The Src homology-2 protein Shb modulates focal adhesion kinase signaling in a BCR-ABL myeloproliferative disorder causing accelerated progression of disease
© Gustafsson et al.; licensee BioMed Central Ltd. 2014
Received: 24 May 2014
Accepted: 19 June 2014
Published: 21 June 2014
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.
KeywordsBCR-ABL Focal adhesion kinase Shb Chronic myeloid leukemia Neutrophilia
Hematopoiesis is a life-long process supported by a finely tuned network of proto-oncogenes and tumor suppressor genes controlling the self-renewal and proliferation of hematopoietic stem and progenitor cells (HSCs and HPCs). Deregulation of any of these elements has the potential to give rise to neoplasms . Leukemic cells thus show characteristics of upregulated signaling cascades promoting self-renewal, increased cell cycle entry as well as prevention of apoptosis [2, 3].
Chronic myeloid leukemia (CML) is myeloproliferative malignancy induced by the translocation between chromosomes 9 and 22 leading to the fusion of the c-ABL gene with the break point cluster region (BCR) gene . The resulting oncogene BCR–ABL is a constitutively active tyrosine kinase with the ability to affect a broad range of signaling pathways including Ras, phosphatidylinositol-3 kinase (PI-3 K), and Rac [5–8]. Hence, cells expressing BCR-ABL display increased proliferative ability combined with reduced apoptotic rates and abnormal migratory characteristics [9–12]. BCR-ABL may, in addition, cause other types of leukemia.
Intracellular signaling events are not the only factors contributing to the progression of the disease. A common feature of most types of tumors is their ability to change the microenvironment to promote neoplastic growth. The tumor cells can either secrete tumor –promoting factors or the surrounding stroma can be induced to generate conditions favorable for expansion of leukemic cells [13, 14]. CML bone marrow secretes increased levels of interleukin -6 (IL -6) and granulocyte colony –stimulating factor (G –CSF), both established as cytokines that stimulate myeloid expansion and differentiation [10, 11, 15–17]. Additionally, in leukemia, the stromal compartment has a reduced ability to support normal hematopoiesis, thus further enhancing the growth advantage of the leukemic cells [10, 11, 18, 19].
The adaptor protein Shb is one of four members in a family of adaptor proteins with homologous tyrosine phosphorylation sites and Src homology 2 (SH2) domains [20–23]. Shb has been shown to operate downstream of tyrosine kinase receptors exerting versatile effects on a number of signaling pathways . The SH2 domain of Shb binds to phosphotyrosines on activated receptors such as the platelet derived growth factor receptor (PDGFR), the IL-2 receptor and the T cell receptor (TCR) . Shb’s various signaling domains further recruit intracellular signaling mediators, including focal adhesion kinase (FAK), Src, phosphatidylinositol 3-kinase (PI3K), Vav-1, and c-Abl [24, 25], thereby regulating cytoskeletal rearrangements, proliferation as well as apoptosis .
Shb’s influence on the hematopoietic system has been documented in a number of studies. Shb knockout embryonic stem cells display reduced colony formation and delayed expression of hematopoietic markers . CD4+ TH cells isolated from a Shb knockout mouse exhibit a TH2 biased cytokine profile upon in vitro stimulation . In HSCs, the loss of Shb results in hyperactivation of FAK leading to impaired HSC proliferation and failure to uphold long –term maintenance of the myeloid compartment . The reduction of HSC proliferation prompted us to investigate Shb’s role in a stem cell mediated myeloproliferative model. BCR-ABL-induced myeloid disease is one of the most established systems to study factors that are known to be coordinated downstream of tyrosine kinase signaling. We observe that Shb-deficiency results in a more rapid progression of disease.
Loss of Shb results in BCR-ABL-driven leukemogenesis with shorter latency
In summary, the loss of Shb expression in malignant hematopoiesis exhibits a disease with shorter latency. Simultaneously, elevated numbers of mature neutrophil granulocytes are observed in peripheral blood.
Shb deletion confers a proliferative advantage and reduces apoptosis in BCR-ABL+ lineage-negative cells
A hallmark of BCR-ABL transformed bone marrow cells is their resistance to apoptosis . To determine if the apoptotic rate was also altered in the absence of Shb, BCR-ABL+ Lin-c-Kit+ bone marrow cells were stained for cleaved caspase-3 when the mice became moribund. A modest, but significant decrease in the percentage of cleaved caspase-3+ cells indicates that BCR-ABL+ Lin- c-Kit+ cells are less apoptotic as a result of Shb deletion (Figure 3a lower panel and Figure 3d). There was no effect on apoptosis in the Lin+ c-Kit+ population (data not shown). In conclusion, the data imply that ablation of Shb expression is associated with an increased expansion of the leukemic lineage-negative progenitor cell population through enhancement of proliferation and survival.
Shb knockout leukemic bone marrow is hypersensitive to cytokine stimulation and expresses increased levels of G-CSF and IL-6
To further explore the cytokine signaling networks in our model, the expression levels of a number of cytokines, known to regulate BCR-ABL-induced leukemogenesis and hematopoietic cell proliferation and differentiation, were determined. In order to facilitate a distinction between cytokine production within the bone marrow as a whole and the hematopoietic compartment, bone marrow was first fractionated based on c-Kit expression, as most HSCs and HPCs are c-Kit+. Transcription of GM-CSF was unchanged in both the c-Kit+ compartment and in unfractionated bone marrow (Figure 4c and Additional file 4: Figure S4). The expression of G-CSF and IL-6 was on the other hand significantly elevated in c-Kit+ cells (Figure 4c) but not in total bone marrow (Additional file 4: Figure S4), suggesting that the increased cytokine production is limited to the hematopoietic compartment. This is in line with previous reports suggesting that leukemic progenitors secrete IL-6 and G-CSF in an autocrine fashion [10, 32]. Other factors, such as TNFα, IL-1α, IL-1β, IL-4, MIP-1α, MIP-1β, SCF, IL-3, thrombopoietin and angiopoietin-2, have also been demonstrated to be important factors in promoting the proliferation of leukemic HSCs and HPCs [10, 18]. The transcript levels of these factors were therefore determined in c-Kit enriched and unfractionated bone marrow, but no differences were detected between wild type and Shb deficient samples (Additional file 4: Figure S4).
Additionally, G-CSF has been linked to impaired bone marrow retention of leukemic HSCs due to decreased production of the chemokine CXCL12 by bone marrow stromal cells . There was however no detectable difference in CXCL12 expression in total (unfractionated) bone marrow (relative expression of CXCL12 was 1 ± 0.15 in wild type and 0.73 ± 0.25 in Shb knockout). Reduced expression of CXCL12 in bone marrow has been shown to increase the number of HSCs found in the spleen . Flow cytometric analysis of splenic HSCs revealed no difference between wild type and Shb knockout recipients (data not shown), further supporting the notion that the elevated levels of G-CSF do not appear to affect the invasion of Shb deficient leukemic HSCs to the spleen.
These results thereby indicate that Shb serves as a modulator of cytokine expression levels thus possibly explaining the increased numbers of neutrophils in blood from BCR-ABL transformed Shb deficient bone marrow.
Accelerated BCR-ABL-induced leukemia in Shb knockout recipient mice as a consequence of Shb deficiency
Enhanced FAK and STAT3 activity of Shb deficient leukemic c-Kit+ bone marrow cells
Elevated FAK activity is important for the proliferative ability of BCR-ABL-transformed Shb knockout bone marrow cells
The adaptor protein Shb has been implicated in intracellular signaling events regulating proliferation, apoptosis and differentiation in a number of different cell types. Recently, HSC exhibited less proliferative activity in Shb knockout mice due to alterations in FAK signaling . The present study aimed at relating those observations to conditions of neoplastic hematopoiesis. The results suggest that Shb depletion accelerates BCR-ABL-driven progression of myeloid neoplasia by causing decreased latency and increased numbers of myeloid cells in peripheral blood due to elevated FAK activity. In addition, niche-dependent cues have an impact on disease progression with a bearing on cytokine production in bone marrow cells and neutrophilia in peripheral blood.
FAK activity was recently noted to be significantly increased in normal HSCs in Shb deficient mice . A similar increase in FAK activity was observed in BCR-ABL-transformed Shb knockout c-Kit+ bone marrow. Our current knowledge of FAK’s effects on leukemogenesis is limited although the bulk of data suggest that active FAK promotes leukemogenesis. BCR-ABL appears to induce FAK phosphorylation and siRNA silencing of FAK reduces survival of AML leukemia cell lines [36, 37]. Additionally, observations from murine models and AML patients suggest that FAK may influence leukemia progression [38, 39]. FAK is mainly activated by integrins, thus mediating signals between cells and their respective surroundings. Leukemic cells enhance their own growth by altering the bone marrow microenvironment [10, 11, 18]. FAK signaling could provide a key link between leukemic cells and the stromal cells of the hematopoietic niche. FAK expressing AML cells have been demonstrated to enhance the ability of bone marrow stroma to support leukemic growth through direct contact . Moreover, in a recent study utilizing a murine model of BCR-ABL-induced leukemia, it was shown that soluble factors synergize with an unidentified contact-dependent mechanism to drastically change the hematopoietic niche composition to promote neoplastic progression . The present colony formation experiments adding a FAK inhibitor in the absence of cytokines lend strong support to the notion that the increased tumor burden of transformed Shb knockout bone marrow cells is mainly due to elevated FAK activity. In Shb deficient BCR-ABL-transformed bone marrow, immature lin-c-Kit+ HSCs and HPCs as well as more differentiated Lin+ c-Kit+ cells, were found to proliferate at an increased rate and this finding is likely to reflect the same mechanism as the colony formation assay.
The Shb deficient myelodysplastic neoplasia phenotype is also intriguing as the rapid progression of disease is associated with a pronounced neutrophilia when studied in wild type recipient mice. In most cases, more aggressive forms of leukemia are distinguished by a blood profile dominated by immature blasts . Shb deficient c-Kit+ leukemic bone marrow cells were on the other hand found to express increased levels of granulopoietic factors IL-6 and G-CSF in these experiments. Myeloid differentiation is supported by IL-6 through cell cycle regulation of myeloid progenitors and IL-6 also blocks lymphoid lineage commitment thus further enhancing myeloid expansion [16, 41, 42]. Moreover, leukemia progression is significantly hampered in IL-6 knockout mice directly linking this cytokine to disease . This may involve effects on both malignant and non-malignant cells . Peripheral blood from recipients transplanted with transformed Shb knockout cells contained elevated numbers of BCR-ABL- myeloid cells indicative of an expansion of non-malignant cells, possibly a result of the increased IL-6 expression. Neutrophil differentiation under homeostatic and stress conditions depends on G-CSF providing survival and differentiation signals to granulocytic progenitors [15, 17]. Further demonstrating G-CSF’s effect on neutrophil expansion is the finding that chronic neutrophilic leukemia, a rare myeloproliferative disorder characterized by excessive expansion of the neutrophilic population in blood and bone marrow, is linked to activating mutations in the CSF3R gene, the human receptor for G-CSF .
STAT3 phosphorylation was significantly augmented in c-Kit+ bone marrow cells isolated from Shb knockout recipients on wild type background. Notably, G-CSF and IL-6 signaling pathways converge in the activation of the transcription factor STAT3 and STAT3 is the main mediator of the proliferation and survival signals provided by G-CSF and IL-6 [34, 35, 44, 45]. The hyperphosphorylation displayed by Shb null cells is thus probably a result of the increased production of G-CSF and IL-6.
The data presented suggest that Shb regulates cues in neoplastic bone marrow of importance for leukemic progression and that absence of Shb decreases disease latency. The Shb- dependent effects include bone marrow cell intrinsic pathways (FAK) as well as niche-dependent signals (cytokine production). Both of these components are considered as druggable targets. Further exploration of the effects of Shb deletion in hematopoietic malignancies is therefore of importance to increase our understanding of mechanisms that control leukemogenesis.
The generation of Shb knockout mice has been described previously . The Shb knockout genotype is not viable on the C57Bl/6 background and the animals were therefore maintained on the Balb/c strain. The local animal ethics committee at Uppsala University approved all experiments.
Bone marrow transduction and transplantation assay
The pMIG-p210bcr/abl vector was used to produce retroviruses directing the expression of BCR-ABL-GFP as described previously . Balb/c Shb wild type or knockout mice 8-10 weeks old were treated with 5 –fluorouracil (5-FU) (Sigma-Aldrich, St. Lois, MO) at a dose of 150 mg/kg body weight 6 days prior to bone marrow isolation, in order to enrich for HSCs. Isolated donor bone marrow was stimulated in RPMI 1640 (Sigma Aldrich) supplemented with 10% FCS (Sigma Aldrich), 2 mM L-glutamine, streptomycin (0.1 mg/ml), penicillin (100 U/ml) (All from Gibco, Paisley, UK), IL-3 (10 ng/ml), stem cell factor (SCF) (10 ng/ml) and IL-6 (10 ng/ml) (all cytokines were purchased from PeproTech, Rocky Hill, NJ) for 24 hours. The cells were subsequently subjected to two rounds of spin infections over the following 48 hours as described previously . Briefly, 8 × 106 cells were centrifuged for 90 minutes at 1000 g and 30°C in medium containing the aforementioned supplements as well as 25% viral supernatant, 7.5 mM Hepes (Gibco) and 8 μg/ml Polybrene (Millipore, Watford, UK). Infection efficiency was determined following the second spin inoculation and just prior to transplantation by flow cytometric analysis of green fluorescent protein (GFP) expression on a FACSCalibur (BD Bioscience, Erembodegem, Belgium) and no differences were found between wild type and Shb knockout bone marrow (5.6 ± 1.0% in wt; 4.9 ± 1.1% in knockout). Recipient wild type or Shb knockout mice were irradiated with two doses of 4.5 Gy separated by at least 2 hours in a Nordion Gammacell 40 Exacto 137Cs irradiator (MDS Nordion, Ottawa, ON). Immediately following the second irradiation the recipients were retroorbitally injected with a dose of 0.4 – 1 × 106 cells (equal number of wild type and Shb knockout cells was given per recipient mouse in each experiment, i.e. transfection/transplantation event).
Pathological examination of diseased mice
The mice were monitored daily from day 6 post –transplantation for signs of disease such as a weight loss of more than 15% of initial body weight, lethargy and splenomegaly. Moribund mice were then sacrificed. Blood was collected immediately prior to sacrifice and samples were prepared for blood smears. Spleens, livers, and lungs were fixed in 4% buffered formalin and embedded in paraffin for later histopathological analysis. Iliac bones, femurs and tibias were dissected and bone marrow was collected. Bone marrow cells were extracted from the bones and used for further downstream applications. Single cell suspensions of spleen and bone marrow were also fixed in 4% paraformaldehyde to enable FACS analysis at a later time point.
Fixed and paraffin embedded organs were sectioned in 5 μm sections, mounted on microscope slides (Menzel Gläser, Braunschweig, Germany) and stained with Hematoxylin –Eosin. For differential blood counts, peripheral blood smears were stained with May –Grünwald Giemsa.
Fluorescent activated cell-sorting (FACS) analysis
Paraformaldehyde fixed peripheral blood and bone marrow single cell suspensions were stained with antibodies directed against Gr -1-PE (eBioscience, Hartfield, UK) and rat anti mouse Mac-1 (Invitrogen, Carlsbad, CA) followed by incubation with goat anti-rat PE-Cy5.5 (Invitrogen) secondary antibody.
In order to identify the HSC population, bone marrow and spleen cells were stained with a lineage excluding cocktail consisting of rat anti-mouse antibodies CD3, CD8, CD4, B220, CD19, Gr-1, and Mac-1. The samples were thereafter incubated with goat anti-rat PE-Cy5.5, followed by staining with CD150-PE-Cy7 (BioLegend, San Diego, CA), c-Kit-APC eFluor 780, and CD48-PE (eBioscience).
Proliferative and apoptotic rates were determined by analysis of Ki-67 and cleaved Caspase-3. Paraformaldehyde fixed bone marrow was stained for lineage defining markers and c-Kit as described above. This was followed by permeabilization with BD Cytoperm Buffer (BD Bioscience) and incubation with either Ki-67-PE antibody (BD Bioscience) or cleaved Caspase-3 antibody (Cell Signaling Technology, Beverly, MA). Cleaved Caspase-3 activity was detected by a PE-conjugated donkey anti-rabbit antibody (eBioscience).
All flow cytometric experiments were analyzed with a LSR II (BD Bioscience) and the data was analyzed with FlowJo (TreeStar, Ashland, OR).
Colony forming assays
Freshly isolated bone marrow and spleen cells were plated on methylcellulose medium M3434 (Stem Cell Technologies, Vancouver, BC) at seeding densities of 2 × 104 and 1 × 105 cells, respectively. Bone marrow cells were also seeded onto M3231 supplemented with a gradient of GM-CSF (PeproTech) at concentrations of 0, 0.1 and 1 ng/ml or 10 μM FAK inhibitor 14 (Tocris Bioscience, Bristol, UK). Colonies were scored at day 10.
RNA isolation and real-time reverse transcription PCR
In order to enrich bone marrow for hematopoietic stem and progenitor cells, c-Kit+ cells were isolated by magnetic separation with anti-c-Kit labeled magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), following the instructions provided by the manufacturer. The number of c-Kit+ cells was determined and RNA was subsequently isolated using a RNAeasy mini kit (Qiagen, Solna, Sweden). Analysis of gene expression was performed with one- step real-time reverse transcription PCR using QuantiTect™ SYBR® Green RT-PCR kit (Qiagen). The following PCR conditions were used; reverse transcription at 50°C for 20 minutes, inactivation at 95°C for 15 minutes, 50 cycles of denaturation at 94°C for 15 s, annealing for 25 s at 60°C, and extension at 72°C for 15 s. All primer sequences can be provided upon request. The PCR reactions were all run a LightCycler™ real- time PCR machine (Roche Diagnostics, Basel, Switzerland). The Cycle threshold (CT) values were estimated with the LightCycler Software v 4.1 and transcript levels were normalized by subtracting the corresponding β –actin values. Control was set at one differences and presented as 2-ΔKOCt-WTCt.
Bone marrow samples were enriched for c-Kit+ cells as described above. Promptly after isolation cells were allowed to rest for 1 hour at 37°C in RPMI 1640 medium supplemented with 10% serum. The cells were subsequently lyzed in SDS sample buffer (250 mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% β-mercaptoethanol). Samples were then separated by SDS-PAGE and transferred to a Hybond-P membrane (GE Healthcare, Uppsala, Sweden). Blocking of the membranes were done over night at 4°C in 5% BSA, followed by probing for phospho-STAT3, STAT3, FAK (all from Cell Signaling Technology), and phospho-FAK (Invitrogen).
All values are presented as mean ± SEM. Comparisons of two groups were analyzed by an unpaired Student t-test as all data sets were found to be normally distributed. For comparisons between multiple groups one-way ANOVA was used, followed by post hoc analysis with Bonferroni’s test. Statistical significance was set to p < 0.05.
Focal adhesion kinase
Src homology-2 domain protein B
Granulocyte colony-stimulation factor
Signal-transducer and activator of transcription
Fluorescence-activated cell sorting
Chronic myeloid leukemia
Hematopoietic stem cell
Hematopoietic progenitor cell
Tumor necrosis factor
Stem cell factor
Macrophage inflammatory protein
Chemokine (C-X-C motif) ligand
Green fluorescent protein
Breakpoint cluster region.
The work was supported by grants from the Swedish Cancer Foundation 120831, the Swedish Research Council 54X-10822, The Swedish Diabetes Fund DIA-2012-15 and the Family Ernfors Fund. MK was supported by NIH/NIDDK K01DK084261-01, Sidney Kimmel foundation Foundation for Cancer Research GC2201617 and V Foundation for Cancer Research GC221323.
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