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.