G9a Is SETting the Stage for Colorectal Oncogenesis

Recently, Kato et al. reported recurrent activating mutations in the SET domain of histone methyltransferase G9a, driving an oncogenic cascade in melanoma. The authors also reported correlations between G9a expression and the regulation of the canonical WNT pathway. Although we could not observe such mutations in human colorectal adenocarcinoma, newly reported findings are of high relevance to colorectal cancer, as WNT target gene signatures were closely associated with G9a expression. Here, we put into perspective such new results on G9a expression in colorectal cancers and the potential relationship with tumor heterogeneity and acquisition of neoplastic stemness.

could not identify the recurrent gain-of-function mutations in the G9a SET domain (G1069L and G1069W) reported by Kato et al. in this set of patients ( Figure 1A), as well as in Metastatic Colorectal Cancer samples (MSKCC, Cancer Cell 2018). EHMT2 gains/amplifications (≥3 copies) are found in 25.8% of melanomas and 21.1% of colorectal patients ( Figure 1B, TCGA PanCancer Atlas Colorectal Adenocarcinoma). EHMT2 was found mutated in 1.7% of queried colorectal cancer patients ( Figure  1C). genomic aberrations and mutations vs. mRNA expression (z-scores vs. diploid samples, RNA Seq V2 RSEM) in human colorectal tumors (n = 524 cases). (C) EHMT2 and APC mutation frequency from eight datasets. * A total of 3407 independent patients were queried due to patients overlap between some specific datasets. (D) Distribution of EHMT and/or APC-mutated cases across a dot chart of both genes' expression levels (RSEM batch normalized from Illumina HiSeq_RNASeqV2) (n = 524 cases).
We expanded our analysis to other malignancies using TCGA PanCancer Atlas cohorts and assessed the EHMT2/G9a mutation status in breast (1084 samples), glioblastoma (592 samples), pancreatic (184 samples) and lung tumors (1053 samples). We could not identify recurrent G1069 mutations in the G9a SET domain as reported by Kato et al. Thus, to the best of our knowledge, reported activating mutations in G9a seem to be a distinct characteristic of melanomas. Yet, We expanded our analysis to other malignancies using TCGA PanCancer Atlas cohorts and assessed the EHMT2/G9a mutation status in breast (1084 samples), glioblastoma (592 samples), pancreatic (184 samples) and lung tumors (1053 samples). We could not identify recurrent G1069 mutations in the G9a SET domain as reported by Kato et al. Thus, to the best of our knowledge, reported activating mutations in G9a seem to be a distinct characteristic of melanomas. Yet, overexpression of G9a protein and/or associated H3K9 methylation levels is not anecdotic in colorectal tumors and has been reported by several groups [8][9][10].
Despite clear connections between G9a activating mutations, Kato et al. established a correlation between this histone methyltransferase and repression of DKK1 in colorectal tumors. This complements the body of knowledge on canonical WNT hyperactivity as a hallmark of colorectal cancer linked to tumorigenesis, self-renewal and maintenance of neoplastic stem phenotype. The interplay between G9a and soluble WNT inhibitors was previously documented in neuroendocrine tumors, where DKK1 and DKK3 promoters are silenced by G9a-dependent H3K9 methylation [11,12]. In addition, Kato et al. used ChIP-seq data from primary colon cancer-initiating cells to confirm G9a enrichment at the DKK1 promoter. This further supports the prospect of G9a as a modulator of canonical WNT/β-catenin in neoplastic stem cells. However, oncogenic mutations within the β-catenin destruction complex act downstream of the G9a-DKK1 axis in hyperactivation of the canonical WNT pathway [11]. While APC gene mutations are found in a large proportion of colorectal tumors and are tightly related to WNT hyperactivity, no particular patterns were observed between APC-mutated cases and G9a mutations or expression ( Figure 1C,D). As stated by Kato et al., G9a could drive tumorigenesis by impacting WNT-related gene programs via other pathways, which is likely to be the case in colorectal cancer. Given the importance of G9a in early embryonic gene regulation [13][14][15], it could possibly influence pluripotent-like gene networks linked to cancer stemness [16].
Remarkably, Kato et al. addressed the issue of a significant number of patients experiencing resistance to immunotherapeutic approaches. Indeed, the use of immune checkpoint inhibitors has currently yielded no major clinical benefits for most colorectal cancer patients [17]. The authors described the inverse correlation observed between G9a expression/copy number and T-cell signatures in melanoma patients. Furthermore, the GLP/G9a inhibitor UNC0642 was used in vivo and led to substantial tumor regression when combined with anti-PD1 and anti-CTLA4 treatments in a syngeneic melanoma mouse model. The authors concluded that G9a overexpression promotes a poorly immunogenic tumor microenvironment through DKK1 and WNT signaling. Such a "cold" tumor immune microenvironment could be reversed by G9a inhibition. Tumor immune insusceptibility is another hallmark of cancer stemness, as recently reported by Miranda et al., who described the role of neoplastic stem cells on immune cell exclusion from the tumor microenvironment [18]. Thus, cancer stem cells contributing to intratumoral heterogeneity, are limiting tumor immune responses via silencing of endogenous retroviral elements and up-regulation of immunosuppressive checkpoints [18]. It is noteworthy that G9a is involved in epigenetic silencing of such endogenous retroviral elements in pluripotent stem cells [19]. Taken together, these findings suggest that G9a overexpression, as observed in colorectal cancer, may contribute to poor tumor immunogenicity by inducing a neoplastic stemness phenotype.
The findings reported by Kato et al. are setting the stage for the use of the G9a/GLP inhibitors as an opportunity to evaluate G9a inhibition as a therapeutic avenue to target colorectal cancer-initiating cells in vivo. As aforementioned, the small molecule UNC0642 was used to block G9a histone methyltransferase activity in mouse syngeneic and human-to-mouse xenograft models. The authors highlighted the importance of developing novel and more effective therapeutics targeting epigenetic determinants, emphasizing the particular "druggability" of G9a in cancer. Recently, Casciello et al. also used UNC0642 to show that inhibition of G9a in breast cancer cells decreases tumor growth and lung metastasis in vivo [20]. These are encouraging demonstrations of in vivo potential of UNC0642 to inhibit H3K9 methylation, since it was previously considered to have poor pharmacokinetics [21]. It will be interesting to see if more studies continue to implement a direct-G9a/GLP inhibition strategy in upcoming cancer research.