RESUMEN
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.
Asunto(s)
Estrés Oxidativo , Proteína de la Leucemia Promielocítica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Cuerpos de Inclusión Intranucleares/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Noqueados , Microscopía Confocal , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Proteína de la Leucemia Promielocítica/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)-retinoic acid receptor-α (PML-RARA) fusion protein, which interferes with nuclear receptor signaling and PML nuclear body (NB) assembly. APL is the only malignancy definitively cured by targeted therapies: retinoic acid (RA) and/or arsenic trioxide, which both trigger PML-RARA degradation through nonoverlapping pathways. Yet, the cellular and molecular determinants of treatment efficacy remain disputed. We demonstrate that a functional Pml-transformation-related protein 53 (Trp53) axis is required to eradicate leukemia-initiating cells in a mouse model of APL. Upon RA-induced PML-RARA degradation, normal Pml elicits NB reformation and induces a Trp53 response exhibiting features of senescence but not apoptosis, ultimately abrogating APL-initiating activity. Apart from triggering PML-RARA degradation, arsenic trioxide also targets normal PML to enhance NB reformation, which may explain its clinical potency, alone or with RA. This Pml-Trp53 checkpoint initiated by therapy-triggered NB restoration is specific for PML-RARA-driven APL, but not the RA-resistant promyelocytic leukemia zinc finger (PLZF)-RARA variant. Yet, as NB biogenesis is druggable, it could be therapeutically exploited in non-APL malignancies.
Asunto(s)
Leucemia Promielocítica Aguda/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Trióxido de Arsénico , Arsenicales/farmacología , Biología Computacional , Humanos , Estimación de Kaplan-Meier , Leucemia Promielocítica Aguda/metabolismo , Ratones , Análisis por Micromatrices , Óxidos/farmacología , Proteína de la Leucemia Promielocítica , Proteolisis/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Receptor alfa de Ácido Retinoico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
As(2)O(3) cures acute promyelocytic leukemia (APL) by initiating PML/RARA oncoprotein degradation, through sumoylation of its PML moiety. However, how As(2)O(3) initiates PML sumoylation has remained largely unexplained. As(2)O(3) binds vicinal cysteines and increases reactive oxygen species (ROS) production. We demonstrate that upon As(2)O(3) exposure, PML undergoes ROS-initiated intermolecular disulfide formation and binds arsenic directly. Disulfide-linked PML or PML/RARA multimers form nuclear matrix-associated nuclear bodies (NBs), become sumoylated and are degraded. Hematopoietic progenitors transformed by an As(2)O(3)-binding PML/RARA mutant exhibit defective As(2)O(3) response. Conversely, nonarsenical oxidants elicit PML/RARA multimerization, NB-association, degradation, and leukemia response in vivo, but do not affect PLZF/RARA-driven APLs. Thus, PML oxidation regulates NB-biogenesis, while oxidation-enforced PML/RARA multimerization and direct arsenic-binding cooperate to enforce APL's exquisite As(2)O(3) sensitivity.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/metabolismo , Óxidos/farmacología , Animales , Trióxido de Arsénico , Western Blotting , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Disulfuros/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Leucemia Promielocítica Aguda/patología , Ratones , Ratones Noqueados , Mutación/genética , Proteínas Nucleares/fisiología , Proteínas de Fusión Oncogénica/genética , Proteína de la Leucemia Promielocítica , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Procesamiento Proteico-Postraduccional , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Differentiation therapy for acute myeloid leukemia uses transcriptional modulators to reprogram cancer cells. The most relevant clinical example is acute promyelocytic leukemia (APL), which responds dramatically to either retinoic acid (RA) or arsenic trioxide (As(2)O(3)). In many myeloid leukemia cell lines, cyclic adenosine monophosphate (cAMP) triggers growth arrest, cell death, or differentiation, often in synergy with RA. Nevertheless, the toxicity of cAMP derivatives and lack of suitable models has hampered trials designed to assess the in vivo relevance of theses observations. We show that, in an APL cell line, cAMP analogs blocked cell growth and unraveled As(2)O(3)-triggered differentiation. Similarly, in RA-sensitive or RA-resistant mouse models of APL, continuous infusions of 8-chloro-cyclic adenosine monophosphate (8-Cl-cAMP) triggered major growth arrest, greatly enhanced both spontaneous and RA- or As(2)O(3)-induced differentiation and accelerated the restoration of normal hematopoiesis. Theophylline, a well-tolerated phosphodiesterase inhibitor which stabilizes endogenous cAMP, also impaired APL growth and enhanced spontaneous or As(2)O(3)-triggered cell differentiation in vivo. Accordingly, in an APL patient resistant to combined RA-As(2)O(3) therapy, theophylline induced blast clearance and restored normal hematopoiesis. Taken together, these results demonstrate that in vivo activation of cAMP signaling contributes to APL clearance, independently of its RA-sensitivity, thus raising hopes that other myeloid leukemias may benefit from this therapeutic approach.