Abstract
An activating point mutation in Janus kinase 2 (JAK2 V617F) was recently identified in myelofibrosis with myeloid metaplasia (MMM). To further elucidate the pathogenic significance, we examined the JAK2 mutation burden, phosphorylation of JAK2 substrates and neutrophil apoptotic resistance. Immunoblotting revealed phosphorylation of signal transducer and activator of transcription-3 (STAT3) in all four JAK2 with high V617F mutant allele burden and seven of eight with intermediate mutant allele burden, but only one of eight with wild-type JAK2 (P<0.001). In contrast, STAT5 phosphorylation was undetectable in patient MMM neutrophils; and phosphorylation of Akt and extracellular signal-regulated kinases (ERKs) failed to correlate with JAK2 mutation status. Apoptosis was lower in MMM neutrophils (median 41% apoptotic cells, n=50) compared to controls (median 66%, n=9) or other myeloproliferative disorder patients (median 53%, n=11; P=0.002). Apoptotic resistance in MMM correlated with anemia (P=0.01) and the JAK2-V617F (P=0.01). Indeed, apoptotic resistance was greatest in MMM neutrophils with high mutant allele burden (median 22% apoptosis, n=5) than with intermediate burden (median 39%, n=23) or wild-type JAK2 (median 47%, n=22; P=0.008). These results suggest that mutant JAK2 contributes to MMM pathogenesis by constitutively phosphorylating STAT3 and diminishing myeloid cell apoptosis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Tefferi A . Myelofibrosis with myeloid metaplasia. N Engl J Med 2000; 342: 1255–1265.
Mesa RA . Myelofibrosis with myeloid metaplasia: therapeutic options in 2003. Curr Hematol Rep 2003; 2: 264–270.
Reilly JT . Pathogenesis of idiopathic myelofibrosis: present status and future directions [Review] [114 refs]. Br J Haematol 1994; 88: 1–8.
Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061.
Levine RL, Wadleigh M, Cools J . Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myelofibrosis with myeloid metaplasia. Cancer Cell 2005; 7: 387–397.
Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790.
Schindler CW . JAK-STAT signaling in human disease. J Clin Invest 2002; 109: 1133–1137.
Aaronson DS, Horvath CM . A road map for those who don't know JAK-STAT. Science 2002; 296: 1653–1655.
Hengartner MO . The biochemistry of apoptosis. Nature 2000; 407: 770–776.
White MK, McCubrey JA . Suppression of apoptosis: role in cell growth and neoplasia. Leukemia 2001; 15: 1011–1021.
Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C . Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest 1989; 83: 865–875.
Muta K, Krantz SB . Apoptosis of human erythroid colony-forming cells is decreased by stem cell factor and insulin-like growth factor I as well as erythropoietin. J Cell Physiol 1993; 156: 264–271.
Buettner R, Mora LB, Jove R . Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res 2002; 8: 945–954.
Alvarez JV, Frank DA . Genome-wide analysis of STAT target genes: elucidating the mechanism of STAT-mediated oncogenesis. Cancer Biol Ther 2004; 3: 1045–1050.
Epling-Burnette PK, Zhong B, Bai F, Jiang K, Bailey RD, Garcia R et al. Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte–macrophage colony-stimulating factor-delayed apoptosis in human neutrophils. J Immunol 2001; 166: 7486–7495.
Horita M, Andreu EJ, Benito A, Arbona C, Sanz C, Benet I et al. Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL. J Exp Med 2000; 191: 977–984.
Aichberger KJ, Mayerhofer M, Krauth MT, Skvara H, Florian S, Sonneck K et al. Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides. Blood 2005; 105: 3303–3311.
Silva M, Richard C, Benito A, Sanz C, Olalla I, Fernandez-Luna JL . Expression of Bcl-x in erythroid precursors from patients with polycythemia vera [see comments]. N Engl J Med 1998; 338: 564–571.
Colovic MD, Wiernik PH, Jankovic GM, Vidovic AD, Janosevic S, Basara NM . Circulating haemopoietic progenitor cells in primary and secondary myelofibrosis: relation to collagen and reticulin fibrosis. Eur J Haematol 1999; 62: 155–159.
English D, Andersen BR . Single-step separation of red blood cells. Granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll–Hypaque. J Immunol Methods 1974; 5: 249–252.
Mow BM, Chandra J, Svingen PA, Hallgren CG, Weisberg E, Kottke TJ et al. Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro. Blood 2002; 99: 664–671.
Reeder TL, Bailey RJ, Dewald GW, Tefferi A . Both B and T lymphocytes may be clonally involved in myelofibrosis with myeloid metaplasia. Blood 2003; 101: 1981–1983.
Kaufmann SH, Svingen PA, Gore SD, Armstrong DK, Cheng YC, Rowinsky EK . Altered formation of topotecan-stabilized topoisomerase I-DNA adducts in human leukemia cells. Blood 1997; 89: 2098–2104.
Yamashita K, Takahashi A, Kobayashi S, Hirata H, Mesner Jr PW, Kaufmann SH et al. Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production. Blood 1999; 93: 674–685.
Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reutelingsperger CP, Roos D . Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood 1995; 85: 532–540.
Kobayashi S, Takahashi A, Yamashita K, Takeoka T, Ohtsuki T, Suzuki Y et al. Calpain-mediated XIAP degradation in neutrophil apoptosis and its impairment in chronic neutrophilic leukemia. J Biol Chem 2002; 277: 33968–33977.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150: 76–85.
Kaufmann SH . Reutilization of immunoblots after chemiluminescent detection. Anal Biochem 2001; 296: 283–286.
Al-Shami A, Mahanna W, Naccache PH . Granulocyte–macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Selective activation of Jak2, Stat3, and Stat5b. J Biol Chem 1998; 273: 1058–1063.
Yagisawa M, Saeki K, Okuma E, Kitamura T, Kitagawa S, Hirai H et al. Signal transduction pathways in normal human monocytes stimulated by cytokines and mediators: comparative study with normal human neutrophils or transformed cells and the putative roles in functionality and cell biology. Exp Hematol 1999; 27: 1063–1076.
McClure R, Mai M, Lasho T . Validation of two clinically useful assays for evaluation of JAK2 V617F mutation in chronic myeloproliferative disorders. Leukemia 2006; 20: 168–171.
Dupriez B, Morel P, Demory J, Lai J, Simon M, Plantier I et al. Prognostic factors in agnogenic myeloid metaplasia: a report on 195 cases with a new scoring system [see comments]. Blood 1996; 88: 1013–1018.
de Groot RP, Coffer PJ, Koenderman L . Regulation of proliferation, differentiation and survival by the IL-3/IL-5/GM-CSF receptor family. Cell Signal 1998; 10: 619–628.
McCubrey JA, May WS, Duronio V, Mufson A . Serine/threonine phosphorylation in cytokine signal transduction. Leukemia 2000; 14: 9–21.
Um M, Lodish HF . Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways. J Biol Chem 2006; 281: 5648–5656.
Akgul C, Moulding DA, Edwards SW . Molecular control of neutrophil apoptosis. FEBS Lett 2001; 487: 318–322.
Juvonen E, Ikkala E, Oksanen K, Ruutu T . Megakaryocyte and erythroid colony formation in essential thrombocythaemia and reactive thrombocytosis: diagnostic value and correlation to complications. Br J Haematol 1993; 83: 192–197.
Prchal JF, Axelrad AA . Letter: bone-marrow responses in polycythemia vera. N Engl J Med 1974; 290: 1382.
Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RJ . Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia. Blood 1994; 83: 2038–2044.
McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG . BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood 1994; 83: 1179–1187.
Carlesso N, Frank DA, Griffin JD . Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J Exp Med 1996; 183: 811–820.
Shuai K, Halpern J, ten Hoeve J, Rao X, Sawyers CL . Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene 1996; 13: 247–254.
Ilaria Jr RL, Van Etten RA . P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members. J Biol Chem 1996; 271: 31704–31710.
Lin TS, Mahajan S, Frank DA . STAT signaling in the pathogenesis and treatment of leukemias. Oncogene 2000; 19: 2496–2504.
Benekli M, Baer MR, Baumann H, Wetzler M . Signal transducer and activator of transcription proteins in leukemias. Blood 2003; 101: 2940–2954.
Epling-Burnette PK, Liu JH, Catlett-Falcone R, Turkson J, Oshiro M, Kothapalli R et al. Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression. J Clin Invest 2001; 107: 351–362.
Catlett-Falcone R, Landowski TH, Oshiro MM, Turkson J, Levitzki A, Savino R et al. Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 1999; 10: 105–115.
Darnell Jr JE . Transcription factors as targets for cancer therapy. Nat Rev Cancer 2002; 2: 740–749.
Sordella R, Bell DW, Haber DA, Settleman J . Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 2004; 305: 1163–1167.
Boucher MJ, Morisset J, Vachon PH, Reed JC, Laine J, Rivard N . MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 2000; 79: 355–369.
Kobayashi S, Werneburg NW, Bronk SF, Kaufmann SH, Gores GJ . Interleukin-6 contributes to Mcl-1 up-regulation and TRAIL resistance via an Akt-signaling pathway in cholangiocarcinoma cells. Gastroenterology 2005; 128: 2054–2065.
Alvarez JV, Febbo PG, Ramaswamy S, Loda M, Richardson A, Frank DA . Identification of a genetic signature of activated signal transducer and activator of transcription 3 in human tumors. Cancer Res 2005; 65: 5054–5062.
Kralovics R, Teo SS, Buser AS, Brutsche M, Tiedt R, Tichelli A et al. Altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of Jak2. Blood 2005; 106: 3374–3376.
Tefferi A, Lasho TL, Gilliland G . JAK2 mutations in myeloproliferative disorders. N Engl J Med 2005; 353: 1416–1417.
Darnell JE . Validating Stat3 in cancer therapy. Nat Med 2005; 11: 595–596.
Sun J, Blaskovich MA, Jove R, Livingston SK, Coppola D, Sebti SM . Cucurbitacin Q: a selective STAT3 activation inhibitor with potent antitumor activity. Oncogene 2005; 24: 3236–3245.
Acknowledgements
This work was supported in part by R01 CA69008 (SHK) and K23 CA96780 (RAM).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mesa, R., Tefferi, A., Lasho, T. et al. Janus kinase 2 (V617F) mutation status, signal transducer and activator of transcription-3 phosphorylation and impaired neutrophil apoptosis in myelofibrosis with myeloid metaplasia. Leukemia 20, 1800–1808 (2006). https://doi.org/10.1038/sj.leu.2404338
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.leu.2404338
Keywords
This article is cited by
-
TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms
Cellular Oncology (2018)
-
Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis
Journal of Hematology & Oncology (2012)
-
Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia
Leukemia (2008)
-
Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy
Leukemia (2008)
-
The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2V617F
Leukemia (2008)