Poliovirus receptor-based chimeric antigen receptor T cells combined with NK-92 cells exert potent activity against glioblastoma.

BACKGROUND: Poliovirus receptor interacts with 3 receptors: T-cell immunoglobulin immunoreceptor tyrosine-based inhibitory motif, CD96, and DNAX accessory molecule 1, which are predominantly expressed on T cells and natural killer (NK) cells. Many solid tumors, including IDH wild-type glioblastoma, have been reported to overexpress poliovirus receptor, and this overexpression is associated with poor prognosis. However, there are no preclinical or clinical trials investigating the use of cell-based immunotherapies targeting poliovirus receptor in IDH wild-type glioblastoma. METHODS: We analyzed poliovirus receptor expression in transcriptome sequencing databases and specimens from IDH wild-type glioblastoma patients. We developed poliovirus receptor targeting chimeric antigen receptor T cells using lentivirus. The antitumor activity of chimeric antigen receptor T cells was demonstrated in patient-derived glioma stem cells, intracranial and subcutaneous mouse xenograft models. RESULTS: We verified poliovirus receptor expression in primary glioma stem cells, surgical specimens from IDH wild-type glioblastoma patients, and organoids. Accordingly, we developed poliovirus receptor-based second-generation chimeric antigen receptor T cells. The antitumor activity of chimeric antigen receptor T cells was demonstrated in glioma stem cells and xenograft models. Tumor recurrence occurred in intracranial xenograft models because of antigen loss. The combinational therapy of tyrosine-based inhibitory motif extracellular domain-based chimeric antigen receptor T cells and NK-92 cells markedly suppressed tumor recurrence and prolonged survival. CONCLUSIONS: Poliovirus receptor-based chimeric antigen receptor T cells were capable of killing glioma stem cells and suppressing tumor recurrence when combined with NK-92 cells.

Diffuse Isocitrate Dehydrogenase-Mutant Gliomas With Histone H3 Alterations Are Distinguished by Unique Clinical Characteristics, Molecular Expression Profile, and Survival Prognosis.

BACKGROUND AND OBJECTIVES: Histopathological features and molecular biomarkers have been studied as potential prognostic factors. This study aimed to investigate the clinical features, molecular phenotypes, and survival prognosis of isocitrate dehydrogenase (IDH)-mutant (IDHmt) gliomas with histone H3 alterations (H3-alterations). METHODS: A total of 236 and 657 patients with whole-exome sequencing data were separately collected from the Chinese Glioma Genome Atlas and The Cancer Genome Atlas databases. Survival analysis of patients with glioma was performed using Kaplan-Meier survival curves stratified by histone H3 status. Univariate and multivariate analyses were used to identify the associations between histone H3 status and other clinicopathological factors with survival in patients with IDH-mutant gliomas. RESULTS: Diffuse gliomas with H3 alterations are more likely to be high grade in 2 cohorts ( P = .025 and P = .021, respectively). IDHmt glioma patients with H3-alteration had significantly less life expectancy than histone H3 wild-type ( P = .041 and P = .008, respectively). In the Chinese Glioma Genome Atlas cohort, Karnofsky performance scores ≤ 80 (HR 2.394, 95% CI 1.257-4.559, P = .008), extent of resection (HR 0.971, 95% CI 0.957-0.986, P < .001), high WHO grade (HR 6.938, 95% CI 2.787-17.269, P < .001), H3-alteration (HR 2.482, 95% CI 1.183-4.981, P = .016), and 1p/19q codeletion (HR 0.169, 95% CI 0.073-0.390, P < .001) were independently associated with IDHmt gliomas. In the The Cancer Genome Atlas cohort, age (HR 1.034, 95% CI 1.008-1.061, P = .010), high WHO grade (HR 2.365, 95% CI 1.263-4.427, P = .007), and H3-alteration (HR 2.501, 95% CI 1.312-4.766, P = .005) were independently associated with IDHmt gliomas. CONCLUSION: Identification and assessment of histone H3 status in clinical practice might help improve prognostic prediction and develop therapeutic strategies for these patient subgroups.

Immunological profiles of human oligodendrogliomas define two distinct molecular subtypes.

BACKGROUND: Human oligodendroglioma presents as a heterogeneous disease, primarily characterized by the isocitrate dehydrogenase (IDH) mutation and 1p/19q co-deletion. Therapy development for this tumor is hindered by incomplete knowledge of somatic driving alterations and suboptimal disease classification. We herein aim to identify intrinsic molecular subtypes through integrated analysis of transcriptome, genome and methylome. METHODS: 137 oligodendroglioma patients from the Cancer Genome Atlas (TCGA) dataset were collected for unsupervised clustering analysis of immune gene expression profiles and comparative analysis of genome and methylome. Two independent datasets containing 218 patients were used for validation. FINDINGS: We identified and independently validated two reproducible subtypes associated with distinct molecular characteristics and clinical outcomes. The proliferative subtype, named Oligo1, was characterized by more tumors of CNS WHO grade 3, as well as worse prognosis compared to the Oligo2 subtype. Besides the clinicopathologic features, Oligo1 exhibited enrichment of cell proliferation, regulation of cell cycle and Wnt signaling pathways, and significantly altered genes, such as EGFR, NOTCH1 and MET. In contrast, Oligo2, with favorable outcome, presented increased activation of immune response and metabolic process. Higher T cell/APC co-inhibition and inhibitory checkpoint levels were observed in Oligo2 tumors. Finally, multivariable analysis revealed our classification was an independent prognostic factor in oligodendrogliomas, and the robustness of these molecular subgroups was verified in the validation cohorts. INTERPRETATION: This study provides further insights into patient stratification as well as presents opportunities for therapeutic development in human oligodendrogliomas. FUNDING: The funders are listed in the Acknowledgement.

Enhancing Activities of Salt-Tolerant Proteases Secreted by Aspergillus oryzae Using Atmospheric and Room-Temperature Plasma Mutagenesis.

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.

Separation, biochemical characterization and salt-tolerant mechanisms of alkaline protease from Aspergillus oryzae.

BACKGROUND: The salt tolerance of proteases secreted by Aspergillus oryzae 3.042 closely relates to the utilization of raw materials and the quality of soy sauce. However, little is known about the salt-tolerant proteases and their salt-tolerant mechanisms. RESULTS: In this study, we isolated and identified a salt-tolerant alkaline protease (AP, approximately 29 kDa) produced by A. oryzae 3.042. It was considered as a metal-ion-independent serine protease. The optimum and stable pH values were both pH 9.0 and the optimum temperature was 40 °C. Over 20% relative activity of AP remained in the presence of 3.0 mol L-1 NaCl after 7 days, but its Km and Vmax were only mildly influenced by the presence of 3.0 mol L-1 NaCl, indicating its outstanding salt tolerance. Furthermore, AP was more stable than non-salt-tolerant protease at high salinity. The salt-tolerant mechanisms of AP could be due to more salt bridges, higher proportion of ordered secondary structures and stronger hydrophobic amino acid residues in the interior. CONCLUSIONS: The above results are vital for maintaining, activating and/or modulating the activity of AP in high-salt environments. They would also provide theoretical guidance for the modification of AP and the engineering of A. oryzae 3.042 so as to secrete more AP. © 2018 Society of Chemical Industry.

Characterization of taste and aroma compounds in Tianyou, a traditional fermented wheat flour condiment.

Taste and aroma compounds in Tianyou were determined using HPLC and GC-MS/GC-olfactometry. By comparison with light soy sauce (control), the contents of salt, sugar, total nitrogen and total acid in Tianyou were higher, while the contents of umami, sweet and bitter free amino acids, and the percentage of 1-5kDa peptides in Tianyou were lower. Thirty-one aroma-active compounds were identified in both Tianyou and the control (30 compounds in common). Aroma extraction dilution analysis indicated that most flavor dilution factors of aroma-active compounds were lower in Tianyou than the control. Quantitative descriptive analysis showed that Tianyou had significantly stronger salty and sweet tastes, weaker umami taste and weaker malty, caramel-like and smoky notes when compared to the control (p<0.05), which were in agreement with the analyses of taste and aroma compounds. This confirms that Tianyou has a distinctively different flavor from light soy sauce.

Purification, characterisation and salt-tolerance molecular mechanisms of aspartyl aminopeptidase from Aspergillus oryzae 3.042.

A salt-tolerant aspartyl aminopeptidase (approximately 57kDa) from Aspergillus oryzae 3.042 was purified and identified. Specific inhibitor experiments indicated that it was an aminopeptidase containing Zn2+. Its optimal and stable pH values and temperatures were 7 and 50°C, respectively. Its relative activity remained beyond 30% in 3M NaCl solution for 15d, and its Km and Vmax were slightly affected in 3M NaCl solution, indicating its excellent salt-tolerance. A comprehensive analysis including protein homology modelling, molecular dynamics simulation, secondary structure, acidic residues and hydrophobicity of interior residues demonstrated that aspartyl aminopeptidase had a greater stability than non-salt-tolerant protease in high salinity. Higher contents of ordered secondary structures, more salt bridges between hydrated surface acidic residues and specific basic residues and stronger hydrophobicity of interior residues were the salt-tolerance mechanisms of aspartyl aminopeptidase.