Introduction: Urothelial carcinoma is characterized by a high incidence of molecular alterations in the CDKN2A-RB-E2F axis. Loss of the cyclin-dependent kinase inhibitor p16 encoded by CDKN2A or amplification of CDK4/6 is associated with sensitivity to CDK4/6 inhibitors in various cancers. Functional RB1 is believed to be canonically required for mediating the effects of CDK4/6 inhibition in this setting. However, muscle-invasive urothelial carcinomas harbor RB mutations and copy-number losses in 21% of patients. To extend the activity spectrum of CDK4/6 inhibitors to these RB1-deficient cancers, we investigated the role of pocket protein p130 encoded by the Retinoblastoma-Like 2 (RBL2) gene, in mediating the downstream effects of CDK4/6 inhibition.
Methods: CRISPR-Cas9 was used for CDKN2A knockout in the 5637-urothelial cancer cell line with an RB-mutant background and in TCCSUP cell line with wild-type RB. CDKN2A- cells were subsequently treated with CDK4/6 inhibitor palbociclib. Levels of RB1, phospho-RB1 (at serine 807/ serine 811), p130 and phospho-p130 (specifically, the CDK4/6-dependent serine-672 phosphorylation) proteins were quantified using SDS-PAGE. Cell-cycle phase analysis was performed using flow cytometric measurement of BrdU and PI staining.
Results: CRISPR-induced disruption of CDKN2A was validated using genomic PCR and targeted Miseq sequencing. p16 protein knockdown was validated using SDS-PAGE. CRISPR-induced CDKN2A knockdown resulted in increased cell proliferation and faster G1/S transition even in the absence of functional RB in the 5637-urothelial cancer cell line. A significant increase in the levels of phospho-p130 was observed after p16 knockdown without changes in the levels of unphosphorylated p130. TCCSUP cell line with functional RB demonstrated a similar increase in proliferation, faster G1/S transition and an increase in phospho-p130. Palbociclib was effective in reversing these changes in all the tested cell lines.
Conclusions: the pocket protein p130 plays an important role in mediating downstream effects of CDK4/6 inhibition in urothelial cancer cell lines with both wild-type and more importantly, deficient RB. CDK4/6-dependent phosphorylation of p130 can effectively compensate for the functional loss of RB activating E2F family of transcription factors that control G1/S checkpoint. These findings potentially explain the lack of correlation between RB and response to CDK4/6 inhibitors in clinical trials. These results provide a mechanistic rationale for extending clinical trials of CDK4/6 inhibitors to patients with RB1-deficient tumors.
Citation Format: Bishoy M. Faltas, Ethan Shelkey, Rebecca Meyer, Nathan Young, Mark A. Rubin. Beyond RB1: the pocket protein p130 mediates the effects of CDK4/6 inhibition in RB1-deficient urothelial carcinoma [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 2344. doi:10.1158/1538-7445.AM2017-2344