Co-targeting menin and LSD1 dismantles oncogenic programs and restores differentiation in MLL-rearranged acute myeloid leukemia

Acute myeloid leukemia (AML) with KMT2A-rearrangement carries a poor prognosis, underscoring the urgent need for new therapeutic approaches. Menin inhibitors demonstrate promising anti-leukemic activity in KMT2A-r AML; however, their efficacy as monotherapy may be limited by partial responses and emerging resistance mutations. Combination strategies using epigenetic modulators may enhance their therapeutic potential. In this study, we performed a combination drug screen using an epigenetic compound library in KMT2A-r AML cells to identify synergistic agents that could potentiate menin inhibitor activity. MV4-11 cells were treated with DSP-5336 (menin inhibitor) to optimize assay performance (Z′ > 0.5). Cells were then screened with a 932-compound epigenetic library to assess drug effects alone or with DSP-5336. Follow-up studies used ORY-1001 (LSD1 inhibitor) and SNDX-5613 (menin inhibitor) across dose ranges. Viability, IC₅₀, and synergy were evaluated using SynergyFinder 2.0. Mechanistic analyses included Western blotting, co-IP, ChIP-qPCR, CUT&RUN, and mass spectrometry to examine LSD1 binding, interactions, and genome-wide occupancy with the Menin-MLL complex. Patient-derived xenograft cells were implanted in NSG mice. The combination screen performed robustly and identified LSD1 inhibition as the strongest synergistic enhancer of menin inhibitor activity. LSD1 blockade substantially increased DSP-5336-mediated killing in KMT2A-rearranged AML cells. Mechanistically, LSD1 depletion disrupted key leukemic transcriptional programs and reduced chromatin occupancy at Menin-MLL target loci. LSD1 immunoprecipitation revealed the known MLL interactor PSIP1(LEDGF) as a novel LSD1 interacting partner via mass spectrometry, suggesting cooperative roles of these epigenetic targets in maintaining leukemic gene expression and stem-like properties. The combination of menin and LSD1 inhibition promoted differentiation, shown by an increased proportion of CD11b⁺ cells compared with either single agent. In an aggressive MLL-AF6 AML PDX model, co-treatment with SNDX-5613 and ORY-1001 produced superior in vivo efficacy. Mice receiving the combination showed markedly reduced leukemia burden and significantly prolonged survival relative to monotherapies, as evidenced by decreased hCD45⁺ levels and improved survival. This study identifies LSD1 inhibition as a top synergistic partner for menin-directed therapy in KMT2A-r AML. These findings suggest that combining menin inhibitors with epigenetic modulators such as LSD1 inhibitors may overcome resistance and strengthen anti-leukemic effects. Ongoing in vivo studies aim to further elucidate the underlying mechanisms and therapeutic potential of this combination. Ultimately, this work may guide the development of innovative combination treatment strategies for KMT2A-r AML patients.