March 1, 2017 (Vol. 37, No. 5)

Meghaan M. Ferreira Ph.D. Contributor GEN and Clinical OMICs

Researchers Hope to Play Epigenetic Cards Such as the Bromodomain and Extra-Terminal (BET) Motif

Epigenetic therapies, small-molecule drugs named for their ability to alter gene expression by targeting epigenetic regulators, could enable powerful new strategies to combat diseases marked by aberrant gene expression, like cancer.

Epigenetic regulators fall into three main categories: writers, erasers, and readers. Writers modify histones by adding chemical groups that result in acetylation, phosphorylation, methylation, or other modification types, while erasers remove these groups. The important task of recognizing these modifications falls to reader proteins. Together, these regulators control gene transcription by either directly or indirectly altering chromatin structure; open chromatin configurations facilitate transcription by permitting access to DNA wrapped around histones, like thread around a spool, while closed configurations limit access and therefore impede transcription.

To truly harness the power of epigenetic therapies, scientists need to develop a greater understanding of the role these epigenetic regulators play in disease. To this aim, the Keystone Symposia on Molecular and Cellular Biology hosted a conference focused on “Epigenetics and Human Disease: Progress from Mechanisms to Therapeutics” in Seattle at the end of January.

While many presentations at the conference highlighted the potential impact of epigenetic therapies on cancer, Alexander Tarakhovsky, M.D., Ph.D., professor at Rockefeller University, revealed how biopharmaceutical companies aren’t the only ones taking advantage of epigenetic mechanisms.

It turns out that viruses have evolved histone-like proteins, “histone mimics,” that undergo the same post-translational modifications as histones, and when concentrated in the cell nucleus they can distort gene regulation through competitive inhibition by tricking epigenetic regulators with their clever disguise. In this way, viruses can effectively hijack the cell’s ability to launch a defensive attack by inhibiting expression of antiviral and immune-response genes.

Scientists have known that viral proteins from infectious diseases like Zika, dengue, yellow fever, and influenza accumulate in the nucleus of infected cells, but they did not know what purpose those proteins served. “Histone mimicry helps us to generate a framework for understanding how the virus interferes with gene expression in infected cells,” Dr. Tarakhovsky explained to underscore how histone mimicry has created a new paradigm that explains how the accumulation of these proteins could attenuate the immune response and allow viral replication.

Normally, the host would counteract the invasion by making slight modifications to the mimicked protein. However, “when the virus imitates histones it creates a very interesting conundrum for the infected cell, because it’s really not very good for the cell to start changing the histone sequence to adapt to the virus,” Dr. Tarakhovsky elaborated.

At the conference, Dr. Tarakhovsky discussed the existence of a histone mimic in yellow fever, its contribution to the disease, and its remarkable similarity to an epigenetic drug: “yellow fever has a protein that operates in a fashion very similar to the human-made bromodomain inhibitors.”

BET bromodomain inhibitors (BBIs) bind to the acetyl-lysine binding pockets of proteins within the BET bromodomain subfamily (BRD2, BRD3, BRD4, and BRDT), and they interfere with the protein’s ability to perform its normal function as an epigenetic reader by blocking its interaction with acetylated histone lysine residues. Research has shown that BBIs can cause significant down-regulation in the expression of cancer-related genes such as MYC.


Hematological Malignancies

Constellation Pharmaceuticals represents one of several companies developing BBIs as a cancer therapies, and they currently have a BBI, CPI-0610, in Phase I trials for hematological malignancies. “We have seen remarkable cell-killing activity across panels of cancer cell lines that translated to great antitumor activity in preclinical models representative of lymphoma, acute myeloid leukemia, and multiple myeloma,” remarked Patrick Trojer, Ph.D., vice president of research at Constellation.

Constellation has also developed a drug that targets different epigenetic machinery. Currently in Phase I trials for advanced B-cell lymphomas, Constellation’s EZH2 inhibitor, CPI-1205, promotes transcription by binding to the active site of EZH2, an enzyme that normally represses transcription by “writing” methyl groups to a particular lysine residue on the histone H3 tail.

The presence of activating EZH2 mutations in patient subgroups diagnosed with germinal center B cell-like diffuse large B-cell lymphoma and follicular lymphoma make EZH2 inhibitors attractive therapies for these cases. However, EZH2 inhibitors have also shown therapeutic efficacy in patients without these mutations—indicating that a spectrum of EZH2-dependence exists in these diseases.

“It’s not currently known why some wild-type EZH2-containing cancer cells depend on it for survival,” admitted Dr. Trojer. “If we could learn more about that context, it could help us predict which patients would benefit most from treatment.”

The ability to identify patient populations that will respond well to treatment based on biomarkers will greatly aid in the clinical success of epigenetic therapies—as it has for many preceding therapies. Breast cancer is a well-known example of how biomarkers, such as HER2 (human epidermal growth factor receptor 2), PR (progesterone receptor), and ER (estrogen receptor), can change how clinicians diagnose and treat disease. Named for the absence of these markers, triple-negative breast cancer (TNBC) is an aggressive, highly metastatic form of the disease that accounts for 15–20% of breast cancer cases.


Breast Cancer

Kornelia Polyak, M.D., Ph.D., and her colleagues at the Dana-Farber Cancer Institute have identified BBIs as a potential targeted therapies for TNBC. Preclinical studies in in vitro cell models and in vivo xenograft mouse models of TNBC showed significant growth inhibition with BBI treatment. In fact, TNBC cells exhibited greater sensitivity to BBIs than other types of breast cancer, a response that the scientists attribute to a dependency on bromodomain protein BRD4.

Despite these promising results, TNBC has a reputation for quickly acquiring resistance. “Even if these drugs prove successful, we know that cancer often devises a way to circumvent therapies and resume its growth,” commented Dr. Polyak, the study’s senior author, in a news release published by Dana-Farber.

The study, published in Nature last year, found that TNBC cells did acquire resistance to BBI treatment but not through conventional mechanisms like acquiring new genetic mutations or activating drug pumps. Shaokun Shu, a postdoctoral fellow in Dr. Polyak’s laboratory, explained how BRD4, MED1, and PP2A play pivotal roles in the resistance mechanism they discovered: “BRD4 becomes hyper-phosphorylated and binds more strongly to MED1, which is important for gene transcription.”

By essentially gluing BRD4 and MED1 together through decreased phosphatase activity by PP2A, the cells can overcome the absence of active bromodomains caused by BBI treatment. By dissecting the mechanism behind BBI resistance, Dr. Polyak and her colleagues have exposed vulnerable targets for combination therapies that could slow or prevent acquired drug resistance to BBIs.


Scientists working at the Dana-Farber Cancer Institute, have identified BET bromodomain inhabition as a potential targeted therapy for triple-negative breast cancer.

Ovarian Cancer

In contrast to breast cancer, which has seen substantial advancements in recent years with the introduction of targeted therapies, ovarian cancer has experienced few breakthroughs. An exciting advancement in ovarian cancer therapy, however, is the approval of PARP (poly ADP ribose polymerase) inhibitors for use in women with recurrent disease who have mutations in the BRCA1 or BRCA2 gene.

PARP inhibitors cripple the cell’s ability to repair single-stranded DNA damage. Cells with deleterious BRCA mutations are particularly sensitive to PARP inhibition, because they lack a mechanism to efficiently repair double-stranded DNA breaks and rely heavily on other DNA repair pathways. “PARP inhibitors work in BRCA wild-type cancers, but just not as well,” said Dineo Khabele, M.D., a gynecological oncologist and associate professor at Vanderbilt University Medical Center.

Dr. Khabele started asking the question, “what if we could enhance current therapies to treat ovarian cancer with novel drug combinations” early in her career. She started investigating epigenetic drugs, such as histone deacetylase (HDAC) inhibitors and, most recently, BBIs, as potential combination therapies. Although the precise mechanism of HDAC inhibitors and BBIs remains unclear, Dr. Khabele observed downregulation of homologous recombination repair genes BRCA and RAD51 in preclinical ovarian cancer models after exposing them to these epigenetic drugs. “We’re making wild-type BRCA cells look more like BRCA-mutated cells, and that enhances the efficacy of PARP inhibitors,” commented Dr. Khabele.

Dr. Khabele sees the biggest impact for epigenetic drugs being realized when they can identify women with ovarian cancers that are very efficient in DNA repair, “those cancers have a very poor prognosis, they don’t respond to chemotherapy, and if we can alter the molecular profile and make those tumors more BRCA-like then we could expand options for women diagnosed with those tumor types.”

Occurring in less than 1% of all ovarian cancer, malignant rhabdoid tumor of the ovary (MRTO, aka small cell carcinoma of the ovary-hypercalcemic type) is a rare but particularly aggressive form of the disease that primarily affects young women. “MRTO has a poor prognosis with less than a 35% two-year survival rate,” said Jesse Smith, Ph.D., head of the biological sciences department at Epizyme. “Not only is there a huge unmet need within this tumor type, but it’s also representative of a broader set of tumors that Epizyme is interested in,” Dr. Smith added.

Epizyme’s EZH2 inhibitor, tazemetostat, makes an appealing drug candidate for MRTO because of inactivating mutations characteristic of the disease in SMARCA4, a gene that normally encodes a protein involved in the SWI/SNF chromatin remodeling complex. “You can think of SWI/SNF as having an opposing function to EZH2—the same genes that EZH2 tends to repress, SWI/SNF keeps on. When you lose this particular gene you get aberrant function of SWI/SNF, and these tumors become very dependent on EZH2 for their survival,” Dr. Smith explained.

The same concept applies to malignant rhabdoid tumors—rare, highly aggressive tumors that occur primarily in children. These tumors exhibit loss of function mutations in the INI1 gene, which also encodes a SWI/SNF protein.

Dr. Smith shared that Epizyme “plans to put three new drugs in the clinic by 2020.” While these new therapies may include histone methyltransferases, like Epizyme’s two current clinical candidates (tazemetostat and pinometostat), they also intend to introduce epigenetic therapies with new targets identified using a CRISPR-based screening platform. “We built a CRISPR library covering over 600 targets within the epigenetic landscape, and we used that space to interrogate a very broad cancer space of over 200 tumor lines,” Dr. Smith said.

In contrast to conventional techniques used to modulate gene expression, CRISPR provides robust inhibition of epigenetic targets with greater specificity and less off-target effects. Epizyme’s screening method has enabled the identification of novel targets for epigenetic drugs with selective toxicity, and, in some cases, it has also allowed them to preemptively identify genetic biomarkers indicative of patient response.

“We need more drugs, we need more research,” Dr. Khabele stated passionately. Strategic advances in the war on cancer will require both targeted and combinatorial attacks, and researchers are hoping to add epigenetic therapies to their arsenal. Epigenetic targets could open a new frontier in orally bioavailable, small molecule drugs for the treatment of deadly diseases like cancer, or even sepsis. The clinical success of epigenetic therapies, however, hinges on our ability to uncover the mechanisms that viruses and cancers use to manipulate epigenetic machinery to promote their own survival.


Scientists at Epizyme’s headquarters in Cambridge, MA, are committed to discovering and developing new epigenetic therapies.

For Discussion

  • Histone-like proteins that accumulate in the nucleus may facilitate viral replication by inhibiting expression of antiviral and immune response genes.
  • Epigenetic drugs, like BET bromodomain and EZH2 inhibitors, have promise in the treatment of lymphoma, but determining which patient subsets will benefit the most from treatment remains a significant challenge.
  • BET bromodomain inhibitors have promising preclinical data for use as the first targeted therapy in TNBC, and the discovery of an epigenetic resistance mechanism may help the development of combination therapies that could slow or prevent acquired resistance to BET bromodomain inhibitors.
  • Treatment with HDAC and BET bromodomain inhibitors makes ovarian cancers without BRCA mutations sensitive to PARP inhibitors by down-regulating expression of homologous repair genes.
  • Tumors with loss of function mutations in genes encoding SWI/SNF proteins may respond to EZH2 inhibitors due to the opposing functions of EZH2 and the SWI/SNF chromatin remodeling function.


Evaluating Male Fertility by Examining Sperm DNA Methylation

Officials at Episona, which offers the Seed male fertility test, say the company set out to understand whether male fertility status and embryo quality during in vitro fertilization (IVF) therapy can be predicted based on genome-wide sperm DNA methylation patterns. Currently, doctors’ ability to evaluate male fertility is largely limited to the semen analysis and one in five couples seeking care are diagnosed with unexplained infertility.

The current literature provides strong evidence for the importance of appropriate DNA methylation for both male fertility and IVF outcome, according to the company. Developing technologies that recognize these marks could greatly improve IVF outcome, as well as accurately diagnose men’s fertility status.

“We conducted a retrospective cohort study with the University of Utah and the University of Southern California,” notes Philip Uren, Ph.D., director of data science at the company. “Participants were 127 men undergoing IVF treatment and 54 normozoospermic, fertile men. A comparison was made of DNA methylation patterns of IVF patients vs. normozoospermic, fertile men. Genome-wide sperm DNA methylation analysis was performed to measure methylation at more than 485,000 sites across the genome.”

The study concluded that sperm DNA methylation patterns differ significantly and consistently for infertile vs. fertile, normozoospermic men. In addition, DNA methylation patterns may be predictive of embryo quality during IVF.

“The data from the study, in addition to data from a large, multicenter prospective study (to be published later this year) was used in the development of Episona’s Seed test,” explains Dr. Uren. “A fertility test looking at epigenetic markers has several advantages over the traditional semen analysis to offer insight into sperm function. Additionally, when abnormal methylation is observed in a gene with a known relationship to fertility, it provides doctors with a potential cause for the problems. For example, the test has observed differential methylation in the ID3 gene which is thought to be important for embryo development.”


























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