title: Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system.
abstract: The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.|School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia.|Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Garcia-Bloj
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.|School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia.
authors.forename: Colette
authors.initials: C
authors.lastname: Moses
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.
authors.forename: Agustin
authors.initials: A
authors.lastname: Sgro
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.
authors.forename: Janice
authors.initials: J
authors.lastname: Plani-Lam
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.|School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia.
authors.forename: Mahira
authors.initials: M
authors.lastname: Arooj
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.|School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia.
authors.forename: Ciara
authors.initials: C
authors.lastname: Duffy
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.
authors.forename: Shreyas
authors.initials: S
authors.lastname: Thiruvengadam
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.
authors.forename: Anabel
authors.initials: A
authors.lastname: Sorolla
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.
authors.forename: Rabab
authors.initials: R
authors.lastname: Rashwan
authors.affiliation: School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia.
authors.forename: Ricardo L
authors.initials: RL
authors.lastname: Mancera
authors.affiliation: Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile.
authors.forename: Andrea
authors.initials: A
authors.lastname: Leisewitz
authors.affiliation: School of Nursing, Virginia Commonwealth University, Richmond, Virginia, 23298, USA.
authors.forename: Theresa
authors.initials: T
authors.lastname: Swift-Scanlan
authors.affiliation: Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Católica de Chile, Santiago, RM, 8330034, Chile.|Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile.
authors.forename: Alejandro H
authors.initials: AH
authors.lastname: Corvalan
authors.affiliation: Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia.|School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia.
authors.forename: Pilar
authors.initials: P
authors.lastname: Blancafort
mesh_terms: D017209:Apoptosis; D064113:CRISPR-Cas Systems / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D062085:RNA, Long Noncoding / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D015843:Serpins / Q000235:genetics / Q000378:metabolism; D000071682:Sterile Alpha Motif / Q000235:genetics*; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D006023:Glycoproteins; D018398:Homeodomain Proteins; D062085:RNA, Long Noncoding; C412566:RPRM protein, human; D012097:Repressor Proteins; C085344:SERPIN-B5; D015843:Serpins; C097504:TGIF1 protein, human; D025521:Tumor Suppressor Proteins; C000622084:long noncoding RNA CASC9, human
keywords: CRISPR/dCas9; TALE; ZF; gene reactivation; tumor suppressor genes