ISGylation of γH2AX retains MDC1 and facilitates homologous recombination repair causing radioresistance in esophageal adenocarcinoma.

High expression of interferon-stimulated gene 15 (ISG15) has been associated with poor survival in patients with esophageal adenocarcinoma (EAC). Like ubiquitin, ISG15 utilizes its C-terminal LRGG motif to post-translationally modify target proteins through a process called ISGylation, thereby influencing their stability, function, and interaction networks. Given ISG15's role in the replication stress response, we hypothesized that it may also contribute to DNA repair mechanisms. We found that ISG15 is upregulated following ionizing radiation (IR), and its knockdown disrupts the IR-induced G2/M checkpoint, leading to increased radiosensitivity in EAC cells. In synchronized cells, ISG15 expression peaks during the S/G2 phases. Knockdown of ISG15 impairs homologous recombination repair (HRR) with compensatory upregulation of non-homologous end joining (NHEJ). Similarly, cells expressing an ISGylation-defective ISG15LRAA mutant exhibit reduced HRR activity and elevated NHEJ, highlighting the critical role of ISGylation in the DNA damage response (DDR). Further investigation revealed that IR-induced ISG15 modifies γH2AX at lysine 120 (K120). Overexpression of an H2AXK120R mutant in EAC cells resulted in diminished MDC1 retention at DNA damage sites, mirroring the phenotype observed with ISG15 knockdown. Additionally, depletion of ISG15 delays RAD51 foci formation at damage sites. Using a tissue microarray of chemoresistant EAC patients, we observed that ISG15 is expressed in almost all cases and, along with high RAD51 expression, correlates with poorer prognosis in node-positive patients. Collectively, we identify γH2AX as a novel substrate of IR-induced ISGylation, which facilitates efficient recruitment and retention of downstream HRR proteins and may contribute to radioresistance in EAC.