Abstract 3457: Characterization of acquired resistance mutations to menin inhibitors
Authors: Perner Florian , Cai Sheng F. , Wenge Daniela V. , Kim Jeonghyeon , Cutler Jevon , Nowak Radosław P. , Cassel Joel , Singh Shivendra , Bijpuria Shipra , Miller William H. , Stein Eytan M. , Levine Ross L. , Fischer Eric S. , McGeehan Gerard M. , Armstrong Scott A. 
Published: April 2023

Abstract
The menin-MLL1 interaction is critical for development of acute leukemias driven by MLL1 rearrangements (MLLr) or mutations in the Nucleophosmin 1 gene (NPM1c). Inhibition of the menin-MLL1 interaction by SNDX-5613 (revumenib) has demonstrated robust clinical responses in the current AUGMENT clinical trial (NCT04065399). During the trial, some responders relapsed during treatment due to acquired resistance in MEN1. Somatic MEN1 mutations were found at residues M327, G331 or T349 which diminished SDNX-5613 binding affinity and mediated therapeutic resistance. The presence of acquired resistance validates MEN1 as a therapeutic target in MLLr and NPM1c AML patients.
Here we characterize the effects of these mutations on the activity of 6 menin inhibitor chemotypes currently in clinical trials (NCT04065399/Syndax, NCT04067336/Kura, NCT04811560/JNJ, NCT04988555/Sumitomo, NCT04752163/Daiichi, NCT05153330/Biomea). In vitro activity and binding modes for these compounds were evaluated in wild-type (WT) and mutant menins using (i) competition binding assays, (ii) cell-based proliferation assays and (iii) X-ray co-crystallography.
For binding, His6-tagged MEN1 mutant proteins (G331R, M327I, M327V, T349M) were expressed and purified. Binding affinities were measured in competition binding format. The menin-MLL interaction was monitored by HTRF using Terbium labeled anti-His6 antibody and FITC labeled MLL peptide (4-43). Acquired mutations affected binding affinities (Ki) to varying degrees. Notably, M327I/V mutations reduced binding for all menin-MLL inhibitors ranging from ~30-300, indicating a class effect for this mutation. An irreversible Biomea chemotype did not inhibit menin-MLL binding in our assays.
The decreased binding affinity to M327I was reflected in cell-based proliferation assays. Menin mutations were introduced into MV4;11 cells using CRISPR-Cas9 in conjunction with a homology directed repair template to edit the endogenous MEN1 coding sequence. Clonal lines were established harboring homozygous (homo) and heterozygous (het) M327I mutations. The M327I (het) MV4;11 cells experienced 15-50-fold shifts in IC50 vs WT cells, consistent with the reduced binding affinities.
The molecular basis for sensitivity to M327 acquired resistance was examined by X-ray co-crystallography of inhibitors bound to M327I and WT menin. Both KO-539 and SNDX-5613 show notable changes in binding in M327I vs WT menin. The isoleucine creates a steric clash, displacing their position in the pocket as previously noted. The Janssen chemotype shows a novel binding mode. Although it has 30-fold lower affinity for M327I, it shows little change in its bound position to M327I menin.
Given the clinical validation of menin inhibition in AML, the design of next generation compounds that block MLL1 binding while avoiding acquired MEN1 mutations may be a strategy to overcome acquired resistance to first generation menin inhibitors.
Citation Format: Florian Perner, Sheng F. Cai, Daniela V. Wenge, Jeonghyeon Kim, Jevon Cutler, Radosław P. Nowak, Joel Cassel, Shivendra Singh, Shipra Bijpuria, William H. Miller, Eytan M. Stein, Ross L. Levine, Eric S. Fischer, Gerard M. McGeehan, Scott A. Armstrong. Characterization of acquired resistance mutations to menin inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3457.