Proteomic analysis and efficacy evaluation reveal strong synergistic anti-leukemic activity of lasmotinib combined with menin inhibitors for AML

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy. The interaction between menin ( MEN1 ) and mixed lineage leukemia 1 ( MLL1 , also known as KMT2A ) is a critical dependency for KMT2A-rearranged ( KMT2A -r ) or nucleophosmin 1 ( NPM1 )-mutant AML, providing a strong rationale for therapeutic targeting. Here, we evaluated a novel combination strategy using lasmotinib (PHI-101), a clinical-stage FLT3 inhibitor, together with menin inhibitors that disrupt the menin-MLL interaction. To investigate synergy in leukemic cells harboring FLT3 -Internal Tandem Duplication (ITD) mutation and KMT2A -r, MV4-11 and MOLM-13 cells were co-treated with lasmotinib and multiple clinical-stage menin inhibitors. Cell viability was assessed using the CellTiter-Glo assay, and the drug interaction profiles were quantified with SynergyFinder. Mechanistic studies of the lasmotinib-bleximenib combination in MV4-11 cells were conducted by Astral-based global proteomics, immunoblotting, and flow cytometry. In vivo efficacy of the combination was assessed using a mouse xenograft model of AML. The single-agent treatment with lasmotinib potently suppressed the growth of three AML cells (MV4-11, MOLM-13, and MOLM-14) bearing FLT3 -ITD mutations, with IC 50 values from 0.5 nM to 3 nM. Among five menin inhibitors, bleximenib induced strong cytotoxicity against three KMT2A -r AML cells, including MV4-11, MOLM-13, and MOLM-14, with IC 50 values from 3 nM to 23 nM. After the drug combination, lasmotinib enhanced and accelerated the anti-leukemic effects of menin inhibitor bleximenib in both MV4-11 and MOLM-13 cells. SynergyFinder analysis demonstrated strong synergism across multiple menin inhibitors, with high synergy scores (>20) even at low drug concentrations. Global proteomics revealed that the lasmotinib-bleximenib combination substantially reduced expression of key AML oncogenic proteins including MEIS1, FLT3 and c-Myc. Immunoblotting confirmed complete suppression of downstream signaling pathways including p-FLT3, p-STAT5, p-AKT, and p-ERK, while Annexin V/PI flow cytometry analysis showed profoundly increased apoptosis. In vivo , combined lasmotinib and bleximenib treatment resulted in markedly synergistic anti-tumor effects. In conclusion, lasmotinib is identified as a highly promising combination partner for menin inhibitors in AML. The strong synergism observed, both in vitro and in vivo, supports further development of lasmotinib plus bleximenib as a compelling therapeutic strategy for KMT2A-r and FLT3 mutant leukemia.