Project Leader : Sylvie Hermouet

MPNs are chronic diseases characterized by an excessive production of mature blood cells from the myeloid lineages, frequently associated with myelofibrosis and splenomegaly. One distinguishes 3 MPNs : polycythemia vera (PV), when the erythroid lineage is the most affected lineage; essential thrombocythemia (ET), when the megakaryocytic lineage is affected; and primary myelofibrosis (PMF), characterized by fibrosis of the bone marrow and splenomegaly. MPN patients present with an inflammatory syndrome of variable intensity: moderate in ET and some PV, severe in other PV and in PMF. MPNs are often complicated by arterial or venous thrombosis, myelofibrosis, or acute myeloid leukemia.

In 2005 the activating V617F mutation in exon 14 of the JAK2 gene (JAK2V617F) was identified in 50% MPNs ; JAK2V617F increases the activity of the JAK2/STAT5 and STAT3 pathways. Since 2005, other JAK2 mutations have been described in MPNs, in acute lymphoid leukemia, and in hereditary forms of thrombocytosis. In about 25% of cases, MPN patients present with mutations in the CALR gene, which encodes calreticulin; CALR mutations result in truncated forms of reticulin. The mechanisms of action of CALR mutations are not fully understood; mutant calreticulin may interact with Mpl, the receptor for thrombopoietin (TPO). In a minority of cases (5%), MPN patients present with an activating mutation in MPL, a JAK2-coupled dimeric receptor expressed by hematopoietic progenitors and platelets. After TPO binding to MPL, JAK2 auto-phosphorylates on tyrosine residues and activates STAT5. Importantly, JAK2 transmits signals of several hematopoietic cytokines (not just TPO), including erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF), interleukin-3 (IL-3), and inflammatory cytokines such as IL-6. JAK2 is also important for the correct localisation of MPL at the cell membrane surface. Hence, any aleration of the expression or function of MPL/JAK2 may result in abnormal myelopoiesis and cytokine signalling and subsequently, in abnormal immune and inflammatory response.

Our own studies and the JAK1/2 inhibitor clinical trials indicate that the clinical presentation and disease evolution of JAK2-mutated MPNs cannot be explained solely by the JAK2V617F mutation and that chronic inflammation plays a major role in the pathogenesis of MPNs. Accordingly, an important reduction of the blood levels of inflammatory cytokines is generally observed in MPN patients who respond well to JAK inhibitors, and clinical symptoms correlate with certain inflammatory cytokines rather than the JAK2V617F mutational load.

Our objectives are to determine the role played by cytokines in MPNs, and to identify the causes of cytokine deregulation.

A balanced activation of STAT1, STAT3 and STAT5 is necessary for a normal, controlled production of myeloid cells. Numerous inflammation cytokines activate the Jak1/Stat1 and Jak2/Stat3 pathways, for multiple actions: i) production of mature myeloid cells (IL-11, IL-6) ; ii) facilitation of cell survival (hepatocyte growth factor, or HGF) ; iii) pro- (IL-6, IL-8, IL-15) ou anti- (HGF, IL-11) inflammatory action; iv) pro-fibrosis (FGF) ; v) pro-angiogenesis (VEGF). These cytokines are present at high concentrations in the bone marrow and blood of MPN patients. Team 16 aims to determine whether over-expression of these cytokines is a consequence of mutations in the JAK2, MPL ou CALR genes, or in oxygen-sensing genes. We also study the effect of JAK2, MPL and CALR mutants on the Jak2/Stat5, Stat3 and Stat1 pathways (4); the expression and cellular localisation of Jak2 (6,9); the expression, maturation and fate of MPL after activation ; and the clinical and biological presentation of patients who carry these mutations (2,3,8). For these studies, international collaborations have been established (European network MPN&MPNr-EuroNet; Prof. Bridget S. Wilson, New Mexico University, Albuquerque, NM, USA). We showed that several inflammation cytokines produced at high level by MPN myeloid progenitors and bone marrow fibroblasts contribute to the abnormal proliferation of MPN progenitor cells (1,5,10). We also studied several mutations of JAK2, described their level of expression, their localisation in the cell and their effect on intra-cellular signalling (4,6,7,9). Moreover, we showed that the JAK2V617F mutation is not sufficient to induce a MPN or leukemic transformation (2,3,8).

As shown below, an improved understanding of the causes and molecular mechanisms of the chronic inflammation associated with MPNs should allow new therapeutic protocoles that combining drugs that target not only JAK2, MPL or CALR mutants, but also the signaling and source(s) of production of inflammation cytokines.

Our studies are performed with the support of local platforms (sequencing, transcriptomics), in collaboration with French and international teams, including MPN&MPNr-EuroNet (ex COST Action BM0902 ; see www.mpneuronet.eu ; Chair: Sylvie Hermouet).

We gratefully acknowledge financial support from the Association Recherche contre le Cancer (ARC) (2010), Ligue Nationale contre le Cancer (2011, 2017), Novartis (Basel) (2014-2016), Cancéropôle Grand Ouest (CGO) and Region Pays de la Loire (HII-GO project, for Hematology, Inflammation, Infection – Grand Ouest) (2015-2016).

Representative Publications
1. Corre-Buscail I, Pineau D, Boissinot M, Hermouet S. Erythropoietin-independent erythroid colony formation by bone marrow progenitors exposed to interleukins 11 and 8. Exp Hematol (2005)
2. Lippert E, Boissinot M, Kralovics R et al. The JAK2-V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera. Blood (2006)
3. Theocharides A, Boissinot M, Girodon F et al. Leukemic blasts in transformed JAK2-V617F positive myeloproliferative disorders are frequently negative for the JAK2-V617F mutation. Blood (2007)
4. Cleyrat C, Jelinek J, Girodon F et al. JAK2 mutation and disease phenotype: A double L611V/V617F in cismutation of JAK2 is associated with isolated erythrocytosis and increased activation of AKT and ERK1/2 rather than STAT5. Leukemia (2010)
5. Boissinot M, Cleyrat C, Vilaine M et al. Anti-inflammatory hepatocyte growth factor and interleukin-11 are overexpressed in Polycythemia Vera and contribute to the growth of mutated erythroblasts independently of JAK2V617F. Oncogene (2011)
6. Girodon F, Steinkamp MP, Cleyrat C, Hermouet S, Wllson BW. Confocal imaging studies cast doubt on nuclear localisation of Jak2V617F. Blood (2011)
7. Vilaine M, Olcaydu D, Harutyunyan A et al. Homologous recombination of wild-type JAK2, a novel early step in the development of myeloproliferative neoplasm. Blood (2011)
8. Lippert E, Mansier O, Migeon M et al. Clinical and biological characterization of patients with low (0.1-2%) JAK2V617F allele burden at diagnosis. Haematologica (2014)
9. Cleyrat C, Dareshouri A, Steinkamp MP et al. Mpl traffics to the cell surface through unconventional and conventional routes. Traffic (2014)
10. Hermouet S, Bigot-Corbel E, Gardie B. Pathogenesis of myeloproliferative neoplasms: Role and mechanisms of chronic inflammation. Mediators Inflamm (2015)

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