Abstract
BACKGROUND: DNA damage repair is a crucial mechanism highly related to therapy resistance for various therapeutic strategies. Our previous results have shown that the degree of drug resistance in small-cell lung cancer (SCLC) cell lines was proportional to both the transcription and expression levels of Wee1, indicating that Wee1, an evolutionarily highly conserved kinase, plays a vital role in the therapeutic resistance of SCLC. In the present study, we aim to determine the nonclassical mechanism of Wee1 on DNA repair regulation. METHODS: Western blot was conducted to determine the mono-ubiquitination level of H2Bub. Comet assay was used to evaluate the degree of DNA damage. Immunofluorescence was conducted to determine the DNA repair markers. Co-immunoprecipitation was utilized to assess the potential interactions with H2BY37ph. MTT assays were used to evaluate the survival rates of SCLC cells. RESULTS: Overexpression of Wee1 increases the level of H2BK120ub and alleviates ionizing radiation (IR)-induced DNA damage in SCLC cells. Moreover, H2BK120ub is a crucial molecule in Wee1-mediated double-strain break (DSB) repair in SCLC cells. Mechanisms study indicated that H2BY37ph is involved in Wee1-mediated H2BK120ub through interaction with the E3 ubiquitin ligase RNF20-RNF40 complex and upregulates its phosphorylation, mutation of H2BY37 phosphorylation sites attenuated DSB repair and enhanced the sensitivity of IR-induced SCLC cell death. CONCLUSION: H2BY37ph produces crosstalk with H2BK120ub in an E3 ubiquitin ligase-dependent manner, promoting Wee1-mediated DSB repair in SCLC cells. This study clarifies the nonclassical mechanism of Wee1 regulation of DSB repair, which provides a theoretical basis for the clinical understanding of the regulatory network of Wee1 and its use as a target for overcoming multiple types of therapeutic resistance.