EZH2 inhibition activates the Notch-driven antitumor response in Acute Myeloid Leukemia

The Notch pathway is broadly inactive in acute myeloid leukemia (AML), and its enforced activation counteracts tumor growth by promoting the apoptosis and differentiation of leukemic blasts, thus suggesting a tumor-suppressive function of Notch in this context. However, the mechanisms mediating Notch repression in AML, as well as the downstream effectors engaged upon pathway activation, are largely unknown. Evidence suggests that histone H3K27me3 modifications, regulated by the methyltransferase EZH2 and the demethylase KDM6B, modulate the epigenetic landscape at the loci of Notch receptors and target genes, thereby tuning the levels of activity and functional outcomes of the pathway across different hematologic malignancies. Interestingly, high EZH2 expression has been linked with poor clinical outcomes and therapeutic resistance in AML, and its targeting has been shown to slow disease progression, supporting a potential oncogenic role. Nevertheless, the mechanisms underlying its tumorigenic function in this cancer remain unclear. In our study, we investigated the contribution of EZH2-mediated epigenetic regulation to the repression of the Notch pathway in AML, and we explored the functional relevance of the EZH2/Notch interplay in sustaining this disease. Our results show that EZH2 inhibition, either by siRNA-mediated knockdown or by the selective inhibitor GSK126, upregulated the expression of distinct Notch ligands, receptors and target genes in acute promyelocytic leukemia HL-60 and NB4, myelomonocytic leukemia ME-1 and pro-monocytic leukemia U937 cell lines. Consistent with Notch pathway activation, GSK126 treatment promoted the nuclear translocation of the intracellular domains of Notch1 and Notch2 receptors. Mechanistically, we found that EZH2 directly suppresses Notch signaling by maintaining elevated levels of H3K27me3 at the locus of its canonical ligand Jagged2, and, consistently, pharmacological inhibition of EZH2 restored Jagged2 expression and reactivated the pathway. As expected, GSK126 treatment reduced cell viability while notably increasing the expression of the differentiation marker CEBPα across all AML cell lines examined, thus suggesting a shift toward myeloid differentiation. Notably, Notch blockage, either through the γ-secretase inhibitor DAPT or by genetic depletion of Jagged2, partially reversed the anti-growth effects of GSK126 and CEBPα accumulation in the promyelocytic HL-60 and NB4 cells. Unexpectedly, DAPT treatment was ineffective in reversing these effects in the myelomonocytic ME-1 and pro-monocytic U937 cell lines, thus suggesting that Notch activation may be relevant to the anti-cancer properties of EZH2 inhibition in an AML subtype-specific manner. Additionally, we found that GSK126 antagonized the antitumor activity of Doxorubicin while synergizing with Cisplatin in HL-60 cells, suggesting that combining EZH2 inhibitors with platinum-based chemotherapy could offer a promising therapeutic strategy for AML. Mechanistically, we demonstrated that CRISPR/Cas9-mediated Jagged2 ablation abolished the anti-viability effects of the drug combination, indicating that the synergistic interaction across the two drugs depends, at least in part, on Notch pathway activation mediated by EZH2 inhibition.