Abstract 4683: Poly (ADP-ribose) polymerase inhibitor LT-626: Sensitivity correlates with MRE11 and synergizes with cisplatin, oxaliplatin, and SN-38 in colorectal cancer cells

Abstract

Abstract Approximately 15% of colorectal cancer (CRC) cases display microsatellite instability (MSI) that is caused by defects in genes involved in the DNA mismatch repair (MMR) pathway. MSI leads to secondary mutations in critical genes including the double-strand break repair gene MRE11. Poly (ADP-ribose) polymerase (PARP) inhibitors have shown efficacy in cancer cells containing defects in DNA repair pathways. The aim of this study was to determine whether CRC cell lines that are MSI-High (MSI-H) or contain a MRE11 mutation are more sensitive to the PARP inhibitor, LT-626 (BioMarin Pharmaceutical Inc., Novato, CA) than cells that are microsatellite stable (MSS) with wild-type MRE11. A second aim was to test whether LT-626 in combination with cisplatin (CIS), oxaliplatin (OXA), and SN-38 (the active metabolite of irinotecan) is synergistic, antagonistic, or additive. We performed cytotoxicity assays on a panel of eleven CRC cell lines of varying MMR and MRE11 status (MSI-H/MRE11-/-: HCT116, HCT116/Chr 2, RKO, SW48, and LoVo; MSI-H/MRE11+/−: DLD1; MSS/ MRE11-/-: HCT116/Chr 3; MSS/ MRE11+/+: SW837, HT29, SW480, and SW403). Cells were treated with serial dilutions of LT-626, CIS, OXA, and SN-38 for 12 days and cell viability was measured by MTT assay to determine IC50 for each drug. Combination drug treatment was performed using a constant ratio design based on the IC50 of each drug and combination index was calculated using CalcuSyn Version 2.0. To demonstrate that sensitivity to LT-626 correlates with MRE11 status, stable cell lines were generated to overexpress MRE11 in SW48 cells and to knock-down expression of MRE11 in SW480 cells. MRE11 cDNA and shRNA retroviral constructs were purchased from Open Biosystems (Huntsville, AL) and used to infect SW48 and SW480 cell lines. Stable clones were selected using puromycin and expression of MRE11 mRNA and protein was confirmed by RT-qPCR and Western blot. Stable cell lines were tested in cytotoxicity assays as described above. Although we found no correlation between MSI status and LT-626 sensitivity, we did observe a significant increase in sensitivity to LT-626 in cells harboring a biallelic mutation in MRE11 (p=0.05). When compared to controls, overexpression of MRE11 in SW48 cells resulted in decreased sensitivity to LT-626 (p=0.002) while knock-down of MRE11 in SW480 cells resulted in increased sensitivity to LT-626. In combination assays, synergism was evident between LT-626 and CIS, OXA and SN-38 in the majority of treated CRC cell lines. These data demonstrate that sensitivity of CRC cell lines to LT-626 correlates with MRE11 mutational status. The observation of synergism in a wide range of CRC cell lines, irrespective of MSI and MRE11 status, suggests that PARP inhibitors in combination with DNA damaging chemotherapeutic agents may be a novel and successful strategy for treatment of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4683. doi:1538-7445.AM2012-4683