p27 allosterically activates cyclin-dependent kinase 4 and antagonizes palbociclib inhibition

INTRODUCTION The cyclin D (CycD)–dependent kinases CDK4 and CDK6 (CDK4/6) phosphorylate the retinoblastoma (Rb) tumor suppressor protein to cause entry into the cell cycle in normal cells and in many cancers. Three small-molecule CDK4/6 inhibitors—palbociclib, ribociclib, and abemaciclib—are clinically approved in HER2-negative, ER-positive breast cancer in combination with antiestrogens. Although these drugs are already being used in clinical trials for diverse cancers, ongoing research is needed to better understand the mechanisms of inherent or acquired resistance to CDK4/6 inhibition. RATIONALE The intrinsically disordered proteins p21 and p27 are commonly known as CDK inhibitors, yet CDK4-CycD requires p21 or p27 (p21/p27) for assembly and activity in vivo. Moreover, all approved CDK4/6 inhibitors were developed against purified CDK4/6-CycD complexes that lacked p21/p27. To date, there are no structures or kinetic data explaining whether and how p21/p27 activates CDK4-CycD and influences the response of chemical inhibitors. We therefore set out to structurally and functionally characterize the trimeric p21/p27-CDK4-CycD complexes, as well as to investigate the mechanism of clinically approved CDK4/6 inhibitors. RESULTS We present a crystal structure of the CDK4 holoenzyme, which reveals that p27 allosterically activates CDK4 to phosphorylate Rb by remodeling the adenosine triphosphate–binding site and by promoting release of the kinase activation segment. We find that tyrosine phosphorylation of p27 is required to activate CDK4 and that the lack of a key tyrosine in p21 makes it a poor activator. Surprisingly, we also find that the purified p27-CDK4-CycD1 complex is refractory to inhibition by approved CDK4/6 inhibitors and that endogenous p27-, CDK4-, and CycD-associated activity is also insensitive. Instead, palbociclib primarily targets CDK4/6 monomer in breast cancer cells, and this association indirectly inhibits the downstream Rb-inactivating kinase CDK2. CONCLUSION The success of CDK4/6 inhibitors demonstrates the clinical importance of Rb inactivation in cancer. Although treatment with palbociclib, abemaciclib, or ribociclib leads to low concentrations of phosphorylated Rb, we conclude that these small molecules do not directly inhibit CDK4 kinase activity in the breast cancer cells we examined. The mechanism that we propose, inhibition of complex assembly, parallels that of the endogenous CDK4/6 inhibitor protein p16. We conclude that mechanisms leading to CDK2 inhibition are critical for inducing cell cycle arrest and are likely critical determinants of whether cells are sensitive to the CDK4/6 inhibitors.