A hallmark of cancer is unchecked cell division. Retinoblastoma protein (Rb) is a human tumor suppressor that guards a cell’s entry into S phase by binding E2F transcription factors and keeping them inactive. Many growth-promoting stimuli increase expression of D-type cyclins, which bind to and activate cyclin-dependent kinases 4 and 6 (CDK4/6). The cyclin D–bound CDK4/6 holoenzymes phosphorylate Rb, resulting in release of E2F, which in turn activates genes required for S phase entry and DNA replication. Numerous oncogenic aberrations converge at the CDK4/6–Rb pathway, providing a strong rationale for developing CDK4/6 inhibitors as cancer therapeutics.
Ribociclib (LEE011) is a selective CDK4/6 inhibitor that has received FDA breakthrough therapy and priority review designations for treatment of hormone receptor–positive breast cancer in combination with letrozole and is being tested in additional clinical trials. Here we describe the preclinical selectivity profile of ribociclib in biochemical and cellular assays. Ribociclib inhibits both CDK4–cyclin D1 and CDK6–cyclin D3 kinase activity with nanomolar IC50s in biochemical assays. To comprehensively address the selectivity of ribociclib in direct comparison with 2 other clinical CDK4/6 inhibitors, palbociclib and abemaciclib, we made use of the KINOMEscan assay consisting of >450 kinase active site–directed competition-binding assays. We adjusted the test concentrations in the kinase-selectivity panel per the binding constants for CDK4 and CDK6 to account for the higher potency of abemaciclib. Data showed that both ribociclib and palbociclib have high selectivity for CDK4 (CDK6 was not covered in the panel), with very few distinct additional binding events detected. In contrast, abemaciclib is a much more promiscuous kinase inhibitor.
Next, we sought to determine the relative potencies of the 3 inhibitors against CDK4 vs CDK6 in cellular assays. When testing different routinely used readouts of cellular viability, we found that assays that measured metabolic activity (eg, CTG) tended to underestimate the effects of CDK4/6 inhibition; thus, assays that either directly or indirectly assessed cell number were used instead. We first identified cancer cell lines primarily dependent on either CDK4 or CDK6 as judged by combined RNA expression analysis and shRNA or CRISPR-based functional assays. When determining IC50s of the 3 CDK4/6 inhibitors in these cell lines, we found that ribociclib and abemaciclib demonstrated greater activity in CDK4-dependent cells vs CDK6-dependent cells, whereas palbociclib was similarly active in both cell types.
The high degree of CDK4 selectivity of ribociclib suggests that off-target kinase inhibition is an unlikely complication in patients. Moreover, the apparent preference for CDK4 over CDK6 could be an advantage in certain cancer types that are primarily dependent on CDK4.
Citation Format: Ralph Tiedt, Scott Delach, Steven Kovats, Thomas Horn, Michael Acker, Barbara Schacher Engstler, Giordano Caponigro, Fei Su. Preclinical selectivity profile of the CDK4/6 inhibitor ribociclib (LEE011) compared with that of palbociclib and abemaciclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2346. doi:10.1158/1538-7445.AM2017-2346