MLL1 (KMT2A, or MLL1) is a transcriptional regulator and a histone-lysine-N-methyltransferase. The N-terminus of MLL1 binds to the scaffold protein Menin to form the Menin-MLL1 complex, which regulates the expression of the leukemogenic Homeobox A9 (HOXA9) gene and its co-factor MEIS1 in myeloid stem progenitor cells. In MLL1-rearranged (MLL-r) AML, which represents approximately 5-10% of AML, the N-terminus of the MLL1 protein fuses to the C-terminus of one of over 90 possible fusion partners, including AF4, AF9, ENL and ELL (~70% of MLL-r AML). This creates the MLL1 fusion proteins (MLL-FPs). The MLL-FP causes dysregulated gene expression of downstream HOXA genes and their co-factors MEIS1, PBX3, as well as of MEF2C and CDK6. In AML with mutant cytoplasmic NPM1 (mtNPM1c), Menin-MLL1 is the main oncogenic driver of HOXA9, MEIS1 and FLT3, and promotes self-renewal of myeloid progenitor cells. Previously reported preclinical data showed that treatment with Menin inhibitor (MI), e.g., SNDX-5613 (revumenib) or KO-539 (ziftomenib), disrupts binding of Menin to MLL1/2 and MLL1-FP, leading to reduced MLL1/2 and MLL1-FP target gene expression, as well as induction of differentiation and apoptosis in AML cells expressing MLL-FP or mtNPM1c. In clinical trials, MIs have exhibited encouraging activity with meaningful rates of objective remissions in patients with previously treated relapsed/refractory AML harboring MLLr or mtNPM1c. However, remission durations are short (4-7 months) and most patients eventually relapse.

The focus of the present preclinical studies was to elucidate the gene expression regulation and anti-AML effects of the investigational Menin inhibitor DSP-5336 (Enzomenib) (Sumitomo Pharma), as well as to identify novel synergistic combinations with other targeted agents to achieve superior preclinical activity in AML expressing MLLr or mtNPM1c. Treatment with DSP-5336 dose-dependently induced differentiation (by morphology and increased CD11b expression) and loss of viability of MOLM13 (MLL-AF9 and FLT3-ITD), MV4-11 (MLL-AF4 and FLT3-ITD) and OCI-AML3 (mtNPM1c and homozygous NRAS mutation) cell lines, as well as patient-derived (PD) MLLr or mtNPM1c AML cells. Utilizing RNA-Seq analysis in PD AML cells with MLL-r, we determined that treatment with DSP-5336 negatively enriched gene sets of MYC-targets, E2F-targets, as well as significantly reduced the mRNA expression of HOXA9, MEIS1, REEP3, PBX3, MEF2C, CDK6 and BCL2. Conversely, gene sets for interferon alpha, interferon gamma and inflammatory response were positively enriched. Consistent with this, Western analysis of cell lysates from MOLM13 and OCI-AML3 cells and PD AML cells, following DSP-5336 treatment, showed markedly reduced protein levels of Menin, MEIS1, FLT3, CDK6, and BCL2 but increased levels of CD11b. In phenotypically characterized PD AML stem/progenitor cells (with high expression of CD33, CLEC12A, CD123, CD244, and CD99, but low expression of CD11b), mass cytometry (CyTOF) analysis also confirmed that DSP-5336 treatment attenuated protein levels of MEIS1, HOXA9, PBX3, and CDK6, but induced p-H2AX and cleaved Caspase 3 expression. Notably, in the cell lines and PD AML cells with MLLr or mtNPM1c AML, in vitro treatment with DSP-5336 in combination with CDK9 inhibitor (CDK9i) AZD4573 or BAY1251152 for 48 to 96 hours induced synergistic apoptosis or loss of viability, as discovered by the ZIP method with SynergyFinder. Importantly, synergistic lethal activity of the combinations was also observed in MI-resistant (MITR) MV4-11-MITR and OCI-AML3-MITR cell lines, and PD MLLr or mtNPM1c-expressing AML cells that demonstrated ex vivo resistance to MI treatment. In NSG mice engrafted with OCI-AML3 Menin-resistant mutation M327I-GFP/Luciferase xenografts, we next evaluated the in vivo anti-leukemia activity of DSP-5336 alone (50 mg/kg, PO, BID) or its co-treatment with BAY1251152 (10 mg/kg, IV, 1x per week) for 3 to 4 weeks. In vivo co-treatment with DSP-5336 and BAY1251152 showed significantly greater reduction in AML burden than treatment with each agent alone (p< 0.05), without inducing weight loss or other toxicities.

These preclinical findings underscore the anti-AML activity of DSP-5336 and its molecular correlates, as well as demonstrate synergistic in vitro and in vivo activity of DSP-5336-based combination with CDK9i against AML harboring MLL1-FP or mtNPM1c.

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2025
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