Mild SARS-CoV-2 Infection After Gene Therapy in a Child With Wiskott-Aldrich Syndrome: A Case Report.

In this work we present the case of SARS-CoV-2 infection in a 1.5-year-old boy affected by severe Wiskott-Aldrich Syndrome with previous history of autoinflammatory disease, occurring 5 months after treatment with gene therapy. Before SARS-CoV-2 infection, the patient had obtained engraftment of gene corrected cells, resulting in WASP expression restoration and early immune reconstitution. The patient produced specific immunoglobulins to SARS-CoV-2 at high titer with neutralizing capacity and experienced a mild course of infection, with limited inflammatory complications, despite pre-gene therapy clinical phenotype.

BCR-ABL1-induced downregulation of WASP in chronic myeloid leukemia involves epigenetic modification and contributes to malignancy.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR-ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR-ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott-Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR-ABL1 and is particularly low in blast crisis. Enforced expression of BCR-ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR-ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR-ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR-ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).

WASP-WIP complex in the molecular pathogenesis of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease characterized by recurrent infection, thrombocytopenia, and eczema. The gene responsible for X-linked WAS encodes the Wiskott-Aldrich syndrome protein (WASP), which is expressed in hematopoietic cells and which regulates T-cell activation and cytoskeletal reorganization in T-cell receptor (TCR) signaling. Here, I review my recent research on WASP and the WASP-interacting protein (WIP) complex in T cells. I and my colleagues first established a diagnostic screening method using flow cytometry and genetic analysis, and elucidated the molecular pathogenesis in WAS patients with unique clinical manifestations. We investigated the mechanisms by which WASP is recruited to lipid rafts following TCR stimulation and to immunological synapses between antigen-presenting cells and T cells. Subsequently, we elucidated the molecular mechanisms by which WASP is degraded by calpain and ubiquitinated by Cbl-family proteins, which terminate WASP activation. More importantly, we found that WIP plays a critical role in WASP stability in T cells. These results provide new insights into the molecular pathogenesis of X-linked WAS and have facilitated the identification of WIP deficiency as an autosomal recessive form of WAS.

Wiskott-Aldrich syndrome/X-linked thrombocytopenia in China: Clinical characteristic and genotype-phenotype correlation.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are caused by mutations of the WAS gene. The genotype-phenotype association of WAS and XLT have not been fully elucidated. Here, we established the largest database of WAS in China to further determine the potential correlation between genotype and phenotype and long-term outcome. PROCEDURES: We collected clinical data of 81 WAS/XLT patients, analyzed mutations of WAS gene at the genomic DNA and transcriptional/translational levels, and quantified three different patterns of WAS protein (WASp) expression in PBMCs by flow cytometry. RESULTS: There were 60 unique mutations identified, including 20 novel mutations and eight hotspots, from 75 unrelated families with a total of 81 affected members. Nearly all the patients with XLT had missense mutations and were WASp-positive in the peripheral cells, while only half of the patients with missense mutations exhibited the XLT phenotype and detectable WASp. In contrast, patients with nonsense mutations, deletions, insertions, and complex mutations were WASp-negative and developed the classic WAS phenotype. An equal number of patients with splice anomalies were either WASp-positive or WASp-negative. Long-term survival rates were lower in WASp-negative patients compared to WASp-positive patients. CONCLUSIONS: The clinical phenotype of classic WAS or milder XLT and long-term outcome are potentially influenced by the effect of these defects on gene transcription and translation. Patients with missense mutations allowing expression of mutated WASp and those with splice anomalies, which result in generation of multiple products, including normal WASp, present the attenuated XLT phenotype and show better prognosis.

High mobility group Box-1 inhibits cancer cell motility and metastasis by suppressing activation of transcription factor CREB and nWASP expression.

The ability to metastasize is a hallmark of malignant tumors, and metastasis is the principal cause of death of cancer patients. The High Mobility Group Box-1 (HMGB1) is a multifunction protein that serves as both a chromatin protein and an extracellular signaling molecule. Our current study demonstrated a novel mechanism of HMGB1 in the regulation of cancer cell actin polymerization, cell skeleton formation, cancer cell motility and metastasis. We found that knockdown of HMGB1 in human lung cancer A549 cells significantly increased cell ß-actin polymerization, cell skeleton formation, cancer cell migration and invasion in vitro, as well as metastasis in vivo. And this increase could be inhibited by treatment of HMGB1 knockdown cells with recombinant human HMGB1. Further studies discovered that HMGB1 suppressed phosphorylation, nuclear translocation, and activation of CREB, by inhibiting nuclear translocation of PKA catalytic subunit. This reduces nWASP mRNA transcription and expression, further impairing cancer cell motility. Our findings on the novel mechanism underlying the HMGB1 anti-metastatic effect on cancer provides significant insight into the understanding of the nature of HMGB1 in cancer invasion and metastasis, further serving as key information for utilization of HMGB1 and its regulated downstream components as new targets for cancer therapy.

Ubiquitylation-dependent negative regulation of WASp is essential for actin cytoskeleton dynamics.

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin dynamics during cell motility and adhesion, and mutations in its gene are responsible for Wiskott-Aldrich syndrome (WAS). Here, we demonstrate that WASp is ubiquitylated following T-cell antigen receptor (TCR) activation. WASp phosphorylation at tyrosine 291 results in recruitment of the E3 ligase Cbl-b, which, together with c-Cbl, carries out WASp ubiquitylation. Lysine residues 76 and 81, located at the WASp WH1 domain, which contains the vast majority of WASp gene mutations, serve as the ubiquitylation sites. Disruption of WASp ubiquitylation causes WASp accumulation and alters actin dynamics and the formation of actin-dependent structures. Our data suggest that regulated degradation of activated WASp might be an efficient strategy by which the duration and localization of actin rearrangement and the intensity of T-cell activation are controlled.

Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins.

BACKGROUND: Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. RESULTS: We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. CONCLUSIONS: All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics.

Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells.

Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution.

Influence of phalloidin on the formation of actin filament branches by Arp2/3 complex.

The cyclic peptide phalloidin binds and stabilizes actin filaments. It is widely used in studies of actin filament assembly, including analysis of branch formation by Arp2/3 complex, but its influence on the branching reaction has not been considered. Here we show that rhodamine-phalloidin binds both Arp2/3 complex and the VCA domain of Arp2/3 complex activator, hWASp, with dissociation equilibrium constants of about 100 nM. Not only does phalloidin promote nucleation of pure actin monomers but it also dramatically stimulates branch formation by actin, Arp2/3 complex, and hWASp-VCA more than 10-fold and inhibits dissociation of branches. Therefore, the appearance of more branches in samples treated with rhodamine-phalloidin arises from multiple influences of the peptide on both the formation and dissociation of branches.

The phenomenon of spontaneous genetic reversions in the Wiskott-Aldrich syndrome: a report of the workshop of the ESID Genetics Working Party at the XIIth Meeting of the European Society for Immunodeficiencies (ESID). Budapest, Hungary October 4-7, 2006.

The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disease caused by mutations in the Wiskott-Aldrich Protein (WASP) gene, which typically leads to absent WASP protein expression in WAS leukocytes. However, some patients have been found with small populations of WASP-expressing cells caused by reverse or second-site mutations that allow protein expression. An international consortium was established to further investigate these phenomena. This paper summarizes data collected by this consortium that was presented at a workshop held during the XIIth Meeting of the European Society for Immunodeficiencies (ESID), October, 2006. WASP reversions were noted in approximately 11% of 272 patients tested. Many different cell lineages showed reversions. These data form the foundation for further investigation into this phenomenon, which has implications for therapy of this disease.

The expression of Wiskott-Aldrich syndrome protein (WASP) is dependent on WASP-interacting protein (WIP).

The Wiskott-Aldrich syndrome protein (WASP) is a key molecule for transduction of extracellular signals that induce a variety of critical biological events involving actin cytoskeleton rearrangement. Among the cellular partners of WASP, the Wiskott-Aldrich syndrome protein-interacting protein (WIP) has been speculated to play a critical role in the pathophysiology of Wiskott-Aldrich syndrome since WASP mutation hot spots map to the WIP-binding region. The notion that WIP promotes WASP function, however, conflicts with evidence that WIP inhibits WASP-mediated actin polymerization and IL-2 production and suggests a complex regulation of WASP function by WIP. Here we show that WASP gene transfer results in high WASP expression only when WIP is concomitantly expressed in K562 cells. Furthermore, WIP-knockdown experiments demonstrated that T cells with reduced WIP expression show a concordant reduction of WASP levels. Mapping studies using WIP mutants showed that the minimal WIP region able to rescue WASP expression in WIP-knockdown cells was the WASP-binding domain. However, expression of such a minimal domain of WIP failed to rescue WASP-dependent, nuclear factor of activated T-cells-mediated IL-2 transcriptional activity. These results demonstrate that expression of WIP is necessary for functional WASP expression in human cells and provide a new paradigm for understanding the function of these two molecules.

Developmental expression of neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous protein (WAVE)-related proteins in postnatal rat cerebral cortex and hippocampus.

The actin cytoskeleton plays a critical role in the cellular morphological changes. Its organization is essential for neurite extension and synaptogenesis under the processes of neuronal development. Recently, neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous protein (WAVE) have been identified as key molecules, which specifically participate in regulation of actin cytoskeleton through small GTPases. The functions of these factors have been investigated using cultured cells; however, in vivo developmental changes in these factors are not fully understood. In this study, we examined the expression levels and distributions of N-WASP, WAVE and their related proteins in the rat cerebral cortex and hippocampus during postnatal development. Protein levels of these factors were progressively increased during development, and actin was accumulated in membranous fractions. Immunoreactivities for these factors were widely but differentially observed in entire brain. In the developing brain, N-WASP and WAVE seemed to exist in the synapse-rich areas, such as stratum radiatum of hippocampal CA1 subfield. A similar tendency in the distributions of these factors was observed in the mature brain. Taken together, N-WASP, WAVE and their related proteins may participate in normal brain development and synaptic plasticity by regulating the actin cytoskeleton.