INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors

Proteïnes supressores de tumors; Inhibidors de la quinasa Proteínas supresoras de tumores; Inhibidores de la quinasa Tumor suppressor proteins; Kinase inhibitors Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C–cyclin D–CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. Significance: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6–INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases. The Chandarlapaty lab has received generous funding support for this research from the Cancer Couch Foundation, the Shen Family Fund, the Smith Fund for Cancer Research, the Breast Cancer Research Foundation, an NIH Cancer Center Support Grant (P30 CA008748), and NIH R01234361. Q. Li has received support from Translational Research Oncology Training Fellowship (MSKCC) made possible by the generous contribution of First Eagle Investment Management. V. Serra reports grants from the Susan G. Komen Foundation (CCR15330331) and Instituto de Salud Carlos III (CPII19/00033) during the conduct of the study and grants from Novartis, Genentech, and AstraZeneca outside the submitted work. The Chodera laboratory receives or has received funding from multiple sources, including the NIH and an NIH Cancer Center Support Grant (P30 CA008748), the National Science Foundation, the Parker Institute for Cancer Immunotherapy, Relay Therapeutics, Entasis Therapeutics, Silicon Therapeutics, EMD Serono (Merck KGaA), AstraZeneca, Vir Biotechnology, Bayer, XtalPi, Foresite Laboratories, the Molecular Sciences Software Institute, the Starr Cancer Consortium, the Open Force Field Consortium, Cycle for Survival, a Louis V. Gerstner Young Investigator Award, and the Sloan Kettering Institute. J. Guo acknowledges support from NIH grant R01 GM121505. J.D. Chodera acknowledges support from NIH grant P30 CA008748, NIH grant R01 GM121505, and NIH grant R01 GM132386. A complete funding history for the Chodera lab can be found at http://choderalab.org/funding, including complete funding information and grant numbers. The authors thank Dr. Marie Will and Madeline Dorso for helpful comments on the manuscript and Dr. Zhan Yao for helpful advice on the kinase assays.