An emerging role for retinoid X receptor α in malignant hematopoiesis

Leukemia Research 36 (2012) 1075–1081

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Invited review

An emerging role for retinoid X receptor ␣ in malignant hematopoiesis Mariam Thomas a,b , Mahadeo A. Sukhai a , Suzanne Kamel-Reid a,b,c,∗ a

Princess Margaret Hospital/the Ontario Cancer Institute, University Health Network, Toronto, ON, Canada Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada c Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada b

a r t i c l e

i n f o

Article history: Received 21 August 2011 Received in revised form 13 April 2012 Accepted 21 May 2012 Available online 17 June 2012 Keywords: Acute promyelocytic leukemia Retinoid X receptor Retinoic acid Hematopoiesis

a b s t r a c t The retinoid X receptor alpha is the obligatory heterodimerization partner for a range of nuclear hormone receptors, and is required for signaling through the pathways mediated by those receptors. While RXR alpha has critical roles in embryonic development, it appears to be dispensable in adult hematopoiesis. Strikingly, recent evidence has indicated that proper functioning of RXR alpha is necessary for the pathogenesis of acute promyelocytic leukemia (APL), suggesting a novel avenue that can be exploited in the management and treatment of this disease. In this review we highlight recent studies that clarify the role of RXR alpha in normal and malignant hematopoiesis. © 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

Retinoid signaling in normal hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075 The role of RXR␣ in hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076 Malignant hematopoiesis: acute promyelocytic leukemia (APL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077 3.1. X-RAR␣ bind RXR␣ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077 3.2. X-RAR␣/RXR␣ in transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078 3.3. Direct gene targets of X-RAR␣ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078 3.4. In vivo evidence for the role of RXR␣ in APL pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078 4. Therapeutic potential of rexinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078 5. Targeting the oncogenic PML-RAR␣ in APL therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078 6. Conclusion: toward a new paradigm for RXR␣ in APL: future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079 Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079

1. Retinoid signaling in normal hematopoiesis Retinoid signaling proceeds through two classes of nuclear receptors, retinoid (RAR) and rexinoid (RXR), each of which are encoded by three genes, giving rise to closely related isoforms ␣, ␤, and ␥ [1]. Like other nuclear receptors, RARs consists of six evolutionarily conserved domains A–F, including the DNA binding domain that mediates binding to retinoic acid response elements

∗ Corresponding author at: Princess Margaret Hospital/Ontario Cancer Institute, Room 9-622, 610 University Avenue, Toronto, ON, M5G 2M9, Canada. Tel.: +1 416 946 4501x5039; fax: +1 416 340 3596. E-mail address: [email protected] (S. Kamel-Reid). 0145-2126/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2012.05.022

(RARE) [2]. The A/B (AF1) domain is a ligand-independent, promoter context-dependent transactivation domain [3]. The C domain contains the DNA binding domain, and the E domain contains the all trans retinoic acid (ATRA) binding site, the ligand-dependent transactivation domain (AF2), the retinoid X receptor alpha (RXR␣) dimerization interface and the nuclear co-repressor and coactivator binding sites [3]. RAREs are located in the promoter elements of retinoid target genes and consist of direct repeats (DRs) (A/G)G(G/T)TCA, separated by 2 or 5 nucleotides [4,5]. RAR/RXR heterodimerization is mediated by the DNA binding and ligand binding domains of RARs and is required for binding to RAREs [6]. Retinoid X receptors (RXRs) were identified as coregulators, required for the efficient binding of RARs to their response elements [7–9]. RXRs can affect multiple biological pathways because of

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their unique ability to heterodimerize with different nuclear receptors, including the thyroid hormone receptor (TR), peroxisome proliferator-activated receptor (PPAR), and vitamin D receptor (VDR) [10–13]. The three RXR subtypes (␣, ␤, and ␥) have a high degree of homology, suggesting that they have common target sequences, and respond to common ligands [14]. The subtypes differ in their expression patterns: RXR␣ and RXR␤ can be found in almost every tissue type, while RXR␥ is restricted to muscle and brain cells [14]. Retinoid signaling plays an important role in the development and differentiation of a number of tissues in the body, including the eye, the heart and circulatory systems, the central nervous system, the urogenital and respiratory systems, the musculoskeletal system and hematopoiesis [15,16]. Retinoid signaling has been implicated in hematopoiesis, specifically terminal neutrophil differentiation, through a number of different lines of investigation [17–20]. Additional means of retinoid-mediated control of hematopoiesis include control of retinoic acid metabolism, and non-ligand-mediated crosstalk among hematopoietic signaling pathways. Numerous studies involving vitamin-A deficient animal systems and in vitro culture models clearly establish a role for retinoids in controlling signaling pathways involved in hematopoiesis [21]. Active retinoid signaling starts with cellular enzymes and retinoic acid binding proteins, which mediate the processing of dietary retinol (vitamin A) to retinoic acid [21]. The different rates of retinol metabolism in hematopoietic cells constitute a means of regulating cellular levels of retinoic acid. This ensures that RAR transcriptional activity is differentially regulated in hematopoietic cells in spite of their exposure to uniform physiological concentrations of retinoids (1–10 nM) in the serum [22]. In the absence of the retinoic acid (RA) ligand, the transcriptional activity of the RAR/RXR heterodimer is inhibited by the binding of co-repressors, including nuclear receptor co-repressor (N-CoR), silencing mediator for retinoid and thyroid receptors (SMRT) and histone deacetylase (HDAC)-containing Sin3A complexes [23]. Binding of all-trans retinoic acid (ATRA) to RAR creates a conformational transition in the RAR ligand binding domain [24]. This disrupts the co-repressor interaction and promotes the sequential recruitment of co-activator complexes, which remodel chromatin to facilitate binding of the transcriptional machinery on the promoter. Co-activators like p300/CBP, locally modify chromatin structure through their histone acetylase (HAT) activity, which acetylates lysine residues on the N-terminal tails of histones and weakens their interaction with DNA, allowing for the activation of gene transcription [25]. Non-ligand mediated activation of RARs have also been recorded at different stages of myelopoiesis [22]. These include interactions of RARs with transcription factors like the STAT family members and their synergism with other receptors such as the protein kinase A (PKA) receptor [22]. A number of hematopoietic cytokines including IL-3, GM-CSF, and IL-1, have been reported to enhance transcriptional activity of RA receptors [26]. IL-3 and GM-CSF mediate their cellular effects by activation of the JAK/STAT pathway. Activated cytokine receptors phosphorylate STATs by their association with JAKs. STATs then translocate to the nucleus, where they act as transcription factors [27,28]. Recent observations revealed significant functional crosstalk between RARs and STAT receptors, as seen by the direct role of STAT5 [29] in mediating IL-3 induced enhancement of RAR activity [30]. Furthermore, a number of overlapping STAT/RAR binding sites have been reported in the RAREs of different genes and illustrate the role of non-ligand mediated activation of RAR transcriptional activities [29].

2. The role of RXR␣ in hematopoiesis While the role of RAR␣ in hematopoiesis is clear, the role of RXRs in myeloid cell differentiation is not well understood beyond their function as obligatory heterodimerization partners for RARs [31]. This is partially because of the complexity of the system; different blood cell lineages express different nuclear receptors that heterodimerize with RXR␣, thus making the phenotype of RXR␣ deficient hematopoiesis difficult to analyze. The other major problem is one of construction of the experimental system: the Rxr␣ −/− mouse is embryonic lethal at ED17.5 due to myocardial defects [32]. This is attributed to the critical roles RXR␣ plays throughout the rest of the organism, though it renders it impossible to study the role of RXR␣ in adult hematopoiesis. Some of what is known about the role of RXR␣ in hematopoiesis is summarized in Table 1 [33–40]. Some of the more recent results, in particular, are intriguing: conditional knockout of RXR␣ in the hematopoietic compartment [37] did not lead to an abnormal hematopoietic phenotype in vivo, suggesting that RXR␣ is not critical for normal adult hematopoiesis. However, down-regulation of RXR␣ is essential for terminal neutrophil differentiation, as RXR␣ is highly expressed in granulocyte/monocytes progenitors, and in terminally differentiated monocytes, but not in mature granulocytes [39]. Furthermore, the ectopic over-expression of RXR␣ in granulocyte-monocyte (GM) progenitors resulted in blocked neutrophil differentiation, while the expression of a dominant negative form of RXR␣ did not impair neutrophil differentiation. Taken together, these data raise intriguing possibilities with respect to the role of RXR␣ in myelopoiesis, and suggest that attempts to knock down RXR␣ expression or function in vivo may have the opposite effect to what has previously been hypothesized in the literature. Much of the in vivo effects of RARs in hematopoiesis have been elucidated using RA receptor knock-out, and vitamin A deficient (VAD), mouse models, e.g. [22]. The engineering of RAR and RXR single and compound knockout mice have allowed for the examination of the roles of these receptors in development and differentiation. A list of some of these established models is presented in Table 2 [32,41–48]. A number of studies have reported the creation and phenotype of RXR␣ mutant mice to elucidate the role of rexinoid signaling during development. These studies have reported the use of both Table 1 Evidence for the involvement of RXR␣ in myelopoiesis. Evidence

Study

In HL-60 cells, activation of RXR signaling was shown to be required for the induction of apoptosis, and a similar link was reported in the NB4 cell line.

Mehta et al. [35]; Shiohara et al. [38]; Nagy et al. [36]; Benoit et al. [34] Benoit et al. [33]

Evidence of crosstalk between the RXR and protein kinase A (PKA) signaling pathways, as activation of both RXR and PKA, using specific ligands and agonists, was found to induce cell maturation in NB4 cells Conditional knockout of RXR␣ in the hematopoietic compartment. Strikingly, these mice did not exhibit an abnormal hematopoietic phenotype in vivo, suggesting that RXR␣ is dispensable for normal adult hematopoiesis. Down-regulation of RXR␣ is essential for terminal neutrophil differentiation, as RXR␣ is highly expressed in granulocyte/monocytes progenitors, and in terminally differentiated monocytes, but not in mature granulocytes. Furthermore, the ectopic over-expression of RXR␣ in GM progenitors resulted in a block of neutrophil differentiation, while the expression of a dominant negative form of RXR␣, lacking amino acids 1-197, did not impair neutrophil differentiation.

Ricote et al. [37]

Taschner et al. [39]

M. Thomas et al. / Leukemia Research 36 (2012) 1075–1081 Table 2 In vivo models of RAR and RXR deficiency in hematopoiesis [for a discussion of conditional knockout models, please see the text]. Experiment

Study

VAD mice develop an expansion of myeloid cells with characteristics of terminally differentiated granulocytes, in the bone marrow, spleen and peripheral blood This myeloid expansion was found to be relieved with ATRA treatment, which, the authors suggested, was due the role of ATRA in granulocytic apoptosis Mice with selective knockout of the RAR␣1 isoform develop normally with no defects in hematopoiesis Mice with disruptions in both isoforms of RAR␣ exhibit early postnatal lethality, but no hematopoietic abnormalities RAR␥ homozygous knockout mice also do not have gross hematopoietic disruptions, but have defective stem cell maintenance RAR␣ and RAR␥ double knockouts die in utero, thereby restricting the study of hematopoiesis to the fetal liver in these models These mice have normal granulopoiesis as seen by the presence of mature granulocytes in the fetal liver and did not exhibit a compensatory increase in RXR␤ expression to offset the deficiency in RAR␣ and RAR␥ Targeted loss of function mutation in the RXR␣ gene in the mouse germ line resulted in embryonic lethality between E13.5 and E16.5. This lethality was attributed to defects in the ventricular chamber of the heart leading to extremely thin ventricular walls Mice expressing a dominant negative form of RXR␤ in myeloid cells were reported to have severe arrest in maturation at the promyelocyte stage of myeloid differentiation in 1 of 12 mice used in the study, while 3 other mice exhibited mild perturbations in myeloid development, suggesting a function for RXRs in myelopoiesis

Kuwata et al. [42]

Li et al. [43] Lufkin et al. [46]

Lohnes et al. [44]; Purton [47] Lohnes et al. [45]

Sucov et al. [32]; Kastner et al. [41]

Sunaga et al. [48]

complete knockouts as well as tissue specific, temporally regulated RXR␣ disruption in the mice. Tissue specific functional knockout of RXR␣ was created to study RXR␣ effects specifically in the ventricular chamber of the heart [49], in epidermal and hair follicle keratinocytes [50,51], in thymocytes and T-lymphocytes [52], and in hepatocytes [53,54]. The mouse models used in these studies all employ the cre-loxP strategy to generate tissue specific knockouts. We used this system to selectively knock out RXR␣ in the hematopoietic compartment, using mice with exon 4 of RXR␣ flanked with loxP sites, also expressing cre under the Flk-1 promoter [55]. Kastner et al. [41] reported the use of RXR␣ null mice with a disruption in exon 4. A similar report of a complete RXR␣ knockout using mice that lacked part of exon 3 (encoding part of the DNA-binding domain) was also reported by [32]. Others have looked at the role of other domains of the protein including the AF1 [56], and AF-2 [57], using transgenic mice lacking these domains of RXR␣. Compound knockout mice exhibit a wider array of abnormalities, and are often embryonic lethal.

3. Malignant hematopoiesis: acute promyelocytic leukemia (APL) Acute promyelocytic leukemia (APL) accounts for ∼10% of all acute myelogenous leukemia (AML) cases worldwide. It is characterized by accumulation of abnormal hematopoietic cells with promyelocytic features in the bone marrow, as well as balanced chromosomal translocations involving the retinoic acid receptor alpha (RAR˛) locus on chromosome 17q21 described below. Normal

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hematopoiesis is inhibited, leading to pancytopenia, and diminished immunity. More than 99% of APL cases involve a translocation t(15;17)(q22;q21) between the promyelocytic leukemia (PML) and RAR˛ genes [58–60]. Other “variant” partner genes have been characterized: PLZF [61]; NPM [62,63]; NuMA [64,65]; STAT5b [66]; PRKAR1A [67]; FIP1L1 [68]; and BCOR [69]. These translocations result in the creation of a functional chimeric protein, whose N-terminus is derived from the partner (“X”) gene, and retains the X protein’s oligomerization domain; and whose C-terminus is derived from RAR␣ [70]. X-RAR␣, therefore, can be viewed as aberrant transcription factors, containing heterologous oligomerization and protein-protein interaction domains fused to an N-terminal truncated RAR␣. For a number of years, the prevailing hypothesis was that this forced homo-dimerization of RAR␣ was responsible for the development of APL; however, while the presence of an oligomerization domain confers oncogenic potential on the fusion protein [71], this is not sufficient for leukemogenesis in vivo [72]. It has been hypothesized that APL fusion proteins aberrantly repress gene transcription and hence de-regulate genes important in myeloid differentiation, resulting in the observed block in maturation [70]. In many cases, the differentiation block can be overcome by treatment with pharmacological doses of ATRA (>10−7 M) [70]. The APL fusion proteins have altered DNA binding properties [73] and can bind RAREs as homodimers [74], while wild type RAR␣ does not [8]. The impaired ability of APL fusion proteins to activate certain promoters used to be explained by their increased ability to bind corepressors SMRT and N-CoR, thus requiring pharmacological doses of ATRA for dissociation. Very recent evidence also suggests that PML-RAR␣ and PLZF-RAR␣ can recognize and bind to other splice variants of SMRT and NCoR that are not recognized by wild type RAR␣ [75]. These data suggest that the acquired ability to interact with alternative splice variants of NCoR and SMRT contributes to the oncogenicity of the APL fusion proteins. The presence of RXR␣ in heterotetrameric complexes of APL fusions favored correpressor recruitment and binding, in stark contrast to wild type RAR␣-RXR␣ heterodimers [75]. In addition to release of corepressors and stimulation of genes responsible for myeloid differentiation, ATRA also acts to degrade the PML-RAR␣ protein and upregulate wild-type RARs to restore retinoid signaling. Recent studies have shed light on an increasingly important role for RXR␣ in APL pathogenesis. X-RAR␣ have the capability of forming homodimers, as reported for PML-RAR␣ [74,76], PLZFRAR␣ [77,78], NPM-RAR␣ [79], and NuMA-RAR␣ [[80]; and our own work, unpublished]. In a previous study [81], we showed that XRAR␣ can form heterodimers with RXR␣, as well as the wild-type X protein. Until very recently, this interaction was not thought to be important in leukemogenesis. Recent reports, however, when taken together, allow us to view the role of RXR␣ in APL, and more broadly, AML, from a novel perspective. 3.1. X-RAR˛ bind RXR˛ All APL fusions retain the RAR␣/RXR␣ dimerization interface found in wild-type RAR␣. A physical interaction between X-RAR␣ and RXR␣ is therefore expected, and indeed, observed, for all fusions studied. PML-RAR␣ is associated with nuclear complexes that are much greater in apparent molecular weight than PMLRAR␣ alone [82]. Wild-type PML and RXR␣ were identified as some of the proteins associated with these complexes [74,82]. Our previous studies demonstrated physical interaction between NPM- and NuMA-RAR␣, and RXR␣ [81]. The latter finding was independently corroborated in a separate study [80,83]. This physical interaction between X-RAR␣ and RXR␣ was preserved after ATRA treatment, as one can follow the mobilization of the X-RAR␣/RXR␣ complex

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within the cell after treatment with pharmacological concentrations of RA [83]. 3.2. X-RAR˛/RXR˛ in transcription NPM-RAR␣ and NuMA-RAR␣ form heterodimeric complexes with RXR␣, which are capable of binding to and repressing transcription from the RARE [84]. PML-RAR␣ binds a wider range of DNA response elements in the genome than the retinoic acid response element (RARE) [85]. A number of these elements are required by other nuclear hormone receptors in order to bind DNA. While RAR␣, as a heterodimer with RXR␣, will only bind DR2 and DR5 elements, PML-RAR␣ has been shown to bind a range of Direct Repeat (DR), Everted Repeat (ER) and Inverted Repeat (IR) sequences in vitro [74,76,85]. Our group demonstrated this relaxed DNA binding specificity for all X-RAR␣, specifically for the DR1 PPRE, as well as for NPM-RAR␣ with DR3 and DR4 elements [84]. PML-RAR␣ requires RXR␣ in the transcriptional complex [85], suggesting that the fusion does not merely sequester RXR␣ away from its sites of action within the cell, but instead co-opts it in order to have a more direct effect at the gene expression level. PLZF-RAR␣/RXR␣ heterodimers bind to RAREs with higher affinity than PLZF-RAR␣ homodimers in vitro [86,87]. Furthermore, PLZF-RAR␣/RXR␣ heterodimers have the capacity to bind to non-consensus RAREs, thus suggesting that these heterodimers contribute to APL pathogenesis through the regulation of novel gene expression [88,89]. Recently, it has been demonstrated that STAT5b-RAR␣ requires RXR␣ for strong association with HDAC complexes and transcriptional repression [90]. More recently, it was shown that the capacity for leukemic transformation by the PRKAR1A-RAR␣ variant fusion also requires interaction with RXR␣ [91]. RXR␣ is also required for the formation of the BCOR-RAR␣ complex on RAREs [69]. 3.3. Direct gene targets of X-RAR˛ More recent evidence, employing chromatin immunoprecipitation followed by array analysis (ChIP-Chip) assessing direct gene targets of PML-RAR␣, confirm that the fusion induces a repressive mark on its target promoters, including the recruitment of HDAC1 and aberrant methylation of histones [92]. Interestingly Wang et al. showed that more than half of PML-RAR␣ targeted promoted regions also contained PU.1 motifs [93]. The fusions therefore deregulated PU.1 mediated transactivation of target genes [93]. The presence of RXR␣ in the PML-RAR␣ oncogenic complex has also been confirmed by ChIP-seq and ChIP-Chip studies that show the colocalization of RXR␣ to promoter elements occupied by PMLRAR␣ [93,94]. Both studies showed that the majority of PML-RAR␣ binding sites do not contain canonical RAREs, but instead contain other binding elements and motifs. Rice et al. extended these finding to PLZF-RAR␣, where they observe that only a minor fraction of gene promotors that were bound by PLZF-RAR␣ contained classical RARE sequences [95]. Their dataset also revealed the presence of promoter regions targeted by other nuclear hormone receptors among those regions occupied by the fusion, raising the possibility that the fusion can bind and deregulate expression of other signaling pathways [95]. More recently, Spicuglia et al. conducted similar ChIP-Chip studies to identify sites specifically bound by PLZF-RAR␣, while distinguishing between genomic regions bound by PLZF-RAR␣ and wild type PLZF [96].

not always correlate with their in vivo phenotypes [72]. Two independent studies used different genetic approaches in transgenic mouse models to assess the role of RXR␣ in leukemia in vivo. One study [97] targeted the PML-RAR␣/RXR␣ interaction by creating a PML-RAR␣ mutant mouse model lacking the RXR␣ dimerization interface in the RAR␣ moiety of the fusion. In these mice, PML-RAR␣ would therefore be incapable of dimerization with RXR␣. Our own study [55], undertaken at the same time, created a conditional functionally inert mutant of RXR␣ in mice expressing NuMA-RAR␣. To elucidate the role of RXR␣ in APL, we conditionally knocked out RXR˛ in the hCG-NuMA-RAR˛ APL mouse model [98]. Strikingly, in both in vivo models, mice failed to develop leukemia. We therefore propose that the APL fusion proteins cooperate with RXR␣ in the development of leukemia, and suggest a novel role for RXR␣ in the pathogenesis of APL. 4. Therapeutic potential of rexinoids The basis of differentiation therapy is to force cells along the normal differentiation pathway through the restoration/reactivation of signal transduction pathways that are otherwise suppressed during tumor development. Guiding cells through the differentiation lineage will eventually result in post-differentiation induced cell death. The APL fusion protein complex consists of higher order hetero-oligomers with RXR␣ as described above. In addition to the classically targeted RAR␣ moiety, studies by two groups [90,97] have implicated RXR␣ as a potential therapeutic target. In APL blasts, RA can trigger a death signaling cascade through the activation of IFN regulated factor-1, which is recruited to the promoter elements of TRAIL, which along with Death Receptor 5 (DR5) and DR4, selectively targets and kills tumor cells [99]. As a result of their lower toxicity profile compared to retinoids, rexinoids are preferred as drug candidates [100]. A characteristic feature of RXRs is the inability of RXR agonists to transactivate RAR-RXR signaling when used as a single agent. This process referred to as “RXR subordination” only allows RXR agonists to enhance the retinoid response initiated by an RAR agonist, and not to be able to transactivate signaling from RAR-RXR heterodimers as a single agent [101]. Altucci et al. demonstrated that corepressor complexes can be dissociated from APL heterodimers when PKA is activated, thereby allowing rexinoids to induce transactivation of gene expression through coactivator recruitment [102]. Benoit et al. also showed that raising intracellular cAMP levels allows RXR ligands to induce differentiation in APL cells that have developed RA resistance [33]. Despite RXR subordination, rexinoids can act through different mechanisms to activate RXR signaling. In the presence of increased cAMP levels, rexinoid signaling can induce differentiation and post maturation cell death [33]. This is the case even in ATRA resistant AML cells, suggesting that this mechanism of rexinoid activation is acting independent of retinoid signaling. RXR agonists can however activate other signaling pathways, as RXR is known to heterodimerize with other proteins including VDR and PPAR␥ [101]. Rexinoids induce cell death in AML cells under reduced serum and growth factor conditions [34]. This signaling was shown to be mediated through rexinoid mediated activation of permissive RXR-PPAR␥ heterodimers [103,104]. These studies indicate that rexinoid mediated pathways mediated through RAR-RXR as well as other RXR dimers can be targeted to result in the activation of pathways leading to cellular differentiation, or cell death.

3.4. In vivo evidence for the role of RXR˛ in APL pathogenesis 5. Targeting the oncogenic PML-RAR␣ in APL therapy While the above data, when taken together, present a compelling case for the necessary role of RXR␣ in leukemogenesis, these studies were all performed in vitro. As has been reported in other studies, the in vitro characteristics of the APL fusions do

Induction of cellular differentiation was classically thought to be the basis by which ATRA therapy worked effectively in patients presenting with APL, as RA induces rapid differentiation of primary

M. Thomas et al. / Leukemia Research 36 (2012) 1075–1081

blasts into terminally differentiated granulocytes. Data from recent studies have called for a re-evaluation of the importance of differentiation in mediating treatment efficacy in APL. The classical model of APL implicated the fusion proteins in cellular transformation through transcriptional repression of myeloid specific target genes. In this model, ATRA worked by converting the fusion into a transcriptional activator and restoring gene expression of targets, and therefore clearing the disease by promoting cellular differentiation. It has been pointed out however that, despite ATRA’s success in APL, in most patients, ATRA treatment alone is not effective in inducing complete remission unless combined with chemotherapy [105]. This suggests that the differentiation process alone does not induce stable depletion of APL cells. In vivo, much higher concentrations of ATRA are required for clearing APL cells, compared to smaller amounts used for inducing transcriptional activation [106]. In the case of the RA resistant fusion PLZF-RAR␣, the resistance was thought to result from a stronger repression of target genes by the fusion [77,107,108]. Recent evidence and our own unpublished observations indicate that PLZF-RAR␣ cells can fully differentiate upon RA treatment [95,109]. Arsenic trioxide (ATO) has emerged as a potent anti-leukemia therapy with profound effects on APL cell clearance, even when administered as a single agent [110]. It does little to affect gene expression of PML-RAR␣ target genes [111], and only partially restores differentiation, but is very effective in the treatment of relapsed APL cases. These data suggest that the block in differentiation alone may not be driving leukemogenesis and that only reversing this inhibition may be insufficient for APL therapy. Importantly, both ATRA and ATO result in degradation of the fusion oncoprotein [112]. In addition to inducing a block in differentiation, the oncogenic fusion PML-RAR␣ exerts self-renewal properties on the leukemic cell [113]. Self-renewal is a key feature of leukemia initiating cells (LICs) [114]. The LIC population has to be targeted for effective tumor eradication, as their persistence after conventional therapy strongly contributes to disease recurrence [114]. Studies in mouse models have shown that mutations in PML-RAR␣’s phosphorylation site S873, which interferes with PML-RAR␣ degradation, impaired disease remission and clearance, while differentiation induction by RA treatment remained unaffected [109]. Both ATRA and arsenic trioxide target the LIC self-renewal capability to varying degrees [115], and also degrade the oncogenic PML-RAR␣. They are synergistic in their effects in mouse models, as would be expected from the fact that they target the oncoprotein through two different mechanisms [115]. This evidence suggests that fusion protein degradation may be the primary factor in disease clearance and effective therapy in APL.

6. Conclusion: toward a new paradigm for RXR␣ in APL: future perspectives Initially, during the development of the aberrant transcriptional repression model of APL pathogenesis, the APL fusion proteins were thought to act as homodimers to aberrantly repress retinoid target gene expression, and thus block neutrophil differentiation. However, as discussed above, recent lines of evidence have indicated that X-RAR␣ require RXR␣ in the transcriptional complex, suggesting that the fusion does not merely sequester RXR␣ away from its sites of action within the cell, but instead co-opts it in order to have a more direct effect at the gene expression level. Several significant questions in this area remain to be addressed. Importantly, while the role of RXR␣ in APL is beginning to be unraveled, its role in AML more generally still needs to be elucidated. Furthermore, whether the interaction and sequestration of RXR␣ by the APL fusions extends to direct transcriptional interference

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in RXR␣-mediated signaling pathways also remains to be seen. Finally, the relevance of RXR␣ as a therapeutic target in APL and AML still needs to be addressed using in vivo preclinical models and ultimately in clinical trials. Conflict of interest statement The authors have no conflicts of interest to disclose. Acknowledgments The authors would like to thank Dr. Patricia Reis for careful reading of this manuscript. M.T. is a Canadian Institutes of Health Research (CIHR) Canada Graduate Scholar. M.A.S. is a post-doctoral research fellow of the CIHR. Contributions. MT, MAS, and SKR wrote and edited the manuscript. References [1] Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83(6):841–50. [2] Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J 1996;10(9):940–54. [3] Nagpal S, Friant S, Nakshatri H, Chambon P. RARs and RXRs: evidence for two autonomous transactivation functions (AF-1 and AF-2) and heterodimerization in vivo. EMBO J 1993;12(6):2349–60. [4] Naar AM, Boutin JM, Lipkin SM, Yu VC, Holloway JM, Glass CK, et al. The orientation and spacing of core DNA-binding motifs dictate selective transcriptional responses to three nuclear receptors. Cell 1991;65(7):1267–79. [5] Umesono K, Murakami KK, Thompson CC, Evans RM. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell 1991;65(7):1255–66. [6] Zechel C, Shen XQ, Chen JY, Chen ZP, Chambon P, Gronemeyer H. The dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of the full-length receptors to direct repeats. EMBO J 1994;13(6):1425–33. [7] Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM, et al. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 1991;67(6):1251–66. [8] Leid M, Kastner P, Lyons R, Nakshatri H, Saunders M, Zacharewski T, et al. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell 1992;68(2):377–95. [9] Hallenbeck PL, Marks MS, Lippoldt RE, Ozato K, Nikodem VM. Heterodimerization of thyroid hormone (TH) receptor with H-2RIIBP (RXR beta) enhances DNA binding and TH-dependent transcriptional activation. Proc Natl Acad Sci USA 1992;89(12):5572–6. [10] Berrodin TJ, Marks MS, Ozato K, Linney E, Lazar MA. Heterodimerization among thyroid hormone receptor, retinoic acid receptor, retinoid X receptor, chicken ovalbumin upstream promoter transcription factor, and an endogenous liver protein. Mol Endocrinol 1992;6(9):1468–78. [11] Bugge TH, Pohl J, Lonnoy O, Stunnenberg HG. RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors. EMBO J 1992;11(4):1409–18. [12] Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM. Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 1992;355(6359):446–9. [13] Gearing KL, Gottlicher M, Teboul M, Widmark E, Gustafsson JA. Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proc Natl Acad Sci USA 1993;90(4):1440–4. [14] Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro AE, et al. Characterization of three RXR genes that mediate the action of 9-cis retinoic acid. Genes Dev 1992;6(3):329–44. [15] Mark M, Ghyselinck NB, Wendling O, Dupe V, Mascrez B, Kastner P, et al. A genetic dissection of the retinoid signalling pathway in the mouse. Proc Nutr Soc 1999;58(3):609–13. [16] Ross SA, McCaffery PJ, Drager UC, De Luca LM. Retinoids in embryonal development. Physiol Rev 2000;80(3):1021–54. [17] Labrecque J, Allan D, Chambon P, Iscove NN, Lohnes D, Hoang T. Impaired granulocytic differentiation in vitro in hematopoietic cells lacking retinoic acid receptors alpha1 and gamma. Blood 1998;92(2):607–15. [18] Gratas C, Menot ML, Dresch C, Chomienne C. Retinoid acid supports granulocytic but not erythroid differentiation of myeloid progenitors in normal bone marrow cells. Leukemia 1993;7(8):1156–62. [19] Hodges RE, Sauberlich HE, Canham JE, Wallace DL, Rucker RB, Mejia LA, et al. Hematopoietic studies in vitamin A deficiency. Am J Clin Nutr 1978;31(5):876–85.

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