ABSTRACT Melanoma is the deadliest form of skin cancer, and accounts for nearly 8,000 deaths each year in the United States. Recently developed immune checkpoint therapies including α-CTLA4 and α-PD1 are promising strategies for treating melanoma and have produced impressive clinical results. However, many patients on these therapies eventually develop resistance, and there remains no cure for melanoma. Aberrant methylation has long been recognized as a driver of melanoma progression and is associated with poor survival in patient cohorts. Increased DNMT3B expression is associated with poor survival in patient cohorts, and DNMT3B promotes melanoma progression via miR-196b and the mTORC2 pathway. Preliminary data indicates that DNMT3B modulates phenotypically distinct cell subsets in murine and human melanoma. Furthermore, hypomethylation of the immune checkpoint PD-L1 was recently shown to be associated with improved survival in melanoma patients, suggesting a role for DNA methylation in the anti-tumor immune response. Therefore, we hypothesize that DNMT3B regulates intratumoral heterogeneity and anti-tumor immunity in melanoma. In our first aim, we will test the heterogeneity of melanoma cell lines lacking DNMT3B. We will then use bisulfite sequencing and expression assays to evaluate methylation changes between subsets in knockout- and wild-type cells. We will use CRISPR/Cas9-mediated genetic knockouts to validate putative mechanisms by which DNMT3B regulates heterogeneity in human and murine cell lines. In our second aim, we will assess DNMT3B control of anti-tumor immunity by evaluating growth of DNMT3B-null and wild-type melanoma tumors in immune-competent and deficient host animals, and assess associated tumor microenvironment changes. We will then identify methylation and expression changes in DNMT3B-null tumors, and use genetic knockouts to validate putative mechanisms. Finally, we will use human datasets and cell lines to translate these findings into human melanoma. Together, these studies will identify mechanisms by which DNMT3B promotes melanoma heterogeneity and resistance to immune checkpoint blockade. This may lead to development of therapies targeting these mechanisms, for improving survival of patients with advanced melanoma.