新辅助放疗被用作结直肠癌（CRC）的标准治疗方法。然而，放射治疗抗性往往导致治疗失败。识别放疗耐药基因将为联合治疗和预后标记提供新的靶点。通过对对放疗敏感或耐药的CRC患者的高通量筛选和组织阵列分析，我们鉴定出了一个强效耐药基因SUMO特异性蛋白酶5（SENP5）。然后，通过CCK8、克隆形成、彗星实验、免疫荧光和流式细胞术分析细胞凋亡和细胞周期，研究了SENP5对放射敏感性的影响。结合共免疫沉淀实验，利用SUMO蛋白质组学质谱分析鉴定了SENP5的靶标。利用病人来源的器官样本（PDO）和异种移植（PDX）模型探讨了临床应用的可能性。通过高通量筛选和CRC患者组织阵列分析，我们确认了SENP5作为一个强效耐药基因。高SENP5表达的患者显示出对放射治疗的增加耐药性。体外和体内实验证明，SENP5的沉默显著增加了CRC细胞的放射敏感性。进一步证明SENP5在同源重组（HR）依赖性DNA损伤修复中至关重要。通过SUMO质谱分析，我们确定了H2AZ作为SENP5的去SUMO化底物，并描绘了H2AZ在HR修复和癌症耐药中的SUMO化平衡。使用PDO和PDX模型，我们发现靶向SENP5显著增加了放疗的疗效。我们的发现揭示了SENP5在HR介导的DNA损伤修复和癌症耐药中的新角色，可作为癌症放疗的强效预后标记和干预靶点。© 2023年。意大利国家癌症研究所“Regina Elena”。

Neoadjuvant radiotherapy has been used as the standard treatment of colorectal cancer (CRC). However, radiotherapy resistance often results in treatment failure. To identify radioresistant genes will provide novel targets for combined treatments and prognostic markers.Through high content screening and tissue array from CRC patients who are resistant or sensitive to radiotherapy, we identified a potent resistant gene SUMO specific peptidase 5 (SENP5). Then, the effect of SENP5 on radiosensitivity was investigated by CCK8, clone formation, comet assay, immunofluorescence and flow cytometric analysis of apoptosis and cell cycle to investigate the effect of SENP5 on radiosensitivity. SUMO-proteomic mass spectrometry combined with co-immunoprecipitation assay were used to identify the targets of SENP5. Patient-derived organoids (PDO) and xenograft (PDX) models were used to explore the possibility of clinical application.We identified SENP5 as a potent radioresistant gene through high content screening and CRC patients tissue array analysis. Patients with high SENP5 expression showed increased resistance to radiotherapy. In vitro and in vivo experiments demonstrated that SENP5 knockdown significantly increased radiosensitivity in CRC cells. SENP5 was further demonstrated essential for efficient DNA damage repair in homologous recombination (HR) dependent manner. Through SUMO mass spectrometry analysis, we characterized H2AZ as a deSUMOylation substrate of SENP5, and depicted the SUMOylation balance of H2AZ in HR repair and cancer resistance. By using PDO and PDX models, we found targeting SENP5 significantly increased the therapeutic efficacy of radiotherapy.Our findings revealed novel role of SENP5 in HR mediated DNA damage repair and cancer resistance, which could be applied as potent prognostic marker and intervention target for cancer radiotherapy.© 2023. Italian National Cancer Institute ‘Regina Elena’.