Abstract
In embryonic stem cells, promoters of key lineage-specific differentiation genes are found in a bivalent state, having both activating H3K4me3 and repressive H3K27me3 histone marks, making them poised for transcription upon loss of H3K27me3. Whether cancer-initiating cells (C-ICs) have similar epigenetic mechanisms that prevent lineage commitment is unknown. Here we show that colorectal C-ICs (CC-ICs) are maintained in a stem-like state through a bivalent epigenetic mechanism. Disruption of the bivalent state through inhibition of the H3K27 methyltransferase EZH2, resulted in decreased self-renewal of patient-derived C-ICs. Epigenomic analyses revealed that the promoter of Indian Hedgehog (IHH), a canonical driver of normal colonocyte differentiation, exists in a bivalent chromatin state. Inhibition of EZH2 resulted in de-repression of IHH, decreased self-renewal, and increased sensitivity to chemotherapy in vivo. Our results reveal an epigenetic block to differentiation in CC-ICs and demonstrate the potential for epigenetic differentiation therapy of a solid tumour through EZH2 inhibition.
Highlights
To determine the effect of Indian Hedgehog (IHH) on cancerinitiating cell (CC-IC) self-renewal, we performed an limiting dilution assays (LDAs) on three CC-IC enriched models pretreated with recombinant IHH (Fig. 5e)
The epigenetic probes screen was repeated in a broader range of colorectal cancer (CRC) samples grown as patient-derived organoids (PDO), a system recently shown to maintain the heterogeneity of primary patient samples[20] (Supplementary Table 2)
Given the important role of Enhancer of Zeste Homologue 2 (EZH2) in maintaining selfrenewal of embryonic stem cells (ESCs) through silencing of differentiation programmes, we focused on investigating EZH2 as a potential driver of CC-IC self-renewal and tumour growth
Summary
Targeting EZH2 reduces growth of patient-derived CC-ICs. Utilising a previously established serum-free, growth factorenriched spheroid culture that enriches for CC-ICs in patientderived CRC samples[17], we screened a collection of 22 selective chemical probes that inhibit epigenetic regulatory proteins using viable cell count as a readout (Supplementary Figure 1a). Three chemical probes reduced the growth of a patient-derived CC-IC enriched culture, POP92, by more than 50% (Fig. 1a). These growth inhibitory probes included the BET Bromodomain BRD2/3/4 inhibitor JQ1, an antagonist of the Bromodomains of BRPF1/2/3, and the EZH1/2 inhibitor UNC199919. To assess EZH2 levels in CRC, we analysed EZH2 and H3K27me[3] immunohistochemistry staining from a tumour microarray of 283 patient samples Staining for both EZH2 and H3K27me[3] were significantly higher in CRC compared with normal intestinal tissue (Fig. 2a, b). EZH2 expression is inversely correlated with the majority of genes, expressed in the top of the crypt, Pearson’s correlation
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