Abstract 1353: Role of TLK1B/NEK1 axis in DNA DSB repair: Implications for prostate cancer progression

Abstract

Defects in DNA Damage Response and Repair are linked to the majority of cancers, including Prostate Cancer (PCa). Common genotoxic stresses that cause damage to the DNA, or replication fork collapse are generated when PCa cells face Androgen Deprivation Therapy (ADT), and must be bypassed to allow continuous replication and division. Tousled like kinase (TLK1) splice variant TLK1B is implicated in DNA damage repair pathways and is translationally increased following various stresses, including the DDR. We found that the expression of TLK1B is rapidly increased following a shift of LNCaP cells to charcoal-stripped serum (ADT), via an activation of mTOR and phosphorylation of 4EBP1 (inhibitor of the translation factor eIF4E). We recently uncovered the existence of the important DDR axis TLK1B> NEK1> ATR>Chk1.(1). TLK1 phosphorylates NEK1 at T141 and activates its autophosphorylation of Y315. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks (DSB) after low dose ionizing radiation (IR).(2). Cells repair DSB either via HR or NHEJ. We investigated whether TLK1 phosphorylation is essential for NEK1 activity in HR repair. siRNA-mediated NEK1 deficient cells have decreased kinetics of Rad54-S572 phosphorylation and persistent Rad51 foci when induced with Doxorubicin that creates DSB.(3). We tested a NEK1-T141A overexpressing (Hek293) mutant for its effect on Rad54-S572 phosphorylation (pRad54) during a time course of recovery from Doxorubicin. There was no significant delay in pRad54 kinetics compared to the control, unless endogenous (WT) NEK1 was knocked down, in which case we observed a significant delay in pRad54 in NEK1 mutant cells. When DSBs occur during DNA damage, Rad51 becomes localized at DSB repair foci. Delay in Rad54 phosphorylation results in persistent Rad51 foci since aspects of HR subsequent to Rad51 filament formation and strand invasion are impaired. We observed increased Rad51 foci that persisted in NEK1-T141A mutants following two hours of Doxorubicin treatment and 14 hours of recovery. A quantitative approach to monitor HR in NEK1-T141A mutants was obtained with the generation of a stable DR-GFP recombination reporter substrate (4) in HSG cell line. Transfection with the megabase cutter endonuclease I-SceI, creates a DSB in a defective double GFP cassette which would generate a functional GFP gene conversion product. Results indicate that approximately 14-17 % cells from parent population undergoes HR in WT, NEK1 overexpressing, and NEK1-T141A mutants. We would expect statistically significant conclusion from improvising GFP reporter experiments with cells in which endogenous Nek1 and Rad54 is knocked down, consistent with the results obtained with pRad54. Another approach would be inhibiting TLK1B interaction with NEK1 to block HR repair which is thought to be critical in Prostate Cancer progression to Castration Resistant Prostate Cancer. Citation Format: Ishita Ghosh, Arrigo DeBenedetti, Gulshan Sunavala. Role of TLK1B/NEK1 axis in DNA DSB repair: Implications for prostate cancer progression [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 1353.