Abstract 820: Inhibition of Rad6 sensitizes triple negative breast cancer cells to platinum-based therapy

Abstract

Abstract Treatment of triple negative breast cancers (TNBCs) poses a clinical challenge because they are not treatable with therapies targeting estrogen receptor and Her2/neu as they lack expression of estrogen, progesterone, and Her2/neu receptors. Since TNBCs share several histologic features with BRCA1-related breast cancer, and these breast cancers have aberrant DNA repair, DNA repair pathways are thought to play a role in TNBC development and drug response. Platinum (Pt)-based compounds induce toxic interstrand DNA crosslinks (ICL), the repair of which requires activities of BRCA/Fanconi anemia (FA) network and Rad6 postreplication DNA repair (PRR) pathways. PRR pathway confers tolerance to DNA damage by enabling cells to complete DNA replication in the face of damage and avoid mitotic catastrophe. Rad6 regulates PCNA ubiquitination, a critical event for PRR, and also promotes FancD2 ubiquitination, a critical event for ICL repair. We have recently reported the identification and synthesis of a small molecule inhibitor (SMI) of Rad6 that targets its ubiquitin conjugating activity. We hypothesize that targeting Rad6 will be beneficial to TNBCs treated with Pt-based compounds by preventing acquisition of resistance and overcoming Pt resistance. Treatment with the Rad6 SMI enhanced CDDP sensitivity of BRCA1 wild type MDA-MB-231 TNBC cells that have intrinsic CDDP resistance. Western blot analysis showed that the CDDP-induced DNA damage is accompanied by PCNA and FancD2 monoubiquitination (surrogate markers of Rad6 and FA pathway activation, respectively) and increases in ΓH2AX levels. Treatment of MDA-MB-231 cells with the Rad6 SMI attenuated CDDP-induced PCNA and FancD2 ubiquitination, and ΓH2AX levels. Treatment with Rad6 SMI inhibited formation of CDDP-induced Rad6, PCNA, FancD2, translesion synthesis polymerase eta and ΓH2AX nuclear foci. These data suggest that Rad6 plays an important role in recruitment of repair factors during CDDP-induced DNA damage response. To verify these findings, IdU/CldU double labeling assays were performed to evaluate the impact of Rad6 inhibition on restart of CDDP-induced stalled replication forks. Whereas restarting of CDDP-induced stalled replication forks occurred efficiently in MDA-MB-231 cells treated with nontarget siRNA or vehicle, resumption of stalled forks was severely compromised in MDA-MB-231 cells treated with Rad6B siRNA or Rad6 SMI. Consistent with the in vitro data, orthotopic implantation of MDA-MB-231 cells pretreated with Rad6 SMI or CDDP alone produced significantly smaller tumors compared to vehicle, and tumor growth was further mitigated when cells were pretreated with both CDDP and Rad6 SMI. These data imply a critical role for the Rad6 PRR pathway in repair of Pt-induced DNA damage and that inhibition of Rad6 catalytic activity may be exploited to sensitize TNBCs to Pt therapies. Supported by NIH 1R21CA178117-01. Citation Format: Brittany Haynes, Matthew Sanders, Malathy Shekhar. Inhibition of Rad6 sensitizes triple negative breast cancer cells to platinum-based therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 820. doi:10.1158/1538-7445.AM2014-820