PDGFRB mutants found in patients with familial infantile myofibromatosis or overgrowth syndrome are oncogenic and sensitive to imatinib.

Recently, germline and somatic heterozygous mutations in the platelet-derived growth factor receptor ß (PDGFRB) have been associated with familial infantile myofibromatosis (IM), which is characterized by soft tissue tumors, and overgrowth syndrome, a disease that predisposes to cancer. These mutations have not been functionally characterized. In the present study, the activity of three PDGFRB mutants associated with familial IM (R561C, P660T and N666K) and one PDGFRB mutant found in patients with overgrowth syndrome (P584R) was tested in various models. The P660T mutant showed no difference with the wild-type receptor, suggesting that it might represent a polymorphic variant unrelated to the disease. By contrast, the three other mutants were constitutively active and able to transform NIH3T3 and Ba/F3 cells to different extents. In particular, the germline mutant identified in overgrowth syndrome, P584R, was a stronger oncogene than the germline R561C mutant associated with myofibromatosis. The distinct phenotypes associated with these two mutations could be related to this difference of potency. Importantly, all activated mutants were sensitive to tyrosine kinase inhibitors such as imatinib, nilotinib and ponatinib. In conclusion, the PDGFRB mutations previously identified in familial IM and overgrowth syndrome activate the receptor in the absence of ligand, supporting the hypothesis that these mutations cause the diseases. Moreover, imatinib seems to be a promising treatment for patients carrying these mutations. To our knowledge, these are the first confirmed gain-of-function point mutations of PDGFRB in human cancer.

PDGFRA alterations in cancer: characterization of a gain-of-function V536E transmembrane mutant as well as loss-of-function and passenger mutations.

Activating mutations in the platelet-derived growth factor (PDGF) receptor alpha (PDGFRA) have been described in patients with gastrointestinal stromal tumors or myeloid malignancies associated with hypereosinophilia. These patients respond well to imatinib mesylate, raising the question as to whether patients with a PDGF receptor mutation in other tumor types should receive a tyrosine kinase inhibitor treatment. We characterized 10 novel somatic point mutations in PDGFRA that have been reported in isolated cases of glioblastoma, melanoma, acute myeloid leukemia, peripheral nerve sheath tumors and neuroendocrine carcinoma. The PDGFRA transmembrane domain mutation V536E stimulated Ba/F3 cell growth and signaling via ERK and STAT5 in the absence of ligand. This mutant, identified in glioblastoma, was strongly inhibited by imatinib. Modeling suggested that the mutation modulates the packing of the transmembrane domain helices in the receptor dimer. By contrast, two mutations in highly conserved residues affected the receptor traffic to the cell surface or kinase activity, thereby preventing the response to PDGF. The other mutations had no significant impact on the receptor activity. This functional analysis matched the predictions of SIFT and PolyPhen for only five mutations and these algorithms do not discriminate gain from loss of function. Finally, an E996K variant that had been identified in a melanoma cell line was not expressed in these cells. Altogether, several newly identified PDGFRA mutations do not activate the receptor and may therefore represent passenger mutations. Our results also underline the importance of characterizing novel kinase alterations in cancer patients.

[Role of PDGF and FGF receptors in cancer]. / Role des récepteurs du PDGF et du FGF dans les cancers.

Growth factors of the PDGF and FGF families act through receptor tyrosine kinases. These receptors can be activated by chromosomal rearrangements in myeloid neoplasms associated with hypereosinophilia. We identified a new fusion gene between KANK1 and PDGFRbeta in a patient with thrombocythemia. We showed that such fusion oncoproteins derived from PDGF and FGF receptors escape the normal degradation pathways, leading to their accumulation in cells. This process amplifies signalling leading to cell proliferation. Using microarrays and bioinformatics, we showed that several transcription factors contribute to the control cell growth, including STATS, FOXO and SREBP.

JAK2, the JAK2 V617F mutant and cytokine receptors.

Recently, a unique recurrent somatic mutation was identified as a major molecular event in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Expression of this mutant in cytokine-dependent hematopoietic cell lines induces autonomous growth. This effect is enhanced by overexpression of cytokine receptors, and can be inhibited by co-expression at higher levels of the wild type JAK2, which may compete for a limited pool of receptors. In JAK2-deficient cells, we showed that JAK2 V617F can transmit signals from ligand-activated TpoR or EpoR. Furthermore, the mutant JAK2 can be demonstrated to stimulate traffic of the EpoR. Thus, JAK2 V617F mutant must be able to interact via its intact FERM-SH2 domains with the cytosolic domains of cytokine receptors. A synergy between JAK2 V617F and insulin-like growth factor 1 receptor (IGF1R) can be detected in cytokine-dependent cell proliferation. Once cells are rendered autonomous by expression of JAK2 V617F, IGF1 acquires the ability to activate the JAK-STAT pathway. Thus, expression of JAK2 V617F may explain the described hypersensitivity of PV erythroid progenitors to IGF1. The V617 is conserved in two other mammalian JAKs, JAK1 and Tyk2. The homologous mutants JAK1 V658F and Tyk2 V678F are also active in proliferation and transcriptional assays. Such mutants may be found in human cancers or autoimmune diseases. In contrast, the JAK3 M592F does not lead to activation of JAK3. Current hypotheses on how JAK2 V617F contributes to three myeloproliferative diseases, and which other events may favor one disease versus another, are discussed.

Interleukin-9 (IL-9) induces cell growth arrest associated with sustained signal transducer and activator of transcription activation in lymphoma cells overexpressing the IL-9 receptor.

Murine interleukin (IL)-9 inhibits apoptosis in murine T lymphomas via signal transducer and activator of transcription (STAT) factors. After transfection of the human IL-9 receptor, human IL-9 had a similar antiapoptotic activity, but, unlike the mouse protein, inhibited proliferation. This effect was correlated with the level of receptor expression and the extent of STAT phosphorylation. Expression of a moderate level of suppressor of cytokine signaling 3 (SOCS3) reduced STAT activation by human IL-9 and prevented inhibition of growth but not of apoptosis. Using mutated IL-9 receptors, we showed that inhibition of proliferation was correlated with STAT1 and STAT3 activation by IL-9 and induction of the cell cycle inhibitor p19/ink4d, a STAT3 target gene. Activation of STAT1 by IFN-gamma did not result in cell growth arrest. In this model, cell growth inhibition is therefore associated with a higher number of receptors, a more robust STAT activation, and a greater sensitivity to SOCS3 expression, compared to apoptosis inhibition.

Interleukin 9 induces expression of three cytokine signal inhibitors: cytokine-inducible SH2-containing protein, suppressor of cytokine signalling (SOCS)-2 and SOCS-3, but only SOCS-3 overexpression suppresses interleukin 9 signalling.

Interleukin 9 (IL-9) is a cytokine preferentially produced by T helper type 2 lymphocytes and active on various cell types such as T- and B-lymphocytes, mast cells and haemopoietic progenitors. The IL-9 receptor (IL-9R) belongs to the haemopoietic receptor superfamily and its signal transduction involves mainly the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Here we studied the implication of a novel family of suppressors of cytokine signalling (called CIS, for cytokine-inducible SH2-containing protein, and SOCS, for suppressor of cytokine signalling) in IL-9 signal attenuation. In BW5147 T-cell lymphoma, IL-9 induced the rapid expression of CIS, SOCS-2 and SOCS-3 with a peak after 2 h of stimulation. Using IL-9R mutants, we showed that STAT activation is required for CIS/SOCS induction: CIS and SOCS-2 expression was induced either via STAT1 and/or STAT3 or via STAT5 but only STAT1 and/or STAT3 were involved in SOCS-3 expression. The effect of these three proteins on IL-9 signal transduction was assessed by transient transfection in HEK-293 cells expressing the components of the IL-9 signalling pathway and a STAT-responsive reporter construct. These experiments showed that only SOCS-3 is able to inhibit IL-9-induced signal transduction; neither CIS nor SOCS-2 exerted any effect. Stable transfection of CIS and SOCS-3 in BW5147 lymphoma cells showed that only overexpression of SOCS-3 had an inhibitory activity on STAT activation, gene induction and the anti-apoptotic activity of IL-9. By contrast, CIS failed to affect the IL-9 response.

Role of insulin receptor substrate-2 in interleukin-9-dependent proliferation.

Interleukin-9 (IL-9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL-9 signaling is not clear. We show that IL-9 induces IRS2 phosphorylation and association with phosphatidylinositol-3 kinase (PI 3-K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL-9 stimulation. We observed a PI 3-K-dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL-9-dependent cell lines. Finally, 32D cells that were transfected with the IL-9 receptor but lack IRS expression survived in the presence of IL-9. Ectopic IRS1 expression allowed for IL-9-induced proliferation, in the absence of significant PKB phosphorylation.

STAT5 activation is required for interleukin-9-dependent growth and transformation of lymphoid cells.

Interleukin-9 (IL-9) is a growth factor for T cells and various hematopoietic and lymphoid tumor cells. IL-9 signaling involves activation of Janus kinase (JAK)1 and JAK3 kinases, and signal transducer and activator of transcription (STAT)1, STAT3 and STAT5. Using a dominant negative form of STAT5 (STAT5delta), we demonstrated that this factor is an important mediator of IL-9-dependent Ba/F3 cell growth. Mutation of the STAT binding site of the IL-9 receptor (tyr116phe) results in an important decrease in STAT activation and inhibition of proliferation in the presence of IL-9. A small number of cells escape this inhibition, and IL-9-dependent cell lines could be derived. The selected cells required activation of STAT5 for growth, which was blocked by STAT5delta expression and enhanced by overexpression of wild-type STAT5. In contrast to parental cells, Ba/F3-Phe116 cells growing in the presence of IL-9 further progress to cytokine-independent tumorigenic clones. These tumorigenic clones exhibited a strong cytokine-independent activation of JAK1 and STAT5, which most likely supports their proliferation. Transfection of a constitutively activated variant of STAT5 promoted the growth of wild-type Ba/F3 cells in the absence of cytokine. Finally, the expression of the proto-oncogene pim-1 was correlated with STAT5 activation and cell growth. Our data suggest that STAT5 is an important mediator of IL-9-driven proliferation and that dysregulation of STAT5 activation favors tumorigenesis of lymphoid cells.

Distinct roles for STAT1, STAT3, and STAT5 in differentiation gene induction and apoptosis inhibition by interleukin-9.

Interleukin-9 (IL-9) activates three distinct STAT proteins: STAT1, STAT3, and STAT5. This process depends on one tyrosine of the IL-9 receptor, which is necessary for proliferation, gene induction, and inhibition of apoptosis induced by glucocorticoids. By introduction of point mutations in amino acids surrounding this tyrosine, we obtained receptors that activated either STAT5 alone or both STAT1 and STAT3, thus providing us with the possibility to study the respective roles of these factors in the biological activities of IL-9. Both mutant receptors were able to prevent apoptosis, but only the mutant that activated STAT1 and STAT3 was able to support induction of granzyme A and L-selectin. In line with these results, constitutively activated STAT5 blocked glucocorticoid-induced apoptosis. In Ba/F3 cells, significant proliferation and pim-1 induction were observed with both STAT-restricted mutants, though proliferation was lower than with the wild-type receptor. These results suggest that survival and cell growth are redundantly controlled by multiple STAT factors, whereas differentiation gene induction is more specifically correlated with individual STAT activation by IL-9.

Interleukin-9 regulates NF-kappaB activity through BCL3 gene induction.

BCL3 encodes a protein with close homology to IkappaB proteins and interacts with p50 NF-kappaB homodimers. However, the regulation and transcriptional activity of BCL3 remain ill-defined. We observed here that interleukin-9 (IL-9) and IL-4, but not IL-2 or IL-3, transcriptionally upregulated BCL3 expression in T cells and mast cells. BCL3 induction by IL-9 was detected as soon as 4 hours after stimulation and appeared to be dependent on the Jak/STAT pathway. IL-9 stimulation was associated with an increase in p50 homodimers DNA binding activity, which was mimicked by stable BCL3 expression. This contrasts with tumor necrosis factor (TNF)-dependent NF-kappaB activation, which occurs earlier, involves p65/p50 dimers, and is dependent on IkappaB degradation. Moreover, IL-9 stimulation or BCL3 transient transfection similarly inhibited NF-kappaB-mediated transcription in response to TNF. Taken together, our observations show a new regulatory pathway for the NF-kappaB transcription factors through STAT-dependent upregulation of BCL3 gene expression.

Ly-6A/E induction by interleukin-6 and interleukin-9 in T cells.

Interleukin-6 (IL-6) is active in the early steps of T cell activation and confers IL-2 responsiveness. In this work, we used EL4 T lymphoma to identify IL-6 target genes in T cells. By differential screening of a cDNA library, we found that IL-6 induced the expression of Ly-6A/E, a GPI-anchored cell surface protein reported to be a regulator of T cell activation. In addition to IL-6, IL-9 and IFN-gamma induced Ly-6A/E expression in EL4 and BW5147 cells. We showed that both IL-6 and IL-9 mediated the transcriptional activation of Ly-6A/E through a GAS element in the Ly-6A/E promoter, which was able to bind STAT1 and STAT3, transcription factors activated by these cytokines. IL-6 had a similar effect in freshly isolated normal T cells, and dramatically increased their proliferation upon Ly-6A/E stimulation. Taken together, our data suggest that Ly-6A/E induction takes part in the T cell activation program initiated by IL-6.

Interleukin 9 and its receptor: an overview of structure and function.

Interleukin-9 (IL-9) is a multifunctional cytokine produced by activated TH2 clones in vitro and during TH2-like T cell responses in vivo. Although IL-9 was initially described as a T cell growth factor, its role in T cell responses is still unclear. While freshly isolated normal T cells do not respond to IL-9, this cytokine induces the proliferation of murine T cell lymphomas in vitro, and in vivo overexpression of IL-9 results in the development of thymic lymphomas. In the human, the existence of an IL-9 mediated autocrine loop has been suggested for some malignancies such as Hodgkin's disease. Various observations indicate that IL-9 is actively involved in mast cells responses by inducing the proliferation and differentiation of these cells. Other potential biological targets for IL-9 include B lymphocytes, and hematopoietic progenitors, for which higher responses were observed with foetal or transformed cells as compared to normal adult progenitors. The IL-9 receptor is a member of the hemopoietin receptor superfamily and interacts with the gamma chain of the IL-2 receptor for signaling. Signal transduction studies have stressed the role of the Jak-STAT pathway in various IL-9 bioactivities, whereas the 4PS/IRS2 adaptor protein might also play a significant role in IL-9 signaling.

Signalling by cytokines interacting with the interleukin-2 receptor gamma chain.

The interleukin-2 (IL-2) receptor gamma chain (gammac) is shared by receptor complexes used by IL-2, IL-4, IL-7, IL-9 and IL-15, all of which are cytokines involved in lymphocyte development and/or activation. Gammac is physically and functionally associated with the JAK3 tyrosine kinase. This molecular pair may be considered as the trigger of the signalling cascades, inducing the activation of JAK1 upon heterodimerization with a cytokine-specific receptor component. JAK1, JAK3 and other tyrosine kinases, the nature of which varies between cytokines, phosphorylate the receptor, thereby creating docking sites for signalling molecules. Among them, PI 3-kinase and downstream effectors play a central role in the signalling processes involved in proliferation and inhibition of apoptosis for every gammac-interacting cytokine, although the mechanism of activation may vary between cytokines. Other important mediators--STAT transcription factors--regulate the expression of specific genes. IL-2, IL-7, IL-9 and IL-15 activate STAT3 and STAT5, in contrast to IL-4, which activates STAT6. These cytokines also trigger specific pathways, such as the MAP kinase cascade for IL-2 and IL-15, and the cascade responsible for immunoglobulin gene V-D-J rearrangement in response to IL-7.

A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

Differential activity of dexamethasone on IL-2-, IL-4-, or IL-9-induced proliferation of murine factor-dependent T cell lines.

Mouse helper T cell lines were developed that proliferate permanently without Ag and APCs in response to either IL-2, IL-4, or IL-9, three cytokines whose receptors interact with the IL-2R gamma-chain for signal transduction. Depending on the growth factor, a marked difference was observed regarding the ability of dexamethasone (DEX) to inhibit cell proliferation. In three different cell lines, proliferation induced by IL-2 was completely arrested, while that supported by IL-9 was hardly affected. With IL-4, proliferation was also maintained but less markedly than with IL-9. Although DEX was able to induce apoptosis in these cells, the inhibition of IL-2-induced proliferation was not the result of apoptosis, as this process was equally antagonized by all three factors. Moreover, addition of IL-4 or IL-9 to cultures previously incubated with IL-2 and DEX for several days restored cell proliferation. Finally, autonomous cell variants derived from the factor-dependent cell lines were still protected by IL-4 and IL-9 against growth inhibition by DEX. Together, these results indicate that growth stimulation in the presence of glucocorticoids and inhibition of apoptosis involve distinct aspects of cytokine activities.

Oxidative DNA damage by t-butyl hydroperoxide causes DNA single strand breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes.

In rat hepatocytes, DNA damage by t-butyl hydroperoxide (tBOOH) was measured by using the fluorimetric analysis of alkaline DNA unwinding. The electrophoretic profile of genomic DNA suggests single rather than double DNA strand breaks formation. Oxidative DNA modifications, measured as increased 8-hydroxy-deoxyguanosine content, were not detected. Lysis of hepatocytes and DNA strand breaks induced by tBOOH did not correlate, indicating that both processes are not interconnected. Since o-phenanthroline prevents against tBOOH-mediated effects on both DNA and membrane integrity, we discussed about a putative role of iron.