Metastasis and chemoresistance, the most lethal consequences of cancer progression, are associated with a form of cellular plasticity known as the epithelial-to-mesenchymal transition (EMT). During this process, epithelial cells disassemble their intercellular junctions and acquire morphologic and motile phenotypes reminiscent of fibroblasts. As a form of cell fate change, EMT is likely driven by alterations to the chromatin landscape. Indeed, there is increasing evidence that epigenetic modifiers act at the promoters of key epithelial and mesenchymal genes. However, since much of this evidence is focused on specific, handpicked loci, the true extent to which EMT-mediated transcriptional rewiring depends on epigenetic factors remains largely unexplored. We therefore performed a targeted CRISPR screen in plastic pancreatic ductal adenocarcinoma (PDA) cell lines to unbiasedly identify epigenetic modifiers critical for this process. More specifically, we found that in the absence of the methyltransferase Nsd2, a writer of H3K36me2, our once plastic cells are fixed in the epithelial state, while in the absence of the demethylase Kdm2a, an eraser of H3K36me2, they remain in the mesenchymal state. These genes were also found to have functional implications for invasion and metastasis in PDA. Since loss of Nsd2 and Kdm2a results in global loss and gain of H3K36me2, respectively, we examined the function of the histone mark itself with a mutant histone that can no longer be methylated at K36 (H3K36M). Expression of H3K36M is sufficient to push plastic cells of various cancer types to an epithelial fate, implying that the modification itself, regardless of which writer or eraser is active, is the key regulator of EMT. Furthermore, we performed histone mass spectrometry and found that even without any genetic perturbations, higher global levels of H3K36me2 are indeed associated with the mesenchymal state. Finally, we mapped H3K36me2 across the genome and found that widespread loss of H3K36me2 leads to dramatic alterations in the enhancer landscape, which in turn correlates with the observed transcriptomic changes. While H3K36me2 is known to play important roles in DNA repair and mRNA splicing, our findings demonstrate a novel role for H3K36me2 as a central epigenetic regulator of EMT and metastasis by regulating enhancer activity genome wide. Just as importantly, despite massive changes in global H3K36me2 levels, the cellular phenotypes affected are remarkably specific to cell identity and differentiation. Collectively, these results implicate a novel mechanism by which global epigenomic changes underpin the transcriptional rewiring associated with epithelial plasticity.

Citation Format: Salina Yuan, Ramakrishnan Natesan, Francisco J. Sanchez-Rivera, Jinyang Li, Natarajan V Bhanu, Taiji Yamazoe, Jeffrey H. Lin, Allyson J. Merrell, Stacy K. Thomas, Junwei Shi, Ben A. Garcia, Scott W. Lowe, Irfan A. Asangani, Ben Z. Stanger. CRISPR screen identifies global regulation of H3K36me2 as an epigenomic mechanism underlying epithelial plasticity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A59.