Crystal structure of active CDK4-cyclin D and mechanistic basis for abemaciclib efficacy

Abstract

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors

“…We discovered that, while all three CDK4/6is—abemaciclib, palbociclib, and ribociclib—effectively inhibited p-RB as a readout of reduction in cyclin D–CDK4/6 complex activity in a dose-dependent manner, the three CDK4/6is all increased p-CDK4 at threonine-172 (T172), an indicator of CDK4 activation (fig. S4B), a phenomenon also recently observed in breast cancer cells, which suggests that CDK4/6is stabilize the active p-T172 CDK4–cyclin D complex ( 44 – 46 ). We next tried to validate this observation and focused on ribociclib in an expansion cohort of eight NF1-MPNST cell lines (Fig.…”

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon restoration of RB function in malignant peripheral nerve sheath tumors

“…To understand mechanisms accounting for the insensitivity in some lines, we carried out a time-course combined with dose response study over 96 hours. We discovered that while all three CDK4/6i -abemaciclib, palbociclib, and ribociclibeffectively inhibited p-RB as a readout of reduction in Cyclin D-CDK4/6 complex activity in a dosedependent manner, the three CDK4/6i all increased p-CDK4 at threonine 172 (T172), an indicator of CDK4 activation (Fig S4B ), a phenomenon also recently observed in breast cancer cells which suggests that CDK4/6i stabilize the active p-T172 CDK4-cyclin D complex (37)(38)(39). We next tried to validate this observation and focused on ribociclib in an expansion cohort of eight NF1-MPNST cell lines (Fig 4B and Fig S4C).…”

CDK2 and CDK4: Cell Cycle Functions Evolve Distinct, Catalysis-Competent Conformations, Offering Drug Targets

“…The N-terminus of cyclin-D not only contributes to the stabilization of a β-strand by establishing interactions with the A-loop of CDK4 but also implies a structural dependence that is critical for maintaining the active state of the kinase (Figure A). This is supported by the study in which the N-terminus of cyclin-D3 enhances the activation segment and plays a role in the transition to the active conformation of CDK4 . The distance between the conserved hydrophobic residues Val20 and Leu147 at the top and bottom of the adenosine ring of ATP, which are part of the C-spine residues, shows a divergence between full-length and truncated (without the N-terminus) cyclin-D in complex with active CDK4 (Figure B).…”