RNA methylation as a novel effector of SETD2 in kidney cancer - ABSTRACT Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers and is the 8th leading cause of cancer death in the United States. In addition to near ubiquitous loss of VHL, the landscape of ccRCC is dominated by mutations in epigenetic regulators SETD2, PBRM1, and BAP1. The epigenome is profoundly disrupted in ccRCC with defects in epigenetic marks on DNA, RNA, and histones, culminating in aberrant gene expression. Mutations in the SETD2 histone H3 lysine 36 trimethylase occur in ~25% of primary ccRCC but increase to >60% in ccRCC metastases. Completion of TCGA enabled identification of actionable mutations in virtually every solid tumor and those profiles now drive treatment decisions. One major exception, however, is RCC, where the current standard of care, checkpoint inhibitor and anti-VEGF therapy, does not account for ~50% of RCCs having mutations in chromatin regulators like SETD2. Many ccRCCs display heterogeneous and subclonal intratumor SETD2 mutations, but we do not understand how SETD2 loss deregulates cell growth and what pathways it works through. After first-line therapy, response rates are 20%, highlighting the need to understand and target drivers of metastasis like SETD2. A recent novel finding linked SETD2 activity to the targeting of N6-methyladenosine in mRNA, a key mechanism for controlling many RNA metabolic processes, which has also been shown to be deregulated in cancer and contribute to metastatic processes. Our preliminary data demonstrate that SETD2 loss leads to large-scale re-localization of m6A and an enhanced sensitivity to an m6A inhibitor that is entering clinical trials. This SETD2-m6A link has not been explored in a ccRCC context but could represent a novel SETD2 effector mechanism and drug target in ccRCC. We explore this relationship for the first time with this pilot R21 study, making it directly relevant to NOT-CA-23-060, RNA Modifications in Cancer Biology. Our proposal is significant because ~30% of patients diagnosed with clinically localized RCC experience metastatic progression, and once metastatic disease develops only ~7% survive >5 years. Our central hypothesis is that loss of SETD2 drives aggressive ccRCC in part through deregulated m6A targeting that promotes a pro-metastatic expression program. Our proposal is expected to develop relevant ccRCC cell line models and define the links between SETD2/H3K36me3 and METTL3/m6A in ccRCC through the following specific aims. In aim 1, we will map H3K36me3, m6A, METTL3, and expression patterns using RIP-/ChIP-/RNA-seq in novel SETD2 isogenic renal cell line models. In aim 2, we will perform integrative analysis of omics data to uncover genes/pathways impacted by SETD2 loss in an m6A-dependent manner. Finally, in aim 3 we will genetically and pharmacologically modulate m6A in SETD2 isogenic cell lines and define cancer-relevant cellular growth phenotypes. This work is expected to positively affect human health by yielding a new understanding of the mechanistic underpinnings of SETD2 driven cell growth deregulation and uncovering new ways to therapeutically target this mutation.
