Fusogenic vesicular stomatitis virus combined with natural killer T cell immunotherapy controls metastatic breast cancer.

BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. CONCLUSION: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.

Differential Cellular Sensing of Fusion from within and Fusion from without during Virus Infection.

The physical entry of virus particles into cells triggers an innate immune response that is dependent on both calcium and nucleic acid sensors, with particles containing RNA or DNA genomes detected by RNA or DNA sensors, respectively. While membrane fusion in the absence of viral nucleic acid causes an innate immune response that is dependent on calcium, the involvement of nucleic acid sensors is poorly understood. Here, we used lipoplexes containing purified reovirus p14 fusion protein as a model of exogenous or fusion from without and a cell line expressing inducible p14 protein as a model of endogenous or fusion from within to examine cellular membrane fusion sensing events. We show that the cellular response to membrane fusion in both models is dependent on calcium, IRF3 and IFN. The method of sensing fusion, however, differs between fusion from without and fusion from within. Exogenous p14 lipoplexes are detected by RIG-I-like RNA sensors, whereas fusion by endogenous p14 requires both RIG-I and STING to trigger an IFN response. The source of nucleic acid that is sensed appears to be cellular in origin. Future studies will investigate the source of endogenous nucleic acids recognized following membrane fusion events.

Citral reduces breast tumor growth by inhibiting the cancer stem cell marker ALDH1A3.

Breast cancer stem cells (CSCs) can be identified by increased Aldefluor fluorescence caused by increased expression of aldehyde dehydrogenase 1A3 (ALDH1A3), as well as ALDH1A1 and ALDH2. In addition to being a CSC marker, ALDH1A3 regulates gene expression via retinoic acid (RA) signaling and plays a key role in the progression and chemotherapy resistance of cancer. Therefore, ALDH1A3 represents a druggable anti-cancer target of interest. Since to date, there are no characterized ALDH1A3 isoform inhibitors, drugs that were previously described as inhibiting the activity of other ALDH isoforms were tested for anti-ALDH1A3 activity. Twelve drugs (3-hydroxy-dl-kynurenine, benomyl, citral, chloral hydrate, cyanamide, daidzin, DEAB, disulfiram, gossypol, kynurenic acid, molinate, and pargyline) were compared for their efficacy in inducing apoptosis and reducing ALDH1A3, ALDH1A1 and ALDH2-associated Aldefluor fluorescence in breast cancer cells. Citral was identified as the best inhibitor of ALDH1A3, reducing the Aldefluor fluorescence in breast cancer cell lines and in a patient-derived tumor xenograft. Nanoparticle encapsulated citral specifically reduced the enhanced tumor growth of MDA-MB-231 cells overexpressing ALDH1A3. To determine the potential mechanisms of citral-mediated tumor growth inhibition, we performed cell proliferation, clonogenic, and gene expression assays. Citral reduced ALDH1A3-mediated colony formation and expression of ALDH1A3-inducible genes. In conclusion, citral is an effective ALDH1A3 inhibitor and is able to block ALDH1A3-mediated breast tumor growth, potentially via blocking its colony forming and gene expression regulation activity. The promise of ALDH1A3 inhibitors as adjuvant therapies for patients with tumors that have a large population of high-ALDH1A3 CSCs is discussed.

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor.

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.

Nonclassical resident macrophages are important determinants in the development of myocardial fibrosis.

Macrophages are increasingly recognized as a potential therapeutic target in myocardial fibrosis via interactions with fibroblasts. We have characterized macrophage depletion and inhibition of nonclassical macrophage migration, in addition to direct interactions between nonclassical macrophages and fibroblasts in angiotensin II (AngII)-mediated, hypertensive myocardial fibrosis. Macrophage depletion was achieved by daily i.v. clodronate liposomes (-1 day to +3 days) during AngII infusion. Cx3cr1(-/-) mice were used to inhibit nonclassical macrophage migration. Macrophage phenotype (F4/80, CD11b, Ly6C) was characterized by immunofluorescence and flow cytometry. Collagen was assessed by Sirius Red/Fast Green. Quantitative real-time RT-PCR was performed for transcript levels. AngII/wild-type (WT) mice displayed significant infiltrate and fibrosis compared with saline/WT, which was virtually ablated by clodronate liposomes independent of hypertension. In vitro data supported M2 macrophages promoting fibroblast differentiation and collagen production. AngII/Cx3cr1(-/-) mice, however, significantly increased macrophage infiltrate and fibrosis relative to AngII/WT. AngII/Cx3cr1(-/-) mice also showed an M1 phenotypic shift relative to WT mice in, which the predominant phenotype was Ly6C(low), CD206(+) (M2). Myocardial IL-1ß was significantly up-regulated, whereas transforming growth factor ß down-regulated with this M1 shift. We demonstrated that infiltrating macrophages are critical to AngII-mediated myocardial fibrosis by preventing the development of fibrosis after liposomal depletion of circulating monocytes. Our findings also suggest that some macrophages, namely M2, may confer a protective myocardial environment that may prevent excessive tissue injury.

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation.

The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell-cell, rather than virus-cell, membrane fusion. The 36-40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell-cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome-liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.

Intracellular delivery of bovine lactoferricin's antimicrobial core (RRWQWR) kills T-leukemia cells.

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide with potent cytotoxic activity against cancer cells. The antimicrobial activity of LfcinB resides in its RRWQWR amino acid sequence (referred to here as LfcinB6); however, the anticancer activity of LfcinB6 is not known. Here, we show that free LfcinB6 did not kill T-leukemia or breast cancer cells but LfcinB6 was strongly cytotoxic when delivered to the cytosolic compartment by fusogenic liposomes. LfcinB6 bound weakly to isolated mitochondria but, unlike LfcinB, did not permeabilize mitochondria or cause cytochrome c to be released. Cathepsin B and caspase activity were important for cytotoxicity caused by intracellular LfcinB6 whereas reactive oxygen species were not involved. The mechanism of LfcinB6-induced cytotoxicity is therefore different from that of LfcinB. We suggest that LfcinB6, in combination with a fusogenic liposome delivery system that selectively targets malignant cells, has potential as a novel anticancer agent.

Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria.

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Liposome reconstitution of a minimal protein-mediated membrane fusion machine.

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein-mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion-associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell-cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome-cell and liposome-liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines.