Characterizing the role of RNF25 in repair of DNA alkylation in blood cancers

摘要 氮素芥末是第一种用于抗癌的化疗药物。这些药物，其中一些仍然 今天使用的是一大组治疗性DNA烷基化试剂的成员。烷化剂，如 马法兰、白头翁和环磷酰胺目前用于治疗侵袭性淋巴瘤、多发性 骨髓瘤和急性髓系白血病。虽然其中一些试剂用更长的碳链修饰碱基，但另一些试剂会产生 简单的甲基化。例如，被广泛用于胶质母细胞瘤的替莫唑胺甲基化这两种药物 腺嘌呤和鸟嘌呤在几个位置。甲基化试剂MMS在化学上是一种甲烷磺酸盐 类似于白花丹。我们已经进行了一次无偏见的CRISPR筛查，以检查缺失的敏感性 每一种泛素连接酶和配对到40种不同的小分子抑制剂的广泛的生物途径， 从细胞骨架到线粒体功能再到有丝分裂。我们发现，删除研究较少的 泛素连接酶RNF25使细胞对DNA烷化剂MMS非常敏感，但根本不敏感 对40种其他药物敏感，包括其他DNA损伤剂。鉴于RNF25在 对于MMS的抗性，RNF25很可能在对DNA烷基化的反应或修复中起直接作用。我们 建议探讨RNF25在DNA烷基化修复中的作用。我们将扩展我们的发现，这些发现在 慢性粒细胞白血病来源的细胞系，到其他血癌。我们将确定烷基化碱的去除率。 来自RNF25突变体的DNA，并检查RNF25突变体对其烷基化试剂的光谱 很敏感。最后，我们将确定RNF25的底物以确定其促进机制 对这些药物的抗药性。

Abstract Nitrogen mustards were the first chemotherapeutics used to fight cancer. These drugs, some of which are still in use today, are members of a large group of therapeutic DNA alkylating agents. Alkylating agents such as Melphalan, Busulfan, and Cyclophosphamide are currently employed for aggressive lymphoma, multiple myeloma and AML. While some of these agents modify bases with longer carbon chains, others generate simple methylations. For example, temozolomide, which is widely used for glioblastoma, methylates both adenine and guanine at several positions. The methylating agent MMS is a methane sulfonate chemically very similar to Busulfan. We have carried out an unbiased CRISPR screen to examine the sensitivity of deletions of each ubiquitin ligase and DUB to forty different small molecule inhibitors of a wide array of biological pathways, ranging from the cytoskeleton, to mitochondrial function to mitosis. We found that deletion of the poorly-studied ubiquitin ligase RNF25 rendered cells extremely sensitive to the DNA alkylating agent MMS, but not at all sensitive to forty other drugs, including other DNA damaging agents. Given the specific role of RNF25 in resistance to MMS, it is likely that RNF25 has a direct role in the response to, or repair of, DNA alkylation. We propose to explore the role of RNF25 in the repair of DNA alkylation. We will extend our findings, obtained in a CML-derived cell line, to other blood cancers. We will determine the rate at which alkylated bases are removed from DNA in RNF25 mutants and examine the spectrum of alkylation agents to which RNF25 mutants are sensitive. Finally, we will identify substrates of RNF25 to determine the mechanism by which it promotes resistance to these agents.