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

Charlene Waryah; Joseph Cursons; Momeneh Foroutan; Christian Pflueger; Edina Wang; Ramyar Molania; Eleanor Woodward; Anabel Sorolla; Christopher Wallis; Colette Moses; Irina Glas; Leandro Magalhães; Erik W Thompson; Liam G Fearnley; Christine L Chaffer; Melissa Davis; Anthony T Papenfuss; Andrew Redfern; Ryan Lister; Manel Esteller; Pilar Blancafort

doi:10.1002/advs.202301802

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

Adv Sci (Weinh). 2023 Aug;10(22):e2301802. doi: 10.1002/advs.202301802. Epub 2023 May 22.

Authors

Charlene Waryah^{1

2}, Joseph Cursons^{3

4}, Momeneh Foroutan^{3

4}, Christian Pflueger^{5

6}, Edina Wang^{1

2}, Ramyar Molania^{7

8}, Eleanor Woodward¹, Anabel Sorolla^{1

2}, Christopher Wallis^{1

2}, Colette Moses^{1

9}, Irina Glas¹, Leandro Magalhães^{1

10}, Erik W Thompson^{11

12}, Liam G Fearnley^{7

13}, Christine L Chaffer^{14

15}, Melissa Davis^{7

8}, Anthony T Papenfuss^{7

13}, Andrew Redfern¹⁶, Ryan Lister^{5

6}, Manel Esteller^{17

18

19

20}, Pilar Blancafort^{1

2}

Affiliations

¹ Cancer Epigenetics Group, Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.
² Centre for Medical Research, University of Western Australia, Perth, WA, 6009, Australia.
³ Monash Biomedicine Discovery Institute, Monash University, Melbourne, VIC, 3800, Australia.
⁴ Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia.
⁵ Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, University of Western Australia, Perth, WA, 6009, Australia.
⁶ Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, 6 Verdun St, Perth, WA, 6009, Australia.
⁷ Population Health and Immunity Division, Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, 3052, Australia.
⁸ Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, VIC, 3010, Australia.
⁹ Evolutionary Neurogenomics, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, XH 1098, The Netherlands.
¹⁰ Postgraduate Program of Genetics and Molecular Biology, Institute of Biological Sciences, Federal University of Pará, Belém, 66075-110, Brazil.
¹¹ School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, 4000, Australia.
¹² Translational Research Institute, Brisbane, QLD, 4102, Australia.
¹³ Department of Medical Biology, University of Melbourne, Melbourne, VIC, 3800, Australia.
¹⁴ The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia.
¹⁵ St Vincent's Clinical School, UNSW Medicine, Darlinghurst, NSW, 2010, Australia.
¹⁶ School of Medicine, University of Western Australia, Perth, WA, 6009, Australia.
¹⁷ Josep Carreras Leukemia Research Institute (IJC), Badalona, Barcelona, 08916, Spain.
¹⁸ Centro de Investigacion Biomedica en Red Cancer (CIBERONC), Madrid, 28029, Spain.
¹⁹ Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, 08010, Spain.
²⁰ Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Catalonia, 08007, Spain.

Abstract

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.

Keywords: CRISPR/dCas9 repression; ZEB1; cancer epigenetics; epithelial-mesenchymal transition; triple negative breast cancer.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Clustered Regularly Interspaced Short Palindromic Repeats
Epigenesis, Genetic / genetics
Humans
Neoplasm Recurrence, Local / genetics
Transcription Factors / genetics
Triple Negative Breast Neoplasms* / genetics
Triple Negative Breast Neoplasms* / pathology

Substances

Transcription Factors

Abstract

Publication types

MeSH terms

Substances

Grants and funding