| TCGA profiling of muscle invasive bladder cancers reveals frequent mutations in cell cycle regulators. Cell cycle inhibitors targeting cyclin dependent kinases 4 and 6 (CDK4/6) have recently shown clinical potential as targeted therapies in various tumor types including sarcomas, breast cancer, melanomas, and hematologic malignancies. Previous studies have shown that functional RB1 is required for cell cycle arrest by CDK4/6 inhibitors in virtually all cancer types. However, these preclinical studies focus on short-term drug assessment. It is not known how cells may evolve in response to long-term treatment with CDK4/6 inhibitors. In this study, we profiled a panel of 33 bladder cancer cells by targeted DNA sequencing, array comparative genomic hybridization, and transcriptome sequencing to identify cell lines with genetic alterations in clinically relevant cell-cycle related genes. Our analysis identified previously unreported RB1 mutations in commercially available cell lines studied in large-scale profiling efforts. A subset of these cell lines was screened for sensitivity to the CDK4/6 inhibitors palbociclib (PD-0332991, Pfizer) and ribociclib (LEE011, Novartis). As in other tumor types, sensitivity to CDK4/6 inhibition was increased in cells with genetic loss of CDKN2A, while cells with deleterious alterations in RB1 were intrinsically resistant regardless of CDKN2A status. To identify the mechanisms of acquired resistance to CDK4/6 inhibitors, the palbociclib-sensitive bladder cancer cell line HCV29 was cultured under continuous drug treatment. After five months, we were able to identify sublines with reduced drug sensitivity. Whole exome sequencing revealed de novo loss-of-function mutations in RB1 in four sublines coupled with whole chromosome loss. Our results confirm previous findings that intact RB1 is necessary for response to CDK4/6 inhibition in bladder cancer cells, but sensitive cells can gain novel genetic alterations that evade drug-induced cell cycle arrest. |
