Hugues DE THÉ
Lionel ADES PUPH2
Caroline BERTHIER TCN, P7
Valentina CRIPPA M2 student
Thassadite DIRAMI PhD, IR
Cécile ESNAULT CRCN, INSERM
Pierre FENAUX PUPHCE
Marie-Claude GEOFFROY CRCN, CNRS
Michiko KAWAKITA IR, P7
Fang QIU Postdoc
Hassane SOILIHI TCN, CNRS
COLLÈGE DE FRANCE MEMBERS
Pierre BERCIER PhD Student
Brigitte LAFOND Secretariat
Valérie LALLEMAND-BREITENBACH DR2 – INSERM
Omar FEHRI PhD, IE – INSERM
Domitille REROLLE PhD Student
Annie SITTLER CRCN – CNRS
Hsin-Chieh WU Postdoc
Julien ABLAIN PhD Student
Shirine BENHENDA Postdoc
Juliane HALFTERMEYER PhD Student
Lisa IVANSCHITZ PhD Student
Florence JOLLIVET IE
Morgane LE BRAS MCU
Anne-Lise MAUBERT IE
Muege OGRUNC Postdoc
Laurent PERES TCN, CNRS
Kim RICE Postdoc
Umut SAHIN Postdoc
Adeline VITALIANO-PRUNIER Postdoc
Hao YUAN Postdoc
Acute Promyelocytic Leukaemia, APL, is a simple model of cancer, as it appears to be primarily a monogenic disease. At the biological level, the hallmark t(15,17) translocation leads to fusion between two genes, encoding a PML/RARA fusion protein that initiates APL in mice. Clinical trials have identified two highly efficient drugs, retinoic acid (RA) and arsenic, which we have shown to target PML/RARA and constitute oncogene-targeted therapies (de Thé, 2010). Clinically, RA differentiates APL blasts into granulocytes, a finding that gave rise to the first example of differentiation therapy (de Thé, 2018). The effects of arsenic on APL cells result from a combination of differentiation and apoptosis. PML/RARA is a transcriptional repressor that inhibits myeloid differentiation and enhances the survival and proliferation of early myeloid progenitors. PML/RARA homo-dimerisation through the coiled-coil of PML was proposed to be the primary basis for repression via enhanced binding of co-repressors. Subsequent studies have stressed importance of the following steps for both transcriptional repression and transformation in primary cells:
– sumoylation of the PML moiety of PML/RARA,
– recognition of non-canonical binding sites,
– binding to RXR.
Moreover, as shown below, PML/RARA disrupts PML nuclear bodies, highlighting novel links between nuclear organisation and oncogenesis.
RA-induced reversion of PML/RARA transcriptional repression and gene re-activation were initially thought to be the molecular basis for the therapeutic effect of RA. Yet, this model could not explain the action of arsenic (de Thé and Chen, 2010). In fact, RA both activates transcription and degrades the PML/RARA fusion via the proteasome. Importantly, arsenic also induces proteasome-dependent degradation of both PML and PML/RARA, identifying a major and unexpected similarity between the two agents.
We have demonstrated that while leukemic promyelocytes rapidly differentiate into granulocytes, the leukaemia initiating cells (LIC) only slowly disappear. We have also provided pharmacological and genetic evidence that the fate of these two cell populations can be fully uncoupled (de Thé et al., 2017). While transcriptional activation through PML/RARA clearly accounts for induction of differentiation, PML/RARA degradation by RA and/or arsenic explains LIC clearance and disease eradication. The latter occurs through the reformation of PML Nuclear bodies, the activation of P53 and senescence (Ablain et al., 2014). As we had initially shown in mice (Lallemand-Breitenbach et al., 1999), most APL patients are now cured with the RA/arsenic association, which synergistically degrades PML/RARA and reforms PML NBs (Ablain et al., 2014; Lo-Coco et al., 2013). The key role of PML nuclear bodies in the cure of APL patients was established by the discovery of PML mutations in therapy resistant patients (Lehmann-Che et al., 2014, 2018). Thus, PML is the final effector of targeted cure of APLs.
PML nuclear bodies
PML is the key organiser of PML nuclear bodies (NBs), nuclear matrix-associated small spherical compartments, which recruit a growing number of partner proteins (Lallemand-Breitenbach and de Thé, 2010; Lallemand-Breitenbach and de Thé, 2018). PML and NBs fine-tune a wide variety of processes (senescence, metabolism, self-renewal, apoptosis), most likely through facilitation of partner protein post-translational modifications, resulting in partner sequestration, activation or degradation. PML NBs came to the front stage with our observation that the oncogenic PML/RARA protein disrupts them in a treatment-reversible manner. Our interest was further supported when we found that NBs are indeed required for APL cure by the RA/Arsenic combination (Ablain et al., 2014). PML NBs are regulated by multiple cellular stresses: viral infection, DNA-damage, transformation, oxidative stress. Interestingly, we have identified PML as the first protein degraded by a SUMO-initiated, ubiquitin-mediated proteolysis pathway initiated by direct arsenic binding and disulphide-initiated dimerization (Jeanne et al., 2010; Lallemand-Breitenbach et al., 2008; Lallemand-Breitenbach et al., 2001). These studies highlight the intimate connexions between PML NBs and protein degradation.
From a cell biology perspective, we have dissected the biochemical assembly of PML NBs, we implicated initial PML oxidation, followed by it conjugation by SUMO and partner recruitment through their SIMs (Sahin et al., 2014). In that respect, while Pml-/- mice are devoid of NBs, develop normally and live well, we demonstrated that redox signalling and response to oxidative stress are profoundly altered in Pml-/- mice (Niwa-kawakita et al., 2017).
The current objectives of the group are
i) To clarify the genetic determinants for APL transformation in vivo and therapy response, in particular through the functional analysis of PML knock-in mutants in vivo
ii) To decipher the mechanism of PML-driven senescence in vivo
iii) To explore the implication of PML in interferon response, redox signaling and partner post-translational modifications.
Some key references
► Ablain, J., Rice, K., Soilihi, H., de Reynies, A., Minucci, S., and de Thé, H. Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure. Nat. Med., 2014. 20, 167-174.
► De Thé, H. Differentiation therapy revisited. Nat. Rev. Cancer, 2018. 18, 117-127.
► De Thé, H., and Chen, Z. Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nat. Rev. Cancer, 2010. 10, 775-783.
► De Thé, H., Pandolfi, P.P., and Chen, Z. Acute Promyelocytic Leukemia: A Paradigm for Oncoprotein-Targeted Cure. Cancer Cell, 2017. 32, 552-560.
► Jeanne, M., Lallemand-Breitenbach, V., Ferhi, O., Koken, M., Le Bras, M., Duffort, S., Peres, L., Berthier, C., Soilihi, H., Raught, B., et al. PML/RARA oxidation and arsenic binding initiate the antileukemia response of As2O3. Cancer Cell, 2010. 18, 88-98.
► Lallemand-Breitenbach, V., and de Thé, H. PML nuclear bodies: from architecture to function. Curr. Opin. Cell Biol. 2018. 52, 154-161.
► Lallemand-Breitenbach, V., and de Thé, H. PML nuclear bodies. Cold Spring Harb Perspect Biol 2, 2010. 2(5): a000661.
► Lallemand-Breitenbach, V., Jeanne, M., Benhenda, S., Nasr, R., Lei, M., Peres, L., Zhou, J., Zhu, J., Raught, B., and de Thé, H. Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway. Nat. Cell Biol. 2008. 10, 547-555.
► Lallemand-Breitenbach, V., Zhu, J., Puvion, F., Koken, M., Honore, N., Doubeikovsky, A., Duprez, E., Pandolfi, P.P., Puvion, E., Freemont, P., et al. Role of Promyelocytic Leukemia (PML) Sumolation in Nuclear Body Formation, 11S Proteasome Recruitment, and As(2)O(3)-induced PML or PML/Retinoic Acid Receptor alpha Degradation. J. Exp. Med. 2001. 193, 1361-1372.
► Lallemand-Breitenbach, V., Guillemin, M.-C., Janin, A., Daniel, M.-T., Degos, L., Kogan, S.C., Bishop, J.M., and de Thé, H. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 1999. 189, 1043-1052.
► Lehmann-Che, J., Bally, C., and de Thé, H. Therapy resistance in APL. New Engl. J. Med., 2014. 371, 1171-1172.
► Lo-Coco, F., Avvisati, G., Vignetti, M., Thiede, C., Orlando, S.M., Iacobelli, S., Ferrara, F., Fazi, P., Cicconi, L., Di Bona, E., et al. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. New Engl. J. Med., 2013. 369, 111-121.
► Niwa-Kawakita, M., O. Ferhi, H. Soilihi, M. Le Bras, V. Lallemand-Breitenbach, and H. de Thé. PML is a ROS sensor activating p53 upon oxidative stress. J. Exp. Med., 2017. 214(11): p. 3197-3206.
► Sahin, U., de Thé, H., and Lallemand-Breitenbach, V. PML nuclear bodies: assembly and oxidative stress-sensitive sumoylation. Nucleus, 2014. 5, 499-507.
The group’s primary interest is to explore the connections between genetic events driving cancer development and response to therapy. In acute promyelocytic leukemia (APL), we have made key contributions to the understanding of the basis for leukemic transformation by the PML/RARA fusion protein and arsenic or retinoic acid therapy. Our studies have revealed that both agents bind PML/RARA and thus constitute targeted therapies that both initiate PML/RARA degradation by the proteasome. We then demonstrated the key role of PML/RARA degradation by retinoic acid and arsenic in APL responses. Recently, in work supported by the ERC, we have provided conclusive evidence in mouse models and in patients that, downstream of PML/RARA degradation, re-organization of PML nuclear bodies (NBs) and the subsequent activation of P53 are the key contributors to therapy response by a mechanism with many features of senescence.
The group has thus invested significant efforts to decipher the normal function of PML NBs, as well as the assembly of these membrane-less subnuclear domains. We have demonstrated that PML NBs are directly implicated in response to oxidative stress. This likely reflects the fact that PML is a redox-sensitive protein whose polymerization yields the external scaffolding shell of NBs. The latter drives recruitment of partner proteins by sumoylated PML. The transient sequestration of partners, as well as the UBC9 sumoylation enzyme was then proposed to allow their conjugation by SUMO. These proteins may subsequently be degraded by the SUMO-initiated, ubiquitin activated proteolysis pathway that we discovered when analysis PML degradation by arsenic.