FLT3 Inhibitors In Combination Research Articles

FLT3 Inhibitors in Combination with FLAG-IDA in Relapsed or Refractory Acute Myeloid Leukemia

1. Introduction The addition of a FLT3 inhibitor improves overall survival in combination with front-line intensive chemotherapy in acute myeloid leukemia (AML). In the ADMIRAL trial, gilteritinib - a second-generation, type I FLT3 inhibitor - improved survival in relapsed or refractory AML compared to intensive chemotherapy. However, it is unknown whether the combination of a FLT3 inhibitor and intensive chemotherapy improves survival in the relapsed or refractory setting compared with intensive chemotherapy alone or intensive chemotherapy with venetoclax in FLT3 mut AML. 2. Methods We analyzed 27 patients with FLT3 mut AML treated with FLAG-IDA for relapsed or refractory disease from January 1, 2013 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. Baseline disease characteristics were obtained, including molecular profiling either through PCR, NGS, or both, alongside cytogenetic risk, doses of induction regimens, response, and survival. We separated patients that underwent treatment with FLAG-IDA and a FLT3 inhibitor (gilteritinib, midostaurin, or sorafenib), venetoclax, or FLAG-IDA alone. Measurable residual disease (MRD) assays were PCR-based when available; otherwise, MRD was assessed through multiparameter flow cytometry. We used the D'Agostino & Pearson method for normality testing and the Kruskal-Wallis test for between-group comparisons. We applied the Bonferroni correction if multiple comparisons were made. We analyzed survival by the Kaplan-Meier method with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. 3. Results All 27 patients treated with FLAG-IDA harbored mutations in FLT3-ITD (70.3%) or FLT3-TKD (29.6%). Twelve (44.4%) patients received FLT3 inhibitors with FLAG-IDA: eight (66.7%) with midostaurin, three (25.0%) with gilteritinib, and one (8.3%) with sorafenib. Five (41.6%) patients received a FLT3 inhibitor (FLT3i) for previously untreated disease, then subsequently received a FLT3i at disease progression. Ten (37.0%) patients received FLAG-IDA without a FLT3i, and five (18.5%) received FLAG-IDA and venetoclax. The median FLT3 variant allele frequency was 33.96% (95% CI, 5.1-63.9) in the FLT3i cohort, 18.0% (95% CI, 8.0-27.0) in the FLAG-IDA cohort, and 5.0% (95% CI, 3.0-29.0) in the venetoclax cohort. The median prior lines of therapy were as follows: three for the FLT3i cohort, two for the FLAG-IDA cohort, and three for the venetoclax cohort (p = 0.137). There was no difference in the achievement of CRc between the FLT3i and FLAG-IDA groups (27.3% vs 33.3%, p > 0.999) or between the FLT3i and venetoclax groups (27.3% vs 50.0%, p = 0.560). The proportion of responders that achieved MRD negativity was nonsignificantly higher in the FLT3i cohort (75.0% vs 20.0%, p = 0.206). The median overall survival nonsignificantly favored the FLT3i cohort compared to the FLAG-IDA cohort (7.7 months vs 4.1 months, p = 0.992) and the venetoclax cohort (7.7 months vs 6.3 months, p = 0.902). 4. Discussion The addition of a FLT3 inhibitor resulted in nonsignificantly higher rates of MRD negativity and prolonged overall survival when compared to FLAG-IDA alone or FLAG-IDA and venetoclax. The optimal combination regimen for relapsed or refractory FLT3 mut AML is unknown, but likely should include a FLT3 inhibitor. The limitations of our study include its retrospective nature; subsequent FLT3 inhibitor combinations or maintenance strategies may have provided a survival benefit the FLT3i cohort. Therefore, prospective clinical trial designs are needed to further confirm these findings.

Ferroptosis vulnerability in FLT3-mutant leukemia

Resistance to FLT3 inhibition is a prevalent challenge in managing FLT3-mutant acute myeloid leukemia (AML). A recent study by Sabatier et al. discovered ferroptosis vulnerability in FLT3-mutant AML and they propose a promising therapeutic approach of combining FLT3 inhibitors with ferroptosis inducers for treating this type of cancer.

Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies

Abstract Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT. Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A05.

Improved Post-Transplant Outcomes in Recent Years for AML Patients with Intermediate Karyotype, FLT3-ITD and Wild Type NPM1: A Report from the EBMT Acute Leukemia Working Party

Rationale: Allogeneic hematopoietic cell transplantation (allo-HCT) is recommended in first complete remission (CR1) in acute myeloid leukemia (AML) patients harboring FLT3 internal tandem duplication (FLT3-ITD). However, historically, long-term survival has been poor due to early relapse, particularly for patients with wild-type NPM1. Recently, FLT3 inhibitors in combination with chemotherapy before allo-HCT, or as post-transplant maintenance, have significantly reduced the risk of relapse and improved survival. Here, we assessed real-world changes over time in transplant characteristics and post-transplant outcomes in young AML patients with FLT3 ITD and either NPM-1 mutated or wild type, using a large dataset from the European Society of Blood and Marrow Transplantation (EBMT) registry. Patients and Methods: We identified 1827 adult AML patients (54% female; median age 49 years, range 18-60) with FLT3 ITD, intermediate karyotype and available NPM1 mutation status, allografted between 2012 and 2021 in CR1 from a matched related (32%), matched unrelated (56%) or haploidentical donor (12%). NPM1 was mutated in 72% of patients. At transplant, 834 patients were measurable residual disease (MRD) positive, 464 MRD negative, and 529 were not evaluated or missing. Comorbidity index (CI) was zero in 59% of patients with available data. Conditioning was myeloablative (MAC) in 64% of patients. In vivo T cell depletion (TCD) graft was given to 62% of patients, post-transplant cyclophosphamide (PTCy) to 17%, and 87% received peripheral blood stem cells (PBSC). Most patients (66%) and donors (54%) were cytomegalovirus (CMV) positive. Median follow-up of alive patients was 30 months (IQR 28-32). Results: We compared changes in patient and transplant characteristics over time in 688 (38%) patients transplanted between 2012 and 2016 and 1139 (62%) patients transplanted between 2017 and 2021. Patients transplanted in recent years had a shorter follow up, were more likely to have NPM1 mutation, to be MRD positive, to have a higher CI, to receive a transplant from a haploidentical donor, a CMV positive donor, and to receive PBSC and PTCy. For patients with wild-type NPM1, day 100 acute graft-versus-host disease (GVHD) grade II-IV and grade III-IV were encountered in 29% and 11% of patients transplanted in 2021-2016, respectively, and 21% and 7% for patients transplanted in 2017-2021, respectively. The 2-year cumulative incidence of chronic and extensive chronic GVHD were 32% and 16%, respectively for 2012-2016 and 36% and 20%, respectively for 2017-2021. Over time, the 2-year cumulative incidence of relapse (CIR) significantly decreased from 43% to 31% between the two time periods (p<0.04) and non-relapse mortality (NRM) significantly decreased from 14% to 8% (p<0.05). The 2-year leukemia free survival (LFS) and overall survival (OS) significantly improved over time from 54% to 64% (p<0.02) and from 63% to 71% (p<0.05) respectively, whereas GVHD-free, relapse-free survival (GRFS) improved from 39% to 46% (p=0.08). In multivariate Cox analysis (MVA), transplantation in recent years significantly reduced the CIR (hazard ratio [HR]=0.65; p<0.01), and acute GVHD grade II-IV (HR=0.6; p<0.01) and significantly improved LFS (HR=0.67; p=0.02), OS (HR=0.66; p<0.03), and GRFS (HR=0.77; p<0.05). The use of unrelated donors increased the risk of acute GVHD but reduced the CIR whereas the use of haploidentical donors increased NRM. In vivo TCD reduced the risk of chronic GVHD whereas female donors to male recipients increased it. PTCy decreased the risk of NRM whereas MAC reduced the CIR. For patients with NPM1 mutation, no significant changes over time in post-transplant outcomes were noted except for decreased NRM from 15% for 217 patients transplanted in 2012-2016, to 10% for 288 patients transplanted in 2017-2021 (p<0.02). In MVA, transplantation in recent years did not significantly affect any of the post-transplant outcomes. Conclusion: In AML patients with FLT3 ITD and wild-type NPM1, we noticed a significant decrease over time in the CIR and a significant improvement of LFS, OS and GRFS, likely reflecting the efficacy of FLT-3 inhibitors, including when used as post-transplant maintenance, in this high-risk setting. On the contrary, no significant change over time was noticed in post-transplant outcomes of patients harboring NPM1 mutation.

AML-241 Sequential Venetoclax and FLT3 Inhibitors in Combination With Hypomethylating Agents or Low-Dose Chemotherapy in Newly Diagnosed, Unfit, FLT3-Mutant Acute Myeloid Leukemia Patients

Targetable mutations such as FLT3, a receptor tyrosine kinase, are present in 30% of cases with acute myeloid leukemia (AML). Direct inhibitors of FLT3 are promising therapeutic agents such as midostaurin and gilteritinib. The use of FLT3 inhibitors as single agents remains to be limited with short-lived responses. For treatment-naïve AML patients with FLT3 mutation, the standard treatment entails induction with intensive chemotherapy in physically fit or younger patients and in combination with an FLT3 inhibitor. There is no approved targeted therapy option for treatment-naïve patients with AML harboring an FLT3 mutation ineligible for intensive therapy. To assess the efficacy and feasibility of sequential administration of venetoclax, a selective inhibitor of Bcl-2, and midostaurin in addition to induction chemotherapy in unfit newly diagnosed FLT3-mutant AML patients. This is a case series reviewing the clinical data of four consecutive patients with FLT3-mutant AML, ineligible for standard induction chemotherapy, who received induction treatment consisting of low-dose chemotherapy or hypomethylating agents (HMA) with the sequential use of venetoclax for 10 days followed by midostaurin for 14 days, treated at the American University of Beirut Medical Center. Between November 2021 and February 2022, four patients were included in our study. The median age was 68 years (range 66-70). All patients had FLT3 mutation (three with ITD, one with TKD), and three patients had concomitant NPM1 mutation. One patient had trisomy 8 and another patient had deletion 7 on cytogenetics. All patients achieved complete remission on day 21 and day 28. One patient with underlying myelodysplastic syndrome remained to have detectable FLT3 with deletion 7 at day 28 evaluation and had the longest time for absolute neutrophil count (ANC) and platelet count recovery. The median number of days for ANC and platelet count recovery was 40 days (range 27-59) and 34 days (24-not reached) respectively. The main toxicity was febrile neutropenia in 3 out of 4 patients. The sequential use of venetoclax followed by midostaurin in combination with low dose chemotherapy or HMA for newly diagnosed AML, FLT3-mutant patients who are unfit for intensive chemotherapy seems to be a promising approach with an acceptable toxicity profile.

FLT3-ITD Activation Mediates Resistance to the BCL-2 Selective Antagonist, Venetoclax, in FLT3-ITD Mutant AML Models

FLT3 internal tandem duplication (FLT3-ITD) mutations account for ~20-25% of adult AML cases and are associated with worse prognosis. FLT3-ITD is a constitutively activated kinase with differential signaling compared to wild type (WT) FLT3. Notably, FLT3-ITD activates STAT5, which can regulate the pro-survival proteins BCL-XL and MCL-1. Venetoclax (ABT-199/GDC-0199), a potent, selective inhibitor of the pro-survival protein BCL-2, recently demonstrated monotherapy activity in relapsed/refractory AML (ORR 19%); however, no activity was seen in FLT3 mutant cases. BCL-XL and MCL-1 are known venetoclax resistance factors; therefore targeting their regulation in combination with venetoclax may enhance cell death. Based on this hypothesis, we interrogated the combination of venetoclax and quizartinib (AC220), a potent FLT3 inhibitor, in FLT3-ITD+ AML models.Sensitivity to venetoclax was assessed in FLT3-ITD+ (Molm13 and MV4;11) and FLT3 WT (HL60 and OCI-AML3) cells in vitro . Dose dependent growth inhibition and induction of apoptosis was seen in MV4;11, Molm13 and HL60 cells following 48hr venetoclax treatment, with the MV4;11 cells being the most sensitive to venetoclax. Twenty-four hour quizartinib treatment reduced expression of BCL-XL and MCL-1 protein, but not BCL-2, in FLT3-ITD+ cells, while quizartinib had no effect on BCL-XL, MCL-1 or BCL-2 expression in FLT3 WT cells, confirming FLT3-ITD mediated regulation of BCL-XL and MCL-1. To interrogate combined FLT3-ITD and BCL-2 inhibition, cells were treated for 48hrs with venetoclax, quizartinib or the combination. Combination treatment led to significant reduction in growth and increased apoptosis in FLT3-ITD+ cells compared to either single agent. FLT3 WT cells were not sensitive to quizartinib alone or in combination with venetoclax. To determine the dependence of FLT3-ITD+ cells on BCL-XL and MCL-1, cells were treated with selective BCL-XL (A1331852) or MCL-1 (A1210477) inhibitors in combination with venetoclax. Treatment with either of the selective inhibitors in combination with venetoclax reduced the growth of cells compared to single agents alone, suggesting that all three pro-survival proteins may contribute to the survival of FLT3-ITD+ cells. Consistent with FLT3-ITD regulating BCL-XL and MCL-1, co-treatment of quizartinib with either BCL-XL or MCL-1 inhibitor had no combination effect in FLT3-ITD+ cells. To elucidate the signaling pathway(s) regulated by FLT3-ITD that confer resistance to venetoclax, cells were treated with PI3K, MEK or JAK2 kinase selective inhibitors alone or in combination with venetoclax. Each pathway inhibitor combined with venetoclax to reduce cell growth when compared to each agent alone, indicating that multiple signaling pathways can contribute to venetoclax resistance rather than a single pathway dependence for driving the pro-survival effects of FLT3-ITD. This data underscores the reliance of these models on FLT3-ITD for survival and regulation of multiple survival pathways that confer resistance to venetoclax, suggesting FLT3-ITD itself could be a predictive biomarker of venetoclax resistance.Further, the combination of quizartinib and venetoclax was assessed in vivo utilizing MV4;11 and Molm13 xenograft models grown orthotopically. Mice were treated with venetoclax (100 mg/kg), quizartinib (5 mg/kg) or the combination for 21 continuous days. Quizartinib treatment significantly increased survival compared to vehicle in both models [56 days vs. 26 days (p<0.0001) and 32 days vs. 14 days (p<0.0001) for MV4;11 and Molm13, respectively]. However, venetoclax alone only marginally improved survival [34 days vs. 26 days (p<0.0001)] in the MV4;11 model and was not efficacious in the Molm13 model. Despite lack of single agent activity, addition of venetoclax to quizartinib led to a further improvement in survival when compared to quizartinib alone [91 days vs. 56 days (p<0.0001) and 40 days vs. 32 days (p<0.0001) in MV4;11 and Molm13, respectively]. Monotherapy and combination treatments were tolerated based on minimal changes in animal body weight throughout the duration of the study.Taken together, our data demonstrate that FLT3-ITD inhibition combines with venetoclax in vitro and in vivo in FLT3-ITD+ AML models. These results provide a strong mechanistic rationale to support clinical investigation of FLT3 inhibitors in combination with venetoclax to treat FLT3-ITD+ AML. [Display omitted] DisclosuresMali:Genentech: Employment. Lasater:Genentech: Employment. Doyle:Abbvie: Employment. Malla:Genentech: Employment. Boghaert:Abbvie: Employment. Souers:Abbvie: Employment.

Experience with FLT3 Inhibitor Midostaurin in Newly Diagnosed Acute Myeloid Leukemia Patients

Introduction With the introduction of FLT3 inhibitors in recent years, the presence of FLT3 mutation in newly diagnosed acute myeloid leukemia (AML) patients has become more important. it was observed that the addition of Midostaurin to remission induction and consolidation chemotherapy, brought a 22% reduction in mortality risk in patients with FLT3 mutation. In the light of the above information, we aimed to share our experience regarding the use of Midostaurin in our newly diagnosed AML patients.&#x0D; Methods The data of 20 patients who were diagnosed with AML between April 2020 and November 2020 in the Hematology Department of Bursa City Hospital and who were eligible for standard remission induction therapy were evaluated retrospectively. Standard remission induction chemotherapy consisting of a combination of cytosine arabinoside (ARA-C) and an anthracycline was applied to patients. Consolidation treatment with high dose ARA-C was given to patients in remission; Midosaturin 100 mg was added to High-dose ARA-C between days 8 and 21 in patients with FLT3 mutation.&#x0D; Results: Twenty patients included in the study, 14 were men and 6 were women. There were six patients with the FLT3 mutation. Five patients with FLT3 mutation were male and one was female. The mean age of the patients is 53.8; the mean age of the patients with FLT 3 mutation was 49.8 years. Two patients with FLT 3 mutation died due to sepsis during remission induction treatment; In Four patients, complete response was obtained with remission induction therapy. Four patients had been given midostaurin with high dose ARA-C and referred for bone marrow transplantation. &#x0D; Conclusion The use of FLT3 inhibitors in combination with chemotherapeutic agents in the treatment of AML patients with FLT3 mutation is considered as a standard. However, it takes a certain amount of time for the FLT3 mutation to result. For this reason, Midostaurin treatment could be added not during remission induction but consolidation. Although our study included a limited number of patients it shows the importance of Midostaurin treatment in newly diagnosed AML patients with FLT3 mutation as real-world data.

FLT3 Mutations in Acute Myeloid Leukemia: Key Concepts and Emerging Controversies.

The FLT3 receptor is overexpressed on the majority of acute myeloid leukemia (AML) blasts. Mutations in FLT3 are the most common genetic alteration in AML, identified in approximately one third of newly diagnosed patients. FLT3 internal tandem duplication mutations (FLT3-ITD) are associated with increased relapse and inferior overall survival. Multiple small molecule inhibitors of FLT3 signaling have been identified, two of which (midostaurin and gilteritinib) are currently approved in the United States, and many more of which are in clinical trials. Despite significant advances, resistance to FLT3 inhibitors through secondary FLT3 mutations, upregulation of parallel pathways, and extracellular signaling remains an ongoing challenge. Novel therapeutic strategies to overcome resistance, including combining FLT3 inhibitors with other antileukemic agents, development of new FLT3 inhibitors, and FLT3-directed immunotherapy are in active clinical development. Multiple questions regarding FLT3-mutated AML remain. In this review, we highlight several of the current most intriguing controversies in the field including the role of FLT3 inhibitors in maintenance therapy, the role of hematopoietic cell transplantation in FLT3-mutated AML, use of FLT3 inhibitors in FLT3 wild-type disease, significance of non-canonical FLT3 mutations, and finally, emerging concerns regarding clonal evolution.

Synergistic Effect of SGN-CD33A and FLT3 Inhibitors in FLT3/ITD Acute Myeloid Leukemia

The prognosis of acute myeloid leukemia (AML) is very poor, with overall survival rates of approximately 50% in children and 25% in adults, despite reaching the limits of conventional cytotoxic chemotherapy. Therapeutic targeting in AML through either surface antigens or aberrantly activated cell signaling pathways are two emerging targeted treatment strategies in efforts to further improve outcomes. FLT3/ITD mutations are among the most common somatic mutations in AML and confer an inferior prognosis. Outcomes for this group of patients have recently been shown to be improved with targeting mutant FLT3 with FLT3 inhibitors (FLT3i). Patients with FLT3/ITD mutant AML express high levels of CD33, and the benefit of CD33 targeted agents in this group of patients has been demonstrated. Thus, we sought to investigate the combination strategy of FLT3i with SGN-CD33A (vadastuximab tarline), a CD33-directed antibody drug conjugate (ADC), in FLT3/ITD AML.Combination experiments with 6 FLT3 inhibitors (midostaurin, quizartinib, crenolanib, gilteritinib, AMG925, G-749) and SGN-CD33A were performed in 2 FLT3/ITD positive cell lines (MV4-11, MOLM-13). Five non-FLT3 mutant cell lines (Kasumi1, KG-1, ME-1, TF1, THP1) as well as normal hematopoietic cells (CD34+ HSCs, megakaryocytes) with and without serum were used as controls. In each pairing, a total of 240 individual drug combinations of SGN-CD33A and FLT3i were evaluated at doses spanning 4 and 6 log-folds respectively. Across the matrix of dose combinations, we compared observed cytotoxicity against 3 standard additivity models (Loewe, Bliss, and Highest Single Agent (HSA)) and summarized each experiment using metrics gauging the degree of synergy or antagonism observed based on those additive models. The combination was also evaluated with xenograft experiments. Following transplantation with MOLM-13 cells, mice received no treatment or treatment with FLT3i, SGN-CD33A, or the combination and survival was evaluated using Kaplan-Meier methods.We performed a total of 88 in vitro cell line experiments, 25 in FLT3/ITD positive cell lines. Aggregate statistics over all dose combinations showed significant synergistic effects of the SGN-CD33A/FLT3i combinations in FLT3/ITD mutant cells. Importantly, no synergistic effects were observed in any of the non-mutant cell lines. Moreover, in most of the experiments in FLT3/ITD-positive cell lines the dose combination producing the highest synergistic effect was within one log of the IC50 for both SGN-CD33A and the FLT3i (Fig 1A). These findings indicate that synergy was achieved at clinically meaningful doses. We subsequently evaluated the effect of the combination in vivo and following transplantation of MOLM-13 cells, xenografts were treated with either quizartinib alone, SGN-CD33A alone, quizartinib+SGN-CD33A, or no treatment. Subjects treated with SGN-CD33A/FLT3i combination had improved survival compared to the untreated or single agent mice (Figure 1B).Patients with FLT3/ITD mutations have a poor prognosis, despite intensive therapy including hematopoietic stem cell transplant. Importantly, this group of patients harbors 2 potential therapeutic targets, namely aberrant FLT3 activation and high CD33 surface expression. We have shown combining FLT3 inhibitors with the CD33 directed ADC SGN-CD33A results in a synergistic effect with increased cell killing and prolonged survival in xenografts. This synergistic effect was most pronounced at doses on the lower range of SGN-CD33A, suggesting that a lower dose of CD33-directed ADCs could be used thus mitigating excess hematopoietic toxicity. These treatments have little overlap in toxicity profiles suggesting that they can be safely combined in patients, and further clinical investigation of this strategy is warranted. [Display omitted] DisclosuresThurman:Seattle Genetics: Employment, Equity Ownership. Rohm:Seattle Genetics: Employment, Equity Ownership. Biechele:Seattle Genetics: Employment, Equity Ownership. Arthur:Seattle Genetics: Employment, Equity Ownership.

Abstract 834: FLT3 inhibitors and PP2A-activating drugs synergistically induce cytotoxicity in AML cells with FLT3-ITD through enhanced Pim-1 and Myc proteasomal degradation

Abstract Background: In 30% of acute myeloid leukemia (AML) patients, the presence of fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) causes constitutive FLT3 signaling, and these patients relapse rapidly following response to chemotherapy. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to its proliferative and antiapoptotic effects and also phosphorylates and stabilizes it in a positive feedback loop. FLT3 activation also inhibits the tumor-suppressor protein phosphatase 2A (PP2A), causing stabilization of Pim-1 protein. FLT3 inhibitors and PP2A-activating drugs (PADs) are known to synergistically induce cytotoxicity in cells with FLT3-ITD, and here we studied mechanisms underlying this effect. Methods: Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 cells, with FLT3-ITD, were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and the PAD fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Apoptosis was measured by Annexin V/propidium iodide staining. Drug combination indexes were determined by the Chou-Talalay method with CompuSyn software. Pim-1 kinase, Myc, MycSer62, MycThr58, STAT5, phospho-STAT5, PP2A and phospho-PP2A levels were measured by immunoblotting. Cycloheximide was used to assess protein stability. Results: Concurrent treatment with ASP2215 or AC220 and FTY720 decreased growth and enhanced apoptosis of cells with FLT3-ITD, relative to single drugs, and produced synergistic cytotoxicity. Concurrent treatment sequentially decreased expression of the 44 kDa Pim-1 isoform (Pim-1L) and then c-Myc, via decreased Pim-1L and c-Myc protein stability. Co-incubation with the proteasome inhibitor MG-132 prevented drug-induced degradation and restored Pim-1L and Myc expression in cells co-treated with FLT3 inhibitor and PAD. Drug-induced Myc destabilization/proteasomal degradation was associated with decreased MycSer62 and increased MycThr58 levels. Treatment with the pan-Pim kinase inhibitor AZD1208 prior to ASP2215 or AC220 and FTY720 treatment markedly increased MycThr58 levels but did not affect MycSer62, consistent with both Pim-dependent and -independent effects of FLT3 inhibition and PP2A activation on c-Myc expression. Conclusions: Combined FLT3 inhibitor and PAD treatment synergistically induces cytotoxicity in cells with FLT3-ITD by enhancing Pim-1 and Myc proteasomal degradation, and effect on Myc is both Pim-1-dependent and -independent. The data support in vivo testing of FLT3 inhibitor and PAD combinations. Citation Format: Mario Scarpa, Shivani Kapoor, Sikemi Ibikunle, Andre Rush, Patrick R. Baldwin, Danilo Perrotti, Maria R. Baer. FLT3 inhibitors and PP2A-activating drugs synergistically induce cytotoxicity in AML cells with FLT3-ITD through enhanced Pim-1 and Myc proteasomal degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 834.

Abstract B052: FLT3-ITD activation mediates resistance to the BCL-2 selective antagonist, venetoclax, in FLT3-ITD mutant AML models

Abstract 20-25% of acute myeloid leukemia (AML) patients harbor FLT3 internal tandem duplication (ITD) mutations, which confer poorer prognosis. FLT3-ITD is a constitutively activated kinase with differential signaling compared to wild type (WT) FLT3. Notably, FLT3-ITD activates STAT5, which can regulate the prosurvival proteins BCL-XL and MCL-1. Venetoclax, a potent, selective inhibitor of the prosurvival protein BCL-2, demonstrated monotherapy activity in relapsed/refractory AML (ORR 19%); however, no activity was seen in FLT3 mutant cases. BCL-XL and MCL-1 are known venetoclax resistance factors and targeting their regulation in combination with venetoclax may enhance cell death. Based on this hypothesis, we interrogated the combination of venetoclax and quizartinib, a potent FLT3 inhibitor, in FLT3-ITD+ AML models. In vitro, quizartinib reduced expression of BCL-XL and MCL-1, but not BCL-2, in FLT3-ITD+ cells (Molm13 and MV4;11). However, quizartinib had no effect on BCL-XL or MCL-1 expression in FLT3 WT cells (HL60 and OCI-AML3). Combination treatment with venetoclax led to significant reduction in growth and increased apoptosis in FLT3-ITD+ cells compared to single agents while FLT3 WT cells were not sensitive to quizartinib alone or in combination with venetoclax. Consistent with FLT3-ITD regulating BCL-XL and MCL-1, cotreatment of quizartinib with either BCL-XL or MCL-1 specific inhibitors had no combination effect in FLT3-ITD+ cells. Additionally, quizartinib and venetoclax were assessed in vivo utilizing MV4;11 and Molm13 orthotopic xenograft models. Mice were treated with venetoclax (100 mg/kg), quizartinib (5 mg/kg), or the combination of both drugs for 21 continuous days. Quizartinib treatment significantly increased survival compared to the vehicle group in both xenograft models (56 days vs. 26 days and 32 days vs. 14 days for MV4;11 and Molm13 xenografts, respectively). However, venetoclax only marginally improved survival (34 days vs. 26 days) in the MV4;11 model and was not efficacious in the Molm13 model. Despite lack of single-agent activity, addition of venetoclax to quizartinib led to a further improvement in survival when compared to quizartinib (91 days vs. 56 days and 40 days vs. 32 days in MV4;11 and Molm13 xenografts, respectively). These data underscore the dependency of these models on FLT3-ITD for survival and its regulation of survival pathways that confer resistance to venetoclax, suggesting FLT3-ITD could be a predictive biomarker of venetoclax resistance. To determine mechanisms of venetoclax resistance mediated by FLT3-ITD, mutant cells were treated with PI3K, MEK or JAK2 kinase selective inhibitors alone or in combination with venetoclax. Each pathway inhibitor combined with venetoclax to reduce cell growth compared to single agents. This observation suggests that multiple pathways may contribute to venetoclax resistance rather than a single pathway dependency to confer the prosurvival effects of FLT3-ITD. Additional mechanistic studies are under way to define the role of each pathway in regulating survival. Together, our data demonstrate that FLT3-ITD inhibition combines with venetoclax in vitro and in vivo in FLT3-ITD+ AML models. These results provide a strong mechanistic rationale to support clinical investigation of FLT3 inhibitors in combination with venetoclax to treat FLT3-ITD+ AML. Citation Format: Raghuveer Mali, Elisabeth A. Lasater, Kelly Doyle, Ritu Malla, Erwin Boghaert, Andrew Souers, Joel Leverson, Deepak Sampath. FLT3-ITD activation mediates resistance to the BCL-2 selective antagonist, venetoclax, in FLT3-ITD mutant AML models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B052.

Abstract 1087: CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD

Abstract Introduction: CPX-351 (Vyxeos) is a liposomal combination of cytarabine and daunorubicin at a synergistic 5:1 molar ratio. Recently, CPX-351 has been shown to be significantly more effective than the 7+3 standard of care chemotherapy in treating high-risk AML patients, including patients with the FLT3-ITD mutation. We previously treated primary patient samples with CPX-351 ex vivo and found that FLT3-ITD+ samples were significantly more sensitive to CPX-351 and showed enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in an activation of liposome uptake pathways, leading to increased sensitivity to CPX-351 and ultimately cell death. Furthermore, we examined the effect of combining CPX-351 with existing FLT3 inhibitors (e.g. quizartinib and midostaurin). Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains an activating FLT3 mutation) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We evaluated cell viability using a colorimetric assay and measured intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry. Additionally, we analyzed the synergy of exposing these cell lines to CPX-351 and FLT3 inhibitors in combination and at different dose schedules, followed by measuring cell viability and daunorubicin fluorescence. Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351 and exhibited increased drug uptake compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, suggesting that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses. Conclusions: These data provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351. This is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling. Citation Format: David K. Edwards, Nathalie Javidi-Sharifi, Angela Rofelty, Max Gordon, Riley Roth-Carter, Paul Tardi, Lawrence Mayer, Jeffrey W. Tyner. CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2017-1087

Loss of Src-like Adaptor Protein 2 Expression Increases the Transforming Potential of Oncogenic FLT3-ITD

The receptor tyrosine kinase FLT3 is found to be a mutated oncogene in hematological malignancies including acute myeloid leukemia (AML). FLT3 inhibitors in combination with chemotherapy display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, targeting signaling proteins downstream of FLT3 can be an alternative approach for the treatment of patients carrying mutant FLT3. Activated FLT3 is constitutively phosphorylated on several tyrosine residues. These tyrosine residues facilitate association of SH2 domain-containing signaling proteins. By using a panel of SH2 domain-containing proteins we identified SLAP2 as a potent interaction partner of FLT3. The interaction in between FLT3 and SLAP2 occurs when FLT3 is activated and an intact SH2 domain of SLAP2 is required for the interaction. SLAP2 associates with FLT3 mainly through its SRC binding sites and expression of SLAP2 inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. By analysis of patient expression data, we found that loss of SLAP2 expression correlates with poor prognosis of AML patients carrying FLT3-ITD. SLAP2 inhibits FLT3-mediated downstream signaling such as activation of AKT, ERK, p38 and STAT5. Inhibition is partially mediated through ubiquitination-mediated degradation of FLT3. Taken together our current study demonstrates that SLAP2 is an important regulator of FLT3-mediated oncogenic signaling and thus modulation of the SLAP2 expression levels can be an alternative approach for the treatment of FLT3-ITD positive malignancies. DisclosuresNo relevant conflicts of interest to declare.

Effective Combination of CPX-351 with FLT3 Inhibitors in AML Blasts Harboring the FLT3-ITD Mutation

Introduction: CPX-351 (Vyxeos), a liposomal combination of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio, has recently been shown to be significantly more efficacious than the standard of care, 7+3 administration regimen of free (non-liposomal) cytarabine + daunorubicin in treating elderly high-risk AML patients, including patients with the FLT3-ITD mutation. We previously exposed AML blasts from primary patient samples to CPX-351 ex vivo and found that samples harboring FLT3-ITD were significantly more sensitive to CPX-351 and also demonstrated enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in upregulated liposome uptake pathways, leading to increased cell death and overall sensitivity to CPX-351. Furthermore, we wanted to examine the effect of combining CPX with existing FLT3 inhibitors (e.g. quizartinib or midostaurin).Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains mutant, activated FLT3) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We measured cell viability and intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry and using a fluorescent plate reader. Additionally, we performed synergy analyses on the cytotoxic activity in these cells, adding CPX-351 and FLT3 inhibitors in combination at different exposure sequences followed by measurement of cell viability and daunorubicin fluorescence.Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351, and exhibited increased drug uptake, compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, indicating that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, in synergy studies we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses.Conclusions: These results provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351, which is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling. DisclosuresTardi:Jazz Pharmaceuticals: Employment. Mayer:Jazz Pharmaceuticals: Employment.

Perspectives for therapeutic targeting of gene mutations in acute myeloid leukaemia with normal cytogenetics.

The acute myeloid leukaemia (AML) genome contains more than 20 driver recurrent mutations. Here, we review the potential for therapeutic targeting of the most common mutations associated with normal cytogenetics AML, focusing on those affecting the FLT3, NPM1 and epigenetic modifier genes (DNMT3A, IDH1/2, TET2). As compared to early compounds, second generation FLT3 inhibitors are more specific and have better pharmacokinetics. They also show higher anti-leukaemic activity, leading to about 50% of composite complete remissions in refractory/relapsed FLT3-internal tandem duplication-mutated AML. However, rapid relapses invariably occur due to various mechanisms of resistance to FLT3 inhibitors. This issue and the best way for using FLT3 inhibitors in combination with other therapeutic modalities are discussed. Potential approaches for therapeutic targeting of NPM1-mutated AML include: (i) reverting the aberrant nuclear export of NPM1 mutant using exportin-1 inhibitors; (ii) disruption of the nucleolus with drugs blocking the oligomerization of wild-type nucleophosmin or inducing nucleolar stress; and (iii) immunotherapeutic targeting of highly expressed CD33 and IL3RA (CD123) antigens. Finally, we discuss the role of demethylating agents (decitabine and azacitidine) and IDH1/2 inhibitors in the treatment of AML patients carrying mutations of genes (DNMT3A, IDH1/2 and TET2) involved in the epigenetic regulation of transcription.

Selective FLT3 Inhibitor FI-700 Neutralizes Mcl-1 and Enhances p53-Mediated Apoptosis in Acute Myeloid Leukemia (AML) Cells with Activating Mutations of FLT3 Via Mcl-1/Noxa Axis.

Abstract 2597Poster Board II-573 Introduction:Activating mutations of the Fms-like tyrosine kinase-3 gene (FLT3) occur in approximately 30–40% of acute myeloid leukemia (AML) patients. FLT3 mutations confer numerous oncogenic properties, including dysregulated proliferation, resistance to apoptosis and a block in differentiation. FLT3 mutations result in abnormal activation of the downstream pathways, including signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase kinase (Mek)/extracellular signal–regulated kinase (Erk) and phosphatidylinositol-3 kinase (PI3K)/Akt. Activation of these downstream effectors has been thought to allow leukemia cells to evade apoptosis. Targeting of FLT3 mutations is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML.Results:FI-700 induced G1-phase cell cycle arrest and apoptosis as evidenced by increased sub-G1 DNA content and phosphatidylserine externalization in FLT3/ITD MOLM-13 (FLT3-ITD, wild-type (wt)-p53) and MV4-11NR (FLT3-ITD, mutated-p53) AML cells. FI-700 did not affect cell cycle distribution patterns nor did it induce apoptosis in FLT3/WT OCI-AML-3 (FLT3/WT, wt-p53) and HL-60 (FLT3/WT, del (del)-p53). Wt-p53 MOLM-13 and OCI-AML-3 cells were susceptible to Nutlin-induced apoptosis. FI-700 augmented Nutlin-induced Bax activation, mitochondrial membrane potential (MMP) loss, caspase-3 activation and phosphatidylserine externalization in MOLM-13 cells. FI-700 rapidly reduced Mcl-1 levels in FLT3/ITD cells, mainly by enhancing proteasomal Mcl-1 degradation. Levels of other Bcl-2 family proteins examined did not change significantly. Mcl-1 levels were only modestly reduced upon Nutlin treatment. The FI-700/Nutlin-3 combination profoundly reduced Mcl-1 levels. Immunoprecipitation/ immunoblotting results suggested that the drug combination results in a profound decrease in Mcl-1-bound Bim. FI-700 enhanced doxorubicin-induced apoptosis in FLT3/ITD MOLM-13 and MV4-11NR cells, suggesting that FI-700 can enhance both the p53-dependent and the p53-independent apoptotic effects of doxorubicin. Finally, cooperative apoptotic effects of FI-700/Nutlin-3 were seen in primary AML cells with FLT3/ITD. Conclusion:FLT3 inhibition by FI-700 immediately reduces anti-apoptotic Mcl-1 levels and enhances Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/ITD-expressing AML cells via the Mcl-1/Noxa axis. FLT3 inhibition, in combination with p53-inducing agents, might represent a potential therapeutic approach in AML with FLT3/ITD. Disclosures:No relevant conflicts of interest to declare.

3,4-Diarylmaleimides Effectively Inhibit Proliferation of FLT3-ITD-Positive Leukemic Cells, Induce Apoptosis and Show Additive Effects in Combination with Chemotherapeutic Drugs.

Internal tandem duplication (ITD) mutations of FLT3 are present in leukemic blasts of approximately 30% of AML patients. ITD-mutations of FLT3 confer a worse prognosis and decreased overall survival. Therefore, FLT3-tyrosine kinase is considered an attractive drug target in AML and several FLT3-tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials (CEP701, MLN518, Sorafenib, PKC412). However, using these drugs as monotherapy, against the setting of remarkable efficacy has emerged the problem of short duration of remission indicating rapid development of secondary resistance. In addition, up to 30% of patients may show primary resistance to currently available FLT3-TKIs. Therefore, to overcome these limitations further clinical development requires novel approaches (e.g. combination with chemotherapy) and development of more efficacious FLT3-TKI. Here, we investigated two novel TKIs (3,4-Diarylmaleimides, compounds 53 and 80) previously described as angiogenesis inhibitors (Peifer, J. Med. Chem. 2006), to determine their mechanism of action and efficacy in FLT3-ITD transfected 32D cells as well as in primary ITD-positive AML blasts. Western Blotting experiments of 32D-FLT3-ITD cells confirmed dose dependent inhibition/dephosphorylation of downstream targets as STAT5 (pTyr694/699), ERK (pThr202/pTyr204) and AKT (pSer473) upon incubation with either compound using nanomolar concentrations. Both compounds (cpd.) showed induction of apoptosis with an IC50 of 350nM (cpd. 53) and 800nM (cpd. 80), respectively, as detected by sub G1-fraction in cell cycle analysis. Using colony assays we defined a possible therapeutic window of these inhibitors: Bone-marrow cells of healthy donors were incubated with increasing concentrations of both inhibitors revealing no significant inhibition of colony formation up to a dose of 5 μM. As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both cpds. in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Within a range of 1–50nM DNR and 0.3–10μM Ara-C, both cpds. showed additive effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays. Primary AML blasts harboring the FLT3-ITD length mutation were also investigated to determine the efficacy in patient material. These experiments revealed apoptosis rates of 10–30% upon incubation with 3,4-Diarylmaleimides for 72 hours. Cellular up-take of cpd. 53 in primary AML blasts could be demonstrated within 5 minutes of incubation using FACS analysis. Increased fluorescence (200–400% of baseline values) could be detected for more than 24 hours suggesting stable intracellular concentration of the inhibitor in AML blasts within this time period. To evaluate signaling targets of 3,4-Diarylmaleimides in detail, we applied a kinomics peptide chip containing a set of 960 kinase substrates (human sequences only) in triplicate on a slide. Phosphorylation of peptide substrates was detected by autoradiography. Results of evaluation will be presented. In conclusion, 3,4-Diarylmaleimides show efficacy in FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Currently, experiments evaluating toxicity in murine models are planned to explore a possible clinical application.

Different FLT3 Inhibitors Display Limited, Non-Overlapping Profiles of Specific FLT3-ITD Resistance Mutations in a Cell-Based Screen.

Mutations causing resistance to therapeutic kinase inhibition can be identified in target kinases in various malignant diseases, such as Bcr-Abl in CML, FLT3-ITD in AML, cKit in GIST, EGFR in NSCLC, and FIP1L1-PDGFRalpha in HES/CEL. Thus, mutations in target kinases constitute a general mechanism of resistance to therapeutic kinase inhibiton. It has been shown that sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. We therefore intended to determine, whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3-ITD expressing cell lines resistant to the FLT3 inhibitors su5614 and PKC412. The frequency of resistant clones per million cells in the presence of inhibitor at 10 times the IC50 was 0.17 for su5614 (1,5?M) and 0 with PKC412 (100nM). When FLT3-ITD transformed cells were mutagenized with ENU (ethylnitrosourea) prior to addition of inhibitors, the frequency of resistant clones increased to 2.33 with su5614, and to 0.34 with PKC412. When we analyzed FLT3-ITD TK1 and TK2 for mutations that may confer inhibitor resistance, we found mutations in 127 out of 179 cell clones (71%) with su5614. Nine different exchanges affecting six positions in TK2 were identified, within or shortly behind the FLT3-ITD A-loop, with exchanges of D835 predominating. We did not detect exchanges affecting TK1. With PKC412, four out of 26 resistant clones (15%) contained mutations. In contrast to su5614, no mutations of TK2 were identified. However, three different amino acid exchanges emerging with PKC412 exclusively affected N676, which is located in TK1 of the FLT3-ITD split kinase domain. This is in line with the observation of a FLT3-ITD N676K exchange identified in a single AML patient with clinical resistance to PKC412 (Heidel et al, 2006). When expressed in cell lines, the identified TK2 A-loop mutations shifted dose-response curves to higher concentrations of su5614, but did not affect response to PKC412, su11248 or sorafenib. However, Y842D caused a strong resistance towards sorafenib, but not to su11248 or PKC412. Recent results also demonstrate a differential response of TK1 N676 exchanges towards different FLT3 inhibitors. We are currently analyzing su11248- and sorafenib- resistant FLT3-ITD clones for specific resistance mutations. Our results demonstrate that cell-based resistance screening is a useful and valid tool for prediction of resistance mutations to kinase inhibitors. In contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib and dasatinib, which display highly overlapping resistance profiles, FLT3 kinase inhibitors may generate distinct, non-overlapping profiles. This opens the possibility of using combined FLT3 inhibitor therapy in AML to prevent resistance due to FLT3-ITD kinase domain mutations.

Bis(1H-indol-2yl)methanones Are Effective Inhibitors of Mutated FLT3 Tyrosine Kinase, Overcome Resistance to PKC412A In Vitro and Show Synergy with Chemotherapy.

Inhibition of mutated FLT3 receptor tyrosine kinase has become a promising therapeutic strategy in AML. Currently, a phase III trial testing the tyrosine kinase inhibitor (TKI) PKC412A in combination with induction chemotherapy is under way. Development of resistance to PKC412A mediated by secondary FLT3 mutations has been described recently (Heidel et al., Blood 2006). This observation may have an increasing impact on duration of response in future clinical trials. Herein we investigated two novel TKIs (methanones) to determine their efficacy on FLT3-ITD transfected 32D cells as well as on primary ITD positive AML blasts.The bis(1H-indol-2-yl)methanone compounds Cpd.98 and Cpd.102 (Mahboobi et al. J. Med. Chem. 2006) that have recently shown efficacy in proliferation assays using FLT3-ITD transfected cells were characterized. Western Blotting experiments of murine 32D-FLT3-ITD cells confirmed dose dependent dephosphorylation of FLT3-ITD and its downstream target STAT5 upon incubation with Cpd.98 and Cpd.102, respectively. To define a non-myelotoxic range of these inhibitors, bone-marrow (BM) cells of healthy donors were tested with different methanone concentrations to determine the rate of apoptosis (sub G1 fraction in cell cycle analysis). No significant increase in apoptosis was seen up to 3μM of inhibitor concentration for either compound.Additionally, colony assays of healthy BM-cells also showed no significant reduction of colony formation up to 3μM. To determine the efficacy of these compounds in PKC412A-resistant cells, transfected 32D cells containing the previously described resistance mediating mutation (32D-FLT3-ITD-N676K) were also tested at different concentrations for induction of apoptosis. Resistance in these cells could be overcome by 300nM (Cpd.102) and 1μM (Cpd.98), respectively.As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both compounds in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Both compounds showed synergistic effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays.Primary AML blasts harbouring the FLT3-ITD length mutation were also incubated with both compounds to determine the efficacy in patient material. These experiments revealed apoptosis rates of up to 30% upon incubation for 72 hours. In conclusion bis(1H-indol-2-yl)methanone compounds overcome resistance mediated by the previously described FLT3-ITD mutation N676K in vitro and show efficacy on FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Experiments evaluating toxicity in murine models are currently planned to explore a possible clinical application.

The effects of lestaurtinib (CEP701) and PKC412 on primary AML blasts: the induction of cytotoxicity varies with dependence on FLT3 signaling in both FLT3-mutated and wild-type cases

AbstractThe receptor tyrosine kinase FLT3 is a promising molecular therapeutic target in acute myeloid leukemia (AML). Activating mutations of FLT3 are present in approximately one-third of patients, while many nonmutants show evidence of FLT3 activation, which appears to play a significant role in leukemogenesis. We studied the effects of lestaurtinib (CEP701) and PKC412, 2 small molecule inhibitors of FLT3, on 65 diagnostic AML blast samples. Both agents induced concentration-dependent cytotoxicity in most cases, although responses to PKC412 required higher drug concentrations. Cytotoxic responses were highly heterogeneous and were only weakly associated with FLT3 mutation status and FLT3 expression. Importantly, lestaurtinib induced cytotoxicity in a synergistic fashion with cytarabine, particularly in FLT3 mutant samples. Both lestaurtinib and PKC412 caused inhibition of FLT3 phosphorylation in all samples. Translation of FLT3 inhibition into cytotoxicity was influenced by the degree of residual FLT3 phosphorylation remaining and correlated with deactivation of STAT5 and MAP kinase. FLT3 mutant and wild-type cases both varied considerably in their dependence on FLT3 signaling for survival. These findings support the continued clinical assessment of FLT3 inhibitors in combination with cytotoxic chemotherapy: Entry to future clinical trials should include FLT3 wild-type patients and should remain unrestricted by FLT3 expression level.