Abstract 3166: Loss of PARP1 in human cancer cells confers resistance to PARP inhibition via activation of innate immune signaling
Authors: Ghosh Rajib , Roy Sanchita , Franco Sonia 
Published: July 2017

Abstract
Purpose: PARP inhibitors (PARPi) are FDA-approved for the treatment of ovarian cancer and in clinical trials for the treatment of other malignancies. PARPi block the catalytic site of PARP1, a poly(ADP-ribose) polymerase that catalyzes the repair of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). Although cancer cells harboring defects in Homologous Recombination (HR) are particularly sensitive to PARPi, disease progression is eventually observed due to the emergence of resistant cells. To address the mechanisms leading to PARPi resistance and identify novel preventive strategies, we have generated and characterized multiple human cancer cell lines genetically engineered to acquire PARPi resistance via depletion of PARP1.
Experimental Procedures: We used CRISPR/Cas9D10A (“double nickase”) to introduce DSBs within exon 2 of PARP1 in human cancer cell lines HCT116 and HEK293T. After allowing for repair via endogenous error-prone pathways, single cell-derived subclones were screened by immunoblotting with an antibody to PARP1. PARP1 “knock out” (KO) clones were further analyzed for proliferation, cell cycle distribution and sensitivity to DNA damaging agents, including ionizing radiation (IR) and the PARPi olaparib. Radiation response was assessed using proliferation and clonogenic assays, immunofluorescence (IF) for γ-H2AX and immunoblotting for ATM substrates phospho-KAP1 (Ser824) and phospho-CHK2 (Thr68). Finally, differential gene expression between PARP1-proficient and deficient HCT116 and HEK293T cells was analyzed by RNA-Seq.
Findings: Although genetic depletion of PARP1 initially led to significant death of HCT116 and HEK293T mass cultures, surviving cells could be isolated to establish stable PARP1 KO clones. Relative to PARP1-proficient controls, HCT-116PARP1-/- and HEK293TPARP1-/- cells exhibited decreased proliferation and clonogenic capacity in vitro and decreased growth in vivo. In addition, loss of PARP1 led to radiosensitivity and persistent activation of cell cycle checkpoints, delayed kinetics of γ-H2AX foci resolution and persistent ATM signaling after IR. In contrast, PARP1 loss induced resistance to the PARPi olaparib in both lines. PARP1 reconstitution using either stable or transient approaches resulted in only a partial rescue of these phenotypes. RNA-Seq analyses revealed increased expression of innate immune signaling and inflammatory cascades upon PARP1 loss, including type I/III interferons and their inducible genes.
Conclusions: PARP1 KO human cancer cells may represent a valuable preclinical model to investigate mechanisms underlying resistance to PARP inhibitors observed in the clinical setting. Loss of PARP1 triggers activation of “viral mimicry” in cancer cells, suggesting that drugs targeting this pathway may synergize with PARPi to kill cancer cells and prevent the emergence of resistance.
Citation Format: Rajib Ghosh, Sanchita Roy, Sonia Franco. Loss of PARP1 in human cancer cells confers resistance to PARP inhibition via activation of innate immune signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3166. doi:10.1158/1538-7445.AM2017-3166