When we acquire new skills, we begin simply. We respect the boundaries that we find or that we impose on ourselves. In childhood, for example, we develop motor skills by coloring inside the lines of the pictures within coloring books. Once we are confident of our motor skills, we may become more creative.

Something like this learning process is occurring in epigenetic therapeutics. Initially, epigenetic therapeutics were strictly epigenetic. That is, epigenetic targets were seen in isolation and acted upon accordingly. Alas, epigenetic therapeutics delivered results that were spotty and limited. But what else could have been expected from spotty and limited drug development strategies?

Eventually, drug developers recognized that epigenetic therapeutics could be effective in combination therapies. At that point, drug developers left their crayons behind and began working in oils. Like oil paints, epigenetic mechanisms mix well together. And now, with access to expanding palettes, drug developers are realizing increasingly subtle and powerful effects—including effects that are more true to life because they reflect a new sophistication, an ability to finesse targets rather than bash them. Instead of destroying dysfunctional cells, epigenetic therapeutics may restore them to a healthy hue.

Transcriptional and signaling effects

“The term epigenetic drug is not necessarily correct,” says Will West, PhD, CEO of CellCentric. “There are many drugs that have epigenetic-related elements, but whether or not they truly act in an epigenetic way is less clear.” With that statement, West suggests that drugs may produce epigenetic effects along with other kinds of effects.

twin histone acetyl transferases
CellCentric investigated over 50 potential epigenetic-related drug targets before focusing on the twin histone acetyl transferases p300 and CBP. To inhibit p300/CBP, the company is developing CCS1477, a small molecule that is highly selective for the conserved bromodomain pocket of both proteins. CCS1477 is being evaluated in Phase I/II trials as a treatment for drug-resistant prostate cancer, hematological malignancies, and tumors with specific drivers (that is, tumors with p300 or CBP mutations or driven by AR or MYC).

One such drug is CellCentric’s lead therapeutic candidate, CCS1477. It has epigenetic and cell signaling effects because it targets the paralogue histone acetyltransferases p300 and CREB-binding protein (CBP)—proteins that impact the expression of cancer driving genes, as well as key cancer-related signaling pathways. Specifically, p300 and CBP act as transcription co-activation factors, triggering the expression of certain genes involved in tumor progression such as c-Myc, AR, and IRF4. The proteins also prevent the acetylation of important signaling proteins, consigning them to ubiquitinylation and proteosomal degradation.

Using CCS1477, CellCentric scientists showed for the first time that p300/CBP could be targeted to block signaling through the androgen receptor pathway, including signaling through the receptor’s constitutively active splice variants. CCS1477 is a selective small-molecule bromodomain inhibitor. Initially, CCS1477 demonstrated potency against specific cancer cell lines and in multiple patient-derived xenograft models. Subsequently, CCS1477 became the first p300/CBP inhibitor to be evaluated in clinical trials. CCS1477’s on-target activity was confirmed through paired tumor biopsies obtained from participants enrolled in a Phase I trial.

“Targeting an epigenetic-related mechanism of action is different from targeting a receptor or degrading a specific protein,” West points out. If the mechanisms of action of two therapeutics are distinct, the two therapeutics can be administered together to provide synergistic benefits. Co-administered therapeutics that act on similar cellular pathways may be especially beneficial.

“We have seen [synergistic effects] across the board,” West asserts. “[They have occurred] whether the epigenetic drug is administered] in combination with second-generation antihormonal drugs for prostate cancer that influence the androgen receptor, or with standard-of-care agents used to treat hematological malignancies which are influenced by Myc and Irf4.”

A large, multi-institutional collaborative study recently used a loss-of-function short hairpin RNA (shRNA) screen and showed that CCS1477 and other p300/CBP inhibitors synergistically enhanced the cellular response to azacytidine, which is currently the best therapeutic option for patients with high-risk myelodysplastic syndromes.

As well as clinical trials in prostate cancer and hematological malignancies, scientists at CellCentric anticipate using CCS1477 for tumors that have specific oncogenic drivers overexpressed, such as small-cell lung cancer, breast cancer, and bladder cancer.

At the interface between the epigenetic and genetic mechanisms of action, p300/CBP is emerging as a promising and versatile target to therapeutically modulate dysregulated pathways relevant to disease. “Before homing in on p300/CBP we looked at over 50 different epigenetic target mechanisms,” West recalls. This selection was driven by efforts to align mechanistic understanding though to clinical application, with drug discovery and lead optimization relying heavily on protein crystallography and computer-aided design.

Potential therapeutics from unexpected sources

According to Bryan Oronsky, MD, PhD, chief medical officer at EpicentRx, most new drugs today are repurposed versions or follow-on variants of existing drugs. This view may explain why EpicentRx began looking for inspiration beyond the drug industry. The company eventually found that novel molecular structures sourced from the defense industry have therapeutic potential. By adapting novel structures, EpicentRx hopes to develop therapeutics that offer, in Oronsky’s words, “new and untapped mechanisms of action.”

A small molecule sourced from the defense industry’s aerospace sector is the lead candidate at EpicentRx. The candidate is designated RRx-001. It is in Phase III trials for several malignancies, and it is in preclinical studies for inhibiting the aberrant inflammation that accompanies several chronic diseases.

Molecules sourced from the defense industry’s arsenal present certain advantages. For example, many such molecules have already been evaluated for their toxic effects, including those on flora and fauna. Such molecules, then, can enter clinical studies with known in vivo safety profiles.

RRx-001, a small-molecule anticancer immunotherapeutic
EpicentRx is developing RRx-001, a small-molecule anticancer immunotherapeutic that downregulates the CD47/SIRPα axis and has the potential to convert “treatment resistant” tumors into “treatment sensitive” tumors. For example, RRx-001 could treat target hepatocellular carcinoma (HCC). This possibility will be explored in DELIVER, a Phase I/II trial. According to EpicentRx, RRx-001 may be able to prevent the progression of liver disease through its anti-inflammatory and antioxidant properties, and to treat HCC once it has already developed.

Although target-based screening is the most common approach used for drug discovery, phenotypic screening was used by EpicentRx to evaluate RRx-001 and several other molecules. “We had no idea what these molecules would do or how they would do it, but we had a sense that they would sensitize cells because of their mechanisms of action,” Oronsky says. “It turned out that RRx-001 is a radiosensitizer.”

Subsequent studies by EpicentRx found that RRx-001 acts by several distinct molecular mechanisms. The compound has immunotherapeutic activities; it repolarizes M2 protumor to M1 antitumor tumor associated macrophages; it activates antioxidant signaling pathways; and it resensitizes cancer cells to therapies they previously responded to.

“RRx-001 also has epigenetic properties,” Oronsky adds. “We saw that it inhibits DNA methyltransferases and histone deacetylases, and that it activates the sirtuins.” These multiple mechanisms of modulating the epigenome place RRx-001 in a unique position among the existing drugs that change epigenetic modifications.

EpicentRx determined that RRx-001 can radiosensitize cancer cells. When the company did so, it also evaluated whether the drug could radiosensitize healthy cells. This unwanted effect seemed plausible, given RRx-001’s mechanisms of action. However, EpicentRx found that RRx-001 was actually protective toward healthy cells.

“In mice that underwent total body irradiation, RRx-001 protected the intestinal crypt stem cells against the toxicity of radiation,” Oronsky notes. “And RRx-001 reversed or reduced the toxicity from chemotherapy in healthy tissues.”

The combination of cytotoxic effects on cancer cells, and cytoprotective effects from radiation damage on healthy cells, makes RRx-001 particularly valuable from a safety perspective. Unlike several other epigenetic therapies, RRx-001 has minimal systemic toxicity and does not have myelosuppressive effects.

Toxicity is a particularly acute concern for compounds that modulate epigenetic pathways in a genome-wide fashion and risk inadvertent changes to gene expression. “The future of epigenetic therapies is probably epigenome editing,” Oronsky suggests. In epigenome editing, the epigenome is modified at a specific site to rewrite the local epigenetic landscape.

“We are working on fusing catalytically inactive Cas9 to RRx-001 so that we can demethylate a particular promoter region of a target gene,” Oronsky reports. “We hope to gain the ability to prevent transcription and silence genes.”

A naturally occurring modulator of DNA methylation

Most developers of epigenome-modulating drugs focus on selectively killing dysfunctional cells. At a developer called Durect, however, an alternative approach is being explored. Durect focuses on eliminating the epigenetic faults that cause cells to become dysfunctional. Following this approach, Durect intends to generate drugs that can treat acute organ injury and chronic liver diseases.

“Attempting to restore the epigenome in a cell where it has become dysregulated to restore the cell function is something different, and a naturally occurring molecule represents an ideal approach,” says James E. Brown, DVM, president and CEO of Durect. “We are fortunate to have DUR-928 as a naturally occurring molecule that very safely turns on and off about 1,000 genes.”

DUR-928 is Durect’s lead candidate. It is an endogenous sulfated oxysterol that inhibits DNMT1, DNMT3a, and DNMT3b, the three mammalian DNA methyltransferases, and thus regulates the expression of many genes that control critical cellular activities. DUR-928 is in clinical development for the treatment of alcoholic hepatitis (AH), a highly lethal acute inflammatory liver disease. AH has no current approved treatments and represents a major unmet medical and public health challenge.

Patients with AH exhibit an increase in DNMT1 and DNMT3a and show DNA hypermethylation of many genes. According to Durect, DUR-928 dramatically reduces this aberrant hypermethylation and helps restore the expression of genes involved in lipid metabolism, the inflammatory response, and cellular survival.

A recently completed Phase IIa trial provided DUR-928 infusions on days 1 and 4 to patients with moderate or severe AH. “All 19 patients survived, and 14 of them left the hospital before they got the second dose,” Brown reports. “We believe this could be significant considering that the overall 28-day mortality rate for AH patients is 26%.”

The Phase IIa trial is being succeeded by a much larger Phase IIb trial—a randomized, double-blind, placebo-controlled, international, multicenter study called AHFIRM—that is evaluating the safety and efficacy of DUR-928 in severe AH patients. The new trial will comprise three arms of approximately 100 patients each: DUR-928 (30 mg); DUR-928 (90 mg); and placebo plus standard of care. (The standard of care, such as it is, may include the use of steroids, even though they usually fail to improve long-term survival). The primary outcome measure will be 90-day survival rate for patients treated with DUR-928 compared to those treated with placebo plus standard of care.

Those who would modulate the epigenome must contend with the number and the complexity of epigenetic changes that occur in various cell types. For example, in human hepatocytes, there are 22 million sites of DNA methylation, Brown points out. With such a large number of sites, the possibilities for epigenome regulation are incredibly vast—so vast, Brown suggests, that molecules to regulate gene expression would be all but impossible to develop without big data technology.

BET inhibitors in oncology applications

Inhibitors of bromodomain and extra-terminal motif (BET) proteins represent a prominent class of epigenetic anticancer drugs. Discovered in the early 1990s, they attracted close study for their therapeutic potential only during the past decade. Even though they were found to selectively interfere with gene expression in tumor cells, BET inhibitors were not seen to exhibit much single-agent activity. This shortcoming became evident in several clinical trials that evaluated how well BET inhibitors worked against hematological malignancies and solid tumors.

Preclinical data has emerged indicating that BET inhibitors may be effective in combination with other agents. “Our belief was always in combinations,” declares Sanjay Lakhotia, PhD, chief business officer at Zenith Epigenetics. “We launched into combination studies with small-molecule BET inhibitors that modulate resistance to existing therapies.”

ZEN-3694, Zenith’s lead therapeutic compound, is currently being used in multiple combination programs in the precision oncology market. A key focus of the strategy at Zenith is the effort to identify patient subgroups that respond best to very specific combinations.

“We were the first ones to show clinical efficacy in two different solid tumor types,” Lakhotia asserts. Scientists at Zenith Epigenetics evaluated the safety and efficacy of ZEN-3694 in combination with enzalutamide (an androgen receptor inhibitor developed by Astellas Pharma and Pfizer) in a Phase Ib/II dose escalation/expansion study in metastatic castrate-resistant prostate cancer. The combination showed efficacy, particularly among participants that responded poorly to abiraterone, and were at risk of becoming androgen-receptor independent, leading to a median radiographic progression-free survival of nine months.

Another combination drug development program is exploring ZEN-3694 together with Pfizer’s poly(ADP-ribose) polymerase (PARP) inhibitor talazoparib in patients with locally advanced or metastatic triple-negative breast cancer. Previously, talazoparib as a single agent had been shown to provide benefits with respect to progression-free survival in patients with advanced breast cancer and germline BRCA1/2 mutations. But the drug has very limited efficacy in tumors with wild-type BRCA1/2.

“In early clinical work,” Lakhotia notes, “we showed that ZEN-3694 can reduce the DNA repair capabilities in breast cancer cells with wild-type BRCA.” ZEN-3694 in combination with talazoparib may also translate into a significant clinical response rate. Both the castrate-resistant prostate cancer and triple-negative breast cancer programs are progressing toward registration studies.

A critical aspect of drug discovery in the epigenetics space is the need to redefine some of the desirable characteristics of the lead molecules that are being pursued. “People often think that BET proteins should be inhibited the way that kinases and other targets are inhibited—in the sense that the target has to be hit hard and for a long time,” Lakhotia says. However, that approach can lead to molecules that are long-lived and exceedingly potent, concomitantly increasing the risk of adverse effects.

“Early on, when we selected our development candidates, we performed extensive work to find the pharmacokinetic profile that allows the compound to be efficacious and safe,” Lakhotia continues. That involved selecting molecules that have moderate half-lives and a favorable but not excessive potency. “We have now shown we can chronically administer ZEN-3694 at safe therapeutic doses combined with other drugs,” Lakhotia reports. “We have had patients in prostate cancer trials for over 4 years.”

In 2020, Zenith demonstrated its commitment to expanding the spectrum of combination anticancer therapies. The company entered into a cooperative and research collaboration agreement with the National Cancer Institute to develop ZEN-3694 together with immune checkpoint inhibitors, MEK inhibitors, CDK4/6 inhibitors, and chemotherapy for multiple oncology indications.

BET inhibitors for applications beyond oncology

Oncology-focused Zenith Epigenetics had its BET inhibition origins in Resverlogix, a pioneer of BET epigenetics. Back in 2013, Zenith was spun out of Resverlogix, which was committed to cardiovascular disease. Today, Resverlogix continues to focus on chronic diseases that are outside of the oncology space. Besides finding ways to fight cardiovascular disease, the company is developing drugs for diabetes, chronic kidney disease, and metabolic diseases.

Resverlogix’s lead candidate is apabetalone (RVX-208), an orally available small-molecule BET inhibitor that selectively modulates BRD4 (BD2). Apabetalone reduces the markers of systemic inflammation in cardiovascular disease, ameliorates endothelial dysfunction and atherosclerosis in patients with chronic kidney disease, and attenuates SARS-CoV-2 infection in vitro at levels comparable to antiviral agents. These effects occur by suppressing the BET-mediated transcription that is triggered by pro-inflammatory stimuli.

“We have seen that in patients, whether they have diabetes, kidney disease, or cardiovascular disease, BRD4 is being recruited to the genome and keeps the gene targets turned on,” says Donald McCaffrey, president and CEO of Resverlogix and Zenith Epigenetics. As a selective bromodomain inhibitor, apabetalone inhibits BRD4 binding to its molecular targets and normalizes dysregulated epigenetic changes that are responsible for the pathogenesis of chronic diseases.

“In epigenetics we don’t want to hammer the target,” McCaffrey explains. “We want to tickle the target.”

Historically, many therapeutic leads under development sought to modulate the cellular levels of individual proteins involved in disease. “That only works for diseases that are caused by changes in a single protein,” McCaffrey points out. Many human diseases, including cardiovascular disease, diabetes, chronic kidney disease, and dementia, are complex and multifaceted. As a result, they are not amenable to interventions that target a single protein.

“Each of these conditions is the result of many proteins and entire pathways that are dysregulated,” McCaffrey emphasizes. In these complex medical conditions, transcriptional dysregulation often results from changes in the internal or external environment, such as diet or a viral infection, and can be corrected by BET4 inhibition. “We showed in several studies, including studies in patients with chronic kidney disease and in patients with COVID-19-like cytokine storms, that we can turn gene transcription back to its original pattern,” McCaffrey asserts.

While studying how well apabetalone works as single agent against complex medical conditions, scientists at Resverlogix are also exploring how well the drug performs when it is administered in combination with other therapeutics. “We are conducting studies that combine apabetalone with one of several existing drugs, including statins, beta-blockers, and ACE-inhibitors,” McCaffrey details.

Resverlogix has found that apabetalone may be particularly effective in combination with certain anti-diabetic therapeutics, specifically, sodium-glucose cotransporter-2 (SGLT2) inhibitors or dipeptidyl peptidase 4 (DPP4) inhibitors. Apabetalone in combination with SGLT2 or DPP4 inhibitors improved renal function markers and reduced the risk of major adverse cardiovascular events more than SGLT2 or DPP4 inhibitors alone.