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.

Retinoic acid and arsenic trioxide target the protein stability and transcriptional repression activity of the fusion oncoprotein PML-RARA, resulting in regression of acute promyelocytic leukemia (APL). Phenotypically, retinoic acid induces differentiation of APL cells. Here we show that retinoic acid also triggers growth arrest of leukemia-initiating cells (LICs) ex vivo and their clearance in PML-RARA mouse APL in vivo. Retinoic acid treatment of mouse APLs expressing the fusion protein PLZF-RARA triggers full differentiation, but not LIC loss or disease remission, establishing that differentiation and LIC loss can be uncoupled. Although retinoic acid and arsenic synergize to clear LICs through cooperative PML-RARA degradation, this combination does not enhance differentiation. A cyclic AMP (cAMP)-dependent phosphorylation site in PML-RARA is crucial for retinoic acid-induced PML-RARA degradation and LIC clearance. Moreover, activation of cAMP signaling enhances LIC loss by retinoic acid, identifying cAMP as another potential APL therapy. Thus, whereas transcriptional activation of PML-RARA is likely to control differentiation, its catabolism triggers LIC eradication and long-term remission of mouse APL. Therapy-triggered degradation of oncoproteins could be a general strategy to eradicate cancer stem cells.

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.