Heterologous Ad26/Ad5 adenovirus-vectored vaccines elicited SARS-CoV-2-specific antibody responses with potent Fc activities.

Introduction: Antibodies against the SARS-CoV-2 spike protein are a critical immune determinant for protection against the virus. While virus neutralization is a key function of spike-specific antibodies, antibodies also mediate Fc-dependent activities that can play a role in protection or pathogenesis. Methods: This study characterized serum antibody responses elicited after two doses of heterologous adenovirus-vectored (Ad26/ Ad5) vaccines. Results: Vaccine-induced antibody binding titers and Fc-mediated functions decreased over six months, while neutralization titers remained stable. Comparison of antibody isotypes elicited after Ad26/Ad5 vs. LNP-mRNA vaccination and after infection showed that anti-spike IgG1 were dominant and produced to high levels in all groups. The Ad26/Ad5 vaccines also induced IgG4 but not IgG2 and IgG3, whereas the LNP-mRNA vaccines elicited a full Ig spectrum (IgM, IgG1-4, IgA1-2). Convalescent COVID-19 patients had mainly IgM and IgA1 alongside IgG1. Despite these differences, the neutralization potencies against early variants were similar. However, both vaccine groups had antibodies with greater Fc potencies of binding complement and Fcg receptors than the COVID-19 group. The Ad26/Ad5 group also displayed a greater potency of RBD-specific antibody-mediated cellular phagocytosis. Discussion: Antibodies with distinctive quality were induced by different vaccines and infection. The data imply the utility of different vaccine platforms to elicit antibody responses with fine-tuned Fc activities.

Synergistic Deoxynucleoside and Gene Therapies for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2KI ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2KI mice. METHODS: We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno-associated virus (AAV)-TK2 was administered to Tk2KI , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides. RESULTS: AAV9 delivery of human TK2 cDNA to Tk2KI mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2KI mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2KI mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan. INTERPRETATION: Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2KI mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640-652.

Targeting a Braf/Mapk pathway rescues podocyte lipid peroxidation in CoQ-deficiency kidney disease.

Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.

MET Inhibition Elicits PGC1α-Dependent Metabolic Reprogramming in Glioblastoma.

The receptor kinase c-MET has emerged as a target for glioblastoma therapy. However, treatment resistance emerges inevitably. Here, we performed global metabolite screening with metabolite set enrichment coupled with transcriptome and gene set enrichment analysis and proteomic screening, and identified substantial reprogramming of tumor metabolism involving oxidative phosphorylation and fatty acid oxidation (FAO) with substantial accumulation of acyl-carnitines accompanied by an increase of PGC1α in response to genetic (shRNA and CRISPR/Cas9) and pharmacologic (crizotinib) inhibition of c-MET. Extracellular flux and carbon tracing analyses (U-13C-glucose, U-13C-glutamine, and U-13C-palmitic acid) demonstrated enhanced oxidative metabolism, which was driven by FAO and supported by increased anaplerosis of glucose carbons. These findings were observed in concert with increased number and fusion of mitochondria and production of reactive oxygen species. Genetic interference with PGC1α rescued this oxidative phenotype driven by c-MET inhibition. Silencing and chromatin immunoprecipitation experiments demonstrated that cAMP response elements binding protein regulates the expression of PGC1α in the context of c-MET inhibition. Interference with both oxidative phosphorylation (metformin, oligomycin) and ß-oxidation of fatty acids (etomoxir) enhanced the antitumor efficacy of c-MET inhibition. Synergistic cell death was observed with c-MET inhibition and gamitrinib treatment. In patient-derived xenograft models, combination treatments of crizotinib and etomoxir, and crizotinib and gamitrinib were significantly more efficacious than single treatments and did not induce toxicity. Collectively, we have unraveled the mechanistic underpinnings of c-MET inhibition and identified novel combination therapies that may enhance its therapeutic efficacy. SIGNIFICANCE: c-MET inhibition causes profound metabolic reprogramming that can be targeted by drug combination therapies.

Activation of LXRß inhibits tumor respiration and is synthetically lethal with Bcl-xL inhibition.

Liver-X-receptor (LXR) agonists are known to bear anti-tumor activity. However, their efficacy is limited and additional insights regarding the underlying mechanism are necessary. By performing transcriptome analysis coupled with global polar metabolite screening, we show that LXR agonists, LXR623 and GW3965, enhance synergistically the anti-proliferative effect of BH3 mimetics in solid tumor malignancies, which is predominantly mediated by cell death with features of apoptosis and is rescued by exogenous cholesterol. Extracellular flux analysis and carbon tracing experiments (U-13 C-glucose and U-13 C-glutamine) reveal that within 5 h, activation of LXRß results in reprogramming of tumor cell metabolism, leading to suppression of mitochondrial respiration, a phenomenon not observed in normal human astrocytes. LXR activation elicits a suppression of respiratory complexes at the protein level by reducing their stability. In turn, energy starvation drives an integrated stress response (ISR) that up-regulates pro-apoptotic Noxa in an ATF4-dependent manner. Cholesterol and nucleotides rescue from the ISR elicited by LXR agonists and from cell death induced by LXR agonists and BH3 mimetics. In conventional and patient-derived xenograft models of colon carcinoma, melanoma, and glioblastoma, the combination treatment of ABT263 and LXR agonists reduces tumor sizes significantly stronger than single treatments. Therefore, the combination treatment of LXR agonists and BH3 mimetics might be a viable efficacious treatment approach for solid malignancies.

Metabolic Reprogramming by Dual AKT/ERK Inhibition through Imipridones Elicits Unique Vulnerabilities in Glioblastoma.

Purpose: The goal of this study is to enhance the efficacy of imipridones, a novel class of AKT/ERK inhibitors that displayed limited therapeutic efficacy against glioblastoma (GBM).Experimental Design: Gene set enrichment, LC/MS, and extracellular flux analyses were used to determine the mechanism of action of novel imipridone compounds, ONC206 and ONC212. Orthotopic patient-derived xenografts were utilized to evaluate therapeutic potency.Results: Imipridones reduce the proliferation of patient-derived xenograft and stem-like glioblastoma cell cultures in vitro and in multiple xenograft models in vivo ONC212 displayed the highest potency. High levels of c-myc predict susceptibility to growth inhibition and apoptosis induction by imipridones and increased host survival in orthotopic patient-derived xenografts. As early as 1 hour, imipridones elicit on-target inhibition, followed by dephosphorylation of GSK3ß at serine 9. GSK3ß promotes phosphorylation of c-myc at threonine 58 and enhances its proteasomal degradation. Moreover, inhibition of c-myc by BRD4 antagonists sensitizes for imipridone-induced apoptosis in stem-like GBM cells in vitro and in vivo Imipridones affect energy metabolism by suppressing both glycolysis and oxidative phosphorylation, which is accompanied by a compensatory activation of the serine-one carbon-glycine (SOG) pathway, involving the transcription factor ATF4. Interference with the SOG pathway through novel inhibitors of PHGDH results in synergistic cell death induction in vitro and in vivo Conclusions: These results suggest that c-myc expression predicts therapeutic responses to imipridones and that imipridones lead to suppression of tumor cell energy metabolism, eliciting unique metabolic vulnerabilities that can be exploited for clinical relevant drug combination therapies. Clin Cancer Res; 24(21); 5392-406. ©2018 AACR.

Combined HDAC and Bromodomain Protein Inhibition Reprograms Tumor Cell Metabolism and Elicits Synthetic Lethality in Glioblastoma.

Purpose: Glioblastoma remains a challenge in oncology, in part due to tumor heterogeneity.Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as the primary model systems.Results: Based on a transcriptome and subsequent gene set enrichment analysis (GSEA), we show by using clinically validated compounds that the combination of histone deacetylase (HDAC) inhibition and bromodomain protein (BRD) inhibition results in pronounced synergistic reduction in cellular viability in patient-derived xenograft and stem-like glioblastoma cells. Transcriptome-based GSEA analysis suggests that metabolic reprogramming is involved with synergistic reduction of oxidative and glycolytic pathways in the combination treatment. Extracellular flux analysis confirms that combined HDAC inhibition and BRD inhibition blunts oxidative and glycolytic metabolism of cancer cells, leading to a depletion of intracellular ATP production and total ATP levels. In turn, energy deprivation drives an integrated stress response, originating from the endoplasmic reticulum. This results in an increase in proapoptotic Noxa. Aside from Noxa, we encounter a compensatory increase of antiapoptotic Mcl-1 protein. Pharmacologic, utilizing the FDA-approved drug sorafenib, and genetic inhibition of Mcl-1 enhanced the effects of the combination therapy. Finally, we show in orthotopic patient-derived xenografts of GBM, that the combination treatment reduces tumor growth, and that triple therapy involving the clinically validated compounds panobinostat, OTX015, and sorafenib further enhances these effects, culminating in a significant regression of tumors in vivoConclusions: Overall, these results warrant clinical testing of this novel, efficacious combination therapy. Clin Cancer Res; 24(16); 3941-54. ©2018 AACR.

Inhibition of Bcl-2/Bcl-xL and c-MET causes synthetic lethality in model systems of glioblastoma.

Recent data suggest that glioblastomas (GBM) activate the c-MET signaling pathway and display increased levels in anti-apoptotic Bcl-2 family members. Therefore, targeting these two deregulated pathways for therapy might yield synergistic treatment responses. We applied extracellular flux analysis to assess tumor metabolism. We found that combined treatment with ABT263 and Crizotinib synergistically reduces the proliferation of glioblastoma cells, which was dependent on dual inhibition of Bcl-2 and Bcl-xL. The combination treatment led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, c-MET-inhibition results in significant energy deprivation with a reduction in oxidative phosphorylation, respiratory capacity and a suppression of intracellular energy production (ATP). In turn, loss of energy levels suppresses protein synthesis, causing a decline in anti-apoptotic Mcl-1 levels. Silencing of Mcl-1 enhanced ABT263 and MET-inhibitor mediated apoptosis, but marginally the combination treatment, indicating that Mcl-1 is the central factor for the induction of cell death induced by the combination treatment. Finally, combined treatment with BH3-mimetics and c-MET inhibitors results in significantly smaller tumors than each treatment alone in a PDX model system of glioblastoma. These results suggest that c-MET inhibition causes a selective vulnerability of GBM cells to Bcl-2/Bcl-xL inhibition.

Pediatric patients with inflammatory bowel disease exhibit increased serum levels of proinflammatory cytokines and chemokines, but decreased circulating levels of macrophage inhibitory protein-1ß, interleukin-2 and interleukin-17.

Inflammatory bowel disease (IBD) is a chronic and progressive inflammatory condition of the gastrointestinal tract. Although the causative events that lead to the onset of IBD are yet to be fully elucidated, deregulation of immune and inflammatory mechanisms are hypothesized to significantly contribute to this disorder. Since the onset of IBD is often during infancy, in the present study, the serum values of a large panel of cytokines and chemokines in pediatric patients (<18 years; n=26) were compared with age-matched controls (n=37). While elevations in the serum level of several proinflammatory and immune regulating cytokines were confirmed, such as interleukin (IL)-1ß, IL-5, IL-7, interferon (IFN)-γ-inducible protein-10, IL-16, cutaneous T-cell-attracting chemokine, leukemia inhibitory factor, monokine induced by γ-IFN, IFN-α2 and IFN-γ, notably decreased levels of IL-2, IL-17 and macrophage inhibitory protein-1ß were also observed. Therefore, while a number of proinflammatory cytokines exhibit increased levels in IBD patients, pediatric IBD patients may also exhibit certain aspects of a reduced immunological response.

Lovastatin dose-dependently potentiates the pro-inflammatory activity of lipopolysaccharide both in vitro and in vivo.

Since contradictory findings have been reported on potential effects of statins in modulating the inflammatory response, we have analysed the biological activity of lovastatin both in vitro using the Raw 264.7 murine macrophagic cell line and in vivo using BALB/c mice. When added to Raw 264.7 cells in combination with lipopolysaccharide, lovastatin significantly potentiated the release of interleukin-1ß, interleukin-6 and interleukin-12 with respect to lipopolysaccharide alone and showed an additive effect on the release of nitric oxide. Similarly, when lovastatin was intraperitoneally administrated to BALB/c mice, it did not induce any pro-inflammatory effect when used alone, but it significantly potentiated the pro-inflammatory activity of lipopolysaccharide, in terms of number of intraperitoneal cells and serum levels of serum amyloid A, interleukin-1ß, interleukin-6 and interleukin-12. A potential clinical implication of our study is that lovastatin might exert a pro-inflammatory activity in subjects affected by inflammatory processes, with clinically evident or subclinical infections.

Mouse model of mevalonate kinase deficiency: comparison of cytokine and chemokine profile with that of human patients.

BACKGROUND: Mevalonate kinase deficiency (MKD) is a rare genetic autoinflammatory disease caused by blocking of the enzyme mevalonate kinase in the pathway of cholesterol and isoprenoids. The pathogenic mechanism originating an immune response in MKD patients has not been clearly understood. METHODS: We investigated the dysregulation of expression of selected cytokines and chemokines in the serum of MKD patients. The results have been compared with those observed in an MKD mouse model obtained by treating the mice with aminobisphosphonate, a molecule that is able to inhibit the cholesterol pathway, mimicking the genetic block characteristic of the disease. RESULTS: Interleukin (IL)-1ß, IL-5, IL-6, IL-9, IL-17, granulocyte colony-stimulating factor, monocyte chemotactic protein-1, tumor necrosis factor-α, and IL-4 expression were dysregulated in sera from MKD patients and mice. Moreover, geraniol, an exogenous isoprenoid, when administered to MKD mice, restored cytokines and chemokines levels with values similar to those of untreated mice. CONCLUSION: Our findings, which were obtained in patients and a mouse model mimicking the human disease, suggest that these cytokines and chemokines could be MKD specific and that isoprenoids could be considered as potential therapeutic molecules. The mouse model, even if with some limitations, was robust and suitable for routine testing of potential MKD drugs.

Cytokine levels in the serum of healthy subjects.

Growing knowledge about the cytokine network response has led to a better comprehension of mechanisms of pathologies and to the development of new treatments with biological drugs, able to block specific molecules of the immune response. Indeed, when the cytokine production is deregulated, diseases often occur. The understanding of the physiological mechanism of the cytokine network would be useful to better comprehend pathological conditions. Moreover, since the immune system and response change their properties with development, differences in patients' age should be taken into account, both in physiological and in pathological conditions. In this study, we analyzed the profile of 48 cytokines and chemokines in the serum of healthy subjects, comparing adults (≥18 years) with young children and children (1-6 and 7-17 years). We found that a certain number of cytokines were not being produced in healthy subjects; others showed a constant serum level amongst the groups. Certain cytokines exhibited a downward or an upward trend with increasing age. The remaining cytokines were up- or downregulated in the group of the children with respect to the other groups. In conclusion, we drew some kinds of guidelines about the physiological production of cytokines and chemokines, underling the difference caused by aging.