**4.3 Acetyltransferases and deacetylases**

Acetylation of histones by acetyltransferases (HATs) and removal of these acetyl marks by HDACs are essential events for the maintenance of normal chromatin organization and function [116]. However, as is often the case in cancer, these enzymes are dysregulated, leading to increased chromosomal instability and aberrant gene expression changes [117]. To date, only a handful of HATs have been reported as contributing to the pathogenesis of CRC. Here, we describe the role of a few of these HATs namely *p300*/*CREB-binding protein* (*p300*/*CBP*), *GCN5*, *N-Acetyltransferase 10* (*Nat10*), and *Human males absent on the first* (*hMOF*). Assessment of 262 CRC samples from patients receiving 5-FU treatment demonstrated that low expression of *p300*/*CBP* in CRC tissue was closely associated with poor clinical response to 5-FU based-chemotherapy [118]. Furthermore, low *p300*/*CBP* expression also correlated with poor disease-free survival and increased early disease progression in the same patients [118]. Mechanistic studies also uncovered that 5-FU induced degradation of *p300*/*CBP* which was dependent on chaperone-mediated autophagy involving *heat-shock cognate protein 70 kDa* (*hsc70*) and *lysosomal-associated membrane protein 2A* (*LAMP2A*). In short, degradation of *p300*/*CBP* was found to be relevant to chemoresistance to 5-FU, since blocking this degradation also enhanced 5-FU's cytotoxicity in CRC cells [118].

Conversely, another HAT *GCN5* has been implicated in promoting CRC cell growth via its upregulation rather than downregulation. One study found that *GCN5* overexpression in human colon adenocarcinoma tissues was attributed to the activities of the transcription factors, *c-Myc* and *E2F transcription factor 1* (*E2F1*) [119]. Depletion of *c-Myc* inhibited CRC cell proliferation mainly by downregulating GCN5 transcription, an effect that was rescued by ectopic expression of *GCN*5. However, ectopic overexpression of *E2F1* had the opposite effect by suppressing *GCN5* levels, thus inducing cell death. Furthermore, inhibition of *GCN5* with CPTH2, a HAT inhibitor, also suppressed CRC cell growth, revealing an avenue of great therapeutic potential [119]. Other HATs implicated in CRC include *Nat10* and *hMOF*, which were downregulated in CRC tissues. Particularly, recent studies

*Advances in the Molecular Understanding of Colorectal Cancer*

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**Table 1.**

to the clinical trial phase thus far. AMI-1, which inhibits *PRMT1* and *PRMT5*, demonstrated antiproliferative activity in CRC cells and xenograft mouse models [106]. However, further *in vivo* validation studies are needed, and it has not

*Overview of pre-clinical and clinical drugs that target epigenetic enzymes in CRC.*

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 lymph node metastasis and advanced tumor stage [121].

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

## **4.4 Targeting HATs and HDACs in CRC**

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 sensitize cancer cells to irradiation [125].

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].

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

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].

**93**

*Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer*

ming and/or combinatorial therapies with cytotoxic drugs [138].

CRC and an orally active formulation of 5-azacitidine, cc-486 [140].

**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

Other general limitations regarding the use of epigenetic therapy in solid tumors

deal with the unfavorable pharmacokinetic properties of these drugs, including instability, toxicity, and short half-life [137]. Some of these invariably contribute in some way to common toxicities associated with HDAC and DNMT inhibitors in CRC including thrombocytopenia, neutropenia, diarrhea, nausea, vomiting, and fatigue [139]. Furthermore, maintaining therapeutically relevant levels of the drugs necessary for clinical benefit is particularly difficult, and as of yet, no FDAapproved epigenetic treatments exist for CRC despite promising preclinical studies. This signifies the overall marginal clinically compelling responses to these agents in CRC patients. To overcome some of these limitations, newer formulations have been made to render these inhibitors more bioavailable, stable, and ultimately usable at lower doses with less toxicity and greater therapeutic efficacy. Examples of these include the oral HDAC inhibitor, entinostat, used in *in vitro* and *in vivo* models of

Additionally, many undesired effects such as reactivity or lack of selectivity between different HAT subtypes are often associated with HAT inhibitors [130]. Hence, studies geared towards the development of more potent and selective inhibitors by carefully studying the catalytic mechanism and enzyme kinetics of various HATs are needed. As far as HDAC inhibitors are concerned, they have shown preferential efficacy against hematological malignancies, and therefore, drugs such as vorinostat (SAHA) and romidepsin (FK228) have achieved FDA approval for the treatment of cutaneous T cell lymphoma [131, 132]. Unfortunately, the evidence regarding HDAC inhibitors efficacy for solid tumors has not been as convincing and encouraging although they are well tolerated at low but not high doses. Currently, adverse side effects and inadequate clinical efficacy are the major limitations to their use, and more efforts are underway to generate specific HDAC inhibitors for solid tumors such as CRC [133]. Nonetheless, a few early phase clinical trials using vorinostat in combination with other chemotherapeutic agents have shown some early promise for mCRC patients. These include combinatorial regimens of vorinostat with 5-FU, leucovorin, and oxaliplatin (FOLFOX) as well as randomized phase II trial studies investigating the efficacy of vorinostat and hydroxychloroquine or regorafenib in refractory mCRC patients [134, 135]. Other regimens are outlined in **Table 1**. Similarly, while DNMT inhibitors have also met with some degree of success for treating blood cancers such as myelodysplastic syndrome (e.g., decitabine and 5-azacitidine), the major drawbacks of these compounds in solid tumors are harsh side effects and transient demethylation, which revert after drug removal [136, 137]. Interestingly, however, some studies have suggested that this transient demethylation that occurs with DNMT inhibition (e.g., 5-azadeoxycytidine) potentially creates a therapeutic window that can be leveraged for epigenetic reprogram-

*DOI: http://dx.doi.org/10.5772/intechopen.82572*

## *Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.82572*

Additionally, many undesired effects such as reactivity or lack of selectivity between different HAT subtypes are often associated with HAT inhibitors [130]. Hence, studies geared towards the development of more potent and selective inhibitors by carefully studying the catalytic mechanism and enzyme kinetics of various HATs are needed. As far as HDAC inhibitors are concerned, they have shown preferential efficacy against hematological malignancies, and therefore, drugs such as vorinostat (SAHA) and romidepsin (FK228) have achieved FDA approval for the treatment of cutaneous T cell lymphoma [131, 132]. Unfortunately, the evidence regarding HDAC inhibitors efficacy for solid tumors has not been as convincing and encouraging although they are well tolerated at low but not high doses. Currently, adverse side effects and inadequate clinical efficacy are the major limitations to their use, and more efforts are underway to generate specific HDAC inhibitors for solid tumors such as CRC [133]. Nonetheless, a few early phase clinical trials using vorinostat in combination with other chemotherapeutic agents have shown some early promise for mCRC patients. These include combinatorial regimens of vorinostat with 5-FU, leucovorin, and oxaliplatin (FOLFOX) as well as randomized phase II trial studies investigating the efficacy of vorinostat and hydroxychloroquine or regorafenib in refractory mCRC patients [134, 135]. Other regimens are outlined in **Table 1**. Similarly, while DNMT inhibitors have also met with some degree of success for treating blood cancers such as myelodysplastic syndrome (e.g., decitabine and 5-azacitidine), the major drawbacks of these compounds in solid tumors are harsh side effects and transient demethylation, which revert after drug removal [136, 137]. Interestingly, however, some studies have suggested that this transient demethylation that occurs with DNMT inhibition (e.g., 5-azadeoxycytidine) potentially creates a therapeutic window that can be leveraged for epigenetic reprogramming and/or combinatorial therapies with cytotoxic drugs [138].

Other general limitations regarding the use of epigenetic therapy in solid tumors deal with the unfavorable pharmacokinetic properties of these drugs, including instability, toxicity, and short half-life [137]. Some of these invariably contribute in some way to common toxicities associated with HDAC and DNMT inhibitors in CRC including thrombocytopenia, neutropenia, diarrhea, nausea, vomiting, and fatigue [139]. Furthermore, maintaining therapeutically relevant levels of the drugs necessary for clinical benefit is particularly difficult, and as of yet, no FDAapproved epigenetic treatments exist for CRC despite promising preclinical studies. This signifies the overall marginal clinically compelling responses to these agents in CRC patients. To overcome some of these limitations, newer formulations have been made to render these inhibitors more bioavailable, stable, and ultimately usable at lower doses with less toxicity and greater therapeutic efficacy. Examples of these include the oral HDAC inhibitor, entinostat, used in *in vitro* and *in vivo* models of CRC and an orally active formulation of 5-azacitidine, cc-486 [140].
