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Promise and Progress - The Story of Epigenetics

Reprogramming Cancer Cells - The Story of Epigenetics
Issue No. 1

The Story of Epigenetics

Date: July 16, 2014

steve baylin
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Stephen Baylin

What Is Epigenetics?

Literally translated, epigenetics means around genetics.  It refers to natural control mechanisms that influence gene expression.  Their role is often compared computer software.  Think of DNA, and the genes we are born with, as the human hard drive.  Everything a cell does is controlled by this hard drive.  But, a hard drive cannot work without software.  Epigenetics is the software package.  Researchers believe that every cancer may have 50 to several hundred genes that have working “hard drives,” but their epigenetic “software” is causing them to act in a way that can lead to cancer development.

In the world of cancer, epigenetics is considered an emerging field, but its study is not new.  Investigators Andrew Feinberg and Bert Vogelstein first reported epigenetic changes in human cancer in 1983. Renowned veteran cancer scientists like Donald Coffey, Stephen Baylin, Peter Jones, Jim Herman, William Nelson, and Dr. Feinberg have been studying this biological process for decades, but it did not gain widespread acceptance until the early 2000s. Dr. Nelson, Kimmel Cancer Center Director, recalls in the 1990s when it was difficult to get a scientific paper on the topic published.   Technologies that allowed science to analyze DNA at the molecular level and the tenacity of a relatively small group of scientists proved its validity.  The long concealed mysteries of what some have referred to as the “ghost in our genes,” referring to epigenetic mechanisms’ ability to alter gene expression without leaving a permanent mark on DNA, are beginning to be uncovered. Now, what was once an outcast in cancer research is being heralded as one of the most promising fields of study in cancer medicine.

With a group of epigenetic scientists, who Dr. Nelson characterizes as “second to none,” the Kimmel Cancer Center has become a hub for epigenetic discovery and clinical translation.

Converting Doubters to Believers

If history was to name a founder of epigenetics, C.H. Waddington would likely get that designation. In the early 1940s, Dr. Waddington, an embryologist, put forth a radical idea for its era. With the genetic knowledge we have today, it is difficult to imagine, but at the time, most embryologists did not believe genes were important in human development. Rather, they contended that they played a minor role, controlling inconsequential details like eye color.  Dr. Waddington disagreed and introduced the concept of genes, and their regulation via an epigenetic landscape, as controlling cell differentiation and cell fate.  Of course, we now know that our DNA-- the genes we are born with--control every physical element of who we are.  Not only do they control eye color; they control eye formation.  The second phase of that development—the primitive mechanisms that turn genes on and off and, for example, let an eye cell know it’s an eye cell, and a brain cell know it’s a brain cell, when to start growing, and when to stop growing—that epigenetic landscape that Dr. Waddington first referred to more than a half century ago—is just now beginning to be understood, particularly how it applies to cancer.

Findings by Dr. Feinberg, now director of the Johns Hopkins Center for Epigenetics, and Kimmel Cancer Center epigenetics expert Stephen Baylin were trailblazers in this area of science, helping to garner attention for the field.

Dr. Feinberg described a global demethylation of the cancer genome. To try to better understand how epigenetics contributed to disease, he dug deeper into the role of normal epigenetic controls in cell behavior. In normal human development, when the sperm and egg come together and form that first cell, how that one cell divides and determines what its fate will be to eventually form a complete human body, Dr. Feinberg showed, is controlled through epigenetic mechanisms. Gene expression is what makes a cell behave the way it behaves, but how a cell figures out what proteins to express is controlled through epigenetics.

Dr. Feinberg suspected that this process was somehow getting hijacked in cancer. Corruption of the mechanisms that makes an undifferentiated cell know to become a liver cell could be at the root of the transformation of that same liver cell into a cancer cell. “In cancer, cells are confused about their own identity,” says Dr. Feinberg.  “I think epigenetic instability may be a general, universal feature of solid tumors—not necessarily genes getting turned off or genes getting turned on, but genes getting randomized until a preference for the cancer cell results.”

Dr. Baylin’s focus was on the hyper- or over- methylation of cancer genes, mainly tumor suppressor genes.  The process turned off gene expression, and what’s more it provided a therapeutic target. Drugs that blocked methylation of the gene could, in principle, turn the gene back on.  This work began to inspire the research of young investigators entering the cancer field.

Kimmel Cancer Center Director William Nelson was one of them.  He did not set out to become an epigenetics researcher.  In the early 1990s, he was beginning his career as a prostate cancer clinician and scientist when his research on cancer drug resistance led him to what remains today as one of the most classic examples of gene silencing through hypermethylation driving the development of cancer.

Drs. Baylin and Herman had already introduced a scenario in which tumor suppressor genes could be rendered inactive through the epigenetic process of hypermethylation, but they had not yet uncovered a good real life example of it they could point to.  When Dr. Nelson’s research led him to a gene called GSTP1, which he found was recognizably and verifiably hypermethylated in prostate cancer, still, the scientific world, except for a few, remained unconvinced of this purported epigenetic involvement in cancer.  Dr. Nelson struggled to get his research published. 

In some ways, Dr. Nelson believes it may have been the early detractors that helped propel the field forward.  “It created a higher level of rigor among those of us who believed,” says Dr. Nelson.  They set out to prove what they were convinced—what their research was showing them—that epigenetics played a key role in cancer initiation and progression.

Dr. Nita Ahuja, a Kimmel Cancer Center surgeon and clinician-scientist was among the small group of investigators involved in epigenetics research. “I was not a basic scientist, but I understood enough to know that all of the things we were seeing in medicine couldn’t just be bad genes,” she says.

While Drs. Nelson, Ahuja, and others were having difficulty getting their research published and felt like outcasts in the larger world of academic medicine, it was not that way at Johns Hopkins.  “The unique part of Johns Hopkins and the Kimmel Cancer Center is the collaborative nature.  It is the culture here,” says Dr. Ahuja.  “The exploration of novel ideas is celebrated here, and we are able to develop cooperative relationships spanning many departments and specialties.  This doesn’t exist at other places.  But, it’s what I love about Johns Hopkins.  There are no silos or territories.  I am a cancer surgeon who has an appointment in the Department of Surgery and laboratory space in the Kimmel Cancer Center, and directors of both programs support this.  Money can’t buy that. It has to exist in the fabric of the institution.”

It is this collegiality that maintained the momentum of epigenetics research at the Kimmel Cancer Center despite an inhospitable environment among the greater cancer research community.   Drs. Baylin and Herman built a tool that allowed scientists to look laterally at many genes across many cancers and establish a pattern of silencing through gene methylation. These hypermethylated genes were the subjects of promising innovation in the form of biomarker tests that could tease out aggressive cancers from more indolent forms and provide new targets for novel treatment strategies.  The origination of personalized cancer medicine was at hand.

Dr. Nelson’s colleagues describe him as a problem solver with an uncanny ability to see complex problems in a straightforward and logical way, and he saw an unmet medical need for men at risk of prostate cancer in his epigenetic discovery. In a disease where overtreatment is a major problem, Dr. Nelson’s discovery was used to create the first, noninvasive, epigenetic-based test for the disease.  The GSTP1 gene is only hypermethylated in cancer.  The epigenetic marker is not found in any normal cell, says Dr. Nelson.  As a result, it is being translated into a simple urine test.  It is most useful for men who are suspected to have prostate cancer because of an abnormal exam or rising PSA (prostate specific antigen) levels, despite a negative biopsy.  Prostate biopsies miss about 25 percent of cancers, and the test helps address this uncertainty.  If the urine test is positive, it predicts the presence of prostate cancer with almost 100 percent certainty.  If results are negative, it confirms the biopsy results with less than a five percent error rate. Dr. Nelson is now collaborating with epigenetics expert Vasan Yegnasubramanian to further refine the technology for use as a general prostate cancer screening test and as a tool to identify men who may have lower risk forms of the disease and could safely forgo treatment.  Conversely, such a test, given the right epigenetic markers, could distinguish men who have more aggressive forms of the cancer who would benefit from treatment.

The promising work of this group of researchers was at last gaining acceptance for the field of epigenetics. When Dr. Baylin first began his research some three decades earlier, the term epigenetics did not exist.  Now, he is viewed as the leader in the field, and his research in epigenetics is the most frequently cited in the established and rapidly growing specialty.  The pioneering work of Dr. Baylin and team in this emerging field of epigenetics, coupled with breakthroughs in cancer genetics made by Ludwig Center scientists, resulted in the Kimmel Cancer Center being dubbed in media reports a “cancer research powerhouse.”

But accolades and laboratory success have never been what the Kimmel Cancer Center is about.  Translational science—the ability to transfer laboratory knowledge to the benefit of patients—was the goal. The most exciting aspect for Dr. Baylin was that his life’s work was about to become a treatment for cancer.  The perseverance and innovative thinking by Dr. Baylin and others had laid the groundwork for an Epigenetic Dream Team.

A Dream Team

Few would dispute the amount of epigenetic research talent amassed at the Kimmel Cancer Center. Laboratory findings in leukemia and lung cancer paved the way for clinical trials of a drug that appeared to have the ability to fix some of the epigenetic initiated changes to genetic code that helped cancers grow and thrive. 

The world was beginning to take notice, and Dr. Baylin’s laboratory model was becoming a clinical model.  Crucial to these advances was a type of drug called a demethylating agent.  Too much methylation in the active regions of tumor suppressor genes was found to shut the genes down, giving advantage to one of the cancer cell’s iconic behaviors–uncontrolled growth. Blocking the methylation of the gene turned it back on.

In 1980, another leading epigenetics expert, Peter Jones, a University of Southern California researcher, provided some of the first evidence that demethylating agents might have unique and unexpected anticancer properties. He was studying the drug 5-azacytidine as a potential chemotherapy agent, when he observed that in low doses the drug didn’t brutally destroy cancer cells as most chemotherapy drugs did, but rather gently nudged the cell into behaving differently.

Years later, Dr. Baylin and team decided to take another look at the drug, which had largely been abandoned because of its toxicity.  Laboratory studies in lung cancer and leukemia led to a clinical trial for patients with a pre-leukemia condition called myelodysplastic syndrome (MDS).  It worked so well, with some patients disease-free for ten years and counting, that the drug received FDA approval for treatment of MDS.  Dr. Baylin wondered if the drug might also work against other cancers.

Then in 2008, the Entertainment Industry Foundation and Major League Baseball formed Stand Up To Cancer (SU2C) to mobilize the public to donate money for cancer research and to motivate the scientific community to collaborate on promising areas of research that could quickly be moved to clinical trials.  “Dream Teams” made up of clinicians and scientists from around the country—the best in their fields were formed—selected after rigorous review by another panel of esteemed cancer experts and directed to take on specific cancer research projects. When the epigenetics dream team was announced, Drs. Baylin and Jones were the natural choices to lead it.

Dr. Baylin’s research of the role of DNA methylation in cancer had led him to a molecular co-conspirator.  He observed that it wasn’t just DNA methylation that affected gene expression but also how the DNA was packaged in the cell.  If the DNA contained within one cell was extracted and stretched out, end-to-end it would extend six feet.  Yet all of that molecular material is compacted and packed inside the nucleus of a human cell. The nucleus is a structure so tiny that more than 50,000 of them can fit on the head of a pin. 

Chromatin, a complex combination of proteins, mainly histones, is responsible for compressing the DNA to fit inside a cell.  This packaging also plays a role in gene expression and the copying of DNA as cells divide.  A loose chromatin results in normal gene expression, but add methylation to the mix, and histones hold the DNA together tightly and interfere with the gene expression. This tightened chromatin, Dr. Baylin and team found, can keep genes, including tumor suppressor genes, in a constant state of non-expression.   It can cause cancer cells to behave in a primitive, embryonic-like manner.  Unlike normal embryonic cells, which receive and respond to signals that tell them to stop making new cells, epigenetically altered cancer cells seem to maintain their ability to replicate, renew and divide.

Scientists do not know what prompts the cancer-promoting changes in chromatin structure.  They suspect it may be a repair mechanism engaged in response to cell injury, such as chronic inflammation.  While the cause is uncertain, Dr. Baylin’s research has identified a way that may fix it.  In the laboratory, when he and his team combined a demethylating drug with a histone-blocking drug  (HDAC inhibitor) in human cancer cell lines, the chromatin structure loosened and some gene expression was restored.  This discovery became the focus of the first SU2C Epigenetic Dream Team patient study.

Few scientists have devoted as much time to epigenetic research as Dr. Baylin.  Mild mannered and soft-spoken, he smiles and nods his head in an expression of tempered optimism and wonder.  Belying  his calm exterior is a feeling of intensity and resolve. These trials are the culmination of his life’s work, but that’s not why he so desperately wants what he and his team have found in the laboratory to work in patients. “It’s not about me.  It’s all about the patients,” he says.  “It is a luxury and privilege to get to a point where your research has resulted in something that could help patients.”

It may seem reasonable to suppose that a laboratory researcher would become disconnected from the patient.  After all, the scientist’s day-to-day relationship is with the cancer cell, the mouse model, the experiment.  It is not with the patient.  We hear of academic institutions where science is performed for the sake of science and success is measured by the scope and size of the project. While all learning has value, at the Kimmel Cancer Center, where scientists work side by side with clinicians and many perform both research and patient care, people like Dr. Baylin measure success by the contributions their science makes to advancing patient care.  It is not always a new treatment or grand discovery, but all work is aimed at figuring out, discovery by discovery, how the cancer cell works and how to break its grip.  Far from disconnected from the patient, laboratory research at the Kimmel Cancer Center is inextricably aligned with the patient.

For Dr. Baylin, the roots run deep.  He is a third generation physician. His father was a radiologist at Duke University, and his grandfather was a general practitioner in Baltimore.  He chose his own path in medicine, but his family’s inspiration is evident. Old family photos are found throughout his office.  A microscope and a cherished old, weathered chair that belonged to his father also are there.

A Tale of Three Responses

Dr. Baylin believed that the patients, not the scientists would tell the story of the study of epigenetics.  He was correct, and with twists befitting a mystery novel, the story was about to get very interesting.

The first clinical study of the combined demethylating agent and histone-blocking HDAC inhibitors was in patients with advanced lung, breast, and colon cancers.  The drugs were not given at the highest dose that patients could tolerate, as is usually the case in early studies of anticancer drugs.  Rather, low doses were given.  The goal was to kill the cancer cells by reprogramming their DNA, instead of obliterating them like most chemotherapy agents do.  In essence, the researchers were using the drugs to convert cancer cells back to normal cells—to change their destiny as Dr. Jones had done so many years ago in his laboratory studies of 5-azacytidine.  At high doses, the drug killed cancer cells, but at lower doses over time, it reprogramed cancer cells to behave like normal cells, a much less toxic and more permanent cancer fix. It was a radical departure from the standard approach of blasting cancer cells with as much poison as possible, but there was significant laboratory evidence to show that it could work.

It did, and remarkably, but only in a few lung cancer patients.  The responses, while small in number, were unprecedented.  Patients with resistant, lethal lung cancer that had spread to other organs and was resistant to other treatments were seeing their tumors melt away.   In a few other patients, tumors stopped growing.  The cancers didn’t go away, but they seemed to be dormant. Grow and spread is what cancer cells do best, so this response, though limited to just a handful of patients, made epigenetics fans and detractors alike take notice.

Still, most patients treated did not respond, and responses in breast cancer and colon cancer patients were not nearly as dramatic as those seen in the small group of lung cancer patients.  This did not surprise or deter Dr. Baylin and team.  Earlier work by his epigenetics colleagues James Herman and Malcolm Brock showed that specific epigenetic biomarkers provided a signature that could differentiate patients who were likely to respond from those who would not.   This trial was open to all patients with resistant cancers, and with no analysis for the epigenetic signature of their tumors, the expectation was that a small subset of patients would see results.  The analysis would come later with Dr. Baylin, basic scientist Cynthia Zahnow and cancer surgeon and translational scientist Nita Ahuja taking cells back to the laboratory for gene expression analyses.

What happened next, however, was not expected.  Because of funding provided through SU2C, Dr. Baylin and team had the opportunity to do something they typically never would get the chance to do. That was to follow up on patients who were taken off of the trial because their cancers continued to grow despite treatment with the experimental epigenetic therapy.  The team went back and reviewed the records of these patients. They expected to find that most of them had passed away.  These were patients with end-stage cancers that had spread and had not responded to three different attempts at chemotherapy.  When they learned that many of the lung cancer patients were still alive because their cancers had suddenly begun to respond to a wide variety of anticancer drugs, they were shocked.  Patients who seemed to progress while they were on the experimental therapy, some who had only received two or three treatments, were alive and doing well.  Cancers that had continued to grow and spread despite every effort were suddenly transformed.  Such responses were virtually unheard of, and the research team was eager to figure it out.

They pored over every scan, piece of clinical paperwork, and biopsy report available.  “There could only be two explanations,” says Dr. Baylin.  “Either the epigenetic therapy sensitized the cancers to subsequent treatment with standard drugs, or their improvement was a direct response to the epigenetic therapy.” The team needed to complete further studies in the laboratory to solve the mystery.

These new epigenetic targeted therapies do not work like the old cell-killing cytotoxic chemotherapies that do not discriminate between normal cells and cancer cells.  They specifically seek out and reprogram the epigenetic alterations that are allowing the cancer cell to survive and grow.  Dr. Baylin says the epigenetic therapies work slowly over time as they make repairs and return genes to normal function.  But Drs. Baylin, Herman, Brock, Ahuja and Zahnow, had another hunch.  They suspected that the epigenetic drugs had a priming affect on the tumor.  Altering the cancer cell’s gene expression with the demethylating agent and HDAC inhibitor had made the formerly resistant cells now vulnerable to treatment with anticancer drugs. 

As they began to study the cell lines in the laboratory, they found that the epigenetic drugs had the capability to impact almost every type of cell mechanism, including cell division, cell repair, and cell cycle and death.  Of particular interest to the researchers was the treatment’s affect on genes related to immune response.

Immune cells are on patrol at all times in the human body differentiating between foreign invaders and normal cells. Cancer cells are derived from normal cells, so they can fly beneath the radar of the immune system.  However, as the science of cancer immunology has advanced, researchers are finding that there is more to the cancer cell’s ability to evade the immune system than its similarities to normal cells.  Cancer cells use epigenetic controls to corrupt immune responses to cancer cells.  By hijacking the mechanisms that allow the immune system to differentiate an invading virus cell from a body’s own cells, it causes the immune system to tolerate cancer. 

In their laboratory analyses of gene expression in cell lines derived from patients in the epigenetic treatment studies, one immune target was jumped out at them.  This target was a gene called PD-L1.

Cancer Immunity

Epigenetic treatment turns on a number of silenced genes.  Some of them encode molecules in the immune system that turn on immune responses and some that turn them off and lead to immune evasion. Immune inhibiting genes turned on by epigenetic therapy include PD-1, part of the intricate checkpoint system hardwired into the immune system, and its partner PD-L1. Normal human cells need the ability to communicate with immune cells that they are the good guys and should be left alone. Unfortunately, cancer cells exploit the same process to avoid an immune attack.

Drs. Baylin and Zahnow sought out the help of one of the world’s leading cancer immunology experts and their Kimmel Cancer Center colleague Drew Pardoll.  In some patients in the study, the PD-L1 gene was already active, and laboratory studies indicated that its expression by lung cancer cells might be enhanced by epigenetic therapy. Dr. Pardoll believed that using a drug to block PD-L1 or PD-1 in conjunction with epigenetic therapy could alter the balance of immune effects of the treatment toward an activated immune response right within the tumor. It was worth a try.

For that, Dr. Pardoll recruited the help of his wife and cancer immunology colleague at the Kimmel Cancer Center, Suzanne Topalian, and lung cancer expert Julie Brahmer.  It has been well established that cancer has an immune evasion signal.  To survive, cancer cells need to at least partially adapt to their environment.  They send out a “don’t look at me” signal to immune cells.  Treated with epigenetic drugs however, the ability to evade the immune system is broken and cancer cells send new signals—on one hand, they beckon the immune cells to come and get them, and on the other,  they shield against immune attack by expressing PD-L1. 

Drs. Baylin, Zahnow, Ahuja, and colleague John Wrangle went back to the laboratory to decipher the immune evasion signature for lung, breast, colon, and ovarian cancers.  To do this they looked at all of the genes that get turned on in cancer cells with demethylating drugs.  Lots of genes, they found, get reactivated, but about 20 percent of them are related to immune regulation.  “This is a much bigger component then we thought,” says Dr. Ahuja.  “A significant part of what the epigenome does is regulate the immune system.” 

Their research revealed a set of genes that are epigenetically programmed to evade detection by the immune system.  Using a drug to reverse this programming may force the cancer cells out of hiding and make them more vulnerable to treatment, or even better, allow the immune system to see the cancer and kill it.  “Imagine if we could get the immune system itself to fight the tumors and keep the cancer in check.  Then we might have a permanent cure,” says Dr. Zahnow.

They are now working to verify their laboratory findings by studying tumor samples from patients in the most recent SU2C lung cancer patient study who are receiving the epigenetic combined therapy of a demethylating agent, histone-blocking HDAC inhibitor and anti-PD-1 treatment.  Dr. Ahuja explains the imagery on the gene expression analysis array. Immune genes that are turned on appear red and those that are suppressed are labeled green.  If the epigenetic therapy is working, they should start to turn “green” tumors into “red” tumors.  “The promise is immense,” says Dr. Baylin.  “It’s a beautiful concept, and it represents translational science at its best.  We hope it is as beautiful in patients.  That’s what this trial will show us.”

At the same time, researchers like pediatric oncologist and cancer immunology expert Christopher Gamper are interested in deciphering what epigenetic therapy does to normal immune cells.  His hope is that these drugs may be used to augment the effectiveness of other immune treatments, such as cancer vaccines.  Rather than reprogramming cancer cells to make them more susceptible to immune attack, Dr. Gamper would like to reprogram immune soldiers called T-cells that patrol the body looking for danger to

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