Nuclear Organization and post-translational control in physio-pathology
Principal Investigators:Valérie LALLEMAND-BREITENBACH, DR2 INSERM & Hugues DE THÉ, Pr. Collège de France
PML nuclear bodies (PML NBs) are stress-responsive compartments organized by the scaffolding ProMyelocytic Leukemia protein (PML) (reviewed in Lallemand-Breitenbach 2018). PML polymerizes into the spherical outer shell of the bodies and recruits a variety of unrelated proteins within their inner core. PML NBs have been involved in various biological processes such as senescence, apoptosis, self-renewal and more recently metabolism, raising the question of an unifying function underlying these diverse activities. PML is a tumor suppressor, but may also be essential for maintenance of some cancer stem cells.
PML NBs gained interest after the observation that they were disrupted in a treatment-reversible manner in the leukemic cells of patients with Acute Promyelocytic Leukemia (APL) (reviewed in de Thé 2017). APL is caused by the oncogenic PML/RARA fusion protein. The two clinically highly efficient drugs, all trans-retinoic acid (ATRA) and arsenic trioxide (arsenic) directly target PML/RARA for degradation (reviewed in de Thé 2010, 2017). We unraveled highly synergistic effects of ATRA/arsenic combination in APL mouse model that was successfully transferred to clinic and most APL patients are now cured with combination (Lo-Coco 2013). Our interest for the mechanism of action of these drugs led us to unravel a novel protein degradation pathway based on post-translational modification by SUMOs (Lallemand-Breitenbach 2001, 2008), as well as a new key function of PML NBs in redox sensing (Kawakita 2017). PML function in ROS-sensing may underlie the diversity of the biological and pathological processes in which the protein was involved: inflammation, virus infections or cancers.
PML NBs (red) in the nucleus (blue) before (left) and after (right) arsenic treatment.
Mechanism of PML Nuclear Bodies biogenesis & oxidative stress
PML (red) forms the shell-like scaffold and recruits proteins, as the key SUMO enzyme UBC9 (green) in their core upon arsenic.
PML NBs are 100nm- to 1um-diameter spherical domains proeminent in inflammatory tissues or stressed cells. There is no comprehensive list of PML NBs-associated proteins obtained through PML NBs isolation, due to the insolubility of the PML shell. From a cell biology perspective, we have dissected the biochemical assembly of PML NBs and we unravelled an initial PML multimerization and oxidation, followed by its conjugation by SUMO. SUMOs are ubiquitin-like polypeptides mainly involved in protein complex stabilization, trough their ability to interact with SUMO-interacting Motifs (SIMs). Sumoylation relies on a single E2 conjugating enzyme, UBC9, which is efficiently recruited by PML NBs upon arsenic, explaining PML hyper-sumoylation (Sahin 2014)(Wang 2018). PML sumoylation on specific target lysines subsequently controls the recruitment of partner proteins through their SIMs (Sahin 2014).
PML is a cysteine-rich and oxidation-prone protein. While some of these cysteines participate in Zn-finger domain folding, others are oxidized and bind directly arsenic in a region mutated in therapy-resistant APL patients (Jeanne 2010). We aim at unraveling how oxidative-stress drives PML NB formation and how sumoylation of PML contributes to NB biogenesis and function. Combining mass spectrometry analysis, characterization of PML biochemical and biophysics properties, as well as modeling in collaborations with biophysicists, our work aims at clarifying the assembly and dynamics of these mysterious nuclear membrane-less compartments.
Function of PML Nuclear Bodies
Posttranslational activity of PML NBs upon oxidative stress.
Sumoylation is a posttranslational modification regulating multiple processes including DNA repair, transcription control etc. While investigating arsenic-induced degradation of PML/RARA, we unravelled a new proteolysis mechanism triggered by sequential SUMO and ubiquitin conjugation, thanks to the SIM-containing ubiquitin ligase RNF4 (Lallemand-Breitenbach 2008). PML was the first identified substrate of this ligase discovered in Yeast by other groups. Arsenic induces the sumoylation of SP100 and TDG, two NB-associated proteins, in a PML dependant manner in cells (Sahin 2014). We have now evidence that PML NBs are hubs controlling sumoylation and degradation in response to oxidative stresses in vivo. Such enhanced sumoylation may result from a concentration effect of PML NBs that recruit UBC9 together with SUMOs and partner substrates.
Interferons induce expression of both PML and SUMO which are required for anti-viral effects of interferons (Sahin 2014b). Pml-/- mice are devoid of NBs, develop normally and live well, we demonstrated that redox signaling and response to oxidative stress are profoundly altered in this context (Niwa-kawakita 2017). Altogether, the ROS-sensing role of PML couples subcellular organization to sumoylation control. More broadly, sumoylation of many PTM enzymes present in PML NBs could favor a second set of PTMs (Acetylation, Phosphorylation) onto partner proteins, as we demonstrated for Ubiquitin. We aim at further exploring the role of PML in redox signaling, interferon and stress responses.
Harnessing PML Nuclear Bodies for therapy
The PML/RARA oncoprotein exerts negative effects on PML and RARA proteins encoded by the non-rearranged alleles in APL cells (reviewed in de Thé 2010, 2017). PML/RARA represses RARA target genes and disrupts PML NBs, leading to the inhibition of RARA-mediated meyloid differentiation and PML downstream signaling. As we had initially demonstrated in mice (Lallemand-Breitenbach 1999), most APL patients are now cured with the RA/arsenic association, which synergistically degrades PML/RARA and reforms PML NBs (Ablain 2014; Lo-Coco 2013). We demonstrated that arsenic-induced NB reorganization synergizes with therapy-induced PML/RARA degradation to trigger senescence of APL leukemia-initiating cells and thus APL cure (Ablain 2014). 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 2014).
We now want to attempt to generalize the concept of leukemia eradication through oncogene catabolism and PML NB signaling to other malignancies, particularly those responsive to interferons (a major PML inducer) or where PML NBs are abnormal. Indeed, the curative pathways that we have outlined in APL may be shared by other therapy/malignancies couples.
Selected publications 2008-2021
- Wu, H.C., Rérolle, D., and de Thé, H. (2021). PML/RARA destabilization by hyperthermia: a new model for oncogenic fusion protein degradation? Blood Cancer Discov 2, 300–301.
- Hleihel, R., El Hajj, H., Wu, H.-C., Berthier, C., Zhu, H.-H., Massoud, R., Chakhachiro, Z., El Sabban, M., De The, H., and Bazarbachi, A. (2021). A Pin1/PML/P53 axis activated by retinoic acid in NPM-1c-acute myeloid leukemia. Haematologica.
- Geoffroy, M.-C., Esnault, C., and de Thé, H. (2021). Retinoids in haematology : a timely revival? Blood.
- Esnault, C., Rahmé, R., and de Thé, H. (2021). [Arsenic: The gold standard for acute promyelocytic leukaemia with FLT3-ITD mutation]. Med Sci (Paris) 37, 544–546.
- Esnault, C., Rahmé, R., Rice, K.L., Berthier, C., Gaillard, C., Quentin, S., Maubert, A.-L., Kogan, S., and de Thé, H. (2019). FLT3-ITD impedes retinoic acid, but not arsenic, responses in murine acute promyelocytic leukemias. Blood 133, 1495–1506.
- Wang, L., Gao, S., Wang, H., Xue, C., Liu, X., Yuan, H., Wang, Z., Chen, S., Chen, Z., de Thé, H., et al. (2019). Interferon regulatory factor 2 binding protein 2b regulates neutrophil versus macrophage fate during zebrafish definitive myelopoiesis. Haematologica.
- De Thé, H. (2018). Differentiation therapy revisited. Nat. Rev. Cancer 18, 117–127.
- Lehmann-Che, J., Bally, C., Letouzé, E., Berthier, C., Yuan, H., Jollivet, F., Ades, L., Cassinat, B., Hirsch, P., Pigneux, A., Mozziconacci, M.-J., Kogan, S., Fenaux, P., de Thé, H., (2018). Dual origin of relapses in retinoic-acid resistant acute promyelocytic leukemia. Nat Commun 9, 2047.
- Lallemand-Breitenbach, V., and de Thé, H. (2018). PML nuclear bodies: from architecture to function. Curr. Opin. Cell Biol. 52, 154–161.
- Wang, P., Benhenda, S., Wu, H., Lallemand-Breitenbach, V., Zhen, T., Jollivet, F., Peres, L., Li, Y., Chen, S.-J., Chen, Z., et al. (2018). RING tetramerization is required for nuclear body biogenesis and PML sumoylation. Nat Commun 9, 1277.
- Niwa-Kawakita, M., Ferhi, O., Soilihi, H., Le Bras, M., Lallemand-Breitenbach, V., and de Thé, H. (2017). PML is a ROS sensor activating p53 upon oxidative stress. J. Exp. Med.
- de Thé, H., Pandolfi, P. P., and Chen, Z. (2017). Acute Promyelocytic Leukemia: A Paradigm for Oncoprotein-Targeted Cure. Cancer Cell 32, 552–560.
- Sahin, U., Ferhi, O., Jeanne, M., Benhenda, S., Berthier, C., Jollivet, F., Niwa-Kawakita, M., Faklaris, O., Setterblad, N., de Thé, H., et al. (2014). Oxidative stress-induced assembly of PML nuclear bodies controls sumoylation of partner proteins. J. Cell Biol. 204, 931–945.
- Sahin, U., Ferhi, O., Carnec, X., Zamborlini, A., Peres, L., Jollivet, F., Vitaliano-Prunier, A., de Thé, H., and Lallemand-Breitenbach, V. (2014). Interferon controls SUMO availability via the Lin28 and let-7 axis to impede virus replication. Nat Commun 5, 4187.
- Ablain, J., Rice, K., Soilihi, H., de Reynies, A., Minucci, S., and de Thé, H. (2014). Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure. Nat. Med. 20, 167–174.
- Lehmann-Che, J., Bally, C., and de Thé, H. (2014). Resistance to therapy in acute promyelocytic leukemia.New England Journal of Medicine 371, 1170–1172.
- 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. (2013). Retinoic Acid and Arsenic Trioxide for Acute Promyelocytic Leukemia. New England Journal of Medicine 369, 111–121.
- Jeanne, M., Lallemand-Breitenbach, V., Ferhi, O., Koken, M., Le Bras, M., Duffort, S., Peres, L., Berthier, C., Soilihi, H., Raught, B., et al. (2010). PML/RARA oxidation and arsenic binding initiate the antileukemia response of As2O3. Cancer Cell 18, 88–98.
- Lallemand-Breitenbach, V., and de Thé, H. (2010). PML nuclear bodies. Cold Spring Harb Perspect Biol 2, a000661.
- de Thé, H., and Chen, Z. (2010). Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nat. Rev. Cancer 10, 775–783.
- Zhang, X.-W., Yan, X.-J., Zhou, Z.-R., Yang, F.-F., Wu, Z.-Y., Sun, H.-B., Liang, W.-X., Song, A.-X., Lallemand-Breitenbach, V., Jeanne, M., Zhang Q.-Y., Yang H.-Y., Huang Q.-H., Zhou G. B., Tong J. H., Zhang Y., Wu J. H., Hu H. Y., de Thé H., Chen S. J., Chen Z. (2010). Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML. Science 328, 240–243.
- Lallemand-Breitenbach, V., Jeanne, M., Benhenda, S., Nasr, R., Lei, M., Peres, L., Zhou, J., Zhu, J., Raught, B., and de Thé, H. (2008). Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway. Nat. Cell Biol. 10, 547–555.
- Nasr, R., Guillemin, M.-C., Ferhi, O., Soilihi, H., Peres, L., Berthier, C., Rousselot, P., Robledo-Sarmiento, M., Lallemand-Breitenbach, V., Gourmel, B., et al. (2008). Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation. Nat. Med. 14, 1333–1342.
Lallemand-Breitenbach Valérie, DR2 INSERM
de Thé Hugues, Professor Collège de France
Sittler Annie, CRCN CNRS
Geoffroy Marie-Claude, CRCN CNRS
Postdoctoral fellows & PhD Students:
Bercier Pierre, PhD student
Rerolle Domitille, PhD student
Berthier Caroline, IE CDF
Ferhi Omar, IE INSERM