Janus Kinase 2
One of the proteins implicated in many cellular processes, including cell-cycle progression, recombination, and apoptosis, is Janus kinase 2 (JAK2), an enzyme involved in physiological and leukemic hematopoiesis. Until recently, JAK2 was thought to reside in the cytoplasm and to perform its function via cytoplasmic signal transduction networks, such as the JAK-STAT pathway. However, recent findings challenged this model when they revealed the existence of a previously unrecognized pool of JAK2.
“We found that this signaling pathway extends all the way down to the chromatin, and the enzyme is also in the nucleus,” explains Tony Kouzarides, Ph.D., Royal Society professor at the University of Cambridge and deputy director at the Gurdon Institute.
Dr. Kouzarides and collaborators revealed, by in vitro and in vivo experiments, that JAK2 phosphorylates tyrosine 41 of histone H3. This specific tyrosine is found in a region on the histone tail that is important in shaping the dynamic properties of nucleosomes and influences nucleosome remodeling.
In addition, Dr. Kouzarides and colleagues showed that the phosphorylated histone prevents the binding of a heterochromatin protein, HP1α, to DNA. HP1α normally represses transcription, and when it is excluded from the DNA as a result of this histone modification, it leads to transcriptional activation, providing a fascinating mechanistic insight into the cellular and molecular consequences of this epigenetic change.
“We have shown that some of the genes that this modification regulates are cancer genes. This is a new paradigm,” explained Dr. Kouzarides. This finding has important clinical applicability, because in some leukemia patients the JAK2-STAT5 pathway becomes constitutively activated as a result of a mutation that increases JAK2 activity and causes the unregulated displacement of HP1α from the chromatin.
At the recent Oxford meeting, Kevin Struhl, Ph.D., professor in the department of biological chemistry and molecular pharmacology at Harvard Medical School, talked about an experimental model of oncogenesis that his group developed to dissect the impact of environmental signals on initiating and maintaining epigenetic states.
In this system, the ligand-binding domain of the estrogen receptor and the Src kinase oncoprotein were used to create a fusion protein that is inducible by tamoxifen and activates NF-κB. By using this construct, Dr. Struhl and collaborators revealed that, subsequent to an Src induction as short as five minutes, the nontransformed breast epithelial cell line was becoming transformed 24–36 hours later.
Most importantly, after becoming transformed, the cells could be propagated for many generations and stayed transformed even though Src was no longer induced. This finding, analogous to the one described during development, revealed that a transient inflammatory reaction was able to activate a positive feedback loop and maintain an epigenetic state for several generations in the absence of the originating event, providing a mechanistic link between inflammation and malignant transformation.
“The inducer starts a positive feedback loop, and once the loop is activated the inducer is not needed any longer because there is a self-propagated mechanism,” explains Dr. Struhl. This phenomenon, known as an “epigenetic switch”, becomes an intriguing idea in the context of malignant transformation.
Mutations and DNA methylation are two examples of stably propagated modifications that, even though they are not sufficient as single events, they contribute to tumorigenesis when acting in combination.
“This is different; it is a switch, analogous to switching cell fates, and we think that it can be a step in tumorigenesis—not by itself, but in combination with other events,” says Dr. Struhl. More recently, research in Dr. Struhl’s lab identified several miRNAs that, upon transient upregulation, were able to induce this epigenetic switch and generate a stably transformed state. Also, this appeared to regulate tumor suppressor genes and activate the positive feedback loop induced during Src-initiated transformation.
In molecular oncology, one of the recent landmarks is the identification of epigenetic targets, which have become promising leads for therapeutic drug development. Epigenetic drug targets have been classified into “writers”, which add chemical groups, “readers”, which recognize the epigenetic modifications, and “erasers”, which remove the marks.
“There has been intense research interest in pharmaceutical companies and academia to develop prototype drugs that target the enzymatic components of the epigenetic machinery,” says James E. Bradner, M.D., assistant professor of medicine at Dana Farber Cancer Institute and Harvard Medical School.
Dr. Bradner and colleagues are focusing on epigenetic readers, which transmit signals from chromatin to the gene-expression machinery by recognizing and binding the amino acid site chains on histone proteins. “In our view, epigenetic reader proteins emerged as a potentially fantastic target for discovery chemistry.”
Recent advances in Dr. Bradner’s lab led to the development of a biochemical method to study epigenetic reader proteins, and catalyzed the development of the first potent and selective inhibitor of acetyl-lysine recognition motifs, known as bromodomains.
In collaboration with Steffan Knapp’s group from the University of Oxford, Dr. Bradner’s lab reported that the targeted use of a small molecule, JQ1, which is a competitive bromodomain inhibitor, in a rare and invariably fatal form of squamous carcinoma, displaced an oncoprotein involved in a chromosomal translocation and exhibited antiproliferative effects.
“This work firmly establishes bromodomain inhibition as a therapeutic rationale for targeted therapy and is approximately a year away from reaching patients in the clinic.” More recent work in Dr. Bradner’s lab explores JQ1 derivatives with improved therapeutic properties. “We anticipate a ligand emerging from this research for clinical development within hopefully the next one to two years.”