Introduction: The understanding of DNA repairs pathways and their interactions allowed for the development of several targeted therapies, which sensitize neoplastic cells to the effects of DNA damaging chemotherapy. Poly-ADP ribose polymerase (PARP) inhibitors can increase the cytotoxicity of several anticancer agents by inhibiting various DNA repair pathways including Rad51 related homologous recombinational repair (HRR). However, inhibition of PARP may result in compensatory activation of the ATR pathway partially limiting the sensitization of chemotherapeutic agents seen with PARP inhibitors. Recently, a specific ATR inhibitor, VE-821 has demonstrated excellent sensitization to various chemotherapeutic agents with preferential antitumor activity in tumor cells as compared to normal cells (Reaper PM et al Nature Chem Biol 7: 428-30, 2011)
Colon cancer cell lines: HCT116/Lovo (p53 wild type) and HT29 (p53 mutated) were treated with SN38, the PARP inhibitor (ABT-888) and/or the ATR inhibitor (VE821) at different concentrations. The SRB cytotoxicity assay was used to determine the IC50 of each drug separately and in combination. DNA damage caused by these agents was quantified by phosphorylated-H2AX. Cell cycle alterations and protein levels (western analysis) were determined after drug treatment.
Results: Nontoxic concentrations (0.5-1 μM) of ABT-888 or VE-821 demonstrated a two to three fold decrease in the IC50 of SN38 in the colon cancer cell lines. Significantly, the combination of both inhibitors at the same nontoxic concentrations resulted in a dramatic 4 to 25-fold decrease in the IC50 of SN38 [HCT116 (8.1 nM to 0.3 nM), HT29 (20.5 nM to 3.9 nM) and Lovo (13.5nM to 3 nM)]. Notably, the observed effect was dose-dependent. In the Lovo cell line, the combination of SN38/VE821/ABT888 increased G2/M cell cycle arrest compared to SN38/VE821, SN38/ABT888 and SN38 alone. Similarly, increased phosphorylated-H2AX and apoptosis were seen with SN38/VE821/ABT888 in the Lovo and HCT-116 cell lines. Preliminary data utilizing the annexin-5 apoptosis assay in the HCT116 cell line supports the increased cell killing by the combination of all three agents. In the HCT116 cell line, treated with the SN38/VE821/ABT888 combination as compared to cells treated with SN38 alone, increased expression of p21 along with decreased expression of phospho-Chk1 was observed.
Conclusion: The SN38/ABT888/VE821 combination resulted in maximal synergy/chemosensitivity in all colon cancer cell lines, associated with increased DNA damage and apoptosis. Our data suggests that this combination may effectively overcome colon cancer resistance to irinotecan-based chemotherapy.
Citation Format: Atlal Abu-Sanad, David Davidson, Yunzhe Wang, John Pollard, Raquel Aloyz, Lawrence Panasci. ATR and PARP inhibition enhances topoisomerase I-dependent DNA damage in colon cancer cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B13.