RAS MULTI(ON) Inhibitor RMC-7977 Targets Oncogenic RAS Mutations and Overcomes RAS/MAPK-Mediated Resistance to FLT3 Inhibitors in AML Models
Authors: Popescu Bogdan , Stieglitz Elliot , Smith Catherine C. 
Published: December 2023

FLT3 inhibitors (FLT3i) such as gilteritinib are clinically active in AML, but their use is limited by resistance due to emergence of clones with RAS/MAPK mutations, both alone and in combination with FLT3 mutations, or with other drivers such as KIT mutations. RAS mutations are also commonly associated with relapse on IDH1/2 inhibitors and the BCL2 inhibitor venetoclax. Importantly, multiple heterogeneous resistant clones are identified at the time of relapse. In addition, 5-10% of all de novo patients harbor oncogenic RAS mutations. Patients with RAS mutations or other mutations that activate RAS/MAPK signaling do not benefit from clinically approved targeted therapies. Targeting oncogenic RAS has been historically challenging and inhibition of downstream effectors of the MAPK pathway, such as MEK, demonstrated modest activity and high toxicity in clinical trials. Effective inhibition of the RAS/MAPK pathway is therefore a critical unmet need in AML. RMC-7977 is a potent, oral small molecule inhibitor of both wild-type and mutant GTP-bound RAS oncoproteins (RAS MULTI) and is a preclinical tool compound representative of the clinical candidate RMC-6236, currently in clinical evaluation (NCT05379985). RMC-7977 non-covalently binds to the intracellular chaperone cyclophilin A, generating a neomorphic interface with high affinity for all isoforms of RAS. The resulting tri-complexes sterically block RAS-effector interactions required for propagating oncogenic signals. We report in vitro data supporting preclinical utility of RAS MULTI(ON) inhibition in AML models harboring RAS mutations, including those with resistance to FLT3i due to hyperactive RAS signaling.

RMC-7977 suppressed cell proliferation in AML cell lines driven by FLT3-ITD (Molm-14, MV4-11), KITN822K (Kasumi-1, SKNO-1) and RAS mutations ( NRASQ61L -OCIAML-3, HL-60, KRASG13D - NOMO-1) with IC 50 values between 5 and 33 nM and repressed phosphorylation of downstream effectors of the MAPK pathway MEK, ERK and RSK. We also assessed the activity of RMC-7977 in resistant Molm-14 cells that developed secondary NRASG12C or NRASQ61K mutations after long-term exposure to FLT3i. At concentrations as low as 5 nM, RMC-7977 restored sensitivity to gilteritinib in both NRAS-mutant resistant cell lines. Using a caspase3/7 assay, we observed that RMC-7977 induced apoptosis, but to different extents across AML cell lines. We have previously demonstrated that effective MAPK signaling inhibition increases apoptotic dependency on BCL2. Consequently, the addition of venetoclax to RMC-7977 significantly enhanced caspase activation in cell lines with FLT3, KIT and NRAS mutations. In a cell viability assay, RMC-7977 and venetoclax showed high synergistic activity as assessed by Bliss independence model.

Given that intrapatient tumor heterogeneity is associated with clinical resistance to targeted therapies, we investigated the in vitro activity of RMC-7977 in models that recapitulate patterns of clonal outgrowth observed in patients treated with FLT3i. We mixed fluorescently-tagged cell lines with FLT3-ITD (Molm-14), FLT3-ITD and NRAS co-mutations (Molm-14 NRASQ61K) and NRAS-only mutations (OCIAML-3), treated the mixtures with RMC-7977 alone and in combination (with gilteritinib or venetoclax) for 96 hours, then assessed cell viability via flow cytometry. Gilteritinib and the gilteritinib/venetoclax combination selected for survival of cells harboring NRAS mutations, but RMC-7977 inhibited outgrowth of all cell populations. The combination of RMC-7977 and gilteritinib had superior activity in mixtures containing FLT3 and FLT3- NRAS co-mutations, but no additional benefit over RMC-7977 monotherapy in cells harboring NRAS mutations alone. Strikingly, the combination of RMC-7977 and venetoclax potently suppressed cell viability equally in all cell line models and was significantly superior to RMC-7977 alone. In vivo studies investigating the tolerability and activity of RMC-7977 and RMC-7977 combinations in RAS mutant/FLT3i-resistant patient-derived xenograft models are ongoing and will be presented.

Collectively, our data provide preclinical evidence that combination therapies leveraging RAS MULTI(ON) inhibition are effective in suppressing RAS-mutant AML clones, a common mechanism of resistance to currently approved targeted therapies in AML and a current area of high unmet clinical need.