Abstract B022: C11ORF68 is essential for γH2AX formation after replication stress

Abstract

Abstract Introduction: Unresolved replication stress (RS) often leads to genomic instability – a feature of many diseases including cancer. RS can activate downstream DNA damage response (DDR) phenotypes such as phosphorylation of histone H2AX (γH2AX). Therefore, understanding the molecular foundation of DDR is crucial for identifying potential biomarkers or therapeutic targets. Our lab has identified an uncharacterized protein that is symmetrically dimethylated by PRMT5 under hydroxyurea (HU) induced RS. Therefore, we hypothesize that C11ORF68 is involved in the RS and DNA damage response. Materials and methods: RS and DNA damaging agents including HU and etoposide were used to induce RS or DNA damage in various cell lines. C11ORF68 was depleted using siRNA and dicer siRNA (dsiRNA) systems. Immunofluorescence and immunoblotting were used to evaluate DDR phenotypes. Results: One of the earliest phenomenon in DDR is phosphorylation of histone H2AX (γH2AX). Depletion of C11ORF68 under HU-induced RS reduced γH2AX foci formation as compared to cells treated with control siRNA or dsiRNA. Since HU affects cells entering S-phase, we evaluated if this is a cell cycle-related effect by using other DNA damaging agents that target cells in all phases of the cell cycle. Similarly, depletion of C11ORF68 under DNA damaging conditions showed reduced γH2AX foci formation. This may be due to a lack of upstream PIKK kinases activation as knockdown of C11ORF68 also attenuated phosphorylation of PIKKs important for H2AX phosphorylation after treatment with DNA damaging agents and RS-inducing agents. Conclusions: Our data suggests that C11ORF68 it appears to modulate early DDR even before H2AX phosphorylation and upstream PIKK activation. We are currently investigating if C11ORF68 is involved in initial events after DNA damage or RS such as PARylation events or MRN sensing of DNA breaks. Citation Format: Clarissa Chin Min Toh, Mai Phuong Hoang, Anand Jeyasekharan. C11ORF68 is essential for γH2AX formation after replication stress [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B022.