Allosteric SHP2 inhibition increases apoptotic dependency on BCL2 and synergizes with venetoclax in FLT3- and KIT-mutant AML.

Mutations in the receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although selective FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report the pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppresses proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that pharmacologic SHP2 inhibition unveils an Achilles' heel of RTK-driven AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in AML cell lines and in clinically relevant xenograft models. Our results provide mechanistic rationale and pre-clinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML.

The Irreversible FLT3 Inhibitor FF-10101 Is Active Against a Diversity of FLT3 Inhibitor Resistance Mechanisms.

Small-molecule FLT3 inhibitors have recently improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML) after many years of development, but resistance remains an important clinical problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 which has previously shown activity against FLT3 tyrosine kinase inhibitor resistance-causing FLT3 F691L and D835 mutations. We report that FF-10101 is also active against an expanded panel of clinically identified FLT3 mutations associated with resistance to other FLT3 inhibitors. We also demonstrate that FF-10101 can potentially address resistance mechanisms associated with growth factors present in the bone marrow microenvironment but is vulnerable to mutation at C695, the amino acid required for covalent FLT3 binding. These data suggest that FF-10101 possesses a favorable resistance profile that may contribute to improved single-agent efficacy when used in patients with FLT3-mutant AML.

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

PURPOSE: 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). EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Gilteritinib is a clinically active FLT3 inhibitor with broad activity against FLT3 kinase domain mutations.

Gilteritinib is the first FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitor (TKI) approved as monotherapy in acute myeloid leukemia with FLT3 internal tandem duplication and D835/I836 tyrosine kinase domain (TKD) mutations. Sequencing studies in patients have uncovered less common, noncanonical (NC) mutations in FLT3 and have implicated secondary TKD mutations in FLT3 TKI resistance. We report that gilteritinib is active against FLT3 NC and TKI resistance-causing mutations in vitro. A mutagenesis screen identified FLT3 F691L, Y693C/N, and G697S as mutations that confer moderate resistance to gilteritinib in vitro. Analysis of patients treated with gilteritinib revealed that 2/9 patients with preexisting NC FLT3 mutations responded and that secondary TKD mutations are acquired in a minority (5/31) of patients treated with gilteritinib. Four of 5 patients developed F691L mutations (all treated at <200 mg). These studies suggest that gilteritinib has broad activity against FLT3 mutations and limited vulnerability to resistance-causing FLT3 TKD mutations, particularly when used at higher doses.