**4.6 Emerging immunomodulatory and epigenetic combinatorial therapies**

Compared to MSS tumors, there are an exponentially higher number of mutations acquired in MSI-H CRCs. Interestingly, these mutations have the potential to elevate the production of neo-antigens [141]. The result is increased tumor immunogenicity, which is further complemented by the fact that these tumors also harbor a high number of tumor-infiltrating lymphocytes. Within this context, CRC patients with MSI-H represent a subgroup more likely to benefit from immune checkpoint inhibitors compared to those with MSS tumors. Immune checkpoint inhibitors have shown unprecedented benefit across multiple tumor types. These agents specifically target the proteins *programmed death ligand-1* (e.g., durvalumab) and *programmed death-1* (e.g., nivolumab, pembrolizumab) and are administered

*Advances in the Molecular Understanding of Colorectal Cancer*

lymph node metastasis and advanced tumor stage [121].

other anticancer agents (**Table 1**).

**4.4 Targeting HATs and HDACs in CRC**

sensitize cancer cells to irradiation [125].

showed that *Nat10* downregulation and subcellular redistribution were associated with increased cellular motility and invasion in CRC cells [120]. Meanwhile, low expression of *hMOF* correlated with clinicopathological features of CRC such as

In CRC, HDACs are also frequently overexpressed and represent another attractive class of targets for anticancer therapy. *HDAC1–3* and *HDAC5–8* have emerged as some of the most relevant deacetylases in CRC. Although all are highly overexpressed in CRC, only few studies have explored the relevance of this overexpression to disease [23]. For example, knockdown of *HDAC1*, *2*, and *3* reduced the growth of several CRC cells by largely unknown mechanisms [122]. Interestingly however, a mechanistic link between *HDAC2* expression and sensitivity of CRC cells to other anticancer agents was recently established. Alzoubi et al. demonstrated that depletion of *HDAC2* specifically enhanced the combined anti-tumor effect of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the DNA-damaging agents, 5-FU and oxaliplatin, in SW480 and HT29 cells. On the other hand, overexpression of *HDAC2* conferred resistance to these agents, which were independent of the p53 mutational status [123]. In summary, these findings strongly suggest that HATs and HDACs are critical biomarkers for CRC and influence the sensitivity of CRC cells to certain therapeutics as evidenced by their frequent combination with

Like methylation, several studies have demonstrated that inhibitors targeting HATs and HDACs also induce epigenetic alterations that modulate the expression of genes or pathways critical for CRC treatment. One study showed that direct inhibition of *p300*/*KAT3B* histone acetyltransferase, a coactivator of *β-Catenin* with rimonabant, induced downregulation of *Wnt*/*β-catenin* target genes in HCT116 cells [124]. Furthermore, rimonabant also significantly reduced tumor growth in HCT116 xenografts [124]. The general HAT inhibitors such as garcinol and anacardic acid have also been shown to inhibit CRC tumor growth in mice as well as

Compared to HATs, a far greater number of studies have been dedicated to investigating the efficacy of HDAC inhibitors at both the preclinical and clinical level. Overall, the use of HDAC inhibitors in preclinical solid tumor models has shown some early promise albeit their progress to the clinic has been hindered by serious limitations including ineffective concentrations and cardiac toxicity [126]. In CRC specifically, these inhibitors are mainly being administered as combination therapy with conventional chemotherapy or other agents [127]. In pre-clinical models for instance, treatment of irinotecan-resistant CRC cells with HDAC

inhibitors conferred sensitization of these cells to irinotecan, whereas HDAC inhibitor CG2 showed an additive effect when used with irinotecan, 5-FU, or oxaliplatin in HCT116 xenografts [128]. Meanwhile, a small molecular inhibitor of *HDAC6*, ACY-1215, was able to enhance the anticancer activity of oxaliplatin by promoting apoptosis and blocking cell proliferation in CRC cells and xenograft models [129].

Despite their potential, a large gap still remains between the biological activity of epigenetic enzyme inhibitors in preclinical studies and their potential clinical utility. For example, the development of HAT inhibitors poses several challenges [130]. Because of their function in complexes consisting of many proteins which play multiple roles in HAT target specificity, this significantly limits inhibitor use *in vivo* [130].

**4.5 Benefits and pitfalls of epigenetic enzyme inhibitors**

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

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.