Oncogene (2011) 30, 990–1001 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Anti-inflammatory cytokines hepatocyte growth factor and interleukin-11 are over-expressed in Polycythemia vera and contribute to the growth of clonal erythroblasts independently of JAK2V617F M Boissinot1,3,4, C Cleyrat1,3, M Vilaine1, Y Jacques1, I Corre1 and S Hermouet1,2 1INSERM UMR 892, Institut de Biologie, Centre Hospitalier Universitaire, Nantes, France and 2Laboratoire d’He´matologie, Institut de Biologie, Centre Hospitalier Universitaire, Nantes, France The V617F activating mutation of janus kinase 2 (JAK2), Keywords: Polycythemia vera; JAK2V617F; hepatocyte a kinase essential for cytokine signalling, characterizes growth factor (HGF); interleukin 11 (IL-11); interleukin Polycythemia vera (PV), one of the myeloproliferative 6 (IL-6); inflammation neoplasms (MPN). However, not all MPNs carry mutations of JAK2, and in JAK2-mutated patients, expression of JAK2V617F does not always result in clone expansion. In the present study, we provide evidence that Introduction inflammation-linked cytokines are required for the growth of JAK2V617F-mutated erythroid progenitors. In a first Myeloproliferative neoplasms (MPNs) constitute a series of experiments, we searched for cytokines over- group of three clonal diseases: Polycythemia vera expressed in PV using cytokine antibody (Ab) arrays, and (PV), essential thrombocythemia (ET) and primary enzyme-linked immunosorbent assays for analyses of myelofibrosis. About half of MPN patients present with serum and bone marrow (BM) plasma, and quantitative activating mutations in the janus kinase 2 (JAK2) gene, reverse transcription–PCRs for analyses of cells purified which encodes for a tyrosine kinase essential for the from PV patients and controls. We found that PV patients signalling of many cytokines (Baxter et al., 2005; James over-expressed anti-inflammatory hepatocyte growth fac- et al., 2005; Kralovics et al., 2005). Among MPNs, PV is tor (HGF) and interleukin-11 (IL-11), BM mesenchymal characterized by an excessive production of erythro- stromal cells (BMMSCs) and erythroblasts being the cytes, resulting in an elevated hematocrit associated with main producers. In a second series of experiments, variable leukocytosis and thrombocytosis. Activating autocrine/paracrine cytokine stimulation of erythroblasts mutations of JAK2, most often V617F (JAK2V617F), was blocked using neutralizing Abs specific for IL-11 or c- are found in 495% of PV cases. It is well established MET, the HGF receptor. The growth of JAK2V617F- that erythroid differentiation depends on erythropoietin mutated HEL cells and PV erythroblasts was inhibited, (Epo) and that JAK2 is the main signal transducer indicating that JAK2-mutated cells depend on HGF activated by Epo receptors (Epo-Rs). Yet, in murine and IL-11 for their growth. Additional experiments models, JAK2V617F is associated with polycythemia showed that transient expression of JAK2V617F in only when expressed at high levels (450% JAK2V617F- BaF-3/erythropoietin receptor cells, and invalidation of mutated alleles), whereas a significant proportion of PV JAK2V617F in HEL cells using anti-JAK2 small inter- patients carry o50% JAK2V617F (Lacout et al., 2006; fering RNA, did not affect HGF and IL-11 expression. Lippert et al., 2006; Wernig et al., 2006; Tiedt et al., Thus, anti-inflammatory HGF and IL-11 are upregulated 2008). We now know that the JAK2V617F mutation is in PV and their overproduction is not a consequence of frequently associated with other molecular genetic JAK2V617F. As both cytokines contribute to the abnormalities and that the JAK2V617F mutation can proliferation of PV erythroblasts, blocking the c-MET/ occur several times in certain patients; moreover, the HGF/IL-11 pathways could be of interest as an additional presence of JAK2V617F does not ensure expansion of therapeutic option in PV. mutated progenitors (Nussenzveig et al., 2007; Kralo- Oncogene (2011) 30, 990–1001; doi:10.1038/onc.2010.479; vics, 2008; Lambert et al., 2009; Schaub et al., 2009; published online 1 November 2010 Cleyrat et al., 2010). Hence, the role of JAK2V617F in MPN and the consequences of its expression in hematopoietic progenitors remain incompletely under- Correspondence: Dr S Hermouet, INSERM UMR 892, Institut de Biologie, Centre Hospitalier Universitaire, 9 quai Moncousu, Nantes stood. 44093, France. Recently, a particular haplotype of chromosome 9p E-mail: [email protected] including the gene JAK2, called the ‘46/1’ or ‘GGCC’ 3These authors contributed equally to this work. haplotype, was found to be associated with a predis- 4Current address: LIMM, Experimental Haematology, Wellcome Trust Brenner Building, Leeds, UK. position to MPN, with or without mutation of JAK2 Received 24 March 2010; revised 10 August 2010; accepted 14 September (Jones et al., 2009; Kilpivaara et al., 2009; Olcaydu 2010; published online 1 November 2010 et al., 2009). The ‘46/1’ haplotype is also associated with Role of HGF and IL-11 in Polycythemia vera M Boissinot et al 991 chronic inflammation, notably inflammatory bowel and in pools of BM plasma from PV patients, compared disease (Ferguson et al., 2010). Interestingly, although with SE patients (Supplementary Figure 1). SE and PV most evident in primary myelofibrosis, signs of chronic differed from HD in having high levels of IL-8, leptin inflammation, such as increased production of inflam- and macrophage chemotactic protein-1 (MCP-1) in matory cytokines, interleukin (IL)-6 and vascular serum. PV differed from SE in having high levels of endothelial growth factor, are also observed in PV and hepatocyte growth factor (HGF) in serum, and high in ET (Wickenhauser et al., 1999; Musolino et al., 2002; levels of tissue inhibitor of metalloproteases-1 (TIMP-1) Panteli et al., 2005; Le Bousse-Kerdile´s and Martyre´, in BM plasma. IL-8, leptin, MCP-1, HGF and TIMP-1 1999). Inflammation-associated symptoms were long are molecules linked to inflammation, as was IL-11, considered consequences of activating mutations of previously reported elevated in PV (Hermouet et al., JAK2, but recent clinical trials of JAK2 antagonists 2002; Corre-Buscail et al., 2005). showed that drugs not strictly specific for JAK2 were Second, IL-8, leptin, MCP-1, HGF, TIMP-1, IL-11 most efficient in reducing inflammation-linked cytokine and also IL-6, a major pro-inflammatory cytokine, were levels and symptoms, indicating that molecules other measured in PV and SE using ELISAs and levels were than JAK2 were likely involved (Tefferi, 2010). compared with those of HD. As shown in Table 1, We begun to investigate the possible stimulation of analysis of serum of SE and PV patients confirmed high PV progenitors by inflammation-linked, JAK2-activat- levels of IL-8, leptin, HGF and MCP-1; TIMP-1 levels ing cytokines because of previous observations of high were low. PV differed from SE by lower levels of leptin messenger RNA (mRNA) levels of JAK2 in patients in serum (male patients only) and higher levels of IL-11 with erythrocytosis, either reactive or secondary (sec- and HGF. For comparison, IL-11 and HGF measured ondary erythrocytosis (SE)—high expression of JAK2 in the serum of 15 ET patients were either not detected wild type (JAK2WT)) or primary (PV—high expression (IL-11) or comparable to values observed for control SE of both JAK2WT and JAK2V617F) (Lippert et al., patients: median serum HGF values, in pg/ml, were 2006). In PV, high mRNA expression of JAK2 could be 1380 in SE (n ¼ 34) and 1594 in ET (n ¼ 15) vs 4673 in due to multiple copies (42 per cell) of the JAK2 gene, PV (n ¼ 49). TIMP-1 levels were confirmed as elevated but this concerns a small minority of patients (Najfeld in PV compared with SE only in BM plasma. Although et al., 2007). As JAK2V617F is not sufficient for the serum IL-6 was elevated for some PV patients, overall development of the PV phenotype and by analogy with IL-6 levels were not found to be significantly high in PV. SE with increased Epo secretion, we reasoned that Of the three molecules confirmed as elevated in PV by increased JAK2 mRNA expression in PV could be due ELISA, HGF and IL-11 seemed to be most interesting. to chronic stimulation by proerythroid, JAK2-activating Indeed, it was established that HGF could induce IL-11, cytokines other than Epo, which is low or undetectable an established stimulant of erythropoiesis (Quesniaux in PV. To investigate this hypothesis, we used cytokine et al., 1992; Schwertschlag et al., 1999; Matsuda-Hashii arrays to compare profiles of blood serum, bone marrow et al., 2004; Ishii et al., 2007). In PV patients, serum (BM) plasma and culture supernatants of BM mesench- levels of HGF and IL-11 were correlated with blood ymal stromal cells (BMMSCs) from PV and SE patients. neutrophil counts (n ¼ 21, r ¼ 0.71, Po0.005) and Several proerythroid molecules were found to be over- hematocrit (n ¼ 45, r ¼ 0.43, Po0.01), respectively. expressed in PV: all were known to be linked to Consequently, the rest of the study focused on HGF inflammation and were produced by BMMSCs or/and and IL-11. by hematopoietic progenitors themselves. These mole- cules were studied at the protein and mRNA levels using Sequential production of HGF, IL-11, IL-6 and IL-8 by enzyme-linked immunosorbent assays (ELISAs) and PV BMMSCs quantitative reverse transcription–PCRs in purified According to the literature, HGF induces the produc- cells: BMMSCs, CD34 þ progenitors, glycophorin A- tion of IL-11 and TIMP-1, IL-11 induces expression of positive (GPA þ ) erythroblasts and CD3 þ lympho- IL-6 and IL-8, and TIMP-1 acts on the cleavage and cytes. Relevance to the pathogenesis of PV was activation of HGF. Accordingly, BM plasma levels of investigated by studying the effects on in vitro growth IL-11, IL-6 and IL-8, as well as serum levels of TIMP-1 of JAK2V617F-mutated erythroblasts.