Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation Xiaohui Lu*†, Ross Levine†‡, Wei Tong*, Gerlinde Wernig‡, Yana Pikman‡, Sara Zarnegar*, D. Gary Gilliland‡§¶, and Harvey Lodish*ʈ *Whitehead Institute for Biomedical Research and the Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142; and ‡Division of Hematology, Department of Medicine, and §Howard Hughes Medical Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 Contributed by Harvey Lodish, November 9, 2005 A recurrent somatic activating mutation in the nonreceptor ty- says revealed that JAK2V617F transforms certain lines of hema- rosine kinase JAK2 (JAK2V617F) occurs in the majority of patients topoietic cells that express the erythropoietin receptor (EpoR) to with the myeloproliferative disorders polycythemia vera, essential growth factor independence and induces constitutive phosphory- thrombocythemia, myelofibrosis with myeloid metaplasia, and, lation of the STAT5 transcription factor (9, 11). The specificity of less commonly, chronic myelomonocytic leukemia. We do not the JAK2V617F mutation in clonal disorders of the myeloid, but not understand the basis for the specificity of the JAK2V617F mutation lymphoid, lineage and the pleiotropic phenotype of JAK2V617F- in clonal disorders of the myeloid, but not lymphoid, lineage, nor associated myeloproliferative disorders are not fully understood. has the basis for the pleiotropic phenotype of JAK2V617F-associ- Here, we show that JAK2V617F functions differently from ated myeloproliferative disorders been delineated. However, the constitutively activated kinases, such as TEL-JAK2. In IL-3- presence of the identical mutation in patients with related, but dependent hematopoietic cell lines, the ability of JAK2V617F to clinicopathologically distinct, myeloid disorders suggests that in- induce cytokine-independent activation of the JAK2 and STAT5 teractions between the JAK2V617F kinase and other signaling pathways and transformation to cytokine independence requires molecules may influence the phenotype of hematopoietic progen- the coexpression of homodimeric Type I cytokine receptors, itors expressing JAK2V617F. Here, we show that coexpression of such as EpoR, thrombopoietin receptor (TpoR), or granulocyte the JAK2V617F mutant kinase with a homodimeric Type I cytokine colony-stimulating-factor receptor (GCSFR). Further, a mutant receptor, the erythropoietin receptor (EpoR), the thrombopoietin EpoR that fails to bind JAK2 also fails to support JAK2V617F- receptor, or the granulocyte colony-stimulating-factor receptor, is mediated transformation and constitutive STAT5 activation. An necessary for transformation of hematopoietic cells to growth- EpoR mutant that lacks cytosolic tyrosines and is deficient in factor independence and for hormone-independent activation of Epo-induced STAT5 activation supports cytokine-independent JAK-STAT signaling. Furthermore, EpoR mutations that impair activation of JAK2V617F but is defective in supporting erythropoietin-mediated JAK2 or STAT5 activation also impair JAK2V617F-mediated STAT5 activation and cytokine- transformation mediated by the JAK2V617F kinase, indicating that independent growth. Our results suggest that, unlike other JAK2V617F requires a cytokine receptor scaffold for its transform- constitutively activating kinase mutants previously identified, ing and signaling activities. Our results reveal the molecular basis JAK2V617F depends on cognate cytokine receptors for both its for the prevalence of JAK2V617F in diseases of myeloid lineage activation and the activation of downstream signal-transduction cells that express these Type I cytokine receptors but not in proteins. This finding provides mechanistic insights into the lymphoid lineage cells that do not. prevalence of JAK2V617F in myeloproliferative diseases like essential thrombocythemia, polycythemia vera, and myeloid erythropoietin receptor ͉ myeloproliferative diseases ͉ STAT5 activation metaplasia and reveals the origins of dysregulation in JAK2V617F-mediated cell growth. onstitutive activation of tyrosine kinases by chromosomal Ctranslocation (1), interstitial deletion (2), internal tandem Materials and Methods duplication (3), or amino acid substitution (4) are frequent Expression Vectors. The murine JAK2 cDNA was cloned into the pathogenetic events in hematopoietic malignancies. These con- retroviral vectors MSCV-Neo and MSCV-GFP. The JAK2V617F stitutively activated kinases are disengaged from normal regu- mutation was generated by using site-directed mutagenesis latory mechanisms and activate signal-transduction pathways, (QuikChange-XL, Stratagene) and confirmed by full-length including the STAT, RAS͞MAPK, and PI3K͞AKT pathways DNA sequencing. that confer proliferative and survival advantage to hematopoi- etic progenitors. For example, TEL-JAK2 is a chimeric tyrosine Cell Culture. The 293T cells were grown in Dulbecco’s modified kinase that is expressed in hematopoietic malignancies as a Eagle’s medium with 10% FCS. Transient cotransfection of consequence of the t (9, 12)(p24;p13) translocation (5, 6). JAK2 293T cells and generation of retroviral supernatant were is normally tightly associated with the cytosolic domain of a performed as described in ref. 14. Parental Ba͞F3 cells, Ba͞F3 cytokine receptor and, after cytokine binding to the cognate receptor, becomes activated by transphosphorylation of a ty- rosine residue in the activation loop. In contrast, TEL-JAK2 is Conflict of interest statement: No conflicts declared. localized to the cytosol and activates STAT5 without a require- Abbreviations: Epo, erythropoietin; EpoR, erythropoietin receptor; GCSFR, granulocyte colony-stimulating-factor receptor; PrlR, prolactin receptor; TpoR, thrombopoietin ment for cytokine stimulation or binding to a cytokine receptor receptor. (7). Indeed, the TEL-JAK2 fusion lacks the conserved JH3 to †X.L. and R.L. contributed equally to this work. JH7 domains that are required for JAK2 association with ¶To whom correspondence may be addressed at: Brigham and Women’s Hospital, 1 cytokine receptors (8). Blackfan Circle, Room 5120, Harvard Medical School, Boston, MA 02115. E-mail: ggilliland@ We and others recently reported an association between a rics.bwh.harvard.edu. somatic point mutation of the JAK2 tyrosine kinase (JAK2V617F) ʈTo whom correspondence may be addressed at: 9 Cambridge Center, Cambridge, and the myeloproliferative diseases polycythemia vera, essential MA 02142. E-mail: [email protected]. thrombocythemia, and myeloid metaplasia (9–13). Functional as- © 2005 by The National Academy of Sciences of the USA 18962–18967 ͉ PNAS ͉ December 27, 2005 ͉ vol. 102 ͉ no. 52 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509714102 Downloaded by guest on October 2, 2021 Fig. 1. A homodimeric Type I cytokine receptor is required for JAK2V617F-mediated transformation of Ba͞F3 cells. (a) Parental Ba͞F3 cells stably expressing wild-type JAK2 (Ba͞F3 WT-JAK2, open circles), JAK2V617F (Ba͞F3 JAK2V617F, open triangles), or TEL-JAK2 fusion cDNA (Ba͞F3 TEL-JAK2, open squares) were cultured in RPMI medium 1640͞10% FCS in the absence of IL-3, demonstrating cytokine-independent growth for Ba͞F3 cells expressing TEL-JAK2 but not wild-type JAK2 or JAK2V617F. Each data point represents the average of a duplicate, if not specified otherwise. (b)Ba͞F3 EpoR cells stably expressing wild-type JAK2 (Ba͞F3 EpoR WT-JAK2, filled circles), JAK2V617F (Ba͞F3 EpoR JAK2V617F, filled triangles), or TEL-JAK2 fusion cDNA (Ba͞F3 EpoR TEL-JAK2, filled squares) were cultured in RPMI medium 1640͞10% FCS in the absence of IL-3, demonstrating cytokine-independent growth for Ba͞F3 EpoR cells expressing either TEL-JAK2 or JAK2V617F but not wild-type JAK2. (c)Ba͞F3 cells stably coexpressing TpoR and JAK2V617F (Ba͞F3 TpoR JAK2V617F, filled triangles), JAK2V617F alone (Ba͞F3 JAK2V617F, open triangles), wild-type JAK2 alone (Ba͞F3 WT-JAK2, open circles), or TpoR together with wild-type JAK2 (Ba͞F3 TpoR WT-JAK2, filled circles) were cultured in RPMI medium 1640͞10% FCS in the absence of IL-3, demonstrating IL-3-independent growth for Ba͞F3 cells stably coexpressing TpoR and JAK2V617F but not cells expressing wild-type JAK2 or JAK2V617F alone. (d)Ba͞F3 cells stably coexpressing GCSFR and JAK2V617F (Ba͞F3 GCSFR JAK2V617F, filled triangles), expressing only JAK2V617F (Ba͞F3 JAK2V617F, open triangles), only wild-type JAK2 (Ba͞F3 WT-JAK2, open circles), or GCSFR together with wild-type JAK2 (Ba͞F3 GCSFR WT-JAK2, filled circles) were cultured in RPMI medium 1640͞10% FCS in the absence of IL-3, demonstrating IL-3-independent growth only for Ba͞F3 cells stably coexpressing JAK2V617F and GCSFR. cells expressing the murine EpoR (BaF3 EpoR) (15), and Results Ba͞F3 cells expressing murine TpoR (Ba͞F3 TpoR) (14) were Expression of a Homodimeric Type I Cytokine Receptor Is Required for grown in RPMI medium 1640 containing 10% FCS and 10% JAK2V617F-Mediated Transformation of Ba͞F3 Cells. We demon- WEHI-3B cell supernatant as a source of IL-3 (WEHI media). strated that expression of TEL-JAK2 transforms the murine These cells were transduced with retroviral supernatant con- IL-3-dependent hematopoietic cell line Ba͞F3 to factor- taining either the MSCV-JAK2-Neo or MSCV-JAK2V617F- independent growth (7) (Fig. 1a). These properties are similar to Neo vectors, respectively, and then selected in G418 (1 mg͞ those reported by our group and others for a number of mutant ml). Ba͞F3 cells expressing human GCSFR