Targeting Non-Kinase Function of CDK6 to Overcome CDK4/6 Inhibitor Resistance

Abstract

CDK4/6 inhibitors have revolutionized the care of patients with Estrogen Receptor-positive (ER+) breast cancer (BC). However, most patients develop resistance to these agents, commonly through overexpression of CDK6. The objective of this study is to define the mechanism by which CDK6 overexpression confers resistance to CDK4/6 inhibitors and to develop a new therapeutic strategy to overcome such resistance. ER+ MCF-7 and T-47D cells overexpressing wild-type CDK6 or kinase dead (K43M) mutant of CDK6 (and the corresponding isogenic cell lines expressing an empty vector) were used assess response to palbociclib in vitro and in vivo. pRB phosphorylation was assessed by Western blot analysis. A selective CDK4/6 degrader, BSJ-1-184, was used to evaluate the therapeutic benefit of eliminating both kinase and non-kinase functions of CDK6 (and CDK4) to overcome palbociclib resistance using standard cell growth and tumor growth assays. Surprisingly, analysis of multiple publicly available databases, including the Human Proteome Atlas, revealed that primary ER+ breast tumors show no detectable expression of CDK6. Similarly, Western blot analysis of a panel of human ER+ BC cell lines showed no detectable expression of CDK6. Despite no detectable expression of CDK6 in primary ER+ breast tumors, resistance to CDK4/6 inhibitors in many cases is driven by overexpression of CDK6 but not CDK4. Based on these findings, we hypothesized that CDK6 may carry additional functions (relative to CDK4) that confer evolutionary benefit for survival of BC cells in the face of treatment with CDK4/6 inhibitors. To test this hypothesis, we generated a panel of stable ER+ BC cell lines overexpressing CDK6. As expected, these cells exhibited complete resistance to palbociclib, a CDK4/6 inhibitor. However, palbociclib completely inhibited phosphorylation of pRB (retinoblastoma protein), a primary substrate for phosphorylation for both CDK4 and 6, suggesting that non-kinase function of CDK6 may play a role in conferring palbociclib resistance. Using a kinase dead mutant of CDK6 (K43M), we demonstrated that non-kinase function of CDK6 was sufficient to confer partial resistance to palbociclib both in vitro and in vivo. Furthermore, BSJ-1-184, a selective CDK4/6 degrader (which eliminates both kinase and non-kinase functions of these proteins) completely overcame resistance to CDK6 overexpression both in vitro and in vivo, further highlighting the dual kinase and non-kinase functions of CDK6 in conferring palbociclib resistance. The kinase function of CDK4/6 has been the primary target of current CDK4/6 inhibitors used in clinic. Our studies show that non-kinase function of CDK6 plays a critical role in palbociclib resistance. We also provide a novel therapeutic strategy to overcome CDK4/6 inhibitor resistance, thereby setting the stage for development of next generation drugs that eliminate both the kinase and non-kinase functions of this class of proteins.