Protein degradation of MYC/GSPT1 combined with menin inhibition overcomes resistance to menin inhibition in KMT2A-rearranged Acute Myeloid Leukemia

Abstract BACKGROUND KMT2A rearranged (KMT2Ar) leukemias are an aggressive subset of acute leukemias with poor outcomes. Pharmacological inhibition of the menin-KMT2A interaction using revumenib and ziftomenib has shown encouraging efficacy in clinical trials, and revumenib has been FDA-approved (Aldoss 11 and OCI-AML2 cells were treated with menin inhibitor SNDX-50469 (SNDX) and GT19715 (GT). Cell death was determined by Annexin V/DAPI flow cytometry, and time-course studies were performed. Synergy was defined as Bliss scores (BSS) 10. Quantitative PCR was performed to determine mRNA levels. GFP/luciferase-labeled MOLM-13 TP53 WT cells were injected into NSG mice to investigate the in vivo activity of SNDX and GT. RESULTS Combinatorial treatment of SNDX and GT substantially reduced nascent protein translation determined by OPP incorporation compared to SNDX and GT monotherapy, suggesting the enhanced reduction of nascent protein translation by combined menin inhibition and MYC/GSPT1 protein degradation. The combinatorial treatment induced over 99% cell death in MOLM-13 cells compared to monotherapy, resulting in significantly synergistic cytotoxicity (BSS ~ 80). Mechanistically, SNDX + GT combination reduced c-MYC protein levels compared to monotherapy in a time-dependent manner in MOLM-13 cells. qPCR confirmed marked reductions in MYC mRNA levels after combination treatment in MOLM-13 cells, confirming our hypothesis of inhibiting MYC at transcription and translation. Interestingly, the combination treatment reduced p-ERK and MCL-1 protein levels by more than 90% in MOLM-13 TP53 WT and TP53 Y220C cells before cells underwent apoptosis, suggesting that the combination treatment reduces essential oncogenic proteins in a TP53-independent manner. Interestingly, MCL-1 overexpressing MV4;11 cells were less sensitive to SNDX compared to MV4;11 cells with empty vector control. The combination treatment induced synergistic cell death (BSS 65) in MCL-1 overexpressing MV4;11 cells, accompanied by a 90% decrease in MCL-1 protein levels. MOLM-13 cells with NRAS G12D showed reduced cell death to SNDX compared to MOLM-13 parental cells, but combinatorial treatment induced synergistic cell death in MOLM-13 cells with NRAS G12D (BSS = 64). The data suggest that the combination treatment overcomes MCL-1 and RAS-mediated resistance to menin inhibition. Finally, combinatorial treatment resulted in a significant reduction of tumor burden in mice injected with MOLM-13 cells. CONCLUSIONS The proposed combinatorial approach of menin inhibition and MYC/GSPT1 protein degradation induces highly synergistic cell death in KMT2A-r AML cells in vitro and in vivo, effectively overcoming resistance by MCL-1 overexpression and RAS pathway activation. Investigations using KMT2Ar AML patient-derived xenograft models are ongoing and will be presented.