Identification of novel genes involved in apoptosis of HIV-infected macrophages using unbiased genome-wide screening.

BACKGROUND: Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. METHODS: We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student's t-test from at least four independent experiments. RESULTS: We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. CONCLUSIONS: We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.

Size-tuneable and immunocompatible polymer nanocarriers for drug delivery in pancreatic cancer.

Nanocarriers have emerged as one of the most promising approaches for drug delivery. Although several nanomaterials have been approved for clinical use, the translation from lab to clinic remains challenging. However, by implementing rational design strategies and using relevant models for their validation, these challenges are being addressed. This work describes the design of novel immunocompatible polymer nanocarriers made of melanin-mimetic polydopamine and Pluronic F127 units. The nanocarrier preparation was conducted under mild conditions, using a highly reproducible method that was tuned to provide a range of particle sizes (<100 nm) without changing the composition of the carrier. A set of in vitro studies were conducted to provide a comprehensive assessment of the effect of carrier size (40, 60 and 100 nm) on immunocompatibility, viability and uptake into different pancreatic cancer cells varying in morphological and phenotypic characteristics. Pancreatic cancer is characterised by poor treatment efficacy and no improvement in patient survival in the last 40 years due to the complex biology of the solid tumour. High intra- and inter-tumoral heterogeneity and a dense tumour microenvironment limit diffusion and therapeutic response. The Pluronic-polydopamine nanocarriers were employed for the delivery of irinotecan active metabolite SN38, which is used in the treatment of pancreatic cancer. Increased antiproliferative effect was observed in all tested cell lines after administration of the drug encapsulated within the carrier, indicating the system's potential as a therapeutic agent for this hard-to-treat cancer.

A novel multi-epitope recombinant protein elicits an antigen-specific CD8+ T cells response in Trypanosoma cruzi-infected mice.

About 6.5 million people worldwide are afflicted by Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi. The development of a therapeutic vaccine to prevent the progression of Chagasic cardiomyopathy has been proposed as an alternative for antiparasitic chemotherapy. Bioinformatics tools can predict MHC class I CD8 + epitopes for inclusion in a single recombinant protein with the goal to develop a multivalent vaccine. We expressed a novel recombinant protein Tc24-C4.10E harboring ten nonameric CD8 + epitopes and using Tc24-C4 protein as scaffold to evaluate the therapeutic effect in acute T. cruzi infection. T. cruzi-infected mice were immunized with Tc24-C4.10E or Tc24-C4 in a 50-day model of acute infection. Tc24-C4.10E-treated mice showed a decreased parasitemia compared to the Tc24-C4 (non-adjuvant) immunized mice or control group. Moreover, Tc24-C4.10E induced a higher stimulation index of CD8 + T cells producing IFNγ and IL-4 cytokines. These results suggest that the addition of the MHC Class I epitopes to Tc24-C4 can synergize the antigen-specific cellular immune responses, providing proof-of-concept that this approach could lead to the development of a promising vaccine candidate for Chagas disease.

Cellular senescence in cancer: from mechanisms to detection.

Senescence refers to a cellular state featuring a stable cell-cycle arrest triggered in response to stress. This response also involves other distinct morphological and intracellular changes including alterations in gene expression and epigenetic modifications, elevated macromolecular damage, metabolism deregulation and a complex pro-inflammatory secretory phenotype. The initial demonstration of oncogene-induced senescence in vitro established senescence as an important tumour-suppressive mechanism, in addition to apoptosis. Senescence not only halts the proliferation of premalignant cells but also facilitates the clearance of affected cells through immunosurveillance. Failure to clear senescent cells owing to deficient immunosurveillance may, however, lead to a state of chronic inflammation that nurtures a pro-tumorigenic microenvironment favouring cancer initiation, migration and metastasis. In addition, senescence is a response to post-therapy genotoxic stress. Therefore, tracking the emergence of senescent cells becomes pivotal to detect potential pro-tumorigenic events. Current protocols for the in vivo detection of senescence require the analysis of fixed or deep-frozen tissues, despite a significant clinical need for real-time bioimaging methods. Accuracy and efficiency of senescence detection are further hampered by a lack of universal and more specific senescence biomarkers. Recently, in an attempt to overcome these hurdles, an assortment of detection tools has been developed. These strategies all have significant potential for clinical utilisation and include flow cytometry combined with histo- or cytochemical approaches, nanoparticle-based targeted delivery of imaging contrast agents, OFF-ON fluorescent senoprobes, positron emission tomography senoprobes and analysis of circulating SASP factors, extracellular vesicles and cell-free nucleic acids isolated from plasma. Here, we highlight the occurrence of senescence in neoplasia and advanced tumours, assess the impact of senescence on tumorigenesis and discuss how the ongoing development of senescence detection tools might improve early detection of multiple cancers and response to therapy in the near future.

Vaccine-linked chemotherapy induces IL-17 production and reduces cardiac pathology during acute Trypanosoma cruzi infection.

Chagas disease resulting from Trypanosoma cruzi infection leads to a silent, long-lasting chronic neglected tropical disease affecting the poorest and underserved populations around the world. Antiparasitic treatment with benznidazole does not prevent disease progression or death in patients with established cardiac disease. Our consortium is developing a therapeutic vaccine based on the T. cruzi flagellar-derived antigen Tc24-C4 formulated with a Toll-like receptor 4 agonist adjuvant, to complement existing chemotherapy and improve treatment efficacy. Here we demonstrate that therapeutic treatment of acutely infected mice with a reduced dose of benznidazole concurrently with vaccine treatment - also known as "vaccine-linked chemotherapy"-induced a TH17 like immune response, with significantly increased production of antigen specific IL-17A, IL-23 and IL-22, and CD8 + T lymphocytes, as well as significantly increased T. cruzi specific IFNγ-producing CD4 + T lymphocytes. Significantly reduced cardiac inflammation, fibrosis, and parasite burdens and improved survival were achieved by vaccine-linked chemotherapy and individual treatments. Importantly, low dose treatments were comparably efficacious to high dose treatments, demonstrating potential dose sparing effects. We conclude that through induction of TH17 immune responses vaccine-linked chemotherapeutic strategies could bridge the tolerability and efficacy gaps of current drug treatment in Chagasic patients.

Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity.

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence-associated lysosomal ß-galactosidase (SA-ß-gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose-encapsulated nanoparticles within these cells. Here, we show that galacto-conjugation of the BCL-2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav-Gal), that can be preferentially activated by SA-ß-gal activity in a wide range of cell types. Nav-Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav-Gal enhances the cytotoxicity of standard senescence-inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav-Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto-conjugation reduces Navitoclax-induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities.

Humanized yeast genetic interaction mapping predicts synthetic lethal interactions of FBXW7 in breast cancer.

BACKGROUND: Synthetic lethal interactions (SLIs) that occur between gene pairs are exploited for cancer therapeutics. Studies in the model eukaryote yeast have identified ~ 550,000 negative genetic interactions that have been extensively studied, leading to characterization of novel pathways and gene functions. This resource can be used to predict SLIs that can be relevant to cancer therapeutics. METHODS: We used patient data to identify genes that are down-regulated in breast cancer. InParanoid orthology mapping was performed to identify yeast orthologs of the down-regulated genes and predict their corresponding SLIs in humans. The predicted network graphs were drawn with Cytoscape. CancerRXgene database was used to predict drug response. RESULTS: Harnessing the vast available knowledge of yeast genetics, we generated a Humanized Yeast Genetic Interaction Network (HYGIN) for 1009 human genes with 10,419 interactions. Through the addition of patient-data from The Cancer Genome Atlas (TCGA), we generated a breast cancer specific subnetwork. Specifically, by comparing 1009 genes in HYGIN to genes that were down-regulated in breast cancer, we identified 15 breast cancer genes with 130 potential SLIs. Interestingly, 32 of the 130 predicted SLIs occurred with FBXW7, a well-known tumor suppressor that functions as a substrate-recognition protein within a SKP/CUL1/F-Box ubiquitin ligase complex for proteasome degradation. Efforts to validate these SLIs using chemical genetic data predicted that patients with loss of FBXW7 may respond to treatment with drugs like Selumitinib or Cabozantinib. CONCLUSIONS: This study provides a patient-data driven interpretation of yeast SLI data. HYGIN represents a novel strategy to uncover therapeutically relevant cancer drug targets and the yeast SLI data offers a major opportunity to mine these interactions.