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Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed ‘stemness’). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

High-throughput transcriptional analysis has unveiled a myriad of novel RNAs. However, technical constraints in RNA sequencing library preparation and platform performance hamper the identification of rare transcripts contained within the RNA repertoire. Herein we present targeted-RNA directional sequencing (TARDIS), a hybridization-based method that allows subsets of RNAs contained within the transcriptome to be interrogated independently of transcript length, function, the presence or absence of poly-A tracts, or the mechanism of biogenesis. TARDIS is a modular protocol that is subdivided into four main phases, including the generation of random DNA traps covering the region of interest, purification of input RNA material, DNA trap–based RNA capture, and finally RNA-sequencing library construction. Importantly, coupling RNA capture to strand-specific RNA sequencing enables robust identification and reconstruction of novel transcripts, the definition of sense and antisense RNA pairs and, by the concomitant analysis of long and natural small RNA pools, it allows the user to infer potential precursor-product relations. TARDIS takes ∼10 d to implement.

Disappearance of the Barr body is considered a hallmark of cancer, although whether this corresponds to genetic loss or to epigenetic instability and transcriptional reactivation is unclear. Here we show that breast tumors and cell lines frequently display major epigenetic instability of the inactive X chromosome, with highly abnormal 3D nuclear organization and global perturbations of heterochromatin, including gain of euchromatic marks and aberrant distributions of repressive marks such as H3K27me3 and promoter DNA methylation. Genome-wide profiling of chromatin and transcription reveal modified epigenomic landscapes in cancer cells and a significant degree of aberrant gene activity from the inactive X chromosome, including several genes involved in cancer promotion. We demonstrate that many of these genes are aberrantly reactivated in primary breast tumors, and we further demonstrate that epigenetic instability of the inactive X can lead to perturbed dosage of X-linked factors. Taken together, our study provides the first integrated analysis of the inactive X chromosome in the context of breast cancer and establishes that epigenetic erosion of the inactive X can lead to the disappearance of the Barr body in breast cancer cells. This work offers new insights and opens up the possibility of exploiting the inactive X chromosome as an epigenetic biomarker at the molecular and cytological levels in cancer.

Massive parallel DNA sequencing combined with chromatin immunoprecipitation and a large variety of DNA/RNA-enrichment methodologies is at the origin of data resources of major importance. Indeed these resources, available for multiple genomes, represent the most comprehensive catalogue of (i) cell, development and signal transduction-specified patterns of binding sites for transcription factors (‘cistromes’) and for transcription and chromatin modifying machineries and (ii) the patterns of specific local post-translational modifications of histones and DNA (‘epigenome’) or of regulatory chromatin binding factors. In addition, (iii) the resources specifying chromatin structure alterations are emerging. Importantly, these types of “omics” datasets populate increasingly public repositories and provide highly valuable resources for the exploration of general principles of cell function in a multi-dimensional genome–transcriptome–epigenome–chromatin structure context. However, data mining is critically dependent on the data quality, an issue that, surprisingly, is still largely ignored by scientists and well-financed consortia, data repositories and scientific journals. So what determines the quality of ChIP-seq experiments and the datasets generated therefrom and what refrains scientists from associating quality criteria to their data? In this ‘opinion’ we trace the various parameters that influence the quality of this type of datasets, as well as the computational efforts that were made until now to qualify them. Moreover, we describe a universal quality control (QC) certification approach that provides a quality rating for ChIP-seq and enrichment-related assays. The corresponding QC tool and a regularly updated database, from which at present the quality parameters of more than 8000 datasets can be retrieved, are freely accessible at www.ngs-qc.org.

Retinoic acid receptors (RARs) are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth, differentiation, survival and death. Due to their regulatory potential, these nuclear receptors (NRs) are major drug targets for a variety of pathologies, including cancer and metabolic diseases. A large amount of RAR- and RXR-selective ligands, ranging from (partial) agonists to antagonists and inverse agonists, have been designed and the corresponding structural and functional analyses have provided deep insight into the molecular basis of ligand action. Ligands regulate, via allosteric conformational changes, the ability of these NRs to interact with different sets of coregulators, which in turn recruit enzymatically active complexes/machineries. Here, we describe strategies in the design of selective RXR and RAR modulators and review the structural mechanisms by which the diverse pharmacological classes of compounds modulate receptor functions. Finally, we discuss the perspectives for retinoid- and rexinoid-based therapies.

Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled to massive parallel sequencing (ChIP-seq) has been achieved for transcription factors and epigenetic modification of chromatin histones with 1,000 to 5,000 cells. LinDA largely simplifies reChIP-seq experiments to monitor co-binding at chromatin target sites. The single-tube design of LinDA is ideal for handling ultrasmall amounts of DNA (<30 pg) and is compatible with automation. The actual hands-on working time is less than 6 h with one overnight reaction. The present protocol describes all materials and critical steps, and provides examples and controls for LinDA. Applications of LinDA for genome-wide analyses of biobank samples and for the study of chromatin conformation and nuclear architecture are in progress.

The development of ligands that as single chemical entities are able to modulate multiple epigenetic targets simultaneously (designed epigenetic multiple ligands) is still in its infancy. We are witnessing some advances with combinations of the fused or linked pharmacophores of an epi-drug and other anticancer agents. More recently, however, a very promising approach has been developed in which a single chemical entity exerts several therapeutic activities simultaneously, such as a compound that inhibits several epigenetic enzymes and as a consequence displays multiple biological profiles that address synergistically a particular multifactorial disease. Despite their promiscuity, these multiple epigenetic ligands have exciting conceptual advantages, as they (i) lower the risk of drug-drug interactions compared to cocktails or multicomponent drugs and facilitate ADMET and toxicology studies, (ii) minimize the development of drug resistance, (iii) exploit synergies between the targeted pathways/factors and (iv) can generally be used at lower therapeutically effective concentrations than the single target drugs. The obvious problem with such compounds is to find/design drugs which target multiple effectors with high selectivity and efficiency without displaying extensive off-target effects. In addition, the rational design of multiple epi-ligands is a major challenge. In this review we provide structurally-based principles and the optimization of activities towards the different epigenetic targets.

Importance of the field:Retinoids are currently used in the clinic for the treatment of skin diseases and acute promielocytic leukemia and are known to contribute to early development and organogenesis in embryo and throughout life. Most of these activities are primarily due to the binding of the retinoid to the retinoic acid receptors (RARs, subtypes α, β and γ). Ligand modulates, via allosteric conformational changes, the ability of RARs to interact with different sets of co-regulators. Structure-based insights on the ligand-binding domain of the ligand-bound RARs have clearly linked retinoid function to co-activator (CoA) recruitment for agonists, CoA dissociation for antagonists and corepressor stabilization for inverse agonists.Areas covered in this review:To help understand ligand-modulated RAR action as a consequence of its interaction with different sets of co-regulators, we present the chemical engineering of subsets of retinoid chemotypes (rexinoids, i.e., the ligands of the retinoid X receptors, α, β and γ, with impact in the treatment of cancer and metabolic diseases, are not covered) that display the whole range of ligand functions, including subtype- and isotype-selectivities.What the reader will gain:An understanding of the correlation of retinoid ligand structure and function. Structural insights into ligand action and retinoid chemotypes. Potential for clinical application of retinoid receptor modulators.Take home message:Potential pharmacological/therapeutic applications of these chemical tools extend beyond cancer prevention and therapy to the treatment of autoimmune and neurodegenerative diseases.

PRKAR1A (R1A)–retinoic acid receptor-α (R1A-RARα) is the sixth RARα–containing fusion protein in acute promyelocytic leukemia (APL). Using the murine bone-marrow retroviral transduction/transformation assay, we showed that R1A-RARα fusion protein could transform bone-marrow progenitor/stem cells. In gel-shift assays, R1A-RARα was able to bind to a panel of retinoic acid response elements both as a homodimer and as a heterodimer with RXRα, and demonstrated distinct DNA-binding characteristics compared with wild-type RARα/RXRα or other X-RARα chimeric proteins. The ratio of R1A-RARα to RXRα proteins affected the retinoic acid response element interaction pattern of R1A-RARα/RXRα complexes. Studies comparing R1A-RARα with R1A-RARα(ΔRIIa) demonstrated that the RIIa protein interaction domain located within R1A was responsible for R1A-RARα homodimeric DNA binding and interaction with wild-type R1A protein. However, the RIIa domain was not required for R1A-RARα–mediated transformation because its deletion in R1A-RARα(ΔRIIa) did not compromise its transformation capability. In contrast, introduction of point mutations within the RARα portion of either R1A-RARα or R1A-RARα(ΔRIIa), previously demonstrated to eliminate RXRα interaction or treatment of transduced cells with RXRα shRNA or a RXRα agonist, reduced transformation capability. Thus, leukemic transformation by APL fusion protein PRKAR1A-RARα is critically dependent on RXRα, which suggests RXRα is a promising target for APL.

Background Retinoids have a pronounced antiviral effect against several viruses. In this study we aimed to investigate the effect of retinoids on human herpesvirus 8 (HHV-8).Methods A panel of retinoic acid compounds were tested for their antiviral activity against HHV-8 in human umbilical vascular endothelial cells (HUVECs) and in a human epithelial cell line. The presence, transcription and antigen expression of HHV-8 in infected cells – in the presence or absence of retinoic acid compounds – were evaluated by PCR, reverse transcriptase PCR and immunofluorescence assays; HHV-8 viral load was determined by real-time quantitative PCR. Angiogenesis induced by HHV-8 was also assessed using Cultrex® basement membrane extract.Results The compounds tested specifically inhibited viral promoters, during the early and late phases of infection in both cell systems tested, and resulted in up to 100fold reduction of viral titre and release of progeny virus. The inhibition of viral replication induced by retinoids in endothelial cells, the primary target of HHV-8-driven transformation in Kaposi's Sarcoma, prevented endothelial cells from developing spindle morphology and in vitrotube formation, characteristic changes associated with HHV-8 infection and transformation.Conclusions We show that retinoids inhibit HHV-8 replication and identify new retinoid compounds with a strong antiviral effect. Selective retinoids, particularly those with retinoic acid receptor agonist activity, may be good candidates for the development of antiviral drugs.

The physiological effects of retinoic acids (RAs) are mediated by members of two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which are encoded by three distinct human genes, RXRα, RXRβ, and RXRγ. RARs bind both all-trans- and 9-cis-RA, whereas only the 9-cis-RA stereoisomer binds to RXRs. As RXR/RAR heterodimers, these receptors control the transcription of RA target genes through binding to RA-response elements. This review is focused on the structure, mode of action, ligands, expression, and pharmacology of RXRs. Given their role as common partners to many other members of the nuclear receptor superfamily, these receptors have been the subject of intense scrutiny. Moreover, and despite numerous studies since their initial discovery, RXRs remain enigmatic nuclear receptors, and there is still no consensus regarding their role. Indeed, multiple questions about the actual biological role of RXRs and the existence of an endogenous ligand have still to be answered.

Retinoid is a term for compounds that bind to and activate retinoic acid receptors (RARα, RARβ, and RARγ), members of the nuclear hormone receptor superfamily. The most important endogenous retinoid is all-trans-retinoic acid. Retinoids regulate a wide variety of essential biological processes, such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation and apoptosis, and homeostasis, as well as their disorders. This review summarizes the considerable amount of knowledge generated on these receptors.

Molecular analysis, in vivo and in vitro has identified TRAIL as a central component of anti-cancer networks in the cell. More recently, TRAIL has been shown to be involved in the selective apoptosis of AML cells by histone deacetylase inhibitors (HDACi). Here we summarize findings that the TRAIL pathway is a key factor in the apoptotic action of HDACi, as well as, retinoids and retinoid-combinations.

Nuclear receptors (NRs) comprise a family of 49 members that share a common structural organization and act as ligand-inducible transcription factors with major (patho)physiological impact. For some NRs (“orphan receptors”), cognate ligands have not yet been identified or may not exist. The principles of DNA recognition and ligand binding are well understood from both biochemical and crystal structure analyses. The 3D structures of several DNA-binding domains (DBDs),in complexes with a variety of cognate response elements, and multiple ligand-binding domains (LBDs), in the absence (apoLBD)and presence (holoLBD) of agonist, have been established and reveal canonical structural organization. Agonist binding induces a structural transition in the LBD whose most striking feature is the relocation of helix H12, which is required for establishing a coactivator complex, through interaction with members of the p160 family (SRC1, TIF2, AIB1) and/or the TRAP/DRIP complex. The p160-dependent coactivator complex is a multiprotein complex that comprises histone acetyltransferases (HATs), such as CBP,methyltransferases, such as CARM1, and other enzymes (SUMO ligase,etc.). The agonist-dependent recruitment of the HAT complex results in chromatin modification in the environment of the target gene promoters, which is requisite to, or may in some cases be sufficient for, transcription activation. In the absence of ligands, or in the presence of some antagonists, certain NRs are bound to distinct multiprotein complexes through the interaction with corepressors, such as NCoR and SMRT. Corepressor complexes comprise histone deacetylases (HDACs) that have the capacity to condense chromatin over target gene promoters. Ligands have been designed that selectively modulate the interaction between NRs and their coregulators. Both HATs and HDACs can also modify the acetylation status of nonhistone proteins, but the significance in the context of NR signaling is unclear. NRs communicate with other intracellular signaling pathways on a mutual basis, and their functionality may be altered, positively or negatively, by post-translational modification. The majority of NRs act as retinoid X receptor (RXR) heterodimers in which RXR cannot a priori respond autonomously to its cognate ligand to activate target gene transcription. This RXR subordination allows signaling pathway identity for the RXR partner. The corresponding mechanism is understood and reveals cell and NR selectivity, indicating that RXR can, under certain conditions, act autonomously. NRs are regulators of cell life and death,and NR malfunction can be at the basis of both disease and therapy, as is impressively documented in the case of acute promyelocytic leukemia. Recently, several pathways have been uncovered that link NR action with cell proliferation and apoptosis.

Nuclear receptors recruit coregulator complexes through both their AF-1 and AF-2 activation domains. Here we demonstrate that TIF2, a p160 coactivator, is able to bridge the two activation domains of the retinoic acid (RA) receptor isotype RARα1, resulting in synergistic activation of transcription. Bridging requires the presence of motifs in region A of RARα1 and in the activation domain AD1 of TIF2. Notably, only RARα1 exerted this interaction, which requires additional unknown factors. This is the first observation of a RAR isotype-selective coactivator interaction. Because another p160 coactivator, SRC-1, has no effect, this is also the first demonstration of a difference between the members of this coactivator family.

ABSTRACTHuman TIF2 (hTIF2) is a member of the p160 family of nuclear receptor coactivators, which includes SRC-1 and p/CIP. Although the functions of hTIF2 and of its mouse homolog (GRIP1 or mTIF2) have been clearly established in vitro, their physiological role remains elusive. Here, we have generated mice lacking mTIF2/GRIP1 and examined their phenotype with a particular emphasis on reproductive functions. TIF2−/−mice are viable, but the fertility of both sexes is impaired. Male hypofertility is due to defects in both spermiogenesis (teratozoospermia) and age-dependent testicular degeneration, and TIF2 expression appears to be essential for adhesion of Sertoli cells to germ cells. Female hypofertility is due to a placental hypoplasia that most probably reflects a requirement for maternal TIF2 in decidua stromal cells that face the developing placenta. We conclude that TIF2 plays a critical role in mouse reproductive functions, whereas previous reports have not revealed serious fertility impairment in SRC-1−/−or p/CIP−/−mutants. Thus, even though the three p160 coactivators exhibit strong sequence homology and similar activity in assays in vitro, they play distinct physiological roles in vivo, as their genetic eliminations result in distinct pathologies.

Human TIF2 (hTIF2) is a member of the p160 family of nuclear receptor coactivators, which includes SRC-1 and p/CIP. Although the functions of hTIF2 and of its mouse homolog (GRIP1 or mTIF2) have been clearly established in vitro, their physiological role remains elusive. Here, we have generated mice lacking mTIF2/GRIP1 and examined their phenotype with a particular emphasis on reproductive functions. TIF2−/−mice are viable, but the fertility of both sexes is impaired. Male hypofertility is due to defects in both spermiogenesis (teratozoospermia) and age-dependent testicular degeneration, and TIF2 expression appears to be essential for adhesion of Sertoli cells to germ cells. Female hypofertility is due to a placental hypoplasia that most probably reflects a requirement for maternal TIF2 in decidua stromal cells that face the developing placenta. We conclude that TIF2 plays a critical role in mouse reproductive functions, whereas previous reports have not revealed serious fertility impairment in SRC-1−/−or p/CIP−/−mutants. Thus, even though the three p160 coactivators exhibit strong sequence homology and similar activity in assays in vitro, they play distinct physiological roles in vivo, as their genetic eliminations result in distinct pathologies.

Amphioxus, the closest living invertebrate relative of the vertebrates, has a notochord, segmental axial musculature, pharyngeal gill slits and dorsal hollow nerve cord, but lacks neural crest. In amphioxus, as in vertebrates, exogenous retinoic acid (RA) posteriorizes the embryo. The mouth and gill slits never form, AmphiPax1, which is normally downregulated where gill slits form, remains upregulated and AmphiHox1 expression shifts anteriorly in the nerve cord. To dissect the role of RA signaling in patterning chordate embryos, we have cloned the single retinoic acid receptor (AmphiRAR), retinoid X receptor (AmphiRXR) and an orphan receptor (AmphiTR2/4) from amphioxus. AmphiTR2/4 inhibits AmphiRAR-AmphiRXR-mediated transactivation in the presence of RA by competing for DR5 or IR7 retinoic acid response elements (RAREs). The 5′ untranslated region of AmphiTR2/4 contains an IR7 element, suggesting possible auto- and RA-regulation. The patterns of AmphiTR2/4 and AmphiRAR expression during embryogenesis are largely complementary: AmphiTR2/4 is strongly expressed in the cerebral vesicle (homologous to the diencephalon plus anterior midbrain), while AmphiRAR expression is high in the equivalent of the hindbrain and spinal cord. Similarly, while AmphiTR2/4 is expressed most strongly in the anterior and posterior thirds of the endoderm, the highest AmphiRAR expression is in the middle third. Expression of AmphiRAR is upregulated by exogenous RA and completely downregulated by the RA antagonist BMS009. Moreover, BMS009 expands the pharynx posteriorly; the first three gill slit primordia are elongated and shifted posteriorly, but do not penetrate, and additional, non-penetrating gill slit primordia are induced. Thus, in an organism without neural crest, initiation and penetration of gill slits appear to be separate events mediated by distinct levels of RA signaling in the pharyngeal endoderm. Although these compounds have little effect on levels of AmphiTR2/4 expression, RA shifts pharyngeal expression of AmphiTR2/4 anteriorly, while BMS009 extends it posteriorly. Collectively, our results suggest a model for anteroposterior patterning of the amphioxus nerve cord and pharynx, which is probably applicable to vertebrates as well, in which a low anterior level of AmphiRAR (caused, at least in part, by competitive inhibition by AmphiTR2/4) is necessary for patterning the forebrain and formation of gill slits, the posterior extent of both being set by a sharp increase in the level of AmphiRAR. Supplemental data available on-line

The recent elucidation of several molecular paradigms by which retinoids regulate growth, differentiation, and apoptosis highlights their promise as drugs for cancer therapy and prevention. Several novel signaling pathways by which retinoids induce cell death have been identified recently. They comprise (a) the induction by RARα-selective retinoids of the tumor-selective death ligand TRAIL that kills acute promyelocytic leukemia (APL) cells in a paracrine mode of action, which is the cause of retinoic acid-induced apoptosis after maturation; (b) a novel RARα-independent rexinoid–PKA cross-talk that induces maturation of both ATRA-sensitive and ATRA-resistant APL cells and does not invoke ligand-induced alteration of PML-RARα signaling, stability, or compartmentalization; and (c) a novel rexinoid signaling pathway that triggers apoptosis of immature APL cells and may correspond to a default death pathway that is operative in the absence of “survival” factors. This rexinoid apoptosis is inhibited by RXR but not RAR antagonists and is distinct from that triggered by RAR agonists, which control cell maturation and postmaturation apoptosis. Here we discuss the promise of retinoids for cancer treatment and prevention with an emphasis on the recently identified mechanisms by which they control (cancer) cell proliferation.

The balance between cell proliferation and programmed cell death (apoptosis) determines body patterns during animal development and controls compartment sizes, tissue architecture and remodeling. The removal of primordial structures by apoptosis allows the organism to develop sex specifically and to adapt for novel functions at later stages; apoptosis also limits the size of evolving structures. It is a ubiquitous function that is essential for all cells. Although inappropriate regulation or execution of apoptosis leads to disease, such as cancer, there is now evidence for its great therapeutic potential. This would be particularly true if apoptosis could be targeted at defined cell compartments, rather than acting ubiquitously like chemotherapy. Here, we discuss the potential of nuclear receptor ligands, many of which act through their cognate receptors in defined body compartments as modulators of cell life and death, with special emphasis on the molecular pathways by which these receptors affect cell-cycle progression, survival and apoptosis.

Both the human retinoic acid receptor α (hRARα) and a constitutively active mutant (F318A) of the mouse retinoid X receptor α (mRXRαF318A) ligand-binding domains were separately overexpressed in Escherichia coli,copurified as a heterodimer in a two-step procedure, and cocrystallized with an RARα-specific antagonist by using polyethylene glycol 10,000 as precipitant. The crystals grew in the hexagonal space group P6122 displaying the unit cell parameters a= b= 116.6 Å and c= 207.8 Å. They diffracted X-ray to a limit of 2.2-Å resolution. The asymmetric unit comprises one heterodimer and the crystal contains 60% solvent. The structure was determined by molecular replacement and is currently being refined.

A mouse cDNA that encodes a nuclear DNA binding protein was identified by yeast two‐hybrid screening using the activation domain 2 of the nuclear receptor coactivator TIF2 as a bait. BLAST analysis revealed that the identified cDNA encodes a KDWK domain and contains sequences almost identical to three tryptic peptides of rat GMEB‐1 which together with the GMEB‐2 heterodimeric partner binds to the GME/CRE sequence (glucocorticoid modulatory element) of the tyrosine aminotransferase (TAT) promoter. Mouse GMEB‐1 is ubiquitously expressed in all the tissues examined. In vitro translated mGMEB‐1 bound specifically to GME oligonucleotides, either alone or as a heterodimer with rGMEB‐2. Transient transfection experiments with TAT promoter reporter genes suggest a potential role for mGMEB‐1 as a transcriptional regulator of the TAT promoter.

Many synthetic retinoids have been generated that exhibit a distinct pattern of agonist/antagonist activities with the three retinoic acid receptors (RARα, RARβ and RARγ). Because these retinoids are selective tools with which to dissect the pleiotropic functions of the natural pan-agonist, retinoic acid, and might constitute new therapeutic drugs, we have determined the structural basis of their receptor specificity and compared their activities in animal and yeast cells.

RAR-RXR heterodimeric complexes (RARαΔAB-RXRαΔAB) bound to cognate DR5 DNA response elements were purified to apparent structural and functional homogeneity using a novel versatile immobilized metal affinity chromatography (IMAC) copurification procedure. Dynamic light scattering studies indicated that the complexes were more than 85% monodisperse. By electron microscopy the negatively stained RAR-RXR–DNA complexes appeared homogeneous and corresponded to a dimeric arrangement of the molecules. Using heterodimers purified according to this procedure we demonstrate ligand binding of RXR in the context of the RAR-RXR heterodimer–DNA complex. The present copurification procedure is rapid and has yielded high quality heterodimer–DNA complexes suitable for both structural and biochemical studies.

In vitrobinding sites of retinoic acid receptors (RARs) were isolated from mouse genomic DNA by immunoprecipitation of receptor/DNA complexes. PuG(G/T)TCA half-site motifs, which constitute RA-responsive elements (RAREs), were identified in the immunoselected fragments (ISFs), some of which contained highly repetitive arrangements of this motif. Genomic Southern analysis of a number of ISFs showed them to be of a single or low copy number. Several, but not all, ISFs acted as ligand-dependent RAREs in transient transfection assays. Two ISFs with repetitive RARE motifs responded preferentially to 9-cisretinoic acid-liganded retinoid X receptor in the presence or absence of co-transfected RAR, while little activation was seen with RAR alone in the presence of either all-transor 9-cisretinoic acid. Another ISF, containing consensus TATA and CAAT box motifs, was shown to have RA-inducible promoter activity. The results suggest a high degree of promiscuity in response element recognition by retinoid receptors.

Multiple regulatory mechanisms assure that signal transduction, involving the nuclear receptor interface, results in an accurate regulation of the respective gene networks. These mechanisms involve selective expression of the cognate receptor and its binding to specific hormone response elements of target genes. However, superimposed onto this “simple” control of activity is interpretation of the signal by the multiple functional modules of a given receptor, based on a specific interplay with 1) various factors binding to complex target gene promoters, 2) cell‐specific transcription factors that mediate its enhancer function, and 3) other signaling pathways. This interpretation can be further modulated by the differential target gene specificities of receptor isoforms and, according to in vitro evidence, by factors that increase the efficiency of the receptor to interact with its response element. Thus, steroid hormone‐regulated gene transcription involves a multitude of interactive elements, as is expected from the central role of nuclear receptors in homeostasis, embryonic development, and differentiation.—Gronemeyer, H. Control of transcription activation by steroid hormone receptors. FASEB J.6: 2524‐2529; 1992.

Synthesis of 4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-[1,3]dioxolan-2-yl]-benzoic acid or SR11237, a specific ligand of Retinoid X Receptors, is described in a ³H-labelled version. The labelled retinoid was prepared in 5 steps and tritium was introduced through a final reduction of an aromatic dihalide moiety with ³H2. © 1997 John Wiley & Sons, Ltd.

Nuclear receptors constitute a superfamily of ligand-inducible transcription factors which respond to endocrine, paracrine and, possibly, autocrine signals. Multiple regulatory mechanisms assure that signal transduction results in an accurate regulation of the respective gene networks. Apart from selective expression of the cognate receptor and its binding to specific hormone response elements of target genes, additional mechanisms are responsible for the cell- and promoter-specific transcription activation. They are based on the "interpretation" of the signal by the multiple functional modules of a given receptor and involve a specific interplay with various factors binding to complex target gene promoters and cell-specific intermediary transcription factors that mediate the activity of the two receptor transcription activation functions, as well as homo- and heterodimerization, and interference with other signalling pathways. Moreover, a single ligand may initiate different gene programs due to the differential target gene specificities of nuclear receptor isoforms. Thus, signal transduction by nuclear receptors involves a multitude of interactive elements, as could have been expected from the central role of these signals in homeostasis, embryonic development and differentiation. Two distinct mechanisms are involved in anti-hormone action. Type I anti-hormones impair the activity of the transcription activation function, while type II antagonists impair DNA binding. Experiments aimed at an understanding of these mechanisms are discussed.

Unliganded bacterially expressed RXRα lacking the N-terminal region AB (apo-RXRαΔAB) was found in solution as an apparent mixture of 165 kDa tetramers and 42 kDa monomers which could be quantitatively separated by gel filtration and non-denaturing gel electrophoresis. Under identical conditions both liganded (holo-) and apo-RARαΔAB were present as single monomeric species. apo-RXRαΔAB tetramers, as well as dimers of the apo-RXR ligand binding domain (apo-LBD), dissociated readily into monomers when exposed to their cognate ligand 9-cisretinoic acid (9c-RA). The apo-RXRαΔAB tetramer bound only transiently to a cognate DR1 response element, and was converted into DR1-apo-RXRαΔAB homodimer complexes indistinguishable from those generated by cooperative DNA binding of apo-RXRαΔAB monomers. In the absence of DNA, the addition of 9c-RA greatly accelerated the formation of heterodimers with the apo-RARαΔAB heterodimerization partner. No RXRαΔAB or RARαΔAB homodimers could be observed in solution, but upon mixing of the two receptor monomers stable heterodimers could be isolated which bound to DR5 response elements in a highly cooperative manner. In these heterodimers, RXRαΔAB interacted with its cognate ligand as efficiently as in RXRαΔAB homodimers. The presence of ligand did not alter the stability of RXRαΔAB homodimer or RXRαΔAB-RARαΔAB heterodimer complexes on DR1 and DR5 response elements, respectively. These in vitrodata support a model in which RXR tetramers could serve as an inactive pool with the dual function of: (I) rapidly supplying large amounts of RXR heterodimerization partners upon 9c-RA generation; and (ii) allowing RXR homodimer formation on “accessible” cognate response elements in the absence of 9c-RA. These events may represent a ligand-dependent regulatory mechanism controlling the availability of the promiscuous RXR dimerization partner that is engaged in multiple nuclear receptor signalling pathways.

A 28-kDa fragment (residues 178–423) of the human retinoic acid receptor γ, hRARγ D3E, encompassing the ligand-binding domain (LBD) was overproduced inEscherichia coliand purified as a monomer to more than 95% purity and homogeneity. The Kdfor all-transretinoic acid binding was 0.6±0.1 nM. Crystals of the LBD complexed with all-transretinoic acid were grown at pH 7 from sodium acetate in the presence of detergents using the vapor diffusion method. They diffract to 2.0 Å using a synchrotron radiation (λ=0.91 Å) and belong to the tetragonal space group P41212 with unit cell parameters a=b=60.6 Å and c=155.3 Å, one monomer per asymmetric unit, a solvent content ofca.33%, and a Vmvalue of ≈2 Å3/dalton.

4-[5H-2,3-(2,5-Dimethyl-2,5-hexano)-5-methyldibenzo-[b,e][1,4]diazepin-11-yl]benzoic acid (HX600), as well as its oxa- (HX620) and thia- (HX630) analogs, enhanced the activity of retinoic acid and a receptor α (RARα)-selective agonist Am80 in HL-60 cell differentiation assays. HX600 synergizes with Am80 by binding to, and transactivating through, the RXR subunit of the RXR-RAR heterodimer. HX600 exhibited RXR pan-agonist activity in transient transfections with a DR1-based reporter gene and synergized with RA-bound RARα and RARβ in inducing transcription from a DR5-based reporter. In addition, all three compounds at high concentrations acted as RAR pan-antagonists in stably transfected RAR “reporter cells.” These efficient synergists bind only weakly with RXRsin vitro,suggesting that they are RXR-RAR heterodimer-selective activators. These HX retinoids exhibited dual functionality, since they affected signalling through both retinoid receptor families (RARs and RXRs).

The ability of nuclear receptors (NRs) to activate transcription of target genes requires the binding of cognate ligands to their ligand-binding domains (LBDs). Information provided by the three-dimensional structures of the unliganded RXRα and the liganded RARγ LBDs has been incorporated into a general alignment of the LBDs of all NRs. A twenty amino-acid region constitutes a NR-specif ic signature and contains most of the conserved residues that stabilize the core of the canonical fold of NR LBDs. A common ligand-binding pocket, involving predominantly hydrophobic residues, is inferred by homology modelling of the human RXRα and glucocorticoid receptor ligand-binding sites according to the RARγ holo-LBD structure. Mutant studies support these models, as well as a general mechanism for ligand-induced activation deduced from the comparison of the transcriptionally active RARγ holo- and inactive RXRα apo-LBD structures.

The existence of two progesterone receptor forms present in crude cytosol of chick oviduct has been demonstrated by photoaffinity labelling using [3H]R5020. On SDS—polyacrylamide gels these two forms exhibit app. Mr‐values of 79 000 and 109 000 corresponding to the progesterone receptor forms A and B. Peptide maps of photoaffinity‐labelled steroid receptors have been established by limited proteolysis with α‐chymotrypsin. The peptide map obtained for chick oviduct cytosol progesterone receptor crosslinked with [3H]R5020 proved to be the sum of peptides obtained from partially purified preparations of forms A and B. The peptide maps of both progesterone receptor forms were identical for peptides below the Mr‐value of form A, indicating extensive homology of the two forms. A significantly different peptide pattern was observed for the rat liver glucocorticoid receptor crosslinked with [3H]triamcinolone acetonide. Prolonged proteolysis with chymotrypsin gave rise to peptides with Mr‐values of 6000 and 10 000 from the hormone‐binding domain of progesterone and glucocorticoid receptors, respectively.

Steroid hormone recepTo Whom It May Concern:rs belong to a family of nuclear receptors that trigger transcriptional activation of target genes by specific binding to DNA recognition sequences, usually located in the 5'-flanking region of the target gene. Nuclear receptors appear to be segmented proteins1and extensive structure-function analyses have attempted to elucidate the functional significance of individual segments. Two of these regions have been defined as the domains responsible for recognition of responsive elements of target genes (region C) and hormone binding (region E) (refs 2–7). But the functional significance of the N-terminal region (A/B), which diverges extensively even for a given receptor between different species, has remained obscure. We have previously cloned, expressed and analysed the chicken progesterone receptor (cPR) (ref. 8). This receptor and its human homologue from T47D breast cancer cells are unique among the steroid hormone receptors in that two forms, A and B, are present in equal amounts in cytosolic extracts, the latter having the higher molecular weight9,10. For the chicken progesterone receptor, we have presented evidence suggesting that the cPR form A corresponds to an N-terminally truncated form of B (ref. 8). Here we report on the functional difference between the forms A and B in the transcriptional activation of two target genes.

The ability of nuclear receptors (NRs) to activate transcription of target genes requires the binding of cognate ligands to their ligand-binding domains (LBDs). Information provided by the three-dimensional structures of the unliganded RXRα and the liganded RARγ LBDs has been incorporated into a general alignment of the LBDs of all NRs. A twenty amino-acid region constitutes a NR-specif ic signature and contains most of the conserved residues that stabilize the core of the canonical fold of NR LBDs. A common ligand-binding pocket, involving predominantly hydrophobic residues, is inferred by homology modelling of the human RXRα and glucocorticoid receptor ligand-binding sites according to the RARγ holo-LBD structure. Mutant studies support these models, as well as a general mechanism for ligand-induced activation deduced from the comparison of the transcriptionally active RARγ holo- and inactive RXRα apo-LBD structures.

The existence of two progesterone receptor forms present in crude cytosol of chick oviduct has been demonstrated by photoaffinity labelling using [ 3H]R5020. On SDS—polyacrylamide gels these two forms exhibit app. Mr-values of 79 000 and 109 000 corresponding to the progesterone receptor forms A and B. Peptide maps of photoaffinity-labelled steroid receptors have been established by limited proteolysis with α-chymotrypsin. The peptide map obtained for chick oviduct cytosol progesterone receptor crosslinked with [ 3H]R5020 proved to be the sum of peptides obtained from partially purified preparations of forms A and B. The peptide maps of both progesterone receptor forms were identical for peptides below the Mr-value of form A, indicating extensive homology of the two forms. A significantly different peptide pattern was observed for the rat liver glucocorticoid receptor crosslinked with [ 3H]triamcinolone acetonide. Prolonged proteolysis with chymotrypsin gave rise to peptides with Mr-values of 6000 and 10 000 from the hormone-binding domain of progesterone and glucocorticoid receptors, respectively.

Synthesis of 4‐[2‐(5,5,8,8‐tetramethyl‐5,6,7,8‐tetrahydro‐naphthalen‐2‐yl)‐[1,3]dioxolan‐2‐yl]‐benzoic acid or SR11237, a specific ligand of Retinoid X Receptors, is described in a 3H‐labelled version. The labelled retinoid was prepared in 5 steps and tritium was introduced through a final reduction of an aromatic dihalide moiety with 3H2. © 1997 John Wiley & Sons, Ltd.

Validated therapies for older pts with AML could rely on intensive or low-intensity strategies. Patient selection for these options remains controversial. There is currently no validated biomarker which can been used to guide therapeutic decision. TP53mutations which are known to negatively impact AML pts outcome when treated with ICT, have been recently described as a positive prognosis factor for blast clearance with a 10-days regimen of decitabine (Welch, NEJM 2016). To date, it remains unclear whether AML pts with TP53mutation represent a clinically homogeneous group. Several classification systems of p53 mutant, derived from in vitro or in vivo data, have been validated in solid tumors and aggressive lymphomas as predictors of p53 mutant functional impact or patient outcome. We retrospectively evaluated the impact of TP53mutational status on the outcome of a real-world cohort of pts, treated frontline with standard doses of azacitidine (AZA). We further hypothesized that functional characterization of TP53mutations could define a subgroup of pts with specific outcome with AZA

Fornecker: Takeda: Honoraria; Servier: Honoraria.

Fornecker: Takeda: Honoraria; Servier: Honoraria.

Hypomethylating agents (HMA) azacitidine (AZA) and decitabine are approved low-intensity frontline therapy for AML pts, but patient selection between intensive versus low-intensity therapy remains mater of debate. To date, description of biomarker of response to HMA has brought few convincing findings. Most studies have focused on somatic mutations in candidate epigenetic genes such as DNMT3A, TET2, and IDH1/2or tumor suppressor TP53and led to controversies with none of this gene currently used to guide therapy decision in clinical practice. We performed an hypothesis-free, innovative, exome sequencing study to identify predictive biomarkers of outcome of AML pts treated with AZA.

DNA sequences regulate glucocorticoid receptor activity.

Apart from PML-RARa acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, in our study we show that elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists (“rexinoids”) the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cell lines and patients’ AML blasts. Protein kinase A activation leads to co-repressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in “de-subordination” of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARß is blunted in mouse embryo fibroblasts lacking RARs, but re-introduction of exogenous RARa re-establishes responsiveness, thus confirming that the RARa-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, the tumor necrosis factor-related apoptosis inducing ligand (TRAIL), both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of TRAIL and DR5 in AML patient blasts cultured “ex vivo”. AML patients’ blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and FAB classification. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Indeed, it is known that cAMP levels can be elevated also by treating cells with 3', 5'-cAMP phosphodiesterase inhibitors (PDEi’s). These observation and the clinical availability of the corresponding drugs provide a rationale for initiating clinical studies addressing the efficacy of combinatorial PDEi-rexinoid therapy in AML patients. Together with our recent finding that a very promising class of epigenetic anti-tumor drugs operates through activation of TRAIL expression (Nat Med2005, 11(1):7784), the possibility to target both the ligand (TRAIL) by HDAC inhibitors and the cognate receptors (DR4, DR5) by the above described rexinoid crosstalk may represent a promising therapeutic option which might lead, despite the genetic, morphologic, and clinical variability of AML, to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

Chimeric fusion proteins involving Retinoic Acid Receptor Alpha (RARa) invariantly associate with almost 100% of Acute Promyelocytic Leukemia (APL). While we and others have recently demonstrated an essential role of aberrant self-association in RARa fusions mediated transformation1,2, forced oligomerization of truncated transcription factors including MLL3 and AML14 has emerged as an important oncogenic property shared by these most common chimeric fusion proteins involved in AML. To further characterize the underlying mechanisms and its modes of action, the present study dissects the essential molecular properties and transformation mechanisms by oligomeric RARa-oncoproteins. Firstly, we define the oncogenic threshold of self-association by showing that homo-oligomerization rather than homo-dimerization is required for Stat5b-RARa mediated transformation of primary hematopoietic cells. Although constitutive transcriptional repression is believed to be the underlying mechanism for RARa-mediated transformation, oligomeric RARa fusion proteins can bind retinoic acid response elements (RAREs) either as homo-oligomers or higher order hetero-oligomers with RXRa. To gain further insights into the transcriptional control mediated by RARa fusions, we successfully constructed various Stat5b-RARa point mutants that maintained the homo-oligomerization properties but lost their abilities to bind DNA as homo-oligomers. These resulting mutants could still efficiently transform primary hematopoietic cells in spite of their lack of homo-oligomeric DNA binding ability. To investigate if this is unique to Stat5b-RARa or is a general phenomena that can also apply to other RARa fusions, we further demonstrated that the synthetic FKBP-RARa fusion, which closely mimick both biochemical and transformation properties of bona fide RARa fusion proteins, was incapable of binding DNA as homo-oligomers, suggesting that homo-oligomeric DNA-binding is dispensable for RARa-mediated transformation. Conversely we reveal that the higher-order RARa-fusion/RXRa hetero-oligomeric complex, which aberrantly recruits transcriptional co-repressor SMRT, represents the major functional DNA-binding module. Specific knockdown of RXRa expression using shRNA approach can suppress transformation mediated by both bona fide and synthetic RARa fusion proteins, thus indicating a critical functional role of RXRa in RARa-mediated transformation of primary hematopoietic cells. Finally to assess the possible therapeutic values of targeting RXRa-mediated pathways in RARa-mediated transformation, we also demonstrated specific inhibition of myeloid transformation mediated by various RARa oncoproteins using the panRXR-selective agonist, SR11237. Taken together, these results not only highlight the functional significance of higher-order RARa-fusion/RXRa hetero-oligomeric complex, but also attest RXRa as a potential therapeutic target for APL.

Apart from PML-RARα acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, in our study we show that elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists (“rexinoids”) the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cell lines and patients' AML blasts. Protein kinase A activation leads to co-repressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in “de-subordination” of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARβ is blunted in mouse embryo fibroblasts lacking RARs, but re-introduction of exogenous RARα re-establishes responsiveness, thus confirming that the RARα-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, the tumor necrosis factor-related apoptosis inducing ligand (TRAIL), both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of TRAIL and DR5 in AML patient blasts cultured “ex vivo”. AML patients' blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and FAB classification. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Indeed, it is known that cAMP levels can be elevated also by treating cells with 3′, 5′-cAMP phosphodiesterase inhibitors (PDEi's). These observation and the clinical availability of the corresponding drugs provide a rationale for initiating clinical studies addressing the efficacy of combinatorial PDEi-rexinoid therapy in AML patients. Together with our recent finding that a very promising class of epigenetic anti-tumor drugs operates through activation of TRAIL expression (Nat Med 2005, 11(1):7784), the possibility to target both the ligand (TRAIL) by HDAC inhibitors and the cognate receptors (DR4, DR5) by the above described rexinoid crosstalk may represent a promising therapeutic option which might lead, despite the genetic, morphologic, and clinical variability of AML, to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

Chimeric fusion proteins involving Retinoic Acid Receptor Alpha (RARα) invariantly associate with almost 100% of Acute Promyelocytic Leukemia (APL). While we and others have recently demonstrated an essential role of aberrant self-association in RARα fusions mediated transformation1,2, forced oligomerization of truncated transcription factors including MLL3and AML14has emerged as an important oncogenic property shared by these most common chimeric fusion proteins involved in AML. To further characterize the underlying mechanisms and its modes of action, the present study dissects the essential molecular properties and transformation mechanisms by oligomeric RARα-oncoproteins. Firstly, we define the oncogenic threshold of self-association by showing that homo-oligomerization rather than homo-dimerization is required for Stat5b-RARα mediated transformation of primary hematopoietic cells. Although constitutive transcriptional repression is believed to be the underlying mechanism for RARα-mediated transformation, oligomeric RARα fusion proteins can bind retinoic acid response elements (RAREs) either as homo-oligomers or higher order hetero-oligomers with RXRα. To gain further insights into the transcriptional control mediated by RARα fusions, we successfully constructed various Stat5b-RARα point mutants that maintained the homo-oligomerization properties but lost their abilities to bind DNA as homo-oligomers. These resulting mutants could still efficiently transform primary hematopoietic cells in spite of their lack of homo-oligomeric DNA binding ability. To investigate if this is unique to Stat5b-RARα or is a general phenomena that can also apply to other RARα fusions, we further demonstrated that the synthetic FKBP-RARα fusion, which closely mimick both biochemical and transformation properties of bona fide RARα fusion proteins, was incapable of binding DNA as homo-oligomers, suggesting that homo-oligomeric DNA-binding is dispensable for RARα-mediated transformation. Conversely we reveal that the higher-order RARα-fusion/RXRα hetero-oligomeric complex, which aberrantly recruits transcriptional co-repressor SMRT, represents the major functional DNA-binding module. Specific knockdown of RXRα expression using shRNA approach can suppress transformation mediated by both bona fide and synthetic RARα fusion proteins, thus indicating a critical functional role of RXRα in RARα-mediated transformation of primary hematopoietic cells. Finally to assess the possible therapeutic values of targeting RXRα-mediated pathways in RARα-mediated transformation, we also demonstrated specific inhibition of myeloid transformation mediated by various RARα oncoproteins using the panRXR-selective agonist, SR11237. Taken together, these results not only highlight the functional significance of higher-order RARα-fusion/RXRα hetero-oligomeric complex, but also attest RXRα as a potential therapeutic target for APL.

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