JAK-STAT Activation Research Articles

JAK2-STAT3 signaling

The function of JAK-STAT signaling in the central nervous system has been widely studied in the context of neural cell development and differentiation and in neuronal and glial responses to CNS injury. A study published recently in Neuron by Nicolas et al. now demonstrates that the JAK2-STAT3 pathway also plays an important role in the regulation of synaptic transmission. By using a combination of biochemical, pharmacological and genetic approaches they show that induction of long-term depression (LTD), an activity-dependent rapid and long-lasting decrease in synaptic strength, via NMDA receptors depends on STAT3 activation by JAK2 that can be localized specifically to postsynaptic structures. Most interestingly, they find that induction of LTD requires STAT3 phosphorylation and dimerization but is independent of nuclear translocation and transcriptional activity of STAT3. Although it remains to be clarified how NMDA receptor-mediated postsynaptic processes lead to JAK2-STAT3 activation and how this in turn translates into persistent changes in synaptic strength, these results provide evidence for a novel mechanism of signal transduction.

Mycobacterium avium subsp. paratuberculosis Inhibits Gamma Interferon-Induced Signaling in Bovine Monocytes: Insights into the Cellular Mechanisms of Johne's Disease

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection by M. avium subsp. paratuberculosis depends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by which M. avium subsp. paratuberculosis subverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected and M. avium subsp. paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation of M. avium subsp. paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates that M. avium subsp. paratuberculosis blocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed in M. avium subsp. paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this critical M. avium subsp. paratuberculosis behavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.

JAK-STAT and AKT pathway-coupled genes in erythroid progenitor cells through ontogeny

BackgroundIt has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival have also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny.MethodsHematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells.ResultsIn microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS2, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes are differentially expressed only in some stages of ontogeny (STATs, GRB2, CREBB). Beside the continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH).ConclusionsThis broad overview of gene expression in erythropoiesis revealed transcription factors differentially expressed in some stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway-coupled NFKBIA and YWHAH genes.

Model-based selection of the robust JAK-STAT activation mechanism

JAK-STAT pathway family is a principal signaling mechanism in eukaryotic cells. Evolutionary conserved roles of this mechanism include control over fundamental processes such as cell growth or apoptosis. Deregulation of the JAK-STAT signaling is frequently associated with cancerogenesis. JAK-STAT pathways become hyper-activated in many human tumors. Therefore, components of these pathways are an attractive target for drugs, which design requires as adequate models as possible. Although, in principle, JAK-STAT signaling is relatively simple, the ambiguities in a receptor activation prevent a clear explanation of the underlying molecular mechanism.Here, we compare four variants of a computational model of the JAK1/2-STAT1 signaling pathway. These variants capture known, basic discrepancies in the mechanism of activation of a cytokine receptor, in the context of all key components of the pathway. We carry out a comparative analysis using mass action kinetics. The investigated differences are so marginal that all models satisfy a goodness of fit criteria to the extent that the state of the art Bayesian model selection (BMS) method fails to significantly promote one model. Therefore, we comparatively investigate changes in a robustness of the JAK1/2-STAT1 pathway variants using the global sensitivity analysis method (GSA), complemented with the identifiability analysis (IA). Both BMS and GSA are used to analyze the models for the varying parameter values. We found out that, both BMS and GSA, narrowed down to the receptor activation component, slightly promote the least complex model. Further, insightful, comprehensive GSA, motivated by the concept of robustness, allowed us to show that the precise order of reactions of a ligand binding and a receptor dimerization is not as important as the on-membrane pre-assembly of the dimers in the absence of ligand.The main value of this work is an evaluation of the usefulness of different model selection methods in a frequently encountered, but not much discussed case of a model of a considerable size, which has several variants differing at peripheries. In such situation, all considered variants can reach nearly perfect agreement with respect to their numerical simulations results and, most often, the sufficient experimental data to test against is not available. We argue that in such an adverse setting, the GSA and IA, although not directly corresponding to the model selection problem, can be more informative than the representative, generalizability-based approaches to this task. An additional insight into how the responsibility for the network dynamics spreads among model parameters, enables more conscious, expert-mediated choice of the preferred model.

Molecular Mechanism of Hepcidin-Mediated Ferroportin Internalization Requires Ferroportin Lysines, Not Tyrosines or JAK-STAT

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. Invitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. Invivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

The Small Molecule JAK1/JAK2 Inhibitor INCB16562 Shows Single Agent Activity and Strongly Synergizes with Bcl-2 Inhibitors in Lymphoma

Abstract 2731Constitutive activation of Janus Kinases has been shown to be an important mechanism supporting human lymphomagenesis. Aberrantly activated JAK/STAT signaling has been described in Hodgkin lymphoma (HL), Anaplastic Large T-cell lymphoma and Diffuse large B cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, making it an attractive target for therapy. In this study we report the activity profile of the JAK2 inhibitor INCB16562 in a panel of lymphoma cell lines (n=17) including Hodgkin (HL), Mantle Cell (MCL), Anaplastic Large T-cell (ALCL), and DLBCL. JAK-STAT pathway was found to be constitutively active (baseline p-STAT3 Tyr 705 detected by western immunoblotting) in the HL (HDLM-2, L.-428 and L-540), ALCL (SUP-M2, KARPAS-299, SUDHL-1) and the ABC derived DLBCL cell lines (HBL-1, U2932, TMD8, OCI-LY-3). In contrast, no activation of JAK-STAT was observed in the HL cell line KM-H2, in the MCL cell lines (Mino, Jeko-1, SP-53) and in the GCB-derived cell lines (SUDHL-4, SUDHL-6, BJAB). To assess the effect of INCB16562 on cell proliferation, cells were first incubated with increasing concentrations of INC16562 (from 0.1 to 10 μM) for 24, 48 and 72 hours (hrs) and cell viability was evaluated by MTS assay. The IC50 values at 48 hrs ranged from 1 to 9 μM. The ABC derived DLBCL cell line TMD8 showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with INCB16562 1μM for 24 hours. Of note, INCB16562 demostrated activity also in primary DLBCL cells. pSTAT3 negative cell lines were resistant to treatment (IC50 from 4.8 to 6.5 μM). However pretreatment level of pJAKs and pSTATs did not predict sensitivity to INCB16562. In fact inhibition of STAT3 phosphorylation alone was not sufficient to predict sensitivity to the drug in pSTAT3 expressing cells. Simultaneous inhibition of STAT3 and ERK phosphorylation was observed by western immunoblotting in the sensitive cell lines (TMD8, HBL-1, SUP-M2, KARPAS 299, SUDHL-1) (IC50 from 1 to 3 μM), whereas in the resistant cell lines (U2932, OCI-LY3, HD-LM2, L-540, L-428) a paradoxical hyperactivation of pERK was observed after incubation with INCB16562 (IC50 from 5–9 μM). Since it has been reported that sensitivity to JAK inhibition is associated to downregulation of bcl-2 family genes such as Bcl-xL and MCL-1, we investigated the baseline expression of these bcl-2 family members in our cell lines by western blot. Bcl-xL and MCL-1 were expressed at various levels, but there was no correlation between levels of baseline expression and outcome. Both Bcl-xL and MCL-1 were dowregulated in the sensitive cell lines following treatment with INCB16562. Furthermore The bcl-2 inhibitor ABT-737 synergistically enhanced the effect of INCB16562 particularly in HL cell lines (HDLM-2 and L-540) and DLBCL cells lines (HBL-1, TMD8, SUDHL-4). These data demonstrate that INCB16562 has a promising therapeutic value in lymphoma, and provide a rationale for combining INCB16562 with bcl-2 family inhibitors. Disclosures:No relevant conflicts of interest to declare.

Tissue Factor Promoter Haplotype Amplifies Inflammatory Response Via p38-MAPK in Neonatal Compared to Adult Human Microvascular Endothelial Cells

Abstract 1150Age- and tissue-dependent diversity of the vascular endothelium may contribute to the devastating sequelae of inflammation and thrombosis in newborn infants, who often develop multi-organ failure from excessive coagulation-related tissue necrosis. Our previous studies have demonstrated that cytokine and cytoadhesion molecule expression by IL-1- or TNF-α-activated macrovascular endothelial cells (EC) is altered depending upon their vascular origin. Since the microvasculature is especially prone to thrombotic complications of inflammation, we compared expression of several coagulation factors between neonatal and adult human microvascular EC (HMVEC; Lonza) in response to IL-1, and found only tissue factor (TF) was expressed differentially.In addition to the differences in TF expression between neonatal vs. adult HMVEC, we also found high variability among individual neonatal HMVEC. This finding led us to investigate the possible contribution of two polymorphic TF promoter haplotypes reported to play a role in TF gene regulation. Genotyping revealed that neonatal HMVEC homozygous for an 18 base pair (bp) deletion at −1208 (D/D) expressed much higher levels of TF mRNA in response to IL-1 than those homozygous (I/I) or heterozygous (I/D) for the 18 bp insertion, as had also been previously reported for human umbilical vein EC. Moreover, IL-1-stimulated TF mRNA, protein, and activity levels were significantly (p<0.05) higher (4-fold, 2-fold, and 4-fold) in D/D neonatal than D/D adult HMVEC. Importantly, these results suggested that the D/D haplotype induces a uniquely robust response to IL-1 by the TF promoter in neonatal HMVEC.Signal transduction pathway macroarray studies (SABiosciences), to compare IL-1 response, showed a striking Jak-STAT pathway activation in neonatal compared to adult HMVEC. Further analysis found that IL-1 induced a 5.8-fold increase in phosphorylation of p38-MAPK by neonatal HMVEC, significantly greater than the 1.9-fold increase by adult HMVEC (p<0.05). Inhibition of the p38-MAPK pathway with SB203580 eliminated the differential Jak-STAT activation and significantly reduced TF mRNA by 58% vs. 20% (p<0.01) in IL-1-stimulated D/D neonatal vs. adult HMVEC. These results suggest that IL-1 hyperactivation of p38-MAPK, possibly regulated by the Jak-STAT pathway, plays a role in up-regulating D/D neonatal HMVEC TF expression.The following schematic model summarizes the hypothetical response of the neonatal HMVEC TF gene to IL-1: [Display omitted] This model highlights key components of both the p38-MAPK and NF-κB regulatory pathways. It also illustrates hyperactivation of the p38 pathway (shown as thicker arrows) and its potential up-regulation of one, or more, known, or yet-to-be characterized, D/D promoter haplotype-specific transcription factors (shown as hatched arrows) in neonatal HMVEC.This amplified inflammatory response of neonatal HMVEC appears to represent a critical developmental transition between the fetal and adult states of vascular gene regulation and expression, providing a potential basis for the increased frequency and morbidity associated with microvascular thrombosis in susceptible newborns. Disclosures:No relevant conflicts of interest to declare.

Acute Lymphocytic Leukemia and Down Syndrome

Abstract SCI-8 Children with Down syndrome are at a markedly increased risk for acute lymphoblastic leukemia (DS-ALL). These leukemias are exclusively of the B lymphoid precursor phenotype and occur in a similar age to “common” sporadic ALLs with the striking absence of infant ALL. Recent studies reveal that DS-ALLs are heterogeneous and differ from sporadic ALLs. Only about a fifth of DS-ALLs carry the common cytogenetic aberrations typical to childhood ALL. Genomic rearrangements leading to the expression of a cytokine receptor, CRLF2, are detected in 60% of DS-ALL in comparison with up to 10% of sporadic ALLs. CRLF2 heterodimerizes with Interleukin 7 receptor-α (IL7R) to form the receptor to thymic stromal lymphopoietin (TSLP). This receptor is usually present in macrophages, dendritic cells, and some T lymphocytes and participates in allergic and inflammatory processes. The aberrant expression of the TSLP receptor is DS-ALL (and sporadic ALL) is often associated with additional mutations that cause constitutive activation of the downstream JAK-STAT and mTOR growth signaling pathways. These are either lymphoid specific activating mutations of JAK2 or JAK1 or mutations in CRLF2 or IL7R that cause ligand-independent receptor dimerization. The role of the trisomy in selecting these somatic abnormalities is presently unknown. Clinically, the prognosis of DS-ALL is inferior to sporadic ALL mainly because of increased treatment toxicity. However, recent data suggest that the inferior outcome may also be related to the genetic properties of the leukemic cells and that excessive chemotherapy dose reduction may not be appropriate for these patients. Therefore increased vigilance for infectious complications and optimal supportive care are required during periods of intensive chemotherapy. The common activation of the TSLP signaling pathway in DS-ALLs suggests a future for targeted therapy with JAK and/or mTOR inhibitors. Importantly, research of DS-ALL has proven relevant for the general patient population with ALL, as somatic mutations in the TSLP pathway have been discovered in children and adults with sporadic ALL. A major research challenge is the elucidation of the roles of constitutional and somatic trisomy 21 in leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Discovery of Novel Recurrent Mutations in Childhood Early T-Cell Precursor Acute Lymphoblastic Leukemia by Whole Genome Sequencing - a Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project

Abstract 68Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown.Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort.We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1).We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations.Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR.Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL. Disclosures:Evans:St. Jude Children’s research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity’s Board of Directors or advisory committees.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

JAK inhibitors in myeloproliferative neoplasms: Rationale, current data and perspective

JAK-STAT is an appealing but also problematic drug target in BCR-ABL1-negative myeloproliferative neoplasms (MPN) – it is appealing because the majority of patients with MPN harbor gain-of-function JAK2 or MPL mutations – it is problematic because currently available JAK inhibitors do not distinguish between oncogenic and physiologic JAK-STAT activation. Furthermore, JAK-STAT-relevant mutations in MPN do not always constitute the predominant or ancestral mutant clone. Such complexities undermine the value of JAK-STAT as a robust drug target in MPN and partly explain the hitherto lack of histologic or molecular remissions associated with currently available JAK inhibitors. Most of these drugs were, however, effective in alleviating constitutional symptoms and reducing spleen size; the mechanism of action in this instance includes drug-induced down-regulation of inflammatory cytokine activity. In addition, non-specific myelosuppression contributes to both their salutary and detrimental effects on peripheral blood count. Non-hematologic side effects include gastrointestinal disturbances, asymptomatic elevation of liver and pancreatic enzymes, peripheral neuropathy and hyperacute relapse of symptoms during treatment interruption. It is our impression that many more JAK inhibitors need to be evaluated in order to identify the best-in-class in terms of efficacy, toxicity and suitability for future combination treatment programs.

A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway

AbstractJAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

Magu is required for germline stem cell self-renewal through BMP signaling in the Drosophila testis

Understanding how stem cells are maintained in their microenvironment (the niche) is vital for their application in regenerative medicine. Studies of Drosophila male germline stem cells (GSCs) have served as a paradigm in niche-stem cell biology. It is known that the BMP and JAK-STAT pathways are necessary for the maintenance of GSCs in the testis (Kawase et al., 2004; Kiger et al., 2001; Schulz et al., 2004; Shivdasani and Ingham, 2003; Tulina and Matunis, 2001). However, our recent work strongly suggests that BMP signaling is the primary pathway leading to GSC self-renewal (Leatherman and DiNardo, 2010). Here we show that magu controls GSC maintenance by modulating the BMP pathway. We found that magu was specifically expressed from hub cells, and accumulated at the testis tip. Testes from magu mutants exhibited a reduced number of GSCs, yet maintained a normal population of somatic stem cells and hub cells. Additionally, BMP pathway activity was reduced, whereas JAK-STAT activation was retained in mutant testes. Finally, GSC loss caused by the magu mutation could be suppressed by overactivating the BMP pathway in the germline.

Sam68 mediates leptin-stimulated growth by modulating leptin receptor signaling in human trophoblastic JEG-3 cells

Sam68, a member of the signal transduction and activation of RNA metabolism (STAR) family of RNA-binding proteins, has been previously implicated as an adaptor molecule in different signaling systems, including leptin receptor (LEPR) signaling. LEPR activation is known to stimulate JAK-STAT, MAPK and PI3K signaling pathways, thus mediating the biological effects of leptin in different cell types, including trophoblastic cells. We have recently found that leptin stimulation also promotes the overexpression and tyrosine phosphorylation of Sam68 in human trophoblastic JEG-3 cells, suggesting a role for Sam68 in leptin signaling and action in these cells. In the present work, we have studied the participation of Sam68 in the main signaling pathways activated by LEPR to increase growth and proliferation in trophoblastic JEG-3 cells. We used an antisense strategy to down-regulate Sam68 expression in these cells, and we studied LEPR signaling by immunoprecipitation and poly-U affinity precipitation and by analyzing phosphorylation levels of signaling proteins by immunoblot. The effect of leptin on protein synthesis and proliferation was studied by ³[H]-leucine and ³[H]-thymidine incorporation. Sam68 knockdown impaired leptin activation of JAK-STAT, PI3K and MAPK signaling pathways in JEG-3 cells. We have also found that leptin-stimulated Sam68 tyrosine phosphorylation is dependent on JAK-2 activity, since the pharmacological inhibitor AG490 prevents the phosphorylation of Sam68 in JEG-3 cells. Finally, the trophic and proliferative effect of leptin in trophoblastic cells is dependent on Sam68 expression, since its down-regulation impaired the leptin-stimulated DNA and protein synthesis. These data demonstrate that Sam68 participates in the main signaling pathways of LEPR to mediate the trophic and proliferative effect of leptin in human trophoblastic cells.

Abstract PL02-02: The molecular pathology of acute leukemia

Abstract PL02-02: The molecular pathology of acute leukemia

Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells

Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.

When the brakes are lost: LNK dysfunction in mice, men, and myeloproliferative neoplasms

Aberrant JAK-STAT signaling is a hallmark of myeloproliferative neoplasms (MPNs). These hyperproliferative disorders are classically associated with activating mutations in tyrosine kinases such as JAK2 and the thrombopoietin (TPO) receptor MPL. Activation of JAK-STAT signaling and responses to JAK2 inhibitors have been observed in MPN patients lacking JAK2 or MPL mutations, suggesting that other regulatory elements in the JAK-STAT pathway are altered. However, the molecular basis for this observation has been unclear. Recently, the role of inhibitory regulators of JAK-STAT signaling in MPN pathogenesis has been increasingly recognized. LNK is an adaptor protein that forms a negative feedback loop by binding to MPL and JAK2 and inhibiting downstream STAT activation. Murine models indicate that loss of LNK function can promote the development of a MPN phenotype. Several recent studies have identified novel LNK mutations in MPNs, thus validating this notion in humans. These findings represent a novel genetic paradigm of loss of negative feedback regulation of JAK-STAT activation in MPNs and have implications for the future development of targeted therapies in MPNs.

Colonic soluble mediators from the maternal separation model of irritable bowel syndrome activate submucosal neurons via an interleukin-6-dependent mechanism

Irritable bowel syndrome (IBS) is characterized by episodic bouts of abdominal pain, bloating, and altered bowel habit. Accumulating evidence has linked immune activation with IBS, including reports of increases in circulating levels of the proinflammatory cytokine interleukin (IL)-6. However, it is unknown whether IL-6 contributes directly to disease manifestation. As enteric nervous activity mediates motility and secretory function, the aims of this study were to determine the effects of IL-6 on submucosal neurons and related gastrointestinal (GI) function. In these studies, we examined the colons of maternally separated (MS) rats, which exhibit elevated circulating levels of IL-6 in addition to GI dysfunction. To our knowledge, these studies are the first to provide evidence of the sensitivity of submucosal neurons to colonic secretions from MS rats (n = 50, P < 0.05), thus recapitulating clinical biopsy data. Moreover, we demonstrated that the excitatory action is IL-6 dependent. Thereafter, the impact of IL-6 on neuronal and glial activation and absorpto/secretory function was pharmacologically characterized. Other proinflammatory cytokines including IL-8 (n = 30, P > 0.05), IL-1β (n = 56, P > 0.05), and TNF-α (n = 56, P > 0.05) excited fewer neurons. Both muscarinic and nicotinic cholinergic receptors participate in the effect and cause downstream activation of ERK, JAK-STAT, and NF-κB signaling cascades. Functionally, IL-6 increases transepithelial resistance and enhances neurally and cholinergically mediated ion transport. These data provide a role for IL-6 in colonic secretory functions and relate these effects to GI dysfunction in an animal model of IBS, thereby elucidating a potential relationship between circulating levels of IL-6 and aberrant GI function.

Requirement of Lipid Raft Integrity for Signaling by JAK2-V617F

AbstractAbstract 4191JAK2 is a cytoplasmic tyrosine kinase that plays an important role in signaling following activation of various cytokine receptors. JAK2 activation promotes growth, survival, and differentiation of various cell types. Mutation of JAK2 is seen in numerous hematopoietic diseases, most notably in myeloproliferative neoplasms (MPNs). JAK2-V617F is a frequent mutation found in the classical MPNs: polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The single amino acid change of valine to phenylalanine occurs in the pseudokinase domain of JAK2, relieving auto-inhibition of the kinase domain and allowing constitutive kinase activity. Numerous mouse models have demonstrated that JAK2-V617F can induce MPN-like disorders in mice. Thus, this point mutation, as well as other less common JAK2 mutations, is believed to play an important etiologic role in the development of MPNs in humans. The development and use of JAK2 inhibitors in clinical trials has shown promising results, again demonstrating the important role JAK2 plays in MPNs. While the JAK2-V617F mutation, as well as other JAK2 mutations, decreases auto-inhibition of JAK2 kinase activity, it is clear that mutated JAK2 still requires the expression of cytokine receptors to induce activation of transforming signals in hematopoietic cells. Normally, JAK2 binds to homodimeric and heterodimeric cytokine receptors through specific receptor motifs and is activated by various structural changes induced by cytokine stimulation. Following activation it utilizes receptor tyrosines as substrates for phosphorylation, leading to recruitment of downstream signaling molecules, such as STAT5, among others. JAK2 then activates STAT5 via phosphorylation and STAT5 then translocates to the nucleus to regulate transcription of target genes. JAK2-V617F does not require ligand for activation, but still requires the scaffolding function of cytokine receptors to facilitate its full activation and activation of downstream signaling via phosphorylation. Lipid rafts are microdomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have gained appreciation in signal transduction as sites of localization of signaling mediators, including membrane-bound receptors. Congregation of signaling proteins in lipid rafts within the plasma membrane promotes complex formation and signaling cascade activation. We have recently demonstrated that JAK2 is present in lipid rafts during erythropoietin signaling and that lipid raft integrity is required for erythropoietin-mediated signal transduction (Blood 2009, 114: 292). In our current study, we demonstrate that constitutive JAK-STAT signaling driven by JAK2-V617F is sensitive to lipid raft disruption. Human erythroleukemia (HEL) cells express constitutive activation of JAK-STAT signaling due to the presence of JAK2-V617F. Treatment of these cells with methyl-beta-cyclodextrin to disrupt lipid rafts abolished JAK2, STAT5, and STAT3 activation. Similar results are obtained in other cell lines harboring JAK2-V617F and that exhibit JAK-STAT activation that is dependent on this activated form of JAK2. We also demonstrate that JAK2-V617F co-localizes with lipid rafts, as shown by immunofluorescence, and that this co-localization is abolished by lipid raft disruption. This suggests the loss of JAK2-V617F-mediated JAK-STAT activation we observe following lipid raft disruption may be due to an inhibition of properly localized protein complex formation in the plasma membrane that is necessary for JAK2-V617F signaling. Lipid rafts may provide a site for an accumulation of JAK2-V617F-containing signaling complexes and may be necessary for the cellular signals initiated by JAK2-V617F. Our data show JAK2-V617F-driven JAK-STAT pathway activation is vulnerable to lipid raft disrupting agents and suggest lipid raft integrity as a potential therapeutic target for JAK2-V617F positive neoplasms. Targeting lipid rafts in combination with JAK2 kinase inhibitors may allow for more effective kinase inhibition at lower doses, potentially decreasing undesirable side effects associated with kinase inhibitor treatment. Disclosures:No relevant conflicts of interest to declare.

Identification of Novel LNK Mutations In Patients with Chronic Myeloproliferative Neoplasms and Related Disorders

AbstractAbstract 315 Introduction:Dysregulated JAK-STAT signaling in chronic myeloproliferative neoplasms (MPNs) has primarily been attributed to activating mutations in tyrosine kinases. However, JAK-STAT activation can be demonstrated in some patients lacking JAK2 or MPL mutations, suggesting alteration of other regulatory elements in this pathway. One regulator of JAK-STAT signaling is LNK (SH2B3), an adapter protein that contains a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain (plasma membrane localization), and an SH2 domain. LNK binds to cytokine receptors (e.g. MPL, EPOR) and JAK2 via its SH2 domain, inhibiting downstream STAT activation and providing critical negative feedback regulation. LNK-/- mice exhibit features consistent with an MPN phenotype. We recently reported the first human disease-related LNK mutations in two JAK2 V617F-negative MPN patients (Oh et al, Blood, Aug 12, 2010). One patient with primary myelofibrosis (PMF) exhibited a 5 base-pair (bp) deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. A second patient with essential thrombocythemia (ET) was found to have a missense mutation (E208Q) in the PH domain. Both mutations conferred aberrant JAK-STAT signaling in cell lines and primary patient samples, indicating that loss of LNK negative feedback regulation contributes to MPN pathogenesis. We now report the results of a comprehensive screen of a large cohort of MPN, overlap myelodysplastic syndrome (MDS)/MPN, and post-MDS/MPN acute myeloid leukemia (AML) patients for LNK mutations. Methods:A total of 341 samples were sequenced (Table 1; polycythemia vera (PV)=34, erythrocytosis=7, ET=61, PMF=75, post-PV/ET MF=25, MPN-U=7, chronic myelomonocytic leukemia (CMML)=71, juvenile myelomonocytic leukemia=20, MDS/MPN=8, MDS with fibrosis=2, refractory anemia with ring sideroblasts and thrombocytosis=4, idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia=4, systemic mastocytosis=4, and post MDS/MPN AML=19). A deep sequencing approach (Illumina multiplexing system) was used to evaluate 84 samples, in which all exons of LNK were sequenced. For the remainder of the samples, direct sequencing was performed on exon 2, the region containing the previously reported DEL and E208Q mutations. Results:After excluding variants previously reported in SNP databases, a total of 11/341 (3.2%) patients were found to have non-synonymous mutations, including 3/61 (4.9%) ET, 3/75 (4.0%) PMF, and 5/71 (7.0%) CMML patients (Table 1). Each of the mutations localized to exon 2 of LNK, implicating this region as a possible mutational hotspot. This included the aforementioned patients with the DEL and E208Q mutations, which were confirmed by deep sequencing. In two other patients, sequencing of DNA from cultured skin fibroblasts DNA indicated that the mutations were germline. For the remaining seven patients, germline analysis is currently ongoing. In one patient with CMML, a 1 bp deletion leading to a frameshift and premature stop codon was identified (Q72fs). This mutation localized to the Pro/DD, likely resulting in a complete loss of LNK function. Interestingly, this patient who is wild type for the JAK2 and RAS genes, also carries a heterozygous CBL mutation (C396Y), suggesting that LNK and CBL mutations may have cooperative effects. Four patients (one with PMF, three with CMML) were found to have a missense mutation (S186I) at a highly conserved residue in the Pro/DD. The previously reported E208Q mutation was also found in one patient with ET and one patient with CMML. None of the 81 patients known to be JAK2 V617F-positive exhibited somatic LNK mutations, suggesting that LNK mutations may provide an alternative basis for JAK-STAT activation in the absence of JAK2 V617F. Conclusion:Missense and deletion mutations of the LNK gene occur at a low frequency in MPNs and MDS/MPNs and segregate predominantly in exon 2. Further analysis of post-MPN AML samples (represented at a low frequency in the current cohort) and other subtypes of acute and chronic myeloid malignancies is warranted to better characterize the disease spectrum of LNK mutations and whether they are mutually exclusive of JAK2 V617F. We are currently investigating whether loss of negative feedback regulation of JAK-STAT signaling is related to haploinsufficiency of LNK or dominant negative effects of the mutant protein. [Display omitted] Disclosures:No relevant conflicts of interest to declare.