Acute promyelocytic leukemia: New issues on pathogenesis and treatment response

The International Journal of Biochemistry & Cell Biology 39 (2007) 1063–1070

Medicine in focus

Acute promyelocytic leukemia: New issues on pathogenesis and treatment response Dominique Vitoux ∗ , Rihab Nasr, Hugues de The CNRS UMR 7151, Universit´e Paris 7, Equipe labellis´ee par la Ligue Nationale contre le Cancer, Hˆopital Saint-Louis (APHP), 1 av Claude Vellefaux, 75475 Paris Cedex 10, France Received 21 September 2006; received in revised form 21 December 2006; accepted 1 January 2007 Available online 21 March 2007

Abstract Pathogenesis of acute promyelocytic leukemia appears to be one of the best understood among human malignancies. The ability of retinoic acid (RA) and arsenic trioxide to directly target the oncogenic promyelocytic leukemia-retinoic receptor A (PMLRARA) fusion protein also made this disease the first model for oncogene-targeted therapies. A set of recent data has significantly increased the complexity of our view of acute promyelocytic leukemia pathogenesis, as well as of therapeutic response. This review summarizes and discusses these findings, which yield novels questions and models. © 2007 Elsevier Ltd. All rights reserved. Keywords: Acute promyelocytic leukemia; PML-RARA; Retinoic acid; Arsenic; cAMP

1. Introduction Acute promyelocytic leukemia (APL) is associated with two cardinal features: a granulocytic differentiation block and reciprocal and balanced chromosomal translocations that always imply a rearrangement of the retinoic acid receptor A (RARA) gene. The fact that APL is always associated with structural alterations of the RARA gene points to the importance of this nuclear receptor in the leukemia transformation process. The translocation t(15,17) (q21;q22), which fuses the RARA receptor with the PML gene is by far the most frequent, representing over 98% of APL. Of the other rare translocations, the t(11,17) yielding the PLZF-RARA fusion is by far the most common. The translocations generat-

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1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.01.028

ing NuMa-RARA, NPM-RARA or STAT5b-RARA are exceptional. While there is conclusive evidence that the reciprocal RARA-PLZF fusion is constantly expressed in patients and participates to the determination of the myeloid phenotype in transgenic mice, evidence is much weaker for the other RARA-X fusions (see for review (Pandolfi, 2001)). Retinoid signalling is transduced by two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR) which form RXR/RAR heterodimers. In the absence of its ligand, RXR/RARA binds on the target gene promoters via specific response elements typically composed with two direct repeats (DR) of a consensus hexameric motif 5 -PuG(G/T)TCA spaced by two or five base pairs (DR2 or DR5). Unliganded, DNA-bound RXR/RARA represses transcription by recruiting the corepressors N-CoR, SMRT and histone deacetylase. When ligand binds to the complex, it induces a conformational change allowing the recruitment of co-activators, of histone acetyltransferases and

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of the basic transcription machinery. Although RARA seems dispensable for granulopoiesis, because the rara −/− mice have normal granulocytes (Kastner et al., 2001), RA accelerates myeloid differentiation in only rara +/+ mice. Yet, curiously, enforced expression of RARA in myeloid progenitor cells blocks myeloid differentiation (Du, Redner, Cooke & Lavau, 1999). Over 20 years, several effective pharmacological agents, which induce leukemic blasts differentiation and apoptosis, have been successfully developed to treat this disease. The compounds currently used in clinical practice are not only all-trans retinoic acid (RA), but also arsenic trioxide, although histone deacetylase inhibitors (HDACI), DNA methylase inhibitors, rexinoids and phosphodiesterase or MEKK inhibitors are also in preclinical development, alone or in combination. Interestingly, these molecules appear to directly or indirectly target PML-RARA function. Elucidation of the molecular mechanisms underlying the differentiation block in APL is an important challenge to develop new therapeutic strategies, which could also show some activity in other myeloid leukemias. 2. Pathogenesis That APL incidence does not significantly increase with age, suggests that a single rate-limiting genetic event underlies pathogenesis of the disease (Vickers, Jackson & Taylor, 2000). In fact, studies of secondary leukemias, observed in patients treated with chemotherapy for solid tumours, have shown a mean time of only 1 year between drugs exposure and leukemia development (Mistry et al., 2005). One of the major experimental arguments to establish the role of these fusions in leukemic transformation is that transgenic mice expressing PML-RARA develop a malignancy, which accurately recapitulates the APL phenotype (Brown et al., 1997). Both PML-RARA and PLZF-RARA bind RA, RXR or DNA with a normal affinity. Despite initial discordances, it was later accepted that PML-RARA and PLZF-RARA behave as potent transcriptional repressors of retinoic acid signalling, but that supra-physiological doses of RA can overcome this repression. PML-RARAspecific response elements or RARs null fibroblasts have conclusively demonstrated that PML-RARA can behave as a bonae fidae transcriptional activator in the presence of RA (Zhou et al., 2006). Transcriptional repression was shown to be the consequence of a greatly enhanced binding of X-RARA homodimers to the SMRT/NCOR corepressors (Lin & Evans, 2000). Indeed, all the fusion partners have a dimerisation domain: the domain coiled coil (CC) for PML-RARA,

STAT5b-RARA, NuMa-RARA, or the domain POZ for PLZF-RARA. Mechanistically, enhanced corepressor binding is the consequence of the presence of two strong corepressor binding sites in the X-RARA homodimers, while the normal RXR/RARA complex only harbours a single one. Then, corepressors recruit HDAC, DNA methyltransferases and convert the target gene into repressed heterochromatin status (Di Croce et al., 2002). Many other fusion proteins associated to myeloid leukemias are transcriptional repressors. In APL, the links between repression and transformation came from experiments in which both PML-RARA and PLZFRARA were shown to block differentiation of U937 cells triggered by vitamin D3 or TGFb. In contrast, only upon expression of these fusion proteins, therapeutic doses of RA became capable of triggering granulocytic differentiation. These experiments have led to the proposal that PML-RARA has a dual role: repressor or activator of both target genes and differentiation, depending on the RA concentration (Lin & Evans, 2000; Minucci et al., 2000). Similarly, a specific repression domain at the Nterminus of PLZF was shown to account for RA-resistant transcriptional repression, correlating with the clinical resistance of PLZF-RARA-associated leukemias to RA. A simple model emerged from these pioneering studies in which enforced dimerisation accounted for transcriptional repression and the differentiation block, while the RARA-independent domain of PLZF accounted for RAresistance. Yet, a number of results derived from primary cells have challenged this model. 2.1. Enforced corepressor binding onto RARA fails to initiate APL A set of recent results has shown that transforming RARA into a potent repressor is not sufficient to generate leukemia. Expression of HDAC1-RARA in transgenic mice does not generate leukemia (Matsushita et al., 2006). RARAM4, a mutant receptor which cannot bind RA or well-established dominant-negative RARA mutants, do not induce leukemia either (Matsushita et al., 2006), showing that enforced repression through HDAC1 recruitment or the inhibition of the transactivation function of RARA cannot constitute the single event responsible for transformation. In addition, RARA self-dimerising mutants such as P50-RARA and F3RARA are poor inducers of myeloid leukemia in vivo (Sternsdorf et al., 2006), and do not fully transform in primary hematopoietic progenitors, despite the fact that, as expected, they are very potent repressors of RARA signalling (Kwok, Zeisig, Dong & So, 2006; Zhou et al., 2006; Licht, 2006). Thus, X-RARA fusion proteins

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have other specific properties, which are required for transformation. Several works suggest that specific properties of the fusion partner are involved in the transformation process, likely accounting for the narrow specificity of the fusion partners, rather than any protein with a dimerisation domain. In the case of PML, studies on the molecular basis of arsenic trioxide anti-leukemic properties had identified K160 in the PML moiety of the fusion as an essential residue for arsenic-induced PML-RARA degradation (see below) (Lallemand-Breitenbach et al., 2001). Sumoylation is a post-translational modification which tags an ubiquitin-like peptide, SUMO, on specific lysine sites and is necessary to PML to recruit certain proteins in the nuclear bodies. Unexpectedly, sumoylation of the lysine site K160 is essential to the leukemic transformation of primary progenitors ex vivo (Zhu et al., 2005). Mutant PML-RARA K160R only induces a myeloproliferative syndrome in transgenic mice, and fails to induce the differentiation block and the ability to transplant at high efficiency. Yet, PML-RARA K160R behaves exactly as PML-RARA with respect to its dimerisation and repression of RARA target genes. What then is the role of K160 sumoylation? Many transcription factors become potent repressors upon their sumoylation, often through Sumo-induced binding of Daxx, a major transcriptional repressor. In that respect, fusion of the repression domain of Daxx to the nonsumoylable PMLRARA mutant fully restores transformation ex vivo. The presence of this transcriptional repression domain in the PML part of the fusion is highly similar to the situation encountered with PLZF-RARA where the POZ domain provides an additional repressive function. Is the binding of corepressors onto RARA even required? Cell-line studies demonstrate that it is indeed essential for the differentiation block (Grignani et al., 1998). Studies in primary cells suggest that it is important for full immortalisation, but perhaps dispensable for the differentiation block (Zhou et al., 2006). Similarly, evidence that PMLRARA target genes are silenced by hypermethylation, is currently not confirmed as PML-RARA seems to be rarely associated with RARB2 promoter methylation in primary cell lines (Tabe et al., 2006). Future studies should elucidate the respective importance of these two different repression domains in the establishment of the APL phenotype. 2.2. Relaxed binding site specificity is essential for transformation Apart from enhancing the binding of corepressors, the dimerisation of PML-RARA through the PML coiled

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coil allows the binding of PML-RARA onto DNA without requiring RXR. However, recent biochemical and functional studies have directly demonstrated that RXR is always present within the PML-RARA complex and oligomerization strongly modifies the binding properties of PML-RARA to DNA (Kamashev, Vitoux & De The, 2004). Indeed, in contrast to RARA, PML-RARA binds to the AGGTCA core motif in three possible ways direct, reversed and everted repeats with a spacing between the sequences varying from 0 to 16 (Kamashev et al., 2004). Hence, the spectrum of PML-RARA binding to DNA is thus much broader than that of RARA, identifying a major gain of function of the fusion. Recent studies have identified similar features in other oncogenes associated with myeloid leukemias, such as V-erbA or AML-ETO (Liu et al., 2006; Zubkova & Subauste, 2004). The relaxed DNA-binding specificity of PML-RARA was also confirmed by gene expression analyses in various cellular systems. In cells expressing PML-RARA, 89% of genes controlled by RA are normally not controlled by RA showing that a broad proportion of RA target genes depends on the expression of PML-RARA in APL cells (Meani et al., 2005). Moreover, a systematic search for degenerate PML-RARA binding motifs has shown that, only in APL cells, most of the primary target genes of RA contain widely spaced DRs, but no DR5, consistent with the widely degenerate binding of PML-RARA. So is this gain of function important in the transformation process? Several recently published experiments suggest that it is indeed the case. In particular, a point mutant of PLZF-RARA that has lost the ability to dimerise, but still contains both the POZ and SMRT repression domains, fails to transform primary hematopoietic progenitors (Kwok et al., 2006). In addition, analysis of a set of self-dimerising RARA mutants (which therefore efficiently bind SMRT) fused or not to the repression domain of Daxx, has demonstrated that recruitment of SMRT, or Daxx, as well as recognition of extended DR response elements are all required for the efficient transformation of primary hematopoietic progenitors (Zhou et al., 2006). 2.3. Is the differentiation block the critical event in APL pathogenesis? While ex vivo experiments of transduction of hematopoietic progenitors clearly demonstrate that expression of PML-RARA suffices to induce a differentiation block in methyl-cellulose assays (Du et al., 1999), this does not appear to be the case in transgenic mice where the differentiation block only appears pro-

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gressively. Indeed, during the long preleukemic phase, mice display terminally differentiated granulocytes that express very high levels of PML-RARA. It is only at the stage of full blown leukemia that the blasts acquire the APL differentiation block. This demonstrates that, in vivo, PML-RARA expression alone is not sufficient to trigger the differentiation block. In contrast, one of the first events that is observed in mice is a myeloproliferative syndrome, that likely reflects a major expansion of compartment of myeloid progenitor cells. In contrast to the differentiation block, this appears to be one of the first events triggered by PML-RARA expression and yields a model, in which two different genetic programs are involved in APL pathogenesis: one controlling progenitor cell self-renewal and one implicated in promyelocyte differentiation. Indeed analysis of RA target genes in several systems has identified genes implicated in stem cell selfrenewal (Meani et al., 2005). In conclusion, transcriptional alterations inflicted by X-RARA expression are due to the conjugation of three molecular properties: strong corepressors/HDAC recruitment, relaxed DNA-binding specificity and presence of an extra repression domain (Fig. 1). These may be required to control two different gene networks. Other properties linked to the fusion partners, in particular a dominant-negative role over PML triggered senescence have been discussed. While there are clear indications

Fig. 1. Molecular properties of X-RARA essential for leukemic transformation. The transcriptional alterations inflicted by X-RARA expression reflect the combination of three molecular properties: dimerisation induced strong corepressors/HDAC recruitment, relaxed DNA-binding specificity and presence of an extra repression domain. These result in major gains of function: binding to de novo target genes and strong transcriptional repression yielding to APL.

that this might be important in cell-lines, the available mouse data is still quite controversial (Rego et al., 2001; Sternsdorf et al., 2006). 3. Therapy The therapeutic targeting of PML-RARA by RA has been extensively discussed elsewhere (see for review (Fenaux, Chomienne & Degos, 2001)). APL is sensitive to the treatment by the retinoid acids (RA): 9-cis (9-cis RA) or all-trans retinoic acid (ATRA), inducing a leukemic blast differentiation and a complete remission among certain patients. However, even with a combination of traditional anthracycline-Ara-C chemotherapy with ATRA, secondary acquired ATRA resistance occurs in many patients after two relapses or more. Hypercatabolic response (increased ATRA oxidation, ATRA sequestration by the RA-binding proteins CRABP) and cellular events that limit ATRA uptake or increase its catabolism in the cell or in the nucleus constitute the two major mechanisms of ATRA resistance (see for review (Gallagher, 2002)). Another important mechanism of resistance is the emergence of APL cell clones with PML-RARA mutations in the ligand binding domain of the RARA region which alter the transcriptional function of the nuclear receptor. 3.1. PLZF-RARA transformed cells retain some RA-sensitivity While in cell-lines PLZF-RARA strongly blocks differentiation in a RA-resistant manner, expression of this fusion protein in mice only yields a myeloproliferative syndrome, reminiscent of chronic myelogenous leukemia (CML) (He et al., 2000). In primary cells, transduction of this fusion yields a differentiation block that is indistinguishable from that induced by PMLRARA (Kwok et al., 2006). In mice, co-expression of the reciprocal RARA-PLZF fusion, which retains the ability to bind PLZF sites, induces a differentiation arrest and yields a RA-resistant leukemia (He et al., 2000). The sensitivity of the CML like disease to RA was not assessed. While it is clear that the PLZFRARA-associated leukemias are not as sensitive to RA as the t(15,17) associated ones (Licht et al., 1995), some recent evidence also challenge the idea that they are clinically insensitive to RA. Indeed, in patients or in primary cells ex vivo, some differentiation response to RA was observed and complete differentiation was obtained when cytokines or cytostatics were used in addition to RA (George, Poonkuzhali, Srivastava, Chandy & Srivastava, 2004; Jansen et al., 1999; Koken et al., 1999).

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3.2. Arsenic trioxide, another targeted therapy in APL

3.3. Activation of cyclic AMP signalling favours APL cell differentiation

Arsenic is an extremely potent anti-leukemic agent, significantly better than RA (Shen et al., 2004). The clinical efficacy of arsenic trioxide was demonstrated several years before its mode of action was understood. The clinical and biological data on arsenic therapy was extensively reviewed elsewhere and we will only sum up the facts needed for the mechanistic discussions below. In cell-lines, high doses of arsenic induce apoptosis (Chen et al., 1996), while lower concentrations (10−7 M) trigger a modest differentiation ex vivo, which becomes terminal in the presence of cytokines or cAMP (Guillemin et al., 2002; Muto et al., 2001; Zhu et al., 2002). In vivo, arsenic induces both massive differentiation and some apoptosis. Arsenic trioxide targets PML-RARA through its PML part. Arsenic induces the rapid sumoylation of a specific lysine in PML or PML-RARA, which is followed by their proteasome-dependent degradation (Lallemand-Breitenbach et al., 2001). The changes in PML sumoylation and the degradation of PML-RARA are most likely to contribute to the therapeutic effects of arsenic. Arsenic could also facilitate transcriptional activation by releasing the corepressor from PML-RARA (Hong, Yang & Privalsky, 2001). Other molecular mechanisms, in particular involving CHK2 activation were recently proposed, but their relevance in vivo remains to be demonstrated (Joe et al., 2007). How does arsenic promote PML sumoylation is not currently understood. It was shown that arsenic-activated MAP kinases enhance phosphorylation of both PML and PML-RARA, resulting in their sumoylation (Hayakawa & Privalsky, 2004), although we have not been able to confirm those results. The data presented above carry some expectations: first, arsenic should only be active in PML-RARA APL, which was indeed demonstrated in mice (Rego, He, Warrell, Wang & Pandolfi, 2000). Second, if arsenic induces differentiation, an enhancement of differentiation and leukemia clearance should be observed upon dual treatments. This was also found in mice (Guillemin et al., 2002; Lallemand-Breitenbach et al., 1999), as well as in patients (Shen et al., 2004). One of the first actions of arsenic is to degrade PML-RARA, that RA and arsenic are highly synergistic for differentiation induction and disease eradication. These observations do not favour a model that would rely entirely on transcriptional modulation of PML-RARA. Rather, this synergy suggests that degradation of the fusion protein contributes to disease eradication.

Cyclic AMP derivatives induce a differentiation of acute myeloid leukemias cell-lines HL60 or U937, or strongly act synergically with other differentiating agents (Benoit, Roussel, Pendino, Segal-Bendirdjian & Lanotte, 2001). In APL cells, addition of cAMP derivatives or elevating its cellular level with phosphodiesterase inhibitors (i.e. theophylline, piclamilast) permit to decrease RA concentrations needed to induce differentiation. Ex vivo and in vivo, cAMP synergizes also with arsenic to induce differentiation of APL cells (Guillemin et al., 2002; Zhu et al., 2002). cAMP dramatically enhances transcriptional response to RA on PML-RARA-specific response elements, showing that the RXR/PML-RARA heterocomplex is implicated in the synergy (Kamashev et al., 2004). Alteration of the binding efficiency of corepressors to the RARA moiety of the X-RARA fusion protein is a possible mechanism, as PKA-dependent phosphorylation is able to dissociate the corepressor complex from RARA (Altucci, Rossin, et al., 2005). Thus, cAMP targets PML-RARA dependent transcription. In addition, cAMP can reverse RA-resistance in several models (Guillemin et al., 2002; Kamashev et al., 2004). The sharp synergy between cAMP and either retinoic acid or arsenic, observed both ex vivo and in vivo could favour the use of phosphodiesterase inhibitors in a clinical setting (Guillemin et al., 2002; Parrella et al., 2004). The RXR ligands, rexinoids, do not release RARbound corepressors in the absence of a RAR ligand, thus preventing rexinoid-induced recruitment of co-activators (Germain, Iyer, Zechel & Gronemeyer, 2002). PKA activation by cAMP derivatives leads to corepressor release from the RARA subunit of the RARA/RXR heterodimer, resulting in desubordination of RXR which acquires transcriptional activity in response to specific agonists (Altucci, Rossin, et al., 2005; Kamashev et al., 2004). That RXR desubordination by PKA allows differentiation in the presence of mutated PML-RARA fusion proteins (Benoit et al., 1999; Kamashev et al., 2004) points to the clinical relevance of this transcriptional cross-talk and unambiguously demonstrates that it acts, at least in part, through PML-RARA (Fig. 2). 3.4. Epigenetic therapies? Several classes of HDAC inhibitors (HDACi) have been identified: butyric acid, valproic acid (VPA), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), cyclic tetrapetides and benzamides

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inhibitors or DNA methylase inhibitors as well as the cAMP/Rexinoid association have definitive activity to induce the differentiation and apoptosis of non-APL AMLs ex vivo (Altucci, Rossin, et al., 2005; Insinga et al., 2005), at least in part through activation of the TRAIL/DR5 pathway (Insinga et al., 2005). Other RA or arsenic combinations were also recently pointed out as the combination of myelomonocytic factors (G-CSF, GM-CSF) with RA which activates RARA and induces myeloid leukemia cell differentiation via MAP kinase pathways (MEK-1/-2 and p38) (Glasow, Prodromou, Xu, von Lindern & Zelent, 2005) or arsenic combination with MEK/ERK and JNK inhibitors which potentiates apoptosis in AML cells (Ramos et al., 2006). These associations can be useful in AML therapy. Hopes that these observations can soon be transposed into clinical practice justify the intense efforts carried out in this field. Fig. 2. Targeting of different therapies on PML-RARA.

(i.e. MS-275) (reviewed (Altucci, Clarke, Nebbioso, Scognamiglio & Gronemeyer, 2005)). These drugs, used alone, do not relieve the PML-RARA mediated transcriptional silencing and do not induce differentiation of APL blasts. But, when combined with RA, they lead to in vitro differentiation of RA-resistant APL. In addition, VPA induces apoptosis of APL and AML cells by inducing TRAIL and its death receptors (Insinga et al., 2005) and could constitute an additional treatment to the classical combination RA/chemotherapy in AML. Drugs which decrease DNMT expression and/or activity (DNMTi) constitute another chromatin-targeted treatment in APL and RA-resistant leukemias. Ex vivo in RA-resistant APL cell-line (NB4 MR4), inhibition of DNMT by 5-azacytidine induces specific changes on the chromatin state at RA-target sites and restores the RA effect on promoter activity and differentiation (Fazi et al., 2005). Some of these compounds, including HDACi/DNMTi combinations, are currently in trial, with some very recent success in MDS and non-APL AMLs (Fig. 2). 4. Conclusion The plethora of agents that are active in APL by targeting PML-RARA or its direct effects on its chromatin binding sites have allowed an unprecedented level of understanding the molecular bases of APL pathogenesis and response to therapy. One can hope that those will become progressively applicable to other malignancies. . . In that respect, several recent studies have shown that RA, chromatin modifiers such as HDAC

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