Continuous treatment with abemaciclib leads to sustained and efficient inhibition of breast cancer cell proliferation.

Abemaciclib is an oral, selective cyclin-dependent kinase 4 & 6 inhibitor (CDK4 & 6i), approved for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC) as monotherapy for endocrine refractory disease, and with endocrine therapy (ET) for initial treatment and after progression on ET. Abemaciclib has also shown clinical activity in combination with ET in patients with high risk early BC (EBC). Here, we examined the preclinical attributes of abemaciclib and other CDK4 & 6i using biochemical and cell-based assays. In vitro, abemaciclib preferentially inhibited CDK4 kinase activity versus CDK6, resulting in inhibition of cell proliferation in a panel of BC cell lines with higher average potency than palbociclib or ribociclib. Abemaciclib showed activity regardless of HER2 amplification and phosphatidylinositol 3-kinase (PI3KCA) gene mutation status. In human bone marrow progenitor cells, abemaciclib showed lower impact on myeloid maturation than other CDK4 & 6i when tested at unbound concentrations similar to those observed in clinical trials. Continuous abemaciclib treatment provided profound inhibition of cell proliferation, and triggered senescence and apoptosis. These preclinical results support the unique efficacy and safety profile of abemaciclib observed in clinical trials.

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

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.