Select EZH2 inhibitors enhance the viral mimicry effects of DNMT inhibition through a mechanism involving Calcium-Calcineurin-NFAT signaling [EM-seq]

DNA methyltransferase inhibitors (DNMTi) are FDA-approved for various hematological malignancies but have limited efficacy in solid tumors. DNA hypomethylation with these drugs is associated with elevated lysine 27 tri-methylation on histone H3 (H3K27me3). We hypothesized that this EZH2-dependent repressive mark limits the full potential of DNMTi. Here, we show in cell line and tumoroid models of colorectal cancer, that low-dose DNMTi sensitizes cells to selective EZH2 inhibitors (EZH2i) that have limited single agent toxicity, and that EZH2i enhances DNMTi-driven molecular and therapeutic effects. Through integrative epigenomic analyses, we reveal that DNMTi induces H3K27me3 accumulation at genomic regions poised with EZH2. Unexpectedly, combined treatment alters the epigenome landscape to promote transcriptional upregulation of the Calcium-Calcineurin-NFAT signaling pathway. Blocking this pathway limits the transcriptional activating effects of the drug combination, including expression of transposable elements and innate immune response genes within a viral defense pathway. Consistently, we demonstrate positive correlations between DNMTi- and viral infection-associated transcription profiles and Calcium signal activation in colon cancer patient samples. Collectively, our study demonstrates that compensatory EZH2 activity following DNA hypomethylation presents a barrier to the therapeutic action of DNMTi in colon cancer, reveals a new application of EZH2i beyond cancers associated with PRC2 hyperactivity, and links Calcium-Calcineurin-NFAT signaling to epigenetic therapy-induced viral mimicry. HCT116 cells were treated with two different doses of DAC (30 nM, 300 nM) and DMSO for the Vehicle control in biological duplicate for 72 hours. Cells were then fixed and prepped for chromatin immunoprecipitation of H3K27me3. Purified DNA fragments from the H3K27me3 ChIP assay were then processed through Enzymatic Methyl-seq (EM-seq) to query the DNA methylation status of the H3K27me3 purified fragments from each drug treatment.