Wiskott–Aldrich Syndrome causing mutation, Pro373Ser restricts conformational changes essential for WASP activity in T-cells

AbstractWiskott–Aldrich Syndrome (WAS) is caused by mutations in Wiskott-Aldrich Syndrome Protein (WASP) and majority of the mutations are found in the WASP Homology 1 (WH1) domain which mediates interaction with WIP (WASP Interacting Protein), a WASP chaperone. Two point mutations together in the proline rich region (PRR) domain of WASP (S339Y/P373S) have been reported to cause WAS however the molecular defect has not been characterized. Expression of these mutants separately (WASPRS339Y, WASPRP373S) or together (WASPRSP/YS) did not rescue the chemotaxis defect or membrane projection defect of JurkatWKD T-cells (WASP knockdown). This is not due to the inability of WASP-PRR mutants to form functional WASP–WIP complex in growth rescue experiments in las17Δ yeast strain. Expression of WASPRS339Y but not WASPRP373S or WASPRSP/YS rescued the IL-2 expression defect of JurkatWKD T-cells, suggesting that Pro373Ser mutation alone is sufficient to inhibit WASP functions in T-cell activation. The diffused localization of WASP-PRR mutants in activated Jurkat T-cells suggests that Ser339 and Pro373 are critical for WASP localization. WASP-PRR mutations either together or individually did not abolish interaction of WASP with sixteen WASP binding proteins including Hck, however they caused reduction in Hck mediated tyrosine phosphorylation of WASP which is critical for WASP activity. The auto-inhibitory conformation of WASPP373S mutant was not relieved by the binding of Toca-1 or Nck1. Thus, our results suggest that Pro373Ser mutation reduces Tyr291 phosphorylation and prevents conformational changes required for WASP activity in chemotaxis and T-cell activation. Thus Pro3373Ser is probably responsible for all the defects associated with WAS in the patients

Molecular Profiling of Hepatocellular Carcinoma Using Circulating Cell-Free DNA.

PurposeMolecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies.Experimental designWe analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA).ResultsA total of 153/206 (74.3%) were men; median age, 62 years (range, 18-91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1-13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%-55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20-38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04).ConclusionsThis study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology

X-Linked thrombocytopenia causing mutations in WASP (L46P and A47D) impair T cell chemotaxis

BACKGROUND: Mutation in the Wiskott-Aldrich syndrome Protein (WASP) causes Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT) and X-linked congenital neutropenia (XLN). The majority of missense mutations causing WAS and XLT are found in the WH1 (WASP Homology) domain of WASP, known to mediate interaction with WIP (WASP Interacting Protein) and CIB1 (Calcium and Integrin Binding). RESULTS: We analyzed two WASP missense mutants (L46P and A47D) causing XLT for their effects on T cell chemotaxis. Both mutants, WASP(R)(L46P) and WASP(R)(A47D) (S1-WASP shRNA resistant) expressed well in Jurkat(WASP-KD) T cells (WASP knockdown), however expression of these two mutants did not rescue the chemotaxis defect of Jurkat(WASP-KD) T cells towards SDF-1α. In addition Jurkat(WASP-KD) T cells expressing these two WASP mutants were found to be defective in T cell polarization when stimulated with SDF-1α. WASP exists in a closed conformation in the presence of WIP, however both the mutants (WASP(R)(L46P) and WASP(R)(A47D)) were found to be in an open conformation as determined in the bi-molecular complementation assay. WASP protein undergoes proteolysis upon phosphorylation and this turnover of WASP is critical for T cell migration. Both the WASP mutants were found to be stable and have reduced tyrosine phosphorylation after stimulation with SDF-1α. CONCLUSION: Thus our data suggest that missense mutations WASP(R)(L46P) or WASP(R)(A47D) affect the activity of WASP in T cell chemotaxis probably by affecting the turnover of the protein. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12929-014-0091-1) contains supplementary material, which is available to authorized users

Role of BAR domain proteins in myogenesis

Skeletal muscle formation is a step-wise process of cell proliferation, differentiation and fusion of mono-nucleated myoblasts to multi-nucleated myotubes. The cellular steps involved in myotube formation are cell-extracellular matrix (ECM) adhesion, change of cell shape from fibroblast-like to elongated morphology, migration towards other fusing partners, cell-cell adhesion followed by membrane breakdown. Membrane reorganization and actin cytoskeleton remodelling are two essential factors in cellular steps during myogenic differentiation and fusion. IRSp53 (53 kDa Insulin Receptor Substrate protein) and Toca-1 (Transducer of Cdc42-dependent actin assembly) are members of BAR (BIN/amphiphysin!Rvs) domain proteins which co-ordinate membrane bending with actin cytoskeleton remodeling. BAR domain proteins work as scaffolds to change membrane curvature. IRSp53 through its I-BAR domain (Inverse BAR)/ IMD (IRSp53 and MIM (missing in metastases) homology Domain) can bend membrane in outward direction and Toca-1 through its F-BAR domain can bend membrane in inward direction. IRSp53 is well-known for inducing filopodia in concert with a number of proteins upon activation by small GTPases. Expression of IRSp53 is downregulated during the differentiation of C2C12 cells to myotubes. Knocking down the expression of IRSp53 using shRNA led to increase in the fusion index compared to cells transfected with the scrambled shRNA. In contrast, transfection of plasmid expressing IRSp53 led to inhibition of myogenic differentiation, decrease in ceii-ECM adhesion and increase in the induction of membrane projections. IRSp53 is an adaptor protein with an IMD/1-BAR domain located at the N-terminus, a GTPase Binding Domain (GBD) and a SH3 domain. Mutations which impaired the function of any of the three domains of IRSp53 abolished the ability of IRSp53 to inhibit myogenic differentiation. Expression of IRSp531MD alone inhibited myogeneis and reduced ceii-ECM adhesion . Therefore, IRSp531MD is the functional unit responsible for suppression of differentiation mediated by IRSp53 in C2C12 cells. Expression of another I-BAR domain protein IRTKS (Insulin receptor tyrosine kinase substrate ; also known as BAIAP2L 1) in C2C 12 cells also induced small membrane projections and inhibited myoblast fusion. Expression of Toca-1 , the F-BAR domain protein, in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated. Knocking down Toca-1 expression in C2C12 cells resulted in a significant decrease in myotube formation. Toca-1 knockdown cells displayed elongated morphology and expressed differentiation markers (MyoD and MyHC) suggesting there was no differentiation defect in Toca-1 knockdown cells. In addition, Toca-1 knockdown cells displayed slightly increased migration and decreased vinculin patches which is similar to N-WASP knockout fibroblasts. Moreover, expression of NWASP suppressed the fusion defect of Toca-1 KD C2C 12 cells suggesting that Toca-1 probably works via N- WASP in myoblast fusion. This study has provided molecular and functional insight into the role of BAR domain family of proteins in myogenic differentiation.​Doctor of Philosophy (SBS

X-Linked thrombocytopenia causing mutations in WASP (L46P and A47D) impair T cell chemotaxis

Background: Mutation in the Wiskott-Aldrich syndrome Protein (WASP) causes Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT) and X-linked congenital neutropenia (XLN). The majority of missense mutations causing WAS and XLT are found in the WH1 (WASP Homology) domain of WASP, known to mediate interaction with WIP (WASP Interacting Protein) and CIB1 (Calcium and Integrin Binding). Results: We analyzed two WASP missense mutants (L46P and A47D) causing XLT for their effects on T cell chemotaxis. Both mutants, WASPRL46P and WASPRA47D (S1-WASP shRNA resistant) expressed well in JurkatWASP-KD T cells (WASP knockdown), however expression of these two mutants did not rescue the chemotaxis defect of JurkatWASP-KD T cells towards SDF-1α. In addition JurkatWASP-KD T cells expressing these two WASP mutants were found to be defective in T cell polarization when stimulated with SDF-1α. WASP exists in a closed conformation in the presence of WIP, however both the mutants (WASPRL46P and WASPRA47D) were found to be in an open conformation as determined in the bi-molecular complementation assay. WASP protein undergoes proteolysis upon phosphorylation and this turnover of WASP is critical for T cell migration. Both the WASP mutants were found to be stable and have reduced tyrosine phosphorylation after stimulation with SDF-1α. Conclusion: Thus our data suggest that missense mutations WASPRL46P or WASPRA47D affect the activity of WASP in T cell chemotaxis probably by affecting the turnover of the protein.Published versio

Designing cost-efficient transport solutions for fixed and mobile broadband access network

This thesis undertakes a techno-economic evaluation of transport solutions for fixed and mobile broadband access. In the case of future mobile access networks, it is proposed to make use of backhaul architectures using fiber and microwave applied to Greenfield deployments and a copper-legacy backhaul infrastructure based on Brownfield migration, i.e. finding a way of using a legacy infrastructure to its full capacity. At the same time, protection deployments based on fiber-wireless schemes are recommended for future fixed broadband. The main contribution made by this thesis is to carry out a research investigation into the total investment cost of the broadband transport infrastructure. This will be determined by employing two sets of models to assess the capital and operational expenditures, (CAPEX and OPEX respectively), of mobile and fixed broadband access network operators. First, this involves a set of models for mobile broadband that are summarized in a general methodology that aims at providing: traffic forecasting, wireless deployment, mobile backhaul deployment and total cost assessment. It was found that, fiber-based backhaul through a Greenfield deployment is the most energy-efficient option. Furthermore, Brownfield reveals that copper-based backhaul can still play a key role if used up to its full capacity and sharply reduces the investment costs in infrastructure. Additionally, there is an examination of the main differences in cost and energy values between Greenfield and Brownfield. Finally, a methodology is employed for fixed broadband based on network dimensioning, failure costs and an assessment of the total cost of ownership. The models are used to assess five architectures that represent different protection schemes for fixed broadband. This research shows the economic benefits of using a hybrid protection scheme based on fiber-wireless architecture rather than fiber-based protection options and a sensitivity analysis is conducted to show that the extra CAPEX invested to protect the infrastructure might be recovered through the OPEX after a number of years. The results obtained in the thesis should be useful for network operators to plan both their fixed and mobile broadband access network infrastructure in the future.CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoEsta tese se concentra na avaliação técnico econômica de soluções de transporte para acesso banda larga fixa e móvel. Para futuras redes de acesso móvel, propõem-se arquiteturas de backhaul usando fibra e microondas baseado no desenvolvimento de redes de acesso wireless verde e infraestruturas legadas de backhaul baseada em cobre para migração Brownfield, ou seja, usando infraestrutura existente até o limite de capacidade suportada, enquanto para redes de banda larga fixa são propostas implantações de proteção baseadas em esquemas híbridos, ou seja, fiber+wireless. As principais contribuições desta tese estão relacionadas ao campo de pesquisa do custo total de investimento em infraestrutura de transporte banda larga. Em termos de custo total de investimento, são propostos dois conjuntos de modelos para avaliar as despesas de capital e de operação, CAPEX e OPEX respectivamente, de operadoras de redes de acesso banda larga fixa e móvel. Primeiramente, para banda larga móvel, é apresentado um conjunto de modelos condensado em uma metodologia geral que visa fornecer: previsão de tráfego, implantação de rede sem fio, implantação de backhaul móvel e avaliação do custo total. É mostrado que o backhaul baseado em fibra considerando acesso sem fio verde é a opção mais eficiente em termos de energia. Além disso, Brownfield mostra que o backhaul baseado em cobre ainda pode desempenhar um grande papel se utilizado até a exaustão de sua capacidade e reduz drasticamente os custos de investimentos em infraestrutura. Adicionalmente, são apresentadas as principais diferenças de custos e valores de energia entre redes de acesso sem fio verde e Brownfield. Finalmente, para banda larga fixa, é proposta uma metodologia baseada em dimensionamento de rede, custos associados à falha e avaliação do custo total por assinante. Os modelos são utilizados para avaliar cinco modelos que representam diferentes esquemas de proteção para arquiteturas de banda larga fixa. Esta pesquisa revela os benefícios econômicos do uso de esquema de proteção híbrido baseado em arquitetura fiber+wireless comparado com a opção de proteção baseada totalmente em fibra e é também apresentada uma análise de sensibilidade para provar que o investimento adicional em CAPEX para proteger a infraestrutura pode ser recuperado em alguns anos através da economia em OPEX

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53 kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell–extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes

Ibrutinib Resistance Mechanisms and Treatment Strategies for B-Cell Lymphomas

Chronic activation of B-cell receptor (BCR) signaling via Bruton tyrosine kinase (BTK) is largely considered to be one of the primary mechanisms driving disease progression in B–Cell lymphomas. Although the BTK-targeting agent ibrutinib has shown promising clinical responses, the presence of primary or acquired resistance is common and often leads to dismal clinical outcomes. Resistance to ibrutinib therapy can be mediated through genetic mutations, up-regulation of alternative survival pathways, or other unknown factors that are not targeted by ibrutinib therapy. Understanding the key determinants, including tumor heterogeneity and rewiring of the molecular networks during disease progression and therapy, will assist exploration of alternative therapeutic strategies. Towards the goal of overcoming ibrutinib resistance, multiple alternative therapeutic agents, including second- and third-generation BTK inhibitors and immunomodulatory drugs, have been discovered and tested in both pre-clinical and clinical settings. Although these agents have shown high response rates alone or in combination with ibrutinib in ibrutinib-treated relapsed/refractory(R/R) lymphoma patients, overall clinical outcomes have not been satisfactory due to drug-associated toxicities and incomplete remission. In this review, we discuss the mechanisms of ibrutinib resistance development in B-cell lymphoma including complexities associated with genomic alterations, non-genetic acquired resistance, cancer stem cells, and the tumor microenvironment. Furthermore, we focus our discussion on more comprehensive views of recent developments in therapeutic strategies to overcome ibrutinib resistance, including novel BTK inhibitors, clinical therapeutic agents, proteolysis-targeting chimeras and immunotherapy regimens

Image_3_Blockade of growth hormone receptor signaling by using pegvisomant: A functional therapeutic strategy in hepatocellular carcinoma.tif

Hepatocellular carcinoma (HCC) is an aggressive neoplasm with poor clinical outcome because most patients present at an advanced stage, at which point curative surgical options, such as tumor excision or liver transplantation, are not feasible. Therefore, the majority of HCC patients require systemic therapy. Nonetheless, the currently approved systemic therapies have limited effects, particularly in patients with advanced and resistant disease. Hence, there is a critical need to identify new molecular targets and effective systemic therapies to improve HCC outcome. The liver is a major target of the growth hormone receptor (GHR) signaling, and accumulating evidence suggests that GHR signaling plays an important role in HCC pathogenesis. We tested the hypothesis that GHR could represent a potential therapeutic target in this aggressive neoplasm. We measured GH levels in 767 HCC patients and 200 healthy controls, and then carried out clinicopathological correlation analyses. Moreover, specific inhibition of GHR was performed in vitro using siRNA and pegvisomant (a small peptide that blocks GHR signaling and is currently approved by the FDA to treat acromegaly) and in vivo, also using pegvisomant. GH was significantly elevated in 49.5% of HCC patients, and these patients had a more aggressive disease and poorer clinical outcome (P<0.0001). Blockade of GHR signaling with siRNA or pegvisomant induced substantial inhibitory cellular effects in vitro. In addition, pegvisomant potentiated the effects of sorafenib (P<0.01) and overcame sorafenib resistance (P<0.0001) in vivo. Mechanistically, pegvisomant decreased the phosphorylation of GHR downstream survival proteins including JAK2, STAT3, STAT5, IRS-1, AKT, ERK, and IGF-IR. In two patients with advanced-stage HCC and high GH who developed sorafenib resistance, pegvisomant caused tumor stability. Our data show that GHR signaling represents a novel “druggable” target, and pegvisomant may function as an effective systemic therapy in HCC. Our findings could also lead to testing GHR inhibition in other aggressive cancers.</p

Image_4_Blockade of growth hormone receptor signaling by using pegvisomant: A functional therapeutic strategy in hepatocellular carcinoma.tif

Hepatocellular carcinoma (HCC) is an aggressive neoplasm with poor clinical outcome because most patients present at an advanced stage, at which point curative surgical options, such as tumor excision or liver transplantation, are not feasible. Therefore, the majority of HCC patients require systemic therapy. Nonetheless, the currently approved systemic therapies have limited effects, particularly in patients with advanced and resistant disease. Hence, there is a critical need to identify new molecular targets and effective systemic therapies to improve HCC outcome. The liver is a major target of the growth hormone receptor (GHR) signaling, and accumulating evidence suggests that GHR signaling plays an important role in HCC pathogenesis. We tested the hypothesis that GHR could represent a potential therapeutic target in this aggressive neoplasm. We measured GH levels in 767 HCC patients and 200 healthy controls, and then carried out clinicopathological correlation analyses. Moreover, specific inhibition of GHR was performed in vitro using siRNA and pegvisomant (a small peptide that blocks GHR signaling and is currently approved by the FDA to treat acromegaly) and in vivo, also using pegvisomant. GH was significantly elevated in 49.5% of HCC patients, and these patients had a more aggressive disease and poorer clinical outcome (P<0.0001). Blockade of GHR signaling with siRNA or pegvisomant induced substantial inhibitory cellular effects in vitro. In addition, pegvisomant potentiated the effects of sorafenib (P<0.01) and overcame sorafenib resistance (P<0.0001) in vivo. Mechanistically, pegvisomant decreased the phosphorylation of GHR downstream survival proteins including JAK2, STAT3, STAT5, IRS-1, AKT, ERK, and IGF-IR. In two patients with advanced-stage HCC and high GH who developed sorafenib resistance, pegvisomant caused tumor stability. Our data show that GHR signaling represents a novel “druggable” target, and pegvisomant may function as an effective systemic therapy in HCC. Our findings could also lead to testing GHR inhibition in other aggressive cancers.</p