P410: combining menin and mek inhibition to treat children with poor prognostic kmt2a-r ras-mutant acute leukemia

Background: Approximately 10% of all acute leukemias harbor a chromosomal translocation involving the Mixed Lineage Leukemia 1 (MLL1, KMT2A) gene locus. The resulting KMT2A-r fusion proteins drive leukemogenic gene expression through an interaction with the transcriptional regulator Menin, giving rise to very aggressive acute blood cancers. RAS mutations are frequent co-occurring mutations in this pediatric population and are associated with an even more unfavorable prognosis. Aims: In this study, we investigated the in vitro and in vivo anti-leukemic potential of drugs already available for pediatric patients with the goal of rapid clinical translation. We evaluated the combination of RAS/MAPK pathway inhibition by the MEK1/2 inhibitor selumetinib with the Menin inhibitor VTP-50469 (SNDX-5613) in a genetically defined, poor prognostic subgroup of pediatric leukemia harboring KMT2A-r with RAS mutations. Methods: The preclinical therapeutic experiments were conducted in NSG mice engrafted with pediatric patient-derived xenograft (PDX) models through tail vein injection. The mice were assigned to four treatment groups with chow containing SDNX-5613 (0.03%, daily), selumetinib (50 mg/kg/d, 5 days a week), both treatments and a control group. After treatment for 18-28 days, leukemia burden and target inhibition were assessed via hCD45 staining using flow cytometry and immunoblots for targets such as pERK, MYC, and MEIS1. Results: We observed synergy with Menin and MEK inhibitors in a panel of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines and PDX models with KMT2A-r and RAS mutations (Panel A) in a cell viability assay. The combinatorial effect was also associated with an impairment of colony forming potential, decrease in cell growth with a G0/G1 arrest and promotion of cell death (Panel B). We explored potential mechanisms by which the combination treatment had a synergistic anti-leukemic effect. As expected, selumetinib reduced phosphorylated ERK protein, but surprisingly, Menin inhibition did as well, with the combination having the strongest impact on downregulating the pathway. Furthermore, analysis of RNA sequencing of drug treated AML cells (single agent versus combination) revealed increased change with the combination regarding loss of stem-like signatures, increase in myeloid differentiation programs, and most strikingly, downregulation of targets of MYC signaling. Moreover, MYC transcript and protein levels were reduced by each drug alone and even more effectively with the combination (Panel C). Next, we evaluated this drug combination in multiple orthotopic pediatric AML and ALL PDX models harboring different KMT2A-r and RAS mutations. After 18 to 28 days of treatment, leukemia burden in bone marrow, spleen and peripheral blood was markedly decreased with the combination compared to the single drug-treated cohorts (Panel D). Target inhibition was confirmed in a KMT2A-AF4 ALL model with a striking decrease in levels of MYC, P-ERK and MEIS1 by western immunoblotting (Panel E). Summary/Conclusion: The findings in our preclinical study suggest a promising, readily translatable treatment for patients with KMT2A-r acute leukemias harboring RAS mutations. Mechanistically, enhanced downregulation of both MYC signaling and the RAS/MAPK pathway with the combinatorial therapy is likely a strong contributor to the observed efficacy.Keywords: MYC, KMT2A, Acute leukemia