Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

Chromatin plasticity can be exploited therapeutically for the selective manipulation of the epigenetic state, to ultimately restore transcriptional aberrations driving tumorigenesis. Abnormal epigenetic silencing of tumor suppressors genes (TSGs) is one of the carcinogenesis features in hepatocellular carcinoma (HCC). We computationally identified a panel of 12 putative TSGs exhibiting an anti-correlation between transcript abundance and promoter DNA methylation, while harbouring no genetic alterations. Silencing of at least one of these TSGs was bioinformatically predicted for any given HCC sample case, and encompassing all the subtypes of HCC, making it possible the targeting of any patient in a personalized manner. To specifically reactivate one or more TSGs of this panel we produced CRISPR activators (CRISPRa) multiplexing systems tailored to representative HCC lines. Unlike epigenetic drugs, which lack locus-selectivity, CRISPRa systems enabled a potent and precise (“at will”) reactivation of up to 4 TSG simultaneously. We show that reactivation of HHIP, MT1M, PZP, and TTC36 TSGs in Hep3B cells led to a functional decrease in cell viability, cell proliferation, and cell migration. By combining different effector domains, we identified a toolbox of epigenetic effectors and gRNAs for the functional interrogation and targeting of putative TSGs silenced in liver cancer patients. We outlined a highly personalized precision oncology approach for the treatment of aggressive forms of HCC. Infinium MethylationEPIC (850K) BeadChip data for Hep3B cells with a SpdCas9-VPR MS2-MCP-p65-HSF1 construct and no targeting gRNA (4 replicates), Hep3B cells with a SpdCas9-VPR MS2-MCP-p65-HSF1 construct and a gRNA targeting the HHIP promoter (4 replicates), Hep3B cells with a SpdCas9-TET1-CD MS2-MCP-p65-HSF1 construct and no targeting gRNA (4 replicates), and Hep3B cells with a SpdCas9-TET1-CD MS2-MCP-p65-HSF1 construct and a gRNA targeting the HHIP promoter (4 replicates). Please note that for the SpdCas9-TET1-CD MS2-MCP-p65-HSF1 no gRNA samples, there are 3 biological replicates, one of which has two technical replicates. This is outlined in Supplementary Data S6 of the associated manuscript, but for clarity: GSM6503183 - repL1 (Biol Rep 1) GSM6503184 - repL2_1 (Biol Rep 2, Tech Rep 1) GSM6503185 - repL3 (Biol Rep 3) GSM6503186 - repL2_2 (Biol Rep 2, Tech Rep 2)