Beyond the genetic code, epigenetic changes profoundly impact health and the development of diseases like cancer. Scientists delve into the intricacies of epigenomic regulation to better understand the underlying mechanisms of cancer and develop new treatment strategies.
Download this ebook from Drug Discovery News to learn about the latest advances in epigenetics research and how they improve cancer therapies and diagnostics.
More Than Mutations: Epigenetics in Oncology and Cancer Therapeutics
Damaged DNA and genetic variants are not the only cause for concern when it comes to heritability and cancer.
By Deanna MacNeil, PhD
Many cancers have heritable factors, and researchers have established mutational mechanisms of familial risk for some cancer types through genetic studies. However, a large portion of cancer heritability is unaccounted for by genetic mutations. As such, there is growing interest in the role of nonmutational epigenetic genome reprogramming in cancer susceptibility, development, and treatment (1,2).
The Interplay Between Mutations, Epigenetics, and Disease
Nonmutational genome reprogramming refers to gene expression changes caused by reversible epigenetic modifications. Molecular writers, readers, and erasers deposit, interpret, and remove epigenetic modifications to change gene expression without changing genetic sequences. DNA methylation, chromatin proteins, and noncoding RNAs such as microRNAs act as molecular tools to regulate epigenetic information. Additionally, 3D chromosome folding plays a key role in the epigenetic modulation of gene expression (3).
More than a decade ago, researchers raised the possibility of epigenetic programming in the absence of genetic mutations as a cancer hallmark. Today, there is growing evidence that tumors regulate their gene expression using similar epigenetic mechanisms to those involved in embryogenesis and tissue differentiation (1,4,5). Indeed, many tumors possess altered gene regulatory states and abnormal genome-wide histone and DNA methylation patterns (1,2).
Genetic factors, along with environmental and lifestyle factors including diet, smoking, alcohol consumption, environmental pollutants, and psychological stress influence epigenetic mechanisms that may persist through generations. To make sense of this, researchers study transgenerational epigenetic inheritance in which a parent’s epigenetics transmit to at least two generations of offspring (2,5).
In a paper published in eLife in 2019, Bluma Lesch, a geneticist at Yale School of Medicine, highlighted the importance of examining epigenetic transgenerational inheritance in cancer research (2). “I did my postdoc working on germline biology, and at the time, a huge gap in the field was the global, genome-wide epigenetic states in the germline,” explained Lesch.
To help fill this knowledge gap, she examined the effects of deleting lysine-specific demethylase 6a (Kdm6a) in the male mouse germline. Kdm6a codes for a candidate tumor suppressor protein that helps prevent cells from growing and uncontrollably dividing. It regulates the location of histone demethylation marks, and loss of this gene contributes to tumor initiation or progression.
Lesch genetically engineered a Kdm6a deletion in the germline of male mice and evaluated how this deletion affected two generations of offspring. The offspring of male mice lacking Kdm6a developed more tumors than those produced by healthy male mice. Despite carrying a normal maternally inherited copy of Kdm6a, somatic tissues in both generations of offspring from the genetically engineered male mice had epigenetic marks similar to the sperm of mice lacking Kdm6a. This suggests the possibility of transgenerational epigenetic changes in tumorigenesis.
Developing Epigenetic Therapeutics for Cancer
Kdm6a is not the only tumor suppressor implicated in the epigenetic regulation of cancer pathogenesis. Researchers studying cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, and noncoding RNA expression. Genes encoding epigenetic regulators such as DNA methylases and demethylases, chromatin remodelers, and transcription factors that promote or repress gene expression are frequently mutated or dysregulated in cancer. Such aberrant changes to epigenetic components often result from environmental or physiological factors, such as infections or aging (2,3,6).
The abundant involvement of epigenetic regulation in cancer and the reversible nature of epigenetic marks have led to cancer therapies targeting epigenomic regulation (7). This includes inhibitors of DNA methylation, histone methylation, and histone acetylation. Because of the complicated nature of epigenetic regulation during tumorigenesis, researchers also examine the effects of combining multiple epigenetic-targeting therapies to synergistically inhibit the expression of oncogenes, promote the re-expression of tumor suppressor genes, and overcome therapeutic resistance (8). “There are a lot of epigenetic drugs that inhibit some aspect of epigenetic regulation,” said Lesch. “One of the implications of the eLife paper was that treating people with epigenetic drugs could have epigenetic effects that could be transmitted and that we should be aware of this.”
Epigenome modifying drugs can sensitize cancer cells to anticancer therapies through a variety of mechanisms such as disrupting antiapoptotic signaling, preventing DNA damage repair, suppressing immune system evasion, and increasing protein expression for targeted therapies (9). Conversely, therapeutics that inadvertently induce epigenetic mutations can also change and disrupt essential cellular properties such as cell division, differentiation, adhesion, and proliferation, and can increase gene expression heterogeneity to promote tumorigenesis (1,3).
Commonly used cancer therapeutics such as chemotherapeutic agents that do not specifically target epigenetic regulators can also affect the epigenome. Scientists examine the heritable epigenetic implications for people treated with these therapies. “It's an important question, this issue of the germline and transgenerational effects of chemotherapy,” said Lesch.
Careful Considerations Before Chemotherapy
Common chemotherapeutic agents can cause methylation changes in the sperm and oocytes of animals, adversely affect rodent reproductive development, and induce transgenerational epigenetic effects (10–12). For example, after administering the osteosarcoma therapeutic and alkylating agent ifosfamide in pubertal male rats, scientists observed changes in different organ pathologies and DNA methylation of both the first- and second-generation offspring (13).
While human transgenerational epigenetic studies are less common, scientists have noted epigenomic effects in patients and physiological changes in children born to chemotherapy-exposed parents (14–16). “It's been only more recently that we've been doing human studies and that we've had the types of techniques that allow us to look at DNA methylation defects at a really good level,” explained Jacquetta Trasler, a pediatric researcher and geneticist at McGill University. In a 2023 study published in Clinical Epigenetics, Trasler and her research team examined sperm cell DNA methylation in healthy individuals and patients with Hodgkin’s lymphoma or testicular cancer before and after chemotherapy (16).
Trasler and her team observed distinct sperm DNA methylation signatures for each patient group in their study before chemotherapy, as well as post-treatment epigenetic defects, some of which persisted for up to two years after treatment for some patients. They found that sperm epigenome abnormalities exist before and after treatment, which may affect the reproductive health of patients with cancer.
“There is DNA damage, but there's also epigenetic damage,” Trasler explained. Trasler’s study provides insight into the transgenerational epigenetic effects of even nonepigenetic cancer therapies.
Studies in humans and animals underscore the importance of determining if and how epigenetic therapeutics and other common chemotherapies, as well as cancer itself, affect transgenerational epigenetics. “We know epigenetic dysregulation is big in cancer; it's now one of the new hallmarks of cancer,” said Lesch.
References
Hanahan, D. Hallmarks of cancer: new dimensions. Cancer Discov, 12, 31-46 (2022).
Lesch, B.J. et al. Intergenerational epigenetic inheritance of cancer susceptibility in mammals. eLife, 8, e39380 (2019).
Cavalli, G. & Heard, E. Advances in epigenetics links genetics to environment and disease. Nature, 571, 489-99 (2019).
Kim, S. & Kaang, B. Epigenetic regulation and chromatin remodeling in learning and memory. Exp Mol Med, 49, e281 (2017).
Alegria-Torres, J.A. et al. Epigenetics and lifestyle. Epigenomics, 3, 267-77 (2013).
Lu, Y. et al. Epigenetic regulation in human cancer: the potential role of epi-drug in cancer therapy. Mol Cancer, 19, 79 (2020).
Sharma, S. et al. Epigenetics in cancer. Carcinogenesis, 31, 27-36 (2010).
Cheng, Y. et al. Targeting epigenetic regulators for cancer therapy: mechanisms and advances in clinical trials. Signal Transduct Target Ther, 4, 62 (2019).
Quagliano, A. et al. Understanding the mechanisms by which epigenetic modifiers avert therapy resistance in cancer. Front Oncol, 10, 992 (2020).
Chan, D. et al. Epigenetic alterations in sperm DNA associated with testicular cancer treatment. Toxicol Sci, 125, 532-43 (2012).
E Silva, P.V. et al. Sexual differentiation and reproductive development of female rat offspring after paternal exposure to the anti-tumor pharmaceutical cisplatin. Reprod Toxicol, 60, 112-22 (2016).
Di Emidio, G. et al. Pre-conceptional maternal exposure to cyclophosphamide results in modifications of DNA methylation in F1 and F2 mouse oocytes: evidence for transgenerational effects. Epigenetics, 14, 1057-64 (2019).
Thompson, R. et al. Examination of generational impacts of adolescent chemotherapy: Ifosfamide and potential for epigenetic transgenerational inheritance. iScience, 25, 105570 (2022).
Patel, B.V. et al. Transgenerational effects of chemotherapy: Both male and female children born to women exposed to chemotherapy have fewer children. Cancer Epidemiol, 56, 1-5 (2018).
Patel, B.V. & Hotaling, J.M. Impact of chemotherapy on subsequent generations. Urol Oncol, 38, 10-13 (2019).
Chan, D. et al. Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer. Clin Epigenetics, 15, 5 (2023).
VISUALIZING EPIGENETICS IN CANCER
A clearer look at chromatin structures in super resolution reveals new insights into cancer pathogenesis.
INTERVIEWED BY YUNING WANG, PHD
Microscopy is an essential tool for clinical diagnosis and for understanding the pathogenesis of cancer. By examining alternations in nuclei architectures, a hallmark of cancer cells, pathologists identify and assess tumor progression. Inside the nucleus, genomic DNA folds into compact chromatin structures that regulate DNA transcription and genomic stability, defining cancer development. However, how chromatin structures change over the tumorigenesis process is beyond the detection limits of conventional microscopes.
Yang Liu, an optical bioengineer at the University of Pittsburgh, exploits the power of light and develops advanced imaging technologies and quantitative analysis tools to capture clearer pictures of subcellular structures. Liu and her team specialize in stochastic optical reconstruction microscopy (STORM), a fluorescence imaging technique that visualizes nanoscale molecular organization. Aiming to understand epigenetic regulations in cancer cells, they use STORM to look at chromatin organization directly in patient-derived tissues across different stages of cancer. By improving the imaging capability of STORM, the team has resolved previously invisible chromatin structures down to a resolution of 20–30 nm, gaining valuable information on malignant transformation and opening new opportunities for detecting cancer at earlier stages.
What interested you in using microscopy to study cancer epigenetics?
Pathologists rely on conventional light microscopes to examine distinct morphological features of cell nuclei, such as sizes and shapes, to identify tumors. However, a clinical dilemma is that precancerous lesions that are morphologically similar may have different levels of risk for developing malignant cancers. It is also challenging to detect cancers at earlier stages when cells still appear normal.
During my PhD training in biomedical engineering at Northwestern University, I worked on developing imaging technologies. I wanted to understand epigenetic changes in cancer at the molecular level using advanced microscopes. So, I looked into chromatin organization to identify features that can differentiate high risk from low risk precancerous lesions and allow the early detection of cancer. Although my training was not in chromatin biology, I was motivated to apply cutting-edge imaging technologies to visualize epigenetic changes and eventually improve risk stratification for cancer patients.
What is STORM?
STORM is a type of single molecule localization microscopy; it is a 2014 Nobel Prize-winning technique. The resolution of conventional microscopy is limited by diffraction, which means that it is hard to distinguish individual molecules because their fluorescently-labeled images overlap. Xiaowei Zhuang at Harvard University and Eric Betzig at the University of California, Berkeley developed the approach of activating only a small subset of fluorescent molecules while turning off the rest. Imagine them like stars blinking. At any given frame, we detect a random set of blinking molecules. By repeating the process, we can accumulate tens of thousands of frames to reconstruct the full image in super resolution. STORM combines a traditional wide field fluorescence microscope, imaging buffers that trigger the blinking events, and an image acquisition and localization system. Among various types of super resolution microscopy techniques such as structured illumination microscopy and stimulated emission depletion microscopy, STORM is one of the simplest and most cost effective approaches, so it has great potential in clinical settings.
What is the biggest challenge of studying chromatin structure with STORM?
Unlike many cellular structures, chromatin is dense and compact, so it tends to introduce higher background compared to other signaling molecules. To capture the entire process of cancer development, especially the early stages, we need to use paraffin embedded tissues from patients as our microscope samples. These tissues are notoriously difficult to image because of the high background as well.
Another challenge comes with the slow data acquisition speed. When STORM was first developed in 2015, scientists mostly used it to study monolayer cell cultures or bacteria. But for paraffin embedded tissues, we need to analyze hundreds of cells to achieve statistical significance, which means increasing the imaging throughput.
To overcome these challenges, we developed an algorithm that removes the background from the original image. This significantly improves the image quality and resolution, reduces artifacts, and increases reproducibility. We are also establishing a high throughput and high resolution system to enable high content phenotyping and study heterogeneous biological systems, such as the tumor microenvironment.
What chromatin structural changes have you observed during cancer progression?
We used mouse and human tissues to study the entire carcinogenesis process and found chromatin to be open and fragmented throughout cancer progression. We observed an increased degree of disruption in chromatin compaction at different stages, including during early carcinogenesis when cells still appear normal, as precursor lesions develop that grow into small tumors, and in malignant invasive cancers. To our surprise, although the opening of chromatin structures continues throughout the carcinogenesis process, the biggest changes occur very early, even before the tumor forms. By looking at various tumor types, such as colon, prostate, and lung, we also realized that this pattern of chromatin changes is universal across a wide range of cancers.
What do these changes tell us about cancer?
For many years, people believed that chromatin was more condensed in cancer cells because cancer cells undergo rapid replication and division. But by looking at the expression of histone proteins and heterochromatin marks, scientists in epigenetics research have found evidence suggesting open chromatin structures in cancer cells. In our study, we indeed observed open and disrupted chromatin structures in tumor tissues.
We also learned from many basic biological studies that compacted chromatin protects the cell from environmental insults. Once the protection is broken, the cell transitions from a normal phenotype to a state prone to carcinogenesis more easily. This is in accordance with our observation that the most remarkable chromatin fragmentation occurs at an early stage of tumorigenesis. Chromatin decompaction is an early event that leads to active transcription and facilitates malignant transformation.
How can super resolution imaging of epigenomic signatures help scientists detect cancer earlier?
The current clinical practice for colon cancer diagnosis relies on routine colonoscopy screening that identifies precursor lesions and colon polyps for a large number of patients. Patients need to do the screening annually, but only a small percentage of them eventually develop colon cancer, which makes this process inefficient.
We hope to analyze the chromatin structures of existing clinical samples with super resolution imaging to identify those that might progress into malignant tumors. We are working with a large database of well documented patient samples to correlate their chromatin features with cancer development. In this way, we will be able to stratify the cancer risks of patients and monitor patients with high risks more closely while examining patients with lower risks less frequently. This approach has the potential to improve clinical practice by providing more guided surveillance.
We are applying this technique to lung cancer. We collaborate with researchers at Hillman Cancer Center who collected many sputum samples from patients. Hopefully, by analyzing cells from these samples and looking at the chromatin signatures, we can detect lung cancer at an early stage.
What has been the most rewarding part of your research?
I am passionate about solving problems and excited about new discoveries and technologies that allow us to do what we have not been able to do in the past. Doing research is a long journey with lots of failures and frustrations, but the discoveries made in the end are worth all the effort. I encourage my students to focus less on the final outcome, and to look at the process and solve one problem at a time. We’re driven by the curiosity of exploring new things, and accomplishing high-impact work that can eventually help patients is truly rewarding.
Epigenetic Cancer Therapy: Targets and Strategies
By Yuning Wang, PhD
Designed by Jerry Mensah
Epigenetic events shape cancer initiation and progression. By targeting mechanisms involved in epigenetic regulation, epigenetic therapeutics serve as effective weapons against cancer.
Inhibiting histone deacetylation
Histone deacetylases (HDACs) remove acetyl groups from histone lysine residues, leading to closed chromatin structures and gene silencing. HDAC mutations aberrantly deacetylate or inactivate tumor suppressor genes that slow cell division, promoting cancer development (1). HDAC inhibitor drugs upregulate tumor suppressor genes and inhibit cancer cell growth.
Targeting DNA methylation
DNA methylation by DNA methyltransferases (DNMTs) blocks transcriptional factor binding and disrupts gene activity, triggering many cancer types. DNMT blockade drugs effectively prevent DNA hypermethylation and inhibit tumor progression (2)
Modifying histone methylation
Lysine-specific histone demethylase 1 (LSD1), enhancer of zeste homolog 2 (EZH2), and disruptor of telomeric silencing 1-like (DOT1L) selectively methylate or demethylate specific histone lysine residues, regulating histone-DNA interactions and assorted cellular processes. LSD1, EZH2, or DOT1L overexpression causes cancer, making their inhibitors promising potential therapeutics (1).
Changing RNA modification
As critical epigenetic modulators, noncoding RNAs undergo methylation modification mediated by methyltransferase-like protein 3 (METTL3), affecting RNA structures and functions. Changes to RNA methylation machinery influence tumor cell proliferation and differentiation (3). Reversing abnormal RNA methylation by targeting METTL3 represents a novel approach to cancer treatment.
References
1. Cheng, Y. et al. Targeting epigenetic regulators for cancer therapy: mechanisms and advances in clinical trials. Sig Transduct Target Ther 4, 1–39 (2019).
2. Hu, C., Liu, X., Zeng, Y., Liu, J. & Wu, F. DNA methyltransferase inhibitors combination therapy for the treatment of solid tumor: mechanism and clinical application. Clinical Epigenetics 13, 166 (2021).
3. Yang, X. et al. Epigenetic modulations of noncoding RNA: a novel dimension of Cancer biology. Molecular Cancer 19, 64 (2020).
ADVANCING IMMUNE FORCES AT THE TUMOR FRONTLINES
Epigenetic therapies revive the immune cell offensive in immunotherapy resistant tumors.
BY IRIS KULBATSKI, PHD
In the darkest enclaves of the human body, the immune system wages a silent war to prevent cancer. An army of immune cells, including T effector and natural killer cells, act as frontline soldiers in an ongoing battle. But cancer cells are stealthy. Eventually, malignant cells neutralize the immune system’s efforts by suppressing the immune response and developing immune tolerance (1).
Immunotherapy enlists a biomolecular cavalry to reactivate the immune system’s anticancer offensive. Some cancers build resistance to immunotherapy, and others such as ovarian cancer respond poorly or not at all (2). Researchers are zeroing in on the body’s epigenome — the molecular elements that turn genes on and off — as an additional force to revive the previously repressed battalion of immune cells. Targeting epigenetic modifiers using epigenetic inhibitors or hypomethylating agents is a promising option for treating immunotherapy resistant cancers.
In a recent study published in The Journal of Clinical Investigation, researchers led by Daniela Matei, an oncologist and ovarian cancer researcher at Northwestern University, tested guadecitabine, a hypomethylating agent that targets the cancer cell epigenome and activates immune cells to boost their receptivity to immunotherapy in end stage ovarian cancer patients (3). “Most patients with ovarian cancer eventually reach this point where treatments are limited and their survival is less than a year,” said Matei.
Cancer cells carry tumor antigens on their surfaces that the immune system recognizes. Eventually, tumors supress these surface antigens to evade the immune response. “We try to remove that veil and make the tumors recognizable by the immune system,” Matei explained.
In a Phase II clinical trial, Matei explored if treating patients with the epigenetic inhibitor guadecitabine, followed by pembrolizumab, a common immune checkpoint inhibitor drug that blocks proteins that suppress the immune response, would provide a therapeutic benefit to patients with ovarian cancer. She and her team hypothesized that guadecitabine may reverse the DNA methylation that tumors use to bypass the immune system. Priming tumors in this way would make ovarian tumors, which are usually unreceptive to immunotherapy, responsive to chemotherapy treatment.
Matei’s team evaluated cancer progression and epigenetic and immune status in patients by analyzing tumor biopsies, blood plasma, and peripheral blood mononuclear cells before and after the combination therapy. Of the 35 patients eligible for evaluation, eight had stable disease and three responded partially to the dual therapy. “Even though we didn't have a lot of [responders], we had patients with stable disease for more than six months. It's not perfect, but it was a desirable outcome,” said Matei. After treatment, these patients carried changes in their methylomes and transcriptomes that indicated antitumor immune response activation.
Matei’s findings address an unmet need for ovarian cancer patients and may help researchers identify the mechanisms that underly immune cell reactivation and predict which patients will respond to treatment. “If you can identify these individuals, you're going to enrich some patients’ lives,” said Stephen Baylin, an epigeneticist at the Johns Hopkins University School of Medicine who was not involved in this study.
However, timing is important. Combining epigenetic therapy and immunotherapy at an earlier disease stage may improve patient outcomes. “Patients who had advanced disease often had multiple treatments [that] caused abnormal epigenetic changes,” said Baylin. “[Epigenetic drugs] would be much more effective therapies if we didn't wait for such advanced disease or resistance to previous therapies.”
References
Chiappinelli, K.B. & Baylin, S.B. Inhibiting DNA methylation improves antitumor immunity in ovarian cancer. J Clin Inves 132, e160186 (2022).
Awada, A. et al. Immunotherapy in the treatment of platinum-resistant ovarian cancer: current perspectives. Onco Targets Ther 15, 853-866 (2022).
Chen, S. et al. Epigenetic priming enhances anti-tumor immunity in platinum resistant ovarian cancer. J Clin Invest 132, e158800 (2022).