Abstract 1787: Identification of factors governing sensitivity of NSCLC cell lines to inhibition of Poly (ADP-Ribose) Polymerase (PARP).

Abstract

PARP inhibitors are emerging as important new anticancer agents, especially in cancers defective in homologous recombination (HR) repair, such as those lacking BRCA1 or BRCA2. To date, the potential role of PARP inhibition in NSCLC has not been extensively studied. As previously reported, we examined a panel of 26 genotypically-defined NSCLC cell lines for sensitivity to the PARP-1 inhibitor AG014699 (rucaparib) using colony formation assays. We identified 5 cell lines that are exquisitely sensitive (IC50 ≈ 50nM), 9 cell lines that are moderately sensitive (IC50 ≈ 60-500nM) and 12 cell lines that are resistant (IC50 > 5 μM). These data were confirmed with a second PARP inhibitor, AZD2281 (Olaparib). To determine whether sensitive cell lines have defective HR compared to resistant cell lines, we examined the formation of RAD51 foci. Both sensitive and resistant cell lines demonstrated efficient formation of RAD51 foci as well as gamma-H2AX foci in response to DNA damaging treatments, indicating correct initiation of HR. In resistant cell lines, the repair process following PARP inhibition is completed within 24 hours, with resolution of RAD51 and gamma-H2AX foci. However, in sensitive cell lines, these foci persist greater than 30 hours following DNA damaging treatments. These results suggest that although sensitive lines are able to initiate HR repair, they are unable to complete the process, so that RAD51 foci are not resolved. The persistence of DNA damage in these cell lines is confirmed by persistent gamma-H2AX focus formation as well as S/G2 arrest, the latter representing intact checkpoint control. Our results suggest that proteins required downstream of RAD51, including helicases and Holliday junction resolvases, are lacking in the sensitive lines. Consistent with this hypothesis, NCI-H520 cells, a highly PARP inhibitor-sensitive cell line, lacks expression of WRN, RECQL4 and RECQL5. siRNA-mediated depletion of these proteins individually sensitizes the resistant A549 cell line to PARP inhibition, with reduction in rucaparib IC50 by 100 fold. Our results may define a novel subset of NSCLCs with a late-step defect in HR that can be approached with PARP inhibition as a novel treatment strategy. Citation Format: Naveen Kommajosyula, Ye Cao, Lisa Moreau, Alan D'Andrea, Geoffrey I. Shapiro. Identification of factors governing sensitivity of NSCLC cell lines to inhibition of Poly (ADP-Ribose) Polymerase (PARP). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1787. doi:10.1158/1538-7445.AM2013-1787

Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples

“…Indeed, defects in DNA repair, particularly those associated with HRR, are a common feature of adult cancers, as well as paediatric cancer syndromes ( Kennedy and D'Andrea, 2006 ). We may even have underestimated the proportion of HRR-defective tumours, as recent evidence indicates that some NSCLC cell lines have defects in HRR downstream of RAD51 focus formation, and instead fail to resolve these foci leading to their persistence ( Kommajosyula et al , 2013 ; Postel-Vinay et al , 2013 ). Adaptation of the current HRR functional assay in future studies will allow detection of defects in the resolution of RAD51.…”