The online version of this article (doi:10.1186/1756-8722-7-45) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
KG, MK and MW conceived the experimental design. KG, MJ, CT and MW performed the experiments and analyzed the data. KG, MK and MW interpreted the data. KG and MW wrote the paper. All authors agree on its content.
The Src homology-2 domain protein B (Shb) is an adapter protein operating downstream of several tyrosine kinase receptors and consequently Shb regulates various cellular responses. Absence of Shb was recently shown to reduce hematopoietic stem cell proliferation through activation of focal adhesion kinase (FAK) and thus we sought to investigate Shb’s role in the progression of leukemia.
Wild type and Shb knockout bone marrow cells were transformed with a retroviral BCR-ABL construct and subsequently transplanted to wild type or Shb knockout recipients. Disease latency, bone marrow and peripheral blood cell characteristics, cytokine expression, signaling characteristics and colony formation were determined by flow cytometry, qPCR, western blotting and methylcellulose colony forming assays.
It was observed that Shb knockout BCR-ABL-transformed bone marrow cells produced a disease with death occurring at earlier time points compared with corresponding wild type controls due to elevated proliferation of transformed bone marrow cells. Moreover, significantly elevated interleukin-6 and granulocyte colony-stimulation factor mRNA levels were observed in Shb knockout c-Kit + leukemic bone marrow cells providing a plausible explanation for the concurrent peripheral blood neutrophilia. Shb knockout leukemic bone marrow cells also showed increased ability to form colonies in methylcellulose devoid of cytokines that was dependent on the concomitantly observed increased activity of FAK. Transplanting BCR-ABL-transformed Shb knockout bone marrow cells to Shb knockout recipients revealed decreased disease latency without neutrophilia, thus implicating the importance of niche-derived cues for the increase of blood granulocytes.
Absence of Shb accelerates disease progression by exerting dual roles in BCR-ABL-induced leukemia: increased cell expansion due to elevated FAK activity and neutrophilia in peripheral blood, the latter dependent on the genetic background of the leukemic niche.
Additional file 1: Figure S1: A) Hematoxylin-eosin stained sections of lung, liver and spleen of diseased mice transplanted with BCR-ABL transformed bone marrow cells of wild type or Shb knockout background to wild type recipients. B) Percentage myeloid GFP + and GFP- cells in spleen. Means ± SEM are given and *indicates p < 0.05. (PDF 5 MB)13045_2014_403_MOESM1_ESM.pdf
Additional file 2: Figure S2: Absolute numbers of BCR-ABL+GFP+ and BCR-ABL-GFP- myeloid cells in peripheral blood. GFP+ GR-1Hi Mac-1Hi and GFP- GR-1Hi Mac-1Hi cells were identified with FACS analysis in order to estimate the number of BCR-ABL carrying cells. Means are presented in arbitrary units ± SEM and are based on 6 mice of each genotype in 2 independent experiments. *denotes p < 0.05 as determined by Student’s t-test. (PDF 45 KB)13045_2014_403_MOESM2_ESM.pdf
Additional file 3: Figure S3: Estimation of HSC proportions in murine BCR-ABL transformed bone marrow. The proportions of HSCs were established using FACS based on expression of lineage defining markers, c-Kit, CD48 and CD150 (a). Right panels are Shb knockout. (b) Percentage CD150+/CD48- cells among c-Kit+/Lin- cells and fraction of GFP + cells among the CD150+/CD48- cells. Means ± SEM for 6 experiments are shown. (c) Percentage lineage-/c-Kit + or lineage+/c-Kit + cells in bone marrows of wild type or Shb knockout BCR-ABL transformed bone marrow cells transplanted to wild type recipients. Means ± SEM for 6 mice of each genotype are given. (PDF 227 KB)13045_2014_403_MOESM3_ESM.pdf
Additional file 4: Figure S4: Evaluation of cytokine expression in c-Kit enriched and unfractionated (total) bone marrow. (a) Transcript levels of SCF, IL-3, thrombopoietin (Thpo) and angiopoietin-2 (Angpt2) were evaluated with semi-quantitative real-time RT-PCR using mRNA isolated from c-Kit + leukemic bone marrow samples. The expression of proinflammatory cytokines TNFα, IL-1α, IL-1β, IL-4, MIP-1α and MIP-1β were determined in c-Kit+ (b) and total bone marrow (d). Expression of G-CSF, IL-6, SCF, IL-3, Angpt-2 and GM-CSF were determined in total bone marrow (c). All Ct values were normalized to β-actin and Shb knockout samples were related to corresponding wild type values. Means are presented as 2-ΔCt ± SEM to demonstrate fold change in mRNA content. Data are based on 6 mice of each genotype from 2 independent experiments for c-Kit+ cells and 3 mice of each genotype from 1 experiment for unfractionated bone marrow. (PDF 97 KB)13045_2014_403_MOESM4_ESM.pdf
Additional file 5: Figure S5: STAT5 activity in c-Kit+ bone marrow from leukemic mice. The activation of STAT5 was determined by Western blot analysis of tyrosine phosphorylation by immunoblotting for phospho- and total STAT5 respectively. Protein phosphorylation was related to total protein content on the same blot and signal strength was estimated by densitometric analysis. Means are presented in arbitrary units ± SEM and are based on 6 mice of each genotype in 2 independent experiments. (PDF 34 KB)13045_2014_403_MOESM5_ESM.pdf
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Cancer Genome Atlas Research N: Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013, 368: 2059-2074. CrossRef
Thomas EK, Cancelas JA, Chae HD, Cox AD, Keller PJ, Perrotti D, Neviani P, Druker BJ, Setchell KD, Zheng Y, Harris CE, Williams DA: Rac guanosine triphosphatases represent integrating molecular therapeutic targets for BCR-ABL-induced myeloproliferative disease. Cancer Cell. 2007, 12: 467-478. 10.1016/j.ccr.2007.10.015. CrossRefPubMed
Pendergast AM, Quilliam LA, Cripe LD, Bassing CH, Dai Z, Li N, Batzer A, Rabun KM, Der CJ, Schlessinger J, Gishizky ML: BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein. Cell. 1993, 75: 175-185. 10.1016/S0092-8674(05)80094-7. CrossRefPubMed
Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak MZ, Wen SC, Zon G, Gewirtz AM, Perussia B, Calabretta B: Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells. Blood. 1995, 86: 726-736. PubMed
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. PubMed
Reynaud D, Pietras E, Barry-Holson K, Mir A, Binnewies M, Jeanne M, Sala-Torra O, Radich JP, Passegue E: IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development. Cancer Cell. 2011, 20: 661-673. 10.1016/j.ccr.2011.10.012. PubMedCentralCrossRefPubMed
Pedroza-Gonzalez A, Xu K, Wu TC, Aspord C, Tindle S, Marches F, Gallegos M, Burton EC, Savino D, Hori T, Tanaka Y, Zurawski S, Zurawski G, Bover L, Liu YJ, Banchereau J, Palucka AK: Thymic stromal lymphopoietin fosters human breast tumor growth by promoting type 2 inflammation. J Exp Med. 2011, 208: 479-490. 10.1084/jem.20102131. PubMedCentralCrossRefPubMed
Wang W, Li Q, Yamada T, Matsumoto K, Matsumoto I, Oda M, Watanabe G, Kayano Y, Nishioka Y, Sone S, Yano S: Crosstalk to stromal fibroblasts induces resistance of lung cancer to epidermal growth factor receptor tyrosine kinase inhibitors. Clin Cancer Res. 2009, 15: 6630-6638. 10.1158/1078-0432.CCR-09-1001. CrossRefPubMed
Welsh M, Mares J, Karlsson T, Lavergne C, Breant B, Claesson-Welsh L: Shb is a ubiquitously expressed Src homology 2 protein. Oncogene. 1994, 9: 19-27. PubMed
Gustafsson K, Heffner G, Wenzel PL, Curran M, Grawe J, McKinney-Freeman SL, Daley GQ, Welsh M: The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity. Exp Cell Res. 2013, 319: 1852-1864. 10.1016/j.yexcr.2013.03.020. CrossRefPubMed
Hestdal K, Ruscetti FW, Ihle JN, Jacobsen SE, Dubois CM, Kopp WC, Longo DL, Keller JR: Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. J Immunol. 1991, 147: 22-28. PubMed
Hariharan IK, Adams JM, Cory S: bcr-abl oncogene renders myeloid cell line factor independent: potential autocrine mechanism in chronic myeloid leukemia. Oncogene Res. 1988, 3: 387-399. PubMed
Klein H, Becher R, Lubbert M, Oster W, Schleiermacher E, Brach MA, Souza L, Lindemann A, Mertelsmann RH, Herrmann F: Synthesis of granulocyte colony-stimulating factor and its requirement for terminal divisions in chronic myelogenous leukemia. J Exp Med. 1990, 171: 1785-1790. 10.1084/jem.171.5.1785. CrossRefPubMed
Tian SS, Lamb P, Seidel HM, Stein RB, Rosen J: Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor. Blood. 1994, 84: 1760-1764. PubMed
Lutticken C, Wegenka UM, Yuan J, Buschmann J, Schindler C, Ziemiecki A, Harpur AG, Wilks AF, Yasukawa K, Taga T, Kishimoto T, Barbieri G, Pellegrini S, Sendtner M, Heinrich PC, Horn F: Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science. 1994, 263: 89-92. 10.1126/science.8272872. CrossRefPubMed
Tyner JW, Walters DK, Willis SG, Luttropp M, Oost J, Loriaux M, Erickson H, Corbin AS, O'Hare T, Heinrich MC, Deininger MW, Druker BJ: RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia. Blood. 2008, 111: 2238-2245. 10.1182/blood-2007-06-097253. PubMedCentralCrossRefPubMed
Gotoh A, Miyazawa K, Ohyashiki K, Tauchi T, Boswell HS, Broxmeyer HE, Toyama K: Tyrosine phosphorylation and activation of focal adhesion kinase (p125FAK) by BCR-ABL oncoprotein. Exp Hematol. 1995, 23: 1153-1159. PubMed
Recher C, Ysebaert L, Beyne-Rauzy O, Mansat-De Mas V, Ruidavets JB, Cariven P, Demur C, Payrastre B, Laurent G, Racaud-Sultan C: Expression of focal adhesion kinase in acute myeloid leukemia is associated with enhanced blast migration, increased cellularity, and poor prognosis. Cancer Res. 2004, 64: 3191-3197. 10.1158/0008-5472.CAN-03-3005. CrossRefPubMed
Despeaux M, Labat E, Gadelorge M, Prade N, Bertrand J, Demur C, Recher C, Bonnevialle P, Payrastre B, Bourin P, Racaud-Sultan C: Critical features of FAK-expressing AML bone marrow microenvironment through leukemia stem cell hijacking of mesenchymal stromal cells. Leukemia. 2011, 25: 1789-1793. 10.1038/leu.2011.145. CrossRefPubMed
Maxson JE, Gotlib J, Pollyea DA, Fleischman AG, Agarwal A, Eide CA, Bottomly D, Wilmot B, McWeeney SK, Tognon CE, Pond JB, Collins RH, Goueli B, Oh ST, Deininger MW, Chang BH, Loriaux MM, Druker BJ, Tyner JW: Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML. N Engl J Med. 2013, 368: 1781-1790. 10.1056/NEJMoa1214514. PubMedCentralCrossRefPubMed
Pear WS, Miller JP, Xu L, Pui JC, Soffer B, Quackenbush RC, Pendergast AM, Bronson R, Aster JC, Scott ML, Baltimore D: Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood. 1998, 92: 3780-3792. PubMed
Li S, Ilaria RL, Million RP, Daley GQ, Van Etten RA: The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity. J Exp Med. 1999, 189: 1399-1412. 10.1084/jem.189.9.1399. PubMedCentralCrossRefPubMed
- The Src homology-2 protein Shb modulates focal adhesion kinase signaling in a BCR-ABL myeloproliferative disorder causing accelerated progression of disease
Michael G Kharas
- BioMed Central
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