January 15, 2016 (Vol. 36, No. 2)

Angelo DePalma Ph.D. Writer GEN

Epigenetic Targets Are Plentiful but Well Camouflaged

Epigenetics is poised to become a cornerstone of drug development in oncology, diabetes, inflammation, developmental and metabolic disorders, cardiovascular and autoimmune diseases, pain, and neurological disorders. 

According to citations from PubMed Epigenetics, 40% year-on-year increases in epigenetics-related scientific publications occurred during the last decade, accompanied by a substantial increase in research funding. Data from ClinicalTrials.gov indicate that more than 40 different epigenetics-related drugs are undergoing clinical trials. Epigenetics will also likely affect developments in animal, plant, and environmental health.

Jim Corbett, president of the human health business at PerkinElmer, notes that epigenetics research is currently limited by the number and availability of fully validated targets and preclinical disease models. “Another limitation stems from the relative dearth of fully selective antibodies for some of the writer and eraser targets to elucidate these complex signaling and modification events,” he points out. “Epigenetics research also suffers from a lack of a translational continuum for specific applications and for solutions from bench to bedside.”

Nevertheless, the field is characterized by a high level of optimism. Research by Mordor Intelligence (“North America and Europe Epigenetics Market Growth, Trends and Forecasts, 2014–2020”) estimates that epigenetics will grow in market reach from approximately $2.9 billion in 2012 to about $12 billion in 2018.

“I anticipate the development of second-generation epigenetic inhibitors with increased selectivity and targeting potential, standardization of epigenetic assays, and the validation of preclinical disease models leading to an improved understanding of epigenetic targets and mechanisms,” Corbett ventures. “The emergence of selective genome-editing technologies such as CRISPR will also apply in epigenetics and epigenome editing. I envision the future the emergence of personalized epigenetic profiles in patients.”


Computer Analogy

Randy L. Jirtle, Ph.D., professor of epigenetics at North Carolina State University, describes epigenetics as a type of biological software. He explains an embryo’s combination of paternal and maternal genetic information, and eventual differentiation into 200–300 cell types, on the basis of cells running different programs.

“The cell can be thought of as a programmable computer where the hardware is DNA and the software is the epigenome,” says Dr. Jirtle. “Very shortly after fertilization, this computer tells the cell how to work. And as with actual computers, things can go wrong because of viruses or—in the case of cells—mutations.”

Dr. Jirtle demonstrated in 2003 that epigenetic modifications in utero may determine adult disease susceptibility, a notion that was not welcomed enthusiastically. “If you think of life as hardware, no known mechanism would explain this [connection],” asserts Dr. Jirtle. “But when you consider the ‘software,’ it becomes understandable.”

Epigenetics can bring about positive effects as well. Through a process known as hormesis, low doses of a toxic agent or low doses of radiation can be administered strategically to improve an organism’s subsequent health. For example, mice exposed to low levels of ionizing radiation experienced a positive adaptive effect, which flies in the face of prevailing “no safe dosage” logic.

In one strain of an experimental mouse bred to develop human-like diseases, 1 cGy of exposure—about the dose received from five X-rays—resulted in a decidedly positive hypermethylation of the epigenome. Exposed mice developed obesity, diabetes, and cancer at significantly lower rates than nonexposed mice. Negative effects occur at significantly higher doses as expected.

Similarly positive epigenetic effects have been observed in plants exposed to very low doses of herbicides.

Dr. Jirtle believes that the characterization of the repertoire of genes imprinted in humans, and their regulatory elements, the imprintome, will guide epigenome-based therapies. Imprinting is the process by which one parental copy of a gene is silenced. Thus, depending on the effectiveness of silencing, one could have two copies of a gene or none, either of which could potentially be deadly. In some cancers, for example, the inability to silence one parental gene for IGF2, which influences apoptosis, allows cancer cells to grow out of control.

“There are probably around 150–500 disease-influencing genes that are regulated this way,” Dr. Jirtle points out.


Implementation Hurdles

The connection between dysregulated DNA methylation and cancer is well established. Keith Booher, Ph.D., epigenetic service projects manager at Zymo Research, believes that modifying how methylation patterns change could allow a reset. Essentially, cells destined to become cancerous could be returned to a normal state.

But significant hurdles block straightforward implementation. For example, getting drugs into cells, particularly solid tumors, is not easy. “It’s no coincidence that DNA methylation inhibitors have proved most successful for blood-based cancers, which are easier to target,” Dr. Booher tells GEN.

Another hurdle is drug resistance, an issue with nearly all oncology agents. Moreover, drugs that alter the activity of the ubiquitous DNA methyl transferase will have broad activity on normal as well as abnormal cell processes.

“Normal cells show low and high methylation levels,” Dr. Booher explains. “DNA methylation tends to limit gene expression, so you want to shut down those genes. And where DNA methylation is absent, genes tend to be expressed.

“Methylation will change across the genome at different development stages, but in adult cells or developing blood cell you want methylation patterns to change in a regulated way. It’s difficult to limit the effect to diseased cells.”

Finally, the way methylation inhibitors interact with DNA is in itself harmful. The original epigenetics-based drugs tested as broad chemotherapeutics, but their toxicology was high. It was only later, after the understanding the relationship between DNA methylation and carcinogenesis was better established, that the potential to use these agents at much lower doses became possible.


Diagnostic Relevance

One of the most important advances in epigenetic research is the ability to obtain comprehensive, per-base methylation status of the methylome using next-generation sequencing (NGS). The significant drop in sequencing costs enables both whole-genome bisulfite sequencing and hybridization capture for targeted enrichment of the methylome.

Initially, notes Laurie Kurihara, Ph.D., director of R&D at Swift Biosciences, these techniques were developed for microgram inputs of genomic DNA that undergo standard NGS library preparation followed by bisulfite conversion, a chemical process that converts nonmethylated cytosines to uracil. Subsequently, the polymerase chain reaction (PCR) process can be used to convert uracil to thymidine. “But the methylated cytosines are protected, thus demarcating methylation status when the DNA sequence is determined,” Dr. Kurihara observes. “The drawback is that bisulfite-induced DNA fragmentation destroys the bulk of the prepared NGS library. Hence the requirement for microgram DNA inputs.”

To enable lower DNA inputs and improved methylome coverage and uniformity, Swift Biosciences has developed an NGS library preparation performed on bisulfite-converted DNA fragments. The underlying technology, Adaptase, is a proprietary NGS adapter attachment chemistry for single-stranded DNA.

“By significantly improving sample recovery from bisulfite-converted DNA,” explains Dr. Kurihara, “more complete analysis of clinical samples is possible, particularly cell-free DNA from plasma that is limited to low-nanogram quantities of DNA.”

Dr. Kurihara cites an example provided by Dennis Lo, M.D., Ph.D., professor of chemical pathology at the Chinese University of Hong Kong. Dr. Lo developed a noninvasive test for cancer by detection of genome-wide hypomethylation of cell-free DNA from patient plasma. Although this “liquid biopsy” does not uncover actionable cancer mutations, it may prove to be a sensitive blood test for early cancer detection as well as treatment monitoring.

More recently, Dr. Lo’s group mapped the tissue of origin for cell-free plasma DNA using genome-wide bisulfite sequencing after mapping tissue-specific methylation patterns. Such noninvasive testing from blood may identify tissue- or organ-specific pathologies, including cancer, stroke, myocardial infarction, autoimmune disorders, and transplant rejection.

“Given that advances in epigenetic technologies have enabled per-base methylation status from low DNA input clinical samples, proof of concept has been established that ‘liquid biopsy’ testing of patient blood may be a universal screen for a variety of diseases that may be pinpointed to individual organs or tissues,” Dr. Kurihara tells GEN. “Such universal testing could be particularly advantageous for early detection of cancer and other diseases where noninvasive screening has not previously been possible.”


This Circos plot from Swift Biosciences represents the methylation status of 1 Mb bins across chromo­somes 1–22 for Sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary).

NGS: An Enabling Technology

The widespread adoption of next-generation genomic sequencing means that for the first time scientists can sequence large numbers of cancer patient genomes. Thus far, these studies have demonstrated that a large proportion of mutated cancer genes may be classified as epigenetic modifying factors.

“Chromatin remodeling and modifying factors are involved in the regulation of gene expression,” says Ali Shilatifard, Ph.D., chairman of the department of biochemistry and molecular genetics at Northwestern University’s Feinberg School of Medicine. “The DNA methylation factors are highly mutated in most cancers characterized thus far.”

Dr. Shilatifard provides the example of a family of mixed-lineage leukemia genes within the complexes known as COMPASS (complex proteins associated with Set1), which are highly mutated in a large number of cancers. “We’ve shown that MLL3/4, two members of the COMPASS family, modify regulatory elements known as enhancers,” notes Dr. Shilatifard. “The job of this COMPASS family member is to regulate these cis-regulatory elements during development.”

It has been shown that MLL3/4 and another component of COMPASS, UTX, are some of the most mutated genes in cancer. “We propose that perhaps these mutations function through enhancer malfunction,” Dr. Shilatifard continues. “And enhancer malfunction through these family members could result in miscommunication of the regulatory elements and promoters and mis-regulation of the expression pattern, resulting in tissue-specific cancers. It’s now very clear that epigenetic regulation and enhancer malfunction are key events in cancer pathogenesis.”

Dr. Shilatifard believes that over the next several years, academic labs and pharmaceutical companies will increasingly rely on agents that intervene epigenetically. For example, a recent study indicated that approximately 75% of patients with diffuse intrinsic pontine glioma (DIPG), a rare brain cancer in children, carried a single point mutation on histone H3, transforming lysine 27 into methionine. Many copies of histone H3 exist in these patients, but mutation in just one copy is sufficient to cause DIPG.

After modeling this mutation in Drosophila, Dr. Shilatifard’s laboratory discovered that a single point mutation on one histone was associated with a global loss of histone methylation and an increase in histone acetylation.

“Epigenetic regulators could be central for treating this disease,” comments Dr. Shilatifard. “Numerous examples in the literature suggest that inhibition of epigenetic regulators and interactors could be very important for treating cancer, and this may work for the treatment of DIPG through the inhibition of factors that bind to hyper-acetylated histones.”  



























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