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, we silence 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 transcriptomic and epigenetic changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) in our cell line models enabled the discovery of a ZEB1-dependent-gene-signature associated with transcriptional upregulation, promoter DNA demethylation and enhanced chromatin accessibility in core cell adhesion loci, outlining epigenetic reprogramming towards 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.
To induce a more robust epithelial phenotype, we further expanded the dCas9-KRAB epi-editing to targeting multiple pro-mesenchymal transcription factors, including the SNAI, TWIST and ZEB families (genome multiplexing). Our results illuminated the non-redundant role of individual transcription factors inducing EMT in TNBC, as well as the cooperative interactions of multiple transcription factors. Our data also outline novel therapeutic vulnerabilities to target these poor outcome tumours. Finally, we describe non-toxic targeted formulations tailored for the delivery of dCas9-KRAB in pre-clinical settings for the treatment of TNBC.
In summary, the synthetic epi-silencing of ZEB1 in conjunction with other EMT transcription factors induces stable “lock-in” epigenetic reprogramming of mesenchymal tumors associated with a more epithelial epigenetic landscape. We outline epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers driven by “hard-to-drug” oncogenic transcription factors.