Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer

Epithelial‐mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor‐outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9‐mediated epigenetic editing, resulting in highly‐specific and nearly complete suppression of ZEB1 in vivo, accompanied by long‐lasting tumor inhibition. Integrated “omic” changes promoted by dCas9 linked to the KRAB domain (dCas9‐KRAB) enabled the discovery of a ZEB1‐dependent‐signature of 26 genes differentially‐expressed and ‐methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally‐spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1‐silencing are enriched in a subset of human breast tumors, illuminating a clinically‐relevant hybrid‐like state. Thus, the synthetic epi‐silencing of ZEB1 induces stable “lock‐in” epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome‐engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.