**Acknowledgements**

*Advances in the Molecular Understanding of Colorectal Cancer*

**5. Conclusion and perspectives**

as monotherapies or in combination with other anticancer agents. At the present time, several ongoing early and late-phase II and III clinical trials investigating the efficacy of immune checkpoint inhibitors in MSI-H and MSS CRC patients are being extensively explored including pembrolizumab (Keytruda), which recently

Moreover, the possibility of combining epigenetic therapy and immunotherapy has also been recently explored, and several ongoing clinical trials in CRC investigating the combination strategies of HDACi and DNMTi with checkpoint inhibitors have been undertaken. Specifically, these epigenetic therapies have been shown to augment the effect of checkpoint inhibitors and are currently in early and late phase clinical trials [143]. However, since MSS subtypes represent the larger fraction of CRC cases, the marginal activity displayed by drugs such as pembrolizumab for treating MSS CRCs has been less than encouraging [144]. Hence, overcoming the clinical ineffectiveness of this class of drugs for this subtype remains an important need. Intriguingly, however, recent studies showed that treatment with 5-azacitidine and entinostat in CRC cell lines conferred a shift towards a CIMP+ signature, which would predictively convert them into a more immunogenic state [145]. This increased sensitivity to immunotherapy has prompted a clinical trial evaluating this strategy, with the combination regimen of romidepsin (HDAC inhibitor) and cc-486 with pembrolizumab in MSS-CRC patients (NCT02512172) [145]. Finally, romidepsin was also found to potentiate 5-FU cytotoxicity in HCT-116, HT29, and SW48 cells by inducing apoptosis and cell cycle arrest [146]. Interestingly, MHC class II gene expression was also induced with this combination, once again supporting the possible cooperation of epigenetic therapy with immunomodulatory agents [146]. In summary, the above evidences support a cooperative role between epigenetic and immune therapies, although further efforts to optimize the epigenetic control of immune-related gene expression will be necessary to successfully translate these notions to the clinic.

In this chapter, we have highlighted the pivotal contribution of epigenetic deregulation, specifically, DNA methylation, histone alterations, and miRNAs to the initiation, progression, and prognosis of CRC. We also underscored the relevance of these epigenetic mechanisms in terms of classifying CRC subtypes as well as their importance in guiding strategies for therapeutic intervention. Moreover, we emphasized the epigenetic enzymes that are involved in these aberrant pathways and presented some up-to-date findings on pre-clinical and clinical trials of epigenetic drugs used as single agents or in combination with conventional anticancer agents in CRC. Furthermore, mounting evidence demonstrates that epigenetic drugs are also capable of altering the immunogenicity of the CRC microenvironment and creating

opportunities for potentiating the effects of immune checkpoint inhibitors. Understandably, drugs targeting the cancer epigenome are also plagued with major challenges including lack of specificity, toxicity, and short half-life. Fortunately, these challenges have facilitated re-evaluation of the dosing and formulation strategies for epigenetic drugs, leading to superior therapeutic drugs with lower toxic profiles. Another underexplored avenue includes targeting less commonly manipulated epigenetic mechanisms such as the use of miRNA mimics [147]. Furthermore, in light of the advent of personalized therapies, more intricate studies are also needed to elucidate the relationship between individual driver genetic mutations and epigenetic alterations, thus providing a pathway-driven basis for developing selective therapeutic strategies. This may call for a more stringent control of gene expression in CRC cells via selective targeting of epigenetic regulatory

obtained FDA approval (e.g., NCT01876511 and NCT02060188) [142].

**94**

This publication is made possible, in part, with support from the Indiana Clinical and Translational Sciences Institute (CTSI) funded from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (to TL), V foundation Kay Yow Cancer Fund (Grant 4486242 to TL), NIH-NIGMS Grant (#1R01GM120156-01A1 (to TL), and 100 VOH Grant (#2987613 to TL), as well as NIH-NCI Grant (#1R03CA223906-01).
