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01.12.2014 | Research | Ausgabe 1/2014 Open Access

Journal of Hematology & Oncology 1/2014

The Src homology-2 protein Shb modulates focal adhesion kinase signaling in a BCR-ABL myeloproliferative disorder causing accelerated progression of disease

Journal of Hematology & Oncology > Ausgabe 1/2014
Karin Gustafsson, Maria Jamalpour, Camilla Trinh, Michael G Kharas, Michael Welsh
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1756-8722-7-45) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

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.

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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)
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)
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)
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)
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)
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