Abstract Background: Radiotherapy induces cytotoxicity primarily through DNA double-strand breaks (DSBs), and the efficiency of DSB repair is a key determinant of tumor radiosensitivity. Caspase-4 (CASP4), frequently overexpressed in human malignancies, has been implicated in actin cytoskeletal remodeling; however, its involvement in nuclear actin dynamics and DNA repair remains unclear. Methods: We investigated the functional role of CASP4 in radioresponse using CASP4 knockout (KO) HepG2 and NCI-H292 cells. Colony formation assays, 53BP1 foci analysis, and a newly developed measurement system that enables the quantification of nuclear actin levels were used to assess DNA repair efficiency and actin distribution. Forced nuclear actin expression was used to evaluate its ability to rescue CASP4-dependent defects. Results: CASP4 KO significantly sensitized tumor cells to ionizing radiation, associated with decreased colony formation and persistent 53BP1 foci, indicative of impaired DSB resolution. Although CASP4-deficient cells exhibited elevated cytoplasmic F-actin, nuclear actin translocation was reduced despite comparable total actin levels, suggesting defective actin shuttling. Quantitative imaging confirmed substantially diminished nuclear actin signals in CASP4 KO cells. Notably, enforced nuclear actin expression restored 53BP1 foci resolution in CASP4-deficient cells. Conclusions: These findings identify a previously unrecognized role of CASP4 in promoting DNA DSB repair through regulation of nuclear actin dynamics. CASP4 facilitates the balance between cytoplasmic and nuclear actin to maintain efficient DNA repair and enhance radioresistance. Targeting CASP4 may therefore represent a promising strategy to overcome tumor resistance to ionizing radiation. Citation Format: Shohei Nagasaka, Tomomitsu Doi, Kunie Obayashi, Masaoki Kohzaki, Kazuhiro Sumida, Yosuke Chiba, Junkoh Yamamoto, Motoyoshi Endo. Caspase-4 deficiency impairs nuclear actin-mediated DNA double-strand break repair and increases radiosensitivity in cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 518.
Nagasaka et al. (Fri,) studied this question.