Epigenomics And Cancer Heterogeneity

There can be considerable heterogeneity in cancers, and this can contribute to progression and resistance to therapy.  This heterogeneity can arise not only from DNA sequence changes (i.e., mutation) but also from epigenetic changes - changes in the modifications of chromosomes - on a single cell level, making one cell different than another with respect to gene expression, even if the gene sequences remain the same.  A paper of interest, abstract:

Cell-to-cell heterogeneity is a major driver of cancer evolution, progression, and emergence of drug resistance. Epigenomic variation at the single-cell level can rapidly create cancer heterogeneity but is difficult to detect and assess functionally.

RESULTS: We develop a strategy to bridge the gap between measurement and function in single-cell epigenomics. Using single-cell chromatin accessibility and RNA-seq data in K562 leukemic cells, we identify the cell surface marker CD24 as co-varying with chromatin accessibility changes linked to GATA transcription factors in single cells. Fluorescence-activated cell sorting of CD24 high versus low cells prospectively isolated GATA1 and GATA2 high versus low cells. GATA high versus low cells express differential gene regulatory networks, differential sensitivity to the drug imatinib mesylate, and differential self-renewal capacity. Lineage tracing experiments show that GATA/CD24hi cells have the capability to rapidly reconstitute the heterogeneity within the entire starting population, suggesting that GATA expression levels drive a phenotypically relevant source of epigenomic plasticity.
CONCLUSION: Single-cell chromatin accessibility can guide prospective characterization of cancer heterogeneity. Epigenomic subpopulations in cancer impact drug sensitivity and the clonal dynamics of cancer evolution.

Identifying these epigenetic changes can be the first step in developing novel approaches to reverse the changes and/or target those cells containing such changes, for therapeutic benefit.

Human Embryo Epigenetics: Methylation

Epigenetic programming during human embryonic development.  Abstract:

DNA methylation is a crucial layer of epigenetic regulation during mammalian embryonic development 1-3 . Although the DNA methylome of early human embryos has been analyzed 4-6 , some of the key features have not been addressed thus far. Here we performed single-cell DNA methylome sequencing for human preimplantation embryos and found that tens of thousands of genomic loci exhibited de novo DNA methylation. This finding indicates that genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused remethylation. Furthermore, demethylation of the paternal genome is much faster and thorough than that of the maternal genome. From the two-cell to the postimplantation stage, methylation of the paternal genome is consistently lower than that of the maternal genome. We also show that the genetic lineage of early blastomeres can be traced by DNA methylation analysis. Our work paves the way for deciphering the secrets of DNA methylation reprogramming in early human embryo.

Epigenetics And Stomach (Gastric) Cancer

The importance of epigenetic changes is underscored by its relationship to progression to gastric cancer, observed in a 10-year study.  Abstract;

Intestinal metaplasia (IM) is a pre-malignant condition of the gastric mucosa associated with increased gastric cancer (GC) risk. We performed (epi)genomic profiling of 138 IMs from 148 cancer-free patients, recruited through a 10-year prospective study. Compared with GCs, IMs exhibit low mutational burdens, recurrent mutations in certain tumor suppressors (FBXW7) but not others (TP53, ARID1A), chromosome 8q amplification, and shortened telomeres. Sequencing identified more IM patients with active Helicobacter pylori infection compared with histopathology (11%-27%). Several IMs exhibited hypermethylation at DNA methylation valleys; however, IMs generally lack intragenic hypomethylation signatures of advanced malignancy. IM patients with shortened telomeres and chromosomal alterations were associated with subsequent dysplasia or GC; conversely patients exhibiting normal-like epigenomic patterns were associated with regression.

Epigenetic Cancer Therapy

Possible epigenetic-based cancer therapy for tumors with certain mutations; abstract:

The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers. KMT2C (hereafter referred to as MLL3) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in MLL3 within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated MLL3 mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring BAP1 or MLL3 mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in MLL3 and suggests that restoration of a balanced state of Polycomb-COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.

Chromatin Remodeling And Cancer Immunotherapy

CC BY 3.0, https://en.wikipedia.org/w/index.php?curid=35818927

Immunotherapy against cancer is a promising treatment approach, but resistance is a problem. It has been shown that the expression of certain chromatin remodeling factors – that alter chromatin structure to affect gene expression – are associated with immunotherapy resistance in human cancer.  Inactivating these factors increases sensitivity to treatment, perhaps by altering the expression of genes that influence response to treatment.  This is an avenue of research that has clinical implications.  Abstract:

Many human cancers are resistant to immunotherapy for reasons that are poorly understood. We used a genome-scale CRISPR/Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of anti-tumor immunity. Inactivation of >100 genes sensitized mouse B16F10 melanoma cells to killing by T cells, including Pbrm1, Arid2 and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by cytotoxic T cells.

Epigenetic CRISPR

Epigenetics is the modulation of gene expression through modification of chromatin (e.g., by methylation and/or acetylation) without changing DNA sequences.  The CRISPR system can be modified to activate genes through epigenetic remodeling; in mice, this has been shown helpful against diabetes, muscular dystrophy, and acute kidney disease.  Abstract:

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases.

Also, it is possible to make the CRISPR system temperature-controlled.

Myc Oncogene And Immune Detection Of Cancer

Activation of the Myc oncogene in cancer helps produce immune-suppressed conditions that aids in the evasion of tumor immune surveillance.

…we find that co-activation of Myc drives the immediate transition to highly proliferative and invasive adenocarcinomas marked by highly inflammatory, angiogenic, and immune-suppressed stroma. 

It therefore stands to reason that suppression if the Myc signaling can help restore anti-tumor immune function and that is what has been shown to occur, using agents that modify epigenetic changes in genes – changes that involve how the chromatin is modified (methylation, acetylation) instead of DNA sequence changes, with consequent alteration of gene expression. This is hopeful in the quest to improve anti-cancer immunotherapy.  Abstract:

Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/β-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.

The Vices Of Men

By Vincent van Gogh - Scanned from Smoke: a global history of smoking (2004) ISBN 1-86189-200-4, Public Domain, https://commons.wikimedia.org/w/index.php?curid=3857076

The paper linked here is a fascinating review that looks at three major vices of men – drinking alcohol, being overweight/obese, and smoking tobacco – and how these activities can damage male fertility and also negatively affect the health of their offspring. Mechanisms by which these vices can affect fertility and the health of children include epigenetic changes to sperm DNA and non-coding RNA (changes to the modification to DNA and RNA, not including actual sequence mutation, that can affect gene expression), sperm DNA damage (which can cause actual sequence mutation affecting gene expression), changes in sperm chromatin structure (for example, changes in the chromosome structure that can affect which genes are expressed and which are silenced), and changes in seminal plasma (that can affect the function of the sperm cells themselves).  Abstract:

There is growing evidence from animal and human studies that demonstrate that acquired paternal traits can impair both a male's fertility and the health of his offspring, including advanced age, smoking, stress, trauma, under-nutrition, infection, toxin exposure, and obesity. Curiously, many of these factors manifest as impaired neurological, behavioural, and/or metabolic functioning in offspring. The underlying molecular mechanisms that respond to the paternal environment and act as vectors of intergenerational transmission are beginning to emerge. This review focuses on three vices of men (alcohol consumption, overweight/obesity, and tobacco smoking) that damage fertility and pose risks to offspring health. These vices are not only the three most prevalent but are also leading risk factors for death and disability adjusted life years (DALYs) worldwide. Clearly, any epigenetic/genetic alterations induced by the paternal exposures responsible for transmission need to escape/bypass the substantial post-fertilisation reprogramming that occurs during embryo development. For example paternal obesity alters the molecular composition of sperm, alters the developmental trajectory of resultant embryos, and increase the incidence of obesity and metabolic disorders in offspring. Mechanistic candidates of paternal programming include changes to the sperm epigenome (eg DNA methylation, histone/protamine modifications, and sperm borne small non-coding RNAs), increased sperm DNA damage, aberrant sperm DNA chromatin structure, and components of seminal plasma. Understanding the molecular mechanisms underpinning paternal programming may lead to the development of interventions designed to reduce the disease burden in future generations, who were born to fathers exposed to these initiating factors. Given that these vices are predominantly self-inflicted, interventions aimed at mitigating their consequences are readily identified.

Add this information to all the other reasons to avoid these vices.

Mouse Dietary Epigenetics

Last week I discussed epigenetics inheritance of traits through mechanisms other than differences in DNA sequences); this week we look at a study showing that the diet of male mice before mating can affect the physiology of their offspring - through an epiegenetic type mechanism that involves tRNAs instead of modification of DNA.
 

Two teams of researchers conducting independent experiments have found evidence that indicates that what a male mouse eats prior to mating with a female mouse can have an impact on the offspring that result. The first group, from several institutions in China, ran experiments testing the impact of male mice eating a high fat diet, on offspring, while the second team, with members from the U.S. and Canada, tested the impact of a low-protein diet by male mice prior to siring offspring. Both teams describe their experiments and results in papers published in the journal Science. For many years it has been assumed that the only impact male mammals can have on their offspring, due purely to mating, is from the DNA they carry in their sperm. In recent years, however, some study results have suggested that they can have another impact due to what are known as transfer RNAs (tRNAs). These two new studies add more evidence, suggesting that tRNA fragments can carry information that adversely impacts offspring. In the first study, the team fed one group of mice a high fat diet, while another group was fed a normal diet. Sperm was harvested from both groups and used to impregnate female mice. Offspring had their weight monitored along with their level of glucose intolerance and insulin resistance. The team reports that the offspring of the males fed the high fat diets did not gain more weight than those from the control group, but they did develop an impaired resistance to insulin and glucose intolerance—precursors to diabetes. To ensure that the change was due to tRNA fragments, the team ran the same experiment again, but the second time around they purified the RNA before injection into the eggs. The resulting offspring developed intolerance to glucose but did not develop insulin resistance. In the second study, the researchers conducted the same type of experiment but had the male study group eat a low-protein diet. The team reports they found no differences between the offspring except for changes to a group of genes that are responsible for the development of stem cells. Though not studied yet, it appears likely that the same results would occur with humans, which suggests that couples looking to have children ought to be aware of or modify the diets of both potential parents.

So, it is not only true that "you are what you eat" - it is also true that your children are what you eat. Be advised and choose carefully.

Possible Epigenetic Effects Of Diet On Offspring

Epigenetic changes are alterations affecting gene expression that do not involve actual changes in gene sequence. For example, methylation of DNA can in many cases suppress gene expression. It is possible for these changes to be passed on from parent to child; one possible historical example was with the Dutch famine of 1944, and subsequent health problems of offspring.

Recently, the possibility has been raised that obesity can cause epigenetic changes that can be passed down to children - a sobering thought that adds to the incentive to lose weight and, optimally, never become obese in the first place. The following paragraphs are from the linked article, in which Dr. Barres explains his findings:

He further explains that in moderately obese men, they detected “that methylation (a metabolic process used by cells to control gene expression) of genes was dramatically different compared to lean men, particularly at genes important for brain development and the regulation of appetite. And although we don’t know if the epigenetic marks of obese man are transferred to the child—we have not studied the children—they carry potential to change appetite in the offspring.” 

While it appears that a man’s weight at the time of conception determines the type of DNA he will be passing on to his children, we wondered if a man who was obese years prior to conception could still pass those genes along. “I wish I could answer this question,” states Barrés. “For the reason I explained above, we don’t know if epigenetic marks will be transferred to the child. And also, we don’t know how long Dad’s marks are stable.” 

Barrés also admits he didn’t expect to observe such significant alterations in epigenetic information. “Discovering that lifestyle and environmental factors, such as a person’s nutritional state, can shape the information in our gametes and thereby modify the eating behavior of the next generation is, to my mind, an important find,” he stated in a press release.