G-CSF receptor activation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2 phosphatase.

Src activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF-stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.

The expression of c-Met pathway components in unclassified pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH): a tissue microarray study.

AIMS: Subclassification of undifferentiated pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH) into distinct biological cohorts based on the expression patterns of molecular markers can identify patient subsets with especially unfavourable clinical outcomes. Identification of molecular prognosticators amenable for drug targeting can facilitate rational development of UPS/MFH tailored therapies. The aim was to evaluate expression of c-Met pathway components in a large cohort of UPS/MFH samples. METHODS AND RESULTS: An immunohistochemical analysis for hepatocyte growth factor (HGF), c-Met, phospho-c-Met (pc-Met), phospho-mitogen-activated protein kinase kinase (MAPKK) also known as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (p-MEK) and phospho-protein kinase B (p-AKT) was performed on a clinically annotated tissue microarray of 158 UPS/MFH samples. Univariable and multivariable analyses were conducted to evaluate the correlation of molecular variables with UPS/MFH disease specific survival. All evaluated markers were expressed in UPS/MFH to varying levels. Most importantly, strong HGF, pc-Met, p-MEK and p-AKT expression correlated significantly with dismal patient outcome on univariable statistical analysis. Expression of p-MEK and p-AKT remained statistically significant independent prognosticators on multivariable analysis. CONCLUSIONS: c-Met pathway components and especially p-MEK and p-AKT are potential prognostic biomarkers for UPS/MFH; their inclusion in future molecular-based staging systems should be evaluated. Furthermore, novel approaches targeting HGF, c-Met, MEK/extracellular-regulated kinase (ERK) and/or AKT should be considered for a subset of UPS/MFH patients.

Increased vascular endothelial growth factor-C expression is insufficient to induce lymphatic metastasis in human soft-tissue sarcomas.

PURPOSE: Unlike carcinomas, soft-tissue sarcoma (STS) rarely exhibit lymphatic spread. Consequently, we examined expression and function of vascular endothelial growth factor (VEGF)-C and STS-associated lymphatic vessel density (LVD) components of this process. EXPERIMENTAL DESIGN: VEGF-C and VEGF-A mRNA and VEGF-C protein expression were evaluated in STS, STS cell lines, and breast cancers (reverse transcription-PCR, quantitative reverse transcription-PCR, and ELISA). STS cell conditioned medium after VEGF-C knockdown was examined for endothelial cell proliferation and migration effects (MTS and migration assays). Paraffin-embedded human lymph node-negative and lymph node-positive STS and lymph node-negative and lymph node-positive breast cancers were examined for VEGF-C, D2-40, and CD31 expression (immunohistochemistry). LVD differences were analyzed by Wilcoxon rank-sum tests. RESULTS: STS and breast cancer VEGF-C expression was comparable and higher than normal tissue levels. STS cells secreted functional VEGF-C: STS conditioned medium induced lymphatic endothelial cell proliferation and migration, which was abrogated by STS cell VEGF-C knockdown. STS and breast cancer intratumoral LVD was similar. STS peritumoral LVD (PT-LVD) was reduced versus breast cancer PT-LVD (P < 0.001). Significantly higher PT-LVD was observed in lymph node-positive versus lymph node-negative STS; lymphatic spreading STS subtypes also had higher LVD. STS VEGF-C expression and PT-LVD lacked correlation, and many lymph node-negative STS had high PT-LVD, suggesting complexity in this metastatic process. CONCLUSIONS: Compared with breast cancers, STS exhibited lower PT-LVD independent of VEGF-C expression, which may underlie STS lymph node metastasis rarity. Moreover, lymphatic vessels appear necessary but not sufficient to sustain STS lymphatic spread. Examining STS "nonlymphatic" dissemination may help elucidate mechanisms of lymphatic spread, insights critically important to cancer metastasis control.

Combining PCI-24781, a novel histone deacetylase inhibitor, with chemotherapy for the treatment of soft tissue sarcoma.

PURPOSE: Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin malignancies. Consequently, we investigated the novel HDACi PCI-24781, alone/in combination with conventional chemotherapy, to determine its potential anti-STS-related effects and the underlying mechanisms involved. EXPERIMENTAL DESIGN: Immunoblotting was used to evaluate the effects of PCI-24781 on histone and nonhistone protein acetylation and expression of potential downstream targets. Cell culture-based assays were utilized to assess the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis, and chemosensitivity. Quantitative reverse transcription-PCR, chromatin immunoprecipitation, and reporter assays helped elucidate molecular mechanisms resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone or with chemotherapy, on tumor and metastatic growth was tested in vivo using human STS xenograft models. RESULTS: PCI-24781 exhibited significant anti-STS proliferative activity in vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing apoptosis. Superior effects were seen when combined with chemotherapy. A PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand break homologous recombination repair, was shown and may be a mechanism underlying PCI-24781 chemosensitization. We showed that PCI-24781 transcriptionally represses Rad51 through an E2F binding-site on the Rad51 proximal promoter. Although single-agent PCI-24781 had modest effects on STS growth and metastasis, marked inhibition was observed when combined with chemotherapy. CONCLUSIONS: In light of these findings, this novel molecular-based combination may be applicable to multiple STS histologic subtypes, and potentially merits rigorous evaluation in human STS clinical trials.

Epidermal growth factor receptor blockade in combination with conventional chemotherapy inhibits soft tissue sarcoma cell growth in vitro and in vivo.

PURPOSE: The epidermal growth factor receptor (EGFR) is highly expressed in many human soft tissue sarcomas (STS). However, EGFR blockade has not apparently been used for human STS therapy; therefore, we examined the in vitro and in vivo effects and the underlying mechanisms before considering EGFR blockade as a therapy for STS patients. EXPERIMENTAL DESIGN: Human STS tissues and cell lines were used to study EGFR expression and activation. Western blot analysis was used to evaluate effects of EGFR activation on downstream signaling. Cell culture assays were used to assess the effect of EGF stimulation as well as EGFR blockade (using an EGFR tyrosine kinase inhibitor, Iressa; AstraZeneca) on STS cell growth, apoptosis, and chemosensitivity. An in vivo study (HT1080 human fibrosarcoma cell line in nude/nude mice: Iressa, doxorubicin, Iressa + doxorubicin, vehicle) was used to examine tumor growth; pEGFR, proliferating cell nuclear antigen, and terminal deoxyribonucleotide transferase-mediated nick-end labeling staining helped assess the effect of therapy in vivo on STS EGFR activation, proliferation, and apoptosis. RESULTS: EGFR was expressed and activated in STS cell lines and tumors, probably due to ligand binding rather than EGFR mutation. Stimulation caused activation of AKT and mitogen-activated protein kinase pathways. EGFR blockade inhibited these effects and also caused increased apoptosis, a p53-independent G(0)-G(1) cell cycle arrest, and decreased cyclin D1 expression. In vivo, Iressa + doxorubicin had markedly synergistic anti-STS effects. CONCLUSION: EGFR blockade combined with conventional chemotherapy results in anti-human STS activity in vitro and in vivo, suggesting the possibility that combining these synergistic treatments will improve anti-STS therapy.

Midkine enhances soft-tissue sarcoma growth: a possible novel therapeutic target.

PURPOSE: New therapeutic targets for soft-tissue sarcoma (STS) treatment are critically needed. Midkine (MK), a multifunctional cytokine, is expressed during midgestation but is highly restricted in normal adult tissues. Renewed MK expression was shown in several malignancies where protumorigenic properties were described. We evaluated the expression and function of MK in STS. EXPERIMENTAL DESIGN: Immunohistochemistry, reverse transcription-PCR, and Western blotting (WB) evaluated MK expression in human STS tissues and cell lines. WB and flow cytometry analyzed MK receptor expression. Cell growth assays evaluated the effect of MK on STS cell growth, and WB assessed MK downstream signaling. MK knock-in and knockout experiments further evaluated MK function. The growth of parental versus MK-transfected human fibrosarcoma cells was studied in vivo. RESULTS: MK was found to be overexpressed in a variety of human STS histologies. Using a rhabdomyosarcoma (RMS) tissue microarray, cytoplasmic and nuclear MK was identified; nuclear MK expression was significantly increased in metastases. Similarly, several STS cell lines expressed and secreted MK; RMS cells exhibited nuclear MK. STS cells also expressed the MK receptors protein tyrosine phosphatase zeta and lipoprotein receptor-related protein. MK significantly enhanced STS cell growth potentially via the Src and extracellular signal-regulated kinase pathways. STS cells stably transfected with MK exhibited increased growth in vitro and in vivo. MK-expressing human STS xenografts showed increased tumor-associated vasculature. Furthermore, MK knockdown resulted in decreased STS cell growth, especially in RMS cells. CONCLUSION: MK enhances STS tumor growth; our results support further investigation of MK and its receptors as therapeutic targets for human STS.

Rad51 overexpression contributes to chemoresistance in human soft tissue sarcoma cells: a role for p53/activator protein 2 transcriptional regulation.

We investigated whether Rad51 overexpression plays a role in soft tissue sarcoma (STS) chemoresistance as well as the regulatory mechanisms underlying its expression. The studies reported here show that Rad51 protein is overexpressed in a large panel of human STS specimens. Human STS cell lines showed increased Rad51 protein expression, as was also observed in nude rat STS xenografts. STS cells treated with doxorubicin exhibited up-regulation of Rad51 protein while arrested in the S-G(2) phase of the cell cycle. Treatment with anti-Rad51 small interfering RNA decreased Rad51 protein expression and increased chemosensitivity to doxorubicin. Because we previously showed that reintroduction of wild-type p53 (wtp53) into STS cells harboring a p53 mutation led to increased doxorubicin chemosensitivity, we hypothesized that p53 participates in regulating Rad51 expression in STS. Reintroduction of wtp53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression. Using luciferase reporter assays, we showed that reconstitution of wtp53 function decreased Rad51 promoter activity. Deletion constructs identified a specific Rad51 promoter region containing a p53-responsive element but no p53 consensus binding site. Electrophoretic mobility shift assays verified activator protein 2 (AP2) binding to this region and increased AP2 binding to the promoter in the presence of wtp53. Mutating this AP2 binding site eliminated the wtp53 repressive effect. Furthermore, AP2 knockdown resulted in increased Rad51 expression. In light of the importance of Rad51 in modulating STS chemoresistance, these findings point to a potential novel strategy for molecular-based treatments that may be of relevance to patients burdened by STS.

Src kinases in G-CSF receptor signaling.

The Granulocyte Colony-Stimulating Factor (G-CSF) receptor, a member of the hematopoietin cytokine receptor superfamily, functions as a homodimer and requires the recruitment of cytosolic protein tyrosine kinases (PTKs) to transduce its signal. At least two cytosolic PTKs are primarily involved: Jak2, a member of the Janus family, and Lyn, a member of the Src family. Through poorly understood mechanisms, these kinases functionally interact with the G-CSF receptor. Jak2 primarily enlists members of the signal transducer and activator of transcription (STAT) family and Lyn phosphorylates a number of adaptor molecules, which link the G-CSF receptor to phosphatidylinositol (PI) 3'-kinase and extracellular signal-regulated kinases (Erk) pathways. This review presents evidence that the Src kinases play a major role in the pathways of G-CSF-mediated proliferation, survival, and differentiation. Identification of Src-dependent pathways provides drug targets useful in the treatment of myeloid leukemias.

PMA Stimulated Hydrolysis of Phosphatidylcholine in CRBH7919 Cell and Its Enzymatic Basis.

The effect of phorbol 12-myristate-13-acetate (PMA) on the hydrolysis of phosphatidylcholine (PC) in rat hepatoma cell line CRBH7919 has been studied. It was found that PMA stimulated PC hydrolysis in CRBH7919 cells in a dose-dependent manner after treatment for 15 min. The product of PC hydrolysis was choline, not phosphocholine. The activity of the membrane bound PC-specific phospholipase D (PC-PLD) was determined. The results showed that the activity of PC-PLD increased after 10 min with 100 nM PMA treatment, and reached a level 3.25 times the control after 30 min. The fact that the PC-PLD activation preceded the hydrolysis of PC, suggests that PC-PLD is involved in the PC hydrolysis into phosphatidic acid and choline in CRBH7919 cells in the presence of PMA.

Combining EGFR and mTOR blockade for the treatment of epithelioid sarcoma.

PURPOSE: Molecular deregulations underlying epithelioid sarcoma (ES) progression are poorly understood yet critically needed to develop new therapies. Epidermal growth factor receptor (EGFR) is overexpressed in ES; using preclinical models, we examined the ES EGFR role and assessed anti-ES EGFR blockade effects, alone and with mTOR inhibition. EXPERIMENTAL DESIGN: EGFR and mTOR expression/activation was examined via tissue microarray (n = 27 human ES specimens; immunohistochemistry) and in human ES cell lines (Western blot and quantitative reverse transcriptase PCR). Cell proliferation, survival, migration, and invasion effects of EGFR and mTOR activation treated with erlotinib (anti-EGFR small-molecule inhibitor) alone and combined with rapamycin were assessed in cell culture assays. In vivo growth effects of erlotinib alone or with rapamycin were evaluated using severe combined immunodeficient mouse ES xenograft models. RESULTS: EGFR was expressed and activated in ES specimens and cell lines. EGFR activation increased ES cell proliferation, motility, and invasion and induced cyclin D1, matrix metalloproteinase (MMP) 2, and MMP9 expression. EGFR blockade inhibited these processes and caused significant cytostatic ES growth inhibition in vivo. mTOR pathway activation at varying levels was identified in all tissue microarray-evaluable ES tissues; 88% of samples had no or reduced PTEN expression. Similarly, both ES cell lines showed enhanced mTOR activity; VAESBJ cells exhibited constitutive mTOR activation uncoupled from EGFR signaling. Most importantly, combined erlotinib/rapamycin resulted in synergistic anti-ES effects in vitro and induced superior tumor growth inhibition in vivo versus single agent administration. CONCLUSIONS: EGFR and mTOR signaling pathways are deregulated in ES. Preclinical ES model-derived insights suggest that combined inhibition of these targets might be beneficial, supporting evaluations in clinical trials.

Activated MET is a molecular prognosticator and potential therapeutic target for malignant peripheral nerve sheath tumors.

PURPOSE: MET signaling has been suggested a potential role in malignant peripheral nerve sheath tumors (MPNST). Here, MET function and blockade were preclinically assessed. EXPERIMENTAL DESIGN: Expression levels of MET, its ligand hepatocyte growth factor (HGF), and phosphorylated MET (pMET) were examined in a clinically annotated MPNST tissue microarray (TMA) incorporating univariable and multivariable statistical analyses. Human MPNST cells were studied in vitro and in vivo; Western blot (WB) and ELISA were used to evaluate MET and HGF expression, activation, and downstream signaling. Cell culture assays tested the impact of HGF-induced MET activation and anti-MET-specific siRNA inhibition on cell proliferation, migration, and invasion; in vivo gel-foam assays were used to evaluate angiogenesis. Cells stably transduced with anti-MET short hairpin RNA (shRNA) constructs were tested for growth and metastasis in severe combined immunodeficient (SCID) mice. The effect of the tyrosine kinase inhibitor XL184 (Exelixis) targeting MET/VEGFR2 (vascular endothelial growth factor receptor 2) on local and metastatic MPNST growth was examined in vivo. RESULTS: All three markers were expressed in MPNST human samples; pMET expression was an independent prognosticator of poor patient outcome. Human MPNST cell lines expressed MET, HGF, and pMET. MET activation increased MPNST cell motility, invasion, angiogenesis, and induced matrix metalloproteinase-2 (MMP2) and VEGF expression; MET knockdown had inverse effects in vitro and markedly decreased local and metastatic growth in vivo. XL184 abrogated human MPNST xenograft growth and metastasis in SCID mice. CONCLUSIONS: Informative prognosticators and novel therapies are crucially needed to improve MPNST management and outcomes. We show an important role for MET in MPNST, supporting continued investigation of novel anti-MET therapies in this clinical context.

Vimentin is a novel anti-cancer therapeutic target; insights from in vitro and in vivo mice xenograft studies.

BACKGROUND: Vimentin is a ubiquitous mesenchymal intermediate filament supporting mechano-structural integrity of quiescent cells while participating in adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in activated cells. Soft tissue sarcomas and some epithelial cancers exhibiting "epithelial to mesenchymal transition" phenotypes express vimentin. Withaferin-A, a naturally derived bioactive compound, may molecularly target vimentin, so we sought to evaluate its effects on tumor growth in vitro and in vivo thereby elucidating the role of vimentin in drug-induced responses. METHODS AND FINDINGS: Withaferin-A elicited marked apoptosis and vimentin cleavage in vimentin-expressing tumor cells but significantly less in normal mesenchymal cells. This proapoptotic response was abrogated after vimentin knockdown or by blockade of caspase-induced vimentin degradation via caspase inhibitors or overexpression of mutated caspase-resistant vimentin. Pronounced anti-angiogenic effects of Withaferin-A were demonstrated, with only minimal effects seen in non-proliferating endothelial cells. Moreover, Withaferin-A significantly blocked soft tissue sarcoma growth, local recurrence, and metastasis in a panel of soft tissue sarcoma xenograft experiments. Apoptosis, decreased angiogenesis, and vimentin degradation were all seen in Withaferin-A treated specimens. CONCLUSIONS: In light of these findings, evaluation of Withaferin-A, its analogs, or other anti-vimentin therapeutic approaches in soft tissue sarcoma and "epithelial to mesenchymal transition" clinical contexts is warranted.

Dual targeting of AKT and mammalian target of rapamycin: a potential therapeutic approach for malignant peripheral nerve sheath tumor.

The mammalian target of rapamycin (mTOR) pathway may constitute a potential target for the treatment of malignant peripheral nerve sheath tumors (MPNST). However, investigations of other cancers suggest that mTOR blockade can paradoxically induce activation of prosurvival, protumorigenic signaling molecules, especially upstream AKT. Consequently, we hypothesized that dual phosphatidylinositol 3-kinase (PI3K)/AKT-mTOR blockade might be applicable for MPNST treatment. Expression of activated mTOR downstream targets (p4EBP1 and pS6RP) and pAKT was evaluated immunohistochemically in a tissue microarray of human MPNSTs (n = 96) and benign neurofibromas (n = 31). Results were analyzed by Wilcoxon rank-sum tests. mTOR and AKT pathways in human MPNST cell lines, and the effects of rapamycin (mTOR inhibitor), LY294002 (dual PI3K/mTOR inhibitor), and PI-103 (potent dual PI3K/AKT-mTOR inhibitor) on pathway activation were evaluated by Western blot. Effects on cell growth were evaluated via MTS and colony formation assays. Cell cycle progression and apoptosis were assessed by propidium iodide/fluorescence-activated cell sorting staining and Annexin V assays. Acridine orange staining/fluorescence-activated cell sorting analysis, electron microscopy, and Western blot evaluated autophagy induction. p4EBP1, pS6Rp, and pAKT levels were found to be significantly higher in MPNST versus neurofibroma (P < 0.05 for all markers). mTOR and AKT pathways were found to be highly activated in MPNST cell lines. MPNST cells were sensitive to rapamycin; however, rapamycin enhanced pAKT and peIF4E expression. PI-103 abrogated MPNST cell growth and induced G(1) cell cycle arrest potentially through repression of cyclin D1. PI-103 did not elicit apoptosis but significantly induced autophagy in MPNST cells. These results suggest further study of combined PI3K/AKT and mTOR inhibition as a novel therapy for patients harboring MPNST.

Soft tissue sarcoma cells are highly sensitive to AKT blockade: a role for p53-independent up-regulation of GADD45 alpha.

The AKT signaling pathway is activated in soft tissue sarcoma (STS). However, AKT blockade has not yet been studied as a potential targeted therapeutic approach. Here, we examined the in vitro and in vivo effects of AKT inhibition in STS cells. Western blot analysis was used to evaluate the expression of AKT pathway components and the effect of AKT stimulation and inhibition on their phosphorylation. Cell culture assays were used to assess the effect of AKT blockade (using a phosphatidylinositol 3-kinase inhibitor and a specific AKT inhibitor) on STS cell growth, cell cycle, and apoptosis. Oligoarrays were used to determine gene expression changes in response to AKT inhibition. Reverse transcription-PCR was used for array validation. Specific small inhibitory RNA was used to knockdown GADD45 alpha. Human STS xenografts in nude mice were used for in vivo studies, and immunohistochemistry was used to assess the effect of treatment on GADD45 alpha expression, proliferation, and apoptosis. Multiple STS cell lines expressed activated AKT. AKT inhibition decreased STS downstream target phosphorylation and growth in vitro; G(2) cell cycle arrest and apoptosis were also observed. AKT inhibition induced GADD45 alpha mRNA and protein expression in all STS cells treated independent of p53 mutational status. GADD45 alpha knockdown attenuated the G(2) arrest induced by AKT inhibition. In vivo, AKT inhibition led to decreased STS xenograft growth. AKT plays a critical role in survival and proliferation of STS cells. Modulation of AKT kinase activity may provide a novel molecularly based strategy for STS-targeted therapies.

The Integrin alpha9beta1 contributes to granulopoiesis by enhancing granulocyte colony-stimulating factor receptor signaling.

The integrin alpha9beta1 is widely expressed on neutrophils, smooth muscle, hepatocytes, endothelia, and some epithelia. We now show that mice lacking this integrin have a dramatic defect in neutrophil development, with decreased numbers of granulocyte precursors in bone marrow and impaired differentiation of bone marrow cells into granulocytes. In response to granulocyte colony-stimulating factor (G-CSF), alpha9-deficient bone marrow cells or human bone marrow cells incubated with alpha9beta1-blocking antibody demonstrated decreased phosphorylation of signal transducer and activator of transcription 3 and extracellular signal-regulated protein kinase. These effects depended on the alpha9 subunit cytoplasmic domain, which was required for formation of a physical complex between alpha9beta1 and ligated G-CSF receptor. Integrin alpha9beta1 was required for granulopoiesis and played a permissive role in the G-CSF-signaling pathway, suggesting that this integrin could play an important role in disorders of granulocyte development and other conditions characterized by defective G-CSF signaling.

G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth.

Granulocyte colony-stimulating factor (G-CSF) drives the production, survival, differentiation, and inflammatory functions of granulocytes. Reactive oxygen species (ROSs) provide a major thrust of the inflammatory response, though excessive ROSs may be deleterious. G-CSF stimulation showed a time- and dose-dependent increase in ROS production, correlating with activation of Lyn and Akt. Inhibition of Lyn, PI3-kinase, and Akt abrogated G-CSF-induced ROS production. This was also blocked by DPI, a specific inhibitor of NADPH oxidase. Following G-CSF stimulation, neutrophils from Lyn-/- mice produced less ROSs than wild-type littermates. G-CSF induced both serine phosphorylation and membrane translocation of p47phox, a subunit of NADPH oxidase. Because patients with a truncated G-CSF receptor have a high risk of developing acute myeloid leukemia (AML), we hypothesized that dysregulation of ROSs contributes to leukemogenesis. Cells expressing the truncated G-CSF receptor produced more ROSs than those with the full-length receptor. G-CSF-induced ROS production was enhanced in bone marrow-derived neutrophils expressing G-CSFRdelta715, a truncated receptor. The antioxidant N-acetyl-L-cysteine diminished G-CSF-induced ROS production and cell proliferation by inhibiting Akt activation. These data suggest that the G-CSF-induced Lyn-PI3K-Akt pathway drives ROS production. One beneficial effect of therapeutic targeting of Lyn-PI3K-kinase-Akt cascade is abrogating ROS production.

G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses.

The granulocyte colony-stimulating factor receptor (G-CSFR) transduces intracellular signals for myeloid cell proliferation, survival, and differentiation through the recruitment of nonreceptor protein tyrosine kinases Lyn and janus kinase 2 (Jak2). This results in the tyrosine phosphorylation of a small set of positive and negative adapters and effectors. Grb2-associated binder-2 (Gab2) is a newly described adapter molecule, preferentially expressed in hematopoietic cells and associated with phosphatidylinositol 3 (PI3) kinase. Studies suggest that Gab2 plays both positive and negative roles in cytokine receptor signaling. To investigate the role Gab2 plays in G-CSF receptor-mediated signaling, we have analyzed its activation state and correlated that with wild-type and mutant G-CSF receptors stably expressed in the murine factor-dependent Ba/F3 cell lines. G-CSF-induced tyrosine phosphorylation of Gab2 occurred in the wild-type and single Y-to-F mutants (Y704F, Y729F, and Y744F), but not in the ADA and W650R loss-of-function mutants. Cells expressing truncated proximal G-CSFR, the tyrosine-null (Y4F) G-CSFR, or Y764F mutant receptors had decreased phosphorylation of Gab2. Specific inhibitors of Src kinase (PD173 and PP1) but not Jak2 kinase (AG490) blocked Gab2 phosphorylation. Phosphorylation of Gab2 occurred in wild-type, but not Lyn-deficient, G-CSFR-transfected DT40 B cells. These data propose that Lyn, not Jak2, phosphorylates Gab2 and that maximal phosphorylation of Gab2 requires Y764, a Grb2-binding site. Serine phosphorylation of Akt, a marker of PI3-kinase activity, was detected in both wild-type and truncated proximal domain receptors, but not in the ADA and W650R mutants. Levels of phospho-Akt and phospho-extracellular signal-regulated kinase (phospho-ERK) were greater in proximal truncated than in wild-type G-CSFR cells, suggesting that Gab2 is dissociated from PI3 kinase or ERK activities. Overexpression of Gab2 enhanced the phosphorylation state of Akt, but not of ERK. This inhibited the proliferation of wild-type and truncated G-CSFR-transfected Ba/F3 cells and enhanced their myeloid differentiation. All together, these data indicate that G-CSF treatment leads to Lyn-mediated tyrosine phosphorylation of Gab2, which may serve as an important intermediate of enhanced Akt activity and myeloid differentiation, not growth/survival response.

Deletional mutation of the external domain of the human granulocyte colony-stimulating factor receptor in a patient with severe chronic neutropenia refractory to granulocyte colony-stimulating factor.

Severe chronic neutropenia (SCN) is characterized by a profound neutropenia, which mostly presents during the neonatal period. The precise genetic basis of SCN remains elusive. Acquired somatic mutations involving the carboxy-terminus of the G-CSF receptor (G-CSFR) have been found, often in association with myelodysplastic syndrome. The authors describe a girl with SCN who did not respond to pharmacologic doses of filgrastim. Genetic analysis of bone marrow and germline cells revealed a 182-bp deletion in the extracellular domain of the G-CSFR. Co-precipitation studies showed an association between the wild-type and mutant G-CSFR, confirmed by their co-localization by confocal microscopy. Coexpression of the mutant receptor inhibited the wild-type response in Ba/F3 cells. These findings establish a novel constitutional defect in the G-CSFR that supports a partial dominant negative mechanism for receptor dysfunction in SCN.