KDM4B Regulates Replication Fork Stability During Replication Stress in Concert with Poly(ADP-ribose) Metabolism in Prostate Cancer

Background/Objectives: Poly(ADP-ribose) polymerase-1 (PARP1) and poly(ADP-ribose) glycohydrolase (PARG) coordinately regulate poly(ADP-ribose) (PAR) metabolism, which is essential for maintaining genome integrity during DNA replication. We investigated the role of the histone demethylase KDM4B in replication-fork protection and whether KDM4 inhibition could be exploited therapeutically in prostate cancer. Methods: The role of KDM4B in replication stress was examined in prostate cancer cells using genetic and pharmacological approaches. KDM4B interactions with PARG, stalled replication forks, and DNA fork structures were assessed using biochemical and molecular assays. Replication-fork stability, chromatin-associated proteins, PARylation, and DNA damage were evaluated following KDM4 inhibition. The therapeutic effects of combined KDM4 and PAR metabolism inhibition were assessed using the KDM4 inhibitor B3 together with the PARP inhibitor olaparib or the PARG inhibitor PDD00017273. Results: KDM4B interacted with PARG and localized to replication stress-associated ssDNA regions, where its depletion increased H3K9me3 accumulation and impaired PARG and XRCC1 accumulation. KDM4B directly bound fork-like DNA structures in vitro, and PARylation enhanced this interaction. Genetic depletion of KDM4B or pharmacological KDM4 inhibition disrupted replication stress responses, altered PAR signaling, and increased replication-associated DNA damage. Proteomic analysis using a synthetic Y-DNA probe revealed changes in the association of replication- and repair-related proteins following KDM4 inhibition. Combined treatment with B3 and either olaparib or PDD00017273 produced synergistic cytotoxicity in prostate cancer cells, particularly in the context of TP53/RB1 deficiency, with pronounced activity of the B3–PDD00017273 combination in HRR-proficient PC3 cells. Conclusions: These findings identify KDM4B as a regulator of replication stress responses and support a functional interplay between KDM4B and PAR metabolism in maintaining replication fork integrity. The synergistic cytotoxicity observed with combined KDM4 and PARP/PARG inhibition provides a rationale for further preclinical investigation, although the efficacy and tolerability of these combinations remain to be established in vivo.