Dual EZH1/2 inhibition enhances DNMT inhibitor efficacy in colon cancer through targeting H3K27me1

Our recent work showed that low-dose DNMT inhibitor (DNMTi) treatment sensitizes colon cancer cells to EZH2 inhibitors (EZH2i), synergistically upregulating tumor suppressor genes (TSGs) and transposable elements through activation of the calcium-calcineurin-NFAT signaling pathway. A key observation was that EZH2i displayed variable sensitivities in combination therapy, which could not be explained solely by loss of lysine 27 trimethylation on histone H3 (H3K27me3), the most commonly studied EZH2 product. This led us to perform a comprehensive pharmacologic screen of Polycomb Repressive Complex 2 (PRC2) antagonists. Here, we show that compounds targeting both EZH2 and its interchangeable catalytic subunit, EZH1, achieved superior TSG re-expression when combined with DNMTi. Integrative proteomic and epigenomic analyses revealed that EZH1/2 inhibitors reduce all three H3K27 methylation states, whereas EZH2-selective inhibitors preserve EZH1-dependent H3K27me1 at deeply Polycomb-repressed genomic regions. Notably, H3K27me1 loss coincided with deposition of p300/CBP-dependent lysine 27 acetylation on histone H3 (H3K27ac), which proved essential for TSG re-expression. Paradoxically, blocking p300/CBP activity further enhanced the growth-inhibitory effects of combined DNMT and EZH1/2 inhibition. Mechanistically, we show that EZH1/2 inhibition redistributes p300/CBP activity, reducing H3K27ac from oncogenic loci and redirecting it to bivalent regions that enable TSG re-expression. Collectively, these findings reveal a coordinated role for EZH1-dependent H3K27me1 and DNA methylation in sustaining oncogenic transcriptional programs and provide strong rationale for advancing dual EZH1/2 inhibitors for combination epigenetic cancer therapy.

Highlights

• Dual EZH1/2 inhibitors reduce all three H3K27 methylation states and robustly synergize with DNMT inhibitors.

• EZH1 mediates H3K27me1 deposition at deeply Polycomb-marked regions in the absence of EZH2.

• EZH inhibitors induce a bivalent repressive state marked by DNA methylation and H3K27ac that limits p300/CBP-dependent transcription.

• DNMT inhibitor-induced “epigenetic switching” is more vulnerable to dual EZH1/2 blockade than selective EZH2 inhibition.

• Combined EZH1/2 and DNMT inhibition redistributes H3K27ac away from pro-growth oncogenic signaling genes and towards bivalent genes.

• Suppression of oncogenic signaling by dual DNMTi and EZH1/2 inhibition is a key efficacy signature of this drug combination.