Abemaciclib is an ATP-competitive, reversible kinase inhibitor selective for CDK4 and CDK6 that has shown antitumor activity as a single agent in hormone receptor positive (HR+) metastatic breast cancer in clinical trials. Here, we examined the mechanistic effects of abemaciclib treatment using in vitro and in vivo breast cancer models. Treatment of estrogen receptor positive (ER+) breast cancer cells with abemaciclib alone led to a decrease in phosphorylation of Rb, arrest at G1, and a decrease in cell proliferation. Moreover, abemaciclib exposure led to durable inhibition of pRb, TopoIIα expression and DNA synthesis, which were maintained after drug removal. Treatment of ER+ breast cancer cells also led to a senescence response as indicated by accumulation of β-galactosidase, formation of senescence-associated heterochromatin foci, and a decrease in FOXM1 positive cells. Continuous exposure to abemaciclib altered breast cancer cell metabolism and induced apoptosis. In a xenograft model of ER+ breast cancer, abemaciclib monotherapy caused regression of tumor growth. Overall these data indicate that abemaciclib is a CDK4 and CDK6 inhibitor that, as a single agent, blocks breast cancer cell progression, and upon longer treatment can lead to sustained antitumor effects through the induction of senescence, apoptosis, and alteration of cellular metabolism.

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.

e12506 Background: Breast cancer is the second most common cancer worldwide. Pharmacologically targeting cyclin-dependent kinases 4 and 6 (CDK4 & 6) has proven to be a successful therapeutic approach in patients with estrogen receptor positive (ER+) breast cancer. Differences in both efficacy and toxicity among the available CDK4 & 6 inhibitors has generated interest in a biological explanation. Abemaciclib is an adenosine triphosphate-competitive, reversible, selective inhibitor of CDK4 & 6 that has shown antitumor activity as a single agent and in combination with standard endocrine therapy (ET), in hormone receptor positive (HR+) metastatic breast cancer patients including those with ET resistance, and in combination with ET in high-risk early breast cancer patients. This study examines attributes of abemaciclib and other CDK4 & 6 inhibitors. Methods: The potency of abemaciclib for CDK4 was evaluated using biochemicals and breast cancer cell-based assays. Additionally, different combinations with an anti-estrogen therapy (e.g., tamoxifen, fulvestrant) were analyzed in an in vitro palbociclib (CDK4 & 6 inhibitor)-resistant breast cancer cell model, as well as in a set of CDK4 & 6 sensitive breast cancer cell models. Using cell-free assays, high content imaging and flow cytometry approaches, a subset of markers were monitored to characterize the phenotype of sensitive cell lines in a continuous dose schedule. Results: In in vitro, cell-free assays, abemaciclib shows selectivity for CDK4 over CDK6, and in cell-based assays, abemaciclib preferentially inhibits the proliferation of cells dependent on the presence of CDK4, not CDK6. Abemaciclib inhibits cell proliferation in a wide range of breast cancer cell lines, showing activity regardless of human epidermal growth factor receptor 2 (HER2) and PI3KCA gene mutation status. Furthermore, in a cell line resistant to palbociclib, abemaciclib in combination with fulvestrant (ET) restores CDK4 & 6 sensitivity, leading to cell senescence and cell death. Finally, in human bone marrow progenitor cells, abemaciclib shows a lesser impact on myeloid maturation than other CDK4 & 6 inhibitors, palbociclib and ribociclib, allowing for continuous dosing. Conclusions: In pre-clinical experiments, abemaciclib is a potent cell growth inhibitor, inhibiting preferentially the CDK4/CyclinD1 complex, leading to cell senescence and cell death. These pre-clinical results support the differentiated safety and efficacy profile of abemaciclib observed in clinical trials.

Background: Prostate cancer (PCa) is a leading cause of cancer death in men and represents a substantial public health burden [1]. Most PCa are primarily dependent on androgen receptor (AR) activity and castration is an effective approach to treat PCa patients. Despite the recent significant treatment advances, PCa inevitably becomes androgen-independent and progresses to the castration-resistant disease state (CRPC), the deadliest form of the disease [2]. Progression of the disease to castration-resistance is often mediated by a reactivation of AR signaling pathway [3]. Upon androgen stimulation, expression of D-type cyclin is up-regulated which results in an increased cyclin-dependent kinase 4 and 6 (CDK4/6) activity and stimulation of the cell cycle. [4]; Thus, inhibition of CDK4/6 may represent an effective strategy to delay or overcome primary androgen resistance. Abemacicilb is a CDK4 and CDK6 inhibitor with a clinical safety profile allowing continuous dosing to achieve sustained target inhibition [5]. Abemaciclib is FDA-approved for the treatment of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer [6,7]. Methods: The anti-proliferative activity of the abemaciclib was evaluated using iodide staining in a panel of 15 PCa cell lines. In order to get new insights on abemaciclib effects, deeper in vitro analysis was carried out in LNCaP, PC-3 and 22RV1, as ADT responding and resistant PCa cell models, respectively. Cell cycle analysis was done by FACS and High Content Imaging; cellular signaling was assessed by Western blotting. Apoptosis was measured by detection of caspase 3 and Tunnel assay. 22RV1 xenograft mouse model was used to evaluate abemaciclib efficacy in vivo. Results: Anti-proliferative activity of abemaciclib was observed across a panel of PCa cell lines, mainly in hormone receptor positive (AR+) cell lines. Overall, abemaciclib efficiently inhibited CDK4 and CDK6 which prevented the phosphorylation of Rb with the consequent effect in cell cycle and induced a G1 cell cycle arrest. Prolonged treatment promoted a marked senescence phenotype indicated by an increased b-galactosidase staining and morphological changes to result ultimately in apoptosis. In 22RV1 xenograft models, abemaciclib significantly reduced tumor growth. Taken together these data provide insights on sensitivity of PCa models to abemaciclib and its mode of action, demonstrating the potential of this drug for the treatment of prostate cancer patients. Conclusions: Abemaciclib inhibits proliferation of AR positive prostate cancer cell lines by inducing cell cycle arrest mediated by inhibition of Rb phosphorylation. Abemaciclib is a CDK4/6 inhibitor with potential to treat prostate cancer by blocking cell proliferation leading to induction of senescence and apoptosis. Citation Format: Raquel Torres-Guzmán, Carmen Baquero, Maria Patricia Ganado, Carlos Marugán, Huimin Bian, Yi Zeng, Ramón Rama, Jian Du, Maria José Lallena. Targeting prostate cancer with the CDK4 and CDK6 inhibitor abemaciclib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4850.