June 1, 2009 (Vol. 29, No. 11)

Technique Focuses on Topology of Genome to Lead Way to Better Treatment

Speaking at the American Association of Cancer Research conference held in Denver recently, Stephen B. Baylin, M.D., professor of oncology and medicine at Johns Hopkins University, declared, “We’re at an exciting era. It’s still all about the genome.” But,  rather than focusing on the sequencing, the “era of epigenetics,” as he called it, “focuses on the topology of the genome—how the nucleosomes are arranged in a three-dimensional fashion—to better understand the origins of cancer.”

As Bradley E. Bernstein, director of pathology, Massachusetts General Hospital, later explained, “The term epigenome describes the packaging of the genome. Epigenetic refers to a process that maintains the cell state stably, ideally through mitosis. A classic example is gene repression.” At the biochemical level, they involve contributions from many interacting components, including histome modifications, cancer protein, ncRNAs, and transcriptions factors. “Histome modifications themselves are not sufficient.”

Researchers are finding that “abnormal gene silencing associated with gene promoter DNA hypermethylation is linked to key aspects of chromatin regulation of gene expression, which maintains the state of embryonic stem progenitor cells,” Dr. Baylin said.

“In most of the normal genome, CpG is packaged away, protected from DNA methylation. In the cancer epigenome, however, “many regions ought to be closed but are open and many that should be open have become methylated and, therefore, are closed,” he added. In his work, Dr. Baylin and his lab used a colon cancer cell line to determine genes that appear most frequently. Frequency and DNA hypermethylation were then overlapped.

Individual patients have about 40 mutations, but 300 to 400 instances of DNA hypermethylation, Dr. Baylin reported. “If you only look at the genes that occur with high frequency, you have missed important genes,” he emphasized.

Dr. Baylin’s lab uses Infinium luminescent technology by Illumina for genotyping, querying only a few sets of genes on the genome. “It is a good screen for methylation across the genome,” he pointed out. He added that embryonic stem cells lack methylation. Leukemia and cancer cells, however, have abnormal DNA methylation. “That’s a stark reality in cancer and is a microcosm of the cancer genome.”

DNA hypermethylation seems to play a causative role in the onset of cancer. In fact, he said, “DNA methylation fosters tumorigenesis.” Of 610 candidate genes studied in a colon cancer cell line, 300 were PcG marked in embryonic stem cells or mesenchymal cells. Dr. Baylin plans to study that more broadly using ChIP deep sequencing.

In analyzing tiling arrays for key chromatin marks that are specific to developing cell types, he found that the polycomb (repressive system) mark is really a balance of marks rather than one that is on or off. “It is bivalent chromatic that hold stem cell genes in a poised state.” His working hypothesis is that bivalent chromatin can be remodeled in many cells as they differentiate. “When the system is challenged to renew, the genes are marked by polycomb.”

The epigenetic gatekeepers—like GATA transcription factors and APC—help stabilize stem cells or block cellular renewal. “I propose there are epigenetic steps as well,” Dr. Baylin suggested. “For example, abnormal epigenetic memory could drive cells forward to proliferation.”

Researchers at Johns Hopkins University are using Illumina’s BeadArray technology and the Infinium Assay to conduct cancer DNA methylation studies.

Stem Cell

In terms of regulation in metazoans, which is critical in regulating behavior in cells, Dr. Bernstein stated, “One thinks of polycombs and trithorax proteins,” which help maintain lineage-specific gene-expression proteins.

In mammalian and mouse stem cells, he explained, “there are many bivalent domains, characterized by the juxtaposition of the repressive polycomb mark and the actived trithorax mark found throughout the genome. These bivalent domains are associated with many developmental regulator genes that are inactive in embryonic stem cells but are rapidly induced along specific differentiation pathways. That helps poise genes for future activation.

Dr. Bernstein’s lab looked at other models, including hematopoietin development, focusing on Pax5 master regulator, a B-cell lineage-specific protein that regulates early-stage B-cell differentiation. Looking at in vivo CD34 cells sorted from cord blood, Pax5 is sitting in a bivalent chromatin state, “whereas, in the CD19 B cells, the polycomb is stripped out,” leaving an active promoter and evidence of p36 elongation. Conversely MyoD, in the hematopoietic lineage, is robustly silenced by polycomb repression.

Additional studies of protein complexes in embryonic stem cells found that PRC2, which catalyzes H3K27 mer3, and PRC1, which blocks RNA polymerase elongation, closely interact in epigenetic repression.  All the bivalent domains are contained PRC2, but fewer than half also are contained PRC1. PRC1 and -2 behave differently, with PRC2 triggering a smaller degree of repression than PRC1.

In another in vitro study, all the cancer proteins were lost. “That’s associated with hypermethylation and is not recapitulated in vivo. In vitro is where most of the hypermethylation occurs,” which caused Dr. Bernstein to question whether hypermethylation is, in fact, an artifact. “It may be, but it is very relevant in human cancer.”


Paula Vertino, Ph.D., associate professor of radiation oncology at Emory University School of Medicine and the Winship Cancer Institute, discussed gene silencing at the meeting. She looked at some of the local features that contribute to which genes or CpG islands are subject to epigenetic silencing and new work that involves a novel epigenetic switch that may predispose certain genes to methylation.

“CpG islands tend to remain unmethylated in normal cells,” Dr. Vertino stated, “so the lack of CpG methylation is thought to be a permissive state for active transcription.”

CpG islands that are actively transcribed are associated with strongly positioned nucleosomes. That means, in each cell, the nucleosomes are in the exact same position, in contrast to other parts of the genome where they tend to be more randomly distributed. CpG islands tend to be marked by active histome marks.

“In cancer cells, there tends to be a global loss of DNA methylation from much of the genome,” added Dr. Vertino. “This is accompanied by loss of the more heterochromatic marks, which may contribute to genome instability through chromosomal aberrations and potential reactivation of transposable elements or certain microRNA.

“At the same time, there is a focal gain of methylation at certain CpG islands,” she explained. “This corresponds with the random positioning of nucleosomes around CpG islands, as well as the loss of active histome marks and the gain of repressive marks.”

In examining some 1,700 CpG islands of the 28,000 that exist, she found that between 3% and 8% of CpG islands may undergo methylation. “The vast majority remain completely unmethylated, even when under great duress.”

In distinguishing between CpG islands that are resistant to DNA methylation from those that are prone to DNA methylation, her lab identified certain local features that may put these islands at risk for permanent silencing associated with DNA methylation.

She induced CpG island methylation in human cells by overexpressing one of the DNA methyltransferases, DNMT1. Using a computational approach developed with collaborator Eva Lee associate professor at Georgia Institute of Technology, she applied a machine-learning approach to discriminate between methylation-prone and -resistant CpG islands, looking at DNA sequence patterns in the areas surrounding the regions that are prone to, and resistant to, DNA methylation. The classifier, she explained, is 85% accurate at predicting the epigenetic status of a given sequence.

When applied across the genome to predict hypermethylation, the genes that were predicted to be prone to aberrant methylation were, in general, much more susceptible to aberrant methylation in lung and breast cancer cell lines than those that were predicted to be methylation resistant.

When they developed this classifier, dubbed PatMan, Dr. Vertino and her colleagues noticed overrepresentation of Hox-related genes within the methylation prone class. “Hox genes tend to be targets for the polycomb repressor complex,” she said, which led to further studies. 

Genes that were predicted to be methylation prone also showed an enrichment of H3K27 mer3, the mark imposed by polycomb complex at the boundaries of the CpG island in normal cells. “Genes that were predicted to be methylation-resistant lacked that enrichment.”

A subset of CpG islands predicted to be methylation-prone or methylation-resistant by the PatMan algorithm
(Emory University School of Medicine)

Clinical Implications

Additionally, “DNA methylation has translational implications, possibly also for solid tumors,” Dr Baylin pointed out, notably in the development of biomarkers to predict the likely efficacy of a given therapy. He reported that certain therapies actually increase cell proliferation in glioblastomas. “The thing that predicts sensitivity may breed resistance to therapy, but more work is needed,” he cautioned.

Another possible use for DNA-methylation screening is to molecularly stage tumors. For example, a sample of stage I lung cancer tumor behaves like normal stage I tissue should, if none of the genes were methylated. But, if methylated, the tumor behaves like stage III tissue and there is a dramatic drop in survivability, he explains.

Dr. Baylin explained that the possibility of epigenetic therapy is promising. The renewal of gene silencing leads to the re-expression of key genes and, thus, to key pathway corrections, he added. “Many research groups are working on it.” Combination epigenetic therapy also has possibilities. 

 “In prostate cancer, about 3% acquire a polycomb mark that silences genes that are active in normal cells,” according to Peter Jones, Ph.D., USC/Norris Comprehensive Cancer Center. “The question is,” he asked, “why does this reprogramming take place?”

“The DNA methylation mark is highly stable,” Dr. Jones pointed out. So once it is on, it remains on. “Importantly, chromatine structures established in embryonic stem cells alter the predisposition of genes to become permanently silenced in cancer.”

He and his lab have investigated several therapeutic approaches. “T24 cells treated with decitabine (Aza-CdR) caused rp16 gene induction, but it takes a while for the drug to work. The dose is critical.” His lab found that “the maximum biological response is a low dose that elicits amazing changes in gene expression and allows the silenced genes to switch back to their active state.” Phase III studies have been completed of demethylating agents in humans with myelodysplastic syndrome.

Another drug, S110, which is in development with SuperGen, seems equivalent to decitabine and is far more stable. It also seems to enter the cell nucleus effectively by entering the cell’s cytoplasm and only then breaking down to enter the nucleus. And, he added, “Animal models show less toxicity with this drug.”

“The future of cancer therapy won’t be a monotherapy,” Dr. Jones predicted. Instead, “combination therapy will be the wave of the future. We may use chemotherapy to debulk the tumor and use epigenetics to switch genes back on again.”

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