Abstract 340: CTDP1 regulates FANCI activation and DNA repair

Abstract

Abstract Objective: The modular BRCA1 C-terminal (BRCT) domain plays a central role in the regulation of DNA damage repair (DDR). RNA Pol II C-terminal domain phosphatase 1 (CTDP1), the only phosphatase encoding a BRCT domain, is one of the few BRCT domain-containing proteins without a formal role ascribed to DDR. Thus, the goal of this study is to characterize the DDR-specific role of CTDP1 through interrogation of the BRCT domain-protein interaction with FANCI and the repair of DNA interstrand cross-links (ICL) through the Fanconi anemia pathway. Methods: Novel CTDP1 BRCT domain-protein interactions were identified with mass spectrometry and analyzed with gene ontology enrichment. Molecular biology experiments were conducted to examine the effects of CTDP1 knockdown or overexpression on cell growth, survival, apoptosis, regulation of FANCI activation and localization, FANCD2 foci formation and ubiquitination, and homologous recombination repair. Mass spectrometry was used to determine both CTDP1-regulated FANCI phosphorylation sites and FANCI protein interactions. Results: The BRCT domain of CTDP1 was found to interact with three Fanconi anemia proteins-FANCA, FANCD2, and FANCI. This interaction remains unchanged upon mitomycin C treatment. Loss of CTDP1 induces a four-fold decrease in homologous recombination repair efficiency. CTDP1 prevents ICL-induced apoptosis. Knockdown of CTDP1 negatively affects plating efficiency and decreases the rate of cellular growth in breast cancer cell lines, but not the untransformed MCF-10A breast cell line. CTDP1 prevents sensitivity to DNA-damaging agents repaired by homologous recombination, but not UV damage or treatment with paclitaxel. CTDP1 knockdown negatively impacts FANCD2 activation measured by DNA damage-induced foci formation and monoubiquitination, which is critical for the onset of ICL repair by the ID2 complex and downstream effector proteins of the Fanconi anemia pathway. CTDP1 regulates 15 phosphorylation sites in FANCI determined by mass spectrometry profiling of cells overexpressing wild-type CTDP1 compared to phosphatase-dead D302K mutant. Paradoxically, CTDP1 phosphatase significantly increases SQ motif phosphorylation of S559 and S556, which is important for dormant origin firing and replication fork restart. This suggests that phosphorylation regulation of FANCI at non-SQ motif residues could regulate FANCI activation potential, resulting in increased phosphorylation at the SQ motif. Significance: This is the first demonstration of a formal role of CTDP1 in DDR. CTDP1 is implicated in the regulation of the specific ICL-repair Fanconi anemia pathway, and regulates breast cancer cell survival in vitro and in vivo. Delineating the mechanism by which CTDP1 regulates FANCI activation can provide the basis for preclinical studies targeting CTDP1 in breast cancer to promote sensitivity to DNA-damaging therapies. Citation Format: Kimiko L. Krieger, Wen-Feng Hu, Dragana Lagundzin, Nicholas T. Woods. CTDP1 regulates FANCI activation and DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 340.