We have previously described critical and non-redundant roles for the PI3K p110δ during the activation and differentiation of naïve T cells and p110δ inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110δ activity is required for IFNγ production. Moreover, acute inhibition of p110δ inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110δ played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked TCR-induced PI3K signaling by both naïve and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110δ inhibition as naïve T cells and show that mouse models accurately predict p110δ function in human T cells. There is therefore a strong rationale for p110δ inhibitors to be considered for therapeutic use in T cell-mediated autoimmune and inflammatory diseases.

Mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) occur in most grade 2 and 3 gliomas, secondary glioblastomas, and a subset of acute myelogenous leukemias, but have not been detected in other tumor types. The mutations occur at specific arginine residues, and result in the acquisition of a novel enzymatic activity that converts 2-oxoglutarate to D-2-hydroxyglutarate. This study reports IDH1 and IDH2 genotyping results from a set of lymphomas which included a large set of peripheral T-cell lymphomas (PTCL). IDH2 mutations were identified in approximately 20% of angioimmunoblastic T-cell lymphomas (AITL), but not in other PTCL entities. These results were confirmed in an independent set of AITL patients, where the IDH2 mutation rate was approximately 45%. This is the second common genetic lesion identified in AITL after TET2, and extends the number of neoplastic diseases where IDH1 and IDH2 mutations may play a role.

Currently, there are no reliable red blood cells invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P. vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin treated P. vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P. vivax isolates freshly collected from patients in Thailand.

In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior.

CCRL2 is a heptahelic transmembrane receptor that shows the highest degree of homology with CCR1, an inflammatory chemokine receptor. CCRL2 mRNA was rapidly (30 min) and transiently (2-4 hrs) regulated during dendritic cell (DC) maturation. Protein expression paralleled RNA regulation. In vivo, CCRL2 was expressed by activated DC and macrophages, but not by eosinophils and T cells. CCRL2−/− mice showed normal recruitment of circulating DC into the lung but a defective trafficking of antigen-loaded lung DC to mediastinal lymph nodes. This defect was associated to a reduction in lymph node cellularity and reduced priming of Th2 response. CCRL2−/− mice were protected in a model of OVA-induced airway inflammation with reduced leukocyte recruitment in the BAL (eosinophils and mononuclear cells) and reduced production of the Th2 cytokines IL-4 and IL-5 and chemokines CCL11 and CCL17. The central role of CCRL2 deficiency in DC was supported by the fact that adoptive transfer of CCRL2−/− antigen-loaded DC in wild type animals recapitulated the phenotype observed in knock out mice. These data show a nonredundant role of CCRL2 in lung DC trafficking and propose a role for this receptor in the control of excessive airway inflammatory responses.

Platelets undergo a series of actin-dependent morphologic changes when activated by thrombin receptor activating peptide (TRAP) or when spreading on glass. Polymerization of actin results in the sequential formation of filopodia, lamellipodia, and stress fibers, but the molecular mechanisms regulating this polymerization are unknown, The Arp2/3 complex nucleates actin polymerization in vitro and could perform this function inside cells as well. To test whether Arp2/3 regulated platelet actin polymerization, we used recombinant Arp2 protein (rArp2) to generate Arp2-specific antibodies (αArp2). Intact and Fab fragments of αArp2 inhibited TRAP-stimulated actin-polymerizing activity in platelet extracts as measured by the pyrene assay. Inhibition was reversed by the addition of rArp2 protein. To test the effect of Arp2/3 inhibition on the formation of specific actin structures, we designed a new method to permeabilize resting platelets while preserving their ability to adhere and to form filopodia and lamellipodia on exposure to glass. Inhibition of Arp2/3 froze platelets at the rounded, early stage of activation, before the formation of filopodia and lamellipodia. By morphometric analysis, the proportion of platelets in the rounded stage rose from 2.85% in untreated to 63% after treatment with αArp2. This effect was also seen with Fab fragments and was reversed by the addition of rArp2 protein. By immunofluorescence of platelets at various stages of spreading, the Arp2/3 complex was found in filopodia and lamellipodia. These results suggest that activation of the Arp2/3 complex at the cortex by TRAP stimulation initiates an explosive polymerization of actin filaments that is required for all subsequent actin-dependent events.

The identity of the cells responsible for the initiation and maintenance of multiple myeloma (MM) remains unclear largely because of the difficulty growing MM cells in vitro and in vivo. MM cell lines and clinical specimens are characterized by malignant plasma cells that express the cell surface antigen syndecan-1 (CD138); however, CD138 expression is limited to terminally differentiated plasma cells during B-cell development. Moreover, circulating B cells that are clonally related to MM plasma cells have been reported in some patients with MM. We found that human MM cell lines contained small (< 5%) subpopulations that lacked CD138 expression and had greater clonogenic potential in vitro than corresponding CD138+ plasma cells. CD138− cells from clinical MM samples were similarly clonogenic both in vitro and in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, whereas CD138+ cells were not. Furthermore, CD138− cells from both cell lines and clinical samples phenotypically resembled postgerminal center B cells, and their clonogenic growth was inhibited by the anti-CD20 monoclonal antibody rituximab. These data suggest that MM “stem cells” are CD138− B cells with the ability to replicate and subsequently differentiate into malignant CD138+ plasma cells.

Hepcidin is the master regulator of iron homeostasis. In the liver, iron-dependent hepcidin activation is regulated through Bmp6 and its membrane receptor hemojuvelin (Hjv) whereas, in response to iron deficiency, hepcidin repression seems to be controlled by a pathway involving the serine protease matriptase-2 (encoded by Tmprss6). To determine the relationship between Bmp6 and matriptase-2 pathways, Tmprss6−/− mice (characterized by increased hepcidin levels and anemia) and Bmp6−/− mice (exhibiting severe iron overload due to hepcidin deficiency) were intercrossed. We showed that loss of Bmp6 decreased hepcidin levels, increased hepatic iron and, importantly, corrected hematological abnormalities in Tmprss6−/− mice. This suggests that elevated hepcidin levels in patients with familial iron-refractory iron deficiency anemia are due to excess signaling through the Bmp6/Hjv pathway.

Oncogenic c-Myc is known to balance excessive proliferation by apoptosis that can be triggered by p53-dependent and p53-independent signaling networks. Here, we provide evidence that the “BH3-only” pro-apoptotic Bcl-2 family members Bmf and Bad are potent antagonists of c-Myc-driven B cell lymphomagenesis. Tumor formation was preceded by accumulation of preneoplastic pre-B and immature IgM+ B cells in hematopoietic organs of Eμ-myc/bmf−/− mice, whereas Eμ-myc/bad−/− mice showed an increase of pre-B cells limited to the spleen. While loss of Bad had no impact on the tumor immunophenotype, Bmf-deficiency favored the development of IgM+ B cell over pre-B cell tumors. This phenomenon was due to a strong protection of immature IgM+ B cells from oncogene-driven apoptosis caused by loss of bmf and c-Myc-induced repression of Bmf expression in premalignant pre-B cells. Steady-state levels of B cell apoptosis were also reduced in the absence of Bad, in support of its role as a sentinel for trophic factor-deprivation. Loss of Bmf reduced the pressure to inactivate p53, whereas Bad-deficiency did not, identifying Bmf as a novel component of the p53-independent tumor suppressor pathway triggered by c-Myc.

Constitutively activating internal tandem duplications (ITD) of FLT3 (FMS-like tyrosine kinase 3) are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD–mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapototic mechanisms of 2 small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras–mitogen-activated protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition.

Mouse innate-like B cells are a heterogeneous collection of multifunctional cells that control infection, play housekeeping roles, contribute to adaptive immunity, and suppress inflammation. We show that, amongst leukocytes, chemokine internalisation by the D6 receptor is a unique and universal feature of all known innate-like B cell populations and, to our knowledge, the most effective unifying marker of these cells. Moreover, we identify novel D6active B1 cell subsets, including those we term B1d, which lack CD5 and CD11b but exhibit typical B1 cell properties, including spontaneous ex vivo production of IgM, interleukin-10, and anti-phosphorylcholine antibody. The unprecedented opportunity to examine D6 on primary cells has allowed us to clarify its ligand specificity and show that, consistent with a scavenging role, D6 internalises chemokines but cannot induce Ca2+ fluxes or chemotaxis. Unexpectedly, however, D6 can also suppress the function of CXCR5, a critical chemokine receptor in innate-like B cell biology. This is associated with a reduction in B1 cells and circulating class-switched anti-phosphorylcholine antibody in D6-deficient mice. Thus, we identify a unifying marker of innate-like B cells; describe novel B1 cell subsets; reveal a dual role for D6; and provide the first evidence of defects in resting D6-deficient mice.

Extravascular coagulation leading to fibrin deposition accompanies many immune and inflammatory responses. Although recognized by pathologists for decades, and probably pathologic under certain conditions, the physiologic functions of extravascular coagulation remain to be fully defined. This study demonstrates that thrombin can activate macrophage adhesion and prompt interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production in vivo. Peritoneal macrophages were elicited with thioglycollate (TG) and then activated in situ, either by intraperitoneal injection of lipopolysaccharide (LPS) or by injection of antigen into mice bearing antigen-primed T cells. Others previously established that such treatments stimulate macrophage adhesion to the mesothelial lining of the peritoneal cavity. The present study demonstrates that thrombin functions in this process, as macrophage adhesion was suppressed by Refludan, a highly specific thrombin antagonist, and induced by direct peritoneal administration of purified thrombin. Although recent studies established that protease activated receptor 1 (PAR-1) mediates some of thrombin’s proinflammatory activities macrophage adhesion occurred normally in PAR-1–deficient mice. However, adhesion was suppressed in fibrin(ogen)-deficient mice, suggesting that fibrin formation stimulates macrophage adhesion in vivo. This study also suggests that fibrin regulates chemokine/cytokine production in vivo, as direct injection of thrombin stimulated peritoneal accumulation of IL-6 and MCP-1 in a fibrin(ogen)-dependent manner. Given that prior studies have clearly established inflammatory roles for PAR-1, thrombin probably has pleiotropic functions during inflammation, stimulating vasodilation and mast cell degranulation via PAR-1, and activating cytokine/chemokine production and macrophage adhesion via fibrin(ogen).

Th17 cells have never been explored in human GvHD. We studied the correlation between the presence of Th17 cells in the gastro-intestinal (GI) tract and in the skin with histological and clinical parameters. We first analyzed a cohort of 40 patients with suspected GvHD of the GI tract. TNF, TNF receptors, and Fas expression, and apoptotic cell, CD4+IL-17+ cells (Th17) and CD4+Foxp3+ cells (Treg) were quantified. A Th17/Treg ratio<1 correlated both with the clinical diagnosis (p<0.001), and ≥ 2 pathologic grade (p<0.001). A Th17/Treg ratio<1 also correlated with the intensity of apoptosis of epithelial cells (p=0.03), Fas expression in the cellular infiltrate (p=0.003), TNF and TNF receptors expression (p<0.001). We then assessed Th17/Treg ratio in two other independent cohorts; a second cohort of 30 patients and confirmed that Th17/Treg ratio<1 correlated with the pathological grade of GI GvHD. Finally 15 patients with skin GvHD and 11 patients with skin rash but without pathological GvHD were studied. Results in this third cohort of patients with skin disease confirmed those found in patients with GI GvHD. These analyses in 96 patients suggest that Th17/Treg ratio could be a sensitive and specific pathological in situ biomarker of GvHD.

In the past 5 years we have witnessed significant advances in both the diagnostic process and optimal therapy for patients with essential thrombocythemia (ET). Insights into the underlying molecular mechanisms have been accompanied by the development of new diagnostic tests and by an improved understanding of the relationship between ET and other related myeloproliferative neoplasms, such as polycythemia vera and primary myelofibrosis. In the first part of this review, we describe how recent molecular and histologic studies can be integrated into a streamlined diagnostic process that is applicable to everyday clinical practice. We also address areas of current diagnostic controversy, including heterogeneity within ET and the phenotypic overlap between ET, polycythemia vera, and primary myelofibrosis. In the second part, we provide an overview of our current approach to the treatment of ET, including risk stratification, choice of cytoreductive agent, and a consideration of special situations such as the pregnant or perioperative patient. Areas of controversy discussed include the identification of those at high risk of complications and therapeutic decisions in the younger patient.

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F–positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2V617F mice develop reduced numbers of lineage−Sca-1+c-Kit+ cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2V617F mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

The 46/1 JAK2 haplotype predisposes to V617F-positive myeloproliferative neoplasms, but the underlying mechanism is obscure. We analyzed essential thrombocythemia patients entered into the PT-1 studies and, as expected, found that 46/1 was overrepresented in V617F-positive cases (n = 404) versus controls (n = 1492, P = 3.9 × 10−11). The 46/1 haplotype was also overrepresented in cases without V617F (n = 347, P = .009), with an excess seen for both MPL exon 10 mutated and V617F, MPL exon 10 nonmutated cases. Analysis of further MPL-positive, V617F-negative cases confirmed an excess of 46/1 (n = 176, P = .002), but no association between MPL mutations and MPL haplotype was seen. An excess of 46/1 was also seen in JAK2 exon 12 mutated cases (n = 69, P = .002), and these mutations preferentially arose on the 46/1 chromosome (P = .029). No association between 46/1 and clinical or laboratory features was seen in the PT-1 cohort either with or without V617F. The excess of 46/1 in JAK2 exon 12 cases is compatible with both the “hypermutability” and “fertile ground” hypotheses, but the excess in MPL-mutated cases argues against the former. No difference in sequence, splicing, or expression of JAK2 was found on 46/1 compared with other haplotypes, suggesting that any functional difference of JAK2 on 46/1, if it exists, must be relatively subtle.

According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1–mediated survival was as efficient as that induced by the cytokine IFN-β and provided an additive, increased delay in apoptosis when given in combination with IFN-β. VCAM-1 delivered its antiapoptotic effect through binding the integrin α9β1. The α9β1 signaling pathway shares significant features with the IFN-β survival signaling pathway, requiring PI3 kinase, NF-κB activation, as well as de novo protein synthesis, but the kinetics of NF-κB activation by VCAM-1 were slower and more sustained compared with IFN-β. This study demonstrates a novel functional role for α9β1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues.

The HLX gene encoding a diverged homeobox transcription factor has been found to be up-regulated by vascular endothelial growth factor-A (VEGF-A) in endothelial cells. We have now investigated the gene repertoire induced by HLX and its potential biologic function. HLX strongly increased the transcripts for several repulsive cell-guidance proteins including UNC5B, plexin-A1, and semaphorin-3G. In addition, genes for transcriptional repressors such as HES-1 were up-regulated. In line with these findings, adenoviral overexpression of HLX inhibited endothelial cell migration, sprouting, and vessel formation in vitro and in vivo, whereas proliferation was unaffected. This inhibition of sprouting was caused to a significant part by HLX-mediated up-regulation of UNC5B as shown by short hairpin RNA (shRNA)–mediated down-modulation of the respective mRNA. VEGF-A stimulation of endothelial cells induced elevated levels of HLX over longer time periods resulting in especially high up-regulation of UNC5B mRNA as well as an increase in cells displaying UNC5B at their surface. However, induction of HLX was strongly reduced and UNC5B up-regulation completely abrogated when cells were exposed to hypoxic conditions. These data suggest that HLX may function to balance attractive with repulsive vessel guidance by up-regulating UNC5B and to down-modulate sprouting under normoxic conditions.

It is currently considered that idiopathic minimal change nephrotic syndrome (I-MCNS) is an immune-mediated glomerular disease. Its association with classical Hodgkin lymphoma (cHL-MCNS) suggests a molecular link between these two diseases, which remains to be elucidated. We analyzed the expression of c-mip (cmaf inducing protein) in lymphomatous tissues and kidney biopsies of patients with cHL-MCNS (n=8) and in lymphomatous tissues of isolated cHL (n=9). Because c-mip affects the regulatory loop involving Fyn, we investigated possible structural defects in this signaling pathway, using laser capture microdissection, RT-PCR and Western-blotting. We found that c-mip was selectively expressed in Hodgkin and Reed-Sternberg (HRS) cells and podocytes of patients with cHL-MCNS but is undetectable in patients with isolated cHL. We demonstrated that c-mip was specifically involved in the negative regulation of early proximal signaling through its interaction with PAG and Fyn. We showed that the upregulation of c-mip in cHL-MCNS was associated with a possible Fyn defect in HRS cells and podocytes, while Fyn was normally expressed in isolated cHL and normal podocytes. Moreover, we showed that c-mip was upregulated in Fyn-deficient podocytes. C-mip may be a useful marker of cHL-MCNS and its induction reflects the dysregulation of proximal signaling.

The prognostic impact of minimal residual disease (MRD) was analyzed in 259 patients with mantle cell lymphoma (MCL) treated within two randomized trials of the European MCL Network (MCL Younger and MCL Elderly trial). After rituximab-based induction treatment 106/190 evaluable patients (56%) achieved a molecular remission (MR) based upon blood and/or bone marrow (BM) analysis. MR resulted in a significantly improved response duration (RD) (87% vs. 61% patients in remission at 2 years, p=0.0043) and emerged to be an independent prognostic factor for RD (HR 0.4, 95% CI 0.1–0.9, p=0.027). MR was highly predictive for prolonged RD independent of clinical response (CR, CRu, PR) (RD at 2 years: 100% in BM MRD-negative CR and 88% in BM MRD-negative CRu/PR, compared to 78% in BM MRD-positive CR and 53% in BM MRD-positive CRu/PR, p=0.0015). Sustained MR during the post-induction period was predictive for outcome in MCL Younger after ASCT (RD at 2 years 100% vs. 65%, p=0.0007) and during maintenance in MCL Elderly (RD at 2 years: 76% vs. 36%, p=0.015). ASCT in MCL Younger patients increased the proportion of patients in MR from 55% prior to high dose therapy to 72% thereafter. Sequential MRD monitoring is a powerful predictor for treatment outcome in MCL.

Fibrinolysis and pericellular proteolysis depend on molecular co-assembly of plasminogen and its activator on cell, fibrin or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular co-assembly on the same surface. First, we demonstrate that despite impaired binding of Glu-plasminogen to the cell membrane by ε-aminocaproic acid (ε-ACA) or by a lysine-binding site-specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell-derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A two-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biological surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.

Phosphorylated signal transducer and activator of transcription 5 (STAT5) is a biomarker and potential molecular target for hematologic malignancies. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5aS711F) requires the N-domain, but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5abnull/null primary mast cells that relative to wild-type STAT5a, STAT5a lacking the N-domain (STAT5aΔN) ineffectively protected against cytokine withdrawal-induced cell death. Both STAT5a and STAT5aΔN bound to a site in the bcl-2 gene and both bound near the microRNA 15b/16 cluster. However, only STAT5a could effectively induce bcl-2 mRNA and reciprocally suppress miR15b/16 leading to maintained bcl-2 protein levels. After retroviral complementation of STAT5abnull/null fetal liver cells and transplantation, persistently active STAT5aS711F lacking the N-domain (STAT5aΔNS711F) was insufficient to protect c-Kit+Lin−Sca-1+ (KLS) cells from apoptosis and unable to induce bcl-2 expression, whereas STAT5aS711F caused robust KLS cell expansion, induction of bcl-2, and lethal MPD. Severe attenuation of MPD by STAT5aΔNS711F was reversed by H2k/bcl-2 transgenic expression. Overall, these studies define N-domain–dependent survival signaling as an Achilles heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain–mediated regulation of bcl-2 family members.

Biopsies and cell lines of NK/T-cell lymphoma, nasal-type (NKTCL) were subject to combined gene expression profiling and array-based comparative genomic hybridization analyses. Compared to PTCL, NOS, NKTCL had higher transcript levels for NK-cell markers and cytotoxic molecules, especially granzyme H, a novel sensitive biomarker of NKTCL. Compared to normal NK cells, tumors were closer to activated than resting cells and overexpressed several genes related to vascular biology, EBV-induced genes and PDGFRA. Notably, PDGFRα and its phosphorylated form were confirmed at the protein level, and in vitro the MEC04 NKTCL-cell line was sensitive to imatinib. Deregulation of the AKT, JAK-STAT and NF-κB pathways suggested by bioinformatical analysis, was corroborated by nuclear expression of phosphorylated AKT, STAT3 and RelA in NKTCL, and several deregulated genes in these pathways mapped to regions of recurrent copy number aberrations (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). Several features of NKTCL uncovered by this analysis (overexpression of VEGFA and its receptor KDR by the tumor cells, overexpression of MET-HGF) suggest perturbation of angiogenic pathways. Integrative analysis also evidenced deregulation of the tumor suppressor HACE1 in the frequently deleted 6q21 region. This study highlights emerging oncogenic pathways in NKTCL and identifies novel diagnostic and therapeutic targets.