24 h. Murine WBC count did not change in any group at the time points assessed. These data establish that the profile of GMI-1359 induced tumor cell mobilization is distinct from both plerixafor (rapid mobilization of short duration) and an E-selectin antagonist (gradual mobilization of long duration). As E-selectin but not CXCR4 has been reported to be critical for tumor cell entry into the BM, the sustained presence of tumor cells in circulation after GMI-1359 treatment could be the result of blocking not only CXCR4 but also E-selectin, thereby inhibiting their re-entry into the BM.The therapeutic impact of tumor mobilization by GMI-1359 was evaluated for therapeutic consequence in combination with AraC and DNR in MV4.11 tumor bearing mice. Mice (n=10/group) were treated with saline; GMI-1359 (40 mg/kg) alone; chemotherapy alone; or GMI-1359 and AraC/DNR. GMI-1359 was initiated at onset of chemotherapy and given for 1, 3 or 14 days. Treatment of mice with GMI-1359 alone or together with AraC/DNR was well-tolerated. Median survival times (MST) of mice treated with saline, GMI-1359 or AraC/DNR was 47, 50 and 50 days, respectively; all mice succumbed to progressive disease by study conclusion (Day 71). In contrast, the MST of tumor bearing mice treated with GMI-1359 qdx3 or qdx14 and AraC/DNR was 63.5 days (p<0.001 vs. AraC/DNR) with a 40% survival rate. The MST of mice given a single dose of GMI-1359 at the start of AraC/DNR was statistically indistinguishable from chemotherapy alone (49.5 and 50 days, respectively) even though a 20% survival advantage was noted for the GMI-1359 combination group.GMI-1359 was tested for off-target interactions against 87 potential targets, with no significant activity observed. Toxicology studies showed a safety profile consistent with that of plerixafor. The pharmacokinetic (PK) profile was characterized in mice, rats, dogs, and monkeys, and was linear with t1/2 <2h in monkey and no accumulation in plasma. GMI-1359 is not mutagenic or clastogenic. Based on microsomal and liver S9 stability testing, GMI-1359 is not expected to be highly metabolized in any species tested. GMI-1359 did not inhibit P-gp, is moderately bound to human plasma proteins, and is stable in human plasma at 37C.In summary, GMI-1359, an innovative dual E-selectin and CXCR4 antagonist, more rapidly mobilized tumor cells and maintained them in circulation for a longer time when compared to either an antagonist of E-selectin or CXCR4 alone in a murine model of FLT3 ITD AML. This novel kinetic signature of mobilization by dual inhibition of two important adhesion molecules and accompanying improvement in survival over standard chemotherapy support the potential for combination with current treatment to improve outcomes for patients with FLT3-mutated AML. First-in-human trials are being initiated. DisclosuresFogler:GlycoMimetics, Inc.: Employment. Flanner:GlycoMimetics, Inc.: Employment. Wolfgang:GlycoMimetics, Inc.: Employment. Smith:GlycoMimetics, Inc.: Employment. Thackray:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Palbociclib treatment of FLT3-mutant AML cells reveals a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

Palbociclib treatment of FLT3-mutant AML cells reveals a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

MiR-155 regulative network in FLT3 mutated acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) represents a heterogeneous disorder with recurrent chromosomal alterations and molecular abnormalities. Among AML with normal karyotype (NK-AML) FLT3 activating mutation, internal tandem duplication (FLT3-ITD), is present in about 30% of patients, conferring unfavorable outcome. Our previous data demonstrated specific up-regulation of miR-155 in FLT3-ITD+ AML. miR-155 is known to be directly implicated in normal hematopoiesis and in some pathologies such as myeloid hyperplasia and acute lymphoblastic leukemia. Methods and resultsTo investigate about the potential influence of miR-155 de-regulation in FLT3-mutated AML we generated a transcription factors regulatory network and combined this with data from multiple sources that predict miR-155 interactions. From these analyses, we derived a sub-network, called “miR-155 module” that describes functional relationship among miR-155 and transcription factors in FLT3-mutated AML. We found that “miR-155 module” is characterized by the presence of six transcription factors as central hubs: four miR-155 regulators (JUN, RUNX1, FOSb, JUNB) and two targets of miR-155 (SPI1, CEBPB) all known to be “master” genes of myelopoiesis. We found, in FLT3-mutated AML, a significant down-regulation of miR-155 target genes CEBPB and SPI1 and up-regulation of miR-155 regulator genes JUN and RUNX1. We also showed that PKC412-related FLT3 inhibition, in MV4-11 cell line, causes down-regulation of miR-155 and increased level of mRNA and protein of miR-155 target SPI1. We showed in experiments of miR-155 mimic in K562 cell line, a high increase of miR-155 and an inverse correlation with the mRNA levels of its targets SPI1 and CEBPB. Moreover silencing of miR-155 in primary AMLs causes mRNA up-regulation of its target SPI1 and CEBPB. ConclusionOur results suggest that activating mutation of FLT3 in AML can lead, through the induction of JUN, to an increased expression of miR-155, which then causes down-regulation of SPI1 and CEBPB and consequently may causes block of myeloid differentiation.

Trial Test of Sorafenib Alone or in Combination with Chemotherapy for Treating Refractory and Relapsed Acute Myeloid Leukemia with FLT3 Mutation

Introduction The prognosis was poor and the rate of refractory and relapsed was high for FLT3-mutated AML. The remission durations are shorter. Once relapsed, the disease is rapidly fatal. Some patients were often refractory to induction. While there was some success with immediate allogenic transplantation, it was shown that patients without FLT3 mutations were still prone to high rates of relapsed, posing a tremendous clinical challenge. At the moment there are no clear guidelines for treating these special patients in both the preliminary and induction phase.Method Six of refractory and relapsed AML with FLT3-ITD mutation were evaluated, with a medium age of 43 (33-61 years old) and a male/female ratio of 3/3. Four of the cases were refractory (1 case at M2, 1 case at M4, and 2 cases at M5), while 2 were relapsed (1 case at M1, 1 case at M5). Two cases had FLT3-ITD mutation only, one patient had FLT3-TKD mutation only, 2 cases was coexisted NPM1 mutation, and one patient was combined DNMT3A mutation. Two cases were treated with sorafenib 200 mg bid or 400 mg bid, five days per week, while four cases were treated with sorafenib (same dosage) with combined half-course IAG or AAG chemotherapy (7 days) during induction. Results Four cases with combined therapies resulted in CR, while the symptoms in 2 relapsed patients with sorafenib-only treatment were unable to be alleviated. After the treatment was terminated, these two patients expired as the disease progressed for 6 months. The time range of sorafenib use was between 9 - 260 days, with a medium of 34 days. The 4 cases of CR included 2 cases with only FLT3-ITD mutation, 1 case with only FLT3-TKD mutation, and 1 case of combined NPM1 mutation. Three of the four cases received hematopoietic stem cell transplantation after the symptoms were reduced. The survival periods were 62, 90, and 117 days respectively. The last case exhibited a recurrence after consolidation chemotherapy was applied in place of sorafenib. FLT3-ITD was negative during the relapsed, and the symptoms were controlled after cladribine treatment with large doses of cytarabine. The major toxicities observed during the induction phase were neutropenia, thrombocytopenia and infections. All patients experienced grade IV WHO neutropenia and thrombocytopenia. The medium days of agranulocytosis was 18 days (4 - 27 days), platelets<20*109/L, medium 17 days (0 - 24 day). All four cases exhibited post-treatment infection: 2 were pulmonary infections, and the other two were infections of the upper respiratory tracts. All the infection was successfully treated. One patient showed a side effect of diarrhea, which was promptly alleviated with berberine, and another patient exhibited signs of a skin rash. The other patients were free of side effects.Conclusion The preliminary results of our trial using sorafenib alone or combined chemotherapy for treating refractory and relapsed acute myeloid leukemia with FLT3 mutation showed positive results and was well tolerated. Therefore, this method of treatment is considered to be suitable for establishing better condition for patients to undergo allogenic hematopoietic stem cell transplantation. Whether sorafenib combined with chemotherapy can become a standard theray for newly diagnosed FLT3-mutated AML still needs further investigation, and more samples and controlled trials are required. DisclosuresNo relevant conflicts of interest to declare.

Improvement in Clinical Outcome of FLT3 Mutated AML Patients over the Last One and a Half Decade

Background: FMS-like tyrosine kinase 3 gene (FLT3) mutations occur in approximately 30% of all AML patients. The overall prognosis of FLT3 mutated AML patients (pts) remains poor. As we have learned more about the aggressive nature of this disease our treatment strategies have changed. Incorporating FLT3 inhibitors with chemotherapy and allogeneic stem cell transplant in first remission are routinely pursued in these pts. In this context we reviewed data to evaluate clinical outcome of FLT3-ITD mutated AML pts since 2000 in a single institution.Methods: We retrospectively analyzed 1441 pts referred to our institution between 2000 and 2014. FLT3 internal tandem duplications (FLT3-ITD) were found in 334 pts with AML. After excluding pts with core binding factor leukemia and acute promyelocytic leukemia, 224 pts were included in this analysis. Among these 224 pts, 21 (9%) pts also had tyrosine kinase domain D835 (TKDs) mutated. Patients are evaluated for response to therapy, treatment related mortality and overall survival.Results: Patients were divided into 5 cohorts by era: 2000-02 (Era 1, n=19), 2003-05 (Era 2, n=41), 2006-08 (Era 3, n=53), 2009-11 (Era 4, n=55), 2012-14 (Era 5, n=56). The median age from Era 1-5; 57, 61, 59, 61, and 65 yrs respectively, p= 0.55. None of the pts in Era 1 received FLT3 inhibitor (inh.) therapy, 4 (10%) in Era 2, 17 (32%) in Era 3, 34 (49%) in Era 4, and 34 (61%) in Era 5 received FLT3 inh. Among these 2 (5%) pts in Era 2, 11 (27%) in Era 3, 9 (16%) in Era 4 and 30 (54%) pts in Era 5 received FLT3 inh. with their frontline therapy. SCT in 1st remission occurred in 4 (21%) pts in Era 1, one in Era 2, 10 (19%) in Era 3, 18 (33%) in Era 4, and 14 (25%) in Era 5. Two (5%) pts in Era 2, 5 (9%) pts in Era 3, 3 (5%) pts in Era 4, and 2 (4%) pts in Era 5, had SCT in 2nd remission. The overall response rate (ORR) to induction chemotherapy in the 5 Eras were 74%, 51%, 77%, 84% and 82%, respectively. The rate of complete remission (CR) from Era 1-5 were 63%, 46%, 70%, 65%, and 57% respectively. Complete remission without platelet recovery (CRp) from Era 1-5 was 5%, 2%, 2%, 7% and 18% respectively. The ORR from Era 1-5 in pts who received FLT3 inhibitor combinations induction therapy was 85%; CR 60%, CRp 17%, HI 6% and PR 2%. Whereas ORR to non-FLT3 inhibitor induction therapy from Era 1-5 was 72%; CR 62%, CRp 6%, PR 2% and HI 2%. Among 21 (10%) pts who were FLT3-ITD and TKDs mutated, CR/CRp was achieved in 14 (67%) pts, compare to 139 (69%) pts with only FLT3-ITD mutated, p= 0.84. At 1 year (yr) from diagnosis through Era 1-5 42%, 29%, 58%, 72% and 61% were alive respectively. Four (10%) pts in Era 2, one pt each in Era 4-5 and none in Era 1 and 3 died ≤4 weeks from start of induction therapy. The median time to relapse was 10.6 months (mo) in Era 1, 6.1 mo in Era 2, 9.5 mo in Era 3, and 8.5 mo in Era 4. The median time to relapse was not reached in Era 5 with 63% of patient in CR/CRp at 1 yr, p= 0.45 (Fig.1). The median overall survival (OS) have improved over time, 9.6 mo in Era 1, 7.6 mo in Era 2, 14.4 mo in Era 3, 15.7 mo in Era 4 and 17.8 mo in Era 5, p= 0.0001 (Fig.2). The median OS in all pts who received FLT3 inhibitors in induction chemotherapy from Era 1-5 was 14 mo, compare to 13 mo in pts who had non FLT3 inhibitor induction therapy (p= 0.25). Patients who received FLT3 inhibitors any time in the course of their treatment, the OS was 14.2 mo, compare to 13 mo in pts who never had FLT3 inhibitor therapy (p= 0.941). In the subset analysis, excluding pts who received SCT in 1st CR, the median overall survival from Era 1-5 was 9.2, 6, 11, 13.4 and 17 mo respectively, p= 0.05.Conclusion: Our data suggested some improvement in outcome of FLT3- ITD mutated AML pts over the last decade and a half. This is probably due to more aggressive treatment strategies including integration of FLT3 inhibitors in chemotherapy regimens and increase use of SCT. Efforts are still needed to continue to improve outcome for these subset of poor prognostic AML patients. [Display omitted] DisclosuresCortes:Ambit: Research Funding; Astellas: Research Funding; Ariad: Research Funding; Arog: Research Funding; Novartis: Research Funding; Astellas: Consultancy; Ariad: Consultancy; Novartis: Consultancy.

Role of altered growth factor receptor-mediated JAK2 signaling in growth and maintenance of human acute myeloid leukemia stem cells

AbstractAcute myeloid leukemia (AML) is sustained by small populations of leukemia stem cells (LSCs) that can resist available treatments and represent important barriers to cure. Although previous studies have shown increased signal transducer and activator of transcription (STAT)3 and STAT5 phosphorylation in AML leukemic blasts, the role of Janus kinase (JAK) signaling in primary AML compared with normal stem cells has not been directly evaluated. We show here that JAK/STAT signaling is increased in LSCs, particularly from high-risk AML. JAK2 inhibition using small molecule inhibitors or interference RNA reduced growth of AML LSCs while sparing normal stem cells both in vitro and in vivo. Increased JAK/STAT activity was associated with increased expression and altered signaling through growth factor receptors in AML LSCs, including receptor tyrosine kinase c-KIT and FMS-related tyrosine kinase 3 (FLT3). Inhibition of c-KIT and FLT3 expression significantly inhibited JAK/STAT signaling in AML LSCs, and JAK inhibitors effectively inhibited FLT3-mutated AML LSCs. Our results indicate that JAK/STAT signaling represents an important signaling mechanism supporting AML LSC growth and survival. These studies support continued evaluation of strategies for JAK/STAT inhibition for therapeutic targeting of AML LSCs.

The evolving role of FLT3 inhibitors in acute myeloid leukemia: quizartinib and beyond.

Acute myeloid leukemia remains associated with poor outcomes despite advances in our understanding of the complicated molecular events driving leukemogenesis and malignant progression. Those patients harboring mutations in the FLT3 receptor tyrosine kinase have a particularly poor prognosis; however, significant excitement has been generated by the emergence of a variety of targeted inhibitors capable of suppressing FLT3 signaling in vivo. Here we will review results from preclinical studies and early clinical trials evaluating both first- and second-generation FLT3 inhibitors. Early FLT3 inhibitors (including sunitinib, midostaurin, and lestaurtinib) demonstrated significant promise in preclinical models of FLT3 mutant AML. Unfortunately, many of these compounds failed to achieve robust and sustained FLT3 inhibition in early clinical trials, at best resulting in only transient decreases in peripheral blast counts. These results have prompted the development of second-generation FLT3 inhibitors, epitomized by the novel agent quizartinib. These second-generation inhibitors have demonstrated enhanced FLT3 specificity and have been generally well tolerated in early clinical trials. Several FLT3 inhibitors have reached phase III clinical trials, and a variety of phase I/II trials exploring a role for these novel compounds in conjunction with conventional chemotherapy or hematopoietic stem cell transplantation are ongoing. Finally, molecular insights provided by FLT3 inhibitors have shed light upon the variety of mechanisms underlying the acquisition of resistance and have provided a rationale supporting the use of combinatorial regimens with other emerging targeted therapies.

All-Trans Retinoic Acid Synergizes With FLT3 Tyrosine Kinase Inhibition To Eliminate FLT3/ITD-Expressing Leukemia Cells

FLT3 is one of the most frequently mutated genes in AML with approximately 1/3 of patients affected, and the internal tandem duplication (ITD) mutations portending a poor prognosis. To improve the cure rate for FLT3 mutant AML a number of FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling. While several recent FLT3 TKIs are proving increasingly successful at achieving high levels of FLT3 inhibition, there remain several problems in treating FLT3 mutant AML. One is that these drugs used as monotherapy achieve limited clinical responses and do not cure patients. While there is hope that combination with chemotherapy will achieve an improved cure rate in these patients without the necessity for hematopoietic stem cell transplantion (HSCT), the ultimate goal of studies are to achieve cures without chemotherapy, eliminating the short and long-term side effects that its use engenders. Thus, finding additional molecular targets that might synergize with FLT3 inhibition will move the field towards the goal of eliminating chemotherapy. One pathway known to play important roles in leukemia stem cell (LSC) survival and differentiation is the retinoic acid (RA) pathway. We therefore explored the combination of molecularly targeting the RA pathway together with FLT3 TKIs to determine the effect on FLT3 mutant leukemia cells.FLT3/ITD+ AML cell lines (Molm14 and MV411), along with FLT3/WT cell lines (THP-1, SEMK2 and NB4), were treated with FLT3 TKI alone (AC220 and sorafenib), ATRA alone, FLT3 TKI plus ATRA, or vehicle controls. Proliferative, apoptotic, cell-cycle and differentiation effects on the cells were assessed by MTT, annexin V binding, propidium iodide (PI) staining, CD11b staining and cell count assays. Highly synergistic effects were observed for the combination of ATRA with FLT3 TKIs against FLT3/ITD+ cells, with combination index (CI) values of 0.1-0.6. Colony forming unit (CFU) assays further demonstrated decreased clonogenicity of Molm14 and MV411 cells upon treatment with ATRA and sorafenib. A series of experiments were performed using a genetic model of spontaneous leukemia that we developed in which FLT3/ITD knock-in mice are bred with NUP98- HOXD13 (NHD13) transgenic mice, resulting in AML that is highly penetrant, lethal and transplantable. Cohorts of leukemic mice were generated by transplanting lineage negative (Lin-) BM cells from leukemic FLT3/ITD-NHD13 mice, and treated with either vehicle, sorafenib, ATRA or a combination of both drugs. We determined that treatment with sorafenib plus ATRA greatly decreases the level of engraftment at 2 and 8 weeks, and increases median survival, with some mice even cured of their disease. We also directly assessed the effect of the treatments (FLT3 TKI alone, ATRA alone, FLT3 TKI plus ATRA, or vehicle alone) on the proliferation of LSCs by using the different treatments on Lin- BM cells harvested from sick FLT3/ITD NHD13 mice ex vivo and found that ATRA further increased the anti-proliferative effect of FLT3 TKIs, with additive/synergistic CI values of 0.6-1. Lin- BM cells were also assessed in vitro in CFU assays of differentiation and clonogenicity, and combinatorial effects were observed.The results of these experiments show good synergy for this drug combination in vitro and improved survival/reduced LSC frequency in vivo. We believe these preclinical data are encouraging for the development of a clinical trial of ATRA plus FLT3 TKI in relapsed/refractory FLT3 mutant AML patients. Disclosures:No relevant conflicts of interest to declare.

Mutational Profiling Of Myeloid Malignancies For Prediction Of Disease Relapse Following Allogeneic Stem Cell Transplantation

Disease relapse remains the greatest cause of mortality following allogeneic stem cell transplantation (SCT). Improved predictive markers are needed to identify patients most likely to benefit from SCT. Several mutations reported recently in MDS and AML have potential prognostic importance, however the relevance of these mutations to clinical outcome after SCT is poorly understood.In order to evaluate mutations present in transplant patients and provide an initial assessment of their clinical significance, we performed next-generation sequencing of myeloid malignancies from 55 patients (23 MDS, 32 AML) treated with SCT at MSKCC from 2010-2013. Median recipient age was 56 (20-69); 22/55 patients were transplanted in remission. Stem cell sources were CD34-selected (36) or unmanipulated (14) peripheral blood, unmanipulated marrow (2), or cord blood (3). 40/55 allografts were HLA matched (20 related, 20 unrelated). Sequencing was performed on peripheral blood or marrow aspirates in patients with >20% blasts (AML) or >20% dysplastic cells (MDS). Adaptor ligated sequencing libraries were captured with two custom baitsets targeting 374 cancer-related genes and 24 genes often rearranged for DNA-seq, and 272 genes often rearranged for RNA-seq. Captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA. Statistics included cumulative incidence functions for relapse, Kaplan-Meier estimates for relapse-free survival (RFS), and outcome comparison with a permutation-based logrank test. Only mutations observed in at least 5 patients were analyzed. No adjustments were made for multiple comparisons. Median follow-up of survivors was 16.2 months (5.5-32.8).We detected genetic variants in each patient, suggesting the utility of this approach for identifying somatic mutations to track minimal residual disease (MRD) post-SCT. Six patients had known FLT3 mutations detected by a CLIA-certified test; all 6 of these mutations were identified by the sequencing platform. In addition, 3 FLT3 mutations and 1 FLT3 amplification were identified in patients who previously tested FLT3 negative. We identified 13 patients with IDH mutations (5 IDH1, 8 IDH2), eight with NPM1 mutations (all in AML), and 10 with DNMT3A mutations. We identified MAP kinase pathway mutations in 12 patients, including NRAS (7), KRAS (5), and NF1 (4), and we identified mutations in novel genes previously implicated in MDS/AML, including STAT4, CASP8, APC, and ALK.We next evaluated if specific mutations were associated with SCT outcome. Patients with IDH mutations (all of whom had normal karyotype) demonstrated significantly less relapse than patients with wild-type (WT) IDH (1 yr incidence: 0% vs 29%, p=.027, Fig 1). This translated into improved RFS (p=.037) in patients with IDH mutant AML (Fig 2). Treatment-related mortality (TRM) was similar with and without IDH mutations, suggesting the improved outcome was due to reduced relapse. For FLT3, 5/10 patients with FLT3 abnormalities relapsed. All 5 that relapsed were IDH WT. In contrast, IDH mutations were present in 4/5 FLT3+ AMLs that did not relapse, suggesting that IDH mutations may predict for improved SCT outcomes in patients with intermediate cytogenetic risk AML and in patients with FLT3 mutations. Mutant KRAS correlated with reduced overall survival in AML (p=.008), but the significance of this was unclear due to the absence of relapses and high TRM, including infection and GVHD, in this group. [Display omitted] [Display omitted] We also evaluated disease progression in 2 AML patients who relapsed post-transplant with archived samples collected pre-SCT and at relapse. In both patients we observed a distinct mutational profile pre and post-transplant consistent with clonal evolution. Of note, 1 patient gained a NF1 mutation post-SCT, while the other patient lost a NF-1 mutation, although when detected, both mutations were present at a frequency less than 10%.In summary, we performed mutational profiling in SCT patients using a novel high throughput platform, which allowed us to identify clinically relevant mutations, including some not detected by clinical laboratory testing. Notably, we found that IDH mutations may predict for favorable outcome after SCT, even in FLT3 mutant AML. These data suggest that mutational profiling can identify clinically relevant biomarkers pre-SCT and identify mutations for tracking MRD. Disclosures:Miller:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Axl, a prognostic and therapeutic target in acute myeloid leukemia mediates paracrine crosstalk of leukemia cells with bone marrow stroma

AbstractAcute myeloid leukemia (AML) represents a clonal disease of hematopoietic progenitors characterized by acquired heterogenous genetic changes that alter normal mechanisms of proliferation, self-renewal, and differentiation.1 Although 40% to 45% of patients younger than 65 years of age can be cured with current therapies, only 10% of older patients reach long-term survival.1 Because only very few novel AML drugs were approved in the past 2 decades, there is an urgent need to identify novel targets and therapeutic strategies to treat underserved AML patients. We report here that Axl, a member of the Tyro3, Axl, Mer receptor tyrosine kinase family,2-4 represents an independent prognostic marker and therapeutic target in AML. AML cells induce expression and secretion of the Axl ligand growth arrest–specific gene 6 (Gas6) by bone marrow–derived stromal cells (BMDSCs). Gas6 in turn mediates proliferation, survival, and chemoresistance of Axl-expressing AML cells. This Gas6-Axl paracrine axis between AML cells and BMDSCs establishes a chemoprotective tumor cell niche that can be abrogated by Axl-targeting approaches. Axl inhibition is active in FLT3-mutated and FLT3 wild-type AML, improves clinically relevant end points, and its efficacy depends on presence of Gas6 and Axl. Axl inhibition alone or in combination with chemotherapy might represent a novel therapeutic avenue for AML.

FLT3-ITD Interacts with and Phosphorylates IL-3β, and JAK1/2 Dependent IL-3β Activation Bypasses FLT3-ITD in FLT3 Kinase Independent Inhibitor Resistance in Vitro: Evidence for the Significance of IL-3β for FLT3-ITD Dependent Oncogeneic Signaling in AML.

AbstractAbstract 2423Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) display limited activity in FLT3 mutant AML. Most patients experience primary or secondary TKI resistance. Only single cases of FLT3 kinase-dependent TKI resistance by secondary FLT3-ITD kinase domain mutations were reported. We therefore examined the mechanism of FLT3-ITD kinase-independent TKI resistance using an in vitro model. FLT3-ITD cell lines resistant to PKC412 or Sorafenib in 50% of the cases did not harbor secondary FLT3-ITD kinase domain mutations. However, 30% of these lines displayed a persistent phosphorylation of JAK2, IL-3βc and STAT5, while FLT3 phosphorylation was suppressed, indicating that these cell lines bypassed FLT3-ITD by activation of IL-3β by JAK2. Strikingly, TKI sensitive FLT3-ITD cells also displayed IL-3β phosphorylation, which in contrast to TKI resistant cells was JAK2 independent and was suppressed upon FLT3 TKI treatment. Expression of FLT3-ITD in IL-3β and JAK2 deficient g2A cells was sufficient for IL-3β phosphorylation. Co-IP experiments indicated a direct interaction of IL-3β and FLT3-ITD in TKI-sensitive and -resistant cells, which did not require interaction of IL-3β with IL-3a. This indicates that FLT3-ITD “physiologically” uses IL-3β as signaling intermediate. Mapping experiments showed that FLT3-ITD binding occurs in the membrane proximal region of IL-3β, independent of 8 tyrosines present in the cytoplasmic part. In search of the kinase bypassing FLT3-ITD in TKI-resistant cell lines, we identified an activating JAK1 mutation V658F in all TKI resistant FLT3-ITD cell lines displaying phosphorylation of JAK2, IL-3β and STAT5. JAK1 V658F is homologous to JAK2 V617F present in PV, phosphorylates IL-3β and STAT5, and confers TKI resistance in FLT3-ITD positive cells, which could be overcome by the JAK1/2 inhibitor Ruxolitinib. In summary, these data indicate that IL-3β not only mediates “physiological” FLT3-ITD dependent STAT5 activation, but also serves as a signaling module mediating JAK1/2-dependent TKI resistance. Thus, JAK inhibitors might represent an attractive therapeutic option in addition to FLT3 inhibitors in FLT3-ITD positive AML. Disclosures:Duyster:Novartis: Honoraria, Speakers Bureau. von Bubnoff:Novartis: Honoraria, Research Funding, Speakers Bureau.

TTT-3002 Is a Novel FLT3 Tyrosine Kinase Inhibitor That Has the Potential to Overcome Some of the Limitations of Current FLT3 Inhibitors in Treatment of Acute Myeloid Leukemia

Abstract 866Acute myeloid leukemia (AML) is a hematologic malignancy characterized by increased myeloproliferation and a block in differentiation of hematopoietic stem/progenitor cells, leading to infiltration of immature blasts in the bone marrow and peripheral blood. FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic stem/progenitor cells. It can activate cell growth/proliferation pathways via STAT5, AKT/PI3K, and RAS/MAPK signaling. Greater than 35% of AML patients harbor a constitutively activating mutation in the FLT3 gene, and the most common type, an internal tandem duplication (ITD), confers poor prognosis. These ITD mutations typically occur in the juxtamembrane domain and consist of variable length sequence repeats. In addition, activating mutations in the kinase domain are observed in 7–10% of patients. Thus, FLT3 is a validated target for the treatment of AML. Towards this end there have been a number of clinical trials testing the clinical efficacy of FLT3 tyrosine kinase inhibitors (TKIs) in FLT3 mutant AML, either alone or combined with chemotherapy. While some patients have responded, there have been many others who fail to demonstrate significant improvement. Additionally, many patients who initially respond to FLT3 TKI have developed resistance. Inadequate achievement of FLT3 inhibition has been one of the factors limiting efficacy in these trials. This appears most often to be due to a combination of insufficiently potent drugs, decreased efficacy against some FLT3 activating mutations, high plasma protein binding, and the selection for resistance-conferring point mutations within the FLT3/ITD gene. For all of these reasons, there is the need to develop additional FLT3 TKIs able to overcome some of these limitations.We report for the first time on TTT-3002, a kinase inhibitor that has activity against FLT3 and may overcome some of the limitations that current FLT3 TKIs have demonstrated in clinical trials. TTT-3002 is one of the most potent FLT3 inhibitors discovered to date with regard to both inhibition of FLT3 autophosphorylation and cell proliferation. Utilizing human FLT3/ITD mutant leukemia cell lines the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation was less than 250 pM. The IC50 for TTT-3002 in inhibiting proliferation in these same FLT3/ITD expressing cells was 490–920 pM. TTT-3002 also showed potent activity when tested against a broad spectrum of known, non-ITD FLT3 activating mutations, including the most frequently occurring D835Y mutation. It also shows potent activity against a number of FLT3/ITD resistance mutations that have been selected for in patients from clinical trials of other FLT3 TKI or through in vitro drug screening.Studies utilizing human plasma samples from healthy donors and AML patients determined that TTT-3002 is only moderately protein bound, resulting in high levels of free drug able to bind target, and thus maintain activity, in the presence of physiological concentrations of major human plasma proteins including alpha-1-acid glycoprotein and human serum albumin. Therefore, relatively low levels of total drug would need to be achieved in vivo to achieve an effective concentration of free drug in clinical trials. These encouraging findings have been validated both ex vivo and in vivo utilizing mouse models of FLT3-associated AML. Survival and tumor burden of mice in a number of FLT3/ITD transplant models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrate that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD expressing AML patients, while displaying minimal toxicity against normal hematopoietic stem/progenitor cells from healthy bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI that may overcome some of the limitations of other TKI in the treatment of FLT3-mutant AML. Disclosures:No relevant conflicts of interest to declare.

FLT3 Mutation Predicts Worse Clinical Outcome of Relapsed Acute Myeloid Leukemia (rAML) Patients (Pts): Mayo Clinic Experience.

AbstractAbstract 2525 Background:Patients with relapsed AML have a very poor outcome. The molecular signature at diagnosis has emerged as a significant marker for prognostic, and possibly therapeutic, values in newly-diagnosed AML. FLT3 mutations are found in about 25–30% of AML pts. In the absence of FDA-approved agents for FLT3, the prognostic value of FLT3 mutation on clinical outcome for relapsed AML (rAML) is not well documented. Aim:To study clinical implications of FLT3 status on rAML. Methods:All adult pts with AML diagnosed and treated at Mayo Clinic between 2003–2011were studied. Retrospective data collection included pts’ demographics, laboratory tests, bone marrow biopsies, FLT3 mutation (internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutation), and survival information. IRB approval was obtained in accordance with Helsinki declaration. Chi-Square and T-test were used for comparatives between groups and Kaplan-Meier test was applied for survival estimates using JMP 9.0 software. Results:A total of 192 pts with newly-diagnosed with AML were identified. Sixty percent (115) were males, 88% (168) were Caucasians, 26% (49) had antecedent hematological disease (AHD), and 13% (25) had previous exposure to chemo- or radiotherapy. Forty-seven percent had a normal chromosomal analysis, and 18% (35) had FLT3 mutation (28 ITD, 7 TKD). In FLT3-mutated pts, median WBC was 70 x109/L (p=0.0016), hemoglobin 9.5 g /dL, and platelets 48 x109/L, compared to 8.8 x109/L, 9.1 g/dL, 50 x109/L in the rest of pts, respectively. Peripheral blood blasts were elevated in FLT3-mutated group compared to rest of pts (p 0.0059).A total of 128 pts achieved complete remission (CR), with 79 (62%) subsequently relapsed. Of the relapsed patients, 93% (74) were Caucasians, 54% (43) males, 22% (17) had AHD, and 8% (6) had prior chemo- or radiotherapy. Cytogenetics were diploid in 49% (39) and complex in 15% (12). Hematopoietic stem cell transplantation was performed in 30% (24) of pts. Eleven of 79 (14%) pts were FLT3-mutated at relapse (Group 1); with 1 pt having TKD-mutation. Forty-eight percent (38) of pts had unmutated-FLT3 (Group 2), and 38% (30) pts were FLT3-unknown (Group 3).In group 1, median WBC was 9.9 x109/L, hemoglobin 10.55 g /dL, platelets 55 x109/L, compared to 3.2 x109/L (p=0.0034), 10.55 g/dL, and 58 x109/L in group 2, respectively. Peripheral blasts were significantly elevated in group 1 compared to group 2 (38% vs 4%, p <0.0001). Surprisingly, 3/11 pts (27%) with FLT3 mutation at relapse were unmutated at diagnosis. CR was achieved in 2/11 (18%) pts in group 1 compared to 15/38 (39%) in group 2 (p=0.19). Median overall survival in rAML was significantly lower in group 1 compared to group 2 (90 days vs 229 days, p 0.0076). This survival difference was statistically more significant in pts with diploid cytogenetics at relapse compared to the rest (p 0.0009 vs 0.48, respectively). Within 1.5 years, all pts in group 1 died, compared to 21% survived in group 2. Conclusion:FLT3-mutated AML pts had a significantly higher peripheral WBC and blasts at both diagnosis and relapse. Importantly, FLT3 at relapse was found to be mutated in one quarter of pts who were wild-type FLT3 at diagnosis; raising the importance of assessing FLT3 gene on all relapses. FLT3 mutation significantly predicted worse median overall survival in rAML raising the need for novel agents, such as FLT3 inhibitors, in this population. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Mechanisms of apoptosis induction by simultaneous inhibition of PI3K and FLT3-ITD in AML cells in the hypoxic bone marrow microenvironment

We investigated the antileukemia effects and molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in AML cells grown under hypoxia in co-cultures with bone marrow stromal cells. Combined treatment with selective class I PI3K inhibitor GDC-0941 and sorafenib reversed the protective effects of bone marrow stromal cells on FLT3-mutant AML cells in hypoxia, which was associated with downregulation of Pim-1 and Mcl-1 expression levels. These findings suggest that combined inhibition of PI3K and FLT3-ITD may constitute a targeted approach to eradicating chemoresistant AML cells sequestered in hypoxic bone marrow niches.

Phase I dose escalation study of TG02 in patients with advanced hematologic malignancies.

6577 Background: TG02 is a novel multikinase inhibitor with a unique spectrum of activity, targeting both the cell cycle regulatory cyclin-dependent kinases (CDKs) 1 and 2 and the transcriptional regulators CDKs 7 and 9. TG02 also inhibits the emerging oncogenic MAPK ERK5 and the DNA damage response mediator CDK5. TG02 kills primary blasts from a variety of hematologic cancers and is curative in the MV4-11 model of FLT3-mutant AML. Methods: This is a first-in-man,single arm, open label, phase I dose escalation trial. The primary endpoints are dose-limiting toxicity (DLT), maximally tolerated dose (MTD ) and recommended phase 2 dose (RP2D). Patients (pts) ≥ 18 years with advanced hematological malignancies or newly diagnosed AML pts ≥ 65 years unfit for intensive therapy were enrolled onto daily (A) and intermittent (B, 5 days on 2 days off X 2 weeks) schedules. Pts had acceptable organ function and ECOG PS 1-2. Definition of DLT was G3-4 AST or ALT ≥7 days, G4 AST or ALT, G4 hyperbilirubinemia, any other NCI CTC G3-4 events not due to underlying disease. Dose levels on arm A were 10mg to 70mg and 15mg-150 mg on arm B. Results: Forty-five pts have received at least one dose of study drug. Median age was 66 years (range, 37-87) and 80% were ECOG 0-1. Disease types enrolled included: AML (80%), high-risk MDS (22%), and CML-BC (3%). The median number of previous regimens was 3 (range, 1-12). The MTD on arm A was defined at 50 mg daily based on 2 DLTs at the 70mg dose level (G4 hyperbilirubinemia, G4 fatigue). Enrollment to arm B has competed dose levels 15 (N=3), 30 (N=3), 50 (N=3), 70 (N=3), 100mg (N=3), and enrollment at 150mg is ongoing without DLT to date. Common drug related adverse events were nausea (42%), vomiting (23%), fatigue (18%), decreased appetite (15%), constipation and diarrhea (13% each). Preliminary PK demonstrated dose proportional increases in exposure and a T1/2 , supporting once daily dosing. Conclusions: The MTD for TG02 has been determined for the daily schedule at 50mg. Enrollment continues on the intermittent schedule. Schedules of every other day and week on/week off dosing will also be evaluated.

Impact of Combining Targeted Agents with High-Dose Cytarabine-Based Induction Chemotherapy on Acute Myeloid Leukemia Outcomes: The M.D. Anderson Cancer Center Experience

Abstract 1539 Background:Standard AML therapy with cytarabine and an anthracycline produces a complete remission (CR) rate of 60%–70% but cure rates of only 15%–25%. AI (ara-C 1.5g/m2x3d and idarubicin 12mg/m2x3d), the standard induction chemotherapeutic backbone used at M.D. Anderson Cancer Center for AML, consists of idarubicin 12 mg/m2/day on days 1–3 and cytarabine 1.5 g/m2 IV over 24 hours daily on days 1–4 (days 1–3 only if age >60 years). In recent years several targeted agents have been combined with IA in an attempt to improve outcomes. Patients & Methods:We reviewed all the AI-based protocols used at our institution over the last decade to identify combinations that may improve outcomes over standard AI. Only trials that enrolled >20 pts (age 15–70 years) were analyzed, including AI alone (n=486) and in combination with sorafenib (BAY; n=56), interleukin-11 (IL-11; n=37), lisofylline (Liso; n=24), vorinostat (SAHA; n=58), tipifarnib (Zarn; n=84), G-CSF+/−ATRA (G+/−ATRA; n=115). Patients with core-binding factor AML or acute promyelocytic leukemia were excluded. Results:The combination of AI with BAY (84%), lisofylline (67%), SAHA (76%) and G+/−ATRA (76%; no difference regardless of the addition of ATRA) produced higher CR rates than IA alone (61%; p=0.0001), which for BAY and SAHA was also accompanied by improved early death (ED; 8-wk mortality) rate and median overall survival (OS) (5%/not reached [NR] & 5%/68wks) compared to AI alone (15%/48wks) (Table 1, Figure 1). AI+Zarn produced similar CR (61%) but improved ED rate & OS (8%/54wks) while AI+G+/−ATRA also rendered a better OS (83wks) than AI. Of note, both BAY (80%) and SAHA (79%) but not G+/−ATRA (57%) included a higher number of pts younger than 60 yrs compared to AI (55%). However, when only pts <60 years were considered, the CR/OS rates were still superior for BAY (87%/NR), SAHA (76%/68wks), and G+/−ATRA (86%/115wks) compared with AI (71%/67wks). A similar trend was observed for pts ≥60 years (73%/64wks for BAY, 67%/32wks for SAHA, 61%/34wks for G+/−ATRA, and 49%/29 for AI) but the low number of pts in the BAY (n=11), and SAHA (n=12), albeit not in the G+/−ATRA group (n=49), limits the value of this comparison.Table 1Outcomes of pts with AML treated with AI-based regimensRegimensAIAI+BAYAI+IL11AI+LISOAI+SAHAAI+ZARNAIG+/-ATRAp valueN%N%N%N%N%N%N%Response RateCR29661%4784%1951%1667%4374%5161%8776%0.001CRp214%24%13%00%610%810%00%ED7415%12%719%625%35%78%2017%NR9520%611%1027%28%610%1821%87%Total4865637245884115Pts age 0–5926655%4580%924%1250%4679%8095%6657%0.00001Pts age ≥6022045%1120%2876%1250%1221%45%4943%0.0001Median CRD (Weeks)635839264272820.0852-year39%34%29%13%50%46%42%5-year28%0%44%31%Median OS (Weeks)48NR2033685483<0.0012-year32%63%12%21%32%39%42%5-year18%8%31%20% [Display omitted] When the analysis was limited to pts with unfavorable cytogenetics (−5, −7, complex) BAY (75%/NR), SAHA (69%/43wks), G+/−ATRA (80%/23wks) also improved the outcomes produced by AI (50%/21wks) with better ED rates (0%, 5%, 12.5% and 19%, respectively).Of the latter targeted agents, sorafenib and SAHA showed remarkable activity in FLT3-mutated AML. CR/OS rates for pts with FLT3-ITD were 94%/67wks for BAY, 91%/77wks for SAHA (no FLT3 mutational data for G+/−ATRA) and 64%/48wks for AI (AI vs BAY or SAHA, p=0.001 for CR and p=0.001 for OS). Conclusion:Long-term follow-up suggests that IA combined with sorafenib, SAHA, or G+/−ATRA may improve outcomes in pts with AML, including those with adverse karyotypes and those older than 60 years, compared with AI alone. AI+Sorafenib and (surprisingly) AI+SAHA showed remarkable activity against FLT3-mutated AML. Randomized studies are warranted to confirm these observations. Disclosures:No relevant conflicts of interest to declare.

FLT3 Inhibitor Treatment in FLT3-Mutated AML Is Associated with Development of Secondary FLT3-TKD Mutations

Abstract 1493 Background:The two most frequent mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor are internal tandem duplication (ITD) of the juxtamembrane region and mutations involving the D835/I836 residues of the tyrosine kinase domain (TKD). They are present in approximately 30% and 7% of AML cases respectively, and have been associated with higher relapse rate and worse overall survival. ITD and TKD mutations produce constitutive activation of the FLT3 receptor; however they appear to have different downstream effectors and different clinical significance. Currently, several FLT3 kinase inhibitors are in development and although some have shown promising clinical activity, responses tend to be transient and insufficient to induce a durable response. Sensitivity of different mutants to diverse FLT3 inhibitors is variable (Zhang et al. JNCI 2008; 100: 184). Aim: To determine the change in mutation status of patients with FLT3 ITD treated with FLT3 inhibitors. Methods: We analyzed 67 patients with FLT3 mutation positive AML, treated in our institution in clinical trials with different FLT3 inhibitors from October 2002 to July 2011 and in whom we obtained mutational assessment before and after treatment. Results: At baseline 58 (87%) patients had an ITD mutation, 5 (7%) had a D835/I836 mutation and 4 (6%) had combined ITD and codon D835/I836 mutations. Thirty three patients were male, median age was 55 years (range 18–87), and the median number of prior leukemia treatments was 2 (range 1–6). Karyotype was diploid in 36%, miscellaneous in 17%, complex in 14%, not done in 23%, and insufficient in 9% of patients. Concomitant NPM1, RAS, and CEBPA mutations were observed in 21%, 13%, and 4% of the patients, respectively. Patients remained on therapy for a median time of 50 days (range 18–561) before progression, 45 (67%) did not achieve response, 13 (19%) cleared their bone marrow blasts, 6 (9%) achieved a partial response, 4 (6%) a complete response and 1 (1%) patient died during treatment. None of the patients with D835/I836 or ITD+D835/I836 achieved a response. Treatment was always discontinued due to progressive disease, except in 9 (13%) patients, 6 who cleared bone marrow blasts and proceeded to hematopoietic stem cell transplantation and 3 who discontinued due to toxicity. One of them was in complete response but progressed within 4 months, and 2 who cleared their bone marrow blasts and were also bridged with other treatments to transplant. All patients in this cohort had assessment of their FLT3 mutation status at the time FLT3 inhibitor was discontinued, 14 (21%) patients progressed from a single ITD mutation to have combined ITD+D835/I836 mutations, 7 (10%) patients (6 ITD and 1 D835/I836) were negative for FLT3 mutation, and 46 (69%) patients had unchanged mutation status. At the time of this analysis, 10 patients were alive, 1 was lost to follow and 56 had died. The median survival in the entire group from the time of FLT3 inhibitor start was 5.3 months, and from time of treatment discontinuation 3.2 months. In those patients in whom the FLT3 mutation became negative the median survival was 7 months, in those with unchanged mutation 4.5 months, and in those with ITD-D835/I836 mutations the median survival was 6.1 months. Further therapy with another FLT3 inhibitor was attempted in 6 out of 14 patients with ITD-D835/I836 mutations, 5 failed to respond and 1 had a transient decrease in bone marrow blasts and peripheral white blood cell count (2 months) with a combination of a FLT3 inhibitor and a hypomethylating agent. Conclusion: There is evidence that a secondary TKD mutations arise after the use of FLT3 inhibitors in patients with single FLT3-ITD mutated AML, a phenomenon that is associated with a poor prognosis. Disclosures:Ravandi:Bayer: Research Funding; Onyx: Research Funding. Cortes:Ambit: Research Funding; Novartis: Research Funding.

The Efficacy of the FLT3 Inhibitor Lestaurtinib in AML Depends on Adequate Plasma Inhibitory Activity (PIA), and Is Unaffected by Rising FLT Ligand Levels: An Update of the NCRI AML15 & 17 Trials

Abstract 421The receptor tyrosine kinase FLT3 remains the subject of intense clinical interest as a therapeutic target in AML. FLT3-activating mutations are seen in one-third of adults at diagnosis and are associated with adverse prognosis. The multi-targeted kinase inhibitor lestaurtinib (CEP701) has potent activity against FLT3. In monotherapy studies, sustained plasma drug activity sufficient to inhibit FLT3 by >85% was found to be a prerequisite for clinical response (1,2). A recently-reported randomized study of lestaurtinib combined with chemotherapy in relapsed FLT3-mutated AML failed to demonstrate improved clinical outcome with lestaurtinib, but definitive conclusions were limited by the relatively low rate (58%) of patients achieving >85% FLT3 inhibition (3). In that study, rising levels of FLT3 ligand (FL) were noted during chemotherapy; raised FL levels have been reported to impede the in vitro efficacy of FLT3 inhibitors (4).As part of an ongoing randomised assessment (NCRI AML15 and 17 Trials) 118 newly-diagnosed FLT3-mutated non-APL AML patients (median age 49 [range 16–66], 86 ITD, 30 TKD point mutation, 2 both FLT3 mutations, 87% intermediate risk karyotype) received lestaurtinib for up to 28 days following each of 4 courses of chemotherapy. Based on early pharmacokinetic data, starting dose of lestaurtinib was adjusted according to concomitant anti-fungal drug use: 40mg bid in azole treated patients, 80mg bid in other patients. Patients received a median 3 courses of lestaurtinib (range 0–4); no excess severe adverse events have been reported in lestaurtinib-treated patients. Median follow-up is now 30 months (range 9–47). 49 patients underwent allogeneic SCT (37 in 1st CR, 28 myeloablative conditioning, 21 RIC). In a non-randomised comparison of clinical outcome with a matched historical age-matched control population of 118 FLT3-mutated patients entering the AML15 Trial prior to the opening of the lestaurtinib randomisation, there was a modest improvement in complete remission (CR/CRi) rate (92% vs 90% historical control), 2-year cumulative relapse rate (47% vs 55%) and 2-year overall survival (50% vs 47%).Trough FLT3 plasma inhibitory activity (PIA) was measured on day 14 of each lestaurtinib course. 169 timepoints were assessed in 84 patients with 88% of patients achieving at least 1 FLT3 PIA measurement of >85%. FLT3 PIA of >85% was seen at 75% of individual timepoints (127/169). Rates of relapse were significantly lower in patients that achieved FLT3 PIA>85% at all evaluated timepoints: the 2-year relapse rate was 38% in inhibited vs 61% in non-inhibited patients (22/58 vs 16/26) (p=0.01) where results were available for 1 or more timepoints, 23 vs 58% (6/28 vs 10/18)(p=0.01) for 2 or more timepoints and 15% vs 64% (2/14 vs 7/11)(p=0.007) for 3 or more timepoints. Significantly increased 2-year overall survival was seen in patients that achieved >85% PIA at 1 or more timepoints, 64% in inhibited vs 40% in non-inhibited patients (37/58 vs 10/26)(p=0.01).Mean day 14 FL concentrations rose through successive courses of lestaurtinib treatment from 529 pg/ml during course 1 to 1350 pg/ml, 2575 pg/ml and 2467 pg/ml respectively during courses 2,3 and 4. No increase was seen in alpha-1-acid glycoprotein levels. Despite rising FL levels, no fall off was seen in achievement of FLT3 inhibition: day 14 FLT3 PIA>85% was achieved by 75% of assayed patients during course 1 (51/68), 80% during course 2 (39/49), 78% during course 3 (25/32) and 60% during course 4 (12/20). No difference was seen in FLT3 PIA between patients grouped according to high or low FL concentration.These data provide evidence of an emerging relationship between sustained FLT3 inhibition and improved clinical outcome when tyrosine kinase inhibition is combined with standard chemotherapy in newly diagnosed AML. Rising FL levels through successive courses of therapy failed to impede target inhibition. The sequential combination of lestaurtinib with chemotherapy continues to be evaluated in the ongoing randomized NCRI AML17 Trial. Disclosures:Off Label Use: The presentation will include discussion of the off-label use of lestaurtinib (CEP701) in the treatment of newly-diagnosed AML patients with FLT3 mutations.

Abstract B74: The dual FLT3-Aurora inhibitor CCT241736 overcomes resistance to selective FLT3 inhibition driven by FLT3 ligand and FLT3 point mutations in acute myeloid leukemia.

Abstract Internal tandem duplication or kinase domain point mutations of the fms-like tyrosine kinase 3 gene (FLT3-ITD, -TKD) result in constitutive FLT3 kinase activation and promote leukemic proliferation. FLT3-ITD occurs in 20–35% of adults and 15% of children with acute myeloid leukemia and confers a poor prognosis in both age groups. Activated FLT3 therefore represents a valid therapeutic target and several FLT3 inhibitors are currently in phase II-III clinical trials. The clinical impact of FLT3 inhibitors has thus far been limited by transient responses when used as single agents due to lack of sustained FLT3 inhibition and the emergence of secondary acquired resistance following treatment. Aurora kinases are a family of serine-threonine kinases that play a key role in several stages of mitosis. Although studies in leukemia have been limited to cell lines and small patient cohorts, over-expression of Aurora A has been consistently demonstrated and Aurora kinase inhibitors show potential in the treatment of AML. Our hypothesis is that simultaneous FLT3 / Aurora inhibition will improve single-agent efficacy and reduce susceptibility to FLT3-ligand-driven and FLT3 mutation-driven resistance in FLT3-ITD positive AML. In addition, we have generated a model of FLT3 inhibitor resistance by prolonged treatment of MOLM-13 cells with the selective FLT3 inhibitor MLN518. These resistant cells, termed MOLM-13-RES, are doubly-mutated with FLT3-ITD and a FLT3-TKD (D835Y) point mutation and over-express Survivin. Here we demonstrate that such doubly-mutated AML cells are sensitive to the dual FLT3-Aurora inhibitor CCT241736. CCT241736, an imidazo[4,5-b]pyridine derivative discovered at our Institution, is a novel, orally bioavailable, potent and highly selective FLT3 and pan-Aurora inhibitor [S(10) = 0.057; a fraction of 386 kinases inhibited &amp;gt;90% when screened at 1 μM of CCT241736]. In biochemical assays, CCT241736 has IC50 values against FLT3, Aurora A and Aurora B of 0.035, 0.015 and 0.1 μM respectively. Furthermore, CCT241736 inhibits a wide range of FLT3 mutants, including FLT3-ITD, -D835Y, -D835H, -K663Q and -N841I. In cellular assays, CCT241736 inhibits viability of the human FLT3-ITD positive AML cell lines MOLM-13 and MV-4;11 with EC50 values of 0.1 and 0.27 M respectively. In vivo, mouse tumor xenograft models of MOLM-13, MV-4;11 and the doubly-mutated cell line, MOLM-13-RES, are also sensitive to CCT241736, with biomarker modulation consistent with dual inhibition of FLT3 and Aurora kinases. In summary, dual inhibition of FLT3 and the critical mitotic kinase Aurora by CCT241736 may represent a novel treatment strategy for FLT3-mutated AML by overcoming the effects of high FL levels and limiting resistance caused by secondary mutations of the FLT3 gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B74.

Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104

AbstractAbstract 868Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups.Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. [Display omitted] Disclosures:Wilson:PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.