Second German-Catalan workshop on epigenetics & cancer

Abstract

The Second German-Catalan Workshop on Epigenetics and Cancer was held in Barcelona on November 19–21, 2014. The workshop brought together, for the second time, scientists from 2 German and 2 Catalan research institutions: the DKFZ, from Heidelberg, the CRCME, from Freiburg, and the IMPPC and PEBC/IDIBELL, both from Barcelona. The German-Catalan Workshops are intended to establish the framework for building a Research School to foster collaborations between researchers from the different institutions. Exchange programs for graduate students are among the activities of the future School. The topics presented and discussed in 33 talks were diverse and included work on DNA methylation, histone modifications, chromatin biology, characterization of imprinted regions in human tissues, non-coding RNAs, and epigenetic drug discovery. Among novel developments from the previous Workshop are the report of the epigenetics angle of the Warburg effect and the long-range trans-acting interaction of DNA methylation and of nucleosome remodeling. A shift in the view on DNA methylation became apparent by the realization of the intertwined interplay between hyper- and hypo-methylation in differentiation and cancer.

Abbreviations

DKFZ

German Cancer Research Center

CRCME

Research Center for Collaborative Medical Epigenetics

IMPPC

Institute of Predictive and Personalized Medicine of Cancer

PEBC/IDIBELL

Program on Cancer Epigenetics and Biology at Institute of Biomedical Research of Bellvitge

Introduction and Executive Summary

The Second German-Catalan Workshop on Epigenetics and Cancer was hosted by the IMPPC. Its director, Manuel Perucho, welcomed the workshop's attendants and encouraged the continuation of the ongoing collaborations initiated since the First Workshop held in Heidelberg in 2013, and the establishment of new ones between the participants. The Workshop was organized in 4 sessions that grouped the presentations by institution. Several of the talks focused on cancer, some others on epigenetics, and the majority on cancer epigenetics. Some of the topics and highlights are summarized in Figures 1 and 2. In his closing remarks at the end the 3-day workshop, Manuel Perucho highlighted the perceived shift on the view of DNA methylation alterations in development and cancer (Fig. 1) and the symmetry between the origins of somatic genetic and epigenetic alterations contributing to carcinogenesis, as well as their reciprocal causal relationships (Fig. 2, middle panel).

Figure 1.

Ubiquitous and genome-wide DNA methylation changes in colorectal cancer. From Illumina HumanMethylation 450 BeadChip (HM 450K) data on somatic DNA methylation alterations, the number of hyper- and hypo-methylations are similar. However, these arrays only cover 1.6% of the genomic CpG sites. When considering all CpG sites, somatic alterations corresponding to hypomethylation are relatively more abundant than those of hypermethylation in tumor cells.

Figure 2.

Selected highlights. Top Left panel: Waddington's landscape and DNA methylation changes during the differentiation process in the haematopoietic system. Top right panel: Methylation status of specific genes associates with the Warburg effect, suggesting new diagnostic and therapeutic targets. Middle panel: The interplay of genetics and epigenetics in (colon) cancer. The diagram illustrates the symmetry of the origins of these oncogenic alterations with both exogenous and endogenous sources generating alterations ultimately activating oncogenes and inactivating tumor suppressors. The diagram also depicts the reciprocal causal relationships between germ-line and/or somatic genetic and epigenetic alterations. The current experimental evidence for the genetic→epigenetic directionality is in its infancy compared with the examples of epigenetic alterations underlying the generation of genetic changes. Bottom left panel: Non-coding RNAs (ncRNAs) play diverse and heterogeneous roles in the RNA world as pivotal in multiple cellular processes as the central dogma's coding RNAs. Bottom right panel: Summary of a fluorescence-based assay for discovery of new inhibitors of histone deacetylase SIRT2 as research tool for potential new epigenetic cancer treatments.

IMPPC researchers reported a long-term population cohort study for genomic characterization in Catalonia; the involvement of DNA methylation alterations in colorectal cancer; the impact of DNA demethylation drugs on chromatin states including genome distribution of histone variants and histone marks; the identification of new polycomb binding sites in differentiating embryonic stem cells; and topological interactions of cis-regulatory networks in human pancreatic islets.

The importance of DNA methylation in development, gene regulation, and cancer, was discussed during the session by IDIBELL researchers. The contribution of epigenetics to the Warburg effect in several tumors was also communicated. Other talks focused on the characterization of imprinted regions in a range of human tissues using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays; and two talks focused on sirtuins histone deacethylases (HDACs).

The DKFZ presented 9 talks. All of them were related to chromatin biology, with 5 focusing in DNA methylation profiles during differentiation and cancer, indicating that it contributes to shape cellular behavior. Two talks showed how other chromatin players, such as nucleosome remodeling enzymes and histone modifications, contribute as well to the epigenetic landscape of a cell. Lastly, data on non-coding RNAs as emerging functional players of gene expression was presented in 2 talks.

The CRC from Freiburg was represented by 7 talks discussing how specific histone modifications, nuclear factors, and chromatin enzymes are crucial for correct neurodevelopment, trophoblast stem cell differentiation, or the control of metabolism. Drugs that may revert aberrant epigenetic profiles associated with myeloproliferative and other neoplasms hold great promise for future therapeutic interventions.

Institute of Predictive and Personalized Medicine of Cancer

Rafael de Cid presented the only talk that was not focused on cancer or epigenetics, he introduced the GCAT Project “GCAT|Genomes for Life: Cohort Study of the Genomes of Catalonia” and the GCAT BioBank located at the IMPPC. The GCAT is a large population cohort study that prospectively collects epidemiological data and biological samples from 40 to 65 year-old individuals in Catalonia. The project contemplates a continuous follow-up of lifestyle habits and health status of the participants. GCAT represents a powerful tool for the community to explore the genomics of chronic disorders in a prospective way involving both scientists and patients. The GCAT project will enable to study the role of genomic factors in the development and treatment of major common diseases in the general population to define the requirements for management and individualized treatment of the patients.

Yulia A. Medvedeva from Tanya Vavouri's group ¹ presented her unpublished work on the epigenetic changes in different genomic contexts induced by treatment with the DNA-demethylating drug Decitabine, which is currently used for the treatment of acute myeloid leukemia and myelodysplastic syndromes. Medvedeva unveiled how Decitabine treatment dramatically changes the chromatin states of the cell, including genome distribution of histone variants and histone marks, even in regions that do not show DNA methylation changes. The results suggest that Decitabine alters chromatin through pathways that are independent of its DNA methyltransferase blocking activity.

Izaskun Mallona from Miquel A. Peinado's group presented their unpublished design for an online resource to check all the correlations between scrutinized CpGs, regardless of their distance, using multi-cancer datasets obtained from The Cancer Genome Atlas (TCGA). As a first approach, they explored the colorectal cancer data set for DNA methylation (HumanMethylation450 BeadChip array). ² The correlations of each probe against every other probe in the chip (over 2.2 × 10¹¹ computations) were calculated exploring the co-methylation panorama in a comprehensive way. Preliminary results described functional and structural properties of genomic elements displaying correlated DNA methylation profiles, the vast majority of them linking probes located in different chromosomes. A web tool will facilitate data access through a user-friendly interface without requiring bioinformatics expertise (http://www.maplab.cat/pacco).

Sergio Alonso, from the group of Manuel Perucho, highlighted the importance of genome-wide DNA hypomethylation in the etiology and prognosis of gastrointestinal cancers. At genome-wide scale, hypomethylation is prevalent over hypermethylation (Fig. 1), and has a stronger association with cancer phenotype (copy number alterations) and poor prognosis, including the risk to develop 2 independent colonic lesions, synchronous or metachronous. ³ He also presented data on DNA methylation alterations in colon cancer patients and ulcerative colitis patients using commercial- and custom-arrays,^4,5 and unpublished observations showing that H. pylori-associated gastritis patients undergo irreversible genome-wide hypomethylation to an extent similar to that found in gastric cancer patients, suggesting a link between chronic inflammation and cancer through the erosion of the normal methylation of gastrointestinal mucosa.

Gabrijela Dumbovic, also from Manuel Perucho's group, presented her unpublished results on the demethylation of a pericentromeric repetitive element, SST-1, in colorectal cancers. In 15% of the patients demethylation was moderate and associated with patient age, but drastic in around 7% without association with patient age. ⁵ Hypomethylated SST-1 elements correlated with reduced levels of H3K9me3 and increased levels of H3K27me3 by ChIP analysis, and correlated with aberrant over-expression of these repeats in primary tumors. Moreover, treatment with Decitabine led to increased expression of SST-1, an effect greatly amplified by treatment with the histone deacetylase trichostatin A. Ongoing work aims to characterize the functions of this aberrantly expressed non-coding RNA, under the hypothesis that it might affect chromosomal stability during mitosis.

Anna Palau from Marcus Buschbeck's group presented their recently published work on the identification of new binding sites of the Polycomb mouse ortholog of Cbx8 in a model of differentiating embryonic stem cells. ⁶ Genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq), identified 171 high confidence peaks. Several differentiation genes transiently recruit Cbx8 during their early activation affecting the activation of these genes. Interaction analysis and ChIP experiments suggest that activating Cbx8 acts in the context of an intact PRC1 complex. The exchange of Cbx7 for Cbx8 seems to be required for the effective activation of differentiation genes. These results establish a function for a Cbx8-containing complex facilitating the transition from a Polycomb-repressed chromatin state to an active state.

Lorenzo Pasquali from the Health Sciences Research Institute of the Germans Trias i Pujol Foundation (IGTP), in which the IMPPC is integrated, presented his data on the topological interactions of cis-regulatory networks in the insulin-producing human pancreatic islets ⁷,⁸ in an effort to dissect the transcriptional programs and molecular mechanisms undelying Type 2 diabetes (T2D). Using 4C-Seq, he found that transcription factor binding events driving islet-specific genes are located at clusters of enhancers, sometimes far away from the genes. This reveals an architectural role of enhancer multiplicity by creating active chromatin structure domains. Some of these enhancer regions overlap previously known single nucleotide polymorphisms (SNPs) associated with T2D. These findings illuminate how tissue-specific transcription factor networks interact functionally with the epigenome, and how sequence variation in the human pancreatic islet regulome contributes to T2D susceptibility. He finally illustrated how all the generated data and the integrative analyses are publicly available and stored at the web site www.isletregulome.com.

Cancer Biology & Epigenetics Program, Bellvitge Biomedical Research Institute

The importance of DNA methylation in development, gene regulation, and cancer was discussed during the session by the IDIBELL researchers. DNA methylation is a key epigenetic mechanism during development, and several examples of the role of hyper- and hypo-methylation were presented. Historically, more attention has been placed on hypermethylation of CpGs localized at CpG islands at promoters regions. In this workshop, however, several speakers (see also talks by IMPPC and DKZF) remarked the importance of hypomethylation and its role in different processes.

As a novel example of DNA hypermethylation driving tumor phenotype, Paula López-Serra (from Manel Esteller's group) reported how the promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3) leads to SLC2A1 overexpression and contributes to the Warburg effect (the use of glycolysis even in the presence of oxygen) in a subset of human tumors with greater vulnerability to drugs targeting glycolysis. Hypermethylation of DERL3 in primary colorectal tumors was associated with shorter relapse-free survival (Fig. 2, top right panel).⁹

The work from Roser Vento-Tormo (Esteban Ballestar's group) focused on high-throughput analysis of DNA methylation changes during monocyte to dendritic cell and macrophage differentiation, analyzing both immature and mature phenotypes, after stimulating with lipopolysaccharide (LPS). DNA demethylation changes were predominant vs. hypermethylation changes, and took place mostly during the differentiation process, whereas very few of them occurred following LPS treatment.

Esteban Ballestar studied DNA methylation during osteoclastogenesis, a terminal differentiation process within the haematopoietic system relevant to autoimmune disease and cancer. There were key changes in DNA methylation during monocyte-to-osteoclast differentiation, whereby hypomethylation associated with changes in 5-hydroxymethylcytosine. The data revealed novel roles for PU.1 transcription factor. Over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs was observed. Among those, just PU.1 motifs were significantly enriched in both hypo- and hyper-methylated genes.¹⁰ PU.1 interacts with DNMT3b and TET2, suggesting its participation in driving hypermethylation and oxidation-mediated demethylation.

The group of David Monk presented studies aimed to characterize imprinted regions in a range of human tissues using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. The data defined methylation profiles of known imprinted domains at base-pair resolution and identified 21 novel loci harboring parent-of-origin methylation, some restricted to the placenta.¹¹ Marta Sanchez-Delgado and Alex Martin-Trujillo reported how the majority of placenta-specific imprinted differential methylated regions (DMRs) are unmethylated in sperm and all human embryonic stem cells.¹² The placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci.

David Monk presented the characterization of a region on chromosome 8q24, which is the human orthologous of the mouse chromosome 15 imprinted cluster, containing multiple brain-specific maternally expressed transcripts.¹³ This region has been associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome. There was reciprocal expression of the novel paternally expressed PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and biallelic expression of flanking transcripts in a range of tissues. A tandem-repeat region overlapping PEG13 transcript was methylated exclusively on the maternal allele. Chromatin immunoprecipitation analysis showed that CTCF-cohesin binds to this regions and exerts an enhancer blocker activity.

Sonia Guil from Manel Esteller's group, continuing with the non-coding RNA (ncRNA) topic, reported that ncRNA Uc.283+A, controls pri-miRNA processing.¹⁴ There is increasing evidence suggesting the involvement of ncRNAs in a network of ncRNA-ncRNA interactions. They proposed a model in which lower-stem strand invasion by Uc.283+A impairs microprocessor recognition and efficient pri-miRNA cropping. This regulation requires complementarity between the lower stem region of the pri-miR-195 transcript and an ultraconserved sequence in Uc.283+A, which prevents pri-miRNA cleavage by Drosha. The study reveals regulatory networks involving different ncRNA classes of importance in cancer (Fig. 2, bottom left panel).

The group of Alejandro Vaquero presented several of their investigations exploring the role of Sirtuins, from the family of NAD+-dependent histone deacetylases (HDACs). These proteins play a major role in stress response, genome stability and cell cycle control.¹⁵ Some Sirtuins, such as SirT2 and SirT6, work as tumor suppressors, but others, such as SirT1 operate as both tumor suppressors and oncogenic factors depending on the context.¹⁶ Among the 7 mammalian Sirtuins, Irene Santos-Barriopedro focused her presentation on SirT6 and its interaction with the H3K9 methyltransferase Suv39h1. In this work, they characterized the unusual cysteine monoubiquitination of Suv39h1 induced by SirT6 through the ubiquitin ligase SKP2 and its implication in NF-kB pathway regulation.

Nuria Sima explored the antagonistic role of MOF and SIRT2 in the regulation of H4K20me1, as well as in genome stability and the control of the cell cycle.¹⁷ Using a double knockout (KO) MOF/SIRT2 they established in vivo the functional implications of this antagonism in genome stability, cancer, and aging.

German Cancer Research Center

Christoph Plass addressed whether DNA methylation may contribute to lineage specification during hematopoiesis. His results indicated that during initial haematopoietic commitment, DNA methylation is lost in 71% of DMRs, whereas later steps of commitment under the Waddington's landscape are characterized by an increased methylation (Fig. 2, top left panel). The mechanisms that regulate these methylation changes are not understood, but may include transcription factor associated mechanisms. Thanks to a new technology called tagmentation-based whole genome bisulfite sequencing (TWGBS), a genome-wide approach that allows single CpG-resolution, 13,000 DMRs that are putative regulatory regions were identified.¹⁸ This technique is expensive but allows interrogate every CpG in the genome, and new regions that previously were undetected are now identified as DMRs.

Dieter Weichenhan further explained in more detail the technical characteristics of TWGBS, which combines the best attributes of previous methods, a genome-wide approach like in whole-genome bisulfite sequencing (WGBS) and small amounts of starting DNA, as in reduced representation bisulfite sequencing. The novelty is that there is no need to prepare the conventional library and instead the DNA is fragmented by a hyperactive Tn5 transposase, which appends methylated sequencing adapters at the same time. This strategy is much more efficient than the ligation chemistry used in classical WGBS library preparations, generating reliable sequencing data from small input DNA amounts (10–30 ng). Therefore, it represents a huge advance in analyzing DNA methylation from precious or unique samples that contain low cell populations, such as isolated stem cell progenitors from adult tissues or some cancer biopsies.¹⁹

Christopher Charles Oakes analyzed genome-wide DNA methylation profiles, gene expression patterns, and ChIPseq data in chronic lymphocytic leukemia (CLL), by next generation sequencing approaches. He compared these profiles to those of B cells isolated at several maturation steps, to identify similarities in the epigenomic/transcriptomic features between these populations to trace a “cell of origin” for CLL. The data indicated that CLLs arise from a continuum of maturation stages that ranged from early memory-type to mature memory-type B cells. The majority of tumor populations showed highly stable DNA methylation profiles during many years of disease development. They were characterized by relative hypomethylation, including a proportion (˜10%) of CpGa methylated on one allele. The methylation subgroup displaying the most mature methylation patterns show low expression of ZAP70, BTK, TCL1a, and other CLL cell survival genes. Above-median levels of methylation heterogeneity were observed in a subset of patients, associated with poor prognosis.²⁰

Marina Laplana, from Angela Risch's laboratory, described methylation profiles in lung cancer. Instead of a genome-wide approach, they pursued an elegant targeted enrichment (Sure Select technology) of regions that had previously been shown by genome-wide association studies (GWAS) ²¹ to contain SNPs for cancer susceptibility. Their undergoing analysis has a final goal to understand whether methylation could also be linked to cancer susceptibility in many SNP-containing regions. Their preliminary data indicates that methylation changes may be associated to SNPs in many cases.

Understanding nuclear architecture is one of the major issues to figure out how processes such as transcription, DNA repair, and DNA replication are tightly regulated. Chromatin remodelers are multicomplexes that use ATP energy to alter chromatin structure by repositioning nucleosomes and therefore are central players on regulating accessibility to chromatin.²² Karsten Rippe reported data that shed new light on chromatin remodelers, the molecular machines that can translocate, evict, or assemble nucleosomes. Using novel fluorescence microscopy-based method termed pixel-wise photobleaching profile evolution analysis (3PEA) they analyzed the localization and mobility of 2 remodelers of the ISWI family (Snf2H and Snf2L) that were GFP-tagged.²³ Their data indicated that these remodelers continuously sample nucleosomes in the genome with surprisingly short average binding times of only 1–2 ms. Together with their relatively high nuclear abundance, this leads to fast cell response time in the order of tens of seconds for a remodeler like Snf2H to ‘find’ a given nucleosome in the genome. Probably, different signaling pathways that impact epigenetic marks as well as other chromatin bound factors are able to modulate the binding affinity of the remodeling complex to select nucleosomes that are to be translocated.

Katharina Müller-Ott, from Karsten Rippe's group, addressed how the structure of pericentric heterochromatin is maintained and transmitted through the cell cycle. Combining ChIPseq and advanced fluorescence microscopy data, she proposed a predictive mathematical model where chromatin-bound SUV39H1/2 acts as a “nucleation site” to propagate a spatially confined H3K9me3 domain. In this model, HP1 dimer bound to H3K9me3-modified nucleosomes, methyl binding proteins (MBPs) that recognize 5meC and perhaps other proteins stabilize SUV39H binding. Elegant experiments where a GFP-SUV39H1 was tethered to the nuclear lamina via a GFP binding protein that is fused to Lamin B1 demonstrated that stably tethered SUV39H is able to generate a de novo H3K9me3 domain. This and other assays suggest that due to chromatin looping, the chromatin-bound SUV39H complexes can methylate adjacent nucleosomes or, in a 3-dimensional fashion, at other loci in spatial proximity.²⁴ The conceptual features of this model might also be relevant for chromatin states at other genomic loci.

Frank Westermann integrated information on epigenetics, genetics, and transcriptomics, to classify neuroblastoma into subtypes that could help clinicians to choose the appropriate therapy. Neuroblastoma is a very aggressive extra-cranial solid tumor that represents a high proportion of childhood cancer-related deaths. It is characterized by MYCN amplification, 1p chromosome deletion, 17q gain and very few recurrent somatic mutations. This work indicated that a newly built gene-expression classifier can distinguish 2 major neuroblastoma subgroups, one with spontaneous regression potential or differentiation ability after treatment, and another with a high tendency to progress or relapse after therapy.²⁵

Sven Diederichs pointed out that deep sequencing technologies revealed that around 70% of our genome is transcribed into non-coding RNA (ncRNA), and that growing evidence indicates that many of these ncRNAs are also functional (Fig. 2, bottom left panel). He showed data on the long non-coding RNA (lncRNA) MALAT1 (Metastasis-associated lung adenocarcinoma transcript-1), one of the first lncRNA's associated with cancer. After silencing MALAT-1 in human lung cancer cells by genomic editing using Zinc Finger Nucleases cells were unable to migrate and form tumor nodules in mouse transplantation models.²⁶ MALAT-1 expression induced epigenetic changes that lead to expression of genes involved in metastasis. MALAT-1 lncRNA does not contain homology sequence stretches that could pair with its targeted metastatic genes, and proteins bound to MALAT-1 may interact with the targeted loci and thus recruit it. Beyond MALAT1, genome-wide analyses show that several lncRNAs are downregulated in lung cancer, correlating with increased levels of DNA methylation. These new lncRNAs may be a new category of tumor suppressors.^27,28

Holger Bierhoff, from Ingrid Grummt's laboratory, showed how upon starvation or in terminal differentiation, silencing of interspersed repetitive sequences, i.e., genes encoding rRNA (rRNA) and intracisternal A-particle (IAP) retrotransposons, helps maintain genomic stability in post-mitotic cells. At the rDNA locus, the mechanism to achieve this repression requires the involvement of a heterogeneous population of lncRNA, which he termed PAPAS (promoter and pre-rRNA antisense), as it covers the pre-rRNA coding region and the rRNA gene promoter in an antisense direction. Upon growth arrest, PAPAS recruits the histone methyltransferase Suv4-20h2 to rDNA loci, promoting H4K20me3 and thus compaction of the chromatin resulting in prevention of transcription at these loci. In a similar mechanism, a lncRNA originated in the long terminal repeat (LTR) regions recruits Suv4-20h2 and H4K20me3 to silence IAP retroelements. In cancer cells, H4K20me3 levels are progressively lost, correlating with increased chromosomal aberrations present in tumors.²⁹

Collaborative Research Center Medical Epigenetics

Before recent gene editing approaches such as TALEN and CRISP-CAS9, mutations or alterations in the DNA sequence were difficult to tackle, seeming unlikely to be able to reverse a mutation. In contrast, epigenetic modifications are catalyzed by enzymes, which can be targeted by drugs. Therefore, efforts toward epigenetic drug discovery are aimed to identify new therapeutic approaches for cancer chemotherapy and chemoprevention.

Felix Krombholz, from Christian Flotho's group, presented the use of Rag2^null/Il2rg^null mice to study juvenile myelomonocytic leukemia (JMML), a rare myeloproliferative malignancy that affects children, to test epigenetic drugs for this disease. These mice harbor genetically engineered null alleles for recombination-activated gene 2 (Rag2) and interleukin-2 receptor gamma chain (Il2rg), thus being immunodeficient with lack of B, T, and NK cell function. The investigators transplanted JMML cells from 3 patients into mice by intrahepatic injection. The long-term presence in murine bone marrow and blood of human CD45+ cells carrying leukemia-specific mutations demonstrated successful engraftment. Several characteristics of JMML disease were recapitulated, such as splenomegaly, lung infiltration, cachexia, myelomonocytic differentiation, and the presence of immature leukemic progenitors in haematopoietic organs. The model will be used to determine the stability of aberrant epigenetic patterns in xenotransplanted JMML, providing robust evidence of the fundamental role of epigenetics in JMML stem cell biology. Importantly, the model will be instrumental in preclinical tests with epigenetic drugs to determine their therapeutic efficacy for JMML.

Soeren Swyter from Manfred Jung's laboratory described a drug discovery approach to identify inhibitors for Sirtuin 2 (Sirt-2). Inhibiting SIRT activity has been shown to result in beneficial effects in a model of Parkinson disease.³⁰ In cancer, Sirtuins have been shown to play a tumor suppressor role as well as to promote oncogenesis.³¹ Therefore, modulating the activity of Sirt2 by specific drugs would be an approach to better understand its functionality as well as a new tool for pharmacological interventions. By screening compound libraries and using several fluorescent assays to monitor the deacetylase activity (Fig. 2, bottom right panel),³² they identified several inhibitors for Sir2 that were termed SirReals (Sirtuin-rearranging ligands). One of these Sirt2 inhibitors, SirReal2, showed the highest inhibition activity, and it was validated in HeLa cells, resulting in hyperacetylation of Tubulin and BubR1 destabilization, both results that also occur when inhibiting Sirt2 in vivo.³³

Tanja Vogel showed unpublished data on how aberrations in AF9 and DOT1L chromatin modifier genes are involved in promoting leukemia and also they contribute to neurodegeneration in central nervous system (CNS) diseases. A translocation in the AF9 gene [t(9;11)(p22;q23)] with KMT2A occurs in acute leukemias; however, other aberrations, such as variations in the CAG repeat length in the AF9 gene could also contribute to leukemia and neurodevelopmental disorders. DOT1L is a histone methyltransferase that targets lysine 79 of histone H3 and has been shown to interact with MLL fusion proteins and be recruited aberrantly to several chromatin loci in leukemias.³⁴ Inhibition of DOT1L activity in vitro reduced proliferation and increased apoptosis, but did not impair neuronal differentiation. However, in vivo results of Dot1l absence using KO mice for Dot1l showed differentiation impairment in cerebral cortex and hippocampus. These results suggest that DOT1L and AF9 are both crucial factors to control neurogenic differentiation.³⁵

Roland Schule laboratory was represented by 2 talks that addressed the role of Lsd1, a mono- and di-methyl demethylase of H3K4 and H3K9. Milica Tosic presented how Lsd1 could be viewed as a sensor of environmental cues (nutritional and temperature changes) in the nucleus and promote oxidative metabolism in white adipose tissue. Lsd1 would be promoting this adaptive metabolic response by upregulating the expression of genes involved in oxidative phosphorylation (OXPHOS) and, therefore, increasing the mitochondrial activity. When Lsd1 is overexpressed in mice and those are fed with high-fat diet, they gain less weight than the controls, and their white adipose tissue (WAT) shows groups of metabolically active brown-like adipocytes. The development of these beige adipocytes has been shown to improve metabolic health and may be a mechanism to reduce adverse effects of WAT excess.³⁶ Lsd1 plays a critical role in this conversion and its targeting may lead to new therapeutic protocols for preventing obese-associated diseases such as insulin-resistant diabetes.

Josefina Castex also showed how Lsd1 controls lineage commitment, but, in this case of trophoblast stem cells (TSCs). Absence of Lsd1 in mice results in fewer TSC, impaired formation of trophectoderm tissues and early embryonic death. Lsd1 deficient TSCs are characterized by higher migration and invasive features. The possible mechanism points to direct Lsd1 repression in stemness conditions over Ovol2 expression, a transcription factor that controls cell motility.³⁷ Therefore, in the absence of Lsd1, Ovol2 is overexpressed impairing the maintenance of the trophoblast stem cell niche, and therefore, aberrant formation of trophectoderm-derived tissues is observed.

Christoph Koellerer, from Heike L. Pahl's laboratory, reported the identification by ChIP that CDK4, CDK6, Cyclin D3, MLL4 and MLL2 are new target genes of the transcriptional factor NF-E2 (nuclear factor erythroid-2). NF-E2 is overexpressed in myeloproliferative neoplasms (MPN) and the group had previously identified that MPN patients present in-del mutations in NF-E2 that result in truncated proteins with impaired binding to target DNA sequences.³⁸ However, the activity of wild type NF-E2 was increased by the presence of mutant NF-E2. In vitro and in primary patient cells, the mutant NF-E2 conferred a proliferative advantage and increased expression of key cell cycle regulator proteins. Therefore, these authors suggest that elevated NF-E2 levels or activity contribute to overexpression of cell cycle regulators and methyltransferases that contribute to increased proliferation. Inhibitors for these CDKs or the methyltransferases could represent new drugs for MPN patients.

Gabriele Greve, from Michael Lübbert's group, showed that although manipulating the activity of chromatin enzymes modifications may not restore the normal epigenetic profiles, it can nevertheless sensitize the chromatin to better respond to traditional medications in non-small cell lung cancer (NSCLC). Treatment of NSCLC cells wild type for tyrosine kinase (TK) receptor EGFR with panobinostat (a pan-HDAC inhibitor) sensitized them to respond to Erlotinib (a TK receptor inhibitor) resulting in reduced proliferation. Erlotinib is used in a subset of NSCLC that bear activating mutations in the EGFR.³⁹ EGFR wild type tumors do not respond to treatment. However, the combination of both drugs resulted in enhancement of Erlotinib effects by decreasing even further the proliferation in one of the cell lines.⁴⁰ The proposed mechanism is that HDACi increases the levels of H3 acetylation resulting in upregulation of p21WAF and p53 expression, whereas Erlotinib also targets JAK2 resulting in decreased levels of pERK, pAKT, and pEGFR.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The workshop was sponsored by the Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain. Financial support: M. Perucho's group is supported by Plan Estatal de I+D+I, ISCIII, FEDER, PI12/00511.