Valérie Lallemand-Breitenbach

Arsenic Trioxide Controls the Fate of the PML-RARα Oncoprotein by Directly Binding PML

Arsenic on the Fingers Arsenic, an ancient drug used in traditional Chinese medicine, has attracted wide interest because it has therapeutic activity in patients with acute promyelocytic leukemia (APL). The drug acts by promoting degradation of an oncogenic protein, PML-RARα, a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor α, which is found specifically in APL cells and helps drive their growth. Zhang et al. (p. 240; see the Perspective by Kogan) now explain how arsenic initiates the molecular events leading to PML-RARα degradation. Arsenic was found to bind directly to cysteine residues within zinc finger domains of PML. Arsenic binding then induced oligomerization of PML, which in turn enhanced its association with an enzyme that helps catalyze SUMOylation, a posttranslational modification that can target proteins for degradation. Arsenic, a drug used clinically for leukemia, binds directly to an oncogenic protein, thereby promoting its degradation. Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARα (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARα degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARα and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)–conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug’s mechanism of action and its specificity for APL.

PML Nuclear Bodies

PML nuclear bodies are matrix-associated domains that recruit an astonishing variety of seemingly unrelated proteins. Since their discovery in the early 1960s, PML bodies have fascinated cell biologists because of their beauty and their tight association with cellular disorders. The identification of PML, a gene involved in an oncogenic chromosomal translocation, as the key organizer of these domains drew instant interest onto them. The multiple levels of PML body regulation by a specific posttranslational modification, sumoylation, have raised several unsolved issues. Functionally, PML bodies may sequester, modify or degrade partner proteins, but in many ways, PML bodies still constitute an enigma.

PML nuclear bodies and apoptosis

Promyelocytic leukaemia nuclear bodies (PML NBs) are structured protein complexes associated with the nuclear matrix. PML constitutes the scaffold component of NBs and recruits onto these domains a striking variety of proteins, many of which are involved in apoptosis control. Several reports have directly implicated PML in apoptosis and senescence, but the mechanisms by which these are conveyed are still largely unsettled. Recruitment of partner proteins onto NBs is regulated by PML sumolation, a specific post-translational modification also found in many NB-associated proteins. Among these, several are implicated in transcription repression or activation, like the transcriptional repressor Daxx or the transcriptional activator P53. Whether NBs constitute platforms where active sites of enzymatic modifications are carried out, as suggested for P53, sites of intranuclear protein sequestration, as proposed for Daxx or organelles specialized in catabolism, is still debated. A variety of stress-related signalling pathways dramatically modulate the formation of PML NBs, which may provide a clue as to their physiological function.

Pathways of retinoic acid- or arsenic trioxide-induced PML/RARα catabolism, role of oncogene degradation in disease remission

Although there is evidence to suggest that PML/RARα expression is not the sole genetic event required for the development of acute promyelocytic leukemia (APL), there is little doubt that the fusion protein plays a central role in the initiation of leukemogenesis. The two therapeutic agents, retinoic acid and arsenic, that induce clinical remissions in APL, both target the oncogenic fusion protein, representing the first example of oncogene-directed cancer therapy. This review focuses on the molecular mechanisms accounting for PML/RARα degradation. Each drug targets a specific moiety of the fusion protein (RARα for retinoic acid, PML for arsenic) to the proteasome. Moreover, both activate a common caspase-dependent cleavage in the PML part of the fusion protein. Specific molecular determinants (the AF2 transactivator domain of RARα for retinoic acid and the K160 SUMO-binding site in PML for arsenic) are respectively implicated in RA- or arsenic-triggered catabolism. The respective roles of PML/RARα activation versus its catabolism are discussed with respect to differentiation or apoptosis induction in the context of single or dual therapies.

Acute promyelocytic leukemia, arsenic, and PML bodies

Acute promyelocytic leukemia (APL) is driven by a chromosomal translocation whose product, the PML/retinoic acid (RA) receptor α (RARA) fusion protein, affects both nuclear receptor signaling and PML body assembly. Dissection of APL pathogenesis has led to the rediscovery of PML bodies and revealed their role in cell senescence, disease pathogenesis, and responsiveness to treatment. APL is remarkable because of the fortuitous identification of two clinically effective therapies, RA and arsenic, both of which degrade PML/RARA oncoprotein and, together, cure APL. Analysis of arsenic-induced PML or PML/RARA degradation has implicated oxidative stress in the biogenesis of nuclear bodies and SUMO in their degradation.

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.

Oxidative stress–induced assembly of PML nuclear bodies controls sumoylation of partner proteins

PML multimerization into nuclear bodies following its oxidation promotes sumoylation and sequestration of partner proteins in these structures.

PML nuclear bodies: regulation, function and therapeutic perspectives.

PML nuclear bodies (NBs) were first described by electron microscopy and rediscovered through their treatment‐reversible disruption in a rare leukaemia. They recruit multiple partner proteins and now emerge as interferon‐ and oxidative stress‐responsive sumoylation factories. NBs mediate interferon‐induced viral restriction, enhance proteolysis, finely tune metabolism and enforce stress‐induced senescence. Apart from being markers of cellular stress, PML NBs could be harnessed pharmacologically in a number of conditions, including cancer, viral infection or neurodegenerative diseases. Copyright

PML nuclear bodies: assembly and oxidative stress-sensitive sumoylation.

PML Nuclear Bodies (NBs) have fascinated cell biologists due to their exquisitely dynamic nature and their involvement in human diseases, notably acute promyelocytic leukemia. NBs, as well as their master organizer - the PML protein - exhibit multiple connections with stress responses. Initially viewed as a tumor suppressor, PML recently re-emerged as a multifaceted protein, capable of controlling numerous aspects of cellular homeostasis. NBs recruit many functionally diverse proteins and function as stress-regulated sumoylation factories. SUMO-initiated partner retention can subsequently facilitate a variety of other post-translational modifications, as well as partner degradation. With this newly elucidated central role of stress-enhanced sumoylation, it should now be possible to build a working model for the different NB-regulated cellular activities. Moreover, pharmacological manipulation of NB formation by interferons or oxidants holds the promise of clearing many undesirable proteins for clinical management of malignant, viral or neurodegenerative diseases.

ATL response to arsenic/interferon therapy is triggered by SUMO/PML/RNF4-dependent Tax degradation

The human T-cell lymphotropic virus type I (HTLV-1) Tax transactivator initiates transformation in adult T-cell leukemia/lymphoma (ATL), a highly aggressive chemotherapy-resistant malignancy. The arsenic/interferon combination, which triggers degradation of the Tax oncoprotein, selectively induces apoptosis of ATL cell lines and has significant clinical activity in Tax-driven murine ATL or human patients. However, the role of Tax loss in ATL response is disputed, and the molecular mechanisms driving degradation remain elusive. Here we demonstrate that ATL-derived or HTLV-1-transformed cells are dependent on continuous Tax expression, suggesting that Tax degradation underlies clinical responses to the arsenic/interferon combination. The latter enforces promyelocytic leukemia protein (PML) nuclear body (NB) formation and partner protein recruitment. In arsenic/interferon-treated HTLV-1 transformed or ATL cells, Tax is recruited onto NBs and undergoes PML-dependent hyper-sumoylation by small ubiquitin-like modifier (SUMO)2/3 but not SUMO1, ubiquitination by RNF4, and proteasome-dependent degradation. Thus, the arsenic/interferon combination clears ATL through degradation of its Tax driver, and this regimen could have broader therapeutic value by promoting degradation of other pathogenic sumoylated proteins.