Abstract 2876: Targeting VCP to enhance pancreatic cancer response to DNA damaging agents
Authors: Astuti Yuliana , Lam Charlene , Wasan Harpreet S. , Maginn Elaina N. 
Published: August 2018

Abstract
Dysregulation of DNA damage response and repair (DDR) is common in pancreatic and other cancers, and impairs their sensitivity to DNA damaging agents. Targeting DDR mediators is therefore an attractive strategy to improve response to DNA damaging agents. We have identified the AAA ATPase VCP as a lead candidate for this via siRNA screening. VCP-chemotherapy synergistic combinations have not as yet been explored in solid tumours. To investigate the mechanism by which VCP modulates response to cisplatin-induced DNA damage, the effect of VCP functionality loss in decreasing cell viability was validated using two pharmacological VCP inhibitors, NMS-873 and CB-5083, in a panel of pancreatic cancer cell lines. Effects on ovarian cancer cell lines were also assessed for comparison, and IC50 values (72 hours) were found to range from 0.03 - 1.4μM. Greater sensitivity to both inhibitors corresponded with accumulation of K48-ubiquitinated protein levels, and increased expression of the unfolded protein response-related genes BiP, DDIT3, and the spliced form of XBP1. NMS-873 (10nM) or CB-5083 (50nM) were also found to increase cisplatin sensitivity (decreased IC50 values, 48 hours) in MIA-PaCa-2, SKOV-3, PEO1, and PEO4 cell lines, but not in AsPC-1 or PANC-1 cells. To investigate if cisplatin sensitization relates to changes in DDR activity, DNA double strand break repair (DSB) assays, using GFP-reporter constructs, were carried out but lower efficiency of homologous recombination (HR) and non-homologous end-joining pathways were found in both PANC-1 and SKOV-3 cells following VCP inhibition. Consistent with decreased HR activity, VCP inhibition reduced numbers of cisplatin-induced Rad51 foci in both cell lines. Unexpectedly, this was associated with increased proteasomal degradation of Rad51, as indicated by western blotting. Phosphorylation of VCP at serine 784 was detected following cisplatin treatment, and interestingly was found to be reduced following inhibition of DNA-PKcs, one of the main PI3K-related kinases (PIKKs) which directs DSB repair. To explore the role of this modification, a phospho-deficient VCP, with mutations in 4 predicted PIKK-targeted sites (including serine 784), was generated. A significant increase in apoptosis, as measured by Caspase-Glo®3/7 assay, was observed in cisplatin-treated SKOV-3 cells, as an initial model system, expressing this phospho-deficient VCP. Studies are underway to further elucidate the importance of each individual phosphorylation site on cisplatin response and DSB repair. Our findings suggest VCP could be a potential therapeutic target for pancreatic and other cancers. Confirming how VCP regulates DSB repair will allow development of novel strategies to enhance the efficacy of DNA damaging agents in cancer.
Citation Format: Yuliana Astuti, Charlene Lam, Harpreet S. Wasan, Elaina N. Maginn. Targeting VCP to enhance pancreatic cancer response to DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2876.