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

Cancer Discov. 2022 Feb;12(2):356-371. doi: 10.1158/2159-8290.CD-20-1726. Epub 2021 Sep 20.


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.This article is highlighted in the In This Issue feature, p. 275.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / chemistry*
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / therapeutic use
  • Breast Neoplasms / drug therapy*
  • Breast Neoplasms / pathology
  • Cell Line, Tumor / drug effects
  • Cyclin-Dependent Kinase Inhibitor Proteins / administration & dosage
  • Cyclin-Dependent Kinase Inhibitor Proteins / chemistry*
  • Cyclin-Dependent Kinase Inhibitor Proteins / therapeutic use
  • Drug Resistance, Neoplasm*
  • Female
  • Humans
  • Piperazines / chemistry*
  • Piperazines / pharmacology
  • Piperazines / therapeutic use
  • Protein Kinase Inhibitors / chemistry*
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinase Inhibitors / therapeutic use
  • Pyridines / chemistry*
  • Pyridines / pharmacology
  • Pyridines / therapeutic use
  • Tumor Suppressor Proteins / metabolism


  • Antineoplastic Agents
  • Cyclin-Dependent Kinase Inhibitor Proteins
  • Piperazines
  • Protein Kinase Inhibitors
  • Pyridines
  • Tumor Suppressor Proteins
  • palbociclib