Methyltransferase Inhibition Enables Tgf Driven Induction of CDKN2A and B in Cancer Cells

CDKN2A/B deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three CDKN2A/B transcripts are independently expressed to varying degrees, but one, ARF, is uniformly silenced. Although TGF beta induces certain CDKN2A/B transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. CDKN2A/B induction by TGF beta correlated with de novo appearance of three H3K27Ac peaks within a 20 kb cis element similar to 150 kb proximal to CDKN2A/B. Deleting that segment prevented their induction by TGF beta but not a basal increase driven by methyltransferase inhibition alone. Expression of two CDKN2A/B transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb cis elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGF beta-dependent activation of a remote enhancer to reverse CDKN2A/B silencing. Though focused on CDKN2A/B here, such crosstalk may apply to other TGF beta-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.