Liquid biopsies to detect DNA methylation driven EMT as a mechanism underlying therapy resistance in cancer

We here study 10 cell lines from diverse tissues of origin that acquired resistance to various targeted and chemotherapy-based treatments through EMT and explore whether and how DNA methylation changes causally underlie the EMT phenotype. We show that common DNA methylation dynamics mediated by the Ten-Eleven-Translocation (TET) demethylating enzymes drive a reversible EMT-mediated phenotype of therapy resistance, both to the drugs the cells had originally been made resistant to, as well as to other targeted and chemotherapy-based treatments. Overexpression of the miRNA-200 cluster, exposure to 5-azacytidine and knock down of the DNA methyltransferases (DNMT) are used to revert EMT and DNA methylation changes, respectively, and all result in the reversal of EMT and the associated therapy resistance.Finally, we clinically validate that DNA methylation changes drive EMT-mediated resistance to sorafenib in patients with advanced HCC. Specifically, we develop a capture-based protocol to interrogate DNA methylation in low amounts of circulating tumour DNA (ctDNA) in patients with advanced hepatocellular carcinoma (HCC) treated with sorafenib. By interrogating the methylation status of several thousands of CpGs (n=47,569) in liquid biopsies, longitudinally collected during treatment, we assess whether DNA methylation changes also drive EMT and therapy resistance in a clinical setting. Particularly, by monitoring methylation changes in EMT genes in serially-sampled ctDNA, we are able to predict tumour response and acquired resistance to sorafenib.