Co-targeting menin and RAS in KMT2A-r/NPM1c AML with activated RTK//RAS/MAPK signaling

Abstract Alteration in hematopoietic transcription programming and activation of signaling transduction pathways frequently co-occur to drive leukemogenesis and disease progression. KMT2A gene rearrangement (KMT2A-r) or mutations in nucleophosmin (NPM1c), one of the most common alterations in AML, induce aberrant HOX/MEIS1 gene signature and initiate leukemia. Menin inhibition that targets menin-KMT2A interactions and suppresses HOX/MEIS1 signaling has shown promising clinical efficacy in these AML subtypes. However, patients develop resistance and responses can be short lived. Therefore, combination strategies are needed to overcome resistance and prolong survival. Mutations in receptor tyrosine kinases (RTK) such as FLT3, PTPN11, NF1, and RAS leading to activation of the RTK/RAS/MAPK/MCL1 cascade are common in KMT2A-r and NPM1c AML. We previously reported that combined inhibition of menin and BCL2 had superior activity against AML stem/progenitor cells (Carter et al. Blood 2021), and co-targeting menin, BCL2 and FLT3 eliminated AML cells, significantly prolonged survival, and cured mice (Carter et al. Haematologica 2023). We here investigate the therapeutic efficacy of co-targeting menin and RAS in KMT2A-r/NPM1c AML with activated RTK//RAS/MAPK signaling. We treated various leukemia cells with KMT2A-r/NPM1c and FLT3/PTPN11/NF1/RAS co-mutations, including OCI-AML3 (NPM1c, NRAS), MV4-11 (KMT2A::AF4, FLT3-ITD), MOLM-13 (KMT2A::AF9, FLT3-ITD), THP1 (KMT2A::AF9, NRAS), and KOPN8 (KMT2A::ENL, KRAS G12D) with menin inhibitor SNDX-50469 or pan-RASon inhibitor RM-028. We observed that SNDX-50469 or RM-028 variably induced cell death and decreased viability in these cells. Menin inhibition (SNDX-50469) decreased menin target protein MEIS1. It also partially decreased anitapoptotic MCL1 or BCL2. Whereas RAS inhibition (RM-028) decreased pERK and partially MCL1. Next, we treated KMT2A-r/NUP98-r/NPM1c and FLT3/PTPN11/NF1/RAS co-mutant leukemia cells with SNDX-50469, RM-028, or both and found that the combination of SNDX-50469 and RM-028 was highly synergistic even in leukemia cell lines resistant to one or each single agent and in blasts and stem/progenitor cells from patient samples. Western blot analyses showed that the combination is more effective in general to reduce antiapoptotic proteins, especially MCL1. Additionally, we generated OCI-AML3 cells with acquired resistance to menin inhibitor SNDX-50469 (SNDX-R). RNA-seq analysis revealed that SNDX-R cells expressed increased RAS signaling and were more sensitive to RM-028. The combination of SNDX-50469 and RM-028 synergistically induced cell death not only in parental but also in SNDX-R OCI-AML3 cells. To understand the potential mechanisms of synergistic effects, we treated MOLM-13 cells with RM-028, SDNX-50469, and both and then performed reversed-phase protein arrays which detect >400 proteins. RAS and/or RAS/menin inhibition targets multiple signaling pathways that support protein synthesis, cell proliferation, and survival. The combination is more effective in decreasing antiapoptotic proteins, increasing proapoptotic proteins and DNA damage responses, and activating caspases. Collectively, we demonstrate that co-targeting menin and RAS inhibits HOX/MEIS and RAS signaling, more effectively decreases antiapoptotic/increases proapoptotic proteins, and synergistically induces cell death in KMT2A-r/NPM1c AML with activated RTK//RAS/MAPK signaling, with the potential of enhancing venetoclax activity.