FLT3 Internal Tandem Duplication Mutations Research Articles

Impact of FLT3 Mutation Clearance after Front-Line Treatment with Gilteritinib Plus Azacitidine, or Gilteritinib or Azacitidine Alone in Patients with Newly Diagnosed AML: Results from the Phase 2/3 Lacewing Trial

Impact of FLT3 Mutation Clearance after Front-Line Treatment with Gilteritinib Plus Azacitidine, or Gilteritinib or Azacitidine Alone in Patients with Newly Diagnosed AML: Results from the Phase 2/3 Lacewing Trial

Multi-Recombinase Mouse Models of Flt3-Driven Leukemia Identifies Distinct Trajectories of Mutational Cooperativity and Leukemic Transformation

Genomic studies in acute myeloid leukemia (AML) have generated a near complete catalogue of genes mutated at varying frequencies both across patients and in individual leukemias. The high variability of mutation burden within a given leukemia is suggestive of a stepwise evolutionary process composed of early, clonal, mutations and subsequent subclonal events. The receptor tyrosine kinase, FLT3, is the most commonly mutated gene in AML, with mutations frequently manifesting as internal tandem duplications (ITDs) in the juxtamembrane domain leading to constitutive kinase activation. Although FLT3 is commonly a subclonal mutational event, FLT3 ITD mutations portend a poor prognosis particularly when combined with DNMT3A and NPM1, earlier mutations that drive clonal expansion. Notwithstanding its role as a subclonal driver, previous preclinical FLT3 models have utilized retroviral overexpression or germline mutant expression at the endogenous locus precluding accurate temporal modeling of disease. These efforts have prohibited evaluation of FLT3 mutational acquisition in the context observed in AML patients.Here, we report the development of an endogenously targeted, Flp inducible, Flt3 ITD mouse allele which can be somatically activated subsequent to cooperating disease alleles. When activated with a tamoxifen inducible FlpoER, Flt3 mutant mice developed rapid leukocytosis peaking at 4-6 weeks post activation and resolving by 8-10 weeks, a finding not previously observed in constitutive models. This leukocytosis was disproportionately monocytic and accompanied by pronounced anemia and thrombocytopenia. Long term, these mice develop a myeloproliferative disease , reminiscent of previously reported constitutive alleles. In competitive transplantation studies, Flt3 mutant cells initiated disease and outcompeted wild-type cells. Despite this competitive advantage, disease was incapable of transplanting into secondary recipients. We further observed a non-cell autonomous depletion of SLAM+ LSKs suggesting the Flt3 mutant cells cannot propagate disease in self-renewing stem cells.To evaluate how this allele influenced leukemic evolution we crossed this Flt3 ITD allele to a Flp inducible Npm1 c mouse where a pulse of tamoxifen simultaneously activated both alleles. The combination of mutant Npm1 and Flt3 resulted in progressive leukocytosis which did not resolve. Within 6 weeks of mutational activation, these mice developed a lethal AML with robust anemia, thrombocytopenia, leukocytosis and expanded cKIT+ blasts in the blood. RNA-sequencing and immunophenotyping by CyTOF revealed distinct patterns of differentiation, gene-expression and downstream signaling.In an effort to model sequential mutational acquisition, we crossed the Flp Flt3 ITD allele to a Cre-inducible Dnmt3a R878H. Cre mRNA was electroporated into lineage negative bone marrow cells to activate the Dnmt3a R878H allele and transplanted into lethally irradiated recipients. Four weeks post engraftment, Flt3 ITD was activated with a pulse of tamoxifen. In contrast to the Flt3-Npm1 model, we observed an increase and subsequent decrease in WBC similar to the kinetics observed in Flt3 ITD only mice. However, by 20 weeks we observed a robust and consistent increase in WBC accompanied by an emergence of cKIT+ cells in the blood. Histopathology indicated that >50% of mice expressing both alleles in sequence developed AML marked by increased blasts in the marrow, with moderate anemia and thrombocytopenia compared to the Flt3-Npm1 models. Critically, in contrast to Flt3 ITD only mice, acquisition of the Flt3 ITD in Npm1 or Dnmt3a mutant HPSCs induced fully transplantable AML with immunophenotypic characteristics seen in human AML with these same genotypes. Collectively these results demonstrate that different co-occurring mutations are capable of transforming Flt3 ITD mutant cells, albeit with distinct latencies and mechanisms of cooperativity.In summary, our studies utilizing novel multi-recombinase models of leukemogenesis reveal new insights into the early phase of oncogene activation, and how cooperating alleles influence this response. This inducible Flt3 ITD allele represents a significant advance in modeling clonal evolution in myeloid malignancies and provides a critical isogenic platform for preclinical development of novel leukemia therapeutic regimens. [Display omitted] DisclosuresBowman: Mission Bio: Honoraria, Speakers Bureau. Xiao: Stemline Therapeutics: Research Funding. Miles: Mission Bio: Honoraria, Speakers Bureau. Trowbridge: Fate Therapeutics: Patents & Royalties; H3 Biomedicine: Research Funding. Levine: Amgen: Honoraria; Lilly: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Ajax: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Zentalis: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Incyte: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Gilteritinib Inhibits Glutamine Uptake and Utilization in FLT3-ITD-Positive AML.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib's antileukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. In addition, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Finally, we found that the glutaminase inhibitor CB-839 enhanced antileukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD-positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.

Abstract 1050: Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is the most common and aggressive acute leukemia in adults, with a 25% 5-year survival rate. FLT3 is the most frequently mutated gene in AML. About 25% of AML patients harbor FLT3 internal tandem duplication (ITD) mutations, and about 8 % of patients harbor FLT3 tyrosine kinase domain (TKD) mutations. Both FLT3-ITD and FLT3-TKD mutations constitutively activate the protein, which causes poor survival. Gilteritinib is a highly potent and selective oral FLT3 inhibitor recently approved by the FDA. Although gilteritinib showed strong single agent activity in AML patients with FLT3 mutations, the development of gilteritinib resistance limits the long-term efficacy of this treatment, indicating that combination therapy may be advantageous for AML patients with FLT3 mutations. FLT3 mutations are known to be anti-apoptotic. Myeloid cell leukemia-1 (MCL-1), an anti-apoptotic protein, expressed in a large percentage of the AML patient population, plays a critical role in AML cell survival and drug resistance. AMG 176 is a potent, selective and orally bioavailable MCL-1 inhibitor, which induces rapid commitment to apoptosis in AML. Here we demonstrated that AMG 176 and gilteritinib combination treatment synergistically targeted FLT3-ITD mutated AML. A strong synergistic effect (Combination index <0.2) was observed in two FLT3-ITD mutated AML cell lines treated with the AMG 176 plus gilteritinib combination. Increased Bcl-2-like protein 11 (BIM) expression and decreased BIM/MCL-1 interaction was observed under the combination treatment, coupled with a significant increase in apoptosis. In the MV4-11 AML xenograft model, mice treated with two cycles of AMG 176 (2 days on 5 days off) at 30 mg/kg or with 3 mg/kg gilteritinib daily showed 76% or 74% tumor growth inhibition respectively. A statistically significant increase in efficacy was observed in the combination treatment group, with 92.8% tumor regression, including 50% of mice being tumor free. No significant difference in body weight was found between groups. Our results show that AMG 176 plus gilteritinib treatment was effective and well tolerated in mouse models of AML, which supports further evaluation in a clinical setting. Citation Format: Xiaoyue Chen, Sean Caenepeel, Brian Belmontes, Patricia L. McElroy, Karen Rex, Tao Osgood, Paul Hughes. Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1050.

Abstract 1257: HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors

Abstract Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduces overall survival. Approximately 30 % of AML patients carry FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutations. Sustained FLT3 inhibition can result in the emergence of resistance-conferring genetic alteration in TKD domain, usually at residues D835 and F691. Therefore, acquired TKD mutations has become a critical therapeutic target in AML therapy. On the other hand, SYK is one of kinases deeply implicated in many hematologic malignancies and highly activated in FLT3 mutation AML. SYK overexpression is known to promote over transformation of FLT3 driven AML and induce resistance to FLT3 targeted therapy. In this study, we characterized HM43239, a novel FLT3 inhibitor with SYK inhibitory activity, and assessed its potential as a novel therapeutic agent to overcome the resistances against AML therapy using current FLT3 inhibitors. HM43239 is an orally active small molecule inhibitor and it exhibited sub-nanomolar potency on binding affinity for FLT3 wild type, ITD, TKDs and ITD/TKDs mutants. HM43239 potently inhibited phosphorylation of FLT3 and its downstream such as p-STAT3/p-STAT5 dose dependently in both MOLM-14 cells harboring FLT3 ITD/F691L and FLT3 ITD/D835Y. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. Moreover, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG-1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Furthermore, HM43239 showed to be exhibited good inhibitory activity against FLT3 mutated AML cell lines and effectively regress the tumors in MOLM-14 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. On the other hand, it was confirmed that HM43239 strongly inhibited the phosphorylation of SYK in the medium of co-culture of stromal cells and AML cells. As a result, HM43239 alone more effectively induced tumor regression and prolonged the survival duration of animals than an approved FLT3 inhibitor (e.g. gilteritinib) in resistant FLT3 ITD/D835Y or ITD/F691L mutated MOLM-14 xenograft mice models. These results suggest that HM43239 could overcome the resistance induced by bone marrow microenvironment in AML patients. Taken together, HM43239 showed strong anticancer activity through various in vitro and in vivo preclinical models of AML, implicating the mechanism of overcoming resistance and preventing relapse. The effect of HM43239 in human would be demonstrated in ongoing Phase I/II clinical trials (NCT03850574) to develop promising therapeutics for patients with AML. Citation Format: Inhwan Bae, Jaeyul Choi, Jiyoung Song, Joo-Yun Byun, Eunyoung Lee, Taehun Song, Yu-Yon Kim, Yesol Bak, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1257.

Abstract 1096: FMS-like tyrosine kinase 3 (FLT3) inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 in preclinical models of FLT3-mutant acute myeloid leukemia (AML)

Abstract AML accounts for 80% of acute leukemias in adults. FLT3 gene mutations are observed in approximately 30% of patients with AML and augur a poor prognosis. Despite antitumor effects of selective FLT3 inhibitors, resistance to these agents continues to pose a formidable clinical challenge. The expression of pro-survival protein BCL-2 is frequently dysregulated, conferring resistance to FLT3 inhibitors, in AML. In this study, we explored the combination of clinical stage multikinase inhibitor HQP1351 (which also targets FLT3) and BCL-2-selective inhibitor APG-2575 in preclinical models of FLT3-mutant AML. Antileukemic activity of HQP1351 was first examined using AML cell lines MV-4-11 and MOLM-13, which harbor FLT3-internal tandem duplication (FLT3/ITD) mutations. In both cell lines, HQP1351 dose-dependently induced cellular apoptosis, APG-2575 also induced apoptosis, and their combination synergistically induced this process. In particular, treatment of subcutaneous MV-4-11 xenograft tumors in nude mice with HQP1351 10 mg/kg significantly suppressed tumor growth, with a tumor growth inhibition (TGI) of 72.4%. This antitumor effect was potentiated when APG-2575 was combined with HQP1351, resulting in a TGI of 97.2%. The combination also improved survival of tumor-bearing mice with a systemic MOLM-13 model compared to either agent alone. Mechanistically, HQP1351 likely inhibited phosphorylation of FLT3 and its downstream signaling pathways, including phosphorylation of AKT, ERK1/2 and STAT5. Taken together, these findings indicate that the antileukemic activity of olverembatinib can be ascribed to on-target inhibition. Single-agent APG-2575 agent triggered cellular apoptosis, as evidenced by cleavage of caspase-3 cleavage and poly(ADP-ribose) polymerase-1 (PARP-1) activation, which are considered hallmarks of apoptosis. Consequently, APG-2575 treatment also increased expression of another antiapoptotic protein (MCL-1), upregulation of which is a pivotal mechanisms of resistance to BCL-2 inhibitors. Notably, HQP1351 substantially decreased cellular MCL-1 expression when combined with APG-2575, further enhancing apoptosis.Taken together, our data suggest that FLT3 inhibition by HQP1351 downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 to potentiate cellular apoptosis in FLT3-mutant AML. The results provide scientific rationale for clinical development of HQP1351 combined with APG-2575 in patients with FLT3-ITD-mutant AML. Citation Format: Douglas D. Fang, Hengrui Zhu, Qiuqiong Tang, Qixin Wang, Na Li, Xu Fang, Ping Min, Guangfeng Wang, Dajun Yang, Yifan Zhai. FMS-like tyrosine kinase 3 (FLT3) inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 in preclinical models of FLT3-mutant acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1096.

Haploidentical Stem Cell Transplantation Attenuates the Prognostic Impact of FLT3-ITD in Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) patients harboring the fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation are considered a particularly high risk patient subset preferentially allocated for allogeneic stem cell transplantation in first remission. Whether FLT3-ITD retains a prognostic role in haploidentical stem cell transplantation (haplo-SCT) is currently unknown.Patients and methodsTo determine whether FLT3-ITD is prognostically significant in T-cell replete haplo-SCT transplanted AML patients, we performed a comparative analysis between FLT3wt and FLT3-ITD using the multicenter registry of the acute leukemia working party of the European society for blood and marrow transplantation. Patients included in this analysis were AML patients over the age of 18 who underwent a first T-cell replete haplo-SCT in first remission.ResultsWe evaluated 293 de-novo AML patients (202 FLT3wt and 91 FLT3-ITD) transplanted in first remission with T-cell replete haplo-SCT between 2005-2016. Both groups did not differ to a significant degree in terms of patient age, donor age, performance status, cytomegalovirus donor-recipient matching, and conditioning intensity. FLT3-ITD patients were more likely to be nucleophosmin1 (NPM1) mutated as well as be in the Medical Research Council (MRC) intermediate risk cytogenetic risk category. In multivariate analysis, patients with FLT3-ITD had comparable rates of relapse incidence [Hazard ratio (HR)=1.34, confidence interval (CI) 95%, 0.67-2.7; P=0.9] and leukemia-free survival (HR=0.99, CI 95%, 0.62-1.57; P=0.9) to those of FLT3wt patients. Survival was not significantly impacted by FLT3-ITD status (HR=0.96, CI 95%, 0.58-1.59; P=0.8), nor were the incidence of non-relapse mortality (HR=0.78, CI 95%, 0.41-1.46; P=0.44), and graft versus host disease-free/relapse-free survival (HR=0.84, CI 95%, 0.54-1.28; P=0.42). A separate analysis for patients treated either on anti-thymocyte globulin (ATG) protocols or post-transplant cyclophosphamide (PTCy) did not significantly associate FLT3-ITD status with clinical outcome in either of these groups. Finally, a focused subset analysis of patients with MRC intermediate risk cytogenetics confirmed the absence of a prognostic impact of FLT3-ITD also for this group of patients.ConclusionsIn AML patients undergoing T-cell replete haplo-SCT, the FLT3-ITD mutation probably does not retain its prognostic significance, potentially indicative of haplo-SCT's capacity to overcome the negative prognostic consequences of FLT3-ITD in AML. DisclosuresNo relevant conflicts of interest to declare.

Concordance between Bone Marrow and Peripheral Blood Samples for Assessment of FLT3 Internal Tandem Duplication (ITD) Mutations: Data from Patients Screened for Participation in Quantum-R, a Global, Randomized, Open-Label, Phase 3 Study Examining the Effect of Quizartinib Monotherapy Vs Salvage Chemotherapy on Overall Survival in Patients with FLT3 - ITD - Mutated AML Who Are Refractory to or Have Relapsed

Concordance between Bone Marrow and Peripheral Blood Samples for Assessment of FLT3 Internal Tandem Duplication (ITD) Mutations: Data from Patients Screened for Participation in Quantum-R, a Global, Randomized, Open-Label, Phase 3 Study Examining the Effect of Quizartinib Monotherapy Vs Salvage Chemotherapy on Overall Survival in Patients with FLT3 - ITD - Mutated AML Who Are Refractory to or Have Relapsed

MAPKAP1 (Sin1), a Key Component of the MTORC2 Complex, Confers Resistance to Sorafenib and Correlates with Adverse Clinical Outcomes in Acute Myeloid Leukemia

Introduction: The mammalian target of rapamycin (mTOR) functions as a critical catalytic core in two distinct intracellular complexes: the rapamycin-sensitive mTORC1 and the rapamycin-insensitive mTORC2. The latter is capable of activating Akt through phosphorylation at the Ser473 residue. Constitutive activation of Akt is identified in up to 70% of acute myeloid leukemia (AML) patients and has been associated with an adverse impact on survival. Akt activation also mediates, at least in part, the leukemogenic effects of activating FLT3 internal tandem duplication (ITD) mutations, which in turn lead to activation of mTORC1. Mitogen-activated protein kinase-associated protein 1 (MAPKAP1 or Sin1) is required for mTORC2 integrity and activation and is also required for phosphorylation and activation of Akt. In addition, the pleckstrin homology (PH) domain of MAPKAP1 directly binds the PI3K product PtdIns P3 to promote mTORC2 kinase activation and membrane localization, thereby revealing a mechanistic link between PI3K and mTORC2. However, the role of MAPKAP1 in AML pathogenesis and its impact on clinical outcomes are as of yet unknown.Methods: The in vitro system included 5 AML cell lines (2 FLT3 -mutated and 3 FLT3 -unmutated) and 6 BA/F3 (murine pro-B cells) stable clones bearing specific FLT3 mutations. Experiments were performed using transient transfections with siRNA or plasmids, pharmacologic studies, Western blot analysis, q-RT-PCR, flow cytometry, immunofluorescence, confocal microscopy and proteomics. The patient cohort included 224 adult AML patients diagnosed and treated at The University of Texas MD Anderson Cancer Center (Houston, TX) and the National University Hospital (Singapore) with available cytogenetics and mutation status data. Potential associations of MAPKAP1 with clinical outcomes were evaluated using mRNA expression data from the The Cancer Genome Atlas (TCGA) AML dataset and protein expression data assessed by immunohistochemistry on bone marrow biopsy specimens from patients in our study group using a monoclonal anti-MAPKAP1 antibody (Cell Signaling). Overall survival (OS) was defined as time from AML diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from AML diagnosis to relapse, death, or last follow-up. Survival analysis was based on the Kaplan-Meier method using the Log-rank test.Results: MAPKAP1 is frequently expressed in AML cells and regulated by STAT3, which is predominantly phosphorylated at Ser727 residue in AML cell lines and patient samples. Inhibition of STAT3 activity by pharmacologic agents, dominant negative constructs, or STAT3 gene silencing leads to decreased MAPKAP1 levels and MTORC2 activity associated with Akt dephosphorylation. In silico analysis reveals 4 potential STAT3 binding sites on the MAPKAP1 gene promoter suggesting transcriptional regulation. Knocking down the MAPKAP1 gene in AML cell lines results in Akt deactivation associated with decreased cell proliferation, and these effects are mediated by cell cycle regulators. MAPKAP1 also confers resistance to sorafenib in vitro, as MAPKAP1 is shown to be upregulated and more active in BA/F3 clones bearing sorafenib-resistant FLT3 mutations. Conversely, STAT3 or MAPKAP1 inhibition sensitizes resistant BA/F3 clones to sorafenib treatment. Using the TCGA dataset, high MAPKAP1 mRNA levels are significantly associated with poorer EFS and OS (Figure 1); these findings were independent of AML risk group or the presence of FLT3 or NPM1 mutations. Similarly, high MAPKAP1 protein levels assessed by immunohistochemistry on bone marrow specimens prior to treatment correlate with adverse clinical outcomes in a cohort of AML patients treated with equivalent regimens.Conclusions: MAPKAP1 is involved in AML pathogenesis, likely through a STAT3-MTORC2-Akt crosstalk mechanism, and may represent a novel target of investigational therapy. MAPKAP1 mRNA and protein levels are significantly associated with clinical outcomes in AML._ [Display omitted] DisclosuresKhoury:Pfizer: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromics: Research Funding. Kantarjian:Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

The Novel, Irreversible FLT3 Inhibitor FF-10101 Demonstrates Broad in Vitro Activity Against FLT3 Inhibitor Resistance Mutations

Background: Small-molecule FLT3 tyrosine kinase inhibitors (TKIs) have improved remission rates in relapsed/refractory AML driven by FLT3 internal tandem duplication (ITD) mutations, although nearly all responders eventually relapse. Acquired clinical resistance to type II inhibitors such as quizartinib and sorafenib has largely been due to kinase domain (KD) mutations that destabilize drug binding. Early inhibitors, limited by short half-life and fluctuating plasma levels, were also only able to transiently inhibit FLT3. FF-10101 is a first-in-class, type I inhibitor that covalently binds FLT3 irreversibly, suggesting its activity should be limited by the half-life of FLT3 itself. In a profiling assay of 216 human recombinant kinases FF-10101 showed selectivity for wild type FLT3 and FLT3 D835Y. It also demonstrated potent activity against FLT3-ITD AML cell lines harboring TKI-resistant D835Y and Y842C/H mutations. Here, we report in vitro testing of FF-10101 against 19 additional FLT3 TKI-resistant mutations, as well as six cysteine-to-serine mutations within the KD that could impact covalent drug binding. Profiling of this activity is relevant as FF-10101 enters single-agent phase 1/2a clinical trial in relapsed/refractory FLT3 mutant AML.Results: FF-10101 was profiled in an in vitro viability assay against murine BaF3 and human AML cell lines harboring a spectrum of mutations. FF-10101 inhibited the proliferation of FLT3-driven Molm14 and MV4;11 cells (IC50 4.1 and 1.1 nM, respectively). BaF3 FLT3-ITD cells were comparably inhibited (IC50 3.1 ± 1.6 nM), while growth of BCR-ABL-driven K562 cells were unaffected. To confirm covalent binding of FF-10101, we then analyzed its activity against all cysteine residues in the FLT3 KD. Every C->S substitution except C695S remained sensitive to FF-10101, confirming C695 as the FF-10101 binding site and suggesting C695S may impart clinical resistance to FF-10101. We next assessed 10 BaF3 FLT3-ITD cell lines harboring mutations at D835 and the gatekeeper F691, given discovery of resistance mutations at these sites in patients treated with AC220 and sorafenib. FF-10101 inhibited F691L and all substitutions/deletions at the D835 residue. N676K, a mutation found in a patient at relapse on midostaurin, was also potently inhibited. Notably, F691L previously exhibited relative resistance to crenolanib, a reversible, type I FLT3 inhibitor. Beyond clinically described mutants, we also tested FF-10101 against six mutations previously identified in mutagenesis screening assays. A D698N mutation found in separate crenolanib and PLX3397 mutagenesis assays demonstrated relative resistance to crenolanib but was sensitive to FF-10101 (IC50 8.4 ± 7.8 nM), as were four D839 amino acid substitutions identified in the PLX3397 mutagenesis assay alone. Alternatively, a Y693C mutation found in a crenolanib mutagenesis screen remained relatively insensitive to FF-10101. Finally, two resistance mutations co-occurring in the same KD are well-described in chronic myelogenous leukemia (CML) patients treated with sequential TKI therapy. While not yet found in AML patients treated similarly, we hypothesize that analogous compound mutations could emerge. As such, we tested FF-10101 against BaF3 FLT3-ITD cells containing three compound mutations. Unlike crenolanib, FF-10101 potently inhibited D698N/D835V, but F691L/D835V and Y693C/D835V each exhibited relatively moderate resistance to both drugs. Analysis of additional activation loop (AL) mutations (N831H/K, R845G, G846R, Y842S) and biochemical studies of FLT3 phosphorylation and downstream effector pathways in response to FF10101 is ongoing and will be presented.Conclusions: FF-10101 is a type I, irreversible FLT3 inhibitor that has demonstrated potent activity against all mutations associated with clinical resistance to FLT3 TKIs. Expanded profiling shows anti-proliferative activity against nearly all mutations associated with FLT3 TKI resistance except for C695S (the site of FF-10101 binding) and the crenolanib-resistant Y693C mutation, which has yet to be associated with clinical TKI resistance. These data suggest FF-10101 has potential to be clinically active against the majority of FLT3 TKI resistant KD mutations. FF-10101 is currently beginning phase I/2a clinical assessment as a single agent in patients with relapsed/refractory AML harboring a FLT3 mutation. [Display omitted] DisclosuresSmith:Fujifilm: Research Funding; Astellas: Research Funding.

Comparative Assessment of FLT3 Variant Allele Frequency By Capillary Electrophoresis and Next-Generation Sequencing in FLT3mut+ patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Who Received Gilteritinib Therapy

Comparative Assessment of FLT3 Variant Allele Frequency By Capillary Electrophoresis and Next-Generation Sequencing in FLT3mut+ patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Who Received Gilteritinib Therapy

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.

Sex disparity in acute myeloid leukaemia with FLT3 internal tandem duplication mutations: implications for prognosis.

Incidence, molecular presentation and outcome of acute myeloid leukaemia (AML) are influenced by sex, but little attention has been directed at untangling sex‐related molecular and phenotypic differences between female and male patients. While increased incidence and poor risk are generally associated with a male phenotype, the poor prognostic FLT3 internal tandem duplication (FLT3‐ITD) mutation and co‐mutations with NPM1 and DNMT3A are overrepresented in female AML. Here, we have investigated the relationship between sex and FLT3‐ITD mutation status by comparing clinical data, mutational profiles, gene expression and ex vivo drug sensitivity in four cohorts: Beat AML, LAML‐TCGA and two independent HOVON/SAKK cohorts, comprising 1755 AML patients in total. We found prevalent sex‐associated molecular differences. Co‐occurrence of FLT3‐ITD, NPM1 and DNMT3A mutations was overrepresented in females, while males with FLT3‐ITDs were characterized by additional mutations in RNA splicing and epigenetic modifier genes. We observed diverging expression of multiple leukaemia‐associated genes as well as discrepant ex vivo drug responses, suggestive of discrete functional properties. Importantly, significant prognostication was observed only in female FLT3‐ITD‐mutated AML. Thus, we suggest optimization of FLT3‐ITD mutation status as a clinical tool in a sex‐adjusted manner and hypothesize that prognostication, prediction and development of therapeutic strategies in AML could be improved by including sex‐specific considerations.

Recurrent Mutations in Cyclin D3 Confer Clinical Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia.

Biomarkers of response and resistance to FLT3 tyrosine kinase inhibitors (TKI) are still emerging, and optimal clinical combinations remain unclear. The purpose of this study is to identify co-occurring mutations that influence clinical response to the novel FLT3 inhibitor pexidartinib (PLX3397). We performed targeted sequencing of pretreatment blasts from 29 patients with FLT3 internal tandem duplication (ITD) mutations treated on the phase I/II trial of pexidartinib in relapsed/refractory FLT3-ITD+ acute myeloid leukemia (AML). We sequenced 37 samples from 29 patients with available material, including 8 responders and 21 non-responders treated at or above the recommended phase II dose of 3,000 mg. Consistent with other studies, we identified mutations in NRAS, TP53, IDH2, and a variety of epigenetic and transcriptional regulators only in non-responders. Among the most frequently mutated genes in non-responders was Cyclin D3 (CCND3). A total of 3 individual mutations in CCND3 (Q276*, S264R, and T283A) were identified in 2 of 21 non-responders (one patient had both Q276* and S264R). No CCND3 mutations were found in pexidartinib responders. Expression of the Q276* and T283A mutations in FLT3-ITD MV4;11 cells conferred resistance to apoptosis, decreased cell-cycle arrest, and increased proliferation in the presence of pexidartinib and other FLT3 inhibitors. Inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells restored pexidartinib-induced cell-cycle arrest but not apoptosis. Mutations in CCND3, a gene not commonly mutated in AML, are a novel cause of clinical primary resistance to FLT3 inhibitors in AML and may have sensitivity to CDK4/6 inhibition.

Phase 1 first-in-human study of irreversible FLT3 inhibitor FF-10101-01 in relapsed or refractory acute myeloid leukemia.

7008 Background: FF-10101-01 is a selective and irreversible FMS-like tyrosine kinase 3 (FLT3) inhibitor with potent in vitro activity against FLT3-mutated AML. FF-10101-01 is highly active against FLT3 internal tandem duplication (ITD) mutations associated with high relapse and low survival/remission rates, as well as resistance-conferring D835 and F691 tyrosine kinase domain (TKD) and non-canonical FLT3 activating mutations. Here we report on a Phase 1 dose escalation trial examining the safety, efficacy, pharmacokinetics, and pharmacodynamics of FF-10101-01 in patients (pts) with relapsed/refractory primary or secondary AML. Methods: To determine the recommended Phase 2 dose, pts with or without a FLT3 mutation received FF-10101-01 orally once (QD) or twice (BID) daily until unacceptable toxicity was observed or pts had no further clinical benefit (1 cycle = 28 days). Composite complete remission (CRc) and partial remission (PR) rates were assessed. Inhibition of FLT3 phosphorylation was evaluated using a plasma inhibitory activity assay and was correlated with associated FF-10101-01 exposure. Results: Fifty-two pts [median age 61 (range, 21-84); 52% female; FLT3: ITD [22 (42%)], TKD [5 (10%)], ITD+TKD [1 (2%)], Wt [24 (46%)] received continuous dosing of FF-10101-01 at 10 - 225 mg QD or 50 - 100 mg BID. Median number of prior therapies was 3 (range, 0-6) and the majority [23/28 (82%)] of pts with known FLT3 mutations had received prior FLT3 inhibitors. The median duration on study was 5.7 (range, 0.1-36) weeks. FF-10101-01 was generally well-tolerated up to total daily doses of 150 mg. The most common treatment related adverse events included nausea [n = 18 (35%)] diarrhea [14 (27%), 2 Grade (Gr) 3/4], elevations in creatine kinase [CK; 14 (27%), 4 Gr 3/4], vomiting [10 (19%)] and increased AST [10 (19%), 2 Gr 3]. Grade 3/4 differentiation syndrome (n = 4, 8%) was observed at 75 - 150 mg/day. Dose-limiting cardiac toxicity (heart failure with reduced ejection fraction; Gr 3 increased troponin/CK) was observed at total daily doses ≥200 mg. The CRc rate was 13% (4/30 pts evaluable for response): 1 CR at 75 mg BID (FLT3-ITD); 1 CRp at 100 mg BID (Wt-FLT3); and 2 CRi’s at 50 mg BID, one that previously progressed on gilteritinib. The median time to overall response was ̃13.3 weeks. Four pts achieved a PR (≥50% decrease in BM blasts to 5 - 25% abnormal cells) at total daily doses of 50 - 150 mg; 2 had ITD mutations, and all had received prior FLT3 kinase inhibitors. At ≥75 mg BID, trough plasma concentrations were &gt; 90 ng/ml and associated with &gt; 90% p-FLT3 inhibition maintained over the dosing interval. Conclusions: The FF-10101-01 FLT3 inhibitor has shown activity in pts with refractory/relapsed AML, including those with activating FLT3-ITD mutations resistant to gilteritinib and other FLT3 kinase inhibitors. Doses of 50-75 mg BID were well tolerated and resulted in sustained FLT3 inhibition. Clinical trial information: NCT03194685.

FLT3-ITD Allele Frequency Is an Independent Prognostic Factor for Poor Outcome in FLT3-ITD–Positive AML Patients

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a molecular genetic alteration significantly affecting the clinical outcome in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are characterized by variable mutant-to-wild allelic ratios (ARs) and sizes of the duplicated sequences. The size of the inserted sequence may vary from a few to hundreds of nucleotides. The aim of this work was to determine the impact of FLT3-ITD ARs, FLT3-ITD allelic frequency (AF), and allele size in de novo AML. We studied 117 patients with FLT3-ITD gene mutation-positive AML, dividing them into those with low ARs and those with high ARs (>0.64) and examined their prognostic impact. High FLT3-ITD AR ≥ 0.64 and AF ≥ 0.5 were significantly associated with a lower overall survival compared with lower AR (median 0.625 vs. 1.020 months, respectively; P=.041) and AF (median 0.493 vs. 0.954 months, respectively; P=.009). NPM1 mutation had no favorable impact on the low-level FLT3-ITD group. Initial high total leukocyte count, FLT3-ITD AF, and splenomegaly are independent prognostic factors for poor outcome in FLT3-ITD-positive AML.

FLT3 inhibitors in the treatment of FLT3-mutated acute myeloid leukemia

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene represent the most common genetic alteration in acute myeloid leukemia (AML), identified in approximately one third of patients newly diagnosed with AML. FLT3 internal tandem duplication mutations (FLT3-ITD) are associated with increased relapse and inferior overall survival. Multiple inhibitors of FLT3 signaling have been developed in the last few years with variable kinase-inhibitory properties, pharmacokinetics, and toxicity profiles. At present, two FLT3 inhibitors (gilteritinib and quizartinib) have been approved as monotherapies for relapsed/refractory FLT3-mutated AML in Japan, and many more drugs are currently being researched in clinical trials as monotherapies or in combination with conventional chemotherapy or hypomethylating agents and in various settings, including front line, relapsed/refractory disease, and maintenance therapy after consolidation chemotherapy or allogeneic stem cell transplantation. Despite significant advances, some issues need to be overcome, including the resistance to FLT3 inhibitors and controversies regarding the role of FLT3 inhibitors in maintenance therapies and the role of allogeneic stem cell transplantation in FLT3-mutated AML.

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.

Genomic Analysis of FLT3 Mutations in a Comprehensive NGS Multicenter Study of AML: HM-Screen-Japan 01

Genomic Analysis of FLT3 Mutations in a Comprehensive NGS Multicenter Study of AML: HM-Screen-Japan 01

Impact of DNMT3a Status on Post Induction NPM1 MRD Predictive Value on Survival in Elderly AML Patients Treated Intensively

Minimal residual disease is now a powerfull surrogate marker to assess response to chemotherapy in acute myeloid leukemia (AML). In younger adults, NPM1 MRD has recently demonstrated to be a favorable predictive marker for EFS and OS independently of fms-like tyrosine kinase-3 internal tandem duplications (FLT3-ITD) status. However, there is very few datas regarding predictive value of NPM1 MRD in elderly patients treated with intensive chemotherapy. Moreover, numerous studies have suggested the negative impact of DNMT3a mutation in NPM1 AML patients, especially in those with concurrent FLT3-ITD mutation. In this study, we aimed to investigate the impact of DNMT3a status on post induction NPM1 MRD1 predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1 mutated AML in two French institutions (Lyon, Lille) were analyzed retrospectively. Median age of the entire cohort was 66.1 years old (range 60-78.2). An FLT3-ITD mutation was evidenced in 52 of 138 patients (37.6%) with a median FLT3-ITD AR of 0.53 (range, 0.05-3). With a median follow-up of 19.61 months (0.07-128.4), the overall CR rate was 89.9% with no influence of DNMT3a or FLT3 mutational status on the probability of CR. In this elderly cohort of NPM1mut patients, a 4log reduction of NPM1 bone marrow (BM) MRD1 was associated with better outcome (median OS: NR vs 13.4 months, HR=0.35, p&amp;lt;0.01)(Figure A). Overall, DNMT3 status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. However, only 9/44 (20.4%) FLT3-ITD patients reached ≥ 4log MRD1 reduction whereas 38/80 FLT3wt (47.5%) were good molecular responders (p&amp;lt;0.001). FLT3-ITD mutated patients who achieved a 4log reduction had a superior outcome compared to those who did not (HR=0.34; 95% CI, 0.16 to 0.70; P &amp;lt;0.001). Similarly, NPM1mut FLT3wt patients with a 4log reduction in NPM1 BM-MRD1 had a longer OS (3-year OS, 68.1%; 95% CI, 48.8 to 82.9) than those without good molecular response (3-year OS, 46.5%; 95% CI, 30.2 to 61.7)(Figure B). DNMT3a negative patients who achieved a 4log reduction had a superior outcome to those who did not reached at least a 4log reduction (HR=0.23; 95% CI, 0.07 to 0.72; P &amp;lt;0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and leukemia free survival (LFS) in DNMT3amut patients. DNMT3amut patients has a very poor LFS which was even worst in poor NPM1 MRD1 responders compared to those who reached at least 4log reduction (median LFS: 8.3 months vs 17.4 months, HR = 0.48, 95% CI, 0.25-0.91, p=0.023)(Figure C). In multivariate analysis, only DNMT3a mutational status and a 4-log reduction in NPM1 BM-MRD were significantly associated with survival. Based on these results, we identified among NPM1 positive patients 3 groups with distinct prognosis, based on FLT3-ITD, DNMT3a status and NPM1 BM-MRD post induction response (NPM1 scoring system)(Figure D). When compared to ELN 2017 intermediate risk group (AUC=0.695), NPM1 scoring system (NPM1 SS) was more accurate for OS prediction in patients within intermediate (AUC=0.833) and unfavorable (AUC=0.863) NPM1 SS risk group. However, there was no significant difference in AUC between NPM1 SS favorable and ELN 2017 favorable risk group. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3a also identify a subgroup of patients at very high risk of relapase, despite good molecular responses. As hematopoietic stem cell transplantation (HSCT) might improve OS in elderly patients, DNMT3a positive AML elderly patients should be considered for HSCT or post induction maintenance strategies, even within the favorable ELN risk group. Figure Disclosures Sujobert: Gilead/Kyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding.