Using single antigen specificity magnetic beads for the isolation of specific antibodies against HLA antigens.

The presence of multiple donor-specific antibodies (DSAs) targeting HLA antigens poses a challenge to transplantation. Various techniques, including the use of recombinant cell lines and crossmatch cells have been developed to isolate DSAs. To simplify the extraction of HLA-specific DSAs from complex sera, we introduced magnetic beads with single HLA specificity (MagSort). Sera were treated with MagSort, allowing HLA-specific antibodies to bind to the beads, and these specific antibodies were subsequently eluted. MagSort beads, coated with 59 different HLA variants, underwent testing through 1329 adsorption/elution processes, demonstrating their effectiveness and specificity in adsorbing and eluting HLA-specific antibodies. The MagSort method proves comparable to the cell method, showing similar isolated antibody binding patterns. The isolated antibody binding patterns from MagSort reveal both known eplets and unknown patterns, suggesting its utility for eplet discovery. Additionally, MagSort proved effective in extracting signals for flow cytometry cross-matching, offering a means to assess the binding capability of isolated antibodies against specific donor cells.

The predictive and prognostic value of peripheral blood antigen-specific memory B cells in phospholipase A2 receptor-associated membranous nephropathy.

Background: Phospholipase A2 receptor-associated membranous nephropathy (PLA2R-MN) is an anti-PLA2R antibody (PLA2R-Ab) mediated autoimmune kidney disease. Although antibody titer correlates closely with disease activity, whether it can provide longer-term predictions on disease course and progression is unclear. Rituximab, a B-cell depletion therapy, has become the first-line treatment option for PLA2R-MN; however, the response to Rituximab varies among patients. Methods: We developed a flow cytometry-based test that detects and quantifies PLA2R antigen-specific memory B cells (PLA2R-MBCs) in peripheral blood, the primary source for PLA2R-Ab production upon disease relapse. We applied the test to 159 blood samples collected from 28 patients with PLA2R-MN (at diagnosis, during and after immunosuppressive treatment, immunological remission, and relapse) to evaluate the relationship between circulating PLA2R-MBC levels and disease activity. Results: The level of PLA2R-MBCs in healthy controls (n=56) is less than or equal to 1.5% of the total MBC compartment. High circulating PLA2R-MBC levels were detected in two patients post-Rituximab despite achieving immunologic and proteinuric remission, as well as in two patients with negative serum autoantibody but increasing proteinuria. Elimination of these cells with Rituximab improved clinical outcomes. Moreover, five patients exhibited elevated PLA2R-MBC levels before disease relapse, followed by a rapid decline to baseline when relapse became clinically evident. COVID-19 vaccination or SARS-CoV-2 infection significantly affected the dynamics of circulating PLA2R-MBCs. Conclusions: This study suggests that monitoring PLA2R-MBC levels in patients with PLA2R-MN may help refine and individualize immunosuppressive therapy and predict disease course and progression. The technology and findings may also have broader applications in the clinical management of other autoimmune diseases.

Site-directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA-A1/A36-specific monoclonal antibody.

Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 Å radius. Eplet 44KM has been previously redefined to the antibody-verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody-induction and binding are limited. In this proof-of-concept study, we performed site-directed mutagenesis to narrow down the antibody-verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12.

Structural determinants of the dominant conformational epitopes of phospholipase A2 receptor in primary membranous nephropathy.

Anti-phospholipase A2 receptor autoantibody (PLA2R-Ab) plays a critical role in the pathogenesis of primary membranous nephropathy (PMN), an autoimmune kidney disease characterized by immune deposits in the glomerular subepithelial spaces and proteinuria. However, the mechanism of how PLA2R-Abs interact with the conformational epitope(s) of PLA2R has remained elusive. PLA2R is a single transmembrane helix receptor containing ten extracellular domains that begin with a CysR domain followed by a FnII and eight CTLD domains. Here, we examined the interactions of PLA2R-Ab with the full PLA2R protein, N-terminal domain truncations, and C-terminal domain deletions under either denaturing or physiological conditions. Our data demonstrate that the PLA2R-Abs against the dominant epitope (the N-terminal CysR-CTLD1 triple domain) possess weak cross-reactivities to the C-terminal domains beyond CTLD1. Moreover, both the CysR and CTLD1 domains are required to form a conformational epitope for PLA2R-Ab interaction, with FnII serving as a linker domain. Upon close examination, we also observed that patients with newly diagnosed PMN carry two populations of PLA2R-Abs in sera that react to the denatured CysR-CTLD3 (the PLA2R-Ab1) and denatured CysR-CTLD1 (the PLA2R-Ab2) domain complexes on Western blots, respectively. Furthermore, the PLA2R-Ab1 appeared at an earlier time point than PLA2R-Ab2 in patients, whereas the increased levels of PLA2R-Ab2 coincided with the worsening of proteinuria. In summary, our data support that an integrated folding of the three PLA2R N-terminal domains, CysR, FnII, and CTLD1, is a prerequisite to forming the PLA2R conformational epitope and that the dominant epitope-reactive PLA2R-Ab2 plays a critical role in PMN clinical progression.

Molecular Dynamics Simulation and Experimental Studies on the Thermomechanical Properties of Epoxy Resin with Different Anhydride Curing Agents.

An investigation of the relationship between the microstructure parameters and thermomechanical properties of epoxy resin can provide a scientific basis for the optimization of epoxy systems. In this paper, the thermomechanical properties of diglycidyl ether of bisphenol A (DGEBA)/methyl tetrahydrophthalic anhydride (MTHPA) and DGEBA/nadic anhydride (NA) were calculated and tested by the method of molecular dynamics (MD) simulation combined with experimental verification. The effects of anhydride curing agents on the thermomechanical properties of epoxy resin were investigated. The results of the simulation and experiment showed that the thermomechanical parameters (glass transition temperature (Tg) and Young's modulus) of the DGEBA/NA system were higher than those of the DGEBA/MTHPA system. The simulation results had a good agreement with the experimental data, which verified the accuracy of the crosslinking model of epoxy resin cured with anhydride curing agents. The microstructure parameters of the anhydride-epoxy system were analyzed by MD simulation, including bond-length distribution, synergy rotational energy barrier, cohesive energy density (CED) and fraction free volume (FFV). The results indicated that the bond-length distribution of the MTHPA and NA was the same except for C-C bonds. Compared with the DGEBA/MTHPA system, the DGEBA/NA system had a higher synergy rotational energy barrier and CED, and lower FFV. It can be seen that the slight change of curing agent structure has a significant effect on the synergy rotational energy barrier, CED and FFV, thus affecting the Tg and modulus of the system.

Melanopsin-Encoded Response Properties of Intrinsically Photosensitive Retinal Ganglion Cells.

Melanopsin photopigment expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) plays a crucial role in the adaptation of mammals to their ambient light environment through both image-forming and non-image-forming visual responses. The ipRGCs are structurally and functionally distinct from classical rod/cone photoreceptors and have unique properties, including single-photon response, long response latency, photon integration over time, and slow deactivation. We discovered that amino acid sequence features of melanopsin protein contribute to the functional properties of the ipRGCs. Phosphorylation of a cluster of Ser/Thr residues in the C-terminal cytoplasmic region of melanopsin contributes to deactivation, which in turn determines response latency and threshold sensitivity of the ipRGCs. The poorly conserved region distal to the phosphorylation cluster inhibits phosphorylation's functional role, thereby constituting a unique delayed deactivation mechanism. Concerted action of both regions sustains responses to dim light, allows for the integration of light over time, and results in precise signal duration.

Anti-Phospholipase A2 Receptor Autoantibody: A New Biomarker for Primary Membranous Nephropathy.

Primary membranous nephropathy (also known as idiopathic membranous nephropathy, IMN) is an organ specific autoimmune kidney disease characterized by the development of immune complex deposits in the sub-epithelial spaces, podocyte effacement and glomerular capillary wall thickening in the later stages. Clinical studies have demonstrated that over 70% of patients with IMN possess circulating autoimmune antibodies specifically targeting the phospholipase A2 receptor (PLA2R) on the surface of podocytes. The autoantibodies only bind to the extracellular portion of PLA2R under the non-reducing condition, indicating that the epitope in PLA2R is conformational requiring specific disulfide bonds to maintain its structure. We recently have successfully located the dominant epitope in PLA2R to the extreme N-terminus of the receptor. This finding has opened a new direction for understanding the pathogenesis of anti-PLA2R autoantibody induced IMN and offered a strong basis for developing sensitive clinical assays for IMN diagnosis and prognosis, and potentially, new therapeutic approaches for IMN treatment.

Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.

The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.

Identification of the immunodominant epitope region in phospholipase A2 receptor-mediating autoantibody binding in idiopathic membranous nephropathy.

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that >70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A2 receptor-1 (PLA2R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA2R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA2R by probing isolated truncated PLA2R extracellular domains with sera from patients with IMN that contain anti-PLA2R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteine-rich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA2R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA2R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA2R. These results indicate that the immunodominant epitope in PLA2R is exclusively located in the CysR-FnII-CTLD1 region.

Structure, function, and regulation of the SLC4 NBCe1 transporter and its role in causing proximal renal tubular acidosis.

PURPOSE OF REVIEW: There has been significant progress in our understanding of the structural and functional properties and regulation of the electrogenic sodium bicarbonate cotansporter NBCe1, a membrane transporter that plays a key role in renal acid-base physiology. The NBCe1 variant NBCe1-A mediates basolateral electrogenic sodium-base transport in the proximal tubule and is critically required for transepithelial bicarbonate absorption. Mutations in NBCe1 cause autosomal recessive proximal renal tubular acidosis (pRTA). The review summarizes recent advances in this area. RECENT FINDINGS: A topological model of NBCe1 has been established that provides a foundation for future structure-functional studies of the transporter. Critical residues and regions have been identified in NBCe1 that play key roles in its structure, function (substrate transport, electrogenicity) and regulation. The mechanisms of how NBCe1 mutations cause pRTA have also recently been elucidated. SUMMARY: Given the important role of proximal tubule transepithelial bicarbonate absorption in systemic acid-base balance, a clear understanding of the structure-functional properties of NBCe1 is a prerequisite for elucidating the mechanisms of defective transepithelial bicarbonate transport in pRTA.

Small-molecule antagonists of melanopsin-mediated phototransduction.

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image-forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several central nervous system responses, including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. New sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod- and cone-mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses, including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions.

Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis.

Mutations in SLC4A4, the gene encoding the electrogenic Na(+)-HCO3(-) cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ~50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3(-) as a surrogate ion for CO3(2-), our result indicated that NBCe1-A mediates electrogenic Na(+)-CO3(2-) cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3(-), compatible with the hypothesis that it mediates Na(+)-HCO3(-) cotransport. In patients, NBCe1-A-T485S is predicted to transport Na(+)-HCO3(-) in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3(-) absorption, possibly representing a new pathogenic mechanism for generating human pRTA.

Topology of NBCe1 protein transmembrane segment 1 and structural effect of proximal renal tubular acidosis (pRTA) S427L mutation.

In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO3(-) from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389-Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394-Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis.

Proximal renal tubular acidosis mediated by mutations in NBCe1-A: unraveling the transporter's structure-functional properties.

NBCe1 belongs to the SLC4 family of base transporting membrane proteins that plays a significant role in renal, extrarenal, and systemic acid-base homeostasis. Recent progress has been made in characterizing the structure-function properties of NBCe1 (encoded by the SLC4A4 gene), and those factors that regulate its function. In the kidney, the NBCe1-A variant that is expressed on the basolateral membrane of proximal tubule is the key transporter responsible for overall transepithelial bicarbonate absorption in this nephron segment. NBCe1 mutations impair transepithelial bicarbonate absorption causing the syndrome of proximal renal tubular acidosis (pRTA). Studies of naturally occurring NBCe1 mutant proteins in heterologous expression systems have been very helpful in elucidation the structure-functional properties of the transporter. NBCe1 mutations are now known to cause pRTA by various mechanisms including the alteration of the transporter function (substrate ion interaction, electrogenicity), abnormal processing to the plasma membrane, and a perturbation in its structural properties. The elucidation of how NBCe1 mutations cause pRTA in addition to the recent studies which have provided further insight into the topology of the transporter have played an important role in uncovering its critically important structural-function properties.

Integrin αIIbß3 inside-out activation: an in situ conformational analysis reveals a new mechanism.

Integrins are a family of heterodimeric adhesion receptors that transmit signals bi-directionally across the plasma membranes. The transmembrane domain (TM) of integrin plays a critical role in mediating transition of the receptor from the default inactive to the active state on the cell surfaces. In this study, we successfully applied the substituted cysteine scanning accessibility method to determine the intracellular border of the integrin α(IIb)ß(3) TM in the inactive and active states in living cells. We examined the aqueous accessibility of 75 substituted cysteines comprising the C terminus of both α(IIb) and ß(3) TMs, the intracellular membrane-proximal regions, and the whole cytoplasmic tails, to the labeling of a membrane-permeable, cysteine-specific chemical biotin maleimide (BM). The active state of integrin α(IIb)ß(3) heterodimer was generated by co-expression of activating partners with the cysteine-substituted constructs. Our data revealed that, in the inactive state, the intracellular lipid/aqueous border of α(IIb) TM was at Lys(994) and ß(3) TM was at Phe(727) respectively; in the active state, the border of α(IIb) TM shifted to Pro(998), whereas the border of ß(3) TM remained unchanged, suggesting that complex conformational changes occurred in the TMs upon α(IIb)ß(3) inside-out activation. On the basis of the results, we propose a new inside-out activation mechanism for integrin α(IIb)ß(3) and by inference, all of the integrins in their native cellular environment.

The expression of c-Met pathway components in unclassified pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH): a tissue microarray study.

AIMS: Subclassification of undifferentiated pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH) into distinct biological cohorts based on the expression patterns of molecular markers can identify patient subsets with especially unfavourable clinical outcomes. Identification of molecular prognosticators amenable for drug targeting can facilitate rational development of UPS/MFH tailored therapies. The aim was to evaluate expression of c-Met pathway components in a large cohort of UPS/MFH samples. METHODS AND RESULTS: An immunohistochemical analysis for hepatocyte growth factor (HGF), c-Met, phospho-c-Met (pc-Met), phospho-mitogen-activated protein kinase kinase (MAPKK) also known as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (p-MEK) and phospho-protein kinase B (p-AKT) was performed on a clinically annotated tissue microarray of 158 UPS/MFH samples. Univariable and multivariable analyses were conducted to evaluate the correlation of molecular variables with UPS/MFH disease specific survival. All evaluated markers were expressed in UPS/MFH to varying levels. Most importantly, strong HGF, pc-Met, p-MEK and p-AKT expression correlated significantly with dismal patient outcome on univariable statistical analysis. Expression of p-MEK and p-AKT remained statistically significant independent prognosticators on multivariable analysis. CONCLUSIONS: c-Met pathway components and especially p-MEK and p-AKT are potential prognostic biomarkers for UPS/MFH; their inclusion in future molecular-based staging systems should be evaluated. Furthermore, novel approaches targeting HGF, c-Met, MEK/extracellular-regulated kinase (ERK) and/or AKT should be considered for a subset of UPS/MFH patients.

Combining EGFR and mTOR blockade for the treatment of epithelioid sarcoma.

PURPOSE: Molecular deregulations underlying epithelioid sarcoma (ES) progression are poorly understood yet critically needed to develop new therapies. Epidermal growth factor receptor (EGFR) is overexpressed in ES; using preclinical models, we examined the ES EGFR role and assessed anti-ES EGFR blockade effects, alone and with mTOR inhibition. EXPERIMENTAL DESIGN: EGFR and mTOR expression/activation was examined via tissue microarray (n = 27 human ES specimens; immunohistochemistry) and in human ES cell lines (Western blot and quantitative reverse transcriptase PCR). Cell proliferation, survival, migration, and invasion effects of EGFR and mTOR activation treated with erlotinib (anti-EGFR small-molecule inhibitor) alone and combined with rapamycin were assessed in cell culture assays. In vivo growth effects of erlotinib alone or with rapamycin were evaluated using severe combined immunodeficient mouse ES xenograft models. RESULTS: EGFR was expressed and activated in ES specimens and cell lines. EGFR activation increased ES cell proliferation, motility, and invasion and induced cyclin D1, matrix metalloproteinase (MMP) 2, and MMP9 expression. EGFR blockade inhibited these processes and caused significant cytostatic ES growth inhibition in vivo. mTOR pathway activation at varying levels was identified in all tissue microarray-evaluable ES tissues; 88% of samples had no or reduced PTEN expression. Similarly, both ES cell lines showed enhanced mTOR activity; VAESBJ cells exhibited constitutive mTOR activation uncoupled from EGFR signaling. Most importantly, combined erlotinib/rapamycin resulted in synergistic anti-ES effects in vitro and induced superior tumor growth inhibition in vivo versus single agent administration. CONCLUSIONS: EGFR and mTOR signaling pathways are deregulated in ES. Preclinical ES model-derived insights suggest that combined inhibition of these targets might be beneficial, supporting evaluations in clinical trials.

G-CSF receptor activation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2 phosphatase.

Src activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF-stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.

Activated MET is a molecular prognosticator and potential therapeutic target for malignant peripheral nerve sheath tumors.

PURPOSE: MET signaling has been suggested a potential role in malignant peripheral nerve sheath tumors (MPNST). Here, MET function and blockade were preclinically assessed. EXPERIMENTAL DESIGN: Expression levels of MET, its ligand hepatocyte growth factor (HGF), and phosphorylated MET (pMET) were examined in a clinically annotated MPNST tissue microarray (TMA) incorporating univariable and multivariable statistical analyses. Human MPNST cells were studied in vitro and in vivo; Western blot (WB) and ELISA were used to evaluate MET and HGF expression, activation, and downstream signaling. Cell culture assays tested the impact of HGF-induced MET activation and anti-MET-specific siRNA inhibition on cell proliferation, migration, and invasion; in vivo gel-foam assays were used to evaluate angiogenesis. Cells stably transduced with anti-MET short hairpin RNA (shRNA) constructs were tested for growth and metastasis in severe combined immunodeficient (SCID) mice. The effect of the tyrosine kinase inhibitor XL184 (Exelixis) targeting MET/VEGFR2 (vascular endothelial growth factor receptor 2) on local and metastatic MPNST growth was examined in vivo. RESULTS: All three markers were expressed in MPNST human samples; pMET expression was an independent prognosticator of poor patient outcome. Human MPNST cell lines expressed MET, HGF, and pMET. MET activation increased MPNST cell motility, invasion, angiogenesis, and induced matrix metalloproteinase-2 (MMP2) and VEGF expression; MET knockdown had inverse effects in vitro and markedly decreased local and metastatic growth in vivo. XL184 abrogated human MPNST xenograft growth and metastasis in SCID mice. CONCLUSIONS: Informative prognosticators and novel therapies are crucially needed to improve MPNST management and outcomes. We show an important role for MET in MPNST, supporting continued investigation of novel anti-MET therapies in this clinical context.