Human Molecular Genetics

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Human Molecular Genetics, 2001, Vol. 10, No. 7 769-775
© 2001 Oxford University Press

Oncogenes and tumor suppressors in the molecular pathogenesis of acute promyelocytic leukemia

Pier Paolo Pandolfi⁺

Department of Human Genetics and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

Received 30 January 2001; Accepted 30 January 2001.

	ABSTRACT

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations always involving the retinoic acid receptor (RAR) gene on chromosome 17 and variable partner genes (X genes) on distinct chromosomes. RAR fuses to the PML gene in the vast majority of APL cases, and in a few cases to the PLZF, NPM, NuMA and Stat5b genes, respectively, leading to the generation of RAR–X and X–RAR fusion genes. Both fusion proteins can exert oncogenic functions through their ability to interfere with the activities of X and RAR proteins. Here, it will be discussed in detail how an extensive biochemical analysis as well as a systematic in vivo genetic approach in the mouse has allowed the definition of the multiple oncogenic activities of PML–RAR, and how it has become apparent that this oncoprotein is able to impair RAR at the transcription level and the tumor suppressive function of the PML protein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (the AML-M3 subtype according to the French–American–British classification) that accounts for >10% of all AMLs and affects approximately 3000 individuals every year in the US alone (1–3 and references therein), and is therefore a relatively rare form of cancer. Nevertheless, this leukemia has become a unique paradigm for its distinctive biological, molecular and clinical features:

(i) APL is caused by the clonal expansion of tumor cells with promyelocytic morphological characteristics, a feature that gives the leukemia its name. Promyelocytes are present in normal bone marrow and are myeloid progenitor cells from which the granulocytic cells circulating in the peripheral blood are constantly generated. The accumulation of promyelocytic blasts in all organs of APL patients has suggested since the very early stages of APL research that the disease may be the consequence of a block in myeloid differentiation, which makes this leukemia a unique model system for the study of the molecular mechanisms controlling normal and aberrant hemopoieis.

(ii) The second feature of APL is molecular and karyological. In almost 100% of cases, APL blasts harbor-specific reciprocal chromosomal translocations that always involve chromosome 17. In almost 99% of APL patients the translocation observed is a reciprocal and balanced translocation between chromosomes 15 and 17, t(15;17)(22;q11.2–12), which has become the hallmark of the disease. It is interesting to note that from a historical point of view this chromosomal translocation represents the second evidence ever obtained that human cancer indeed has a genetic basis (4). In view of this molecular homogeneity, the molecular cloning of the APL-specific translocation was a long sought achievement and, a decade ago, a major goal for many laboratories.

(iii) The last feature, which makes APL a paradigm from a therapeutic stand-point, is that APL blasts have been proven to be exquisitely sensitive to the differentiating action of retinoic acid (RA). From this perspective APL has become the paradigm for therapeutic approaches for cancer utilizing differentiating agents (differentiation therapy). This therapeutic approach is conceptually novel in that, it does not involve chemical or physical agents to eradicate the tumor by ‘killing’ the neoplastic cells, but rather re-programs these cells to differentiate normally. However, although effective, treatment of APL patients with RA alone induces disease remission transiently and relapse is inevitable if remission is not consolidated with chemotherapy. Based on these findings, the work of many laboratories has focused on understanding why APL cells respond to RA to start with, why RA cannot completely eradicate the neoplastic clone and how to potentiate the effects of RA, thus sparing the patient conventional chemotherapy.

Almost 10 years ago the t(15;17) translocation of APL was molecularly cloned and the two genes involved at the breakpoint identified (5–7). Surprisingly, and paradoxically, the breakpoints were found to lie within the retinoic acid receptor (RAR) gene on 17 and a new gene, originally termed myl and subsequently PML (for promyelocytic leukemia gene) on chromosome 15 (8–11). At the time the translocation was cloned, RAR was, and possibly still is, one of the most well characterized transcription factors (12). Its involvement in the t(15;17) translocation of APL made this leukemia one of the first convincing examples of aberrant transcription in cancer pathogenesis. However, the involvement of RAR also raised a paradox, in that it was very surprising to find the nuclear receptor for the ligand, which is therapeutically effective in the treatment of the disease, involved in the APL translocation. Based on the rearrangement of RAR one would have anticipated that RA does not function in APL. This contradictory observation has also been the subject of intense research in the last few years, and has led to the proposal of a transcriptional model for APL pathogenesis with exciting and straightforward therapeutic implications.

Finally, APL has also become a compelling and reassuring example of the power of modern molecular medicine in conquering cancer. Suffice to say, in the 1960s APL survival rates at MSKCC were, on average, 1–2 weeks from diagnosis depending on the quality of care (B. Clarkson, personal communication). Nowadays, in view of the progress made in understanding the molecular basis of this disease and also because of the advent of specific and effective therapeutic strategies, this disease is now regarded as curable, with success rates in ~70–90% of APL cases (1–3).

Here, the most recent progress made in elucidating the molecular genetics of APL and the mechanisms of action of the PML–RAR fusion protein, and also how this new understanding has allowed the development of novel therapeutic strategies, will be reviewed.

	THE MOLECULAR GENETICS OF APL

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
As mentioned previously, the vast majority of APL patients harbor a chromosomal translocation that involves chromosomes 15 and 17. However, in a few cases, the translocation involves chromosome 11 instead of 15, and the promyelocytic leukemia zinc finger (PLZF) gene. In rare cases the translocation involves chromosomes 5, 11 and 17 and the nucleophosmin (NPM), NuMA and Stat5b genes, respectively (1,2 and references therein; 13). The X–RAR and RAR–X fusion genes (whereby X indicates the various RAR partner genes) generated by the reciprocal translocation in APL encode for structurally different X–RAR and RAR–X products, co-expressed in the leukemic blast, that differ in their X portions, but are identical in their RAR portion, and can therefore be considered as RAR mutants. APL associated with the various translocations results in diseases with similar features but with one very important exception: APL associated with chromosomal translocations between the RAR and the PLZF genes (PLZF–RAR) and possibly (STAT5b–RAR) show a distinctly worse prognosis with poor response to chemotherapy and little or no response to treatment with RA, thus defining a new APL syndrome (13,14). The various partners of RAR are structurally diverse. It may therefore appear that the disruption of RAR function is the major and only cause of APL. Indeed, the X–RAR protein is able to affect the function of RAR at multiple levels, as will be discussed in the following paragraphs. However, the various X–RAR fusion molecules are always able to form heterodimeric complexes with the respective X protein. As a consequence, these fusion products have the potential ability to interfere, at least in theory, with both X and RAR pathways.

	THE MULTIPLE ONCOGENIC FUNCTION OF PML–RAR

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
Both in vitro analysis in cell lines and in vivo analysis in transgenic mice have defined the functional importance and the oncogenic role of the X–RAR fusion protein. Transgenic mice in which PML–RAR was expressed in the myeloid/promyelocytic compartment develop leukemia, with APL features, but only after a long pre-leukemic phase, conclusively demonstrating that PML–RAR is critical, albeit not sufficient, in causing this leukemia (15–18). The transgenic approach in the mouse also demonstrated that RAR–X proteins are not sufficient but do play a crucial role in leukemogenesis, in acting both as tumor modifiers and tumor metamorphosers (19,20). As an example, we have shown that while PML–RAR transgenic mice develop leukemia with APL features, PLZF–RAR transgenic mice develop myeloid leukemias that completely lack the distinctive differentiation block at the promyelocytic stage, which characterizes human APL, thus resembling human chronic myelogenous leukemias (CML). However, double transgenic mice that co-express RAR–PLZF and PLZF–RAR develop leukemia with APL features (20). Thus, RAR–PLZF is capable of metamorphosing a CML-like phenotype in an APL-like phenotype. On the other end RAR–PML can accelerate leukemia onset in PML–RAR transgenic mice (19). In summary, the in vivo analysis in the mouse strongly supports the notion that a key oncogenic event in APL pathogenesis is the expression of the X–RAR fusion protein while the activity of RAR–X cooperates with it, not only in concurring to full-blown leukemogenesis, but in determining the critical feature of the leukemia in its native form.

A detailed analysis of the biological activity of PML–RAR soon rendered obvious that this molecule exerted, both in vivo in transgenic mice and in vitro in hemopoietic cell lines, multiple oncogenic functions such as the ability: (i) to block myeloid differentiation (15–18,21); (ii) to confer a survival advantage and possibly a proliferative advantage to the expressing cell (21,22); and (iii) to render the cells genomically unstable (23,24) (Fig. 1). How PML–RAR could exert so many diverse oncogenic activities at the molecular level remains a puzzling question. Progress in elucidating the molecular functions of PML–RAR will be discussed in the subsequent paragraphs.

View larger version (14K): [in this window] [in a new window]   Figure 1. The multiple oncogenic functions of PML–RAR. As for many oncogenes, the fusion protein of APL can confer a proliferative and survival advantage to the myeloid hemopoietic cells. It can also favor the accumulation of additional genetic lesions rendering the cells genomically unstable. However, PML–RAR also has the ability to perturb myeloid differentiation. The latter function is clearly relevant for the pathogenesis of APL.

 

	PML–RAR IS A DOMINANT NEGATIVE RAR MUTANT

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
RARs are members of the superfamily of nuclear hormone receptors that act as RA-inducible transcriptional activators in their heterodimeric form with retinoid-X-receptors (RXRs), a second class of nuclear retinoid receptors (12 and references therein). In the absence of RA, however, RAR/RXR heterodimers can repress transcription through histone deacetylation by recruiting nuclear receptor corepressors (N-CoR or SMRT), Sin3A or Sin3B, which in turn form complexes with histone deacetylases (HDAC), thereby resulting in nucleosome assembly and transcriptional repression (25 and references therein). The presence of RA at physiological concentrations induces an allosteric change in the receptor, leading to the dissociation of the corepressors complex and the recruitment of transcriptional co-activators to the RAR/RXR complex. This results in the activation of gene expression, which in turn may induce terminal differentiation and growth arrest of cells of various histological origins, including normal myeloid hemopoietic cells (see following paragraphs; 12,26,27 and references therein). PML–RAR can bind to retinoic acid response elements (RARE) and, through the RAR DNA binding domain, can form multimeric complexes with RXRs (1,2 and references therein). Furthermore, PML–RAR binds RA with the same affinity as RAR (28). Therefore, the fusion protein can potentially interfere with the RAR pathway at multiple levels. However, the molecular mechanisms by which PML–RAR would be leukemogenic at physiological doses of RA, and permissive for the differentiating ability of RA at pharmacological doses, remained unexplained until recently. It was also unclear whether APL was caused by the aberrant RA-dependent transactivation of gene expression by PML–RAR proteins since, in this case, APL should always be exacerbated by RA, whereas on the contrary, and paradoxically, RA was extremely effective in APL cases harboring PML–RAR. Recently, the aberrant transcriptional activity of the X–RAR fusion proteins has been molecularly defined in a unified model that provides a transcriptional basis for both the molecular pathogenesis of APL and the differential response to RA in APL (16,29,30). Others and we have found that at physiological concentration of the ligand, the various X–RAR proteins act as potent transcriptional repressors in view of an increased and aberrant affinity for nuclear corepressors and HDACs (16,29,30). At a pharmacological dose of RA, whereas PLZF–RAR, via the PLZF moiety, renders the leukemic cells unresponsive to RA through RA-insensitive corepressor/HDAC interactions, the PML–RAR–corepressor complex is resolved and the fusion protein is able to directly mediate trans-activation of RAR target genes (16,29,30). Indeed, HDAC inhibitors (HDACIs) such as trichostatin A (TSA) could overcome the transcriptional repressive activity of PML–RAR and PLZF–RAR (16,29,30 and our unpublished data). HDACIs could also overcome the unresponsiveness of PLZF–RAR leukemic cells to RA (16). It has also been reported recently that the repressive ability of PML–RAR may strictly depend on its dimerization ability through the PML moiety and that the PML–RAR homodimer could compete with RAR for RXR binding (31–33).

In summary, PML–RAR certainly affects the RAR pathway at multiple levels. However, as will be discussed below, PML–RAR can also impair the function of PML. This PML–RAR activity could be as relevant for APL pathogenesis as its ability to interfere with the RAR pathway and introduces a second level of complexity in the analysis of the molecular mechanisms underlying APL pathogenesis.

	PML–RAR IS A DOMINANT NEGATIVE PML MUTANT

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
As soon as the first anti-PML antibody was obtained, it became apparent that PML–RAR can act as a dominant negative PML mutant. In fact, PML was typically found concentrated in discrete nuclear speckles, termed nuclear bodies (NBs; also called Kremer bodies, ND10 or POD, for PML oncogenic domains; 34 and references therein). Between 10 and 30 of these macromolecular structures are found in the nucleus of each cell, although their number and size change during the cell cycle and upon the introduction of various stimuli, such as interferon (9 and references therein). PML colocalizes in the NB with multiple proteins such as SUMO-1, Sp100, Sp140, CREB-binding protein (CBP), DAXX, RB and p53 (34 and references therein). In APL, PML–RAR physically interacts with PML, disrupts the PML–NB in a dominant negative manner and induces the delocalization of PML as well as the other NB component into aberrant nuclear subdomains, thus potentially impairing PML function (35–37) (Fig. 2). While the PML/PML–RAR interaction provided a straightforward explanation for the delocalization of PML from the NBs, until very recently it was unclear how PML–RAR would be able to induce an aberrant localization of all the other NB components. By studying primary Pml–/– cells in which Pml was inactivated by homologous recombination, it could be demonstrated that PML is essential for the proper formation and stability of the NB since in Pml–/–cells, irrespective of the histological origin, all the other NB components no longer accumulate in NB and display aberrant localization patterns (38). This provided a direct explanation for the reason why PML–RAR through PML can lead to the disruption of the NB and in turn implied that PML–RAR could affect the NB-dependent functions of the various NB components. The fact that PML–RAR could simultaneously affect both PML and RAR functions made it imperative to elucidate the normal function of PML and how this relates to APL pathogenesis.

View larger version (13K): [in this window] [in a new window]   Figure 2. PML–RAR is a dominant negative inhibitor of PML function. PML–RAR can physically interact with PML through the coiled-coil moiety that normally mediates PML homodimerization. The RING finger and the coiled-coil moiety characterize the RBCC PML domain required for the NB localization of PML as well as for its ability to interact with other proteins. In the presence of the fusion protein (i.e. in the APL blasts) PML acquires an aberrant microspeckled nuclear localization pattern. All the proteins that would normally colocalize with PML in the NBs are also delocalized in APL cells. In this way, PML–RAR can possibly affect PML functions as well as the NB-dependent function of multiple NB proteins.

 

	PML–RAR BLOCKS DIFFERENTIATION OF MYELOID HEMOPOIETIC PROGENITORS

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
As mentioned previously, PML–RAR can cause a block in myeloid differentiation. This notion is supported by in vitro experiments in leukemic cell lines such as U937. These cells would normally differentiate in response to RA. The RA differentiating activity in U937 is abrogated as a consequence of PML–RAR expression (21). Not only do PML–RAR transgenic mice develop an APL-like leukemia in vivo, but these leukemias are preceded by a long pre-leukemic phase characterized by a block of differentiation at the promyelocytic stage that results in the slow and progressive accumulation of promyelocytes in the bone marrow and spleen of these transgenic mice (15–18). RA modulates important developmental processes as well as myeloid differentiation in adulthood (12,26,27). Thus, it was logical to hypothesize that PML–RAR could impair myeloid differentiation by blocking RA–RAR responses in a dominant negative fashion. However, while there is little doubt that this is a critical molecular event for APL leukemogenesis, since the various X–RAR fusion proteins appear to be oncogenic in vivo in transgenic mice despite distinct X moieties (15–18,39), it is still bewildering that leukemias in PLZF–RAR mice do not display the block of differentiation that characterize APL, even if PLZF–RAR is the most potent dominant negative RAR mutant identified thus far, in view of its marked transcriptional repressive abilities. This may suggest instead that: (i) the interference with the RAR pathway is not sufficient to cause the block of differentiation; or (ii) the various fusion proteins have different DNA binding specificities and that the subset of genes repressed by the various X–RAR proteins is therefore distinct.

In considering the first hypothesis, it is important to remember that PML–RAR is capable of disrupting the function of PML and, as recently shown, PML can act as a RAR transcriptional co-activator (40). This is in agreement with the marked RA unresponsiveness observed in Pml–/– bone marrow cells and mouse embryonic fibroblasts (MEFs) (41). In contrast PLZF–RAR, which does not cause a differentiation block in leukemic transgenic mice (16), does not seem to perturb PML function (42), nor does it appear to block the function of PLZF (20). In contrast, the RAR–PLZF molecule, which can affect the transcriptional function of PLZF, confers the block of differentiation in leukemias from double PLZF–RAR/RAR–PLZF transgenic mice (20 and references therein). Based on these findings it would seem logical to propose that, in vivo, the concomitant blockade of the X and RAR pathways is important for causing the block of differentiation at the promyelocytic stage observed in APL. On the other hand, microarray analysis will be essential to determine whether the DNA binding specificity of the various X–RAR fusion proteins is distinct.

	THE ROLE OF PML–RAR IN THE CONTROL OF CELLULAR PROLIFERATION AND SURVIVAL

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
Unlike classic oncogenes PML–RAR does not transform NIH 3T3 fibroblasts, but rather displays potent growth inhibitory effects on all non-hematopoietic cell lines and on the majority of the hematopoietic cell lines (43). Inducible expression of PML–RAR in fibroblasts demonstrated that the basis for the growth suppression is induction of cell death (43). This observation may suggest that only a few cell types are permissive for the oncogenic potential of PML–RAR. This tenet is also supported by the fact that, in vivo, PML–RAR causes leukemia only when expressed in the myeloid promyelocytic compartment (1). Furthermore, ubiquitous expression of PML–RAR in transgenic mice results in embryonic lethality (1). While it is still unclear why PML–RAR can lead to oncogenic transfomation only in cells of specific histological origins, the net result of PML–RAR activity cannot rely solely on its ability to affect myeloid differentiation. In fact in transgenic mice, the phase that precedes leukemia onset is characterized by a progressive accumulation of cells with promyelocytic features in the spleen and the bone marrow. In principle, this expansion can be due to an increased proliferative rate and/or to a survival advantage. While the leukemic cells from PML–RAR transgenic mice are found in active proliferation and can form an increased number of colonies in in vitro bone marrow cultures (15,16), the cell cycle profile of PML–RAR promyelocytes prior to leukemia onset has not yet been characterized in detail. Nevertheless, it is tempting to speculate that the dominant negative action of PML–RAR on PML could result in a shortening of the transition through the cell cycle. In fact Pml–/– cells also have an increased proliferative potential in view of a faster transition through the G₁ phase of the cell cycle (41 and our unpublished data). The RA–RAR/RXR and the vitamin D/vitamin D receptor (VDR) pathways can also negatively control cell proliferation (12,26,27,44 and references therein). Thus, PML–RAR can lend a proliferative advantage to the neoplastic cells through the blockade of these pathways. Indeed, PML–RAR can render the cells unresponsive to RA and vitamin D both in vitro and in vivo (21,44).

While the role of PML–RAR in cell cycle control is still unclear, it has been shown conclusively that the fusion protein renders myeloid hemopoietic progenitors, as well as leukemic cells, resistant to multiple apoptotic stimuli (21,22). In particular, we could demonstrate that bone marrow progenitors from transgenic mice harboring the fusion protein are protected from apoptotic stimuli such as Fas, tumor necrosis factor (TNF), and interferons (IFNs) prior to leukemia occurrence (22). The inactivation of PML also results in protection from the aforementioned apoptotic stimuli, amongst others (22). Thus, it is logical to propose that PML–RAR could lend a survival advantage to the leukemic cells, at least in part, through its ability to impair the function of PML (Fig. 3). In this respect, it was recently reported that PML can act as a positive regulator of p53-dependent as well as p53-independent pathways for apoptosis (Fig. 3) (45). We found that PML physically interacts with p53 both in vitro and in vivo. PML is required for proper acetylation of p53 upon -irradiation. p53-dependent DNA damage-induced apoptosis, p53 DNA binding ability and the induction of p53 target genes such as BAX upon -irradiation are impaired in Pml–/– thymocytes (Fig. 4) (45). PML is also essential for p53-induced senescence upon oncogenic transformation (46). This is also in agreement with the surprising observation that in APL, unlike other acute myeloid leukemia subtypes, p53-inactivating mutations are extremely rare since p53 function in APL could be defective as a result of impaired PML function (47). However, PML is also involved in p53-independent pathways such as those that control Fas-induced apoptosis in activated splenocytes (Fig. 4) (48 and references therein). In this respect, we recently reported that Daxx, an important molecule for the transduction of the Fas pro-apoptotic stimulus, accumulates in the PML–NB at the position where PML and Daxx physically interact (48 and references therein). In the absence of PML, Daxx acquires a dispersed nuclear pattern, and activation-induced cell death of splenocytes is profoundly impaired. Furthermore, Pml inactivation results in the complete abrogation of the Daxx pro-apoptotic ability. In APL cells Daxx is delocalized from the NB. Following treatment with RA, which induces disease remission in APL, Daxx relocalizes to the PML–NBs. These results indicate that PML and Daxx cooperate in a novel NB-dependent p53-independent pathway for apoptosis.

View larger version (24K): [in this window] [in a new window]   Figure 3. Consequence of the PML–RAR interference in PML function. PML is essential for the induction of apoptosis and growth arrest upon multiple cellular stresses. PML mediates both p53-dependent and p53-independent apoptotic responses. PML–RAR can block these PML pro-apoptotic activities. Although a thorough cell cycle analysis of hemopoietic cells harboring PML–RAR has not yet been performed, it is logical to propose that the fusion oncoprotein could also lend a proliferative advantage to the APL blasts through its ability to impair the PML function.

 

View larger version (18K): [in this window] [in a new window]   Figure 4. Roles of PML–RAR and PML in the p53 tumor suppressive pathway. PML is required for proper acetylation of p53 upon DNA damage and oncogenic transformation (45,46). Acetylation of p53 may occur in the NB where p53 is found to colocalize with PML and the acetyltransferase CBP (45,46). Acetylation of p53 results, in turn, in its transcriptional activation. These processes may be antagonized by PML–RAR as they depend on the presence and the proper formation of the PML–NB. The blockade of p53 function in APL may explain why p53 is rarely mutated in this form of cancer.

 
Based on these findings it can be hypothesized that PML antagonizes leukemogenesis by the PML–RAR oncoprotein. This may be facilitated in APL by the reduction to heterozygosity of the normal PML allele. Indeed, the progressive reduction of the dose of Pml resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PML–RAR TM. In hemopoietic cells from PML–RAR TM, Pml inactivation resulted in impaired response to differentiating agents such as RA and vitamin D₃ as well as in a marked survival advantage upon pro-apoptotic stimuli (44). Thus, in vivo, PML acts as a tumor suppressor by rendering the cells resistant to pro-apoptotic and differentiating stimuli and it is haploinsufficient in antagonizing the leukemogenic potential of PML–RAR. This also suggests that the reduction of PML to heterozygosity as a consequence of its involvement in the t(15;17) translocation may be a critical event in APL pathogenesis.

	PML–RAR: A GENOME DESTABILIZER?

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
Leukemic cells from PML–RAR transgenic mice display numerous recurrent chromosomal abnormalities (24). A provocative interpretation of these data suggests that PML–RAR favors the accumulation of these genetic lesions by rendering the cells genomically unstable. Indeed, numerous proteins involved in the maintenance of genomic stability accumulate in the PML–NB, including the Bloom syndrome DNA helicase BLM (49). BLM is in fact delocalized in APL cells as well as in Pml–/– cells (23). Furthermore, in Pml–/– cells, as in Bloom cells, the frequency of sister chromatid exchange is greatly augmented, which suggests that the PML-dependent localization of BLM in the PML–NB is required for its normal function (23). Additional proteins important for the maintenance of genomic stability, such as Nibrin/p95 (the protein mutated in Nijmegen breakage syndrome) (50), MRE11 (50), topoisomerase III (also a BLM-interacting protein) (51), are also found in the PML–NB. Thus, PML–RAR could favor genomic instability and the accumulation of additional genetic events selected by the leukemic phenotype through its ability to target the PML–NB.

	IMPLICATION FOR THERAPY

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
In vivo analysis in the mouse has revealed that PML–RAR is a key oncogenic event on which the leukemic cells depend in order to proliferate and thrive, even if additional genetic events do ultimately coexist with it in the leukemic blasts (52). This observation has important therapeutic implications in that drugs that directly target the activity and/or stability of the PML–RAR fusion protein may be extremely effective in t(15;17) APL. Indeed, both RA and arsenic trioxide, another powerful weapon for the treatment of this disease (53), are capable of inducing the proteolytic degradation of the PML–RAR fusion protein (1,2,52 and references therein). RA, as mentioned above, can also overcome the aberrant transcriptional repressive capacity of PML–RAR. RA has already been proven to be effective in the clinical management of the early phase of the disease. This approach followed by conventional chemotherapy has rendered APL curable: ~70–90% of APL cases are now cured (1,2). HDACIs such as sodium phenylbutyrate (SBP) and SAHA are also promising therapeutic options in APL in view of their ability to revert the transcriptional repressive activity of the X–RAR fusion proteins as well as their relatively low toxicity (54 and references therein). Indeed, we have already successfully utilized SPB in combination with RA for the treatment of one case of APL refractory to multiple chemotherapeutic regimes as well as to RA (55). Thus, there is little doubt that in the near future, the utilization of drugs that can specifically target the PML–RAR protein, such as HDACIs and arsenic trioxide, will further enhance cure rates in APL.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
The cloning of the t(15;17) translocation of APL has rendered it possible to form a detailed characterization of the molecular mechanisms underlying the pathogenesis of this leukemia both in vitro and in vivo in K.O. and transgenic mice. While it is becoming apparent that chromosomal translocations involving the RAR gene, such as the t(15;17) translocation, are not the only genetic lesions found in the APL blasts, these translocations and their products do, however, play an essential role in APL pathogenesis. It has also become apparent that the PML–RAR fusion protein can exert multiple oncogenic functions. These aberrant biological activities can be attributed to a larger extent to its ability to concomitantly block PML and RAR pathways (Fig. 5).This new understanding has allowed the development of novel diagnostic and therapeutic strategies, which have dramatically improved APL survival rates over the last 10 years.

View larger version (20K): [in this window] [in a new window]   Figure 5. The dual leukemogenic function of PML–RAR. PML–RAR can affect both PML and RAR functions. This may explain, at least in part its pleiotropic oncogenic activity and also its ability to affect, all at once, such a large number of biological functions, including cellular differentiation, genomic stability, cell proliferation and cell survival.

 

	ACKNOWLEDGEMENTS

 
I would like to thank all the past and present members of the Molecular and Developmental Biology (MADB) laboratory at the Memorial Sloan-Kettering Cancer Center who have or are working on APL and related subjects: Austin Changou, Jose Costoya, Maria Barna, Mantu Bhaumik, Laurent Delva, Mirella Gaboli, Domenica Gandini, Marco Giorgio, Ailan Guo, Carmela Gurrieri, Nicola Hawe, Li-Zhen He, Sundeep Kalantry, Letizia Longo, Taha Merghoub, Francesco Piazza, Daniela Peruzzi, Eduardo Rego, Roberta Rivi, Simona Ronchetti, Davide Ruggero, Paolo Salomoni, Carla Tribioli, Zhu-Gang Wang, Hui Zhang and Sue Zhong. P.P.P. is a scholar of the Lymphoma and Leukemia Society. This work is supported by the NCI, the De Witt Wallace Fund for the Memorial Sloan-Kettering Cancer Center, the Mouse Model of Human Cancer Consortium (MMHCC) and NIH grants to P.P.P.

	FOOTNOTES

 
⁺ Tel: +1 212 639 6168; Fax: +1 212 717 3374; Email: p-pandolfi@ski.mskcc.org

	REFERENCES

TOP ABSTRACT INTRODUCTION THE MOLECULAR GENETICS OF... THE MULTIPLE ONCOGENIC FUNCTION... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} IS A DOMINANT... PML-RAR{alpha} BLOCKS... THE ROLE OF PML-RAR{alpha}... PML-RAR{alpha}: A GENOME... IMPLICATION FOR THERAPY CONCLUSIONS REFERENCES

 
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