Cancer cells acquire genetic and epigenetic alterations that increase fitness and drive progression through multiple steps of tumor evolution. We have limited understanding of functional epigenetic events that are associated with melanoma progression. An in vivo RNAi screen focused on epigenetic regulators identified KMT2D, an H3K4 methyltransferase, as a tumor suppressor in melanoma. KMT2D harbors somatic mutations in various solid and hematological malignancies including in melanoma (~15%). Consistently, loss of KMT2D in a genetically engineered mouse model of melanoma bearing activated BRAFV600E and PTEN inactivation drastically accelerated melanomagenesis. Human mouse melanoma tumors with KMT2D loss displayed drastic alterations in enhancers (H3K4me1 and H3K27Ac) compared to wild type tumors and displayed poor survival. These enhancers targeted immune modulatory genes in the tumor cells and consistently KMT2D KO tumors showed altered tumor microenvironment. These data suggest that enhancer malfunction in melanomas due to mutations in KMT2D may promote highly tumorigenic behavior of melanoma cells. We hypothesize that KMT2D loss reprograms enhancers in melanoma and imparts aggressive properties to cancer cells by misregulation of immune microenvironment. Overall objectives of this grant to understand the define roles of KMT2D in modulating tumor microenvironment, test functionality of enhancer losses and determine molecular mechanism of preferential enhancer regulation by KMT2D in this system.
Aim 1 : To determine the mechanism of immune microenvironment alteration by KMT2D and its mutants in melanoma. In this aim, we will utilize iBIP;KMT2D mouse model and available human tumor data to determine alterations in immune microenvironment in KMT2D deficient conditions.
Aim 2 : To determine the functionality of immune enhancer losses in KMT2D deficient cells. In this aim, we will perform directed tests at functionality of enhancer loss on IFNg receptors and IL11 genes by CRISPR-Cas9 based enhancer deletion and epigenetic editing experiments.
Aim 3 : To determine the molecular mechanism of KMT2D mediated regulation of immune enhancers. Here we will perform mechanistic experiments to determine how KMT2D is recruited preferentially to immune enhancers. Together, our study will determine how enhancer reprogramming in tumors specifically those due to KMT2D loss reshape the tumor microenvironment which could provide insight into new therapeutic strategies in melanoma.
Our studies focus on KMT2D, a histone methyltransferase that harbors genetic alterations in 10-85% of tumors of various solid and hematological malignancies including melanoma. In addition, KMT2D mediated enhancer malfunction has been shown to occur in multiple tumor types. We propose to study in depth mechanism of action of these KMT2D mutants as well as enhancer malfunction seen in melanoma and identify therapeutic vulnerabilities imparted by these mutations. Our work is expected to provide critical molecular and translational insights into the role of KMT2D, its mutant forms and enhancers, which could have broad implications for all stages of melanoma as well as other malignancies.
