Research Article

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

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Science  13 Dec 2019:
Vol. 366, Issue 6471, eaaw2106
DOI: 10.1126/science.aaw2106

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Revised view of anticancer drug mechanism

A crystal structure of the active form of cyclin-dependent kinase 4 (CDK4) provides insight into regulation of the cell cycle and the mechanism of action of a drug used for breast cancer therapy. The protein p27 has been thought to act as a CDK inhibitor. Guiley et al. performed a structural analysis of active complexes of CDK4 with cyclin D1 (CycD1) and p27 (see the Perspective by Sherr). The results showed that p27 actually remodels the active site of CDK4 to allow full activation when p27 is phosphorylated on tyrosine (phosp27). Furthermore, they found that the breast cancer drug palbociclib, a CDK4 inhibitor, doesn't actually interact with active phosp27-CDK4-CycD1 trimers. Instead, it appears that the drug, which shows promise in the clinic, binds to inactive CDK4 monomers and prevents interaction with p27.

Science, this issue p. eaaw2106; see also p. 1315

Structured Abstract

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.

Understanding how p27 mediates cyclin-dependent kinase 4 (CDK4) assembly, activity, and sensitivity to the kinase inhibitor palbociclib.

p27 binds CDK4 together with cyclin D (CycD), helping CDK4 to mature from the Hsp90 chaperone complex into an inhibited, inactive trimer. Phosphorylation of the key tyrosine Tyr74 (Y74) in p27 yields an active trimer capable of phosphorylating critical cell cycle substrates to promote cell division. X-ray crystal structures show how p27 remodels the CDK4-CycD structure to inhibit or activate the enzyme, depending on Y74 phosphorylation (red circle). Surprisingly, the active trimer complex is resistant to the CDK4/6 inhibitor palbociclib. Instead, data from experiments with purified enzymes and cancer cells indicate that palbociclib primarily targets CDK4 monomer and promotes the formation of inactive CDK2 complexes.

Abstract

The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors.

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