Activation Of STAT5 Research Articles

Mechanisms of Ikaros-Mediated Tumor Suppression

Abstract 408 Background:The Ikaros (IKZF1) tumor suppressor is deleted in >80% of the cases of Ph+ ALL. While Ikaros cooperates with pre-B cell receptor signaling to induce cell cycle exit in Ph+ ALL (Trageser et al., J Exp Med, 2009), the mechanism of Ikaros-mediated tumor suppression is poorly understood. Here we report on a series of genetic experiments that show that Ikaros (i) interferes with key survival pathways downstream of the BCR-ABL1 kinase, (ii) inhibits leukemia cell proliferation through interaction with the pre-B cell receptor signaling pathway and (iii) activates the tumor suppressors p53, p21 and p27. Results:To elucidate the mechanism of Ikaros-dependent tumor suppression in BCR-ABL1-driven B cell lineage leukemia, we studied regulation of critical phosphorylation events downstream of the BCR-ABL1 kinase as a central mediators of survival and proliferation. Reconstitution of Ikaros expression in BCR-ABL1-transformed pre-B ALL cells resulted in rapid and global dephosphorylation comparable to the effect of Imatinib. A detailed analysis showed that Ikaros-induced dephosphorylation events affect activation of Stat5 (Y694), AKT (S473), ERK1/2 (T202 and Y204) and SRC (Y416). Interestingly, both Imatinib-treatment and reconstitution of pre-B cell receptor signaling using retroviral vectors for expression of the m heavy chain or the BLNK adapter molecule have the same effects as reconstitution of Ikaros. In fact, a comprehensive gene expression analysis demonstrated that Ikaros reconstitution resulted in similar gene expression changes as reconstitution of pre-B cell receptor signaling (m heavy chain or BLNK), reconstitution of PAX5, Cre-mediated deletion of Stat5 or Myc, or treatment with Imatinib. The signature of common gene expression changes shared between reconstitution of Ikaros, Pax5, m heavy chain, BLNK and inducible deletion of Stat5 or Myc and Imatinib-treatment involves known tumor suppressors including SPIB, BTG1, and BTG2. These findings suggest that reconstitution of tumor suppressive transcription factor (Ikaros, Pax5) converges with pre-B cell receptor-mediated tumor suppression. To better understand how pre-B cell receptor signaling and Ikaros intersect, we combined reconstitution of Ikaros with genetic deletion of either the (more proximal) SYK kinase or the (more distal) BLNK adapter molecule. While inducible Cre-mediated deletion of Syk had no effect on Ikaros-mediated tumor suppression, deletion of the BLNK adapter compromised the ability of Ikaros to function as tumor suppressor. These findings were confirmed in an in vivo transplantation experiment. While mice transplanted with Ikaros+ BLNK+ leukemia cells survived indefinitely, mice transplanted with Ikaros- BLNK+, Ikaros+ BLNK- or Ikaros- BLNK- leukemia cells died after 24 to 31 days post transplantation.While these findings provide genetic evidence for collaboration between the Ikaros and pre-B cell receptor tumor suppressor pathways, Ikaros and pre-B cell receptor signaling differ with respect to activation of classical tumor suppressor pathways. While reconstitution of pre-B cell receptor signaling failed to activate Arf, p53 or p27, protein levels of all these molecules were strongly upregulated by Ikaros. In agreement with these findings, reconstitution of pre-B cell receptor signaling had the same tumor suppressive effect in wildtype leukemia cells as in Arf−/−, p53−/− as well as p27−/− leukemia cells. Conversely, deletion of Arf and p53 significantly diminished the ability of Ikaros to function as tumor suppressor. Conclusion:Ikaros deletion represents a near-obligatory lesion in the pathogenesis of Ph+ ALL. Here we provide genetic evidence for three novel pathways of Ikaros-mediated tumor suppression. Like PAX5, Ikaros reconstitution results in multiple dephosphorylation events (Stat5, AKT, ERK1/2 and SRC are affected). In collaboration with the pre-B cell receptor and its downstream adapter molecule BLNK, Ikaros suppressed MYC and inhibits cell cycle progression. Induction of the Arf/p53 pathway represents a distinct function of Ikaros, which is not shared with the pre-B cell receptor signaling pathway. Disclosures:No relevant conflicts of interest to declare.

Generation of a Transgenic Mouse with Inducible Constitutively Active Stat5a,

Abstract 3399The Jak/Stat signalling pathway is essential for survival and proliferation of haematopoietic cells. The Stat5 transcription factors (Stat5a and Stat5b) play a crucial role in the development of various lineages of the hematopoietic system. Stat5 is activated by many cytokines and growth factors that regulate hematopoiesis. Persistent Stat5 activity is frequently found in hematopoietic cancers due to aberrant tyrosine kinase signalling. We have previously characterized a constitutively activated version of Stat5 (called oncogenic Stat5; cS5F), which mimics persistent tyrosine kinase signalling and promotes multi-lineage leukaemia development in a bone marrow transplantation model.The aim of the study is to generate a novel inducible mouse model for oncogenic Stat5a using the BAC recombineering technology. A cassette expressing cS5F (with a C-terminal FLAG tag) and a reporter gene (truncated human CD2; hCD2) was cloned, sequenced and biochemically verified for persistent tyrosine phosphorylation in absence of cytokines or growth factors. The cassette is flanked by loxP sites in opposite orientation and was used to replace the original Stat5a gene in a BAC carrying the endogenous Stat3/5 locus using BAC recombineering. Therefore, the expression of the transgene is regulated by the endogenous Stat5a promoter. The transgene is in an inverted (off) orientation and can be switched into the transcribing (on) orientation by Cre activity. [Display omitted] Pronuclear injections were performed with the linearized and purified BAC. We obtained 5 transgenic founder lines which showed germ line transmission of the transgene. The copy number of the BAC was estimated to be 2 by Southern blot analysis. The transgenic founder lines were bred with the Rosa CreERT2 mice, and the Cre activity was induced by treating the mice with 1 mg of tamoxifen every day, for 5 consecutive days. The recombination of the transgene into the ‘on’ orientation in liver, lungs, kidney, spleen and bone marrow was demonstrated by Southern blotting. The expression of the hCD2 marker was detected in various hematopoietic lineages by FACS and the expression of the transgenic protein in liver was confirmed by Western blotting with anti-Stat5a and anti-FLAG antibodies.One week after induction of the transgene, all the mice induced with tamoxifen (n=6) developed atrophic thymii with nearly a 4-fold reduction in total thymocyte numbers (p=0.0003). However, the percentage of CD8+ cells was increased more than 3 fold in the thymus (p=0.0002). Moreover, there was a 2-fold reduction in the number of early hematopoietic progenitors; defined as lin−, Sca1+ and c-kit+, in the bone marrow (n=5, p=0.0217).We have established an inducible mouse model for expression of constitutively active Stat5a under the endogenous promoter. We are currently monitoring these mice for development of cancer as they provide a competent tool to study the molecular mechanisms triggered by persistent Stat5a activity, including identification of other cooperating signalling pathways that contribute to generation of cancer. Moreover, it would allow one to test interference strategies, which could lead to potential therapeutics. Disclosures:No relevant conflicts of interest to declare.

Leptin produced by joint white adipose tissue induces cartilage degradation via upregulation and activation of matrix metalloproteinases

ObjectivesTo investigate the effect of leptin on cartilage destruction.MethodsCollagen release was assessed in bovine cartilage explant cultures, while collagenolytic and gelatinolytic activities in culture supernatants were determined by bioassay and...

Cardioplegia and Angiotensin II Receptor Antagonists Modulate Signal Transducers and Activators of Transcription Activation in Neonatal Rat Myocytes

Previous investigations have shown that the signal transducers and activators of transcription (STATs) signaling pathway play an important role in the modulation of apoptosis after ischemia and reperfusion. The mechanism for this enhanced cardioprotection is unknown, but we believe that alterations STATs may play a role. To investigate this hypothesis, we examined the effects of angiotension II type 1 (AT1) and angiotension II type 2 (AT2) receptor antagonist added to cardioplegia on the downstream response of different STATs, connected with proinflammatory pathways (STAT2, STAT5) and prohypertrophic and antiapoptotic pathways (STAT3). Isolated, nonworking hearts (n = 3 per group) from neonatal rats were perfused aerobically (4°C) for 20 min in the Langendorff mode with the modified St. Thomas' Hospital no. 2 (MSTH2) cardioplegic solution (Group 1), the MSTH2 cardioplegic solution + AT1 receptor antagonist (Group 2), and MSTH2 cardioplegic solution + AT2 receptor antagonist (Group 3). Thus, myocytes were isolated by enzymatic digestion, and STAT2, STAT3, and STAT5 were investigated in Western blot studies. Times to arrest after cardioplegia were 8-12 s for all groups. Total cardioplegia delivery volume was about 300 mL for the 20 min. Perfusion with the MSTH2 cardioplegic solution supplemented with AT1 receptor antagonist (Group 2) induced a significant reduction in STAT2 and STAT5 tyrosine phosphorylation (-58 and -63%, respectively, vs. Group 1, P < 0.05). Conversely, STAT2 and STAT5 activation were unaffected by perfusion with the MSTH2 cardioplegic solution supplemented with AT2 receptor antagonist (Group 3). The decreased activation of STAT2 and STAT5 observed in Group 2 was accompanied by reduction of interleukin-1β (-57% in Group 2 vs. Group 1, P < 0.05). There were no significant differences in STAT3 phosphorylation among all groups. Only the addition of AT1 receptor antagonist to MSTH2 cardioplegia significantly decreases the inflammatory response of the neonatal rat cardiomyocytes without affecting antiapoptotic influence provided by tyrosine phosphorylation of STAT3. AT1 receptor antagonist added to cardioplegia represents an additional modality for enhancing myocardial protection during cardiac surgery and could contribute to optimize the ischemia tolerance of the pediatric heart.

Negative Cross Talk between NFAT1 and Stat5 Signaling in Breast Cancer

The molecular mechanisms that modulate the activity of the signal transducers and activators of transcription 5 (Stat5) during the progression of breast cancer remain elusive. Here, we present evidence that the calcineurin/nuclear factor of activated T cells (NFAT) pathway negatively regulates the activation of Stat5, and vice versa in breast cancer. NFAT1 interacts with Stat5 in breast cancer cells, and their physical association is mediated by the DNA binding and transactivation domains of Stat5. Ectopically expressed NFAT1 is capable of inhibiting Stat5-dependent functions, including Stat5 transactivation, Stat5-mediated transcription of the downstream target gene expression, and binding of Stat5a to the Stat5 target promoter. By contrast, overexpression of a selective NFAT inhibitor VIVIT reversed NFAT1-mediated suppression of Stat5-dependent gene expression, whereas silencing of NFAT1 through RNA interference enhanced prolactin-induced, Stat5-mediated gene transcription, and breast cancer cell proliferation. A reciprocal inhibitory effect of Stat5 activity on NFAT1 signaling was also observed, implying these two signaling cascades antagonize each other in breast cancer. Importantly, analysis of a matched breast cancer progression tissue microarray revealed a negative correlation between levels of NFAT1 and Stat5 (pY694) during the progression of breast cancer. Taken together, these studies highlight a novel negative cross talk between the NFAT1- and Stat5-signaling cascades that may affect breast tumor formation, growth, and metastasis.

Opposed regulation of type I IFN‐induced STAT3 and ISGF3 transcriptional activities by histone deacetylases (HDACS) 1 and 2

The antiviral and antiproliferative responses mediated by type I interferons (IFNs) depend on JAK/STAT signaling and ISGF3 (STAT1:STAT2:IRF9)-dependent transcription. In addition, type I IFNs stimulate STAT3 activation in many cell types, an event generally associated with cell cycle progression, survival, and proliferation. To gather more insight into this functionally contradictive phenomenon, we studied the regulation of STAT3 transcriptional activity upon type I IFN treatment. We show that IFNα2 stimulation strongly induces STAT3 phosphorylation, nuclear translocation, and promoter binding, yet the activation of transcription of a STAT3-dependent reporter and endogenous genes, such as SOCS3 and c-FOS, is impaired. Simultaneous treatment with IFNα2 and trichostatin A, as well as combined HDAC1/HDAC2 silencing, restores STAT3-dependent reporter gene and endogenous gene expression, strongly suggesting that HDAC1 and HDAC2 are directly involved in repressing IFNα2-activated STAT3. Of note, single silencing of only one of the two HDACs does not lead to enhanced STAT3 activity, supporting a functional redundancy between these two enzymes. In sharp contrast, HDAC1 and HDAC2 activities are required for ISGF3-dependent gene expression. We conclude that HDAC1 and HDAC2 differentially modulate STAT activity in response to IFNα2: while they are required for the induction of ISGF3-responsive genes, they impair the transcription of STAT3-dependent genes.

Short Stat5-Interacting Peptide Derived from Phospholipase C-β3 Inhibits Hematopoietic Cell Proliferation and Myeloid Differentiation

Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.

Screening for serine/threonine kinases phosphorylating Stat5

Background Stat transcription factors are highly conserved key regulators of cellular processes such as proliferation, differentiation, growth and apoptosis. Stats have been frequently found to be deregulated in cancer. One example is the constitutive activation of Stat5 in chronic myelogenous leukaemia (CML). The disease is caused by a chromosomal translocation resulting in the expression of the Bcr/ Abl fusion kinase. The transcription factor Stat5 is required for Bcr/Abl-induced leukaemic initiation and disease progression. Furthermore, serine phosphorylation of a constitutive active form of Stat5 is crucial for haematopoietic transformation. These findings put Stat5 into the spotlight of new therapeutic tactics. Here, we screen for kinases phosphorylating Stat5 by employing a cell viability-based screening approach.

STAT5A Expression in Swiss 3T3 Cells Promotes Adipogenesis In Vivo in an Athymic Mice Model System

Many studies from our laboratories and others have shown that STAT5 expression and activity are increased during adipogenesis of murine and human adipocytes. Ectopic expression of STAT5A in fibroblasts or preadipocytes can confer or enhance adipogenesis. To determine whether STAT5A also plays a role in adipogenesis in vivo, we injected athymic mice with Swiss 3T3 cells expressing an empty pBABE retrovirus, Swiss cells expressing a pBABE retrovirus-containing STAT5A, or 3T3-F442A preadipocytes. Athymic mice injected with either 3T3-F442A cells or Swiss 3T3 cells expressing STAT5A resulted in fat pad formation at the site of injection. However, mice injected with Swiss cells containing the parent retroviral vector did not have any observable fat pads. An analysis of the ectopic fat pads obtained from the Swiss 3T3 STAT5A mice revealed abundant expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and adiponectin. The protein levels of both of these fat cell markers were comparable to expression levels in epididymal fat pads. These results demonstrate that STAT5A can promote adipogenesis in vivo in this model system which supports a role of this transcription factor in adipocyte development in the whole animal.

Inducible and repressable oncogene-addicted hepatocellular carcinoma in Tet-on xmrk transgenic zebrafish

Liver cancer, mainly hepatocellular carcinoma, is a major malignancy and currently there are no effective treatment protocols due to insufficient understanding of hepatocarcinogenesis. As a potentially high-throughput and cost-effective experimental model, the zebrafish is increasingly recognized for disease studies. Here, we aim at using the zebrafish to generate a convenient hepatocellular carcinoma model. Using the Tet-on system for liver-specific expression of fish oncogene xmrk, a hyperactive version of epidermal growth factor receptor homolog, we have generated transgenic zebrafish with inducible development of liver cancer. Liver tumors were rapidly induced with 100% penetrance in both juvenile and adult xmrk transgenic fish. Histological examination indicated that they all showed features of hepatocellular carcinoma. The induced liver tumors regressed rapidly upon inducer withdrawal. During the tumor induction stage, we detected increased cell proliferation and activation of Xmrk downstream targets Erk and Stat5, which were important for liver tumorigenesis as proved by inhibition experiments. When tumors regressed, there were decreased phosphorylated Erk and Stat5 accompanied with an increase in apoptosis. Our zebrafish model demonstrates the potential of a hyperactivated epidermal growth factor receptor pathway in initiating heptocarcinogenesis. It provides clear evidence for the requirement of only a single oncogene for HCC initiation and maintenance and is thus a convenient model for further investigation of oncogene addiction and future anti-cancer drug screening.

A nonsynonymous LNK polymorphism associated with idiopathic erythrocytosis

AbstractNo abtract.

MiRNA-mediated feedback inhibition of JAK/STAT morphogen signalling establishes a cell fate threshold

Patterns of cell fates generated by morphogens are critically important for normal development; however, the mechanisms by which graded morphogen signals are converted into all-or-none cell fate responses are incompletely understood. In the Drosophila ovary, high and sustained levels of the secreted morphogen Unpaired (Upd) specify the migratory border-cell population by activating the signal transducer and activator of transcription (STAT). A lower or transient level of STAT activity specifies a non-migratory population of follicle cells. Here we identify miR-279 as a component of a feedback pathway that further dampens the response in cells with low levels of JAK/STAT activity. miR-279 directly repressed STAT, and loss of miR-279 mimicked STAT gain-of-function or loss of Apontic (Apt), a known feedback inhibitor of STAT. Apt was essential for miR-279 expression in non-migratory follicle cells, whereas another STAT target, Ken and Barbie (Ken), downregulated miR-279 in border cells. Mathematical modelling and simulations of this regulatory circuit including miR-279, Apt and Ken supported key roles for miR-279 and Apt in generating threshold responses to the Upd gradient.

Progesterone Promotes Differentiation of Human Cord Blood Fetal T Cells into T Regulatory Cells but Suppresses Their Differentiation into Th17 Cells

Progesterone, a key female sex hormone with pleiotropic functions in maintenance of pregnancy, has profound effects on regulation of immune responses. We report in this work a novel function of progesterone in regulation of naive cord blood (CB) fetal T cell differentiation into key T regulatory cell (Treg) subsets. Progesterone drives allogeneic activation-induced differentiation of CB naive, but not adult peripheral blood, T cells into immune-suppressive Tregs, many of which express FoxP3. Compared with those induced in the absence of progesterone, the FoxP3(+) T cells induced in the presence of progesterone highly expressed memory T cell markers. In this regard, the Treg compartment in progesterone-rich CB is enriched with memory-type FoxP3(+) T cells. Moreover, CB APCs were more efficient than their peripheral blood counterparts in inducing FoxP3(+) T cells. Another related function of progesterone that we discovered was to suppress the differentiation of CB CD4(+) T cells into inflammation-associated Th17 cells. Progesterone enhanced activation of STAT5 in response to IL-2, whereas it decreased STAT3 activation in response to IL-6, which is in line with the selective activity of progesterone in generation of Tregs versus Th17 cells. Additionally, progesterone has a suppressive function on the expression of the IL-6 receptor by T cells. The results identified a novel role of progesterone in regulation of fetal T cell differentiation for promotion of immune tolerance.

Prolactin increases SMN expression and survival in a mouse model of severe spinal muscular atrophy via the STAT5 pathway

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease that is characterized by the loss of motor neurons, resulting in progressive muscle atrophy. It is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. A potential treatment strategy for SMA is to upregulate levels of SMN protein. Several agents that activate STAT5 in human and mouse cell lines enhance SMN expression from the SMN2 gene and can compensate, at least in part, for the loss of production of a functional protein from SMN1. Here, we have shown that prolactin (PRL) increases SMN levels via activation of the STAT5 pathway. PRL increased SMN mRNA and protein levels in cultured human and mouse neuronal cells. Administration of STAT5-specific siRNA blocked the effects of PRL, indicating that the PRL-induced transcriptional upregulation of the SMN-encoding gene was mediated by activation of STAT5. Furthermore, systemic administration of PRL to WT mice induced SMN expression in the brain and spinal cord. Critically, PRL treatment increased SMN levels, improved motor function, and enhanced survival in a mouse model of severe SMA. Our results confirm earlier work suggesting STAT5 pathway activators as potential therapeutic compounds for the treatment of SMA and identify PRL as one such promising agent.

Multidimensional Single Cell Based STAT Phosphorylation Profiling Identifies a Novel Biosignature for Evaluation of Systemic Lupus Erythematosus Activity

IntroductionDysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE), a complex autoimmune disease. Comprehensively quantifying basal STATs phosphorylation and their signaling response to cytokines should help us to better understand the etiology of SLE.MethodsPhospho-specific flow cytometry was used to measure the basal STAT signaling activation in three immune cell types of peripheral-blood mononuclear cells from 20 lupus patients, 9 rheumatoid arthritis (RA) patients and 13 healthy donors (HDs). A panel of 27 cytokines, including inflammatory cytokines, was measured with Bio-Plex™ Human Cytokine Assays. Serum Prolactin levels were measured with an immunoradiometric assay. STAT signaling responses to inflammatory cytokines (interferon α [IFNα], IFNγ, interleukin 2 [IL2], IL6, and IL10) were also monitored.ResultsWe observed the basal activation of STAT3 in SLE T cells and monocytes, and the basal activation of STAT5 in SLE T cells and B cells. The SLE samples clustered into two main groups, which were associated with the SLE Disease Activity Index 2000, their erythrocyte sedimentation rate, and their hydroxychloroquine use. The phosphorylation of STAT5 in B cells was associated with cytokines IL2, granulocyte colony-stimulating factor (G-CSF), and IFNγ, whereas serum prolactin affected STAT5 activation in T cells. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the STAT1 response to IFNα in monocytes. The response of STAT3 to IL6 was reduced in SLE T cells.ConclusionsThe basal activation of STATs signaling and reduced response to cytokines may be helpful us to identify the activity and severity of SLE.

IL-15 and dendritic cells induce proliferation of CD4+CD25+ regulatory T cells from peripheral blood

CD4+CD25+ regulatory T cells (Tregs) have recently been the subject of intense research due to their strong immunosuppressive effect. Increasing evidence suggests that IL-15 plays an important role in Tregs biology. Nevertheless, the mechanism by which IL-15 performs this function remains to be fully elucidated. To address this question, we isolated Tregs from human peripheral blood, and utilized IL-15, dendritic cells (DCs), or DCs combined with IL-15, to examine the proliferation of Tregs and to explore related molecular mechanisms. Here, we show that IL-15 can induce the proliferation of Tregs in the presence of DCs. The induction is mediated by DCs presenting IL-15 in trans to Tregs. Simultaneously, DCs-derived IL-2, regulated by IL-15, may also play a supportive role. After IL-15 withdrawal, IL-15 trans-endosomal recycling in DCs contributes to the proliferation of Tregs. The activation of Akt, Erk1/2 and STAT5, and the degradation of p27kip1 may be involved in this process. These findings might explain the proliferation of Tregs in the absence of IL-2 in vivo and provide a novel method to achieve large-scale proliferation of Tregs in vitro in order to obtain cell numbers sufficient for immunotherapy.

R723, a selective JAK2 inhibitor, effectively treats JAK2V617F-induced murine myeloproliferative neoplasm

AbstractThe activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.

Tight Control of STAT5 Activity Determines Human CD34-Derived Interstitial Dendritic Cell and Langerhans Cell Development

Despite the crucial function of dendritic cells (DC) in immunity, the molecular mechanisms regulating human DC development remain poorly defined. STAT5 regulates various hematopoietic lineages and is activated by GM-CSF, a critical cytokine in DC development. In this study, we investigated the role of STAT5 during differentiation of human CD34(+) hematopoietic progenitors into precursor DC (pre-DC) and their subsequent differentiation toward interstitial DC and Langerhans cells. Inhibiting STAT5 activity by dominant-negative STAT5 promoted Langerhans cell commitment of hematopoietic progenitors but resulted in loss of pre-interstitial DC development, showing subset-specific regulation. Increasing the low endogenous STAT5 activity by ectopic STAT5 activation downregulated expression of the critical DC transcription factor PU.1 and abrogated commitment to either DC lineage. In contrast, high STAT5 activity was beneficial in already committed pre-DC: terminal DC differentiation was associated with increased endogenous STAT5 phosphorylation levels, JAK2-STAT5 inhibition reduced terminal DC differentiation, and conditional STAT5 activation in pre-DC improved development of BDCA-1(+), DC-SIGN(+), and Langerin(+) DC with normal maturation and T cell stimulation. These data show that STAT5 critically regulates human DC development, with specific requirements for the level of STAT5 activation at distinct differentiation stages. By regulating STAT5 activity, cytokines present at specific locations and under different pathophysiological conditions can determine the fate of DC precursors.

Selection of targeted agent(s) based on matching differential protein expression and mutational profile in NSCLC patients.

e21085 Background: Treatment of nonsmall-cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs) shows initial response, but most tumors develop acquired resistance to TKIs due to secondary resistance mutations (T790M) and amplification of other RTKs. Patients stratified for mono therapy treatment based on genotype alone is not sufficient as other kinases contribute to survival and necessitate inhibition of multiple kinases. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. CEER is capable of high detection sensitivity from FNA/CTC viable samples (23-35 gage needle). Lysates were prepared from frozen tissues obtained surgically from 71 Caucasian and 29 Asian patients with NSCLC. Expression and activation of EGFR, ErbB2, ErbB3, cMET, IGF1R, cKIT, Shc, PI3K, AKT, ERK, VEGFR2, Src, FAK, Stat5, JAK2, CrKL, and other pathway proteins were profiled. Genotyping on panel of mutations in KRAS, EGFR, and BRAF were performed on all samples. Results: We have observed differential expression patterns between Asian and Caucasian patients. KRAS is mutated in 30% of all the patients. In Asians, EGFR over expression is higher (27%) compared to the Caucasians (3%). Caucasian patients expressed high Her3 (58%), cMET (25%), and co expression of both cMET and HER3 (27%). VEGFR2 is highly expressed in 17% of all patients. Patients over expressing cMET, HER3, and HER2 will benefit from combination of pan-HER and cMET inhibitors. Those over expressing cMET and EGFR will benefit from cMET and EGFR inhibitors. Lastly, patients over expressing VEGFR2 and cMET will benefit from cMET and VEGFR inhibitors. A comprehensive differential biomarker prevalence for each NSCLC sub-populations for all tested RTKs and signaling proteins will be presented. Conclusions: Mono therapy and patient selection based on genotype is not an effective criteria for treatment options. Instead, a comprehensive pathway profile using FNA/CTC should guide selection of appropriate therapy (combined or sequenced), and shifts in pathway profile should be monitored for appropriate response.

Interaction between FGFR-2, STAT5, and Progesterone Receptors in Breast Cancer

Fibroblast growth factor (FGF) receptor 2 (FGFR-2) polymorphisms have been associated with an increase in estrogen receptor and progesterone receptor (PR)-positive breast cancer risk; however, a clear mechanistic association between FGFR-2 and steroid hormone receptors remains elusive. In previous works, we have shown a cross talk between FGF2 and progestins in mouse mammary carcinomas. To investigate the mechanisms underlying these interactions and to validate our findings in a human setting, we have used T47D human breast cancer cells and human cancer tissue samples. We showed that medroxyprogesterone acetate (MPA) and FGF2 induced cell proliferation and activation of ERK, AKT, and STAT5 in T47D and in murine C4-HI cells. Nuclear interaction between PR, FGFR-2, and STAT5 after MPA and FGF2 treatment was also showed by confocal microscopy and immunoprecipitation. This effect was associated with increased transcription of PRE and/or GAS reporter genes, and of PR/STAT5-regulated genes and proteins. Two antiprogestins and the FGFR inhibitor PD173074, specifically blocked the effects induced by FGF2 or MPA respectively. The presence of PR/FGFR-2/STAT5 complexes bound to the PRE probe was corroborated by using NoShift transcription and chromatin immunoprecipitation of the MYC promoter. Additionally, we showed that T47D cells stably transfected with constitutively active FGFR-2 gave rise to invasive carcinomas when transplanted into NOD/SCID mice. Nuclear colocalization between PR and FGFR-2/STAT5 was also observed in human breast cancer tissues. This study represents the first demonstration of a nuclear interaction between FGFR-2 and STAT5, as PR coactivators at the DNA progesterone responsive elements, suggesting that FGFRs are valid therapeutic targets for human breast cancer treatment.