Lucia Altucci

The promise of retinoids to fight against cancer

Retinoids have a reputation for being both detrimental and beneficial: they are teratogens, but they also have tumour-suppressive capacity. Cell biology and genetics have significantly improved our understanding of the mechanisms that underlie the anti-proliferative action of retinoids. Recent elucidation of the pathways that are activated by retinoids will help us to exploit the beneficial aspects of this powerful class of compounds for cancer therapy and prevention.

Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway

Histone deacetylases (HDACs) regulate transcription and specific cellular functions, such as tumor suppression by p53, and are frequently altered in cancer. Inhibitors of HDACs (HDACIs) possess antitumor activity and are well tolerated, supporting the idea that their use might develop as a specific strategy for cancer treatment. The molecular basis for their selective antitumor activity is, however, unknown. We investigated the effects of HDACIs on leukemias expressing the PML-RAR or AML1-ETO oncoproteins, known to initiate leukemogenesis through deregulation of HDACs. Here we report that: (i) HDACIs induce apoptosis of leukemic blasts, although oncogene expression is not sufficient to confer HDACI sensitivity to normal cells; (ii) apoptosis is p53 independent and depends, both in vitro and in vivo, upon activation of the death receptor pathway (TRAIL and Fas signaling pathways); (iii) TRAIL, DR5, FasL and Fas are upregulated by HDACIs in the leukemic cells, but not in normal hematopoietic progenitors. These results show that sensitivity to HDACIs in leukemias is a property of the fully transformed phenotype and depends on activation of a specific death pathway.

RAR and RXR modulation in cancer and metabolic disease

Retinoic acid receptors (RARs) are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth and survival. The success of RAR modulation in the treatment of acute promyelocytic leukaemia (APL) has stimulated considerable interest in the development of RAR and RXR modulators. This has been aided by recent advances in the understanding of the biological role of RARs and RXRs and in the design of selective receptor modulators that might overcome the limitations of current drugs. Here, we discuss the challenges and opportunities for therapeutic strategies based on RXR and RAR modulators, with a focus on cancer and metabolic diseases such as diabetes and obesity.

Retinoic acid-induced apoptosis in leukemia cells is mediated by paracrine action of tumor-selective death ligand TRAIL

The therapeutic and preventive activities of retinoids in cancer are due to their ability to modulate the growth, differentiation, and survival or apoptosis of cancer cells. Here we show that in NB4 acute promyelocytic leukemia cells, retinoids selective for retinoic-acid receptor-α induced an autoregulatory circuitry of survival programs followed by expression of the membrane-bound tumor-selective death ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand, also called Apo-2L). In a paracrine mode of action, TRAIL killed NB4 as well as heterologous and retinoic-acid–resistant cells. In the leukemic blasts of freshly diagnosed acute promyelocytic leukemia patients, retinoic-acid–induced expression of TRAIL most likely caused blast apoptosis. Thus, induction of TRAIL-mediated death signaling appears to contribute to the therapeutic value of retinoids.

Whole-Genome Bisulfite Sequencing of Two Distinct Interconvertible DNA Methylomes of Mouse Embryonic Stem Cells

The use of two kinase inhibitors (2i) enables derivation of mouse embryonic stem cells (ESCs) in the pluripotent ground state. Using whole-genome bisulfite sequencing (WGBS), we show that male 2i ESCs are globally hypomethylated compared to conventional ESCs maintained in serum. In serum, female ESCs are hypomethyated similarly to male ESCs in 2i, and DNA methylation is further reduced in 2i. Regions with elevated DNA methylation in 2i strongly correlate with the presence of H3K9me3 on endogenous retroviruses (ERVs) and imprinted loci. The methylome of male ESCs in serum parallels postimplantation blastocyst cells, while 2i stalls ESCs in a hypomethylated, ICM-like state. WGBS analysis during adaptation of 2i ESCs to serum suggests that deposition of DNA methylation is largely random, while loss of DNA methylation during reversion to 2i occurs passively, initiating at TET1 binding sites. Together, our analysis provides insight into DNA methylation dynamics in cultured ESCs paralleling early developmental processes.

PML-RARα/RXR Alters the Epigenetic Landscape in Acute Promyelocytic Leukemia

Many different molecular mechanisms have been associated with PML-RARalpha-dependent transformation of hematopoietic progenitors. Here, we identified high confidence PML-RARalpha binding sites in an acute promyelocytic leukemia (APL) cell line and in two APL primary blasts. We found colocalization of PML-RARalpha with RXR to the vast majority of these binding regions. Genome-wide epigenetic studies revealed that treatment with pharmacological doses of all-trans retinoic acid induces changes in H3 acetylation, but not H3K27me3, H3K9me3, or DNA methylation at the PML-RARalpha/RXR binding sites or at nearby target genes. Our results suggest that PML-RARalpha/RXR functions as a local chromatin modulator and that specific recruitment of histone deacetylase activities to genes important for hematopoietic differentiation, RAR signaling, and epigenetic control is crucial to its transforming potential.

Design of selective nuclear receptor modulators: RAR and RXR as a case study.

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are members of the nuclear receptor superfamily whose effects on cell growth and survival can be modulated therapeutically by small-molecule ligands. Although compounds that target these receptors are powerful anticancer drugs, their use is limited by toxicity. An improved understanding of the structural biology of RXRs and RARs and recent advances in the chemical synthesis of modified retinoid and rexinoid ligands should enable the rational design of more selective agents that might overcome such problems. Here, we review structural data for RXRs and RARs, discuss strategies in the design of selective RXR and RAR modulators, and consider lessons that can be learned for the design of selective nuclear-receptor modulators in general.

Salermide, a Sirtuin inhibitor with a strong cancer-specific proapoptotic effect

Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2) belong to the family of NAD+ (nicotinamide adenine dinucleotide-positive)-dependent class III histone deacetylases and are involved in regulating lifespan. As cancer is a disease of ageing, targeting Sirtuins is emerging as a promising antitumour strategy. Here we present Salermide (N-{3-[(2-hydroxy-naphthalen-1-ylmethylene)-amino]-phenyl}-2-phenyl-propionamide), a reverse amide with a strong in vitro inhibitory effect on Sirt1 and Sirt2. Salermide was well tolerated by mice at concentrations up to 100 μM and prompted tumour-specific cell death in a wide range of human cancer cell lines. The antitumour activity of Salermide was primarily because of a massive induction of apoptosis. This was independent of global tubulin and K16H4 acetylation, which ruled out a putative Sirt2-mediated apoptotic pathway and suggested an in vivo mechanism of action through Sirt1. Consistently with this, RNA interference-mediated knockdown of Sirt1, but not Sirt2, induced apoptosis in cancer cells. Although p53 has been reported to be a target of Sirt1, genetic p53 knockdowns showed that the Sirt1-dependent proapoptotic effect of Salermide is p53-independent. We were finally able to ascribe the apoptotic effect of Salermide to the reactivation of proapoptotic genes epigenetically repressed exclusively in cancer cells by Sirt1. Taken together, our results underline Salermides promise as an anticancer drug and provide evidence for the molecular mechanism through which Sirt1 is involved in human tumorigenesis.

BLUEPRINT to decode the epigenetic signature written in blood

volume 30 number 3 march 2012 nature biotechnology To the Editor: Last October, scientists gathered in Amsterdam to celebrate the start of BLUEPRINT (http://www.blueprintepigenome.eu/), an EU-funded consortium that will generate epigenomic maps of at least 100 different blood cell types. With this initiative, Europe has pledged a substantial contribution to the ultimate goal of the International Human Epigenome Consortium (IHEC) to map 1,000 human epigenomes. Here, we provide a brief background to the scientific questions that prompted the formation of BLUEPRINT, summarize the overall goals of BLUEPRINT and detail the specific areas in which the consortium will focus its initial efforts and resources. In mammals, nucleated cells share the same genome but have different epigenomes depending on the cell type and many other factors, resulting in an astounding diversity in phenotypic plasticity with respect to morphology and function. This diversity is defined by cell-specific patterns of gene expression, which are controlled through regulatory sites in the genome to which transcription factors bind. In eukaryotes, access to these sites is orchestrated via chromatin, the complex of DNA, RNA and proteins that constitutes the functional platform of the genome. In contrast with DNA, chromatin is not static but highly dynamic, particularly through modifications of histones at nucleosomes and cytosines at the DNA level that together define the epigenome, the epigenetic state of the cell. Advances in new genomics technologies, particularly next-generation sequencing, allow the epigenome to be studied in a holistic fashion, leading to a better understanding of chromatin function and functional annotation of the genome. Yet little is known about how epigenetic characteristics vary between different cell types, in health and disease or among individuals. This lack of a quantitative framework for the dynamics of the epigenome and its determinants is a major hurdle for the translation of epigenetic observations into regulatory models, the identification of associations between epigenotypes and diseases, and the subsequent development of new classes of compounds for disease prevention and treatment. The task, however, is daunting as each of the several hundred cell types in the human body is expected to show specific epigenomic features that are further expected to respond to environmental inputs in time and space. The research community has realized these limitations and the need for concerted action. The IHEC was founded to coordinate large-scale international efforts toward the goal of a comprehensive human epigenome reference atlas (http://www.ihec-epigenomes. org/). The IHEC will coordinate epigenomic mapping and characterization worldwide to avoid redundant research efforts, implement high data quality standards, coordinate data storage, management and analysis, and provide free access to the epigenomes produced. The maps generated under the umbrella of the IHEC contain detailed information on DNA methylation, histone modification, nucleosome occupancy, and corresponding coding and noncoding RNA expression in different normal and diseased cell types. This will allow integration of different layers of epigenetic information for a wide variety of distinct cell types and thus provide a resource for both basic and applied research. BLUEPRINT aims to bridge the gap in our current knowledge between individual components of the epigenome and their functional dynamics through state-of-the-art analysis in a defined set of primarily human hematopoietic cells from healthy and diseased individuals. Mammalian blood formation or hematopoiesis is one of the best-studied systems of stem cell biology. Blood formation can be viewed as a hierarchical process, and classically, differentiation is defined to occur along the myeloid and lymphoid lineages. The identity of cellular intermediates and the geometry of branch points are still under intense investigation and therefore provide a paradigm for delineation of fundamental principles of cell fate determination and regulation of proliferation and lifespan, which differ considerably between different types of blood cells. BLUEPRINT will generate reference epigenomes of at least 50 specific blood cell types and their malignant counterparts and aim to provide high-quality reference epigenomes of primary cells from >60 individuals with detailed genetic and, where appropriate, medical records. To account for and quantify the impact of DNA sequence variation on epigenome differences, BLUEPRINT will work whenever possible on samples of known genetic variation, including samples from the Cambridge BioResource (Cambridge, UK), the International Cancer Genome Consortium and the British Diabetic Twin Study for disease-discordant monozygotic twin samples. The Wellcome Trust Sanger Institute (Hinxton, UK) will also provide full genomic sequencing for up to 100 samples. BLUEPRINT will harness existing proven technologies to generate reference epigenomes, including RNA-Seq for transcriptome analysis, bisulfite sequencing for methylome analysis, DNaseI-Seq for analysis of hypersensitive sites and ChIPSeq for analysis of at least six histone marks. Moreover, BLUEPRINT aims to develop new technologies to enhance high-throughput epigenome mapping, particularly when using few cells. BLUEPRINT is initially focusing on four main areas. One main goal of the project is to comprehensively analyze diverse epigenomic maps and make them available as an integrated BLUEPRINT-IHEC resource to the scientific community. Integration is envisioned for related projects within species (e.g., the 1000 Genomes Project) and between species (e.g., modENCODE) to better understand functional aspects (e.g., shared pathways) and the evolution of cell lineage development. Analysis of the BLUEPRINT data is expected to catalyze a better understanding of the relationship between epigenetic and genomic information and will form the basis for generation of new methods (e.g., epigenetic imputation) for prediction of epigenetic states from epigenomic profiles. Such prediction methods will facilitate a move toward a more quantitative knowledge and modeling of epigenetic mechanisms. As a result, such models could in the future assist in ‘reverse engineering’ of regulatory networks to repair or restore epigenetic codes that have been perturbed by disease. A second goal of BLUEPRINT is to systematically link epigenetic variation with phenotypic plasticity in health and disease. This will be attempted in three ways. First, genetic and epigenetic varation in two blood cell types from 100 healthy individuals will be analyzed. These measurements will be combined with whole-genome and transcriptome sequencing to dissect the interplay between common DNA sequence BLUEPRINT to decode the epigenetic signature written in blood CORRESPONDENCE

HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.