A New Set of in Silico Tools to Support the Interpretation of ATM Missense Variants Using Graphical Analysis.

Establishing the pathogenic nature of variants in ATM, a gene associated with breast cancer and other hereditary cancers, is crucial for providing patients with adequate care. Unfortunately, achieving good variant classification is still difficult. To address this challenge, we extended the range of in silico tools with a series of graphical tools devised for the analysis of computational evidence by health care professionals. We propose a family of fast and easy-to-use graphical representations in which the impact of a variant is considered relative to other pathogenic and benign variants. To illustrate their value, the representations are applied to three problems in variant interpretation. The assessment of computational pathogenicity predictions showed that the graphics provide an intuitive view of prediction reliability, complementing and extending conventional numerical reliability indexes. When applied to variant of unknown significance populations, the representations shed light on the nature of these variants and can be used to prioritize variants of unknown significance for further studies. In a third application, the graphics were used to compare the two versions of the ATM-adapted American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines, obtaining valuable information on their relative virtues and weaknesses. Finally, a server [ATMision (ATM missense in silico interpretation online)] was generated for users to apply these representations in their variant interpretation problems, to check the ATM-adapted guidelines' criteria for computational evidence on their variant(s) and access different sources of information.

(-)-Oleocanthal induces death preferentially in tumor hematopoietic cells through caspase dependent and independent mechanisms.

Olive oil is a key component of the highly cardiovascular protective Mediterranean diet. (-)-Oleocanthal (OLC) is one of the most interesting phenolics present in virgin olive oil, and is formed from secoiridoid ligustroside during the processing of olives to yield the oil. Anti-inflammatory and anti-oxidant properties were identified shortly after OLC isolation, followed by the discovery of anti-tumor activities in a few non-hematopoietic cell lineages. Because of the scarcity of tissues potentially targeted by OLC analyzed so far and the unresolved mechanism(s) for OLC anti-tumor properties, we used a panel of 17 cell lines belonging to 11 tissue lineages to carry out a detailed examination of targets and pathways leading to cell growth inhibition and death. We found that OLC inhibits cell proliferation and induces apoptotic death as revealed by sub-G1 cell cycle analyses and Annexin-V staining in all lineages analyzed except lung carcinoma cell lines. Hematopoietic tumor cell lines, untested until now, were the most sensitive to OLC treatment, whereas non-transformed cells were significantly resistant to cell death. The specificity of OLC-mediated caspase activation was confirmed by blocking experiments and the use of transfectants overexpressing anti apoptotic genes. OLC triggers typical mediators of the intrinsic apoptotic pathway such as production of reactive oxygen species and mitochondrial membrane depolarization (Δψm). Complete blockade of caspases, however, did not result in parallel abrogation of Annexin-V staining, thus suggesting that complex mechanisms are involved in triggering OLC-mediated cell death. Our results demonstrate that OLC preferentially targets hematopoietic tumor cell lines and support that cell death is mediated by caspase-dependent and independent mechanisms.

Synthesis and screening of 6-alkoxy purine analogs as cell type-selective apoptotic inducers in Jurkat cells.

Purines are ubiquitous structures in cell biology involved in a multitude of cellular processes, because of which substituted purines and analogs are considered excellent scaffolds in drug design. In this study, we explored the key structural features of a purine-based proapoptotic hit, 8-tert-butyl-9-phenyl-6-benzyloxy-9H-purine (1), by setting up a library of 6-alkoxy purines with the aim of elucidating the structural requirements that govern its biological activity and to study the cell selectivity of this chemotype. This was done by a phenotypic screening approach based on cell cycle analysis of a panel of six human cancer cell lines, including T cell leukemia Jurkat cells. From this study, two derivatives (12 and 13) were identified as Jurkat-selective proapoptotic compounds, displaying superior potency and cell selectivity than hit 1.

A Collaborative Effort to Define Classification Criteria for ATM Variants in Hereditary Cancer Patients.

BACKGROUND: Gene panel testing by massive parallel sequencing has increased the diagnostic yield but also the number of variants of uncertain significance. Clinical interpretation of genomic data requires expertise for each gene and disease. Heterozygous ATM pathogenic variants increase the risk of cancer, particularly breast cancer. For this reason, ATM is included in most hereditary cancer panels. It is a large gene, showing a high number of variants, most of them of uncertain significance. Hence, we initiated a collaborative effort to improve and standardize variant classification for the ATM gene. METHODS: Six independent laboratories collected information from 766 ATM variant carriers harboring 283 different variants. Data were submitted in a consensus template form, variant nomenclature and clinical information were curated, and monthly team conferences were established to review and adapt American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria to ATM, which were used to classify 50 representative variants. RESULTS: Amid 283 different variants, 99 appeared more than once, 35 had differences in classification among laboratories. Refinement of ACMG/AMP criteria to ATM involved specification for twenty-one criteria and adjustment of strength for fourteen others. Afterwards, 50 variants carried by 254 index cases were classified with the established framework resulting in a consensus classification for all of them and a reduction in the number of variants of uncertain significance from 58% to 42%. CONCLUSIONS: Our results highlight the relevance of data sharing and data curation by multidisciplinary experts to achieve improved variant classification that will eventually improve clinical management.

Reconstitution of the Ataxia-Telangiectasia Cellular Phenotype With Lentiviral Vectors.

Ataxia-telangiectasia (A-T) is a complex disease arising from mutations in the ATM gene (Ataxia-Telangiectasia Mutated), which plays crucial roles in repairing double-strand DNA breaks (DSBs). Heterogeneous immunodeficiency, extreme radiosensitivity, frequent appearance of tumors and neurological degeneration are hallmarks of the disease, which carries high morbidity and mortality because only palliative treatments are currently available. Gene therapy was effective in animal models of the disease, but the large size of the ATM cDNA required the use of HSV-1 or HSV/AAV hybrid amplicon vectors, whose characteristics make them unlikely tools for treating A-T patients. Due to recent advances in vector packaging, production and biosafety, we developed a lentiviral vector containing the ATM cDNA and tested whether or not it could rescue cellular defects of A-T human mutant fibroblasts. Although the cargo capacity of lentiviral vectors is an inherent limitation in their use, and despite the large size of the transgene, we successfully transduced around 20% of ATM-mutant cells. ATM expression and phosphorylation assays indicated that the neoprotein was functional in transduced cells, further reinforced by their restored capacity to phosphorylate direct ATM substrates such as p53 and their capability to repair radiation-induced DSBs. In addition, transduced cells also restored cellular radiosensitivity and cell cycle abnormalities. Our results demonstrate that lentiviral vectors can be used to rescue the intrinsic cellular defects of ATM-mutant cells, which represent, in spite of their limitations, a proof-of-concept for A-T gene therapy.

Regulated expression of murine CD40L by a lentiviral vector transcriptionally targeted through its endogenous promoter.

BACKGROUND: Targeted lentiviral vectors may contribute to circumventing genotoxicity associated with uncontrolled transcription of therapeutic genes. Some vectors replacing strong viral sequences for gene promoters such as ß-globin, CD4, CD19 or Igκ were able to drive tissue-specific expression of the transgene. Gene therapy, however, faces even greater hurdles when the therapeutic transgene is subject to strict regulatory mechanisms. This is the case of the CD40LG gene, which encodes for the CD154 (also known as CD40L) molecule, transiently expressed upon activation on CD4(+) T cells. Mutations in this gene cause the X-linked hyper IgM syndrome (HIGM1) in humans because the interaction of CD40L with its ligand CD40 triggers signals that are critical for the immunobiology of B lymphocytes. METHODS: We developed a lentiviral vector containing the murine Cd40lg cDNA under the control of its endogenous promoter. RESULTS: The CD4(+) BW5147 T cells transduced with the pCd40lg-Cd40lg lentiviral vector express CD40L only upon stimulation. The intensity of the expression correlates with the number of vector integrations per cell and detected molecules rapidly decay after removing the stimulating agent. The tissue-specific, activation-dependent and reversible expression of CD40L fully mimics the physiological induction and disappearance of the molecule from the surface of murine T lymphocytes. The functional activity of the regulated lentiviral vector is demonstrated by the ability of transduced BW5147 cells to promote the proliferation of purified B cell splenocytes. CONCLUSIONS: We have developed a fine-regulated lentiviral vector that can be a model for expressing molecules subject to stringent regulatory mechanisms.

Synthesis of 6,8,9 poly-substituted purine analogue libraries as pro-apoptotic inducers of human leukemic lymphocytes and DAPK-1 inhibitors.

A 18-member library of 6,8,9-poly-substituted purines was prepared from pyrimidines, primary alcohols, and N,N-dimethylamides under basic conditions via a novel one-pot synthetic pathway controlled by amide sizes and the novel analogues were tested against two leukemia cell lines: Jurkat (acute T cell leukemia) and K562 (chronic erythroleukemia) cells. Compounds having a benzoxy group at C6 position of the aromatic ring exhibited antiproliferative activity in Jurkat cells whereas all compounds induced a lower effect on K562 cells. Analysis of cell cycle, Annexin-V staining, and cleavage of initiator caspases assays showed that the active purine analogues induce cell death by apoptosis. Based on these results, a new purine derivative was synthesized, 6-benzyloxy-9-tert-butyl-8-phenyl-9H-purine (6d), which displayed the highest activity of the series against Jurkat cell lines. Finally, (33)P-radiolabeled kinase assays using 96 recombinant human kinases known to be involved in apoptotic events were performed. Just one of the kinases tested, DAPK-1, was inhibited 50% or more by the phenotypic hits at 10 µM, suggesting that the inhibition of this target could be responsible for the induction of cell death by apoptosis. In agreement with the phenotypic results, the most active antiproliferative agent, 6d, displayed also the lowest IC50 value against recombinant DAPK1 (2.5 µM), further supporting the potential role of this protein on the observed functional response. DAPK-1 inhibition led by 6d together with its pro-apoptotic properties against the Jurkat line makes it an interesting candidate to further investigate the role of DAPK1 kinase in triggering apoptosis in cancer cells, a role which is attracting recent interest.

An exopolysaccharide produced by the novel halophilic bacterium Halomonas stenophila strain B100 selectively induces apoptosis in human T leukaemia cells.

Microbial exopolysaccharides (EPSs) are highly heterogeneous polymers produced by fungi and bacteria and have recently been attracting considerable attention from biotechnologists because of their potential applications in many fields, including biomedicine. We have screened the antitumoural activity of a panel of sulphated EPSs produced by a newly discovered species of halophilic bacteria. We found that the novel halophilic bacterium Halomonas stenophila strain B100 produced a heteropolysaccharide that, when oversulphated, exerted antitumoural activity on T cell lines deriving from acute lymphoblastic leukaemia (ALL). Only tumour cells were susceptible to apoptosis induced by the sulphated EPS (B100S), whilst primary T cells were resistant. Moreover, freshly isolated primary cells from the blood of patients with ALL were also susceptible to B100S-induced apoptosis. The newly discovered B100S is therefore the first bacterial EPS that has been demonstrated to exert a potent and selective pro-apoptotic effect on T leukaemia cells, and thus, we propose that the search for new antineoplastic drugs should include the screening of other bacterial EPSs, particularly those isolated from halophiles.

IL-17-producing CD8+ T lymphocytes from psoriasis skin plaques are cytotoxic effector cells that secrete Th17-related cytokines.

IL-17-producing CD4+ T lymphocytes (Th17) are currently considered relevant participants in the pathogenesis of psoriasis skin lesions. However, little is known about the potential role of IL-17-producing CD8+ T cells, which are also present at the psoriatic plaque. We have addressed the functional characterization of this CD8+ subtype of T lymphocytes from psoriasis patients. Our results show that CD8+IL-17+ cells from psoriasis-inflamed skin tissue produce TNF-alpha and IFN-gamma (Th1-related cytokines) as well as IL-17, IL-21, and IL-22 (Th17-related cytokines) efficiently. A significant up-regulation of the RORC transcription factor is also observed. These cells are refractory to Tregs but show a proliferative response to anti-CD3/CD28 stimulation that is enhanced by IL-12 and IL-15. Blocking of TNF-alpha activity inhibits TCR-mediated activation and IL-17 production. CD8+IL-17+ T cells are cytotoxic cells that display TCR/CD3-mediated cytotoxic abilities to kill target cells. Thus, CD8+IL-17+ T cells share some key features with Th17 cells and exhibit remarkable differential abilities attributable to the CD8+ lineage of T lymphocytes, adding new insights into the functional resources of IL-17-producing cells from human epidermis that could be of potential interest to our understanding of the pathogenesis of psoriasis.

Hematopoietic-specific lentiviral vectors circumvent cellular toxicity due to ectopic expression of Wiskott-Aldrich syndrome protein.

Efficient and safe gene modification of hematopoietic stem cells is a requirement for gene therapy of primary immunodeficiencies such as Wiskott-Aldrich syndrome. However, deregulated expression or ectopic expression in the progeny of transduced nonhematopoietic progenitor cells may lead to unwanted toxicity. We therefore analyzed the effect of ectopic expression of Wiskott-Aldrich syndrome protein (WASp) and the potential benefits of hematopoietic-specific lentiviral vectors (driven by the WAS proximal promoter). Overexpression of WASp by constitutive lentiviral vectors is highly toxic in nonhematopoietic cells because it causes dramatic changes in actin localization and polymerization that result in decreased cell viability, as evidenced by a significant growth disadvantage of WASp-overexpressing nonhematopoietic cells and increased cell death. These toxic effects do not affect cells of hematopoietic origin because, remarkably, we found that WASp cannot be readily overexpressed in T cells, even after multiple vector integrations per cell. The adverse cellular effects found after transduction of nonhematopoietic cells with constitutive lentiviral vectors are overcome by the use of transcriptionally targeted lentiviral vectors expressing WASp, which, at the same time, are efficient tools for gene therapy of WAS as demonstrated by their ability to reconstitute cellular defects from WASp-deficient mouse and human cells. We therefore postulate that transcriptionally regulated lentiviral vectors represent a safer and efficient alternative for the development of clinical protocols of WAS gene therapy.