Aberrant JAK-STAT Signaling Research Articles

Aberrant JAK-STAT signaling-mediated chromatin remodeling impairs the sensitivity of NK/T-cell lymphoma to chidamide

BackgroundNatural killer/T-cell lymphoma (NKTL) is a rare type of aggressive and heterogeneous non-Hodgkin's lymphoma (NHL) with a poor prognosis and limited therapeutic options. Therefore, there is an urgent need to exploit potential novel therapeutic targets for the treatment of NKTL. Histone deacetylase (HDAC) inhibitor chidamide was recently approved for treating relapsed/refractory peripheral T-cell lymphoma (PTCL) patients. However, its therapeutic efficacy in NKTL remains unclear.MethodsWe performed a phase II clinical trial to evaluate the efficacy of chidamide in 28 relapsed/refractory NKTL patients. Integrative transcriptomic, chromatin profiling analysis and functional studies were performed to identify potential predictive biomarkers and unravel the mechanisms of resistance to chidamide. Immunohistochemistry (IHC) was used to validate the predictive biomarkers in tumors from the clinical trial.ResultsWe demonstrated that chidamide is effective in treating relapsed/refractory NKTL patients, achieving an overall response and complete response rate of 39 and 18%, respectively. In vitro studies showed that hyperactivity of JAK-STAT signaling in NKTL cell lines was associated with the resistance to chidamide. Mechanistically, our results revealed that aberrant JAK-STAT signaling remodels the chromatin and confers resistance to chidamide. Subsequently, inhibition of JAK-STAT activity could overcome resistance to chidamide by reprogramming the chromatin from a resistant to sensitive state, leading to synergistic anti-tumor effect in vitro and in vivo. More importantly, our clinical data demonstrated that combinatorial therapy with chidamide and JAK inhibitor ruxolitinib is effective against chidamide-resistant NKTL. In addition, we identified TNFRSF8 (CD30), a downstream target of the JAK-STAT pathway, as a potential biomarker that could predict NKTL sensitivity to chidamide.ConclusionsOur study suggests that chidamide, in combination with JAK-STAT inhibitors, can be a novel targeted therapy in the standard of care for NKTL.Trial registration: ClinicalTrials.gov, NCT02878278. Registered 25 August 2016, https://clinicaltrials.gov/ct2/show/NCT02878278

PRIMARY MYELOPHYBROSIS: RECOGNIZING ONCET TO COMPLICATIONS

Myelofibrosis is the accumulation of scar tissue in the bone marrow so that blood cells cannot develop properly. Myeloproliferative disorders, myelofibrosis is classified into two, namely primary myelofibrosis and secondary myelofibrosis. In contrast to secondary myelofibrosis, primary myelofibrosis can occur without being preceded by myeloproliferative disorders or other diseases. In a 2013 meta-analysis study in Europe, the incidence of primary myelofibrosis (PMF) was around 0.3 per 100,000 per year. PMF may result from increased expression of inflammatory cytokines, lysyl oxidase, transforming growth factor-β, impaired megakaryocyte function, and aberrant JAK-STAT signaling. The most common clinical features found in patients with PMF are splenomegaly, hepatomegaly, fatigue, anemia, leukocytosis, and thrombocytosis. Currently the only treatment modality capable of prolonging survival or healing potential in MF is allogeneic hematopoietic stem cell transplantation (AHSCT) especially for high or very high risk patients.

Genetic and epigenetic insights into cutaneous T-cell lymphoma

AbstractPrimary cutaneous T-cell lymphomas (CTCLs) constitute a heterogeneous group of non-Hodgkin T-cell lymphomas that present in the skin. In recent years, significant progress has been made in the understanding of the pathogenesis of CTCLs. Progress in CTCL classifications combined with technical advances, in particular next-generation sequencing, enabled a more detailed analysis of the genetic and epigenetic landscape and transcriptional changes in clearly defined diagnostic entities. These studies not only demonstrated extensive heterogeneity between different CTCL subtypes but also identified recurrent alterations that are highly characteristic for diagnostic subgroups of CTCLs. The identified alterations, in particular, involve epigenetic remodeling, cell cycle regulation, and the constitutive activation of targetable oncogenic pathways. In this respect, aberrant JAK-STAT signaling is a recurrent theme; however, it is not universal for all CTCLs and has seemingly different underlaying causes in different entities. A number of the mutated genes identified are potentially actionable targets for the development of novel therapeutic strategies. Moreover, these studies have produced an enormous amount of information that will be critically important for the further development of improved diagnostic and prognostic biomarkers that can assist in the clinical management of patients with CTCL. In the present review, the main findings of these studies in relation to their functional impact on the malignant transformation process are discussed for different subtypes of CTCLs.

A System Biology Perspective to Overcoming the Challenges of JAK Inhibition Mon Therapy for Myeloproliferative Neoplasms: An Overview

The JAK-STAT pathway mediates signals that are involved in hematopoiesis. Aberrant JAK-STAT signaling has been identified in myeloproliferative neoplasm making the pathway a novel therapeutic target for myeloproliferative neoplasms through the application of JAK inhibitors. A major limitation to therapy with the current JAK inhibitors is a lack of selectivity which results in toxicity to patients. It is thought that increasing the selectivity of inhibitors will reduce toxicity observed with JAK inhibition therapy. System biology is a novel technology that holds great potentials for increasing the selectivity of JAK inhibitors. Its Application to drug designing can broaden the spectrum as well as repurpose the available JAK inhibitors for improved clinical outcome and possible cure for myeloproliferative neoplasms. This review presents an overview on the role of system biology in JAK inhibition therapy for myeloproliferative neoplasms.

An immunoevasive strategy through clinically-relevant pan-cancer genomic and transcriptomic alterations of JAK-STAT signaling components

BackgroundSince its discovery almost three decades ago, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway has paved the road for understanding inflammatory and immunity processes related to a wide range of human pathologies including cancer. Several studies have demonstrated the importance of JAK-STAT pathway components in regulating tumor initiation and metastatic progression, yet, the extent of how genetic alterations influence patient outcome is far from being understood.MethodsFocusing on 133 genes involved in JAK-STAT signaling, we investigated genomic, transcriptomic and clinical profiles of over 18,000 patients representing 21 diverse cancer types. We identified a core set of 28 putative gain- or loss-of-function JAK-STAT genes that correlated with survival outcomes using Cox proportional hazards regression and Kaplan-Meier analyses. Differential expression analyses between high- and low-expressing patient groups were performed to evaluate the consequences of JAK-STAT misexpression.ResultsWe found that copy number alterations underpinning transcriptional dysregulation of JAK-STAT pathway genes differ within and between cancer types. Integrated analyses uniting genomic and transcriptomic datasets revealed a core set of JAK-STAT pathway genes that correlated with survival outcomes in brain, renal, lung and endometrial cancers. High JAK-STAT scores were associated with increased mortality rates in brain and renal cancers, but not in lung and endometrial cancers where hyperactive JAK-STAT signaling is a positive prognostic factor. Patients with aberrant JAK-STAT signaling demonstrated pan-cancer molecular features associated with misexpression of genes in other oncogenic pathways (Wnt, MAPK, TGF-β, PPAR and VEGF). Brain and renal tumors with hyperactive JAK-STAT signaling had increased regulatory T cell gene (Treg) expression. A combined model uniting JAK-STAT and Tregs allowed further delineation of risk groups where patients with high JAK-STAT and Treg scores consistently performed the worst.ConclusionProviding a pan-cancer perspective of clinically-relevant JAK-STAT alterations, this study could serve as a framework for future research investigating anti-tumor immunity using combination therapy involving JAK-STAT and immune checkpoint inhibitors.

Targeting compensatory MEK/ERK activation increases JAK inhibitor efficacy in myeloproliferative neoplasms.

Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT, and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling, suggesting that cell-extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK inhibition. PDGF-BB maintained ERK activation in the presence of ruxolitinib, consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.

Bone marrow fibrosis – the basis of mielofibrosis: pathogenesis, prognostication and antifibrogenic targeted strategies.

Bone marrow fibrosis is a key patological feature and major diagnostic criterion of mielofibrosis. Although bone marrow fibrosis is manifested in a variety of malignant and non-malignant disease states, the deposition of reticulin and collagen fibrosis in the bone marrow of patients with myelofibrosis is believed to be mediated by the mielofibrosis of hematopoietic stem/progenitor cells, contributing to an impaired microenvironment toward malignant over normal hematopoiesis. The increased expression of pro­inflammatory cytokines, transforming growth factor-β, impaired megakaryocyte function and aberrant JAK-STAT signaling are the peculiarities of pathogenesis of bone marrow fibrosis. Hematopoietic stem cell transplantation remains the only therapeutic approach that reliably results in resolution of bone marrow fibrosis in patients with mielofibrosis. In the work we review the pathogenesis, biological consequences and prognostic results of impact of bone marrow fibrosis. We discuss the rationale of various anti-fibrogenic treatment strategies targeting at clonal hematopoietic stem/progenitor cells, aberrant signaling pathway, fibrogenic cytokines, and tumor microenvironment.

Loss of NF-κB1 Causes Gastric Cancer with Aberrant Inflammation and Expression of Immune Checkpoint Regulators in a STAT-1-Dependent Manner

Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.

Overview of Transgenic Mouse Models of Myeloproliferative Neoplasms (MPNs).

Myeloproliferative neoplasms (MPNs) are a class of hematologic diseases characterized by aberrant proliferation of one or more myeloid lineages and progressive bone marrow fibrosis. In 2005, seminal work by multiple groups identified the JAK2V617F mutation in a significant fraction of MPN patients. Since that time, murine models of JAK2V617F have greatly enhanced the understanding of the role of aberrant JAK-STAT signaling in MPN pathogenesis and have provided an in vivo pre-clinical platform that can be used to develop novel therapies. From early retroviral transduction models to transgenics, and ultimately conditional knock-ins, murine models have established that JAK2V617F alone can induce an MPN-like syndrome in vivo. However, additional mutations co-occur with JAK2V617F in MPNs, often in proteins involved in epigenetic regulation that can dramatically influence disease outcomes. In vivo modeling of these mutations in the context of JAK2V617F has provided additional insights into the role of epigenetic dysregulation in augmenting MPN hematopoiesis. In this overview, early murine model development of JAK2V617F is described, with an analysis of its effects on the hematopoietic stem/progenitor cell niche and interactions with downstream signaling elements. This is followed by a description of more recent in vivo models developed for evaluating the effect of concomitant mutations in epigenetic modifiers on MPN maintenance and progression. Mouse models of other driver mutations in MPNs, including primarily calreticulin (CALR) and Tpo-receptor (MPL), which occur in a significant percentage of MPN patients with wild-type JAK2, are also briefly reviewed. © 2017 by John Wiley & Sons, Inc.

Oncogenic activation of STAT3 pathway drives PD-L1 expression in natural killer/T cell lymphoma.

7549 Background: Natural killer/T-cell lymphoma (NKTL) is a rare type of non-Hodgkin lymphoma that occurs more frequently in East Asia and Latin America and is associated with Epstein–Barr virus infection. Recent whole-exome sequencing studies in NKTL have reported recurrent somatic mutations in genes associated with JAK-STAT pathway, however the role of aberrant JAK-STAT signaling in tumor immune escape through PD-L1 regulation is unclear. Methods: To determine the prevalence of JAK-STAT pathway alteration in NKTL, we performed targeted sequencing of 188 genes associated with JAK-STAT pathway in 109 NKTL (22 Singapore cases, 79 China cases and 8 cell lines). Single nucleotide variants and micro-indels were called using Freebayes and candidate variants annotated using ANNOVAR. Ba/F3 model system was used to test the transformation capacity of identified variants. Cell lines were evaluated for PD-L1 expression by immunoblotting and flow cytometry. Tissue microarrays were examined for p-STAT3 and PD-L1 expression by immunohistochemistry. Results: We identified a total of 284 non-synonymous somatic mutations candidates in 114 genes, including 243 missense, 10 nonsense, 4 splice-site and 27 indel mutations. Recurrent mutations were most frequently located in STAT3 (25/109 cases, 23%) followed by TP53 (16/109 cases, 16%) and JAK3 (8/109 cases, 7%). A total of 18 STAT3 variants were identified including known hotspot mutations and novel mutations in the SH2, coiled coil and DNA-binding domains. Characterization of novel E616K mutant residing in the SH2 domain showed that E616K conferred IL3 independent growth to Ba/F3 cells, increased STAT3 phosphorylation and PD-L1 expression. Consistent with these findings, PD-L1 was over expressed in cell lines harboring STAT3 mutations. A positive correlation between PD-L1 and p-STAT3 expression was also observed in tumor tissue (R = 0.51, P = 0.02). Conclusions: We characterized a novel activating STAT3 mutant and demonstrated its ability to drive PD-L1 expression, which may promote tumor evasion from the antitumor immune response. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising and novel therapeutic approach for NKTL in the future.

JAK-STAT signaling in the therapeutic landscape of myeloproliferative neoplasms

Myeloproliferative neoplasms (MPN) are a group of disorders defined by clonal proliferation of mature myeloid cells with overlapping clinical features. The driver mutations of these disorders, namely JAK2 (Janus Kinase), MPL (Myeloproliferative Leukaemia Virus) and CALR (Calreticulin) upregulate JAK-STAT signaling with increase in downstream transcription and gene expression. Epigenetic mutations are prevalent in MPNs but their interplay with aberrant JAK-STAT signaling is not known. This understanding lead to development of first targeted treatment in MPN; ruxolitinib for primary myelofibrosis. This has shown clinical benefit in overall survival and symptoms improvement but has yet to show significant disease modifying effects. This review will focus on contemporaneous understanding of altered JAK-STAT signaling in MPN and targeted treatments in clinical practice.

Bone marrow fibrosis in myelofibrosis: pathogenesis, prognosis and targeted strategies.

Bone marrow fibrosis is a central pathological feature and World Health Organization major diagnostic criterion of myelofibrosis. Although bone marrow fibrosis is seen in a variety of malignant and non-malignant disease states, the deposition of reticulin and collagen fibrosis in the bone marrow of patients with myelofibrosis is believed to be mediated by the myelofibrosis hematopoietic stem/progenitor cell, contributing to an impaired microenvironment favoring malignant over normal hematopoiesis. Increased expression of inflammatory cytokines, lysyl oxidase, transforming growth factor-β, impaired megakaryocyte function, and aberrant JAK-STAT signaling have all been implicated in the pathogenesis of bone marrow fibrosis. A number of studies indicate that bone marrow fibrosis is an adverse prognostic variable in myeloproliferative neoplasms. However, modern myelofibrosis prognostication systems utilized in risk-adapted treatment approaches do not include bone marrow fibrosis as a prognostic variable. The specific effect on bone marrow fibrosis of JAK2 inhibition, and other rationally based therapies currently being evaluated in myelofibrosis, has yet to be fully elucidated. Hematopoietic stem cell transplantation remains the only curative therapeutic approach that reliably results in resolution of bone marrow fibrosis in patients with myelofibrosis. Here we review the pathogenesis, biological consequences, and prognostic impact of bone marrow fibrosis. We discuss the rationale of various anti-fibrogenic treatment strategies targeting the clonal hematopoietic stem/progenitor cell, aberrant signaling pathways, fibrogenic cytokines, and the tumor microenvironment.

Clinical Significance of LNK (SH2B3) Expression in Pediatric B Cell Precursor Acute Lymphoblastic Leukemia

Background: In pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), aberrant JAK-STAT signaling is one of the key leukemogenic mechanisms. Although activating mutations of JAK2 are found in high-risk BCP-ALL patients in western countries, such mutations are rare in Japanese cohorts, led to speculation of other factors contributing abnormal activation of JAK-STAT pathway. The adaptor protein LNK (SH2B3) is one of the negative regulator of JAK-STAT signaling, and its loss of function mutations have been identified in myeloproliferative neoplasms and high-risk BCP-ALL. In addition, the loss of function of LNK has been demonstrated to increase proliferation of B cells in vivo. Based on these findings, we conducted genetic analysis to determine the prognostic impact of LNKin BCP-ALL, and functional analysis to investigate its possible mechanisms.Methods: For genetic analysis, we evaluated diagnostic bone marrow or peripheral blood samples of 164 pediatric BCP-ALL patients treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol along with peripheral blood samples from 9 healthy volunteers. The LNK expression level was determined by qRT-PCR. Deletion of IKZF1 was determined by MLPA, and direct sequencing was employed to detect LNK mutations in patients with IKZF1 deletion. For functional analysis, thrombopoietin (TPO)-dependent BaF3-MPL cells expressing LNK (BaF3-MPL-LNK) was established by retroviral transduction to investigate if LNKexpression levels impact on drug sensitivity for prednisolone (PSL), doxorubicin (DOX) or vincristine (VCR). For growth assay, BaF3-MPL cells with or without LNK were cultured for four days in the presence of TPO and the viable cell number was counted. For drug sensitivity test, BaF3-MPL cells with or without LNK were treated with each drug for 48 hours in the presence of TPO, then the IC50 was calculated. Phospho-specific flow cytometory (Phos-Flow) was performed to measure JAK-STAT activation.Results: The LNK expression levels in pediatric BCP-ALL patients’ samples were significantly lower than in samples from healthy donors (P < 0.01). When analyzing all 164 cases, the expression level of LNK was decreased in relapsed patients but there was no statistical significance (P = 0.067). IKZF1 deletion was found in 25 (15% of all) patients, and in these patients, LNK expression level dose not relate to relapse (P = 0.39). Intriguingly, when patients with known high-risk factor (i.e., IKZF1 deletion or poor response to PSL) were excluded, the expression level of LNK was significantly higher in non-relapsed patients (P < 0.05). In functional assay, we observed inhibition of TPO dependent growth of BaF3-MPL cells by expression of LNK (P < 0.01), consistent with previous reports. Phos-Flow analysis revealed that LNK expression suppressed TPO–induced phosphorylation of STAT5 in BaF3-MPL cells. In drug sensitivity test, we found that IC50 of PSL and DOX were substantially lower in BaF3-MPL-LNK cells from in BaF3-MPL-mock cells (0.70 vs 3.93 nM, P < 0.01 and 0.61 vs 1.14 nM, P < 0.05, respectively). Decline in IC50 of VCR by LNK expression was not statistically significant (1.38 vs 2.45 nM, P = 0.056). We next compared the impact of LNK with Ruxolitinib (RUX), a potent synthesized JAK2 inhibitor. The diminution in IC50 of PSL in BaF3-MPL-mock cells treated with RUX (50 nM) was comparable of that in BaF3-MPL-LNK cells (0.31 nM, combination index (CI) = 0.39), consistent with our hypothesis that LNK is working as a JAK2 inhibitor. Since we identified two amino-acid substitutions in N-terminal proline-rich dimerization domain (R139H) and PH domain (P242S), we also examined their function. Transductions of these genes in BaF3-MPL cells, however, did not alter cell growth, suggesting they are single nucleotide variants.Discussions: Our findings that high LNK expression is associated with low relapse rate in intermediate risk (IKZF1 intact, good PSL response) patients indicate potential of LNK to restrain relapse in such patients, presumably by suppressing JAK-STAT signaling. Since we proved the impact of LNK expression to improve sensitivity of PSL in vitro which was comparable to RUX, RUX could compensate lack of internal LNK expression to induce cell death of BCP-ALL cells. Collectively, targeting JAK-STAT pathway will be promising therapeutic option for intermediate risk BCP-ALL patients with low expression level of LNK. DisclosuresNo relevant conflicts of interest to declare.

Inhibition Of JAK-STAT Pathway As a Therapeutic Option For Myelofibrosis Associated Pulmonary Hypertension

Pulmonary hypertension (PH) is an under-recognized complication of myelofibrosis (MF) occurring in 30% of MF patients and associated with poor survival. Echocardiographic diagnostic findings include; elevated right ventricular systolic pressure (RVSP)>35 mmHg, right atrial (RA) enlargement and increased tricuspid regurgitation velocity (TRV) ≥2.5 m/sec. The pathophysiology of PH in MF has not been elucidated, although in idiopathic PH, the proliferation of pulmonary artery endothelial cells has been linked to activation of STAT3 pathway. Dysregulation of JAK-STAT pathway has been implicated in the pathogenesis of MF. Ruxolitinib, a JAK1/2 inhibitor, was approved for management of splenomegaly and cytokine-mediated symptoms in MF. Furthermore, no specific therapy in the management of MF-associated PH has been established. Given the association between MF and PH and the possible pathophysiologic link mediated by JAK signaling, we prospectively followed 19 patients with MF-associated PH and compared their echocardiographic findings and PH relevant serum biomarker levels (nitric oxide [NO], NT-pro brain natriuretic peptide [NT-proBNP], von Willebrand antigen (vWB), ristocetin co-factor (RCA), and uric acid (UA) pre- and post-ruxolitinib therapy. All categorical data were summarized for frequency, counts and percentages, and the comparison between two groups was performed by two-sample Wilcoxon signed rank test. Among 19 patients (pts), 9 had PMF, 5 post-ET MF, 4 post-PV MF and one CMML-1. In this cohort, 11 were females and 8 were males. The median age of the cohort was 68 years (range, 50-81 years). Fifteen pts were JAK2 V617F positive and 4 were wild-type, 8 were intermediate-1, 4 intermediate-2 and 6 high risk per Dynamic International Prognostic Scoring System-Plus risk grouping. The mean ruxolitinib dose was 10 mg BID (range: 5 mg QOD-20 mg BID]. Median duration of disease was 32 mos (6-164 mos), ruxolitinib duration of treatment was 10 mos (4 -17 mos) and follow-up was 11 mos (6-22 mos). Prior to the initiation of ruxolitinib treatment, NT-pro BNP levels, were measured and found to be elevated in 90% (17/19) of pts. In addition, UA, vWB, and RCA levels were all elevated in 47% (9/19), 24% (4/17), and 12% (2/17) of pts respectively. The strongest correlation among serum biomarkers was between plasma vWB and RCA levels (r2=-0.89, P=<.001). The biomarker most closely associated with elevated NT-pro BNP was UA both in the pre- (r2=-0.53, P=.065) and post-treatment (r2=-0.64, P=.019) settings. Echocardiographic findings by TTE pre- and post ruxolitinib therapy were available for 12 pts (63%). All 12 had documented PH with a mean RVSP of 47.5 mm Hg (42-68) [normal pressure ≤30 mmHg]. Echocardiographic evidence correlated with RCA (r2=-0.64, P= .045) and plasma NT-pro BNP levels (r2=-0.8, P=.013). Ruxolitinib resulted in reductions in NT-pro BNP level (88%) (p=.013), plasma UA levels in (71%), vWB (71%), and RCA (71%) (P=.045). Nitric oxide, a primary regulator of vascular endothelial function is reduced in MF patients with PH compared to normal individuals (median NO, 36 vs 65 pM). Treatment with ruxolitinib resulted in marked increase in NO levels compared to baseline (68 pM vs 36 pM; P=0.04) while no changes in NO levels were observed after treatment with hydroxyurea and lenalidomide (N=10). Treatment with ruxolitinib also resulted in reduction of key cytokines (TNF-α, IL-4, IL-10) that inhibit NO production and induction of cytokines (IFN-γ) that lead to increase in NO synthesis supporting the role of cytokines in PH pathogenesis in MF. Murine studies further supported the role of ruxolitinib in induction of NO levels. Eight normal CD-1 mice were treated with ruxolitinib (50 mg/kg p.o. daily for 5 days for three consecutive cycles with 14 day intervals between each cycle). After the first cycle, NO levels were significantly higher compared to baseline followed by significant increase compared to baseline at cycle 3 (P=.04). In addition, PH mice (Caveoline-1 mice) have been bred and undergoing treatment with ruxolitinib to assess changes in NO levels and its impact in improving of PH. In conclusion, aberrant JAK-STAT signaling in MF mediates PH by dysregulation of NO and cytokines levels which can be restored by therapy with JAK inhibitors. This suggests that inhibition of the JAK-STAT signaling pathway is a novel and viable target for the management of patients with MF-associated PH. Disclosures:No relevant conflicts of interest to declare.

Implication of the JAK-STAT Pathway in Gliomagenesis: A Target for Therapy?

Gliomas, being the most prevalent and aggressive malignancy of the central nervous system, are associated with poor prognosis and very low survival rates. Failure of malignant glioma cells to respond to conventional cancer therapies is attributed to their infiltrating and immunosuppressive phenotype along with increased molecular heterogeneity. Current evidence implicates aberrant JAK-STAT signaling and expression of STAT inhibitors in the molecular pathogenesis of gliomas. This review provides a critical account of recent evidence regarding JAK-STAT signaling components in gliomagenesis, highlighting the potential therapeutic benefits and perspectives of targeting this pathway.

Fine Tuning of Surface CRLF2 Expression and Its Associated Signalling Profile in Childhood B Cell Precursor Acute Lymphoblastic Leukemia

Abstract 1409▪▪This icon denotes a clinically relevant abstractCytokine receptor-like factor 2 (CRLF2) alterations occur in 8% of unscreened children with B cell precursor acute lymphoblastic leukemia (BCP-ALL). The prognostic impact is still controversial and likely to be dependent on different clinical protocols. Biologic studies may facilitate the identification of factors contributing to these discrepant results. Moreover a better understanding of the biochemical mechanism(s) underlying such aberrancies may provide a more effective selection of patients that could benefit of signal transduction inhibitors treatments. In the present study we evaluated CRLF2 genomic rearrangements and the signalling profile involving pJAK, pSTAT5 and pS6 signalling pathways, known to be associated with CRLF2 rearrangements (Tasian SK et al, Blood 2012).A total of 60 (54 consecutive fresh and 6 thawed) diagnostic bone marrow samples from children with BCP-ALL enrolled in AIEOP-BFM-ALL trials were analyzed for CRLF2 surface expression by flow cytometry (FCM). CRLF2 transcript levels as well as CRLF2 aberrations (i.e. P2RY8-CRLF2) were analyzed by molecular methods as recently described (Palmi C et al, Leukemia 2012). TSLP-induced (10 ng/mL for 30 minutes) pSTAT5 and pS6 response were evaluated in CD7-/CD19+/CD10+/CD45low blasts by phosphoflow cytometry in thawed mononuclear cells obtained after ficoll gradient centrifugation.Eight out of 60 (13.3%) samples were FCM CRLF2 ’positive’ (mean value of CRLF2+ cells = 87.5%) with a bright or intermediate fluorescence distribution. However, further cases showed peculiar CRLF2 expression: 2/60 (2.25%) were ’partially positive’ (mean CRLF2+ cells = 2.25%), with two clearly distinguished blast populations (one negative and one positive); 9/60 (15.00%) were ’dim positive’ (mean CRLF2+ cells = 3.24%) showing a clear shift-to-right distribution compared to negative control (normal B lymphocytes CD7-/CD19+/CD10-/CD45++), and 41/60 (68.30%) were fully ’negative’ (CRLF2+ cells <1%). Parallel RT-PCR analysis of CRLF2 expression was performed in 51 samples: all FCM-positive samples were over expressed by PCR (i.e. fold change of >20 compared to the median value of almost 500 samples tested) whilst CRLF2 ’dim positive’ or ’partially positive’ had a PCR fold change of < 20. Interestingly, 29/32 CRLF2 ’negative’ were concordantly negative by PCR, however, in 3/32 samples a fold change of >20 was obtained. Of note, in these 3 discordant cases P2RY8-CRLF2 was detected as a very weak PCR amplification (n=2) or PCR negative (n=1), suggesting the presence of minor rearranged cell populations.Aberrant pSTAT5 response was observed in all CRLF2 ‘positive' analyzed samples (n = 8) compared with CRLF2 ’negative’ cases (n = 9) showing 59.7% and 5.4% of pSTAT5+ cells (means), respectively (p =0.00001). Interestingly CRLF2 ’dim positive’ cases (n = 5) had an intermediate level of pSTAT5 response (mean 18.1%) significantly different compared to both CRLF2 ’positive’ and CRLF2 ’negative’ cases (p <0.05). Our results are concordant with recent findings by Taisan et al, although with some differences. We found that IL-7 (100 ng/mL) induced p-STAT5 response at variable levels in BCP-ALL blasts regardless of CRLF2 expression. Also TSLP-induced pS6 response, in addition to pSTAT5, was aberrant in CRLF2 ’positive’ cases compared to CRLF2 ’negative’ ones (mean 26.9 vs. 0.2, p = 0.0001), yet CRLF2 ’dim positive’ cases showed intermediate mean level of pS6 response being 8.3%. All CRLF2 ’dim positive’ cases were negative (or undetectable) for P2RY8-CRLF2 deletion.In conclusion, we confirm and further extend that TSLP mediated signal transduction induces aberrant JAK-STAT and PI3K-mTOR signaling pathways in CRFL2 mutated or over expressed BCP-ALL cases. Of note, cases with dim positivity of CRLF2 (usually considered negative by standard flow cytometry criteria) and negativity for P2RY8-CRLF2 deletion showed an active pSTAT5 and pS6 signaling at intermediate level between CRLF2 'positive' and CRLF2 'negative'. Whether it refers to the pattern of minor clones or to the presence of additional mechanism(s) driving aberrant signal transduction, are still under investigation. Overall, these findings may be relevant for diagnosis and prognosis in CLRF2 positive BCP-ALLs; they further support that signal transduction inhibitors may have therapeutic relevance in this setting. Disclosures:No relevant conflicts of interest to declare.

The interplay between inhibition of JAK2 and HSP90

A recent article by Weigert et al. published in The Journal of Experimental Medicine described the in vitro generation of synthetic mutations in Janus kinase 2 (JAK 2) that decreased the potency of JAK2 (or JAK1/JAK2) inhibitors in artificial systems. The authors found that heat shock protein 90 (HSP90) inhibitors circumvented the potency shift and suggested that HSP90 inhibition may abrogate JAK inhibitor resistance in these experimental systems. However, the clinical relevance of these laboratory-generated JAK2 mutations, which have not been identified to-date in patients treated with JAK inhibitors, and the therapeutic potential of HSP90 inhibitors in diseases involving aberrant JAK-STAT signaling remain to be determined.

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.

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.

HSP90 is essential for Jak-STAT signaling in classical Hodgkin lymphoma cells

In classical Hodgkin lymphoma (cHL) chemotherapeutic regimens are associated with stagnant rates of secondary malignancies requiring the development of new therapeutic strategies. We and others have shown that permanently activated Signal Transducer and Activator of Transcription (STAT) molecules are essential for cHL cells. Recently an overexpression of heat-shock protein 90 (HSP90) in cHL cells has been shown and inhibition of HSP90 seems to affect cHL cell survival. Here we analysed the effects of HSP90 inhibition by geldanamycin derivative 17-AAG or RNA interference (RNAi) on aberrant Jak-STAT signaling in cHL cells. Treatment of cHL cell lines with 17-AAG led to reduced cell proliferation and a complete inhibition of STAT1, -3, -5 and -6 tyrosine phosphorylation probably as a result of reduced protein expression of Janus kinases (Jaks). RNAi-mediated inhibition of HSP90 showed similar effects on Jak-STAT signaling in L428 cHL cells. These results suggest a central role of HSP90 in permanently activated Jak-STAT signaling in cHL cells. Therapeutics targeting HSP90 may be a promising strategy in cHL and other cancer entities associated with deregulated Jak-STAT pathway activation.