Epigenetic therapy in gastrointestinal cancer: the right combination

Abstract

Epigenetics is a relatively recent field of molecular biology that has arisen over the past 25 years. Cancer is now understood to be a disease of widespread epigenetic dysregulation that interacts extensively with underlying genetic mutations. The development of drugs targeting these processes has rapidly progressed; with several drugs already FDA approved as first-line therapy in hematological malignancies. Gastrointestinal (GI) cancers possess high degrees of epigenetic dysregulation, exemplified by subtypes such as CpG island methylator phenotype (CIMP), and the potential benefit of epigenetic therapy in these cancers is evident. The application of epigenetic drugs in solid tumors, including GI cancers, is just emerging, with increased understanding of the cancer epigenome. In this review, we provide a brief overview of cancer epigenetics and the epigenetic targets of therapy including deoxyribonucleic acid (DNA) methylation, histone modifications, and chromatin remodeling. We discuss the epigenetic drugs currently in use, with a focus on DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and explain the pharmacokinetic and mechanistic challenges in their application. We present the strategies employed in incorporating these drugs into the treatment of GI cancers, and explain the concept of the cancer stem cell in epigenetic reprogramming and reversal of chemo resistance. We discuss the most promising combination strategies in GI cancers including: (1) epigenetic sensitization to radiotherapy, (2) epigenetic sensitization to cytotoxic chemotherapy, and (3) epigenetic immune modulation and priming for immune therapy. Finally, we present preclinical and clinical trial data employing these strategies thus far in various GI cancers including colorectal, esophageal, gastric, and pancreatic cancer.

Keywords

CIMP colorectal cancer DNMT inhibitor epigenetic therapy gastrointestinal cancer HDAC inhibitor chemosensitization immune therapy radiosensitization

Introduction

Epigenetics is a relatively recent field of molecular biology that has arisen over the past 25 years. However, in that time there has been remarkable progress in our understanding of how epigenetic mechanisms contribute to differentiation, aging, and disease development. Cancer is now understood to be a disease of widespread epigenetic dysregulation that interacts extensively with underlying genetic mutations. As our understanding of epigenetic processes grows, the development of drugs targeting these processes has rapidly progressed. Several drugs have already been FDA approved and incorporated into treatment algorithms as first-line therapy in hematological malignancies.

However, the optimal strategy of incorporating these drugs into the treatment of solid tumors is still being elucidated. GI cancers are recognized as possessing high degrees of epigenetic dysregulation. This has implications in pathogenesis, pathophysiology and therapeutic approaches, and the potential benefit of epigenetic therapy in these cancers is evident. Prior clinical trials have been hindered by inconsistent or improper dosing regimens. Additionally, the enrollment of end-stage patients who have failed multiple regimens may not be optimal for epigenetic agents that may require a long duration of treatment to achieve a therapeutic response. In this review, we will describe cancer epigenetics and the targets for therapy, the drugs currently being used and developed, and the strategies for incorporating these therapies to achieve the optimal benefit in gastrointestinal malignancies.

Epigenetic regulation and targets

Epigenetics is defined as the collection of heritable changes on deoxyribonucleic acid (DNA) that affect the packaging of chromatin [Jones and Baylin, 2007; Baylin and Jones, 2011; Dawson and Kouzarides, 2012; Azad et al. 2013]. These changes either increase or decrease the ability of the cell’s transcription machinery to express the genes in that section of chromatin [Schuebel et al. 2007; Baylin and Jones, 2011; Dawson and Kouzarides, 2012]. Epigenetic changes are different from mutations that change the underlying structure of the DNA [You and Jones, 2012]. Epigenetic regulation of chromatin is a normal and vital process of cell differentiation and development. It explains the ability of stem cells from a single origin to differentiate into all of the necessary cells of the human body; then remain differentiated without reverting back to a stem cell or other cell type [Arney and Fisher, 2004; Lunyak and Rosenfeld, 2008]. In tumorigenesis however, there is dysregulation of the physiologic epigenetic milieu, and this contributes to the development of malignancy [Jones and Baylin, 2007; Dawson and Kouzarides, 2012; Reddy et al. 2015].

There are multiple epigenetic ‘marks,’ or changes on the chromatin. The ones that are best understood, and are the targets of most epigenetic drugs, are DNA methylation and histone lysine acetylation. However, there are many others including histone lysine methylation, chromatin protein modifications, and noncoding ribonucleic acids (RNAs), all of which are under investigation for their role in tumorigenesis and as potential therapeutic targets.

DNA methylation was the first type of epigenetic mark that was discovered and has been the focus of much of the attention in epigenetic therapeutics [Herman and Baylin, 2003; Feinberg and Tycko, 2004; Jones and Baylin, 2007]. DNA methylation occurs when a methyl group is added to cytosine residues in CpG islands (cytosine nucleotide bases followed by guanine bases) at the 5-carbon position. This affects the ability of DNA to coil around histone proteins and causes a condensed heterochromatin conformation, which prevents genes from being transcribed. This is in contrast to the open euchromatin conformation to which the cell’s transcription machinery can bind more readily [Herman and Baylin, 2003] (Figure 1). The enzyme involved in this process is DNA methyltransferase (DNMT), of which there are several subtypes (1, 3A, and 3B). These subtypes have different roles either in maintaining methylation during DNA replication (DNMT1) or as de novo DNA methylators (DNMT 3A and 3B) [Dawson and Kouzarides, 2012]. DNA methylation was first observed and described in X chromosome-related gene silencing [Mohandas et al. 1981; Feinberg and Tycko, 2004]. Later, it was discovered that cancer caused severe alterations in the normal methylation patterns of DNA and that these changes occur early in tumorigenesis, having been observed as early as in the malignant precancerous stages of at-risk cells [Esteller et al. 1999, 2000; Fleisher et al. 2001; Kang et al. 2001]. The story of DNA methylation in cancer is complicated, however. The cancer epigenome overall is not hypermethylated. Rather, there is overall relative hypomethylation interspersed with hypermethylation of CpG islands at the promoter regions of genes, silencing transcription of those genes [Jones and Baylin, 2007; Baylin and Jones, 2011]. This is especially relevant when the genes involved are tumor suppressor genes. Recent evidence also points to the important interaction between DNA methylation and certain mutations. For example, mutation of isocitrate dehydrogenase 1 (IDH1) has been shown to contribute to a hypermethylator phenotype in gliomas [Turcan et al. 2012] and cholangiocarcinomas [Wang et al. 2013]. In colorectal cancer, a strong association has been demonstrated between a hypermethylator phenotype known as CIMP (CpG island methylator phenotype) and mutation of the oncogene BRAF, a protein kinase involved in the EGFR pathway downstream of KRAS [Weisenberger et al. 2006].

Figure 1.

The landscape of epigenetic modification of chromatin. Chromatin is composed of deoxyribonucleic acid (DNA) coiled twice around a set of eight histone proteins, forming a nucleosome. This is the fundamental subunit of chromatin. Promoter hypermethylation of DNA at CpG islands causes a condensed heterochromatin structure that silences transcription of genes. Demethylation and acetylation at histone lysine tails contributes to an open euchromatin conformation that is amenable to binding by the transcription machinery of the cell. Methylation at histone lysine tails also influences gene transcription, but these marks can be either repressive or activating depending on which lysines of which histone proteins are methylated. Repressive histone lysine methylation marks interact with proteins that make up the polycomb repressive complex that can interact with multiple genes and nucleosomes to repress gene transcription. Some of these genes are bivalent and can quickly revert from a repressed to an active state. Noncoding RNAs can interact with genes to either silence or activate transcription.

Histone lysine acetylation is another epigenetic mark that has been a major target of epigenetic therapy. Acetylation of lysine residues on histone proteins neutralizes lysine’s positive charge and leads to a weakened electrostatic interaction between histone proteins and the negatively charged DNA [Dawson and Kouzarides, 2012]. When this acetylation occurs at enhancer and promoter regions, a more open euchromatin conformation occurs, which leads to more active transcription [Dawson and Kouzarides, 2012] (Figure 1). Conversely, removal of acetylation by the enzyme histone deacetylase (HDAC) causes transcriptional silencing. HDAC and lysine acetyl transferase (KAT) are competing enzymes involved in dynamic regulation of histone lysine acetylation and transcriptional regulation [Muntean and Hess, 2009].

Many other epigenetic marks have been discovered in recent years, with some becoming the focus of new investigations and possible future targets for therapy, as well as informing our strategy towards current therapies. This includes methylation of histone lysine residues, which may be activating, such as H3 trimethylation on lysine 4 (H3K4me3), or inhibitory, such as H3 trimethylation on lysine 27 (H3Kme27) [Dawson and Kouzarides, 2012]. These histone alterations, along with the previously mentioned histone acetylation, undergo a complex interaction with transcription promoter regions and proteins of the polycomb repressor complex (PRC). The PRC marks genes that are prone to cancer-specific DNA hypermethylation and transcriptional silencing [Baylin and Jones, 2011]. These interactions can result in bivalent states that allow regulatory flexibility and rapid activation of those genes when required [Baylin and Jones, 2011; Dawson and Kouzarides, 2012]. Post-translational histone modifications also interact with nucleosomes that are the fundamental subunit of chromatin and consist of DNA wrapped around a histone protein octamer [Muntean and Hess, 2009]. The PRC can interact with multiple nucleosomes at once [Baylin and Jones, 2011] (Figure 1). This adds yet another level to chromatin modification and demonstrates further how the interaction between all of the epigenetic marks creates a dynamic state of transcriptional regulation that becomes hijacked in cancer.

Finally, it is important to note that evidence is emerging regarding noncoding RNAs and their effects on transcriptional regulation. These include micro-RNAs, long noncoding RNAs, small interfering RNAs, small nucleolar RNAs, and piwi-interacting RNAs [Esteller, 2011]. These noncoding RNAs have also been found to interact with previously described epigenetic marks [Khalil et al. 2009; Dawson and Kouzarides, 2012] and are being investigated as new forms of epigenetic therapy for cancer [Petrocca and Lieberman, 2011; Wang et al. 2011b].

Epigenetic drugs and gastrointestinal malignancies

The classes of epigenetic therapy that we have focused on in this review are the DNMT and the HDAC inhibitors. These are the drugs that have undergone the most investigation and have the largest amount of data regarding their usage in cancer.

DNMT inhibitors are the class of drugs that inhibits the enzyme DNA methyltransferase that is responsible for the initiation and maintenance of DNA methylation. These are cytosine analogs that incorporate into DNA and RNA molecules then, after one cycle of DNA replication, form covalent adducts with DNMT, thereby trapping and depleting the enzyme after several cycles, causing inhibition of genome-wide methylation [Azad et al. 2013; Juo et al. 2015]. Because these drugs require integration into the genome during the S-phase of the cell cycle to become active, there is preferential incorporation into rapidly diving cells, such as cancer cells [Momparler, 2005]. In addition to this epigenetic mechanism, these drugs also have direct cytotoxic effects. Covalent linkage of the incorporated drug to DNMT causes induction of cell death, and DNA damage occurs as a result of structural instability at the site of incorporation of the DNMT inhibitors [Goffin and Eisenhauer, 2002]. The DNMT inhibitor 5-azacitidine (5-AC) incorporates preferentially into RNA while 5-Aza-2’-deoxycytodine (DAC) incorporates mainly into DNA [Derissen et al. 2013]. This difference between incorporation into RNA and DNA leads to increased potency with DAC and may result in increased secondary or off-target effects with 5-AC due to integration into the RNA of quiescent, nondividing cells [Gravina et al. 2010].

These drugs were first discovered in the 1960s and were initially developed and tested as cytotoxic agents, but showed poor efficacy and tolerability given the excessively high doses being used [Sorm and Vesley, 1968; Azad et al. 2013]. However, in the 1980s they were found to have demethylating activity, and investigation into utilizing them with this strategy began [Jones and Taylor, 1980; Feinberg and Tycko, 2004]. A key concept for incorporation of these drugs into the clinical arena has been the use of lower doses, allowing the drugs to function more as epigenetic agents, as opposed to traditional cytotoxic drugs. The earliest trials employing this concept were primarily in hematologic malignancies, in which they have been particularly effective [Issa et al. 2004; Kantarjian et al. 2006]. 5-AC received FDA approval in 2004 for the treatment of myelodysplastic syndrome (MDS), with approval for DAC following shortly after in 2006. The two drugs are now first-line therapy in the treatment of MDS, [Sekeres and Cutler, 2014] and are being actively investigated in leukemia. [Dombret et al. 2015]

HDAC inhibitors are another class of epigenetic therapeutics, and these molecules directly inhibit the enzyme HDAC. There are 18 HDACs in human cells; these enzymes are not redundant in function and are typically divided into four classes [Xu et al. 2007]. Different types of HDAC inhibitors are grouped based on which HDAC enzymes they target, their chemical structure, and their composition [Dokmanovic et al. 2007; Dawson and Kouzarides, 2012]. Some of these drugs, such as valproic acid, have long been in use clinically for other purposes and were later discovered to have HDAC activity. Classical HDAC inhibitors, such as SAHA, inhibit Class I, II and IV HDACs, but not NAD⁺-dependent class III HDACs. Because of the wide range of HDAC targets and their various functions, these drugs act not only through an epigenetic mechanism of increased histone acetylation causing selective alteration of gene expression, but also through a plethora of cytotoxic and antitumor mechanisms [Xu et al. 2007]. These antitumor effects include induction of cell cycle arrest, activation of extrinsic and intrinsic apoptotic pathways, induction of mitotic cell death, accumulation of reactive oxygen species, disruption of chaperone proteins important for stability, and inhibition of angiogenesis [Xu et al. 2007]. However, it does appear that, like DNMT inhibitors, lower doses have a more prominent epigenetic effect [Robert and Rasool, 2012; Azad et al. 2013]. Certain HDAC inhibitors exhibit more cytotoxic effects than others, and their clinical efficacy may in fact be due to one of these other pathways that does not involve histone deacetylation [Marks and Xu, 2009; Marks, 2010]. Parsing out the degree to which the effect of HDAC inhibitors is truly due to epigenetic mechanisms is still a work in progress. Nevertheless, these drugs have shown efficacy in hematologic malignancies, and two HDAC inhibitors (vorinostat in 2006 and romidepsin in 2009) have been approved for use in cutaneous T-cell lymphoma and relapsed peripheral T-cell lymphoma [Mann et al. 2007; Bertino and Otterson, 2011].

The utility of these drug classes, both DNMT inhibitors and HDAC inhibitors, for epigenetic therapy in solid tumors is just emerging. Earlier trials used both classes of drugs at high doses that were poorly tolerated in patients. Their poor tolerability at high doses is partly due to the significant off-target effects of both DNMT inhibitors and HDAC inhibitors. The global genomic demethylation and acetylation that occurs is not selective for cancer cells or aberrantly silenced genes [Marks, 2010; Gnyszka et al. 2013]. This has led to a recent re-evaluation of the optimal strategy in dosing epigenetic drugs. It has been shown that concentrations of DNMT inhibitors in the nanomolar range are not cytotoxic to cells, but still cause genome-wide promoter DNA demethylation and reprogramming of regulatory pathways in tumor cells through re-expression of key silenced genes [Tsai et al. 2012]. These pathways are involved in apoptosis, cell-cycle regulation, immune modulation, interferon signaling, and other processes vital to maintaining malignancy [Wrangle et al. 2013; Li et al. 2014]. In addition, this reprogramming creates a ‘memory’ response whereby the effects of the drug continue for days, well after its removal from the tumor environment [Tsai et al. 2012].

Therefore, to utilize DNMT and HDAC inhibitors through their epigenetic mechanisms, the focus in solid tumors has shifted away from single- or double-agent epigenetic therapy towards combination therapies to ‘reprogram’ tumor cells that could also make them more susceptible to existing cytotoxic agents. In hematopoietic malignancies, in addition to apoptosis and growth arrest, a major part of the mechanism of epigenetic therapy is to cause terminal differentiation of malignant myeloid precursor cells [Pinto et al. 1984; Guo et al. 2006]. It has been proposed that solid tumors are made up of heterogeneous populations of malignant cells that include cancer ‘stem cells’ that are responsible for recurrence and the development of resistance to chemotherapy [Feinberg et al. 2006; Jones and Baylin, 2007]. Epigenetic drugs may be able to cause a ‘differentiation’ of sorts in these cancer stem cells by reprogramming them to sensitize to cytotoxic chemotherapy and reverse chemo resistance [Azad et al. 2013; Juo et al. 2015]. Clinical trials have been initiated that utilize this treatment paradigm, and these are discussed in more detail below. Finally, the importance of upregulation of immunomodulatory pathways after 5-AC treatment cannot be understated [Wrangle et al. 2013; Li et al. 2014] as this creates the potential for combination therapy with immune-checkpoint blockade, vaccines, and other immune therapy.

Some of the limitations regarding the use of epigenetic therapy relate to the pharmacokinetic properties of these drugs. DNMT inhibitors are unstable in aqueous solutions, undergoing spontaneous hydrolysis especially at room temperature or 37°C [Derissen et al. 2013]. In vivo, they are rapidly and irreversibly degraded by cytidine deaminase, a nucleotide salvage enzyme, because these drugs are cytosine analogs [Derissen et al. 2013]. The half-life in plasma has been measured at 15–25 minutes for DAC [Karahoca and Momparler, 2013] and 22 minutes for 5-AC [Kaminskas et al. 2005]. While it has been accepted that the most beneficial application of these drugs is through administering prolonged lower doses and avoiding high cytotoxic doses, translating this principle into clinical trials and accurately determining the doses that achieve nanomolar epigenetic-range intratumoral concentrations has not been as simple. Dosing schedules have varied widely across trials, ranging from 10 to 75 mg/m² daily for 5-AC, and this difference has had significant effects on plasma concentration and degree of demethylation in tumor biopsies [Rudek et al. 2005; Derissen et al. 2013]. Higher doses also increase toxicities that limit tolerability, the most important toxicities being myelosuppression and neutropenia [Kaminskas et al. 2005]. HDAC inhibitors also cause severe dose-limiting fatigue and GI toxicity at higher doses [Dokmanovic et al. 2007]. Because of these limitations, newer formulations of DNMT inhibitors have been developed that attempt to address them. These drugs are designed to be more specific in targeting tumor DNMTs while being less rapidly degraded. These include the small molecules hydralazine, already in use for many years as an antihypertensive, and zebularine, a nucleoside analog DNMT inhibitor that also inhibits cytidine deaminase [Zhou et al. 2002]; MG98, an antisense oligonucleotide that binds directly to DNMT mRNA to prevent its translation [Stewart et al. 2003]; and SGI-110, a complex of DAC with deoxyguanosine which causes resistance to degradation by cytidine deaminase [Srivastava et al. 2014].

Gastrointestinal CpG island methylator phenotype and epigenetic therapy

Gastrointestinal malignancies are excellent candidates for treatment with epigenetic therapy. In fact, it was in colorectal cancer tumor samples that CIMP was first observed and defined [Toyota et al. 1999a]. When observing promoter methylation patterns of multiple known genes at the time, it was noted that a subgroup of colorectal tumors was noted to have higher degrees of aberrant promoter hypermethylation. Five of the most frequently methylated genes in this subgroup (MINT1, MINT2, MINT31, MLH1, p16) were used to create a panel that defined a CIMP [Toyota et al. 1999a]. The CIMP colorectal cancers were also found to correlate with microsatellite instability (MSI) due to promoter hypermethylation and silencing of the mismatch repair gene MLH1 that was one of the genes in the CIMP gene panel [Toyota et al. 1999a]. Later studies showed that 70–80% of sporadic-MSI tumors (i.e. not related to familial hereditary nonpolyposis colorectal cancer syndrome, also known as Lynch syndrome) are a result of CIMP and MLH1 methylation [Herman and Baylin, 2003]. A subsequent study that looked at genome-wide methylation markers in 295 primary human colorectal cancer tumors developed a new five-gene CIMP panel (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1) that more accurately reflected genome-wide aberrant methylation [Weisenberger et al. 2006]. Again, MSI status was found to correlate closely with CIMP. However, it was also noted that nearly all patients with BRAF mutation were CIMP positive while KRAS mutation was correlated with CIMP-negative tumors [Weisenberger et al. 2006].

This finding, as well as others, lends credence to the suggestion that colorectal cancer has disparate epigenetic and genetic-pathogenesis pathways that ultimately lead to different diseases that are all called ‘colorectal cancer.’[Ogino and Goel, 2008] This is noteworthy because the end result of each respective pathway behaves differently. There is a continuum in the lower GI tract with CIMP and MSI being most common in the cecum and ascending colon, and decreasing in likelihood as one travels distally, making CIMP or MSI much more likely to arise in right-sided colon cancers. Their precursor lesions are sessile-serrated adenomas that are similar to hyperplastic lesions and may be misdiagnosed on screening colonoscopy [Snover et al. 2005; Farris et al. 2008]. While nearly all sporadic-MSI tumors are CIMP positive, CIMP can be either MSI or microsatellite stable (MSS), so one can evaluate MSI and CIMP as independent factors (Table 1). A recent meta-analysis of CIMP in colorectal cancer showed significantly shorter disease-free survival and overall survival for CIMP colorectal cancers both when ignoring MSI status and when looking only at MSS CIMP tumors [Juo et al. 2014]. Another study found that while CIMP positivity did not correlate with survival in proximal tumors, CIMP had significantly worse disease-free and overall survival as the location became more distal [Bae et al. 2013]. MSI tumors, on the other hand, had better survival in proximal locations but did not correlate with survival distally [Bae et al. 2013]. Generally speaking, CIMP positivity is a negative prognostic factor in colorectal cancer, while MSI is a positive factor, and the tumors that are CIMP positive but MSS are the ones with the most dismal prognosis.There are also therapeutic implications for CIMP and MSI positivity. Several retrospective studies have evaluated whether CIMP confers a survival benefit in stage III colorectal cancer patients receiving adjuvant chemotherapy with 5-fluorouracil (5-FU). The conclusions of these studies were disparate, with some showing that CIMP positivity led to improved survival, while others showed no difference or worse survival in CIMP positive patients receiving 5-FU compared with those undergoing surgery alone [Van Rijnsoever et al. 2003; Shen et al. 2007; Iacopetta et al. 2008; Jover et al. 2011; Min et al. 2011]. A recent study evaluated CIMP and MSI status in tumor samples from stage III colorectal cancer patients who had been enrolled in the CALBG trial and randomized to receive either 5-FU/leucovorin (LV) alone or in combination with irinotecan (IFL) [Shiovitz et al. 2014]. Their analysis appears to shed some light on the controversy because they found that CIMP-positive MSI tumors had excellent response with 5-FU/LV alone. However, CIMP-positive MSS tumors had significantly worse survival with 5-FU/LV alone which improved dramatically with the addition of irinotecan, while the CIMP-positive MSI tumors did the same or slightly worse [Shiovitz et al. 2014]. This suggests that it is necessary to consider both CIMP and MSI status when making treatment decisions regarding adjuvant chemotherapy (Table 2).

Table 1.

CpG island methylator phenotype (CIMP) and microsatellite instability in colorectal cancer.

	CIMP+	CIMP-
MSS	BRAF⁺	KRAS⁺
	Poor response to 5-FU/LV	Descending colon
	Improved response to	AIM-low signature
	5-FU/LV + irinotecan	PD-1 Ab nonresponsive
	PD-1 Ab nonresponsive
MSI	BRAF⁺	Extremely rare for sporadic; most likely familial
	Cecum and ascending colon
	Sessile-serrated precursor adenomas
	Good response to 5-FU/LV alone
	PD-1 Ab responsive
	AIM-high signature

CIMP, CpG island methylator phenotype; 5-FU, 5-fluorouracil; AZA, azacitidine; AIM, AZA-immune gene set; CRC, colorectal cancer; LV, leucovorin; MSI, microsatellite instability; MSS, microsatellite stable.

Table 2.

Modalities for epigenetic therapy.

	Epigenetic agent(s)	Proposed mechanism of action	Clinical trials	References
Single-agent therapy	DNMT inhibitorsHDAC inhibitorsHistone methyltransferase inhibitorsHistone demethylase inhibitorsncRNAs	Target DNA demethylation or histone acetylation in isolation; terminal differentiation in hematological malignancies; tumor stasis	NSCLC: DACAML/MDS: DACCTCL: vorinostatCTCL: romidepsin	Momparler and Ayoub [2001] Issa et al. [2004] Kantarjian et al. [2006] Olsen et al. [2007] Piekarz et al. [2009]

Combinatorial therapy	DNMT inhibitorsHDAC inhibitors	Multiple epigenetic pathways for increased efficacy; induction of apoptosis	NSCLC: AZA/entinostatSolid tumors/NSCLC: romidepsin/AZACRC: AZA/entinostat	[Juergens et al. 2011][ClinicalTrials.gov identifier: NCT01537744][ClinicalTrials.gov identifier: NCT01105377]

Epigenetic priming	RadiosensitizationHDAC inhibitors	Hyperacetylation of histone using HDAC inhibitors leads to: induction of DNA double strand breaks; inhibition of DNA repair mechanisms	GI cancer: vorinostat/radiotherapyPancreatic Cancer; vorinostat/gemcitabine/paclitaxel/sorafenib toyslatePancreatic cancer: capecitabine/vorinostat/radiotherapy	[Ree et al. 2010][ClinicalTrials.gov identifier: NCT02349867][ClinicalTrials.gov identifier: NCT00983268]
	ChemosensitizationDNMT inhibitorsHDAC inhibitors	Reprogramming of tumor cells; re-expression of tumor suppressor genes involved in cytotoxic drug pathway; reversal of chemoresistance; differentiaion of cancer stem cells	CRC: vorinostat/5-FU/leucovorinCRC: SGI-110/irinotecanCervical cancer: valprolic acid/hydralazine/cisplatinGastric cancer: vorinostat/capecitabine/cisplatinOvarian cancer: carboplatin/DACCRC: vorinostat/hydroxychloroquinePancreatic cancer; AZA/abraxane/gemcitabine	[ClinicalTrials.gov identifier: NCT00942266][ClinicalTrials.gov identifier: NCT01896856][Coronel et al. 2011][ClinicalTrials.gov identifier: NCT01045538][Matei et al. 2012][ClinicalTrials.gov identifier: NCT02316340][ClinicalTrials.gov identifier: NCT01845805]
	Immune modulationDNMT inhibitorsHDAC inhibitors	Increase tumor associated antigens; upregulation of immunomodulatory pathways and AIM genes; inhibition of MDSCs; induction of apoptosis	CRC: AZA/romidepsin/PD-1 AbCRC: SGI-110/GVAX/CY	[ClinicalTrials.gov identifier: NCT02512172][ClinicalTrials.gov identifier: NCT01966289]

5-FU, 5-fluorouracil; AML, acute myeloid leukemia; AZA, azacitidine; AIM, AZA-immune gene set; CRC, colorectal cancer; CTCL, cutaneous T-cell lymphoma; CY, cyclophosphamide; DAC, decitabine; DNA, deoxyribonucleic acid; DNMT, DNA methyltransferase; HDAC, histone deacetylase; GI, gastrointestinal; GVAX, gene-transduced tumor vaccine; MDS, myelodysplastic syndrome, MDSCs, myeloid-derived suppressor cells, ncRNA, noncoding RNA; NSCLC, non-small cell lung cancer.

The potential for epigenetic therapy in CIMP tumors is self-evident, as this is the most obvious subtype of cancer where the pathophysiology and tumorigenesis depends on the overwhelming accumulation of aberrant promoter hypermethylation. While pathologists now frequently test colorectal cancer specimens for microsatellite instability, KRAS, and BRAF mutations, CIMP is not routinely tested [Shi and Washington, 2012]. Only if microsatellite instability is identified will MLH1 methylation status be evaluated, and this is the only gene whose methylation status is routinely assessed clinically [Shi and Washington, 2012]. Epigenetic clinical trials in solid tumors have rarely targeted CIMP or MSI tumors specifically. However, one such trial, a phase I/II study of 5-AC and capecitabine/oxaliplatin in refractory CIMP-high metastatic colorectal cancer, has been performed [Overman et al. 2014]. These were metastatic colorectal cancer patients who had previously progressed on 5-FU/oxaliplatin-based therapy. The 5-AC was tolerated at doses of 75 mg/m², and while no objective response was seen, 10 of 11 CIMP-high patients had stable disease, with stable disease for longer than 6 months observed in two patients [Overman et al. 2014].

CIMP has been largely defined and studied in colorectal cancer. However, there has been some evidence that this hypermethylator phenotype exists in other GI malignancies as well, although the data is not as robust. A CIMP phenotype was identified in gastric cancer that also correlated with MLH1 methylation and MSI [Toyota et al. 1999b]. A later study provided an additional factor more specific to gastric cancer, showing that nearly all Epstein-Barr Virus (EBV)-associated gastric cancers are CIMP high and possess higher degrees of aberrant CpG island methylation than EBV-negative CIMP-high gastric cancers [Chang et al. 2006]. A CIMP subtype has also been proposed in hepatocellular carcinoma, duodenal adenocarcinoma, pancreatic adenocarcinoma, and esophageal squamous cell carcinoma, and was shown to be a negative prognostic factor for disease-free and overall survival in all of these cancer types [Ueki et al. 2000; Cheng et al. 2010; Ling et al. 2011; Fu et al. 2012]. However, in many of these studies, CIMP was also found to be highly correlated with factors that confer a worse prognosis independently, such as lymph-node metastasis and distant metastasis. This raises the question of whether CIMP in these cancers is a marker of a distinct pathophysiological pathway dependent on promoter hypermethylation, as in colorectal cancer, or merely a secondary result of late-stage cancers successively accumulating promoter hypermethylation during progression of tumorigenesis

Radiosensitization

Radiosensitization of cancer cells using HDAC inhibitors is a strategy that has been investigated in esophageal and colorectal cancer. Studies began in colorectal cancer-cell lines, and it was observed that pretreatment with HDAC inhibitors followed by radiation therapy after a several-hour delay caused a synergistic radiosensitization response both in vitro and in mouse xenografts [Flatmark et al. 2006; Folkvord et al. 2009]. The mechanism of radiosensitization with HDAC inhibitors has been proposed as hyperacetylation of histone proteins leading to chromosomal instability, cell-cycle arrest, and induction of DNA double-strand breaks, which occurs in concurrence with inhibition of proteins involved in DNA repair [Flatmark et al. 2006; Folkvord et al. 2009; Ree et al. 2010; Shoji et al. 2012]. The p53 status of the tumor is also thought to play a role in the mechanism of HDAC radiosensitization, as it is a frequently mutated cell-cycle regulator of the G1 checkpoint and affects the apoptotic response after DNA damage induced by radiation and HDAC treatment. [Fei and El-Deiry, 2003; Flatmark et al. 2006]. However, there have been disparate results as to whether p53 mutation is a positive or negative predictor of HDAC radiosensitization. [Shoji et al. 2012] The approach of employing HDAC inhibitors prior to radiotherapy was extended to esophageal cancer, where radiation plays an important role in the treatment algorithm. A similar radiosensitization effect was seen in four esophageal cancers after pretreatment with the HDAC inhibitor valproic acid (VPA) [Shoji et al. 2012]. This was taken one step further in combining vorinostat with radiotherapy and capecitabine in colorectal cancer xenografts, and the combination showed a synergistic response and further increasing radiosensitization [Saelen et al. 2012]. The PRAVO (pelvic radiation and vorinostat) trial was a phase I study which combined palliative radiotherapy with the HDAC inhibitor vorinostat in patients with bulky pelvic GI tumors [Ree et al. 2010]. While the higher doses of vorinostat in this study were tolerated as single therapy when assessing for maximum tolerated dose, the combination with a 2-week course of palliative radiotherapy caused severe dose-limiting GI toxicities and fatigue. The authors concluded that this regimen and the dosages employed were not a feasible approach [Ree et al. 2010], however further trials have been initiated incorporating this strategy in multiple GI malignancies, including pancreatic and gastric cancer [ClinicalTrials.gov identifiers: NCT00983268, NCT02349867] (Table 2).

Chemosensitization

Because of the failure of single-agent epigenetic therapy in solid tumors and increasing recognition of the reprogramming of intracellular pathways that these drugs cause, the focus has shifted to combination therapy. In a phase I/II trial in non-small cell lung cancer that used a combination epigenetic regimen of 5-AC with entinostat, 2 out of 45 patients experienced a response to combination epigenetic therapy alone and 4 of 19 patients (21%) experienced major responses and prolonged survival after going on to subsequent treatment with cytotoxic chemotherapy [Juergens et al. 2011]. A similar effect had been seen in a prior trial using DAC in advanced non-small cell lung cancer [Momparler and Ayoub, 2001]. Therefore, there appeared to be a benefit and improved response to cytotoxic chemotherapy in some patients after receiving pretreatment with epigenetic drugs.

Chemosensitization of GI cancers, and solid tumors in general, using epigenetic therapy is an exciting approach. The development of drug resistance in tumor cells is due at least in part to epigenetic regulation, and by treating with DNMT and HDAC inhibitors to re-express tumor-suppressor genes such as p16, RASSF1A, DAPK, as well as methylated genes involved in pathways of specific chemotherapeutic agents, we can cause a ‘reprogramming’ of tumor cells to sensitize them to cytotoxic agents [Plumb et al. 2000; Glasspool et al. 2006; Jones and Baylin, 2007; Steele et al. 2009; Sharma et al. 2010; Zeller et al. 2012]. Studies in colorectal cell lines have shown that epigenetic therapy with 5-AC or DAC improves sensitivity to both irinotecan and 5-fluorouracil (5-FU) both in vitro and in xenografts [Morita et al. 2006; Ishiguro et al. 2007; Crea et al. 2009; Miyaki et al. 2012; Vatapalli et al. 2014]. One study showed that MSS-cell lines were more likely to show chemosensitization to irinotecan after pretreatment with 5-AC, while MSI tumors were already responsive to irinotecan and did not demonstrate further synergy with 5-AC pretreatment [Vatapalli et al. 2014]. Another study showed that p53 mutation status was a predictive factor for chemosensitization in the cell lines [Crea et al. 2009].

Mechanistic studies evaluating this effect on irinotecan have uncovered multiple genes involved in the pathways of irinotecan uptake, function and metabolism that are epigenetically regulated via CpG island methylation. These include UGT1A1 and HNF1, which are involved in irinotecan elimination and display increased expression in irinotecan-resistant cell lines [Gagnon et al. 2006; Bélanger et al. 2010], TOPORS, which is involved in topoisomerase I and p53-binding [Saleem et al. 2004], and DEXI, which is hypermethylated and showed demethylation with improved response to irinotecan after DAC treatment [Miyaki et al. 2012]. Conversely, there was decreased sensitivity to irinotecan when DEXI was silenced with siRNA [Miyaki et al. 2012]. Other hypermethylated genes involved in the irinotecan mechanism of topoisomerase inhibition and apoptosis include Top-1, p16 and Sp1, which showed demethylation, increased expression, and chemosensitization after treatment with 5-AC [Crea et al. 2009].

Clinical trials utilizing exclusively epigenetic therapy in colorectal cancer have not shown responses to epigenetic therapy alone thus far. For example, in the largest such trial, a combination of DNMT inhibitor with HDAC inhibitor was attempted in a phase II study of azacitidine with entinostat in metastatic colorectal cancer patients who had already failed two or more chemotherapy regimens [ClinicalTrials.gov identifier: NCT01105377]. No objective response by response evaluation criteria in solid tumors (RECIST) or improvement in progression-free survival was appreciated in these heavily pretreated patients. RECIST is a set of guidelines used to objectively determine whether there has been a response to cancer therapy based on tumor burden from imaging results. However, building on the chemosensitization data outlined above, multiple clinical trials were developed and are underway in solid tumors and GI cancers which incorporate epigenetic therapy with established cytotoxic chemotherapeutics. In a completed phase II trial of vorinostat with 5-FU and leucovorin in metastatic colorectal cancer that failed other treatments, half of the patients experienced stable disease or objective response with progression-free survival of 2.4–2.9 months and overall survival of 6.5–6.7 months [ClinicalTrials.gov identifier: NCT00942266]. A phase I/II trial is also underway which is applying the results from data in our laboratory showing chemosensitization to irinotecan in colorectal cancer [Vatapalli et al. 2014]. This study has combined the novel DNMT inhibitor SGI-110 with irinotecan in metastatic colorectal cancer and will compare outcomes against treatment with the multikinase inhibitor regorafenib [ClinicalTrials.gov identifier: NCT01896856] (Table 2).

Another promising result in solid tumor epigenetic chemosensitization has come out of a double-blind, phase III, randomized, controlled trial of valproic acid with hydralazine (a DNMT inhibitor) and cisplatin in metastatic cervical cancer [Coronel et al. 2011]. The progression-free survival in the epigenetics + cisplatin group was 10 months, compared with 6 months in the cisplatin + placebo group (p < 0.05), and there was a trend towards greater rates of overall survival, objective response, and stable disease in the epigenetics group [Coronel et al. 2011]. In another gynecological malignancy, a phase II trial in 17 heavily pretreated and platinum-resistant ovarian cancer patients demonstrated resensitization to carboplatin after treatment with DAC [Matei et al. 2012]. The number of demethylated genes from tumor biopsies was greater in patients with progression-free survival of longer than 6 months than in those of less than 6 months, and methylation of four genes was shown to correlate to survival (MLH1, RASSF1A, HOXA10, HOXA11) [Matei et al. 2012].

Immune modulation

While epigenetically modulated re-expression of silenced genes to induce chemosensitization is a promising strategy, another exciting approach has emerged: the use of epigenetic therapy to sensitize for treatment with immune therapy. A recent study showed that after treatment with low dose 5-AC in 63 breast, ovarian, and colorectal cancer-cell lines as well as primary tumors, 20% of all upregulated gene sets belonged to immunomodulatory pathways [Li et al. 2014]. These pathways included interferon signaling, cytokines, antigen presentation, inflammation and cancer testes antigens, and the commonly upregulated gene sets between the 3 cancer types (breast, ovarian, and colorectal) were grouped into an azacitidine-immune gene set (AIM). This AIM panel was also seen to be present in lung cancer and melanomas. Interestingly, although the increased expression occurred after treatment with 5-AC, most of these AIM genes do not have CpG islands in their promoters [Li et al. 2014]. This suggests that the changes that cause transcriptional upregulation of these genes may lie downstream of a hub that is itself triggered by promoter demethylation. This emphasizes the concept that the epigenetic chromatin changes involved in transcriptional regulation interact on a level that affects multiple pathways and genes simultaneously. The cell lines also appeared to cluster into AIM-high and AIM-low signatures. AIM-high had high baseline expression of the AIM genes and a lower degree of upregulation than the AIM-low signature, which had low baseline expression and showed greater upregulation [Li et al. 2014]. This suggests that there is a subgroup of cancers that have decreased immunomodulatory gene expression and could benefit from treatment with 5-AC or other epigenetic drugs to upregulate the AIM genes, followed by treatment with immune therapy. Additionally, CIMP-positive colon cancers appeared to correlate with an AIM-high immune signature [Li et al. 2014] (Table 1). A follow-up study demonstrated that a major mechanism underlying the 5-AC-triggered immune response is induction of a cytosolic double-stranded RNA (dsRNA)-sensing pathway used by epithelial and other cell types as a viral defense mechanism that triggers a type I-interferon response [Chiappinelli et al. 2015]. The genes of this pathway, termed ‘viral-defense genes,’ were analyzed in tumor samples from multiple cancers in The Cancer Genome Atlas, including colon cancer, and were found to divide into high and low basal-expression groups. CIMP-high tumors correlated with high and intermediate basal expression of the viral-defense genes [Chiappinelli et al. 2015]. Additionally, high viral-defense signature expression correlated with improved response to immune-checkpoint therapy with anti-CTLA-4 antibodies in melanoma patients [Chiappinelli et al. 2015].

The rapidly expanding field of cancer immunology has recognized the role of the immune system in inhibiting tumorigenesis, and the ability of cancer to avoid immune-mediated destruction through alterations to the tumor microenvironment [Adam et al. 2003; Sharma and Allison, 2015]. Multiple strategies have been developed to block tumoral immune suppression and allow the body’s innate anticancer immune response to combat malignant cells [Sharma and Allison, 2015]. One of these strategies is immune-checkpoint blockade. The PD-1 receptor on CD8⁺ cytotoxic T cells downregulates the function of these cells and causes immune suppression. Cancer cells have been found to upregulate expression of PD-L1, the ligand that binds to PD-1, to cause adaptive immune resistance [Tumeh et al. 2014]. PD-1 inhibitors, which are antibodies to the PD-1 receptor, have seen success in clinical trials in melanoma, renal cell and non-small cell lung cancer [Topalian et al. 2012]. Two drugs, nivolumab and pembrolizumab, have received FDA approval for metastatic melanoma, and the indication for nivolumab was recently expanded to include metastatic non-small cell lung cancer after a randomized controlled trial demonstrated improved survival compared with current therapy [Brahmer et al. 2015].

A phase II trial of pembrolizumab in metastatic cancers with microsatellite instability, the majority of which were colorectal, introduced the importance of MSI status in immune-checkpoint blockade [Le et al. 2015]. MSI colorectal cancers showed a 40% objective response rate and 78% progression-free survival at 20 weeks, while MSI noncolorectal cancers showed 71% objective response and 67% progression-free survival. This is compared with 0% objective response and 11% progression-free survival in the MSS tumors. Median progression-free survival and overall survival was not reached in the MSI tumors, while MSS tumors showed 2.2 months and 5.0 months progression-free and overall survival, respectively [Le et al. 2015]. It is important to note that most of the MSI colorectal cancers (9/11) had Lynch syndrome or a detected germline mutation in a mismatch repair gene, while 2/11 were sporadic MSI. In total, there were six sporadic-MSI tumors of all types, but all showed an objective response by RECIST criteria [Le et al. 2015]. The authors proposed that MSI tumors were more susceptible to PD-1 checkpoint blockade due to increased immune-cell infiltration in the tumor microenvironment as a result of neoantigens caused by microsatellite mutations.

Combining these results with the results of the AIM gene panel and the association of AIM-high with CIMP-high tumors, one can propose a new epigenetic approach to this immune-checkpoint strategy. Sporadic MSI is associated with CIMP, and although the trial in MSI tumors enrolled mostly nonsporadic-MSI cancers, all of the patients who did have sporadic-MSI cancers showed an objective response. These tumors would also likely have had high viral-defense gene signature expression. However, the CIMP status of these tumors was not assessed in the study. Therefore, it follows that if CIMP-low tumors display a low AIM gene signature, and these tumors are most likely MSS and nonresponsive to PD-1 therapy, then utilizing epigenetic therapy with DNMT inhibitors to increase the immunogenicity of these tumors followed by treatment with immune-checkpoint blockade could be the key to unlocking the full potential of this immune strategy.

A preclinical study has employed this strategy with a combination of four drugs: CTLA-4 antibody (an immune-checkpoint inhibitor similar to the PD-1 inhibitors), PD-1 antibody, 5-AC and entinostat [Kim et al. 2014]. The cell lines used were CT26, a colon-cell line, and 4T1, a breast-cell line, both aggressive murine lines. Results showed that treatment with the checkpoint inhibitors alone was unable to eradicate large CT26 tumors and metastatic 4T1 tumors due to their modest immunogenicity. However, when combined with 5-AC and entinostat, there was striking improvement in mouse survival and tumor-growth inhibition, with complete eradication of large CT26 tumors [Kim et al. 2014]. The mechanism proposed was a downregulation of immune inhibitory myeloid-derived suppressor cells (MDSCs) by the epigenetic drugs. Although there was not significant toxicity reported in the study, the doses of entinostat used and the combination of four drugs would likely be a difficult regimen to implement in humans due to difficult tolerability. Nevertheless, this study showed that epigenetic therapy significantly improves the immunogenicity of tumors and their susceptibility to immune-checkpoint inhibitors. A clinical trial in colorectal cancer testing this concept is now underway by our group combining 5-AC and romidepsin with PD-1 antibody therapy [ClinicalTrials.gov identifier: NCT02512172] (Table 2).

Epigenetic therapy in noncolorectal gastrointestinal cancers

Esophageal cancer

Epigenetic therapy in esophageal cancer is still largely in the preclinical phase. A combination of 5-AC and different HDAC inhibitors (vorinostat, entinostat, romidepsin) in six esophageal cancer cell lines was shown to be selective for cancer cells, and the combination inhibited growth in a synergistic fashion superior to either drug alone [Ahrens et al. 2015]. Mechanistic studies showed induction of DNA damage, loss of cell viability and increased apoptosis in cancer-cell lines compared to non-neoplastic cells treated with the drug combination [Ahrens et al. 2015]. However, the drug doses used were high and may have relied more on cytotoxicity than epigenetic mechanisms.

Another study used low-dose DAC treatment followed by the HDAC inhibitor desipeptide in lung and esophageal cancer-cell lines and found induction of MAGE-3 and NY-ESO-1 cancer testes antigens that augment the immunogenicity of cancer cells [Weiser et al. 2001a, 2001b]. Taking another combination approach, studies have combined HDAC inhibitors with adenovirus or coxsackievirus-mediated p53 gene therapy in esophageal squamous cell carcinoma cell lines and found improved transduction efficacy of the virus-to-cancer cells with improved antitumor activity [Hoshino et al. 2008; Ma et al. 2012]. A phase I clinical trial of single-agent DAC in thoracic malignancies evaluating dose-limiting toxicity and maximum tolerated dose included some esophageal cancers [Schrump et al. 2006]. No objective treatment response was appreciated, and one esophageal adenocarcinoma patient showed stable disease, however one in three tumors showed induction of NY-ESO-1, MAGE-3, and p16 expression, providing proof of concept that DAC modulates gene expression in solid tumors through DNA demethylation [Schrump et al. 2006]. Other trials that include esophageal cancer in combination-epigenetic-therapy regimens are ongoing, and these mainly employ HDAC inhibitors [ClinicalTrials.gov identifiers: NCT00537121, NCT00670553, NCT00413075].

Gastric cancer

Single-agent and combination-epigenetic therapy has been investigated in gastric cancer in preclinical studies. The gastric cancer-cell line AGS exhibits increased promoter DNA methylation of somatostatin (SST) that may possess potent antitumor activity, and treatment with DAC showed promoter demethylation and re-expression of SST [Jackson et al. 2011]. Combination with trichostatin-A, a selective HDAC inhibitor, led to a synergistic increase in mRNA expression of SST [Jackson et al. 2011]. Another study showed that treatment with the DNMT inhibitor zebularine causes increased, dose-dependent mitochondrial-mediated apoptosis in gastric BGC823 cells in vivo [Tan et al. 2013]. Integrating the concept of CIMP with epigenetic therapy, one study treated 17 gastric cell lines (7 CIMP positive, 10 CIMP negative) with DAC in vitro and showed significantly reduced proliferation in the CIMP-positive lines [Zouridis et al. 2012]. The CIMP-positive AZ-521 cell line also demonstrated significant tumor-growth reduction in a mouse model with DAC, and cisplatin + DAC combination, compared with control [Zouridis et al. 2012]. However the in-vitro and in-vivo doses of DAC used in this study were high. Further work on combination strategies has also evaluated suberoylanilide hydroxamic acid (SAHA, or vorinostat), an HDAC inhibitor, in combination with taxanes [Chang et al. 2010]. This combination demonstrated synergistic tumor-growth inhibition in taxane-resistant gastric cancer-cell lines, but resulted in antagonism in some taxane-susceptible lines [Chang et al. 2010]. A phase I clinical trial of FOLFIRI (folinic acid, 5-fluorouracil, irinotecan) with vorinostat demonstrated stable disease in five patients, partial response in two patients, and no dose-limiting toxicity out of eight evaluable patients [Fetterly et al. 2009]. Other clinical trials are ongoing that evaluate combination therapy with vorinostat in gastric cancer [ClinicalTrials.gov identifier: NCT01249443] and vorinostat with radiotherapy [ClinicalTrials.gov identifier: NCT01045538].

Pancreatic cancer

Preclinical data for epigenetic therapy in pancreatic cancer has shown inhibition of pancreatic cancer both in vitro and in vivo. Multiple HDAC inhibitors have been investigated and responses have been seen in pancreatic-cell lines and mouse xenograft models [Van Kampen et al. 2014]. Recently, the HDAC inhibitor mocetinostat has been shown to chemosensitize to gemcitabine in pancreatic cancer-cell lines with ZEB1-associated drug resistance [Meidhof et al. 2015]. Another recent study utilized the HDAC inhibitor SAHA in combination with the small molecule inhibitor JQ1, which inhibits the bromodomain and extraterminal (BET) family of proteins, in genetically modified murine pancreatic models [Mazur et al. 2015]. They found a synergistic effect with the combination that greatly decreased tumor volume and was dependent on derepression of the gene p57 (CDKN1C). This approach is intriguing because bromodomain proteins are the principal readers of acetylated histone lysine residues [Muller et al. 2011]. Therefore, the investigators have combined two drugs that target separate mechanisms along the pathway of histone acetyl-mediated chromatin regulation.

Different studies have shown the effect of DNMT inhibitors in inhibition, chemosensitization, and immune sensitization of pancreatic cancer-cell lines in vitro [Missiaglia et al. 2005; Cecconi et al. 2009; Hollevoet et al. 2015]. An important feature of pancreatic cancer that is receiving a great deal of attention is its low cellularity and surrounding desmoplastic reaction. This quality highlights the importance of the tumor microenvironment or stroma in the initiation and progression of pancreatic cancer. Therefore, the use of DNMTs in a recent study to successfully inhibit tumor progression in a stroma-rich mouse model of pancreatic cancer is particularly notable [Shakya et al. 2013]. Clinical trials are ongoing using HDAC or DNMT inhibitors in combination with chemotherapeutic agents and surgery [ClinicalTrials.gov identifiers: NCT02349867, NCT01845805]. However, the aggressive nature and rapid progression of pancreatic cancer in combination with the longer time needed for epigenetic reprogramming to take effect has made design of dosing schedules in clinical trials a challenge. In our institution’s phase II clinical trial, 5-AC is administered after surgical resection for pancreatic cancer until there is evidence of tumor recurrence, followed by standard chemotherapy with abraxane or gemcitabine [ClinicalTrials.gov identifier: NCT01845805].

Future directions and conclusions

The field of epigenetics and epigenetic therapy is rapidly expanding. Early successes in hematologic malignancies have led to the approval of several epigenetic drugs in this group of diseases. Success in solid tumors so far has been less robust due to multiple issues including the pharmacokinetics of these compounds and the long duration required for treatment effect to be appreciated. In addition, multiple early trials used higher doses of these compounds at cytotoxic levels and as our understanding of epigenetic drugs has grown, we have made important adjustments to our strategies that we continue to assess and reassess to formulate our optimal plan of attack with epigenetic therapy. These re-evaluations, with the implementation of low-dose epigenetic therapy, epigenetic chemosensitization, and epigenetic-immune priming, have led to promising results in solid malignancies both in vitro and in early clinical trials.

Newer, more specific drugs that avoid the pharmacokinetic and stability issues of epigenetic therapy may prove superior to the first generations of DNMT inhibitors and HDAC inhibitors. Our application has also progressed, using the low-dose reprogramming effects to sensitize and weaken tumor defenses for the final kill with radiation, cytotoxic chemotherapy and immune therapy. These combinations are the key to unlocking the true potential of epigenetic therapy, applying it as the jab in a one–two punch. However, the recognition must be made that these drugs require time and patience for reprogramming effects to occur, and clinical trials must be designed with this in mind, applying epigenetic therapy to earlier stages and with lower tumor burden. These drugs have frequently been applied in clinical trials in metastatic solid-tumor patients with extensive tumor burdens who have failed multiple prior therapies. Survival for these patients is expected to be short, and due to the fact that a prolonged duration of treatment for epigenetic priming may be required, these patients may not have adequate time to allow for the epigenetic reprogramming of these drugs to fully take effect. Additionally, due to the previously discussed pharmacokinetics of these drugs and their short half-lives, the question remains: how much penetration really takes place in large, widely disseminated solid tumors? These important limitations must be recognized and addressed and may delay the incorporation of these drugs into clinical practice.

Treatment regimens are constantly being modified to find not only the ideal dosing and combinations, but also the groups of patients that will benefit from epigenetic therapy, as we move closer towards the ideal of truly personalized cancer medicine. With the introduction of methylation biomarkers into clinical practice through the Cologuard test [Imperiale et al. 2014], epigenetic markers will continue to be developed and utilized not only for diagnosis but also prognosis and clinical decision-making regarding treatment decisions. Microsatellite status has already shown potential as a biomarker that can predict 5-AC sensitization to irinotecan or immune-checkpoint therapy, as described above [Vatapalli et al. 2014; Le et al. 2015]. Further biomarkers that predict response to epigenetic therapy will provide another tool for personalization of oncological therapy. Additionally, there are already several epigenetic biomarkers which have shown not only prognostic significance in predicting patient outcomes [Kato et al. 2008; Wang et al. 2011a; Yi et al. 2011; Oguejiofor et al. 2013], but also in predicting response to various treatments as well. CIMP has been associated with response to treatment with 5-FU in colon cancer, although results have been disparate [Van Rijnsoever et al. 2003; Shen et al. 2007; Iacopetta et al. 2008; Jover et al. 2011; Min et al. 2011; Shiovitz et al. 2014]. LINE-1 methylation has also been associated with response to oral fluoropyrimidines (capecitabine) in colon cancer [Kawakami et al. 2011]. These epigenetic biomarkers will allow for improved, more precise, and more effective treatment while limiting overtreatment and adverse effects.

Finally, new drugs targeting different epigenetic marks are in development and may become more prominent in the coming years. Histone lysine methylation, as described earlier, can cause either transcriptional repression or activation depending on which lysines of which histone proteins are methylated, and there is a wide variety of enzymes involved in this regulation [Yoshimi and Kurokawa, 2011]. Therefore, both histone methyltransferase inhibitors as well as histone demethylase inhibitors have been developed, targeting the enzymes responsible for the methylation of specific histone lysine residues [Højfeldt et al. 2013; Zagni et al. 2013; Maes et al. 2015]. Some of these drugs have reached phase I clinical testing in hematological malignancies [ClinicalTrials.gov identifiers: NCT01897571, NCT02261779]. Research into the transcriptional regulation effects of noncoding RNAs has led to investigation into their application as biomarkers as well as therapeutic targets or effectors [Petrocca and Lieberman, 2011; Wang et al. 2011b]. Several phase I clinical trials are in progress, applying noncoding RNA therapy to silence specific oncogenes [ClinicalTrials.gov identifiers: NCT 01829971, NCT01061840, NCT02314052]. If these new drugs show efficacy, then their combination with other chromatin-modifying agents such as DNMT inhibitors and HDAC inhibitors, as well as with cytotoxic agents and immunotherapy, could lead to epigenetic reprogramming on a whole new scale.

Footnotes

Acknowledgements

Portions of Nita Ahuja’s work cited in this invited review were supported by K23 CA127141 and R01 CA185357, both from the National Institutes of Health and National Cancer Institute, and by the American College of Surgeons. Eihab Abdelfatah’s work is supported by NIH T32 Ruth L. Kirschstein National Service Research Award (T32CA126607). Nainika Nanda’s work was supported by the Johns Hopkins Summer Translational Oncology Program (STOP), RR25CA171973. Anup Sharma helped in formatting the figures.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest statement

Dr Ahuja has grant funding from Astex Inc. and has licensed biomarkers for early detection of pancreatic cancer.

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