Abstract 2356: Functional characterization of CDKN1A loss in bladder cancer and effects on cisplatin sensitivity

Abstract

Abstract Introduction Muscle-invasive bladder cancer (MIBC) is most optimally treated with cisplatin-containing combination chemotherapy, and if clinically localized, bladder removal. With recent discoveries delineating aberrant driver genes involved in MIBC, it is now possible to personalize treatment approaches. Development of a therapeutic rationale for personalized therapies which is based on mutational landscape or other characteristic of each tumor may prospectively identify patients prone to chemoresponse, thus maximizing therapeutic index. Inactivating mutations in CDKN1A, the gene encoding cyclin dependent kinase inhibitor p21, occur in about 14% of MIBCs, with a majority of these truncating the peptide. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell cycle to repair the damage, and subsequently proceed down an apoptotic pathway, potentially serving as a therapeutic vulnerability. Methods RT4 and SW780 cell lines were chosen for study based on chemoresistance to cisplatin and WT status of CDKN1A and TP53. Cell-cycle arrest and apoptotic pathways were evaluated with Western blot after treatment with IC50 doses. CRISPR sgRNAs were generated to introduce frameshifts 5’ to the regions encoding the cyclin binding domain (CDI), within the CDI, and 5’ to the PCNA binding domains. Knock-outs were confirmed by immunoblot. MTT was used to assess cipslatin response. Results p53 increased in both cell lines 24 hours after a 3h pulse of cisplatin exposure. However, p53 peaked at 24 hours and then diminished in RT4 but continued to rise in SW780. p21 was basally expressed in both cell lines, decreased at 24 hours, then increasing at 72 hours. In RT4, peak Rb phosphorylation (pRb) occurred at 24 hours, indicating cell cycle arrest, and gradually decreased at 72 hours. In SW780 pRb remained unchanged during the course of the treatment. In RT4, PARP cleavage was observed at 24 hours and increased at 48 hours indicating apoptosis. RT4 cells harbored extensive DNA damage as indicated by persistent phosphorylated γH2AX. In contrast a smaller fraction of SW780 cells underwent PARP cleavage at 48 and 72 hours and there was less phosphorylated γH2AX. Single cell CDKN1A knockout clones were generated and confirmed by immunoblotting. sgRNAs targeting amino acids 12 and 59 completely abolished p21 detection, and an sgRNA targeting amino acid 109 generated a truncated peptide, confirming p21’s apparent stability. CDKN1A disruption at any of the three sites did not reverse chemorensitance to cisplatin in either cell line. Conclusion Cisplatin resistant cell lines use a combination of arrest and repair to survive an apoptotic insult. Although p21 loss did not reverse cisplatin resistance, it may impact sensitivity to other agents. Other mechanisms of chemo- and radio-sensitization are being explored. Citation Format: Philip H. Abbosh, Rahmat K. Sikder, Wafik S. El-Deiry. Functional characterization of CDKN1A loss in bladder cancer and effects on cisplatin sensitivity [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 2356. doi:10.1158/1538-7445.AM2017-2356