**3. The clinical application of epigenetic therapies**

## **3.1 DNA methylation inhibitors (DNMTis)**

DNA methylation inhibitors (DNMTis) are deoxycytosine analogs. DNMTis prevent methyl group transfer by covalently binding to and trapping methyltransferases [63]. The simplest way to understand the effect of DNMTis is through their effect on oncogenes and tumor suppressor genes [64]. BRCA1 and BRCA2 are oncogenes that when hypermethylated, can lead to a variety of cancers including ovarian cancer [65]. In a similar way, demethylation of tumor suppressor genes like p53, MLH1, H1C1, p16, E-cadherin and APC, can also play a role in the genetic instability that leads to the development of ovarian cancer, its propagation and chemoresistance [64]. Indeed, both demethylation and hypermethylation of the genome have been associated with the development of platinum resistance in ovarian cancer [64]. Consequently, DNMTis have been shown in preclinical models to restore chemosensitivity and restore normal epigenetics [66].

The most commonly utilized DNA methyltransferase inhibitors are 5-azactidine (AZA) and decitabine (5-aza-2'deoxycytidine) [63]. Both were developed in the 1960s for the treatment of hematologic malignancies and are currently FDA approved for myelodysplastic syndromes. Both AZA and decitabine have demonstrated some efficacy in clinical and pre-clinical ovarian cancer studies, however, their dose-limiting myelotoxicity limits their practical use. As they can be toxic,

other DNMTis are currently under investigation: zebularine, procaine epigallocatechin-3-gallate (EGCG) (from green tea extracts), and RG 108 [64].

## **3.2 Histone deacetylase inhibitors (HDACis)**

Histone deacetylase inhibitors (HDACis) act by targeting the zinc ion required for the catalytic function of the class I, II and IV HDACs [64]. The class III HDACs are not zinc dependent and are not inhibited by any of the current HDACis. HDACis are stratified by activity and chemical structure. There are pan-HDAC inhibitors, which affect classes I, II and IV, as well as class-specific inhibitors [67]. The chemical structure of HDACis include: hydroxamic acids, cyclic tetrapeptides, benzamides, and short-chain aliphatic acids [67]. They act on ovarian cancer in the alteration of gene transcription and chromatin remodeling [64]. In doing so, HDACis arrest cell growth, promote apoptosis, and inhibit angiogenesis [64].

The largest group of HDACis are the hydroxamic acids: vorinostat (suberanilohydroxamic acid or SAHA), belinostat, and panobinostat, all of which are pan-HDAC inhibitors FDA approved for hematologic malignancies [64]. Romidepsin, a tetrapeptide, has specific activity against Class I HDAC and is currently FDA approved for the treatment of cutaneous t-cell lymphoma [64]. Another HDACi in this group is etinostat [64]. Valproic acid is a short-chain aliphatic acid and is overall a weak HDACi with little clinical utility [64].

Since aberrant DNA methylation and histone acetylation contribute to the progression, metastasis and chemoresistance of high grade serous ovarian cancer, epigenetic drugs are thought to have the capability of reversing these effects (**Figure 2**).

### **3.3 Other epigenetic therapies**

While DNMTis and HDACis have been more extensively studied, other epigenetic therapies are on the horizon. These drugs target methylation and

### **Figure 2.**

*The Role of DNA methyltransferase inhibitors (DNMTis) and histone deacetylase inhibitors (HDACis) in halting tumorigenesis.*

**241**

*Novel Indications of Epigenetic Therapy in Ovarian Cancer*

hold promise, but still need further investigation.

toxicities seen in the clinical trials done with epigenetic therapies.

**4. Relevant clinical trials using epigenetic therapy in ovarian cancer**

Clinical translation studies with epigenetic therapy have had mixed results, but the most success with epigenetic therapy appears to be when it is used in combination with other agents and at the lowest effective dose [64]. This was discovered with one of the first epigenetic clinical trials in 2008, when the Gynecologic Oncology Group learned that as a single agent, SAHA is not very effective. They conducted a phase II study of vorinostat (SAHA) in the treatment of 27 platinum resistant patients. While 9 of 27 patients had stabilization of their disease, only 1 of 27 had a partial response and only 2 patients had a progression free survival of greater than 6 months [70]. In 2013, Mendivil and colleagues conducted a study where vorinostat was given in combination with paclitaxel and carboplatin to 18 patients as upfront therapy. The investigators reported a 50 percent total response rate, however, the study was closed prematurely due to safety concerns. Patients suffered grade 3 and 4 neutropenia. Additionally, three bowel perforations effected closure of the study [71]. Matulonis et al. in 2015 conducted a phase 1 trial of platinum sensitive patients at their first recurrence again using vorinostat. In this trial, vorinostat was given with gemcitabine and carboplatin. This combination has also demonstrated some efficacy in the recurrent setting but had significant hematologic toxicity, namely, thrombocytopenia

Fu and colleagues used azacitidine (AZA) to re-sensitize 17 platinum resistant patients to carboplatin in a phase Ib-II trial [73]. While the numbers were small, a partial response was noted in 70 percent of patients with an overall response rate of 22 percent [73]. Notably, these investigators gave their patients 5 days of AZA prior to carboplatin [73]. As it appears, epigenetic therapies may be most advantageous when used to augment classic chemotherapy and even immunotherapy, as opposed

Oza and colleages recently conducted a larger study with 103 patients [74]. It randomized patients to guadecitabine and carboplatin versus investigator's choice

to being given in isolation or in combination with an existing regimen.

**3.4 Efficacy of different inhibitors**

**4.1 Success and failures**

and neutropenia [72].

phosphorylation of the cancer genome. Examples are small molecule inhibitors targeting the histone lysine methyltransferases EZH2 and inhibitors of bromodomain proteins, BET inhibitors [64]. G9A is one such target. It is a histone methyltransferase that demethylates H3K9 and is detected in 71.6% of metastatic high grade serous cancers [68]. JQ1 is an agent that targets the bromodomain and extraterminal (BET) protein BRD4 [68]. In preclinical models, JQ1 has suppressed BRD4 and restored cisplatin sensitivity in ovarian cancer [68]. Furthermore, JQ1 has been shown by other researchers to synergize with PARP inhibitors in ovarian cancer cells that are proficient in homologous recombination [68, 69]. These newer epigenetic therapies

It is important to note that in pre-clinical models, epigenetic therapies are more active against tumor cells, while normal cells appear to be resistant to their effects. [64] Yet, this is a double-edge sword. Because epigenetic regulators have a broad impact over the entire genome, there will be great anti-tumor effects, but also unintended nonspecific consequences [68]. These nonspecific effects explain the

*DOI: http://dx.doi.org/10.5772/intechopen.98187*

*Novel Indications of Epigenetic Therapy in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.98187*

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

**3.2 Histone deacetylase inhibitors (HDACis)**

a weak HDACi with little clinical utility [64].

**3.3 Other epigenetic therapies**

other DNMTis are currently under investigation: zebularine, procaine epigallocate-

Histone deacetylase inhibitors (HDACis) act by targeting the zinc ion required for the catalytic function of the class I, II and IV HDACs [64]. The class III HDACs are not zinc dependent and are not inhibited by any of the current HDACis. HDACis are stratified by activity and chemical structure. There are pan-HDAC inhibitors, which affect classes I, II and IV, as well as class-specific inhibitors [67]. The chemical structure of HDACis include: hydroxamic acids, cyclic tetrapeptides, benzamides, and short-chain aliphatic acids [67]. They act on ovarian cancer in the alteration of gene transcription and chromatin remodeling [64]. In doing so, HDACis arrest cell growth, promote apoptosis, and inhibit angiogenesis [64].

The largest group of HDACis are the hydroxamic acids: vorinostat (suberanilohydroxamic acid or SAHA), belinostat, and panobinostat, all of which are pan-HDAC inhibitors FDA approved for hematologic malignancies [64]. Romidepsin, a tetrapeptide, has specific activity against Class I HDAC and is currently FDA approved for the treatment of cutaneous t-cell lymphoma [64]. Another HDACi in this group is etinostat [64]. Valproic acid is a short-chain aliphatic acid and is overall

Since aberrant DNA methylation and histone acetylation contribute to the progression, metastasis and chemoresistance of high grade serous ovarian cancer, epigenetic drugs are thought to have the capability of reversing these effects (**Figure 2**).

While DNMTis and HDACis have been more extensively studied, other epigenetic therapies are on the horizon. These drugs target methylation and

*The Role of DNA methyltransferase inhibitors (DNMTis) and histone deacetylase inhibitors (HDACis) in* 

chin-3-gallate (EGCG) (from green tea extracts), and RG 108 [64].

**240**

**Figure 2.**

*halting tumorigenesis.*

phosphorylation of the cancer genome. Examples are small molecule inhibitors targeting the histone lysine methyltransferases EZH2 and inhibitors of bromodomain proteins, BET inhibitors [64]. G9A is one such target. It is a histone methyltransferase that demethylates H3K9 and is detected in 71.6% of metastatic high grade serous cancers [68]. JQ1 is an agent that targets the bromodomain and extraterminal (BET) protein BRD4 [68]. In preclinical models, JQ1 has suppressed BRD4 and restored cisplatin sensitivity in ovarian cancer [68]. Furthermore, JQ1 has been shown by other researchers to synergize with PARP inhibitors in ovarian cancer cells that are proficient in homologous recombination [68, 69]. These newer epigenetic therapies hold promise, but still need further investigation.

### **3.4 Efficacy of different inhibitors**

It is important to note that in pre-clinical models, epigenetic therapies are more active against tumor cells, while normal cells appear to be resistant to their effects. [64] Yet, this is a double-edge sword. Because epigenetic regulators have a broad impact over the entire genome, there will be great anti-tumor effects, but also unintended nonspecific consequences [68]. These nonspecific effects explain the toxicities seen in the clinical trials done with epigenetic therapies.

## **4. Relevant clinical trials using epigenetic therapy in ovarian cancer**

### **4.1 Success and failures**

Clinical translation studies with epigenetic therapy have had mixed results, but the most success with epigenetic therapy appears to be when it is used in combination with other agents and at the lowest effective dose [64]. This was discovered with one of the first epigenetic clinical trials in 2008, when the Gynecologic Oncology Group learned that as a single agent, SAHA is not very effective. They conducted a phase II study of vorinostat (SAHA) in the treatment of 27 platinum resistant patients. While 9 of 27 patients had stabilization of their disease, only 1 of 27 had a partial response and only 2 patients had a progression free survival of greater than 6 months [70]. In 2013, Mendivil and colleagues conducted a study where vorinostat was given in combination with paclitaxel and carboplatin to 18 patients as upfront therapy. The investigators reported a 50 percent total response rate, however, the study was closed prematurely due to safety concerns. Patients suffered grade 3 and 4 neutropenia. Additionally, three bowel perforations effected closure of the study [71]. Matulonis et al. in 2015 conducted a phase 1 trial of platinum sensitive patients at their first recurrence again using vorinostat. In this trial, vorinostat was given with gemcitabine and carboplatin. This combination has also demonstrated some efficacy in the recurrent setting but had significant hematologic toxicity, namely, thrombocytopenia and neutropenia [72].

Fu and colleagues used azacitidine (AZA) to re-sensitize 17 platinum resistant patients to carboplatin in a phase Ib-II trial [73]. While the numbers were small, a partial response was noted in 70 percent of patients with an overall response rate of 22 percent [73]. Notably, these investigators gave their patients 5 days of AZA prior to carboplatin [73]. As it appears, epigenetic therapies may be most advantageous when used to augment classic chemotherapy and even immunotherapy, as opposed to being given in isolation or in combination with an existing regimen.

Oza and colleages recently conducted a larger study with 103 patients [74]. It randomized patients to guadecitabine and carboplatin versus investigator's choice (topotecan, pegylated liposomal doxorubicin, paclitaxel or gemcitabine) until disease progression or unacceptable toxicity. Cross-over was allowed from the standard arm to the experimental arm and 27 patients crossed-over. The combination of guadecitabine and carboplatin was found to be effective, however the median progression free survival of 16 weeks when compared to the 9 weeks in the standard treatment arm was not found to be statistically significant [74].

### **4.2 The administration of epigenetic therapy – better together?**

One approach to utilizing epigenetic therapy effectively up front is in alternating treatments of classic chemotherapy and epigenetic therapy. This method was found to be effective and less toxic in clinical translational studies [73, 74]. Sequential administration of classic chemotherapy and epigenetic drugs not only suppresses ovarian cancer growth *in vitro*, but also spares toxicity to normal cells and preserves the healing ability of stem cells [75]. Furthermore, chemotherapy and epigenetic therapy act synergistically allowing smaller doses of both to be administered. In turn, this decreases the toxicity of both chemotherapy and epigenetic therapy [69]. This methodology has yet to be broadly adopted in clinical trials involving epigenetic therapy.

For recurrent disease, epigenetic therapy may have utility. Epigenetic therapy restores platinum sensitivity as both hypermethylation and histone modification contribute to chemoresistance, reversing these epigenetic changes, should reverse the chemoresistance [64]. This has been borne out in the literature as less than 10 percent of platinum resistant patients would be expected to respond to platinum again, yet pretreatment with AZA yields a 22 percent response and decitabine, a 35 percent response [64]. Taxol resistance has not been as heavily explored in the literature as platinum resistance, however, epigenetic therapy, may re-sensitize ovarian cancer to paclitaxel as it does cisplatin. In one preclinical study, the HDACi panobinostat was used to re-sensitize ovarian cancer cell lines that had become resistant to paclitaxel [76]. These researchers were further able to demonstrate that when human ovarian cancer xenografts were implanted in a murine model, panobinostat in combination with cisplatin and paclitaxel was superior in efficacy to cisplatin-paclitaxel or panobinostat alone [76]. Thus, epigenetics may possibly be used upfront to "prime" or increase the efficacy of classic chemotherapy. Additionally, they may be sequenced in between classic chemotherapy and again when patients recur to re-sensitize them to platinum and taxol agents.
