Curing APL through PML/RARA degradation by As2O3

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by the PML/RARA oncogene. The prognosis for patients with APL was revolutionized by two treatments: retinoic acid (RA) and As(2)O(3) (arsenic trioxide). These were both shown a posteriori to target PML/RARA, explaining their exquisite specificity for APL. Arsenic, as a single agent, cures up to 70% of patients, whereas APL patients treated with the combination of RA and As(2)O(3) reach a stunning 90% cure rate. Recent physiopathological models highlight the key role of RA- and As(2)O(3)-triggered PML/RARA degradation, and the molecular mechanisms underlying As(2)O(3)-induced PML/RARA degradation have been recently clarified. As discussed below, arsenic binding, oxidation, sumoylation on PML nuclear bodies, and RNF4-mediated ubiquitination all contribute to the As(2)O(3)-triggered catabolism of PML/RARA.     

The PML and PML/RARα Domains: From Autoimmunity to Molecular Oncology and from Retinoic Acid to Arsenic

Acute promyelocytic leukemia (APL) is specifically associated to a t(15; 17) translocation which fuses a gene encoding a nuclear receptor for retinoic acid, RARα, to a previously unknown gene PML. The PML protein is localized in the nucleus on a specific domain of unknown function (PML nuclear bodies, NB) previously detected with autoimmune sera from patients with primary biliary cirrhosis (PBC). These bodies are nuclear matrix-associated and all of their identified components (PML, Sp100, and NDP52) are sharply upregulated by interferons. We show that autoantibodies against both PML and Sp100 are usually associated in sera with multiple nuclear dot anti-nuclear antibodies and demonstrate that PML is an autoantigen, not only in PBC, but also in other autoimmune diseases. In APL, the PML/RARα fusion interferes with both the retinoic acid (RA) response and PML localization on nuclear bodies, but the respective contribution of each defect to leukemogenesis is unclear. RA induces the terminal differentiation of APL blasts, yielding to complete remissions, and corrects the localization of NB antigens. Arsenic trioxide (As₂O₃) also induces remissions in APL, seemingly through induction of apoptosis. We show that in APL, As₂O₃leads to the rapid reformation of PML bodies. Thus, both agents correct the defect in NB antigen localization, stressing the role of nuclear bodies in the pathogenesis of APL.     

A New Role for C/EBP-beta in Acute Promyelocytic Leukemia

The differentiating properties of retinoic acid (RA) have been used beneficially for the treatment of Acute Promyelocytic Leukemia (APL) for more than a decade. However, the molecular mechanisms of how RA induces APL cell differentiation are still poorly understood. In our previous work, we provided a novel mechanism to explain the unique sensitivity to RA of APL cells. We proposed that C/EBPb is an ATRA-dependent PML/RARA target gene and that its activation is critical during ATRA-induced differentiation of APL cells. Here, I discuss how C/EBPb could be an important gene in APL pathogenesis.     

A new role for C/EBPbeta in acute promyelocytic leukemia

The differentiating properties of retinoic acid (RA) have been used beneficially for the treatment of Acute Promyelocytic Leukemia (APL) for more than a decade. However, the molecular mechanisms of how RA induces APL cell differentiation are still poorly understood. In our previous work, we provided a novel mechanism to explain the unique sensitivity to RA of APL cells. We proposed that C/EBPbeta is an ATRA-dependent PML/RARA target gene and that its activation is critical during ATRA-induced differentiation of APL cells. Here, I discuss how C/EBPbeta could be an important gene in APL pathogenesis.     

Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus.

The PML protein, identified first as part of the oncogenic PML-RARalpha chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML-RARalpha and provokes the restoration of intact nuclear bodies. In non-APL cells, the ubiquitin-like protein SUMO-1 is covalently attached to a subset of wild-type PML in a reversible and phosphorylation-dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1-polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO-1-PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML-RARalpha, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO-1 modulates its intracellular localization and suggest that post-translational modification by SUMO-1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of 'ubiquitin-like' post-translational modification is not limited to a degradation signal.     

Characterization of cryptic rearrangements, deletion, complex variants of PML, RARA in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation t(15;17)(q22;q21) leading to the disruption of Promyelocytic leukemia (PML) and Retionic Acid Receptor Alpha (RARA) followed by reciprocal PML-RARA fusion in 90% of the cases. Fluorescence in situ hybridization (FISH) has overcome the hurdles of unavailability of abnormal and/or lack of metaphase cells, and detection of cryptic, submicroscopic rearrangements. In the present study, besides diagnostic approach we sought to analyze these cases for identification and characterization of cryptic rearrangements, deletion variants and unknown RARA translocation variants by application of D-FISH and RARA break-apart probe strategy on interphase and metaphase cells in a large series of 200 cases of APL. Forty cases (20%) had atypical PML-RARA and/or RARA variants. D-FISH with PML/RARA probe helped identification of RARA insertion to PML. By application of D-FISH on metaphase cells, we documented that translocation of 15 to 17 leads to 17q deletion which results in loss of reciprocal fusion and/or residual RARA on der(17). Among the complex variants of t(15;17), PML-RARA fusion followed by residual RARA insertion closed to PML-RARA on der(15) was unique and unusual. FISH with break-apart RARA probe on metaphase cells was found to be a very efficient strategy to detect unknown RARA variant translocations like t(11;17)(q23;q21), t(11;17)(q13;q21) and t(2;17)(p21;q21). These findings proved that D-FISH and break-apart probe strategy has potential to detect primary as well as secondary additional aberrations of PML, RARA and other additional loci. The long-term clinical follow-up is essential to evaluate the clinical importance of these findings.     

PIC-1/SUMO-1-Modified PML-Retinoic Acid Receptor α Mediates Arsenic Trioxide-Induced Apoptosis in Acute Promyelocytic Leukemia

Fusion proteins involving the retinoic acid receptor alpha (RARalpha) and PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemia (APL). APLs with PML-RARalpha or PLZF-RARalpha fusion protein differ only in their response to retinoic acid (RA) treatment: the t(15;17) (PML-RARalpha-positive) APL blasts are sensitive to RA in vitro, and patients enter disease remission after RA treatment, while those with t(11;17) (PLZF-RARalpha-positive) APLs do not. Recently it has been shown that complete remission can be achieved upon treatment with arsenic trioxide (As2O3) in PML-RARalpha-positive APL, even when the patient has relapsed and the disease is RA resistant. This appears to be due to apoptosis induced by As2O3 in the APL blasts by poorly defined mechanisms. Here we report that (i) As2O3 induces apoptosis only in cells expressing the PML-RARalpha, not the PLZF-RARalpha, fusion protein; (ii) PML-RARalpha is partially modified by covalent linkage with a PIC-1/SUMO-1-like protein prior to As2O3 treatment, whereas PLZF-RARalpha is not; (iii) As2O3 treatment induces a change in the modification pattern of PML-RARalpha toward highly modified forms; (iv) redistribution of PML nuclear bodies (PML-NBs) upon As2O3 treatment is accompanied by recruitment of PIC-1/SUMO-1 into PML-NBs, probably due to hypermodification of both PML and PML-RARalpha; (v) As2O3-induced apoptosis is independent of the DNA binding activity located in the RARalpha portion of the PML-RARalpha fusion protein; and (vi) the apoptotic process is bcl-2 and caspase 3 independent and is blocked only partially by a global caspase inhibitor. Taken together, these data provide novel insights into the mechanisms involved in As2O3-induced apoptosis in APL and predict that treatment of t(11;17) (PLZF-RARalpha-positive) APLs with As2O3 will not be successful.     

Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.     

The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion.

Nuclear bodies (NBs) are ultrastructurally defined granules predominantly found in dividing cells. Here we show that PML, a protein involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), is specifically bound to a NB. PML and several NB-associated proteins, found as auto-antigens in primary biliary cirrhosis (PBC), are co-localized and co-regulated. The APL-derived PML-RAR alpha fusion protein is shown to be predominantly localized in the cytoplasm, whereas a fraction is nuclear and delocalizes the NB antigens to multiple smaller nuclear clusters devoid of ultrastructural organization. RA administration (which in APL patients induces blast differentiation and consequently complete remissions) causes the re-aggregation of PML and PBC auto-antigens onto the NB, while PML-RAR alpha remains mainly cytoplasmic. Thus, PML-RAR alpha expression leads to a RA-reversible alteration of a nuclear domain. These results shed a new light on the pathogenesis of APL and provide a molecular link between NBs and oncogenesis.     

Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway

In acute promyelocytic leukaemia (APL), arsenic trioxide induces degradation of the fusion protein encoded by the PML-RARA oncogene, differentiation of leukaemic cells and produces clinical remissions. SUMOylation of its PML moiety was previously implicated, but the nature of the degradation pathway involved and the role of PML-RARalpha catabolism in the response to therapy have both remained elusive. Here, we demonstrate that arsenic-induced PML SUMOylation triggers its Lys 48-linked polyubiquitination and proteasome-dependent degradation. When exposed to arsenic, SUMOylated PML recruits RNF4, the human orthologue of the yeast SUMO-dependent E3 ubiquitin-ligase, as well as ubiquitin and proteasomes onto PML nuclear bodies. Arsenic-induced differentiation is impaired in cells transformed by a non-degradable PML-RARalpha SUMOylation mutant or in APL cells transduced with a dominant-negative RNF4, directly implicating PML-RARalpha catabolism in the therapeutic response. We thus identify PML as the first protein degraded by SUMO-dependent polyubiquitination. As PML SUMOylation recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, these domains could physically integrate the SUMOylation, ubiquitination and degradation pathways.     

Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix- associated PML bodies

The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4- ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.     

New insights into the role of PML in tumour suppression

The PML gene is involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), which generates the oncogenic fusion protein PML (promyelocytic leukaemia protein)-retinoic acid receptor alpha. The PML protein localises to a subnuclear structure called the PML nuclear domain (PML-ND), of which PML is the essential structural component. In APL, PML-NDs are disrupted, thus implicating these structures in the pathogenesis of this leukaemia. Unexpectedly, recent studies indicate that PML and the PML-ND play a tumour suppressive role in several different types of human neoplasms in addition to APL. Because of PML's extreme versatility and involvement in multiple cellular pathways, understanding the mechanisms underlying its function, and therefore role in tumour suppression, has been a challenging task. In this review, we attempt to critically appraise the more recent advances in this field and propose new avenues of investigation.     

The nuclear bodies inside out: PML conquers the cytoplasm

The promyelocytic leukemia (PML) protein is the core component of nuclear substructures that host more than 70 proteins, termed nuclear domains 10 or PML-nuclear bodies. PML was first identified as the gene participating in the translocation responsible for the pathogenesis of acute promyelocytic leukemia (APL). The notion that PML is a tumor suppressor gene was soon extrapolated from leukemia to solid tumors. The last decade has radically changed the view of how this tumor suppressor is regulated, how it can be therapeutically targeted, and how it functions. Notably, one of the most recent and striking features uncovered is how PML regulates cellular homeostasis outside its original niche in the nucleus. These new findings open an exciting new area of research in extra-nuclear PML functions.     

The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML.

Acute promyelocytic leukaemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and the retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex (termed ND10, Kr bodies, nuclear bodies, PML oncogenic domains or PODs) which is disrupted in the APL disease state. PML contains a number of characterized motifs including a Zn2+ binding domain called the RING or C3HC4 finger. Here we describe the solution structure of the PML RING finger as solved by 1H NMR methods at physiological pH with r.m.s. deviations for backbone atoms of 0.88 and 1.39 A for all atoms. Additional biophysical studies including CD and optical spectroscopy, show that the PML RING finger requires Zn2+ for autonomous folding and that cysteines are used in metal ligation. A comparison of the structure with the previously solved equine herpes virus IE110 RING finger, shows significant differences suggesting that the RING motif is structurally diverse. The role of the RING domain in PML nuclear body formation was tested in vivo, by using site-directed mutagenesis and immunofluorescence on transiently transfected NIH 3T3 cells. Independently mutating two pairs of cysteines in each of the Zn2+ binding sites prevents PML nuclear body formation, suggesting that a fully folded RING domain is necessary for this process. These results suggest that the PML RING domain is probably involved in protein-protein interactions, a feature which may be common to other RING finger domains.     

PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, beta-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders.