PDGFRß promotes oncogenic progression via STAT3/STAT5 hyperactivation in anaplastic large cell lymphoma.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRß. Blocking PDGFRß kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. METHODS AND RESULTS: In a transgenic mouse model that mimics PDGFRß-driven human ALCL in vivo, we identify PDGFRß as a driver of aggressive tumor growth. Mechanistically, PDGFRß induces the pro-survival factor Bcl-xL and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. CONCLUSIONS: We therefore propose PDGFRß as a novel biomarker and introduce PDGFRß-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRß or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK+ ALCL patients.

Activation of different Stat5 isoforms contributes to cell-type-restricted signaling in response to interferons.

Tyrosine phosphorylation and activation of the transcription factor Stat5 occur in response to stimuli like granulocyte-macrophage colony-stimulating factor, interleukin-3, or erythropoietin that stimulate both proliferation and differentiation of hematopoietic cells. It is unclear whether Stat5 is part of a proliferative response or part of the events leading to cellular differentiation. Here we report that agents promoting differentiation but not proliferation of hematopoietic cells, like phorbol ester or both types of interferons (IFNs), activate Stat5 in promonocytic U937 cells. Both IFN types caused tyrosine phosphorylation and DNA binding of predominantly one Stat5 isoform (Stat5a) despite expression of both Stat5a and Stat5b proteins. Monocytic differentiation of U937 cells led to a strong decrease in IFN-gamma-mediated activation of Stat5 but not of Stat1. Transactivation of Stat5-target genes occurred in response to IFN-gamma, which activates both Stat5 and Stat1, but not in response to granulocyte-macrophage colony-stimulating factor, which activates only Stat5. Tyrosine phosphorylation of Stat5 is not generally part of the IFN response. IFN-gamma did not cause Stat5 activation in HeLa cells, despite the expression of both Stat5 isoforms at similar levels. By contrast, IFN-alpha caused tyrosine phosphorylation and DNA binding of exclusively the b isoform of Stat5, and activated Stat5b formed a DNA binding activity previously found in HeLa cells and designated IFN-alpha activation factor 2. Taken together, our results demonstrate that ligand binding of IFN receptors leads to an isoform-specific activation of Stat5 in a restricted number of cell lineages. Moreover, they suggest that Stat5 might be part of the differentiation response of myeloid cells.

Platelet dysfunction associated with cardiopulmonary bypass.

The clinical significance and pathogenesis of the platelet dysfunction following cardiopulmonary bypass were studied in conjunction with the degree of functional impairment associated with the use of membrane and bubble oxygenators. Forty consecutive patients had the following tests preoperatively and postoperatively: complete blood count (CBC), platelet count, prothrombin consumption time, bleeding time, prothrombin time, partial thromboplastin time, fibrinogen, euglobulin clot lysis, fibrin degradation products, and platelet aggregation tests. Six patients were given 14C-serotonin tests before and after operation, and preoperative and postoperative electron micrographs were made of the platelets of 3 patients. The amount of blood lost, the blood transfused, and plasma hemoglobin levels were also measured. Abnormal aggregation of platelets was found, with no difference between the membrane and bubble oxygenators. In vitro aggregation tests with protamine sulfate and hemoglobin solutions, as well as the 14C-serotonin studies and electron micrographs, suggest that platelets acquire storage pool deficiency and an abnormal membrane during cardiopulmonary bypass.

Silencing of ETV6/RUNX1 abrogates PI3K/AKT/mTOR signaling and impairs reconstitution of leukemia in xenografts.

The ETV6/RUNX1 (E/R) gene fusion is generated by the t(12;21) and found in approximately 25% of childhood B-cell precursor acute lymphoblastic leukemia. In contrast to the overwhelming evidence that E/R is critical for the initiation of leukemia, its relevance for the maintenance of overt disease is less clear. To investigate this issue, we suppressed the endogenous E/R fusion protein with lentivirally transduced short hairpin RNA in the leukemia cell lines REH and AT-2, and found a distinct reduction of proliferation and cell survival. In line with the observed concurrent inactivation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, pharmacological inhibition diminished the phosphorylation of AKT and ribosomal protein S6, and significantly increased the apoptosis rate in E/R-positive leukemias. Moreover, PI3K/mTOR inhibitors sensitized glucocorticoid-resistant REH cells to prednisolone, an observation of potential relevance for improving treatment of drug-resistant relapses. Of note, knockdown of the E/R fusion gene also severely impaired the repopulation capacity of REH cells in non-obese deficient/severe combined immunodeficient mice. Collectively, these data demonstrate that the E/R fusion protein activates the PI3K/AKT/mTOR pathway and is indispensible for disease maintenance. Importantly, these results provide a first rationale and justification for targeting the fusion gene and the PI3K/AKT/mTOR pathway therapeutically.

Stat1 combines signals derived from IFN-gamma and LPS receptors during macrophage activation.

Complete activation of macrophages during immune responses results from stimulation with the activating cytokine interferon-gamma (IFN-gamma) and a second stimulus, usually a microbial product. Bacterial infection of macrophages, or treatment with bacterial lipopolysaccharide (LPS), resulted in rapid Stat1 phosphorylation on Ser727 (S727) independently of concomitant tyrosine phosphorylation. IFN-gamma also caused rapid phosphorylation of S727. In both situations, S727 phosphorylation was reduced by pre-treatment of cells with the serine kinase inhibitor H7. When macrophages were treated sequentially or simultaneously with LPS and IFN-gamma, the pool of molecules phosphorylated on both Tyr701 (Y701) and S727 was strongly increased. Consistently, Stat1-dependent transcription in response to IFN-gamma was significantly enhanced if the cells were pre-treated with bacterial LPS. The relative amount of S727-phosphorylated Stat1 in the non-tyrosine phosphorylated fraction was considerably smaller than that in the tyrosine-phosphorylated fraction. No evidence was found for an effect of S727 phosphorylation on the phosphorylation of Y701 by IFN-gamma. Thus, serine and tyrosine phosphorylation of Stat1 are caused independently of each other, but the serine kinase may recognize tyrosine-phosphorylated Stat1 preferentially in the course of an IFN-gamma response. The data suggest Stat1 to be a convergence point for immunological stimuli in a macrophage proinflammatory response.

Listeria monocytogenes modulates macrophage cytokine responses through STAT serine phosphorylation and the induction of suppressor of cytokine signaling 3.

Macrophage activation as part of natural resistance to infection is caused by stimulation with IFN-gamma and by the invading microorganisms or microbial products. Infection of macrophages with the Gram-positive bacterium Listeria monocytogenes for short periods before activation with IFN-gamma increased the phosphorylation of transcription factor STAT1 at S727 and thereby the expression of IFN-gamma-induced genes. By contrast, persistent infection with viable bacteria or treatment with heat-killed Listeria diminished IFN-gamma-stimulated transcription and the phosphorylation of STAT1 at Y701. Decreased IFN-gamma signaling correlated with the induction of suppressor of cytokine signaling 3 (SOCS3) mRNA and protein. Contrasting our previous findings with LPS, maximal synthesis of SOCS3 required both the immediate signals from Listeria receptors on the cell surface and the activity of a polypeptide secreted in response to bacterial infection. SOCS3 induction by the secreted protein could not be blocked by neutralizing Abs to IL-10 and it did not require the presence of STAT1. Consistent with the induction of SOCS3 activity, Listeria also inhibited activation of STAT5 by GM-CSF. The p38 mitogen-activated protein kinase was rapidly activated upon infection of macrophages with L. monocytogenes. Inhibition of p38 mitogen-activated protein kinase with the pyridinyl imidazol SB203580 abrogated both STAT1 S727 phosphorylation and the expression of SOCS3. The data suggest that STAT1 serine kinase and SOCS3 activity are hallmarks of immediate and delayed phases of influence by bacterial signals on signal transduction in response to IFN-gamma.

Lipopolysaccharide induces in macrophages the synthesis of the suppressor of cytokine signaling 3 and suppresses signal transduction in response to the activating factor IFN-gamma.

The goal of this study was to investigate how bacterial LPS affects macrophage responsiveness to the activating factor IFN-gamma. Pretreatment of macrophages with LPS for <2 h increased the transcriptional response to IFN-gamma. In contrast, simultaneous stimulation with IFN-gamma and LPS, or pretreatment with LPS for >4 h, suppressed Stat1 tyrosine 701 phosphorylation, dimerization, and transcriptional activity in response to IFN-gamma. Consistently, the induction of MHCII protein by IFN-gamma was antagonized by LPS pretreatment. Neutralizing Abs to IL-10 were without effect on LPS-mediated suppression of Stat1 activation. Decreased IFN-gamma signal transduction after LPS treatment corresponded to a direct induction of suppressor of cytokine signaling3 (SOCS3) mRNA and protein. Under the same conditions socs1, socs2, and cis genes were not transcribed. In transfection assays, SOCS3 was found to suppress the transcriptional response of macrophages to IFN-gamma. A causal link of decreased IFN-gamma signaling to SOCS3 induction was also suggested by the LPS-dependent reduction of IFN-gamma-mediated Janus kinase 1 (JAK1) activation. Further consistent with inhibitory activity of SOCS3, LPS also inhibited the JAK2-dependent activation of Stat5 by GM-CSF. Our results thus link the deactivating effect of chronic LPS exposure on macrophages with its ability to induce SOCS3.

Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-gamma uses a different signaling pathway.

STAT1 is an essential transcription factor for macrophage activation by IFN-gamma and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-alpha occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-gamma-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-gamma-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-alpha caused activation of p38 MAPK whereas IFN-gamma did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-alpha production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKalpha and beta but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-gamma-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.