Background: The cyclin-dependent kinase (CDK) 4/6 inhibitors ribociclib, abemaciclib, and palbociclib have been established as effective treatment options for hormone receptor–positive (HR+) human epidermal growth factor receptor 2–negative (HER2–) breast cancer. While these agents have not been evaluated head-to-head in clinical studies, they have all demonstrated significant progression-free survival improvements; however, only two of these, abemaciclib plus endocrine therapy (ET; MONARCH 2) and ribociclib plus ET (MONALEESA-3 and MONALEESA-7), achieved significant overall survival (OS) to date prompting closer examination of how these CDK4/6 inhibitors are distinct. Preclinical studies revealed differences in how these molecules interact with various kinases. These studies indicated that ribociclib and palbociclib exhibited greater selectivity for CDK4 and CDK6 relative to other human kinases than abemaciclib. In addition, studies suggested that these molecules displayed different relative activity against their primary targets CDK4 and CDK6. Although intriguing, these latter studies either used purified proteins without the accompanying cellular context or used proliferation as a proxy for target engagement. Here, we sought to extend the body of evidence created by the prior analyses by constructing cellular model systems where the effects of each CDK4/6 inhibitor on CDK4 or CDK6 could be studied in isolation.Methods: MEL-JUSO (an NRAS mutant melanoma cell line) and MIA PaCa-2 (a KRAS mutant pancreatic ductal adenocarcinoma cell line) cells were selected for this analysis after being identified as lacking dependence on either CDK4 or CDK6, as indicated by short hairpin RNA knockdown. Isogenic variants of each cell line lacking either CDK4 or CDK6 expression were generated by ablating CDK4 or CDK6 expression using CRISPR/CAS9 genome editing techniques and single-cell clonal selection. Levels of phosphorylated RB (phospho-RB) protein were used as a readout for target inhibition and were measured by Fast Scan phospho-RB (ppRB807/811) enzyme-linked immunosorbent assay (ELISA). Half-maximal inhibitory concentrations (IC50) for each CDK4/6 inhibitor were calculated in parental cells, as well as variant cell lines expressing only CDK4 or CDK6. Additionally, measurement of phospho-RB was performed in T47-D cells (an estrogen receptor–positive breast cancer cell line) to confirm results of prior analyses.Results: The level of phospho-RB inhibition in T47-D cells was similar to what has been described previously (Chen P, et al. Mol Cancer Ther. 2016). In MEL-JUSO cells, ribociclib, abemaciclib, and palbociclib inhibited CDK4 at 11-, 22-, and 2-fold lower drug concentrations than CDK6, respectively. In MIA PaCa-2 cells, ribociclib, abemaciclib, and palbociclib inhibited CDK4 at 9-, >47-, and 2-fold lower drug concentrations than CDK6, respectively. Conclusions: Consistent with prior biochemical studies and cell proliferation assays, our findings indicate that both ribociclib and abemaciclib more potently inhibit CDK4 than CDK6, whereas palbociclib has similar activity against both targets in cells. Understanding the importance of CDK4 relative to CDK6 in the etiology of breast cancer and possible implications for increased CDK4 target engagement is an emerging topic of discussion in the field. Given that CDK4 has been shown to be expressed at higher levels in breast tumor samples, and many breast cancer cell lines have demonstrated greater dependence on CDK4 vs CDK6, the differential inhibition of the CDK4/6 inhibitors may have important implications.

Citation Format: Scott Delach, Giordano Caponigro. Preclinical head-to-head comparison of CDK4/6 inhibitor activity toward CDK4 vs CDK6 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-10.