Discovery of CNP200137, a second-generation menin-MLL inhibitor with superior preclinical properties and anti-leukemic activity against acquired resistance

Targeting the menin-mixed lineage leukemia (MLL, also known as KMT2A) interaction is a promising therapeutic strategy for treating acutemyeloid leukemia (AML). Oncogenic KMT2A-fusion proteins in AML recruit menin to chromatin, a critical step for activating leukemogenic target genes such as MEIS1 and HOX clustergenes. This menin-dependent leukemogenic program is also shared by mutant nucleophosmin 1 (NPM1) AML. Menin inhibitors recently demonstrated substantial clinical activity inKMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1m) leukemias, as well as broader subsets exhibiting menin-dependent biology-collectively representing approximately 40% of AML cases. However, the emergence of on-target MEN1 resistance mutations has limited the long-term therapeutic benefits of first-generation agents such as revumenib and ziftomenib. Herein, we report the discovery of CNP200137, a next-generation menin-MLL inhibitor designed to effectively overcome MEN1-mediated resistance.Menin-MLL disruption was measured using and HTRF assay. Anti-proliferative activity was evaluated in KMT2A-r AML cells (MV4-11, MOLM-13), NPM1m AML cells (OCI-AML3), MLL wild type cells (MOLT-4), CRISPR-engineered MEN1-resistant mutants (M327I, T349M, G331R, E368K), and revumenib-resistant MOLM-13 cells. Cellular target engagement was assessed using qPCR, immunoblotting, and Astral-based global proteomics, which sensitively quantified robust menin protein degradation. Following pharmacokinetic (PK) characterization in mice, in vivo anti-tumor efficacy in mice bearing parental MV4-11 and resistant MV4-11(M327I) mutant was evaluated. The novel menin-MLL inhibitor CNP200137 effectively disrupted the menin-MLL interaction with nanomolar potency, exhibiting an IC₅₀ value of 2 nM against wild-type MEN1 and maintaining strong activity against the clinically relevant resistance mutants M327I with an IC₅₀ value of 13 nM and T349M with an IC₅₀ value of 11 nM. CNP200137 selectively exhibited strong anti-proliferative activity against AML cells harboring KMT2A rearrangements, with IC₅₀ values of 19 nM and 35 nM in MV4-11 and MOLM-13 cells, respectively, and an IC₅₀ value of 31 nM in NPM1-mutant OCI-AML3 cells. Notably, CNP200137 displayed superior anti-leukemic activity against both engineered MV4-11 cells with clinically relevant acquired resistance and revumenib-resistant MOLM-13 cells, compared to approved or clinical-stage menin inhibitors. Notably, CNP200137 displayed superior anti-leukemic activity against engineered MV4-11 cells with clinically relevant acquired resistance, compared to approved or clinical-stage menin inhibitors. Proteomic analysis revealed that these inhibitors induced stronger and more consistent degradation of menin proteins in MV4-11, MOLM-13 and OCI-AML3 cells, with the downregulation of MEIS1 genes. In mice, the compounds exhibited an improved PK profile and strongly suppressed tumor growth in both parental and resistant MV4-11 xenografts. This novel, potent, and orally available second-generation menin-MLL inhibitor, CNP200137, represents a promising therapeutic strategy for overcoming acquired resistance in KMT2A-rearranged and NPM1-mutant acute leukemias.