DNA损伤修复是与多种治疗策略的治疗抵抗性密切相关的关键机制。我们先前的研究结果表明，小细胞肺癌（SCLC）细胞系的耐药程度与Wee1的转录和表达水平成正比，这表明高度保守的激酶Wee1在SCLC的治疗抵抗性中发挥着至关重要的作用。在本研究中，我们旨在确定Wee1对DNA修复调控的非经典机制。进行Western blot以确定H2Bub的单泛素化水平。使用Comet试验评估DNA损伤程度。进行免疫荧光检测以确定DNA修复标记物。利用共免疫沉淀评估与H2BY37ph的潜在相互作用。使用MTT试验评估SCLC细胞的生存率。Wee1的过表达增加了H2BK120ub的水平，并减轻了SCLC细胞受到电离辐射（IR）引起的DNA损伤。此外，在SCLC细胞中，H2BK120ub是Wee1介导的双链断裂（DSB）修复的关键分子。机制研究表明，H2BY37ph通过与E3泛素连接酶RNF20-RNF40复合物相互作用并上调其磷酸化参与Wee1介导的H2BK120ub，突变H2BY37磷酸化位点弱化DSB修复并提高IR引起的SCLC细胞死亡的敏感性。H2BY37ph以E3泛素连接酶依赖性方式与H2BK120ub产生交叉作用，促进Wee1介导的SCLC细胞DSB修复。本研究阐明了Wee1对DSB修复的非经典调控机制，为临床理解Wee1的调控网络并将其用作克服多种治疗抵抗性的靶点提供了理论基础。© 2023作者。《胸腔肿瘤》由中国肺癌学组和John Wiley＆Sons Australia，Ltd.发表。

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.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.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.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.© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.