Abnormal Expression and Function of the Lnk Adaptor Protein in Myeloproliferative Neoplasms (MPN).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2897-2897
Author(s):  
Jean-Jacques Kiladjian ◽  
Fanny Baran-Marszak ◽  
Christophe Desterke ◽  
Bruno Cassinat ◽  
Hajer Magdoud ◽  
...  
Keyword(s):  
Cell Lines ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Terminal Region ◽  
Interaction Site ◽  
Cd34 Cells ◽  
And Function ◽  
Mutant Forms ◽  
Jak2v617f Allele Burden

Abstract Abstract 2897 Poster Board II-873 Introduction: We have shown in a deficient mouse model that the adaptor protein Lnk had an important role as negative regulator of cytokine signaling during hematopoiesis (Velazquez et al., J. Exp Med 2002). Lnk-/- animals display abnormal megakaryopoiesis sharing many features with that found in MPN patients. This phenotype is due to loss of Lnk inhibition of thrombopoietin (TPO)-mediated JAK2 activation (Tong et al., J Exp Med 2004). Recent studies have shown that Lnk, when expressed in hematopoietic cell lines, could bind and regulate two mutant proteins found in MPNs, JAK2V617F and MPLW515L. However, the role of Lnk in MPN pathogenesis is still unclear. In the present study, we studied in detail both Lnk expression and function in MPNs. Patients and methods: The study included a total of 82 MPN patients (pts), including 41 essential thrombocytemia (ET), 29 polycythemia vera (PV) and 12 primary myelofibrosis (PMF). Lnk expression was assessed by quantitative RT-PCR. Biochemical and cellular analyses of Lnk and JAK2 interaction were carried out using co-immunoprecipitation, GST pull-down and proliferation assays on primary hematopoietic cells and cell lines expressing either wild-type (WT) or mutant forms of both Lnk and JAK2. Results: Lnk mRNA was clearly overexpressed in platelets and CD34+ cells of most MPN pts compared to controls (P=0.005 and P=0.03, respectively). Moreover, this increased Lnk expression strongly correlated with JAK2V617F allele burden (P=0.02). In contrast, Lnk mRNA levels were reduced in the 18 pts treated with interferon-α compared to the 34 pts treated with hydroxyurea (P=0.04). TPO specifically upregulated Lnk expression at both mRNA and protein levels in both primary and UT7/Mpl megakaryocytic (MK) cells. Analysis of TPO-stimulated platelets from ET patients revealed the existence of a new interaction site between Lnk and JAK2 located in the N-terminal region of Lnk, in addition to the previously known interaction mediated by Lnk SH2 domain. This interaction resulted in Lnk phosphorylation. In JAK2V617F expressing platelets or cell lines, we observed both increased phosphorylation of Lnk, and stronger binding of JAK2 to the N-terminal region of Lnk compared to WT-JAK2 cells. Overexpression of Lnk in JAK2V617F cells showed a dose dependent growth inhibition, as seen in JAK2 WT cells. In addition, overexpression of various mutant forms of Lnk showed that this inhibition required a fully functional SH2 domain. Finally, expression of either WT or mutant forms of Lnk also demonstrated the crucial role of Lnk SH2 domain in growth inhibition of myeloid and MK progenitors in Lnk-/- hematopoietic cells. Conclusion: This first study of a large cohort of 82 patients allowed us to investigate the role of Lnk in MPN: (1) Lnk mRNA was found to be significantly overexpressed in MPN derived platelets and CD34+ cells, and correlated with JAK2V617F allele burden. (2) Lnk expression is upregulated by TPO, an effect likely mediated by JAK2 activation. (3) The Lnk SH2 domain plays a major role in the down-regulation of both normal and MPN-derived hematopoiesis. (4) We describe here a novel interaction site between the N-terminal region of Lnk and JAK2. Stronger interaction of the JAK2V617F mutant form with this N-terminal binding site may account for the dysregulated hematopoiesis observed in MPN patients despite Lnk overexpression. Disclosures: No relevant conflicts of interest to declare.

The transmembrane adaptor protein NTAL limits mast cell chemotaxis toward prostaglandin E2

2018 ◽  
Vol 11 (556) ◽  
pp. eaao4354 ◽  
Author(s):  
Ivana Halova ◽  
Monika Bambouskova ◽  
Lubica Draberova ◽  
Viktor Bugajev ◽  
Petr Draber
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
And Function ◽  
Cell Chemotaxis

Chemotaxis of mast cells is one of the crucial steps in their development and function. Non–T cell activation linker (NTAL) is a transmembrane adaptor protein that inhibits the activation of mast cells and B cells in a phosphorylation-dependent manner. Here, we studied the role of NTAL in the migration of mouse mast cells stimulated by prostaglandin E2 (PGE2). Although PGE2 does not induce the tyrosine phosphorylation of NTAL, unlike IgE immune complex antigens, we found that loss of NTAL increased the chemotaxis of mast cells toward PGE2. Stimulation of mast cells that lacked NTAL with PGE2 enhanced the phosphorylation of AKT and the production of phosphatidylinositol 3,4,5-trisphosphate. In resting NTAL-deficient mast cells, phosphorylation of an inhibitory threonine in ERM family proteins accompanied increased activation of β1-containing integrins, which are features often associated with increased invasiveness in tumors. Rescue experiments indicated that only full-length, wild-type NTAL restored the chemotaxis of NTAL-deficient cells toward PGE2. Together, these data suggest that NTAL is a key inhibitor of mast cell chemotaxis toward PGE2, which may act through the RHOA/ERM/β1-integrin and PI3K/AKT axes.

scholarly journals OsJAZ13 Negatively Regulates Jasmonate Signaling and Activates Hypersensitive Cell Death Response in Rice

2020 ◽  
Vol 21 (12) ◽  
pp. 4379
Author(s):  
Xiujing Feng ◽  
Lei Zhang ◽  
Xiaoli Wei ◽  
Yun Zhou ◽  
Yan Dai ◽  
...  
Keyword(s):  
Cell Death ◽  
Splice Variants ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Hypersensitive Cell Death ◽  
Cell Death Response ◽  
Jaz Proteins ◽  
Localization Analysis ◽  
And Function

Jasmonate ZIM-domain (JAZ) proteins belong to the subgroup of TIFY family and act as key regulators of jasmonate (JA) responses in plants. To date, only a few JAZ proteins have been characterized in rice. Here, we report the identification and function of rice OsJAZ13 gene. The gene encodes three different splice variants: OsJAZ13a, OsJAZ13b, and OsJAZ13c. The expression of OsJAZ13 was mainly activated in vegetative tissues and transiently responded to JA and ethylene. Subcellular localization analysis indicated OsJAZ13a is a nuclear protein. Yeast two-hybrid assays revealed OsJAZ13a directly interacts with OsMYC2, and also with OsCOI1, in a COR-dependent manner. Furthermore, OsJAZ13a recruited a general co-repressor OsTPL via an adaptor protein OsNINJA. Remarkably, overexpression of OsJAZ13a resulted in the attenuation of root by methyl JA. Furthermore, OsJAZ13a-overexpressing plants developed lesion mimics in the sheath after approximately 30–45 days of growth. Tillers with necrosis died a few days later. Gene-expression analysis suggested the role of OsJAZ13 in modulating the expression of JA/ethylene response-related genes to regulate growth and activate hypersensitive cell death. Taken together, these observations describe a novel regulatory mechanism in rice and provide the basis for elucidating the function of OsJAZ13 in signal transduction and cell death in plants.

scholarly journals The N-Terminus ofDictyosteliumScar Interacts with Abi and HSPC300 and Is Essential for Proper Regulation and Function

2007 ◽  
Vol 18 (5) ◽  
pp. 1609-1620 ◽  
Author(s):  
Diana Caracino ◽  
Cheryl Jones ◽  
Mark Compton ◽  
Charles L. Saxe
Keyword(s):  
Actin Polymerization ◽  
Terminal Region ◽  
Wild Type ◽  
Large Molecular Weight ◽  
Weight Protein ◽  
And Function ◽  
Necessary And Sufficient

Scar/WAVE proteins, members of the conserved Wiskott-Aldrich syndrome (WAS) family, promote actin polymerization by activating the Arp2/3 complex. A number of proteins, including a complex containing Nap1, PIR121, Abi1/2, and HSPC300, interact with Scar/WAVE, though the role of this complex in regulating Scar function remains unclear. Here we identify a short N-terminal region of Dictyostelium Scar that is necessary and sufficient for interaction with HSPC300 and Abi in vitro. Cells expressing Scar lacking this N-terminal region show abnormalities in F-actin distribution, cell morphology, movement, and cytokinesis. This is true even in the presence of wild-type Scar. The data suggest that the first 96 amino acids of Scar are necessary for participation in a large-molecular-weight protein complex, and that this Scar-containing complex is responsible for the proper localization and regulation of Scar. The presence of mis-regulated or unregulated Scar has significant deleterious effects on cells and may explain the need to keep Scar activity tightly controlled in vivo either by assembly in a complex or by rapid degradation.

HSP27 Protects Malignant Hematopoietic Cells Against VP-16-Induced Apoptosis through Modulation of P38 and C-JUN.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1276-1276
Author(s):  
Hein Schepers ◽  
Marjan Geugien ◽  
Marco van der Toorn ◽  
Anton L. Bryantsev ◽  
Harm H. Kampinga ◽  
...  
Keyword(s):  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
Hsp27 Expression ◽  
Cd34 Cells ◽  
Induced Apoptosis ◽  
And Function ◽  
Jnk Activation

Abstract In the present study, expression and function of Heat Shock Protein 27 (HSP27) was analyzed in acute myeloid leukemia (AML), since HSP27 expression is linked to unfavourable prognosis. HSP27 protein was predominantly expressed in monocytic blasts (M4-M5, 91%, N = 11) and absent in myeloid leukemic blasts (M1-M2, N = 5). A similar lineage restricted expression was observed in normal hematopoietic cells: high expression in normal CD34+ cells and monocytes, and absent in granulocytes. To study the functional role of HSP27, RNA interference (RNAi) studies were performed in the leukemic TF-1 cell line. These experiments demonstrated a twofold increase in VP-16-induced apoptosis after HSP27 siRNA. In contrast, CD95 Fas-induced apoptosis remained the same, as a result of CD95 Fas-mediated upregulation of HSP27. Additional investigations demonstrated that the increased VP-16-induced apoptosis after HSP27 RNAi, was associated with an enhanced phosphorylation of p38 and c-Jun. This VP-16-induced phosphorylation was subsequently followed by the release of cytochrome c into the cytoplasm, which increased twofold after siRNA treatment. These results indicate that HSP27 plays an important role in the protection against VP-16-induced apoptosis through the modulation of p38 and JNK activation, probably through interference with DAXX-mediated ASK1 activation. This was further underscored by co-immunoprecipitation studies, demonstrating complex formation of DAXX and HSP27 in an ASK1-dependent manner. However, in the investigated AML samples, VP-16-mediated apoptosis correlated moderately with HSP27 expression, although HSP27 was highly expressed and phosphorylated and activated in primitive monocytic AML blasts. This is likely due to the co-expression of p21Waf1/Cip1, which is in the majority of the monocytic AML M4-M5 blasts constitutively localised in the cytoplasm and interferes with apoptosis via the DAXX-ASK1-dependent pathway. Preliminary data indicate that overexpression of a cytoplasmic form of p21 is able to reduce the VP-16-induced apoptosis after RNAi for HSP27 as compared to controls, suggesting a predominant anti-apoptotic role of p21 over HSP27. In summary, we demonstrate a role for HSP27 in the survival of leukemic cells by modulation of the DAXX/p38/JNK apoptosis pathway. This survival advantage can further be promoted by the co-expression of cytoplasmic localised p21Waf1/Cip1 protein, indicating that strategies in AML treatment should be focused on targeting multiple signal transduction pathways.

scholarly journals Disabled Is a Putative Adaptor Protein That Functions during Signaling by the Sevenless Receptor Tyrosine Kinase

1998 ◽  
Vol 18 (8) ◽  
pp. 4844-4854 ◽  
Author(s):  
Ngocdiep Le ◽  
Michael A. Simon
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathway ◽  
Receptor Tyrosine Kinase ◽  
Biochemical Analysis ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Sevenless Receptor Tyrosine Kinase ◽  
Ptb Domain

ABSTRACT DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by thesevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

A decreased Level of Shp1 provides an additive survival advantage to the Ph+ Cells of CML Patients and may account for Resistance to Imatinib Treatment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3186-3186
Author(s):  
Nicola Esposito ◽  
Irene Colavita ◽  
Fabrizio Quarantelli ◽  
Barbara Izzo ◽  
Luigia Luciano ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Mapk Pathway ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
The Other ◽  
Receptor Protein ◽  
Imatinib Treatment ◽  
Tyrosine Residues ◽  
Knock Down

Abstract Although rare in chronic phase myeloid leukemia (CML), primary or acquired resistance to the treatment with tyrosine kinase inhibitors (TKI) may be observed in the advanced phases of disease. Bcr/Abl related resistance has been well described, while the other mechanisms of resistance are poorly understood. In this study, we investigated the role of two SH2-containing, non-receptor protein tyrosine phosphatases (Shp1 and Shp2) in the resistance to Imatinib (Ima). To this aim, we have first used, as model system, a couple of Ima-sensitive (KCL22s) and Ima-resistant (KCL22r) KCL22 cell lines. In these cells, Ima resistance is independent by the oncogenic Bcr/Abl activity. We have found a very low level of Shp1 (both mRNA and protein), a protein with a tumour suppressor activity, in the KCL22r resistant cells, when compared to KCL22s sensitive cells. We have al shown the down-regulation of this gene to be related to the methylation level of SHP1 promoter. Indeed, 5-Azacytidine (5-AC) treatment, along with demethylation of the promoter region, re-induced expression of Shp1 in KCL22r. That treatment also re-established the Ima sensitivity, i.e. Ima growth inhibition, in these cells. At molecular level, the restored Ima sensitivity was associated to a significant reduction of phosphorylation of both STAT3 and ERK1/2. To better understand the functional role of Shp1, we carried out mass spectrometry to search for Shp1-binding proteins, and found that Shp1 interacts in these cells with Shp2, a protein phosphatase well known as positive regulator of oncogenic pathways, including the Ras/MAPK pathway. Gain-of-function mutations have been described in various hemopoietic neoplasias including Juvenile Chronic Myelomonocytic Leukemia. In Ph+ cells, oncogenic Bcr/Abl protein activates Shp2 through Gab2, an adaptor protein that, once phosphorylated is able to bind SH2 domain of Shp2. Through complex interactions that may involve the two carboxy-terminal tyrosine residues (542 and 580) Shp2 is also a signal transducer of growth factor receptor. We hypothesized that, Shp1, through dephosphorylation, might modulate the activity of Shp2 and constitute an important mechanism of Ima resistance. Knock-down of Shp1 in KCL22s cell line resulted in complete phosphorylation of Shp2 both 542 and 580 tyrosine residues and in its reduced sensitivity to the drug, thus supporting the role of this protein in Ima sensitivity. On the other hand, knock-down of Shp2 in KCL22r, that shows low Shp1 level, resulted in growth inhibition, restored Ima sensitivity and is associated to a significant reduction of phosphorylation of both STAT3 (60%) and ERK1/2 (70%). The data on primary cells support the role of Shp1 in Ima resistance in patients. Indeed, we analyzed 60 CML patients classified, according to the ENL definitions, as optimal (n =35), suboptimal (n=17) Ima responder, and primary (n=5) or secondary resistant (n=3) to Ima. The levels of Shp1 mRNA were significantly reduced in resistant patients [ratio of SHP1/ABL 3.2 ± 1.04, (mean±SD), *p<0.05] when compared to the suboptimal (3.8±1.54) and optimal responders (5.8±1.77). Moreover, the Shp1 decrease was observed in CD34+ cells isolated from 6 resistant patients in comparison to 6 optimal responders. In conclusion, our study suggests that an aberrant balance between the Shp1 and 2 levels play a role in the Bcr-Abl independent resistance to Ima through activation of Ras/MAPK pathway and that lower levels of Shp1 are associated with non responsive patients.

The Role Of SOCS1 In BCR-ABL Mediated Transformation and Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2507-2507
Author(s):  
Özlem Demirel ◽  
Olivier Balló ◽  
Hubert Serve ◽  
Christian H. Brandts
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Sh2 Domain ◽  
General Mechanism ◽  
Cytokine Signaling ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Murine Bone Marrow ◽  
Socs Proteins

Abstract The BCR-ABL oncogene activates several signaling pathways, most notably by constitutive phosphorylation of the signal transducer and activator of transcription protein 5 (STAT5). After phosphorylation and nuclear translocation, STAT5 transcriptionally activates numerous genes responsible for proliferation, survival and differentiation of hematopoietic stem and progenitor cells. Among the STAT5 target genes are suppressor of cytokine signaling (SOCS) proteins. SOCS proteins inhibit JAK kinases by multiple mechanisms, thereby terminating cytokine signaling in a classical negative feedback loop. The SOCS family of proteins comprises eight members: cytokine-inducible SRC homology 2 (SH2) domain protein (CIS) and SOCS1–SOCS7. SOCS1 is frequently silenced by hypermethylation in multiple myeloma and inactivating mutations have been found in Hodgkin lymphoma with consecutive increase in JAK2 kinase activity. More recently, we identified SOCS1 as a “conditional oncogene” in the context of the FLT3-ITD oncogene (Reddy et al, Blood 2012): SOCS1 significantly enhanced FLT3-ITD-mediated myeloid transformation, both in vitro and in vivo. We hypothesized that this may be a more general mechanism of transformation and therefore analyzed the role of SOCS proteins in BCR-ABL mediated transformation and leukemogenesis. First, we investigated gene expression levels of SOCS proteins in BCR-ABL positive (versus BCR-ABL negative) cell lines and primary ALL long term-cultured cells. Upon treatment with the BCR-ABL inhibitor imatinib, mRNA expression levels of CIS and SOCS1-4 were reduced. SOCS5-7 did not exhibit any changes and were non-responsive to ABL-kinase inhibition. In lineage-depleted primary murine bone marrow retrovirally transduced with BCR-ABL, high induction of CIS and SOCS1-3 mRNA was detected, while SOCS4-7 showed only minor changes. When overexpressed in IL-3 dependent cell lines, SOCS1 led to a very rapid cell death within few days. Similar effects were demonstrated for CIS and SOCS2 overexpression, however, with a slower kinetics. In contrast, BCR-ABL transduced cells were insensitive to SOCS1 overexpression. In colony formation assays performed with primary hematopoietic cells, expression of SOCS1 led to a significant decrease of colony numbers. Interestingly, co-expression of SOCS1 and BCR-ABL (hereafter abbreviated as SOCS1/BCR-ABL) also lowered colony numbers compared to cells expressing BCR-ABL alone. However, when cells were subjected to interferon alpha or interferon gamma treatment, SOCS1/BCR-ABL positive cells displayed higher colony numbers and gained a growth advantage over BCR-ABL expressing cells, since anti-proliferative effects of the cytokines were inhibited by the presence of SOCS1. A careful analysis of the downstream signaling cascade of BCR-ABL and SOCS1/BCR-ABL expressing cells did not demonstrate any differences in the phosphorylation of AKT, ERK1/2 and STAT5. However, when BCR-ABL was inhibited by imatinib, STAT5 phosphorylation was significantly decreased in SOCS1/BCR-ABL transduced cells. Finally, the influence of SOCS1 in BCR-ABL mediated leukemia was investigated in a murine bone marrow transplantation model. BCR-ABL or SOCS1/BCR-ABL expressing cells led to disease formation with a chronic myeloid leukemia-like phenotype. Interestingly, the co-expression of SOCS1 and BCR-ABL prolonged disease latency, as opposed to the phenotype observed with FLT3-ITD (where SOCS1 co-expression shortened latency). In this setting SOCS1 acts as a tumor suppressor, protecting BCR-ABL transformed cells from rapid disease development, and a molecular analysis will be presented. Disclosures: No relevant conflicts of interest to declare.

Targeting CD99 in T-Cell Neoplasms with Monoclonal Antibodies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2749-2749
Author(s):  
Montreh Tavakkoli ◽  
Dong H. Lee ◽  
Benjamin Durham ◽  
Stephen S. Chung ◽  
Christopher Y. Park
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
T Cell ◽  
Cell Lines ◽  
Functional Role ◽  
Normal Peripheral Blood ◽  
T Cell Lymphomas ◽  
Cd34 Cells

Abstract CD99 is a 32-kDa glycoprotein involved in leukocyte migration and homotypic cell aggregation. Since its initial discovery as a marker on acute lymphoblastic leukemia (ALL), few studies have investigated its potential targeting and biological role in this disease. We have shown that CD99 is up-regulated in malignant stem cells in acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS), and that monoclonal antibodies (mAbs) targeting CD99 induce cell death. Given that targeting CD99 holds promise in AML/MDS, we sought to determine whether it is an effective target in other hematologic malignancies. We began by screening 15 T-, B-, and plasma cell lines as well as normal peripheral blood and umbilical cord CD34+ cells for CD99 expression by flow cytometry. CD99 expression was 7- and 10-fold higher on 1/1 T-ALL and 1/2 anaplastic large cell lymphoma (ALCL) cell lines compared with CD34+ cells, and 2- and 3-fold higher relative to normal peripheral blood T cells, respectively. However, it was minimally expressed in 11/12 B cell lymphomas, plasma cell dyscrasias, and peripheral T cell neoplasms. CD99 expression (degree, localization) was also assessed on 264 lymphoma patient samples by immunohistochemistry (IHC) using the CD99 mAb, 12E7. We found that 11/20 (55%) T-lymphoblastic lymphomas, 7/16 (44%) angioimmunoblastic T-cell lymphomas, 4/13 (31%) ALCLs, 10/63 (16%) peripheral T-cell lymphomas, and 0/3 (0%) of NK/T cell lymphomas express CD99 by IHC, while only 1/70 (1.4%) diffuse large B cell lymphomas, 2/24 (8%) mantle cell lymphomas, 2/17 (12%) follicular lymphomas, 4/22 (18%) chronic lymphocytic leukemias, and 3/16 (19%) marginal zone lymphomas express CD99. Staining was predominately moderate and cytoplasmic. Using a BioGPS dataset from T-ALL patient bone marrow samples, CD99 transcript was found to be up-regulated in T-ALL bone marrow (n=117) relative to normal bone marrow (n=7) (p<0.0001), and was expressed at similar levels at diagnosis (n=14) and relapse (n=14), suggesting it is stably expressed and may be a candidate therapeutic target. To test whether CD99 mAbs are cytotoxic to T-ALL and ALCL cell lines, cells were incubated with 5µg/ml CD99 mAb in the presence of 7µg/ml anti-IgG antibody, and cell survival was assessed by flow cytometry following 72-hours relative to IgG isotype control. 4/5 T-ALL cell lines (KOPTK1, Loucy, CCRF HSB-2, PF283) were sensitive to the cytotoxicity of CD99 mAb, mediating 30-96% cell death (p≤0.003), with 2/4 cell lines displaying 90-96% cytotoxicity. Remarkably, incubating CD99 mAb with a primary T-ALL patient sample induced 100% cell death within 48 hours of treatment (p<0.0001). 1/2 ALCL cell lines (Karpas-299) were sensitive to cytotoxic CD99 mAb (46% cell death, p=0.02). Furthermore, CD99 mAb treatment induced Annexin V positivity, and cell death occurred independent of complement and within 3 hours of treatment. To determine whether CD99 mAb cytotoxicity depends on the level of CD99 expression, we stably transduced KOPTK1 cells with an optimized CD99 shRNA (199-fold reduction in CD99 mean fluorescence intensity [MFI]), stably transduced CD99-low Mac2A (ALCL) cells with TetOn CD99 (17-fold increase in CD99 MFI), and analyzed the cells for cytotoxicity following 24-hour incubations with CD99 mAbs. CD99 mAb-induced cell death increased from 4.4% to 88% upon overexpressing CD99, and decreased from 89% to 20% upon knocking down CD99, suggesting that cell death is dependent on the level of CD99 expression. To elucidate the functional role of CD99 in T-cell neoplasms, we xenografted KOPTK1 cells expressing CD99 shRNA into sublethally irradiated NOD/SCID/IL-2Rgc-null (NSG) mice. Animals transplanted with CD99 knockdown showed no improved survival compared to controls (n=4 and 5 in each group, respectively). We further evaluated the potential oncogenic role of CD99 in vitro, and observed no effect of CD99 knock down in KOPTK1 or overexpression in Mac2A on cell cycle status or proliferation by PI staining and cell counting. Our data indicate that CD99 is expressed in a subset of T-lineage neoplasms. While there is no evidence for a functional role of CD99 in the growth or survival of T-ALL and ALCL, CD99 can be targeted by CD99 mAbs to induce apoptosis with rapid kinetics and in a manner that is dependent on levels of CD99 expression and independent of complement. Thus, CD99 is a promising target in the treatment of a subset of T-cell neoplasms. Disclosures No relevant conflicts of interest to declare.

scholarly journals Allelic Exclusion of the T Cell Receptor β Locus Requires the Sh2 Domain–Containing Leukocyte Protein (Slp)-76 Adaptor Protein

1999 ◽  
Vol 190 (8) ◽  
pp. 1093-1102 ◽  
Author(s):  
Iannis Aifantis ◽  
Vadim I. Pivniouk ◽  
Frank Gärtner ◽  
Jacqueline Feinberg ◽  
Wojciech Swat ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Allelic Exclusion ◽  
Double Positive ◽  
Thymic Development ◽  
Developmental Progression

Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4−CD8− double-negative (DN) thymocytes into CD4+CD8+ double-positive (DP) cells and for TCR-β allelic exclusion. The adaptor protein SH2 domain–containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76−/− mice are arrested at the CD25+CD44− DN stage. Here we show that SLP-76−/− DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-α/β transgene into the SLP-76−/− background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-β rearrangement in SLP-76−/− TCR-transgenic mice or in single CD25+CD44− DN cells from SLP-76−/− mice indicates an essential role of SLP-76 in TCR-β allelic exclusion.

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