A powerful new set of scientific tools developed by Weill Cornell Medicine and New York Genome Center (NYGC) researchers enables them to track the molecular evolution of cancers.
The tools should enable a better understanding of how cancers arise and spread in the body, and how they respond to different therapies.
Described in papers on May 15 in Nature and April 23 in Nature Communications, the new approach allows researchers to isolate individual cancer cells sampled from patients and map the epigenetic marks on the cells’ chromosomes.
Epigenetic marks are chemical marks on DNA or on DNA-support proteins called histones that help control which genes are switched “on” and which are switched “off” in a cell.
They essentially program what the cell does and what it doesn’t do.
Epigenetic mechanisms in normal cells
Chromatin is made of repeating units of nucleosomes, which consist of ∼146 base pairs of DNA wrapped around an octamer of four core histone proteins (H3, H4, H2A and H2B) (9).
Epigenetic mechanisms that modify chromatin structure can be divided into four main categories: DNA methylation, covalent histone modifications, non-covalent mechanisms such as incorporation of histone variants and nucleosome remodeling and non-coding RNAs including microRNAs (miRNAs).
These modifications work together to regulate the functioning of the genome by altering the local structural dynamics of chromatin, primarily regulating its accessibility and compactness.
The interplay of these modifications creates an ‘epigenetic landscape’ that regulates the way the mammalian genome manifests itself in different cell types, developmental stages and disease states, including cancer (4,10–14).
The distinct patterns of these modifications present in different cellular states serve as a guardian of cellular identity (Table I).
Here, we will discuss the important aspects of the key epigenetic mechanisms present in normal cells.
Epigenetic mechanisms involved in regulating gene expression and chromatin structure in normal mammalian cells
|All cell types|
|Stable heritable modification|
|Imprinting, X-chromosome inactivation, silencing of repetitive elements|
|Mediated by DNMTs|
|Bimodal distribution pattern|
|Global CpG methylation|
|CpG islands unmethylated|
|Pluripotency gene promoters unmethylated|
|Tissue-specific methylation of some CpG islands and most non-CpG island promoters|
|Pluripotency gene promoters methylated|
|Covalent histone modifications|
|All cell types|
|Labile heritable modification|
|Both gene silencing (H3K9me, H3K27me etc.) and gene activation (H3K4me, acetylation etc.)|
|Specific distribution patterns of histone marks contribute to chromatin organization|
|Mediated by HMTs, HDMs, HATs and HDACs etc.|
|Bivalent domains—coexistence of active and repressive marks (H3K4me and H3K27me) at promoters of developmentally important genes|
|Loss of bivalency and restricted epigenome|
|Establishment of tissue-specific monovalent H3K27me and H3K4me domains|
|Presence of large organized chromatin K9 modifications|
|Nucleosome positioning and histone variants|
|All cell types|
|Labile epigenetic regulatory mechanism|
|Both gene silencing and gene activation by modulating chromatin accessibility|
|Mediated by ATP-dependent chromatin-remodeling complexes|
|Both sliding of existing and incorporation of new nucleosomes|
|H2A.Z and H3.3 preferentially localized to gene promoters that are active or poised for activation|
|Acetylated H2A.Z associates with euchromatin and ubiquitylated H2A.Z with facultative heterochromatin|
|All cell types|
|Labile epigenetic regulatory mechanism|
|Can be epigenetically regulated|
Epigenetic mechanisms including DNA methylation, covalent histone modifications, nucleososme positioning and miRNAs are essential for normal mammalian development and regulation of gene expression. These epigenetic modifications display unique properties and distribution patterns in different mammalian cells. The distinct combinatorial patterns of these modifications, collectively termed the epigenome, are key determinants of cell fate and gene activity. ES cells maintain a more plastic epigenome required for developmental processes. In contrast, the epigenome of differentiated tissue displays a relatively restricted structure that is stably maintained through multiple cell divisions.
DNA methylation is perhaps the most extensively studied epigenetic modification in mammals.
It provides a stable gene silencing mechanism that plays an important role in regulating gene expression and chromatin architecture, in association with histone modifications and other chromatin associated proteins.
In mammals, DNA methylation primarily occurs by the covalent modification of cytosine residues in CpG dinucleotides.
CpG dinucleotides are not evenly distributed across the human genome but are instead concentrated in short CpG-rich DNA stretches called ‘CpG islands’ and regions of large repetitive sequences (e.g. centromeric repeats, retrotransposon elements, rDNA etc.) (15,16).
CpG islands are preferentially located at the 5′ end of genes and occupy ∼60% of human gene promoters (17).
While most of the CpG sites in the genome are methylated, the majority of CpG islands usually remain unmethylated during development and in differentiated tissues (11).
However, some CpG island promoters become methylated during development, which results in long-term transcriptional silencing.
X-chromosome inactivation and imprinted genes are classic examples of such naturally occurring CpG island methylation during development (15).
In contrast, the repetitive genomic sequences that are scattered all over the human genome are heavily methylated, which prevents chromosomal instability by silencing non-coding DNA and transposable DNA elements (11).
DNA methylation can lead to gene silencing by either preventing or promoting the recruitment of regulatory proteins to DNA.
Thus, DNA methylation uses a variety of mechanisms to heritably silence genes and non-coding genomic regions.
The precise DNA methylation patterns found in the mammalian genome are generated and heritably maintained by the cooperative activity of the de novo methyltransferases – DNMT3A and DNMT3B, which act independent of replication and show equal preference for both unmethylated and hemimethylated DNA and the maintenance DNA methyltransferase – DNMT1, which acts during replication preferentially methylating hemimethylated DNA (24,25).
While the role of CpG island promoter methylation in gene silencing is well established, much less is known about the role of methylation of non-CpG island promoters.
Recent studies have shown that DNA methylation is also important for the regulation of non-CpG island promoters. For example, tissue-specific expression of MASPIN, which does not contain a CpG island within its promoter, is regulated by DNA methylation (26).
Similarly, methylation of the non-CpG island Oct-4 promoter, strongly influences its expression level (27). Since CpG islands occupy only ∼60% of human gene promoters, it is essential to elucidate the role of non-CpG island methylation in order to fully understand the global role of DNA methylation in normal tissue (17).
Covalent histone modifications
Histone proteins, which comprise the nucleosome core, contain a globular C-terminal domain and an unstructured N-terminal tail (9).
The N-terminal tails of histones can undergo a variety of posttranslational covalent modifications including methylation, acetylation, ubiquitylation, sumoylation and phosphorylation on specific residues (12).
These modifications regulate key cellular processes such as transcription, replication and repair (12).
The complement of modifications is proposed to store the epigenetic memory inside a cell in the form of a ‘histone code’ that determines the structure and activity of different chromatin regions (28).
Histone modifications work by either changing the accessibility of chromatin or by recruiting and/or occluding non-histone effector proteins, which decode the message encoded by the modification patterns.
The mechanism of inheritance of this histone code, however, is still not fully understood.
Unlike DNA methylation, histone modifications can lead to either activation or repression depending upon which residues are modified and the type of modifications present.
For example, lysine acetylation correlates with transcriptional activation (12,29), whereas lysine methylation leads to transcriptional activation or repression depending upon which residue is modified and the degree of methylation.
For example, trimethylation of lysine 4 on histone H3 (H3K4me3) is enriched at transcriptionally active gene promoters (30), whereas trimethylation of H3K9 (H3K9me3) and H3K27 (H3K27me3) is present at gene promoters that are transcriptionally repressed (12).
The latter two modifications together constitute the two main silencing mechanisms in mammalian cells, H3K9me3 working in concert with DNA methylation and H3K27me3 largely working exclusive of DNA methylation (Figure 1).
Genome-wide studies showing distinct localization and combinatorial patterns of these histone marks in the genome have significantly increased our understanding of how these diverse modifications act in a cooperative manner to regulate global gene expression patterns (31,32).
Such bivalent domains are established by the activity of two critical regulators of development in mammals: the polycomb group that catalyzes the repressive H3K27 trimethylation mark and is essential for maintaining ES cell pluripotency through silencing cell fate-specific genes and potentially the trithorax group that catalyzes the activating H3K4 trimethylation mark and is required for maintaining active chromatin states during development (34).
This bivalency is hypothesized to add to phenotypic plasticity, enabling ES cells to tightly regulate gene expression during different developmental processes. Differentiated cells lose this bivalency and acquire a more rigid chromatin structure, which may be important for maintaining cell fate during cellular expansion (33).
This hypothesis is supported by the recent discovery of large condensed chromatin regions containing the repressive H3K9me2 mark, termed ‘LOCKs’ (large organized chromatin K9 modifications), in differentiated ES cells that can maintain silencing of large genomic regions in differentiated tissues (37).
Histone modification patterns are dynamically regulated by enzymes that add and remove covalent modifications to histone proteins.
Histone acetyltransferases (HATs) and histone methyltransferases (HMTs) add acetyl and methyl groups, respectively, whereas HDACs and histone demethylases (HDMs) remove acetyl and methyl groups, respectively (38,39).
A number of histone-modifying enzymes including various HATs, HMTs, HDACs and HDMs have been identified in the past decade (12).
These histone-modifying enzymes interact with each other as well as other DNA regulatory mechanisms to tightly link chromatin state and transcription.
Previous studies have shown that cancers are driven in part by epigenetic changes to cells, but relatively little research has been done in this area; cancer-driving genetic mutations have received much more attention.
In the new studies, the scientists mapped the set of epigenetic marks – the “epigenomes” – in thousands of cancerous cells from patients with chronic lymphocytic leukemia (CLL).
This slowly progressing malignancy affects white blood cells called B cells.
The scientists analyzed this enormous set of epigenomic data to show how the patients’ cancers had evolved at the epigenetic level, and how they responded to standard treatment with the drug ibrutinib.
“With this epigenetic information we were able to trace with high resolution the lineage of these cancerous cells and the evolution of the cancer cell populations, in a way that couldn’t have been done previously on human samples,” said Dr. Dan Landau, an assistant professor of medicine and a member of the Sandra and Edward Meyer Cancer Center at Weill Cornell Medicine, and a core member of the NYGC.
“This kind of analysis can give us insights into how different cancers adapt to challenges such as drug treatments.”
For the Nature study, Dr. Landau and his team used their new technique to map epigenetic marks called methylations on more than 800 normal B cells from six healthy people, and on more than 1,800 cancerous cells from 12 CLL patients.
They found that in the CLL patients’ cell populations, the rate of epimutation – the average change to the epigenome with each cell division – was abnormally and uniformly high, resulting in a great variety of epigenetic patterns among the cells.
Dr. Landau and other researchers have previously shown how a diversification occurs at the genetic level in cancers, such that different cells within a tumor contain distinct sets of gene mutations.
“This enormous diversity within each cancerous cell population means that in each patient, we’re dealing with thousands of variants of the cancer rather than just one entity, and all this variation increases the cancer’s potential to adapt to challenges such as drug therapy,” said Dr. Landau, who is also an oncologist at NewYork-Presbyterian/Weill Cornell Medical Center. “Here we’re extending that concept to show that there is epigenetic diversity as well.”
The team was able to analyze the epigenomic data to trace the lineage of each CLL cell back to its cancerous origin and show how it had evolved in the course of the disease.
The scientists also classified the CLL cells into different groups, or “clades,” of closely related cells based on their epigenomic patterns, and showed that some clades but not others appeared to be sensitive to ibrutinib treatment.
This type of analysis, taken further in future studies, could clarify, for example, how well a drug or drug-combination works against a given cancer, and could lead to better ways of monitoring cancer progression and detecting drug resistance.
For the Nature Communications study, Dr. Landau and colleagues showed that with the same single-cell precision they could map epigenetic marks called histone modifications, in addition to methylations, on the DNA of patients’ CLL cells.
DNA is packaged within chromosomes in part by spooling it around histone proteins; tiny chemical changes to histones can loosen or tighten this spooling at various points to permit or suppress gene activity.
The study, a collaboration with the laboratory of Dr. Omar Abdel-Wahab at Memorial Sloan Kettering Cancer Center, showed that CLL cell populations diversify their sets of histone modifications along with other epigenetic marks.
Both studies confirmed that at the epigenetic level, CLL cell populations lose the highly defined order seen in healthy B cell populations.
“Normal human cells are programmed to function in a precisely defined way within a multicellular organism,” said Dr. Landau, who is also a paid scientific advisory board member for Pharmacyclics, an AbbVie company focused on the treatment of cancers and immune-mediated diseases.
“Cancerous cells devolve into something more like a one-celled life form, like bacteria – in which you also see this diversifying tendency that makes the cell population more resilient.”
Both studies also demonstrated that the approach developed by Dr. Landau and colleagues can be used to map not only different kinds of epigenetic information, but also genetic – DNA sequence – information, and patterns of gene expression, all in single cells.
“Understanding the epigenetic changes that occur in individual cells will provide the best insights into how cells develop resistance to our treatments, and how these changes might be prevented in the future,” said co-author Dr. Richard Furman, the Morton Coleman, M.D. Distinguished Professor of Medicine and director of the CLL Center at Weill Cornell Medicine, and an oncologist at NewYork-Presbyterian/Weill Cornell Medical Center. “This information will be critical to helping CLL patients achieve normal longevity.”
“We hope to use this ability to capture multiple layers of information from individual cells to understand all the different molecular relationships that determine how cancers evolve and how they develop resistance to therapies – and how we can prevent that resistance,” Dr. Landau said.
More information: Federico Gaiti et al. Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia, Nature (2019). DOI: 10.1038/s41586-019-1198-z
Alessandro Pastore et al. Corrupted coordination of epigenetic modifications leads to diverging chromatin states and transcriptional heterogeneity in CLL, Nature Communications (2019). DOI: 10.1038/s41467-019-09645-5
Journal information: Nature , Nature Communications
Provided by Weill Cornell Medical College