Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation Rihab Nasr 1,7,8 , Marie-Claude Guillemin 1,8 , Omar Ferhi 1 , Hassan Soilihi 1 , Laurent Peres 1 , Caroline Berthier 1 , Philippe Rousselot 2 , Macarena Robledo-Sarmiento 1 , Vale ´rie Lallemand-Breitenbach 1 , Bernard Gourmel 1 , Dominique Vitoux 1 , Pier Paolo Pandolfi 3 , Ce ´cile Rochette-Egly 4 , Jun Zhu 1,5 & Hugues de The ´ 1,5,6 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. APL is characterized by a specific t(15;17) translocation that encodes a fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-a (RARA) proteins. PML-RARA is a transcriptional repressor with both gain-of-function and dominant-negative properties, result- ing in transcriptional repression of retinoic acid and non–retinoic acid target genes 1,2 and culminating in differentiation arrest. Gene silencing involves enhanced recruitment of nuclear receptor co-repressors, the polycomb complex or Daxx onto PML-RARA or its obligatory retinoid X receptor-a (RXRA) partner, resulting in changes in chro- matin organization and DNA methylation 3–9 . APL is a unique model in cancer biology in that two therapeutic agents, retinoic acid and arsenic trioxide, both target PML-RARA 10,11 . In vivo, both drugs induce leukemia differentiation to varying extents, making APL the only cancer that can be treated through differentia- tion therapy 12 . High plasma concentrations of retinoic acid are essential for prolonged clinical responses in patients 3,13 . At the molecular level, retinoic acid binds PML-RARA and turns it into a transcriptional activator, but also triggers its degradation 3,14,15 . PML-RARA degradation depends on PML-RARA cleavage by differentiation-activated proteases 14,16 and on recruitment of protea- somes by the retinoic acid–bound RARA moiety of PML-RARA 15,17 . Arsenic activates kinases targeting PML-RARA, or its RXRA partner, and induces PML-RARA degradation through specific SUMOylation and subsequent ubiquitination of its PML moiety 5,18–22 . The respec- tive contributions of drug-induced differentiation, apoptosis, tran- scriptional activation and PML-RARA degradation to APL eradication have been a matter of debate. The molecular pathogenesis of APL and the bases for its response to therapy have been studied in PML-RARA transgenic mice, which recapitulate responses to therapy in individuals with APL 23–26 . In particular, the combination of retinoic acid and arsenic triggers prolonged remission in mouse models and humans. However, although the two drugs synergize to effect APL eradication in vivo, this does not seem to result from enhanced differentiation 23,24,27–29 . Retinoic acid–induced APL differentiation and PML-RARA trans- activation are enhanced by cAMP signaling, and many retinoic acid– resistant APLs differentiate after exposure to retinoic acid and cAMP 1,30,31 . At the molecular level, cAMP activates protein kinase A (PKA), which dissociates RARA from the SMRT co-repressor, allow- ing transcriptional activation by retinoic acid metabolites through RXRA 1,32 . RARA phosphorylation by cAMP-activated PKA on Ser369, within the ligand binding domain, also allows recruitment Received 24 July 2008; accepted 10 October 2008; published online 23 November 2008; corrected after print 7 January 2009; doi:10.1038/nm.1891 1 Universite ´ de Paris 7/CNRS UMR 7151, Equipe labellise ´e N111 Ligue Nationale Contre le Cancer, Service de Biochimie, Ho ˆpital St. Louis, 1, Av. C. Vellefaux, 75475 Paris CEDEX 10, France. 2 UFR de Me ´ decine Universite ´ de Paris 5 & Hematology, Ho ˆpital Mignot, 177 rue de Versailles, 78157 Le Chesnay, France. 3 Department of Pathology, Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 4 Institut de Ge ´ne ´ tique et de Biologie Mole ´culaire et Cellulaire, Department of Functional Genomics, 1 rue Laurent Fries, BP 10142, Illkirch F-67400, France. 5 CNRS laboratoire associe ´ MPC, Shanghai Institute of Hematology, Rui Jin Hospital, 197 Rui Jin Road, 200025 Shanghai, China. 6 Institut Universitaire de France. 7 Current address: Department of Internal Medicine, American University of Beirut, Bliss Street, P.O. Box 11-0236, Beirut, Lebanon. 8 These authors contributed equally to this work. Correspondence should be addressed to H.d.T. (dethe@univ-paris-diderot.fr). NATURE MEDICINE VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 1333 ARTICLES © 2009 Nature America, Inc. All rights reserved.