Engineering Electrophile-Sensitive Kinase Mutants To Accelerate Oncology Target Validation.

Abstract Nomination of new oncology targets has been greatly aided by advances in genetic screening and profiling, but developing potent, selective small molecule inhibitors against these targets remains a resource intensive pursuit. To significantly de-risk this process we applied a chemical biology strategy to model pharmacological inhibition of the MASTL kinase and showed that selective enzymatic inhibition accurately mimicked targeted genetic perturbation. Specifically, we engineered an electrophile-sensitive version of MASTL through a single amino acid substitution, Asp117Cys, within the ATP-binding site hinge region. Only 11 human kinases contain a cysteine handle in this particular hinge position (H10), which drastically limits the scope of off-targets prone to any H10 Cys-selective covalent inhibitors but also demonstrates that an H10 Cys is not incompatible with kinase activity. Furthermore, endogenous H10 Cys kinases include several targets of successful covalent inhibitor development campaigns (e.g. EGFR, JAK3 and BTK). This broad availability of optimized compounds allowed us to quickly screen and identify potent inhibitors of the MASTL Asp117Cys recombinant mutant. MASTL overexpression is observed across a broad spectrum of solid tumors, so to directly examine how its inhibition would impact cancer cell growth, Asp117Cys was created in the endogenous MASTL gene through CRISPR-directed gene editing. Pancreatic cancer cells homozygous for MASTL Asp117Cys showed clear dose-dependent growth inhibition when treated with a T790M mutant-specific EGFR covalent inhibitor (PF-06459988). Moreover, inhibitor treatment induced a visible phenotype of large multi-nucleated cells that phenocopies genetic perturbation of MASTL and aligns with MASTL's functional role regulating mitotic division. These inhibitor induced effects were not observed in unmodified parental cells and affinity purification experiments utilizing an alkyne probe of PF-06459988 confirmed binding specifically in engineered Asp117Cys mutant cells. When these Asp117Cys mutant cells are propagated as mouse xenografts, their in vivo tumor growth rate is indistinguishable from parental, but PF-06459988 treatment selectively induces tumor growth inhibition of mutant cells, thus demonstrating that selective enzymatic inhibition is sufficient to drive anti-tumor effects. Herein our application of an electrophile-sensitive mutant demonstrates how existing, optimized covalent small molecule inhibitors can be repurposed as chemical probes against engineered kinase domains. This engineered system provides a valuable orthogonal benchmark in advance of a drug discovery campaign, reveals target-dependent biology, and is likely to translate to additional kinase targets due to structural conservation within this enzymatic domain. Citation Format: Jon A. Oyer, Ted W. Johnson, Andrew C. Wang, Michael F. Maestre, Ana Flores-Bojorquez, Roksolana Melnychuk, Sergei Timofeevski, Sherry Niessen, Zhenxiong Wang, Jian Li, Wade C. Diehl, Koleen J. Eisele, Nathan V. Lee, Aihua Zou, Carl Davis, Eric C. Greenwald, Jacob DeForest, Martha Ornelas, Bryan Li, Stephanie Scales, Penney L. Khamphavong, Catherine M. Ambler, Yun Huang, Romelia Salomon-Ferrer, Samantha E. Greasley, Ben Bolanos, Neil Grodsky, Lawrence Lum, Todd L. VanArsdale, Indrawan J. McAlpine. Engineering electrophile-sensitive kinase mutants to accelerate oncology target validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 330.