Epigenetic Marks and Cancer Drugs 2013

Keystone Symposia

Highlights

Over 350 scientists from 22 countries gathered in the USA oldest capital city, Santa Fe in New Mexico on March 2013, 20th-25th, for the Keystone Symposia meeting “Epigenetic Marks and Cancer Drugs”. The small but always vibrant city of Santa Fe, a place of great historic and cultural significance, with enormous density of museums and galleries, offered a stimulating atmosphere for vivid interactions among participants, whereas the well-structured meeting program offered the opportunity for short walks around and visits to the Sante Fe ski resort or Bandelier National Monument, without missing any of the excellent sessions.

Overall, in 4 full days, 52 outstanding talks were given in 8 plenary sessions and 2 workshops, along with 3 poster sessions with approximately 200 posters. As stated by the meeting organizer, Ali Shilatifard (Stowers Institute for Medical Research), the ultimate goal of this keystone symposium was to brindge the gap between academics and scientists from pharmaceutical industry towards a more systematic and efficient exchange of scientific knowledge and a reinforcement of strategic collaborations in the field of epigenetics and drug development.

Scientific sessions covered a broad spectrum of recent intriguing findings at epigenetics field ranging from basic molecular biology studies addressing fundamental mechanistic questions in eukaryotic cells or model organisms to highly disease-orientated and cancer-therapy epigenetics. The variety of studies presented concerned also the scale of research focus: from global epigenetic patterns to single genes or proteins that had gained attention due to their “master regulator” properties and the consequent therapeutic opportunities.

More specifically, two sessions were devoted to chromosomes, chromatin and transcription, one in the Polycomb and Trithorax, one session focused on the role of Myc in transcriptional regulation, whereas three sessions addressed the role of (i) histone marks, (ii) DNA methylation, (iii) ncRNAs in development and cancer, respectively. Finally, the last session, chaired by Robert Eisenmann (Fred Hutchinson Cancer Research Center), included also presentations by scientists from major pharmaceutical companies (Epizyme Inc., Constellation Pharmaceuticals, Genentech Inc.) and the status quo of epigenetic-based drug discovery.

 

Linking Genetic Features of Human Cancers and Histone-Modifying Enzymes for Future Cancer Therapies

Stuart L. Schreiber, Harvard University, USA

Stuart L. Schreiber gave the keynote address on the first day of the Keystone Symposia meeting with a focus on the genetic traits of human cancers and histone-modifying enzymes (HME)-based cancer therapeupies. Stuart L. Schreiber is well known for having developed systematic ways to explore and modulate biology of cancer cell by small molecules. Importantly, his lab has contributed to the diversity-oriented synthesis (DOS) approach for small-molecule screening towards drug discovery. Among the FDA-approved anti-cancer drugs discovered by the Schreiber group are the epigenetic drugs for cutaneous T-cell lymphoma vorinostat and romidepsin, which inhibit HDACs.

In his talk Dr Schreiber pointed out the role of cancer genetic dependencies involving chromatin, as well as the creation of new cancer dependencies by altering cell states through chromatin modifications, and finally he summarized the current concept on discoveries of novel small molecules that alter chromatin states.  A key message from his work was that inhibiting chromatin-modifying enzymes with small molecules does not always cause dramatic changes on a transcriptome-wide scale. On the contrary, a certain molecule can modulate very specific pathway.

Certain cancer-relevant paradigms of gene regulation by chromatin-modifying histone deacetylases, small-molecule dimerizers that activate cellular processes by enforced proximity, and small-molecule probes of “hard targets” were also mentioned. Among others, one interesting paradigm concerned beta-catenin pathway and potential therapeutic targets for endometrial, vaticellular, colorectal cancers that are frequently mutated in beta-catenin.  Schreiber lab identified a molecule that targets the multi-protein complex involved in ROS dissipation.  Drug-sensitive cell lines have mutations in beta-catenin.

The drugs that Schreiber lab works with are one telomerase reverse transcriptase inhibitor and two molecules that target the BCL-2 family of proteins. Importantly: (i) canonical WNT signaling stabilizes beta-catenin and turns on a WNT gene-dependent expression program; (ii) beta-catenin is also activated by ROS and it partners in nucleus with FOXO and GSTpi-mediated phenomena. Based on these facts, the hypothesis is that beta-catenin in mutant tumors may be dependent on the FOXO/GSTpi program in a synthetic lethal interaction between GSTpi and beta-catenin. However, the picture is more complicated since: (i) TERT is required for beta-catenin to mediate its oncogenic program; (ii) beta-catenin is known to be a survival gene and it does so by down-regulating BAX and up-regulating BCL2 family members.

Overall, Dr Schreiber concluded that there are at least two distinct approaches on the way to explore HME and develop HME-based drugs. Both targeting non-oncogenes, creation of new cancer dependancies and interrupt oncogene-dependancies can be exploited for modulating the epigenome in a therapeutic setting.

 

New Insights into the Mechanisms of Transcription and its Regulation

Patrick Cramer, Gene Center, Ludwig-Maximilians University of Munich, Germany

In the session Chromosomes, Chromatin and Transcription Prof. Patrick Cramer from University of Munich (LMU, Germany) presented his fascinating work on transcription regulation. Cramer lab addresses fundamental concepts of the mechanisms of transcription process by combining structural biology, functional analysis in vitro, and system-wide analysis in vivo. In his talk, Prof. Cramer presented an overview of  key aspects of RNA polymerase (Pol) II initiation and elongation. In a movie published by Cramer group at Cell (Cheung and Cramer, Cell 149, 22 June 2012) seven functional states for RNA polymerase II are described: (i) initiation-competent complete Pol II-TFIIF complex; (ii), minimal closed promoter complex; (iii), minimal open promoter complex; (iv), initially transcribing complex; (v), Pol II-Spt4/5 elongation complex; (vi), arrested complex; (vii) Pol II-TFIIS reactivation intermediate.

From a mechanistic point of view, In the different phases of the transcription cycle, RNA pol II recruits various factors via its C-terminal domain (CTD). The CTD was found by Cramer lab to consist of conserved heptapeptide repeats (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7). In yeast, CTD is phosphorylated at Tyr1, in addition to Ser2, Thr4, Ser5, and Ser7. Tyr1 phosphorylation was shown to stimulate binding of elongation factor Spt6 and impairs recruitment of termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation levels rise downstream of the transcription start site and decrease before the polyadenylation site, largely excluding termination factors from gene bodies. Overall, the work of Cramer lab has shown that CTD modifications trigger and block factor recruitment and lead to an extended CTD code that explains transcription cycle coordination.

The CTD code is based on differential phosphorylation of Tyr1, Ser2, and Ser5. Cramer lab also elucidated the crystal structures of the Pol II–TFIIB complex from the yeast S. cerevisiae at 3.4 Å resolution and of an initially transcribing complex that additionally contains the DNA template and a 6-nucleotide RNA product. The main message of this work was summarized as follows: TFIIB and its counterparts may account for primer-independent chain initiation and product separation from the template.

In the second part of his talk, Patrick Cramer presented techniques, which were developed, in his lab for monitoring the dynamics of mRNA metabolism system-wide. One technique is called “dynamic transcriptome analysis” or DTA, and is based on non-perturbing metabolic RNA labeling coupled to microarray analysis. This method also provides the mRNA half-lives and thus enables studies of post-transcriptional regulatory events such as mRNA decay.

Another approach is to map gene-regulatory proteins over the genome in living cells by chromatin immunoprecipitation coupled to high-resolution tiling microarrays (ChIP-chip). This technique provides occupancy profiles for RNA polymerases and their associated factors over the entire yeast genome.

 

Chromatin Modifications, Transcriptional Elongation Control, and Childhood Leukemia

Ali Shilatifard, Stowers Institute for Medical Research, USA

The third session, entitled “Polycomb and Trithorax in Gene Expression and Cancer” started with the talk by Dr. Ali Shilatifard with a focus on the mixed lineage leukemia (MLL) protein family and leukemogenesis. MLL positively regulates multiple HOX genes, and several chromosomal translocations resulting in chimeric proteins and to leukemic phenotype. ELL is the first and best characterized MLL partner (RNA polymerase II enlogation factor). In addition to ELL, Shilatifard lab identified the S. cerevisiae Set1 as a MLL homologue and purified Set1/COMPASS as the first histone H3 lysine 4 (H3K4) methylase.

Interestingly, Set1/COMPASS and its enzymatic product, H3K4 methylation, are highly conserved across species. Although there is only one COMPASS in yeast, Shilatifard lab has shown that Drosophila possesses three COMPASS family members, and humans bear six COMPASS family members, each capable of methylating H3K4 with non redundant functions. Dr Shilatifard also presented unpublished data regarding the COMPASS family and COMPASS family members’ activities involved in the regulation of gene expression and enhancer promoter communication.

In the second part of his talk, Dr. Ali Shilatifard presented a proposed model suggesting the regulation of the rate of transcription elongation by Pol II could have a central role in leukemogenesis. Biochemical studies from Shilatifard laboratory have demonstrated that many of the MLL translocation partners (in addition to P-TEFb) are found within a biochemically distinct protein complex, the so-called “ELL-containing super elongation complex” -SEC. Importantly, the translocation of MLL into SEC is involved in the misrecruitment of SEC to MLL target genes, perturbing transcriptional elongation checkpoint control (TECC) at these loci and mediating leukemogenesis.

Further studies also showed that not only is SEC involved in the regulation of transcription of the MLL target genes in leukemia, but this elongation complex plays a central role in regulating transcription elongation control and gene expression during development in response to environmental stimuli. Finally, Dr Shilatifard presented recent findings regarding the identification of the SEC family of Pol II elongation factors (SEC1-3) and their diverse roles in the regulation of gene expression in embryonic stem cells.

 

Role of Tet1-Mediated 5mC Oxidation in PGC Reprogramming and Meiosis

Yi Zhang Harvard Medical School, USA

The session for “DNA methylation in Development and Cancer” was chaired by Shuv. I.S. Grewal (NCI, National Institues of Health, USA) and started by Prof. Yi Zhang’ talk (Howard Hughes Medical Institute, Harvard Medical School, USA). Mammalian germ cells originate from the pluripotent epiblast and therefore, they need to erase their “somatically-derived” epigenetic modifications in order to acquire an epigenetic state compatible with the germ cell program. The epigenetic reprogramming occurring in the Primordial germ cells (PGCs) includes the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA.

Although enzymes responsible for DNA methylation have been well characterized, enzymes that responsible for active DNA demethylation in mammalian cells have remained elusive. Recent studies have demonstrated that a novel family of proteins, the so-called Tet family (ten-eleven translocation-Tet) have the capacity to convert 5mC (5-methylcytosine) to 5hmC (5-hydroxymethylcytosine), 5fC (5-formylcytosine), and 5caC (5-carboxylcytosine) raising the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation. In his talk, Prof.Yi Zhang, presented recent findings on the mechanism and functions of Tet-catalyzed dynamic regulation of DNA methylation.

More specifically, Zhang group has shown that Global DNA demethylation in PGCs takes place in three steps: (i) the first step involves a massive Tet-independent loss of 5mC around E8.5 as 5mC levels in PGCs at this time is significantly lower than that in somatic cells; (ii) the second step involves oxidation of the remaining 5mC to 5hmC by Tet proteins; (iii) the final step features the loss of 5hmC in a replication-dependent manner that takes place for specific period E10.5 to E13.5.

In another study Zhang group used loss-of-function approach in mice and showed that the 5mC-specific dioxygenase Tet1 has an important role in regulating meiosis in PGCs. Importantly, Tet1 deficiency significantly reduces female germ-cell numbers and fertility. Univalent chromosomes and unresolved DNA double-strand breaks are also observed in Tet1-deficient oocytes. Although, Tet1 deficiency did not greatly affect the genome-wide demethylation of PGCs, it led to defective DNA demethylation and decreased expression of a subgroup of meiotic genes suggesting a function for Tet1 in meiosis and meiotic gene activation.

 

Cooperative Transcriptional Regulation of Metabolism Mediated by the Myc-Max/Mlx-Mondo Network

Robert N Eisenman, Fred Hutchinson Cancer Research Center, Seattle, USA

The Myc oncogene, a member of the bHLHZip family of transcription factors, is well known for its contribution to cancer via numerous protein/protein and protein/DNA interactions. Myc forms heterodimers with Max, another bHLHZip factor, to bind in the promoters of pro-growth and proliferation targets and thereby activating their expression. Activation of these Pol II targets is mediated by the recruitment of different chromatin modifying activities. Myc/Max complex also interact with other proteins, including members of the Mad family and the Max-like protein Mlx. However, the role of Myc in oncogenesis is much more complex since: (i) transcriptional repression at some targets, is achieved by Myc’s direct participation in Pol I- and Pol III-dependent transcription; (ii) Myc has a role in global chromatin structure; (iii) Myc most likely exhibit also Max-independent capacities. Due the importance of Myc-dependent effector pathways in cancer and the complex epigenetic phenomena in which Myc is involved, the morning session on Sunday, March 24th was devoted to Myc and its role in transcriptional regulation and development.

Myc’s role in tumor metabolism was the focus of Dr Rober N Eisenman talk. Myc drives the aerobic glycolysis as well as mitochondrial biogenesis and glutaminolysis. Tumor progression is achieved in part by a shift to aerobic glycolysis coupled with increased anabolic metabolism. Although Myc is considered a central regulator of intermediary metabolism required to support the demands of fast proliferating malignant cells, nowdays we know that it does not act alone. Apart from the Myc/Max complex, an extended Myc-related transcriptional network controls the metabolic status of tumor cells. This network includes the Max-like protein Mlx, as well as its heterodimerization partners MondoA  and ChREBP. Importantly, both MondoA and ChREBP have been shown to be nutrient-responsive transcriptional regulators.

MondoA-Mlx arm of the extended Max-Mlx network is required for deregulated Myc to drive metabolic reprogramming and maintain survival and growth of tumors.  In addition, the Eisenman laboratory has shown that a subgroup of Myc-Induced genes involved in metabolism requires MondoA for full expression. Overall, the main message of Dr Eisenman talk was that Myc-Max/MondoA-Mlx network regulates the transcriptional activity of several genes critical for Myc’s ability to alter metabolic status during tumor progression. Dr Eisenman concluded that the integration of Myc and MondoA functions might serve to link Myc to nutrient sensing and to augment metabolic flexibility for the tumor cells.

 

Epigenetic Marks and Cancer Drugs Summary

The principal aim of the Keystone Symposia meeting “Epigenetic Marks and Cancer Drugs” was to present to the scientific community cutting edge developments in cancer epigenetics with a focus on drug discovery. As epigenetics research is rapidly evolving, the meeting provided a constructive platform for knowledge exchange between scientists from both basic as well as applied side of epigenetics. Furthermore, the meeting provided a unique bridge to bring together researchers from academia and pharmacological industry allowing the development of strategic collaborations.

Scientific contributions by participants and invited speakers, spread the message that epigenetic therapy is a rapidly evolving and promising field, which is not limited to the current FDA-approved epigenetic drugs for cancer treatment. The more our knowledge for cancer epigenetics and cell reprogramming grows; the more challenges for novel therapies are created. Recent studies presented during the meeting underscored the extensive reprogramming of every component of the epigenetic machinery in cancer including DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs. A comprehensive understanding of the numerous and diverse molecular phenomena occurring in the epigenome of normal and malignant cells will hopefully provide novel targets for more effective epigenetic cancer treatment strategies.

 

**EpiGenie would like to extend big thanks to  Dr. Efterpi Kostareli out of the German Cancer Research Center (DKFZ) in Heidelberg for this amazing write up.