Activation Of STAT5 Research Articles

Neuromedin U Peptide Activates STAT5 and S6 in a JAK-2 Dependent Manner and Promotes Erythroid Cell Growth in Primary Erythroid Progenitor Cells

AbstractAbstract 1241Erythropoiesis is a multi-step process during which hematopoietic stem cells terminally differentiate into red blood cells (RBCs). Erythropoietin (EPO) is the only known cytokine regulator of terminal erythroid differentiation. Previously, we reported that the neuropeptide, neuromedin U (NmU), which interacts with NmU receptor type 1 (NMUR1), functions as a novel extracellular cofactor with EPO to promote the expansion of early erythroblasts, which are CD34−, CD71+, glycophorin A (GlyA)dim(Gambone et al, Blood. 2011). Here, we describe studies to understand the mechanism whereby NmU augments EPO effects on erythroid cell growth. EPO triggers Janus kinase (Jak)-2 dependent activation of signal transducer and activator of transcription (STAT) 5 and phosphatidylinositol 3-kinase (PI3K) to promote the proliferation and/or survival of erythroid progenitor cells. We hypothesized that NmU peptide would cooperate with EPO to promote the proliferation of early erythroblasts through STAT5 and/or PI3K activation. To address this hypothesis, we cultured primary human CD34+ cells in 2-stage liquid culture with IL-3, IL-6, and stem cell factor (SCF) from day 0 to day 6. On day 6, 2U/mL of EPO was added, and the cells were cultured for an additional 5 days to expand erythroid progenitors. On day 11, cells were briefly serum starved and then stimulated with EPO and/or NmU in the absence or presence of a Jak-1/2 inhibitor. Activation of STAT5 and S6, a surrogate marker for PI3K activation, were assessed by phospho-flow in ERY3 (CD34−, CD71+, GlyA+) and ERY4 (CD34−, CD71dim, GlyA+) cells. As expected, EPO alone activated STAT5 and S6 in ERY3 cells only, and the presence of a Jak-1/2 inhibitor diminished STAT5 activation. Interestingly, STAT5 and S6 were activated by NmU peptide alone in ERY3 and ERY4. Surprisingly, in the presence of a Jak-1/2 inhibitor, NmU peptide, which binds to NMUR1 a G-protein coupled receptor, did not activate STAT5 or S6 in ERY3 or 4 cells, suggesting that NmU functions through a JAK kinase in erythroid cells. No additive or synergistic activation of STAT5 and S6 is observed in the presence of both EPO and NmU peptide when EPO was used at a dose of 2 U/mL. The mechanism whereby NmU activates a JAK dependent signaling pathway is under investigation. Preliminary evidence suggests that EPO induces the physical association of NMUR1 with EPO receptor (EPOR). Taken together, we propose that NmU is a neuropeptide expressed in bone marrow cells that cooperates to regulate erythroid expansion during early erythropoiesis through the activation of cytokine receptor like signaling pathways and perhaps through direct interaction with EPOR. NmU may be useful in the clinical management of anemia in patients unresponsive to EPO or other erythroid-stimulating agents. Disclosures:No relevant conflicts of interest to declare.

Targeting STAT5 in Leukemia Through Inhibition of Bromodomain Proteins

Abstract 399 Introduction:The transcription factor STAT5 is constitutively activated in many forms of hematologic malignancies, including chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). STAT5 can be activated by constitutively activated tyrosine kinases or autocrine and paracrine secretion of cytokines signaling through Jak kinases. STAT5 is essential for the pathogenesis of neoplasms induced by BCR-ABL1 and Jak2V617F, as well as for leukemia stem cell self-renewal. Development of tyrosine kinases inhibitors (TKIs), such as imatinib, has greatly improved the outcome of patients with leukemias harboring aberrantly activated oncogenic tyrosine kinases. However, TKIs used as a single agent only achieve significant success in CML, with very limited benefit in the more aggressive ALL. Moreover, patients with CML who initially respond well may acquire resistance to TKIs with the progression of their disease. In fact, increased activity of STAT5 is often associated with CML progression and may underlie resistance to TKIs. Importantly, leukemia cells that are resistant to TKIs remain sensitive to STAT5 inhibition, and dual inhibition of both tyrosine kinases and STAT5 leads to more efficient reduction of leukemia cell viability. Thus targeting STAT5 alone or in combination is a promising therapeutic strategy for many hematological malignancies. While many strategies directly inhibit STAT5, we considered the possibility that STAT5 association with co-regulatory proteins is essential for STAT5 function and therefore targeting this association may be a suitable therapeutic strategy. Methods and Results:Given the importance of BET bromodomain proteins in chromatin remodeling necessary for transcription, we tested the activity of the BET bromodomain inhibitor JQ1 on STAT5-dependent transcriptional activity. Using both heterologous reporter systems and endogenous STAT5 target genes, we found that JQ1, but not its inactive enantiomer, potently and specifically inhibited STAT5-dependent gene expression. Inhibition of STAT5 dependent gene regulation was also replicated by another BET bromodomian inhibitor, iBET, further demonstrating that BET inhibition inhibits STAT5. Since JQ1 inhibits BET family members Brd2, Brd3, Brd4, and BrdT, we asked which BET family member is specifically associated with STAT5 transcriptional function. To do this, we utilized shRNA to knock-down each bromodomain protein and determined the effect on STAT5 activity. We found that knocking-down Brd2, but not Brd3 or Brd4, reduces STAT5 target gene expression, indicating that Brd2 is specifically involved in regulating STAT5 transcriptional function. JQ1 can reduce STAT5 transcriptional activity without inhibiting STAT5 phosphorylation or STAT5 binding to its genomic binding sites. Similarly, knocking-down Brd2 can reduce STAT5 target gene expression without influencing STAT5 phosphorylation. We hypothesize that Brd2 regulates STAT5 transcriptional function by acting as a co-activator for STAT5. Thus through blocking Brd2, JQ1 can inhibit STAT5 transcriptional function without directly targeting STAT5 itself. In a group of aggressive T cell acute lymphoblastic leukemia (T-ALL) cell lines, where constitutively activated STAT5 contributes to leukemia cell survival, knocking-down Brd2 renders leukemia cells more sensitive to TKI induced apoptosis. In addition, combined treatment with TKIs and JQ1 showed strong synergy in inducing T-ALL leukemia cells apoptosis and reducing viability. Overexpressing a constitutively active form of STAT5 rescues these leukemia cells from death induced by TKIs and JQ1, indicating an important role of STAT5 as a target for TKI and JQ1 induced cell death in T-ALL cells. Conclusion:We found that the BET bromodomain inhibitor JQ1 can reduce STAT5 transcriptional function by blocking Brd2 without reducing STAT5 phosphorylation or STAT5 DNA binding. In addition, the combination of TKIs and JQ1 induces T-ALL leukemia cell apoptosis and reduces survival in a synergistic manner, and represents a rational drug combination for treating this sub-group of highly aggressive leukemias. Disclosures:Bradner:Tensha Therapeutics: Consultancy, Equity Ownership, Scientific founder Other.

Effects of Ponatinib and Other Novel TKI On Growth, Survival, and Function of Neoplastic Eosinophils Carrying FIP1L1/Pdgfra

AbstractAbstract 1760In chronic eosinophilic leukemia (CEL), the transforming oncoprotein FIP1L1-PDGFRA (F/P) is a major target of therapy. In most patients, the PDGFRA-targeting tyrosine kinase inhibitor (TKI) imatinib induces complete and durable molecular remissions. For patients who are intolerant or resistant against imatinib, novel TKI may serve as potential alternative therapy. Indeed, several different TKI have been described to act on Ba/F3 cells transfected with F/P, and some even block the activity of imatinib-resistant F/P mutants. However, little is known about the effects of novel TKI on growth and survival of primary neoplastic eosinophils. In the current study, we examined the in vitro effects of 12 kinase blockers on growth and viability as well as cytokine-induced migration of EOL-1 cells, a human F/P+ eosinophil leukemia cell line. In addition, we examined TKI effects on primary human neoplastic eosinophils obtained from a patient with F/P+ CEL, one with aggressive systemic mastocytosis and massive eosinophilia (ASM-eo) and one with reactive hypereosinophilia (HE). In EOL-1 cells, major growth-inhibitory effects were seen with all PDGFRA-blocking agents, with IC50 values in the low nM-range: ponatinib: 0.1–0.2 nM, sorafenib: 0.1–0.2 nM, masitinib: 0.2–0.5 nM, nilotinib: 0.2–2 nM, dasatinib: 0.5–2 nM, sunitinib: 1–2 nM, and midostaurin: 5–10 nM. These drugs were also found to block the activity of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. Targeting of individual downstream molecules with specific inhibitors (PI3-kinase: NVP-BEZ235; mTOR: everolimus; STAT5: pimozide and piceatannol) also induced growth-inhibition in EOL-1 cells, although IC50 values were higher compared to that obtained with PDGFR-blocking TKI. All effective TKI produced dose-dependent apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI staining, and staining for active caspase-3. In a next step, we applied the most effective TKI on primary neoplastic eosinophils. In these experiments, ponatinib, dasatinib, and nilotinib were found to suppress the growth of primary neoplastic eosinophils obtained from a patient with F/P+ CEL and one with ASM-eo, in a dose-dependent manner (IC50 <0.5 μM). In the patient with reactive HE, the TKI also produced growth inhibition, but IC50 values were higher compared to neoplastic eosinophils. We also examined drug effects on growth of Ba/F3 cells expressing the imatinib-resistant F/P mutants T674I and D842V. In these experiments, sunitinib was found to inhibit the growth of Ba/F3 cells expressing the T674I mutant of F/P. By contrast, no substantial effects of masitinib or nilotinib on Ba/F3 cells expressing this mutant were found, and Ba/F3 cells expressing F/P D842V were found to be resistant against sunitinib and masitinib. Strong inhibitory effects on both mutants were only seen with ponatinib. We next examined the effects of various TKI on cytokine-induced migration of neoplastic eosinophils. Unexpectedly, of all cytokines tested including IL-5 and eotaxin, only SDF-1A was found to induce in vitro migration of EOL-1 cells. We found that imatinib, nilotinib, dasatinib, ponatinib, sorafenib, and masitinib inhibit SDF-1A-induced migration of EOL-1 cells in a dose-dependent manner (effective range: 10–100 nM). Finally, we analyzed TKI effects on expression of activation-linked cell surface antigens on EOL-1 cells. In these experiments, we found that ponatinib and sorafenib downregulate expression of CD25 and CD63 in EOL-1 cells, whereas the other TKI tested showed no effects. By contrast, no effects of ponatinib or sorafenib on expression of HLA-DR, CXCR4 and CD95 on EOL-1 cells were seen. We were also unable to detect any significant effects of the other TKI on expression of activation-linked cell surface antigens in EOL-1 cells. In summary, our data show that various novel TKI counteract growth, survival, activation, and migration of neoplastic human eosinophils. The most potent agent that also blocks all known mutant-forms of F/P appears to be ponatinib. Novel PDGFR-targeting TKI, such as ponatinib, may be attractive alternative drugs for the treatment of imatinib-resistant or intolerant CEL. Disclosures:Valent:Phadia: Research Funding.

Displaced Niche Location and Decreased Quiescence Maintenance of Hematopoietic Stem Cells Due to Dysregulation of Notch Adhesive Interaction with Stromal Environment in Mice with Notch O-Fucosylation Deficiency

Abstract 29O-fucose modification and Fringe mediated O-fucose extension of Notch EGF-like repeats is essential for Notch binding with Notch ligand and Notch signaling transactivation. Previously we have shown that mice with conditional deficiency of Notch O-fucose modification develop a myeloproliferative disorder (MPD) with some mice displaying features of MPD-like leukemia. We disclosed that this MPD is mainly contributed by the cell-autonomous loss of response of myeloid progenitors bearing non-fucosylated Notch to Notch ligand induced suppression of granulo-monocytic differentiation. More recently, several Notch loss-of-function mutations have been identified in human chronic myelomonocytic leukemia (CMML). To gain a better insight of the role of Notch loss-of-function in stem cell dysfunction and MPD progression, here we studied the significance of O-fucose deficiency of Notch on progenitor proliferation and survival, and on HSC quiescence maintenance and niche location.We used a mouse model of pan-Notch signaling loss-of-function by Mx-Cre1 induced deficiency of Pofut1, an enzyme that modifies EGF O-fucosylation of all 4 Notch receptors. Pofut1-null hematopoietic stem and progenitor cells (HSPCs) had enhanced myeloid specification and proliferation in vitro, and displayed an increased activation of ERK and Stat5 in response to IL3 and GM-CSF when compared to the control HSPCs. The enhanced myeloid specification of Pofut1-null HSCs could be rescued by either activated Notch1 or Notch2. In addition, the HSPCs from Pofut1-null marrow and spleen displayed a 30% reduction of apoptosis. However, the increased proliferation and survival of Pofut1-null HSPCs were only partially reversed by the blocking of G-CSF, a cytokine that was up-regulated in the serum of Pofut1-null mice, supporting a role of cell-autonomous mechanism in its contribution to the increased proliferation and survival of Pofut1-null HSPCs. In line with this notion, we found that Pofut1-null mice had ∼ 50% increase in frequencies of the multi-potential progenitors (MPP) and the short-term self-renewable HSC (ST-HSC) but a 70% reduction of the more primitive long-term self-renewable HSC (LT-HSC). This change of HSC frequency was accompanied by an increased HSC cell cycling and a loss of adhesion to Notch ligand-expressing stromal cells despite that the Pofut1-null HSCs had a normal chemotactic response to SDF-1 and normal expression of CXCR4 as well as integrin adhesion molecules. Consistent with these findings, frequencies of circulating and splenic-residing HSCs were increased in Pofut1-null mice. To explore the mechanism by which loss of O-fucose of Notch regulates the stem cell activity in the bone marrow niches, we performed two-photon intravital microscopy to visualize the niche location of transplanted HSCs. We found that the Pofut1-null HSCs were positioned further from the endosteal niche and the niche supporting osteoblasts, when compared to control HSCs. In addition, Pofut1-null HSCs were not responsive to the inhibition of HSC expansion imparted by the osteoblasts in an in vitro co-culture assay.In summary, loss of O-fucosylation of Notch not only results in skewed myeloid specification and differentiation, but also promotes HSC proliferation and suppresses HSC quiescence. We conclude that the HSC phenotypes observed in mice with Pofut1 deficiency result as a consequence of the displacement of HSCs expressing non-fucosylated Notch from the suppressive endosteal niche that is otherwise enhanced by the adhesion between HSCs with the niche supporting cells through Notch and Notch ligand interaction. Disclosures:No relevant conflicts of interest to declare.

The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

AbstractAbstract 5120STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal.Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR.The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table.Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression.Table:The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50.DRUGSCELL LINEIC50APOPTOSIS (%)STAT3 mRNA Down Regulation (%)STAT5A mRNA Down Regulation (%)STAT5B mRNA Down Regulation (%)ENALAPRILHL-607 μM2020*765*ZOLEDRONATEK56260 μM34633157BORTEZOMIBK562177 μM379810099DASATINIBK5623,314 nM75NA3378NA: not applied*: Not significant Disclosures:No relevant conflicts of interest to declare.

PCM1-JAK2 Fusion Product Selectively Activates MAP-Kinase Pathway and Fails to Phosphorylate JAK2 and STAT5: Implications for Therapeutic Strategies.

AbstractAbstract 2858Janus-activated kinase 2 (JAK2) gene mutations and translocations are involved in the pathogenesis of a variety of hematologic malignancies. Among different translocation partners, pericentriolar material 1 (PCM1)-JAK2 fusion products have been described in rare cases of both lymphoid and myeloid neoplasms characterized by morphological (myeloproliferaton, eosinophilia, myelofibrosis) and clinical (striking male predominance, aggressive course) similarities.We recently identified a new case of the rare translocation PCM1-JAK2 in a 29-year-old man presenting with atypical chronic myeloid leukemia (aCML) and peculiar aspects of diserythropoiesis in the bone marrow (BM): abundant paratrabecular clusters of proerythroblasts associated with marked reduction of mature erythroid compartment (Sammarelli et al., S.I.E.S. 12th Meeting, 2012).For the first time we describe here the erythroid differentiation capacity of ex-vivo expanded CD34+ cells from this PCM1-JAK2 fusion case, as well as the signaling pathways activated in peripheral blood neoplastic cells (PBNC) harboring the translocation.Presence of the PCM1-JAK2 fusion transcript in PBNC was confirmed by nested RT-PCR using primers derived from PCM1 exon 25 and JAK2 exon 9 described in Reiter et al. Cancer Res. 2005 (Figure 1).CD34+ cells were isolated from the peripheral blood (PB) of the patient and cultured in serum-free medium supplemented with erythropoietin (EPO), interleukin-3 (IL-3) and stem cell factor (SCF) to induce erythroid differentiation; erythroid cell output [evaluated in terms of fold increase (FI) and glycophorin-A (GlyA) expression at day 14 of culture] was compared to the one obtained from PB CD34+ cells from a polycythemia vera patient (PV), in which JAK2 is constitutively activated by V617F point mutation, and to CD34+ cells from a G-CSF-mobilized donor (M).As shown in Figure 2, FI and GlyA expression were significantly lower in our patient compared to M and PV (FI: 0.63, 6.47 and 7.92 respectively; GlyApos cells: 4.2%, 51.6% and 64.2%, respectively) consistently with the diserythropoietic picture in the BM.We then investigated the activation of the 3 main signaling pathways associated to Receptor tyrosine kinases and most commonly turned on in cancer: Mitogen-activated protein (MAP) kinase pathway, JAK/Signal transducer and activator of transcription (STAT) pathway and phosphatidylinositol 3-kinase (PI3K)/AKT pathway, evaluating, by Western Blot analysis, levels of phosphorylation of Extracellular signal-Regulated Kinase (ERK1/2), JAK2, STAT5 and AKT in PBNC from our PCM1-JAK2 case and in PB mononuclear cells (PBMC) from 5 healthy control subjects (C1-C5).Although these signaling cascades are deeply interconnected, we surprisingly found a selective activation of the sole MAP-kinase pathway in PBNC (Figure 3).These data suggest that, while presence of JAK2V617F mutation leads to ligand-independent activation of STAT5, AKT and ERK1/2 (Laubach et al. Exp. Hematol. 2009), PCM1-JAK2 fusion product fails to activate JAK/STAT and PI3K/AKT axis. Specifically, reduced STAT5 activation might explain impaired erythroid differentiation of CD34+ cells in vitro as well as the marked aspects of diserythropoiesis in the BM.The signaling signature of PMC1-JAK2 neoplastic cells described here has also relevant implications on the treatment strategy for these patients. In particular, given the lack of activation of JAK2 and its down-stream partner STAT5, JAK-inhibitor therapy does not seem the ideal candidate in this specific setting. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Commitment of CML Cells to Apoptotic Cell Death Depends On the Length of Exposure to Das and the Level of STAT5 Activity

AbstractAbstract 3736Despite the 3–5h half-life of the tyrosine kinase inhibitor (TKI) dasatinib (Das), patients with chronic myeloid leukemia (CML) receiving a once daily dose of 100mg achieved similar cytogenetic and molecular responses as patients on 50mg twice a day. We previously reported that high dose short-term dasatinib exposure (100nM for 30 min) induces cell death and results in complete inhibition of Bcr-Abl, which is reversed within 2h of drug washout. Recently, it has been suggested that retention of low Das levels, following transient exposure to high drug doses, is responsible for the observed in vitro effect on cell survival. Here we investigate the mechanism of cell death in the setting of incomplete Bcr-Abl kinase inhibition.Das treatment was investigated in KU812 and Meg01 BCR-ABL-positive CML cell lines using Annexin V/7-AAD staining and cell proliferation assays (at 72h, n=3). Continuous low dose (LD) Das (1nM) induced cell death (83% and 88%) which was accompanied by an immediate loss of pSTAT5 and pErk, and reduction of Bcl-XL, Mcl-1 and Bcl-2 expression. Initial inhibition of pBcr-Abl was poor, however, it was fully inhibited by 48 hours, suggesting that loss of survival signalling through pSTAT5 and pErk prior to inhibition of pBcr-Abl promotes cells death in this setting. Transient exposure to higher dose Das (100nM) for 30min followed by drug washout (STD wash, 3 × 10ml PBS, at 37°C) and re-culture to 72h in the absence of drug, induced cell death (90.2% and 91%) similar to continuous LD culture. Inhibition of pSTAT5 and pErk also preceded inhibition of p-Bcr-Abl, suggesting the presence of residual Das after washout.Adoption of an optimal drug washout approach, where cells equilibrate for 1h between washes following 30min exposure to 100nM Das, resulted in a significant reduction in cell death (13.6% and 5.9%). Following this optimal washout (OPT wash), no inhibition of pBcr-Abl was observed and transient inhibition of pErk and pSTAT5 was reversed by 4h. Association of cell survival with recovery of pSTAT5 and pErk suggests that inhibition of these signals by low level Das, whether in continuous LD or the STD wash setting, is critical to Das-induced cell death.STAT5 and Erk activation occur through various pathways, therefore we interrogated pathways known to facilitate escape from cell death. We and others have previous shown that inhibition of the JAK/STAT pathway prevents cytokine survival signalling, and that inhibition of TKI-induced autophagy induces cell death. We therefore assessed cell survival and proliferation in the presence of a JAK1/2 inhibitor (INCB-018424, Active Biochem) or pSTAT5 inhibitor (pimozide, Sigma) to target cytokine signalling pathways and chloroquine, an autophagy inhibitor, in the presence and absence of 100nM Das (OPT wash) to assess their potential to sustain the otherwise reversible effect of short-term Das priming. Interestingly, neither inhibition of JAK1/2 nor autophagy enhanced cell death, however pimozide plus Das eradicated live cells after OPT wash (1.6%) while pimozide alone had minimal impact on cell survival (89%) emphasising the key role of STAT5 in CML survival signalling.To translate these observations to a more clinically relevant setting, Das exposures of 100nM were prolonged to 4 and 8 h (analogous to the in vivo half life and activity of the drug) prior to OPT wash. Both conditions resulted in significant cell death in KU812 and Meg01 cells comparable to continuous treatments (Figure 1), which was associated with pBcr-Abl inhibition contrary to the 30 min drug treatment. [Display omitted] This study demonstrates that a low level of Das (either 1nM continuous dose or residual Das following 100nM transient exposure) induces apoptosis in CML cells despite minimal Bcr-Abl inhibition. Cell death induced by low level Das may be tightly coupled to inhibition of pSTAT5 and pErk achievable with only partial kinase inhibition. Removal of residual Das using an optimal washout reverses apoptosis, but can be overcome by longer, clinically relevant Das exposure or concomitant inhibition of pSTAT5. Our findings enable a better understanding of the potential clinical effectiveness of low dose Das treatment and will help establish the critical CML signalling components which may be targeted in combination therapeutic approaches. Disclosures:Nievergall:CSL: Research Funding. Hiwase:CSL: Research Funding. White:Novartis Oncology: Honoraria, Research Funding; BMS: Research Funding; CSL: Research Funding. Hughes:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A non-cell-autonomous tumor suppressor role for Stat in eliminating oncogenic scribble cells

Elucidating signaling events between tumor cells and their microenvironment is a major challenge in understanding cancer development. Drosophila melanogaster has emerged as an important tool for dissecting the genetic circuits tumors depend on because their imaginal discs, simple epithelia present in the larva, can be genetically manipulated to serve as models to study cancer mechanisms. Imaginal disc cells mutant for the tumor-suppressor gene scribble (scrib) lose apical-basal polarity and have the potential to form large neoplastic tumors. Interestingly, when scrib mutant (scrib(-)) cells are surrounded by normal cells the scrib(-) population is eliminated. However, the signals and mechanisms that cause the elimination of clones of scrib(-) cells are poorly understood. Here, we analyzed the role of Stat, a component of the JAK/STAT signaling pathway, in tissues with clones of scrib(-) cells. We found that Stat activity is required in normal cells for the elimination of neighboring scrib(-) cells. Importantly, these competitive defects of stat mutant cells are not simply due to defects in cell proliferation because even stat(-) cells manipulated to hyperproliferate are unable to eliminate scrib(-) cells. These data identify Stat activity as a critical determinant of whether or not a tissue can eliminate abnormal cells and provide an important step forward in understanding the complex network of signals operating in and around tumorigenic cells.

Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis

Cell competition is a conserved mechanism that regulates organ size and shares properties with the early stages of cancer. In Drosophila, wing cells with increased Myc or with optimum ribosome function become supercompetitors that kill their wild-type neighbors (called losers) up to several cell diameters away. Here, we report that modulating STAT activity levels regulates competitor status. Cells lacking STAT become losers that are killed by neighboring wild-type cells. By contrast, cells with hyper-activated STAT become supercompetitors that kill losers located at a distance in a manner that is dependent on hid but independent of Myc, Yorkie, Wingless signaling, and of ribosome biogenesis. These results indicate that STAT, Wingless and Myc are major parallel regulators of cell competition, which may converge on signals that non-autonomously kill losers. As hyper-activated STATs are causal to tumorigenesis and stem cell niche occupancy, our results have therapeutic implications for cancer and regenerative medicine.

The insect peptide CopA3 inhibits lipopolysaccharide‐induced macrophage activation

We recently demonstrated that the insect peptide CopA3 (LLCIALRKK), a disulfide-linked dimeric peptide, exerts antimicrobial and anti-inflammatory activities in a mouse colitis model. Here, we examined whether CopA3 inhibited activation of macrophages by LPS. Exposure of an unseparated mouse peritoneal cell population or isolated peritoneal macrophages to LPS markedly increased secretion of IL-6 and TNF-α; these effects were significantly inhibited by CopA3 treatment. The inhibitory effect of CopA3 was also evident in murine macrophage cell line, RAW 264.7. Western blotting revealed that LPS-induced activation of STAT1 and STAT5 in macrophages was significantly inhibited by CopA3. Inhibition of JAK (STAT1/STAT5 kinase) with AG490 markedly reduced the production of IL-6 and TNF-α in macrophages. Collectively, these observations suggest that CopA3 inhibits macrophage activation by inhibiting activating phosphorylations of the transcription factors, STAT1 and STAT5, and blocking subsequent production of IL-6 and TNF-α and indicate that CopA3 may be useful as an immune-modulating agent.

C/EBPβ promotes BCR–ABL-mediated myeloid expansion and leukemic stem cell exhaustion

The BCR-ABL fusion oncoprotein accelerates differentiation and proliferation of myeloid cells during the chronic phase of chronic myeloid leukemia (CP-CML). Here, the role of CCAAT/enhancer binding protein β (C/EBPβ), a regulator for 'emergency granulopoiesis,' in the pathogenesis of CP-CML was examined. C/EBPβ expression was upregulated in Lineage(-) CD34(+) CD38(-) hematopoietic stem cells (HSCs) and myeloid progenitors isolated from bone marrow of patients with CP-CML. In EML cells, a mouse HSC line, BCR-ABL upregulated C/EBPβ, at least in part, through the activation of STAT5. Myeloid differentiation and proliferation induced by BCR-ABL was significantly impaired in C/EBPβ-deficient bone marrow cells in vitro. Mice that were transplanted with BCR-ABL-transduced C/EBPβ knockout bone marrow cells survived longer than mice that received BCR-ABL-transduced wild-type (WT) bone marrow cells. Significantly higher levels of leukemic stem cells were maintained in BCR-ABL-transduced C/EBPβ-deficient cells than in BCR-ABL-transduced WT cells. These results suggest that C/EBPβ is involved in BCR-ABL-mediated myeloid expansion. Further elucidation of the molecular mechanisms underlying the C/EBPβ-mediated stem cell loss might reveal a novel therapeutic strategy for eradication of CML stem cells.

P067 JAK2 V617F mutation causes cytokine-independent growth and tumorigenesis – Roles of STAT5 and c-Myc

Introduction JAK2 is an essential signal transducer for various cytokine signaling including IL-3 or Epo. Mutation at V617F of JAK2 promotes cell growth and prevents apoptosis which causes a variety of myeloproliferative neoplasms (MPNs). How the JAK2 V617F mutation causes dysregulated proliferation, resistance to anti-tumor drugs and tumorigenesis in nude mice is to be investigated. Methods Ba/F3 cells harboring JAK2 V617F were established to study the constitutive activation of JAK2 and cytokine-independent growth. Tumorigenesis was tested by the inoculation of JAK2 V617F mutant in nude mice. DNA array was done to search genes expressed predominantly by JAK2 V617F and STAT5. Results (1) JAK2 V617F mutant caused aberrant activation of STAT5 and Akt when EpoR was expressed, which is critical for the JAK2 V617F-induced anti-apoptotic and oncogenic activities. (2) In search of genes induced by JAK2 V617F mutant, we observed significant induction of c-Myc. (3) While the phosphorylation at Thr58 by GSK-3b induced the proteasome-mediated degradation of c-Myc, GSK-3b was inactivated in JAK2 V617F mutant, which resulted in the enhancement of c-Myc expression. The enforced expression of c-Myc accelerated not only the cell proliferation but also apoptotic cell death; however, the inhibition of GSK-3b completely inhibited c-Myc-induced apoptosis. (4) JAK2 V617F mutant also induced expression of a target gene of c-Myc, ornithine decarboxylase (ODC) known as a rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO) prevented the proliferation of the JAK2 V617F mutant cells. Furthermore, administration of DFMO effectively delayed tumor formation in nude mice, resulting in a prolonged survival. Conclusion JAK2 V617F mutant caused aberrant activation of Akt and STAT5 pathways. ODC expression induced by c-Myc is critical for JAK2 V617F mutant-driven transformation and that targeted disruption of c-Myc-ODC axis may provide therapeutic utility for the treatment of MPNs. STAT5 and c-Myc have critical roles for JAK2 V617F mutant-induced tumorigenesis, which help clarify the mechanism how JAK2 V617F mutant causes MPNs.

IL-6 activates STAT5 in T cells

BackgroundIL-6 is a pleiotropic cytokine which emerged recently as a key regulator of CD4 T cell function. IL-6 alone or in combination with other cytokines promotes T helper 1, T helper 17 and T follicular helper cell differentiation whilst inhibiting the induction of regulatory T cell generation. IL-6 activates multiple pathways among which JAK/STAT3 is the most clearly validated in the control of CD4 T helper differentiation. Activation of STAT5 by cytokines such as IL-2 can counteract IL-6-induced T helper 17 and T follicular helper cell differentiation and promote the induction of regulatory T cell generation. STAT5 and STAT3 are known to compete for promoter binding sites in CD4 T cells and the two transcription factors are believed to have opposite functions in the control of CD4 T cell differentiation. MethodsWe analyzed IL-6-induced STAT1, 3 and 5 activation by flow cytometry (phosflow) in mouse mononuclear cells and its effect on the level of the mRNA coding for cytokine-inducible SH2-containing protein (CIS). ResultsThe results show that IL-6 also induces STAT5 activation in both CD4 and CD8 T as well as NK cells. Analysis of STAT5 phosphorylation in CD4 T cells indicates that it is transient and requires higher cytokine concentrations than that of STAT3. CD4 T cell stimulation with IL-6 induces the synthesis of CIS, which is encoded by a gene known to be regulated by STAT5. ConclusionsThus, IL-6 at concentrations corresponding to levels observed in the serum during inflammation may activate, in CD4 T cells, a STAT5-negative feedback loop which alters the balance between STAT3-dependent pro-inflammatory helper T cells and STAT5-induced T regulatory cells. STAT5 activation may modulate the differentiation of T helper cells through attenuation of TGF-β stability and production. Since STAT5 is directly activated by Janus kinases, therapeutic approaches designed to inhibit STAT3 activation or to recruit STAT3 phosphatases may be useful in altering the balance of activated STAT3 and STAT5 in favor a profile that would be beneficial in pathologies involving IL-6.

Expression of phosphorylated signal transducer and activator of transcription 5 is associated with an increased risk of death in acute myeloid leukemia

Constitutive activation of STAT5 (by phosphorylation) has been identified in a number of malignancies, including acute myeloid leukemia (AML). We investigated whether the level of phosphorylated STAT5 (pSTAT5) expression correlates with clinical outcome in AML. Adult patients with newly diagnosed AML receiving induction chemotherapy and with an available diagnostic bone marrow were evaluated. Forty-two percent of patients had pSTAT5 expression >0 on immunohistochemical analysis of fixed bone marrow core biopsies. In multivariable analyses, controlling for age, history of antecedent hematologic disorder, cytogenetic risk, and WBC at diagnosis, pSTAT5 expression was significantly associated with an increased risk of death (HR 1.96, 95% CI 1.19-3.23, P = 0.008) and of relapse after achieving complete remission (HR 2.31, 95% CI 1.16-4.63, P = 0.018). Validation of pSTAT5's prognostic value requires additional study in a larger group of uniformly treated patients. However, our data suggests that targeting this signaling pathway in AML may improve the outcome of patients.

The STAT5 Inhibitor Pimozide Displays Efficacy in Models of Acute Myelogenous Leukemia Driven by FLT3 Mutations

Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myelogenous leukemia (AML) containing the FLT3 internal tandem duplication (ITD) mutation. FLT3 ITD is a constitutively active tyrosine kinase that drives the activation of STAT5, leading to the growth and survival of AML cells. Although there has been some success in identifying tyrosine kinase inhibitors that block the function of FLT3 ITD, there remains a continued need for effective treatment of this disease. We have identified the psychotropic drug pimozide as an effective inhibitor of STAT5 function. Pimozide inhibits the tyrosine phosphorylation of STAT5, leading to the death of AML cells through the induction of apoptosis. Pimozide shows a combinatorial effect with the tyrosine kinase inhibitors midostaurin (PKC412) and sunitinib in the inhibition of STAT5 tyrosine phosphorylation and the induction of apoptosis. Significantly, pimozide reduces the tumor burden in a mouse model of FLT3-driven AML. Therefore, identifying STAT5 inhibitors may provide a new avenue for the treatment of AML, and these may be effective alone or in combination with tyrosine kinase inhibitors.

A Requirement for SOCS-1 and SOCS-3 Phosphorylation in Bcr-Abl-Induced Tumorigenesis

Suppressors of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3) are inhibitors of the Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (STAT) pathway and function in a negative feedback loop during cytokine signaling. Abl transformation is associated with constitutive activation of JAK/STAT-dependent signaling. However, the mechanism by which Abl oncoproteins bypass SOCS inhibitory regulation remains poorly defined. Here, we demonstrate that coexpression of Bcr-Abl with SOCS-1 or SOCS-3 results in tyrosine phosphorylation of these SOCS proteins. Interestingly, SOCS-1 is highly tyrosine phosphorylated in one of five primary chronic myelogenous leukemia samples. Bcr-Abl-dependent tyrosine phosphorylation of SOCS-1 and SOCS-3 occurs mainly on Tyr 155 and Tyr 204 residues of SOCS-1 and on Tyr 221 residue of SOCS-3. We observed that phosphorylation of these SOCS proteins was associated with their binding to Bcr-Abl. Bcr-Abl-dependent phosphorylation of SOCS-1 and SOCS-3 diminished their inhibitory effects on the activation of JAK and STAT5 and thereby enhanced JAK/STAT5 signaling. Strikingly, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 impaired the expression of Bcl-XL protein and sensitized K562 leukemic cells to undergo apoptosis. Moreover, selective mutation of tyrosine phosphorylation sites of SOCS-1 or SOCS-3 significantly blocked Bcr-Abl-mediated tumorigenesis in nude mice and inhibited Bcr-Abl-mediated murine bone marrow transformation. Together, these results reveal a mechanism of how Bcr-Abl may overcome SOCS-1 and SOCS-3 inhibition to constitutively activate the JAK/STAT-dependent signaling, and suggest that Bcr-Abl may critically requires tyrosine phosphorylation of SOCS-1 and SOCS-3 to mediate tumorigenesis when these SOCS proteins are present in cells.

Progress towards direct inhibitors of Stat5 protein.

Abstract Molecular approaches to inhibit STAT5 signaling have been hailed as a viable targeted anticancer therapy. In particular, many drugs and drug candidates have been developed to successfully inhibit upstream effectors of STAT5 by indirectly targeting cell surface receptors and protein kinases (FLT-3, JAK2, and BCR-ABL). Indirect strategies have yielded potent agents, such as imatinib, AC2207, and EXEL823 which effectively silence STAT5 activity but which suffer from off-target effects and toxicity. This article will focus on reviewing the current literature pertaining to direct inhibitors of STAT5 protein and assess the prospects for a future STAT5-targeting therapeutic.

Modulation of Microenvironment Acidity Reverses Anergy in Human and Murine Tumor-Infiltrating T Lymphocytes

Stimulating the effector functions of tumor-infiltrating T lymphocytes (TIL) in primary and metastatic tumors could improve active and adoptive T-cell therapies for cancer. Abnormal glycolysis, high lactic acid production, proton accumulation, and a reversed intra-extracellular pH gradient are thought to help render tumor microenvironments hostile to roving immune cells. However, there is little knowledge about how acidic microenvironments affect T-cell immunity. Here, we report that lowering the environmental pH to values that characterize tumor masses (pH 6-6.5) was sufficient to establish an anergic state in human and mouse tumor-specific CD8(+) T lymphocytes. This state was characterized by impairment of cytolytic activity and cytokine secretion, reduced expression of IL-2Rα (CD25) and T-cell receptors (TCR), and diminished activation of STAT5 and extracellular signal-regulated kinase (ERK) after TCR activation. In contrast, buffering pH at physiologic values completely restored all these metrics of T-cell function. Systemic treatment of B16-OVA-bearing mice with proton pump inhibitors (PPI) significantly increased the therapeutic efficacy of both active and adoptive immunotherapy. Our findings show that acidification of the tumor microenvironment acts as mechanism of immune escape. Furthermore, they illustrate the potential of PPIs to safely correct T-cell dysfunction and improve the efficacy of T-cell-based cancer treatments.

STAT5 activity downstream of IgE and IgG receptors in mast cells (177.8)

Abstract Removal of STAT5 impairs mast cell development, survival, and IgE-mediated cytokine production, justifying a need to closely investigate its activity downstream of Fc receptors. We found that STAT5 tyrosine phosphorylation occurs shortly after FcϵRI crosslinking in a Fyn-dependent manner, while the suppression of Lyn, and other negative regulators of IgE signaling, appears to enhance STAT5 activation. Tyrosine phosphorylation of STAT5 also occured during IgG-mediated activity, suggesting some overlap in the signaling proximal to different Fc receptors in mast cells. Additionally, IgE- and IgG-mediated signaling elicited serine phosphorylation of both STAT5A and STAT5B, possibly in a divergent manner. Inhibition of Syk ablated IgE-mediated pS-STAT5, but affected to a lesser extent, if at all, pS-STAT5B and IgG-mediated total pS-STAT5. Furthermore, inhibition of JNK reduced pS-STAT5 during FcγR crosslinking. Collectively, these data highlight similarities and differences from variable receptor inputs as well as the different STAT5 proteins, supporting their essential roles in mast cell biology.

Development of a novel method for in vitro analysis of γc-cytokine effects on CD8 T cells positive selection (111.15)

Abstract Interleukin (IL)7 is the only γc-cytokine known to support double-positive thymocytes differentiation to the single-positive (SP) stage. To determine if additional γc-cytokines can support T-cell development, we studied their differential effects on positive selection in vitro using a monolayer of OP9 bone marrow-derived stromal cells. We chose the OTI transgenic TCR as our working model due to the previous identification of positively selecting peptides in FTOC assays. In addition to rIL7, rIL4 and rIL13 (but not rIL2, rIL9, rIL15 or rIL21) were able to induce SP CD8 T-cell development. Interestingly, rIL4 induces the de novo expression of EOMES and triggers the development of 2 distinct SP CD8 T cell populations, which we termed CD8int and CD8hi. Flow-cytometry analysis showed that ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44 whereas rIL7 or rIL13 treatments did not up regulate these markers. Furthermore, rIL4 has a unique ability to enhance expression of FOXO1, KLF2 and S1PR1. Flow-cytometry analysis of signaling events demonstrated that every γc-cytokine leads to differential activation of AKT, ERK, STAT3, STAT5 and STAT6. Taken together, our findings show that CD8 T-cell positive selection can be studied with an in vitro model that is more convenient than FTOC assays and can be customized to a high-throughput format. Notably, we validate a novel role for rIL4 and rIL13 in supporting positive selection of thymocytes.