In the early 20th century, German researcher Theodor Boveri described the basic tenets of tumor biology: Cells can turn cancerous when they lose the programming that controls their division and death. This loss usually results from a mutation that disrupts a cell’s internal “checks and balances” and changes the order of nucleotides within the DNA code itself.
Later, other researching pioneers discovered that cells can become malignant through other routes that are not directly the result of changes in DNA sequence. If the genetic code is the cell’s “hardware,” there is a second code – or a cell’s “software” – that dictates when and how genes are turned on or off.
This second code, typically called epigenetics, provides the cell with information on how the genetic code should be read and accessed by the cell’s machinery. The epigenetic code is written on top of the DNA code, designating some genes to be active while silencing others.
Epigenetics is a complex topic, and what constitutes epigenetics has evolved since 1939, when British developmental biologist C.H. Waddington introduced the term. Now, the modern definition covers heritable gene expression changes that are not caused by alterations in the DNA sequence.1
“Epigenetics is not just the study of a discrete mechanism used by certain cells under specific conditions, but a fundamental property of life that explains multicellular organisms,” said Ari M. Melnick, MD, professor of hematology and oncology at Weill Cornell Medicine in New York City, whose lab studies epigenetic programming is disrupted in hematologic malignancies. “There are more than 3,000 proteins that directly mediate epigenetic programming in the cell, which constitutes the largest gene functionality category,” he told ASH Clinical News.
The U.S. Food and Drug Administration (FDA) approved several cancer therapies that target epigenetic mechanisms, including the hypomethylating agents (HMAs) azacitidine and decitabine, which are indicated for the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with low blast percentage. Yet, the full scope of how these agents work and whether they have effects beyond their epigenetic functions is not yet clear.
“The more we know about epigenetics, the more questions we have,” said Thomas Prebet, MD, PhD, associate professor of hematology and oncology at the Yale Cancer Center in New Haven, Connecticut, who studies epigenetic deregulation in hematologic malignancies. “We are not at a point where we have fully deciphered the exact mode of action of these agents,” he noted.
How does epigenetic deregulation occur in hematologic malignancies, and how are these mechanisms being targeted by investigational treatment agents for blood cancers? ASH Clinical News spoke with Drs. Melnick, Prebet, and other researchers specializing in the epigenetics of hematologic malignancies for answers.
The DNA sequence of the genome is the same throughout an individual’s cells, but what makes one cell a heart, liver, or a skin cell is the specific pattern of gene expression that arises from the genome. The sequence of events is as follows: DNA wraps around proteins called histones to form chromatin. Epigenetic marks on the genome are contained in chemical modifications of DNA and the histones. Certain amino acids on histones can be modified by methyl, acetyl, and other chemical groups. All these modifications constitute “the epigenetic code,” which dictates whether, when, and how genes are expressed in a cell.
Mutations in the genes that encode the enzymes that regulate both histone and DNA modifications have emerged as the genetic hallmark of most hematologic malignancies, but also occur in many solid tumors. “In blood tumors, we have a more precise idea of the key players in epigenetic dysregulation, and so far, we have among the most compelling evidence that targeting epigenetic alterations can make a difference for patients,” said Dr. Prebet.
For example, mutations in the enzyme DNMT3A – which encodes a DNA methyltransferase to silence surrounding genes – are found in up to 25 percent of patients with AML.2
That information has translated to the development of several treatments that target epigenetic mechanisms. The drugs azacitidine and decitabine, for example, are thought to decrease DNA methylation, which might restore expression of abnormally silenced genes in MDS and AML cells. However, the drugs also may have a general cytotoxic effect, depending on the dose at which they are administered.
“We know that these drugs cause [a decrease in methylation] of thousands of genes, including genes that are important in cancer prevention, such as immune-related and tumor suppressor genes,” Naval Daver, MD, associate professor in the Department of Leukemia at MD Anderson Cancer Center in Houston, told ASH Clinical News. “But we don’t know much about whether their efficacy is due to their epigenetic effect or their direct cytotoxic effects.”
The FDA approved two oral histone deacetylase (HDAC) inhibitors, vorinistat and romidepsin, for the treatment of patients with progressive or recurrent cutaneous T-cell lymphoma (CTCL). While most physicians think of these HDAC inhibitors as epigenetic therapies,” Dr. Melnick noted that “no one knows if their effects on disease are due to epigenetic mechanisms.”
Two other epigenetic-targeting drugs also are approved for the treatment of hematologic malignancies: In 2017, the FDA approved enasidenib, an IDH2 inhibitor, for patients with relapsed or refractory AML whose disease harbors specific mutations in the IDH2 gene. This year, the FDA also approved ivosidenib for the same indication, in patients whose tumors harbor specific mutations in the IDH1 gene. Normally, both the IDH1 and IDH2 genes produce enzymes that regulate DNA cytosine modifications; mutations in either gene result in increased DNA methylation and gene silencing.
But, as with the HMAs, IDH1 and IDH2 inhibitors likely have multiple effects beyond just acting on cells’ epigenetic modifications. These agents affect the epigenome but do not directly target an epigenetic-modifying enzyme “The chemical produced by the mutant IDH1 and IDH2 enzymes, 2 hydroxyglutarate, acts as an oncometabolite,” Dr. Prebet explained, “so the influence of these drugs on epigenetic modifications is only part of the story.”
Hematologic malignancies are characterized by mutations in multiple epigenetic regulators, with some that result in disruption of chromatin throughout the genome. “What we know – and what is easy to demonstrate – is that the genes that encode many of these epigenetic enzymes are mutated, but what has been harder to demonstrate is what that does to the chromatin,” said Lucy A. Godley, MD, PhD, professor of hematology and oncology at the University of Chicago who studies how epigenetic alterations affect the development of hematologic malignancies.
Two recent studies underscore the likely importance of epigenetic-functioning genes in the development of AML. Both teams of researchers identified mutations in the blood cells that increased individuals’ risks of developing AML a decade later.3,4 These genetic changes, according to both groups, are likely the initial steps necessary for normal blood cells to transform into AML cells.
“Epigenetics is not just the study of a discrete mechanism used by certain cells under specific conditions, but a fundamental property of life.”
—Ari M. Melnick
In the first study, the authors found that people diagnosed with AML a median of 9.6 years from baseline (range not reported) were four times as likely as those who did not develop AML to have at least one AML-associated mutation.3 All participants with TP53 mutations (n=21/21) and IDH1 or IDH2 mutations (n=15/15) eventually developed AML, suggesting that these two mutations confer a high probability of developing subsequent AML. Mutations in the DNMT3A gene and the TET2 gene also increased the odds of developing AML.
“These and other studies give us a picture of how the average person, by accumulating certain mutations over time, particularly in chromatin-modifying genes, could develop leukemia,” said Dr. Godley. Still, she acknowledged that the exact mechanisms through which these mutations facilitate leukemia development are a mystery. “We think the mutations result in global chromatin structure and organization changes, but we don’t know the molecular details.”
Both activating and inactivating mutations in the EZH2 methyltransferase enzyme, which maintains gene silencing by adding mono-, di-, or trimethylation of a specific amino acid on histone H3, have been identified in several hematologic malignancies. “EZH2-activating mutations are rare in solid tumors, but quite common in B-cell malignancies such as follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL),” Dr. Melnick said, noting that another type of EZH2 mutation, those that inactivate the enzyme, have been identified in patients with MDS and myelofibrosis.5-7
Again, this discovery has led to the development of an epigenetic-targeting treatment, the EZH2 inhibitor tazemetostat, which is being studied as a monotherapy in ongoing phase I and II trials of FL and DLBCL. However, on April 23, 2018, the FDA placed the agent on partial clinical hold and halted enrollment in the clinical trials following a report that a pediatric patient participating in a phase I study developed a secondary lymphoma.
“EZH2 mutations are a promising target in non-Hodgkin lymphoma, but this area is still a work in progress,” Dr. Prebet acknowledged.
Dr. Melnick agreed. “The most recent data demonstrate that EZH2 inhibitors have shown fairly dramatic activity for a monotherapy in lymphomas.” However, he cautioned that these agents are not going to cure patients, particularly when used as a single agent. “Lymphomas are genetically complex, and hence will require combination approaches to eradicate the disease fully,” he said.
Trials of combination regimens that incorporate an agent targeting an epigenetic mechanism are already underway – and the results are mixed. For example, in a trial evaluating the combination of an HMA (either azacitidine or decitabine) plus the BCL2 inhibitor venetoclax in older patients with AML considered unfit for standard chemotherapy, 61 percent of participants experienced a complete remission, while earlier research has shown that single-agent HMAs result in response rates of 30 percent or lower.8 A second study of venetoclax plus low-dose cytarabine presented similar results, prompting the manufacturers to submit this combination for FDA review.9
However, results from a phase II trial comparing azacitidine alone or in combination with lenalidomide or with vorinostat in patients with higher-risk MDS and chronic myelomonocytic leukemia showed no significant advantage with the combination approaches.10 At a median follow-up of 23 months (range = 1-43 months), the overall response rate was 38 percent for single-agent azacitidine, 49 percent for azacitidine plus lenalidomide (p=0.14), and 27 percent for azacitidine plus vorinostat (p=0.16).
Much to Study, Many Unknowns
One of the difficulties of studying epigenetic changes in cells is that many of the epigenetic marks, on both histones and DNA, are transient and not readily captured by available technologies.
“As a field, epigenetics is running behind DNA-sequencing technologies,” said Dr. Prebet. “For now, we don’t have a gold-standard, genome-wide method for evaluating the epigenetic code, which is multilayered. We need a different technique for each type of epigenetic mark (e.g., methylation or acetylation), so a primary goal of epigenetics research is defining a reliable method for capturing these throughout a person’s genome.”
“Many of the assays we are using to capture epigenetic marks are technically challenging to perform and have limitations,” Dr. Godley agreed. “Some of the assays do not provide enough resolution and detail, which adds to the mystery of what the chromatin-modifying mutations are doing in hematologic malignancies and in cancers in general.”
“The epigenome is hardly understood at all,” Dr. Melnick said. “There likely are thousands of ways that covalent histone modifications of histones combine to regulate the epigenome; so far, researchers have studied only a couple of them. And, of these, we still know very little about how they work. Epigenetics research is still in its early stages.”—By Anna Azvolinsky
- Esteller M. Epigenetics in cancer. N Engl J Med. 2008;358:1148-59.
- Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363:2424-33.
- Desai P, Mencia-Trinchant N, Savenkov O, et al. Somatic mutations precede acute myeloid leukemia years before diagnosis. Nat Med. 2018;24:1015-23.
- Abelson S, Collord G, Ng SWK, et al. Prediction of acute myeloid leukaemia risk in healthy individuals. Nature. 2018;559:400-4.
- Ernst T, Chase AJ, Score J, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet. 2010;42:722-6.
- Morin RD, Mendez-Lago M, Mungall AJ, et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature. 2011;476:298-303.
- Guglielmelli P, Biamonte F, Score J, et al. EZH2 mutational status predicts poor survival in myelofibrosis. Blood. 2011;118:5227-34.
- DiNardo CD, Pratz KW, Letai A, et al. Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: a non-randomised, open-label, phase 1b study. Lancet Oncol. 2018;19:216-28.
- Wei A, Strickland S, Roboz G, et al. Safety and efficacy of venetoclax plus low-dose cytarabine in treatment-naive patients aged ≥65 years with acute myeloid leukemia. Abstract #102. Presented at the 2016 ASH Annual Meeting, December 3, 2016; San Diego, CA.
- Sekeres MA, Othus M, List AF, et al. Randomized phase II study of azacitidine alone or in combination with lenalidomide or with vorinostat in higher-risk myelodysplastic syndromes and chronic myelomonocytic leukemia: North American Intergroup Study SWOG S1117. J Clin Oncol. 2017;35:2745-53.