Abstract 1488: DNA polymerase kappa slows replication fork speed by promoting fork reversal in glioblastoma

Abstract Replication fork stalling in response to replication stress triggers the activation of replication stressresponse (RSR) pathways to facilitate repair and restart replication processes, including fork reversal andtranslesion DNA synthesis (TLS). During fork reversal, the replication fork undergoes a structural rearrangementto form a four-stranded structure, known as a “chicken foot, ” which helps regulate fork speed by recruitment ofdifferent fork reversal factors, including HLTF, and SMARCAL1. Optimal replication conditions ensure a relativelyconstant and rapid fork progression rate, which is essential for timely genome duplication. TLS requiresspecialized DNA polymerases that assist fork progression by promoting direct bypass of replication blocks. Inglioblastoma, the most aggressive and resistant form of brain cancer, the TLS polymerase kappa (Pol κ) isoverexpressed. This overexpression causes resistance to standard therapeutics and is associated with poorprognosis. A major goal of our study is to understand how Pol κ protects glioma cells from endogenous oncogenicstressors and the DNA-damaging effects of chemotherapeutics. Results from Eoff lab indicate that Pol κ slowsreplication fork speed in glioma cells (T98G, U118-MG) without significantly affecting non-glioma cells (HAP-1, U2OS, RPE). We hypothesized that Pol κ slows replication fork speed by promoting fork reversal in glioma cells.To test this hypothesis, we used a proximity ligation assay (PLA) to verify the colocalization of fork reversalfactors SMARACL1, and HLTF to sites of DNA synthesis in glioma cells. PLA measures the proximity betweenproteins and newly synthesized DNA labeled with nucleotide analog 5-ethynyl-2′-deoxyuridine (EdU), indicatingtheir potential interaction or colocalization, as evidenced by the appearance of fluorescent foci. EdU-SMARCAL1, or EdU-HLTF PLA foci were increased in T98G WT without a significant increase in POLK KO T98G cellsfollowing treatment with 50 nM CPT with no considerable increase in POLK KO T98G cells. These resultssuggest that Pol κ has a role in promoting the colocalization of fork reversal proteins to sites of DNA synthesisin T98G (glioma) cells. To support this conclusion and to study how this affects fork speed, we used DNA fiberspreading (DFS) to determine fork speed with/without depletion of the fork reversal factor SMARCAL1 and HLTF.Depletion of SMARCAL1 and HLTF produced longer IdU tracts in T98G WT cells, indicating faster fork speed.The fork acceleration phenotype in POLK-KO T98G cells remained unaffected by the knock-down ofSMARCAL1, suggesting that these proteins work in the same pathway to regulate fork dynamics in glioblastomacells. These results are the first to establish a link between Pol κ and fork reversal, helping to explain how glioma-specific mechanisms tolerate high rates of DNA damage in the tumor microenvironment. Citation Format: Reham S. Sewilam, Megan R. Reed, Robert L. Eoff. DNA polymerase kappa slows replication fork speed by promoting fork reversal in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1488.