Discovery On Target – Histone Methyltransferases & Demethylases 2012



While weather in Boston left much to be desired, the Discovery On-Target: Histone Methyltransferases and Demethylases conference that took place on October 2-3 did not disappoint. Brigham and Women’s Hospital’s Miriam Enos was able to attend and cover the meeting for the EpiGenie readers, so take a few minutes to find out what happened in the following meeting report.

Discovery On-Target: Histone Methyltransferases and Demethylases Summary

Talks by the investigators from both academia and industry presented novel methods and mechanisms for screening, identifying and validating potential medicines of tomorrow.  Each branch was impressive in its own right: industry with its blitzkrieg approach to finding a perfect drug, and academics, with their ability to see the big picture and connect the chemistry behind drug synthesis to the deep understanding of the biology behind every disease.

In 2003, a year that marked the 50th anniversary of the publication of DNA’s double stranded structure in “Nature”, James Watson stated that epigenetic studies will become the center of attention in the new era of life sciences. This conference, amongst many others that take place every year around the world, has been a living manifestation of Watson’s insightful prediction.

High-throughput Screening for Demethylase Inhibitors

Ji-Hu Zhang, Novartis

Dr. Zhang talked about the challenges associated with developing inhibitors for methylases and demethylases. First there are difficulties associated with establishing good cell-based methylation and demethylation assays. Among some of the obstacles he mentioned were: our lack of complete understanding of methylases/ demethylases interactome, no good pathway that can be used as a read-out, high endogenous histone background and lack of tool components for aiding assay development. Additionally there are difficulties with  biochemical assays: enzymes (HMT and HDMs) are slow and there is no perfect detection system. Coupled assay and Western blotting or LC/MS, radioactive or microfluidic approaches at detection are the only ones available at this time and all have serious short-comings. The goal of the project was to develop high-throughput assay for screening of H3K4 demethylase inhibitors.

Time Resolved Flourescence Resonance Energy Transfer (TR-FRET) was selected as the technique of choice. A perfect antibody was developed that recognized specifically H3K4 in its unmethylated state only, without any affinity for other lysines on H3. These properties made it an ideal candidate for a readout for demethylase activity.

In order to estimate its efficacy TR-FRET was compared to LC/MS and to Coupled Assays side-by-side. It was shown to be as sensitive as LC/MS and much more sensitive than Coupled assays. Additionally, unlike LC/MS it was very robust, making it a front-runner in the competition for the perfect cell based detection assay.

Dr. Zhang also emphasized that when a known inhibitor profile was tested with TR-FRET assay – it proved to be remarkably sensitive and accurate and correlated well with what is already known about the inhibitor.

JARID1 Demethylases in Cancer

Qin Yan, Memorial Sloan-Kettering Cancer Center

Dr. Yan told a story that began with the description of how RPB2, a JARID1 H3K4 histone demethylase, is involved in a number of human malignancies, specifically in breast cancer. RBP2 – a protein that interacts with a well-known tumor suppressor RB and is known to block differentiation and senescence. RBP2 loss rescues aberrant proliferation of RB null cells in vitro and extends life span of RB null mice in vivo. Importantly, Dr. Yan highlighted, RBP2 is overexpressed in breast cancer and its levels correlate highly with the incidence of distal metastasis.

Thus, having identified PRB2 as an important target, Dr. Yan then moved on to the practical part of the problem: drug discovery. His group had carried out Alpha Screens to identify the potential inhibitors for RBP2. The initial screen yielded a number of molecules and was followed by a series of validation assays that narrowed the number of effective molecules down to two. Dr. Yan’s lab went on to successfully confirm the efficacy of the final compound in the in vivo setting.

Demethylation and Heterochromatin Balance in Development and Disease

Johnathan R. Whetstine, Harvard Medical School

Dr. Whetstine, the man behind the discovery of the first demethylase, a finding that forever changed the way we think about epigenetic modifications, started his presentation by talking about JMJD2 H3K9 and K36 demethylase and its role in maintenance of cellular homeostasis. When disregulated JMJD2 is capable of changing cell cycle pace and influencing disease progression. Increase in JMJD2 expression speeds up S phase progression and makes transformed cells more chemo-resistant. Conversely loss of JMJD2 slows down S phase and increases sensitivity to chemotherapy.

Dr. Whetstine reminded us that state of chromatin is a balance between different groups of opposing forces: acetylases and deacetylases, methylases and demethylases and so on.

Thus, he mentioned, opposing the function of JMJD2 are G9a/GLP and SUV39H1, which catalyze H3K9 mono-, di- and tri- methylation.  Their overexpression is also associated with transformation and disease, specifically colorectal cancer.

Using a library of small molecule inhibitors a compound UNC0638 was identified as a potent and highly specific in vitro inhibitor of G9a/GLP. However, due to poor pharmacokinetics it was not useful in the in vivo setting. Based on a known structure of G9a methyltransferase and the mode of its activity another compound UNC0642 was synthesized to accommodate the in vivo requirements.

In his talk that was akin to a good lecture Dr. Whetstine has showed us how and why a cell balances its act and how we can exploit its vulnerabilities when things go astray.

Histone Methyltransferases Targets for Cancer Therapy

Roy Pollock, Epizyme

Dr. Pollock’s talk was focused on two cases: EZH2 and DOT1.Both so far are success stories. They are also the “poster children” of how one goes from understanding the biology behind the disease, finding relevant targets, identifying an unmet medical need and coming up with an effective drug that quickly moves from the bench to the mouse room and, if all goes well, to the bed-side.

The first story is that of an infamous polycomb catalytic subunit EZH2. Over the past decade it was shown to be disregulated in a variety of human cancers. High levels of EZH2 expression and tri-methylated H3K27 correlate tightly with the late stage, aggressive disease and their abundance in a primary tumor is usually a bad omen. In his talk Dr. Pollock focused on a role of EZH2 in non-Hodgkin’s lymphomas.

EZH2 is a H3K27 methyltransferase that has a highest affinity for unmethylated and mono-methylated H3K27, and much lower affinity for di-methylated H3K27. Recently it was shown that a subset of non-Hodgkin’s lymphomas carries mutations in the Y641 residue, endowing a protein with a new function: increased affinity for di-methylated H3K27. This mutation is always found in a heterozygous state with WT allele, making it a“killer” combination. Wild type EZH2 takes care of un- and to some degree mono-methylated H3K27 and Y641 mutant completes the job by methylating  di-methylated species. This results in the increased levels of tri-methylated H3K27, which is a mark of an aggressive disease. Knocking-down of EZH2 by RNAi results in a slowed proliferation rate, showing that it is not merely a passenger mutation. Until a week ago there were no effective inhibitors available for EZH2.  This month Epizyme published a study  in Nature Chemical Biology where such an inhibitor was revealed.

Dr. Pollock presented this new compound, called EPZ005687, at the conference and described its high selectivity for EZH2, with Ki of 20nM and over 1000 fold more affinity for EZH2 than any other protein with the exception of EZH1.

DOT1 became a second major hit for Epizyme. DOT1 was implicated in AML and ALL, however its role in these diseases is much more complex than simple overexpression. In a very elegant study conducted by the Epizyme investigators it was shown that DOT1 is mis-localized and MLL fusion to DOT1 shows that it is found at the aberrant locations on the chromatin, affecting local H3K79 methylation status. Thus the global levels of H3K79 methylation were not affected, but the local and specific regions were aberrantly altered, resulting in the disregulated transcription of what is likely to  be the key players in AML and ALL.  The group went on to show that DOT1 inhibitor blocks expression of many MLL fusion target genes, but not any of the genes expressed in a healthy cell, thus confirming DOT1 candidacy as a perfect therapeutic target.

**EpiGenie would like to pass along a major thank you to Miriam Enos, who is a  Post-doctoral Fellow at the Brigham and Women’s Hospital in Boston, for providing this conference coverage.