PML Nuclear bodies: the cancer connection and beyond.

Promyelocytic leukemia (PML) nuclear bodies, membrane-less organelles in the nucleus, play a crucial role in cellular homeostasis. These dynamic structures result from the assembly of scaffolding PML proteins and various partners. Recent crystal structure analyses revealed essential self-interacting domains, while liquid-liquid phase separation contributes to their formation. PML bodies orchestrate post-translational modifications, particularly stress-induced SUMOylation, impacting target protein functions. Serving as hubs in multiple signaling pathways, they influence cellular processes like senescence. Dysregulation of PML expression contributes to diseases, including cancer, highlighting their significance. Therapeutically, PML bodies are promising targets, exemplified by successful acute promyelocytic leukemia treatment with arsenic trioxide and retinoic acid restoring PML bodies. Understanding their functions illuminates both normal and pathological cellular physiology, guiding potential therapies. This review explores recent advancements in PML body biogenesis, biochemical activity, and their evolving biological roles.

Structural Basis of PML-RARA Oncoprotein Targeting by Arsenic Unravels a Cysteine Rheostat Controlling PML Body Assembly and Function.

PML nuclear bodies (NB) are disrupted in PML-RARA-driven acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) cures 70% of patients with APL, driving PML-RARA degradation and NB reformation. In non-APL cells, arsenic binding onto PML also amplifies NB formation. Yet, the actual molecular mechanism(s) involved remain(s) elusive. Here, we establish that PML NBs display some features of liquid-liquid phase separation and that ATO induces a gel-like transition. PML B-box-2 structure reveals an alpha helix driving B2 trimerization and positioning a cysteine trio to form an ideal arsenic-binding pocket. Altering either of the latter impedes ATO-driven NB assembly, PML sumoylation, and PML-RARA degradation, mechanistically explaining clinical ATO resistance. This B2 trimer and the C213 trio create an oxidation-sensitive rheostat that controls PML NB assembly dynamics and downstream signaling in both basal state and during stress response. These findings identify the structural basis for arsenic targeting of PML that could pave the way to novel cancer drugs. SIGNIFICANCE: Arsenic curative effects in APL rely on PML targeting. We report a PML B-box-2 structure that drives trimer assembly, positioning a cysteine trio to form an arsenic-binding pocket, which is disrupted in resistant patients. Identification of this ROS-sensitive triad controlling PML dynamics and functions could yield novel drugs. See related commentary by Salomoni, p. 2505. This article is featured in Selected Articles from This Issue, p. 2489.

Exploration of nuclear body-enhanced sumoylation reveals that PML represses 2-cell features of embryonic stem cells.

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml-/- mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.

Sumoylation in Physiology, Pathology and Therapy.

Sumoylation is an essential post-translational modification that has evolved to regulate intricate networks within emerging complexities of eukaryotic cells. Thousands of target substrates are modified by SUMO peptides, leading to changes in protein function, stability or localization, often by modulating interactions. At the cellular level, sumoylation functions as a key regulator of transcription, nuclear integrity, proliferation, senescence, lineage commitment and stemness. A growing number of prokaryotic and viral proteins are also emerging as prime sumoylation targets, highlighting the role of this modification during infection and in immune processes. Sumoylation also oversees epigenetic processes. Accordingly, at the physiological level, it acts as a crucial regulator of development. Yet, perhaps the most prominent function of sumoylation, from mammals to plants, is its role in orchestrating organismal responses to environmental stresses ranging from hypoxia to nutrient stress. Consequently, a growing list of pathological conditions, including cancer and neurodegeneration, have now been unambiguously associated with either aberrant sumoylation of specific proteins and/or dysregulated global cellular sumoylation. Therapeutic enforcement of sumoylation can also accomplish remarkable clinical responses in various diseases, notably acute promyelocytic leukemia (APL). In this review, we will discuss how this modification is emerging as a novel drug target, highlighting from the perspective of translational medicine, its potential and limitations.

Actinomycin D Targets NPM1c-Primed Mitochondria to Restore PML-Driven Senescence in AML Therapy.

Acute myeloid leukemia (AML) pathogenesis often involves a mutation in the NPM1 nucleolar chaperone, but the bases for its transforming properties and overall association with favorable therapeutic responses remain incompletely understood. Here we demonstrate that an oncogenic mutant form of NPM1 (NPM1c) impairs mitochondrial function. NPM1c also hampers formation of promyelocytic leukemia (PML) nuclear bodies (NB), which are regulators of mitochondrial fitness and key senescence effectors. Actinomycin D (ActD), an antibiotic with unambiguous clinical efficacy in relapsed/refractory NPM1c-AMLs, targets these primed mitochondria, releasing mitochondrial DNA, activating cyclic GMP-AMP synthase signaling, and boosting reactive oxygen species (ROS) production. The latter restore PML NB formation to drive TP53 activation and senescence of NPM1c-AML cells. In several models, dual targeting of mitochondria by venetoclax and ActD synergized to clear AML and prolong survival through targeting of PML. Our studies reveal an unexpected role for mitochondria downstream of NPM1c and implicate a mitochondrial/ROS/PML/TP53 senescence pathway as an effector of ActD-based therapies. SIGNIFICANCE: ActD induces complete remissions in NPM1-mutant AMLs. We found that NPM1c affects mitochondrial biogenesis and PML NBs. ActD targets mitochondria, yielding ROS which enforce PML NB biogenesis and restore senescence. Dual targeting of mitochondria with ActD and venetoclax sharply potentiates their anti-AML activities in vivo. This article is highlighted in the In This Issue feature, p. 2945.

JAK2V617F myeloproliferative neoplasm eradication by a novel interferon/arsenic therapy involves PML.

Interferon α (IFNα) is used to treat JAK2V617F-driven myeloproliferative neoplasms (MPNs) but rarely clears the disease. We investigated the IFNα mechanism of action focusing on PML, an interferon target and key senescence gene whose targeting by arsenic trioxide (ATO) drives eradication of acute promyelocytic leukemia. ATO sharply potentiated IFNα-induced growth suppression of JAK2V617F patient or mouse hematopoietic progenitors, which required PML and was associated with features of senescence. In a mouse MPN model, combining ATO with IFNα enhanced and accelerated responses, eradicating MPN in most mice by targeting disease-initiating cells. These results predict potent clinical efficacy of the IFNα+ATO combination in patients and identify PML as a major effector of therapy, even in malignancies with an intact PML gene.

PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers.

High-grade serous ovarian cancer (HGSOC) remains an unmet medical challenge. Here, we unravel an unanticipated metabolic heterogeneity in HGSOC. By combining proteomic, metabolomic, and bioergenetic analyses, we identify two molecular subgroups, low- and high-OXPHOS. While low-OXPHOS exhibit a glycolytic metabolism, high-OXPHOS HGSOCs rely on oxidative phosphorylation, supported by glutamine and fatty acid oxidation, and show chronic oxidative stress. We identify an important role for the PML-PGC-1α axis in the metabolic features of high-OXPHOS HGSOC. In high-OXPHOS tumors, chronic oxidative stress promotes aggregation of PML-nuclear bodies, resulting in activation of the transcriptional co-activator PGC-1α. Active PGC-1α increases synthesis of electron transport chain complexes, thereby promoting mitochondrial respiration. Importantly, high-OXPHOS HGSOCs exhibit increased response to conventional chemotherapies, in which increased oxidative stress, PML, and potentially ferroptosis play key functions. Collectively, our data establish a stress-mediated PML-PGC-1α-dependent mechanism that promotes OXPHOS metabolism and chemosensitivity in ovarian cancer.

PML nuclear bodies: from architecture to function.

PML nuclear bodies are nucleated by the PML protein, which polymerizes into spherical shells where it concentrates many unrelated partner proteins. Emerging data has connected PML bodies to post-translational control, notably conjugation by SUMOs. High concentrations of SUMO-bound proteins were proposed to condense into liquid-like droplets and such phase transition may occur within NBs. Many stress pathways modulate NB formation and recent findings have directly implicated PML in oxidative stress response in vivo. PML may also undergo SUMO-dependent ubiquitination/degradation. We highlight recent advances linking PML to partner degradation and other adaptative post-translational modifications in the context of chromatin remodeling, telomere biology, senescence or viral infections.

Stability of HTLV-2 antisense protein is controlled by PML nuclear bodies in a SUMO-dependent manner.

Since the identification of the antisense protein of HTLV-2 (APH-2) and the demonstration that APH-2 mRNA is expressed in vivo in most HTLV-2 carriers, much effort has been dedicated to the elucidation of similarities and/or differences between APH-2 and HBZ, the antisense protein of HTLV-1. Similar to HBZ, APH-2 negatively regulates HTLV-2 transcription. However, it does not promote cell proliferation. In contrast to HBZ, APH-2 half-life is very short. Here, we show that APH-2 is addressed to PML nuclear bodies in T-cells, as well as in different cell types. Covalent SUMOylation of APH-2 is readily detected, indicating that APH-2 might be addressed to the PML nuclear bodies in a SUMO-dependent manner. We further show that silencing of PML increases expression of APH-2, while expression of HBZ is unaffected. On the other hand, SUMO-1 overexpression leads to a specific loss of APH-2 expression that is restored upon proteasome inhibition. Furthermore, the carboxy-terminal LAGLL motif of APH-2 is responsible for both the targeting of the protein to PML nuclear bodies and its short half-life. Taken together, these observations indicate that natural APH-2 targeting to PML nuclear bodies induces proteasomal degradation of the viral protein in a SUMO-dependent manner. Hence, this study deciphers the molecular and cellular bases of APH-2 short half-life in comparison to HBZ and highlights key differences in the post-translational mechanisms that control the expression of both proteins.

RING tetramerization is required for nuclear body biogenesis and PML sumoylation.

ProMyelocyticLeukemia nuclear bodies (PML NBs) are stress-regulated domains directly implicated in acute promyelocytic leukemia eradication. Most TRIM family members bind ubiquitin E2s and many acquire ligase activity upon RING dimerization. In contrast, PML binds UBC9, the SUMO E2 enzyme. Here, using X-ray crystallography and SAXS characterization, we demonstrate that PML RING tetramerizes through highly conserved PML-specific sequences, which are required for NB assembly and PML sumoylation. Conserved residues implicated in RING dimerization of other TRIMs also contribute to PML tetramer stability. Wild-type PML rescues the ability of some RING mutants to form NBs as well as their sumoylation. Impaired RING tetramerization abolishes PML/RARA-driven leukemogenesis in vivo and arsenic-induced differentiation ex vivo. Our studies thus identify RING tetramerization as a key step in the NB macro-molecular scaffolding. They suggest that higher order RING interactions allow efficient UBC9 recruitment and thus change the biochemical nature of TRIM-facilitated post-translational modifications.

PML nuclear bodies, membrane-less domains acting as ROS sensors?

PML Nuclear bodies (PML NBs) are spherical domains associated with a broad range of activities upon stress responses such as apoptosis, senescence DNA repair, epigenetic control, as well as control of oncogenesis. These bodies are considered as privileged sites for post-translational modifications, where sumoylation plays a key role. Here we summarize recent in vitro and in vivo findings on the link between PML NBs and ROS, in particular PML contributions to oxidative stress response. We discuss how it may regulate switch from cell protection against stress to cell arrest/cell death.

PML is a ROS sensor activating p53 upon oxidative stress.

Promyelocytic leukemia (PML) nuclear bodies (NBs) recruit partner proteins, including p53 and its regulators, thereby controlling their abundance or function. Investigating arsenic sensitivity of acute promyelocytic leukemia, we proposed that PML oxidation promotes NB biogenesis. However, physiological links between PML and oxidative stress response in vivo remain unexplored. Here, we identify PML as a reactive oxygen species (ROS) sensor. Pml-/- cells accumulate ROS, whereas PML expression decreases ROS levels. Unexpectedly, Pml-/- embryos survive acute glutathione depletion. Moreover, Pml-/- animals are resistant to acetaminophen hepatotoxicity or fasting-induced steatosis. Molecularly, Pml-/- animals fail to properly activate oxidative stress-responsive p53 targets, whereas the NRF2 response is amplified and accelerated. Finally, in an oxidative stress-prone background, Pml-/- animals display a longevity phenotype, likely reflecting decreased basal p53 activation. Thus, similar to p53, PML exerts basal antioxidant properties but also drives oxidative stress-induced changes in cell survival/proliferation or metabolism in vivo. Through NB biogenesis, PML therefore couples ROS sensing to p53 responses, shedding a new light on the role of PML in senescence or stem cell biology.

Promyelocytic Leukemia Protein (PML) Controls Listeria monocytogenes Infection.

The promyelocytic leukemia protein (PML) is the main organizer of stress-responsive subnuclear structures called PML nuclear bodies. These structures recruit multiple interactors and modulate their abundance or their posttranslational modifications, notably by the SUMO ubiquitin-like modifiers. The involvement of PML in antiviral responses is well established. In contrast, the role of PML in bacterial infection remains poorly characterized. Here, we show that PML restricts infection by the pathogenic bacterium Listeria monocytogenes but not by Salmonella enterica serovar Typhimurium. During infection, PML undergoes oxidation-mediated multimerization, associates with the nuclear matrix, and becomes de-SUMOylated due to the pore-forming activity of the Listeria toxin listeriolysin O (LLO). These events trigger an antibacterial response that is not observed during in vitro infection by an LLO-defective Listeria mutant, but which can be phenocopied by specific induction of PML de-SUMOylation. Using transcriptomic and proteomic microarrays, we also characterized a network of immunity genes and cytokines, which are regulated by PML in response to Listeria infection but independently from the listeriolysin O toxin. Our study thus highlights two mechanistically distinct complementary roles of PML in host responses against bacterial infection. IMPORTANCE: The promyelocytic leukemia protein (PML) is a eukaryotic protein that can polymerize in discrete nuclear assemblies known as PML nuclear bodies (NBs) and plays essential roles in many different cellular processes. Key to its function, PML can be posttranslationally modified by SUMO, a ubiquitin-like modifier. Identification of the role of PML in antiviral defenses has been deeply documented. In contrast, the role of PML in antibacterial defenses remains elusive. Here, we identify two mechanistically distinct complementary roles of PML in antibacterial responses against pathogens such as Listeria: (i) we show that PML regulates the expression of immunity genes in response to bacterial infection, and (ii) we unveil the fact that modification of PML SUMOylation by bacterial pore-forming toxins is sensed as a danger signal, leading to a restriction of bacterial intracellular multiplication. Taken together, our data reinforce the concept that intranuclear bodies can dynamically regulate important processes, such as defense against invaders.

Promyelocytic Leukemia Protein, a Protein at the Crossroad of Oxidative Stress and Metabolism.

SIGNIFICANCE: Cellular metabolic activity impacts the production of reactive oxygen species (ROS), both positively through mitochondrial oxidative processes and negatively by promoting the production of reducing agents (including NADPH and reduced glutathione). A defined metabolic state in cancer cells is critical for cell growth and long-term self-renewal, and such state is intrinsically associated with redox balance. Promyelocytic leukemia protein (PML) regulates several biological processes, at least in part, through its ability to control the assembly of PML nuclear bodies (PML NBs). Recent Advances: PML is oxidation-prone, and oxidative stress promotes NB biogenesis. These nuclear subdomains recruit many nuclear proteins and regulate their SUMOylation and other post-translational modifications. Some of these cargos-such as p53, SIRT1, AKT, and mammalian target of rapamycin (mTOR)-are key regulators of cell fate. PML was also recently shown to regulate oxidation. CRITICAL ISSUES: While it was long considered primarily as a tumor suppressor protein, PML-regulated metabolic switch uncovered that this protein could promote survival and/or stemness of some normal or cancer cells. In this study, we review the recent findings on this multifunctional protein. FUTURE DIRECTIONS: Studying PML scaffolding functions as well as its fine role in the activation of p53 or fatty acid oxidation will bring new insights in how PML could bridge oxidative stress, senescence, cell death, and metabolism. Antioxid. Redox Signal. 26, 432-444.

Comment on "SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML".

Fasci et al proposed that a SENP1-mediated switch from SUMO2 to SUMO1 conjugation on Lys(65) in promyelocytic leukemia protein (PML) is required for arsenic-induced PML degradation, the basis for the antileukemic activity of arsenic. We found that PML or PML/RARA (retinoic acid receptor α) mutants that cannot be SUMO-conjugated on this specific site nevertheless underwent immediate arsenic-triggered SUMO modification. Moreover, these mutants were efficiently degraded in cells and even in vivo, demonstrating that SUMOylation of Lys(65) was dispensable for arsenic response. The existence and putative role of a SUMO switch on PML should thus be reassessed.

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.

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.

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.

Interferon controls SUMO availability via the Lin28 and let-7 axis to impede virus replication.

Small ubiquitin-related modifier (SUMO) protein conjugation onto target proteins regulates multiple cellular functions, including defence against pathogens, stemness and senescence. SUMO1 peptides are limiting in quantity and are thus mainly conjugated to high-affinity targets. Conjugation of SUMO2/3 paralogues is primarily stress inducible and may initiate target degradation. Here we demonstrate that the expression of SUMO1/2/3 is dramatically enhanced by interferons through an miRNA-based mechanism involving the Lin28/let-7 axis, a master regulator of stemness. Normal haematopoietic progenitors indeed display much higher SUMO contents than their differentiated progeny. Critically, SUMOs contribute to the antiviral effects of interferons against HSV1 or HIV. Promyelocytic leukemia (PML) nuclear bodies are interferon-induced domains, which facilitate sumoylation of a subset of targets. Our findings thus identify an integrated interferon-responsive PML/SUMO pathway that impedes viral replication by enhancing SUMO conjugation and possibly also modifying the repertoire of targets. Interferon-enhanced post-translational modifications may be essential for senescence or stem cell self-renewal, and initiate SUMO-dependent proteolysis.

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

The promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are stress-responsive domains where many partner proteins accumulate. Here, we clarify the basis for NB formation and identify stress-induced partner sumoylation as the primary NB function. NB nucleation does not rely primarily on intermolecular interactions between the PML SUMO-interacting motif (SIM) and SUMO, but instead results from oxidation-mediated PML multimerization. Oxidized PML spherical meshes recruit UBC9, which enhances PML sumoylation, allow partner recruitment through SIM interactions, and ultimately enhance partner sumoylation. Intermolecular SUMO-SIM interactions then enforce partner sequestration within the NB inner core. Accordingly, oxidative stress enhances NB formation and global sumoylation in vivo. Some NB-associated sumoylated partners also become polyubiquitinated by RNF4, precipitating their proteasomal degradation. As several partners are protein-modifying enzymes, NBs could act as sensors that facilitate and confer oxidative stress sensitivity not only to sumoylation but also to other post-translational modifications, thereby explaining alterations of stress response upon PML or NB loss.