Effects of NRF2 polymorphisms on safety and efficacy of bardoxolone methyl: subanalysis of TSUBAKI study.

BACKGROUND: In the TSUBAKI study, bardoxolone methyl significantly increased measured and estimated glomerular filtration rates (GFR) in patients with multiple forms of chronic kidney disease (CKD), including Japanese patients with type 2 diabetes and stage 3-4 CKD. Since bardoxolone methyl targets the nuclear factor erythroid 2-related factor 2 pathway, this exploratory analysis of the TSUBAKI study investigated the impact of the regulatory single nucleotide polymorphism, rs6721961, on the effects of bardoxolone methyl. METHODS: Japanese patients aged 20-79 years with type 2 diabetes and stage 3-4 CKD were randomized to bardoxolone methyl 5-15 mg/day (titrated as tolerated) or placebo for 16 weeks. Genotype frequency, clinical characteristics, renal function, and adverse events were primarily assessed. RESULTS: Of 104 patients (bardoxolone methyl n = 55, placebo n = 49); 57% were genotype C/C, 32% C/A and 12% A/A. The frequency of the A/A genotype was higher among patients with diabetic kidney disease than in the general Japanese population (~ 5%). Measured and estimated GFRs increased from baseline in all genotypes receiving bardoxolone methyl. There were no significant differences between genotypes for safety parameters, including blood pressure, bodyweight, and levels of B-type natriuretic peptide, or in the type and frequency of adverse events, suggesting that the efficacy and safety of bardoxolone methyl are unaffected by the rs6721961 polymorphism-617 (C→A) genotype. CONCLUSIONS: Our approach of combining genome analysis with clinical trials for an investigational drug provides important and useful clues for exploring the efficacy and safety of the drug. TRIAL REGISTRATION: ClinicalTrials.gov; NCT02316821.

Non-catalytic conversion of chitin into Chromogen I in high-temperature water.

The non-catalytic conversion of chitin into N-acetyl-ᴅ-glucosamine (GlcNAc) derivatives such as 2-acetamido-2,3-dideoxy-ᴅ-erythro-hex-2-enofuranose (Chromogen I) was investigated in high-temperature water at 290-390 °C and 25 MPa with a reaction time of 0-180 min. High-temperature water treatment is a promising method for chitin conversion as it does not require the use of any additional organic solvents or ionic liquids. A semi-batch reactor was developed to control the reaction temperature and time. It was found that the chitin powder could be converted into a water-soluble fraction in ~90% yield, with Chromogen I being obtained in a maximum yield of 2.6%. Furthermore, a kinetic model was developed to estimate the reaction rate for the conversion of the chitin powder to the water-soluble fraction.

Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.

tRNase-Z(L)-utilizing efficacious (TRUE) gene silencing is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the property of tRNase Z(L) that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA). To search for novel potential therapeutic sgRNAs for hematological malignancies, we screened a library composed of 156 sgRNAs, and found that 20 sgRNAs can efficiently induce apoptosis in leukemia and/or myeloma cells. Furthermore, we demonstrated that 4 of the 20 sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models.

miR-142-3p enhances FcεRI-mediated degranulation in mast cells.

Mast cells are immune cells derived from hematopoietic progenitors. When they are activated by stimuli, they immediately release granule-associated mediators, leading to allergic inflammation. Several factors controlling mediator release have been identified; however, little is known whether microRNAs (miRNAs) are involved in this process. miRNAs are a small class of non-coding RNAs that negatively regulate gene expression. In this study, we investigated the relationship between miRNAs and degranulation in LAD2 cells, a human mast cell line. We demonstrated that silencing of Dicer, a key enzyme of miRNA biogenesis, attenuates degranulation, indicating that miRNAs are involved in mast cell degranulation. We furthermore discovered that the overexpression of miR-142-3p enhances FcεRI-mediated degranulation and that miR-142-3p rescues the reduction of degranulation by silencing Dicer. Similar effects were observed in bone marrow-derived mast cells obtained miR-142-3p-deficient mice. Our studies suggest that miR-142-3p is a potential therapeutic target in pathological conditions caused by mast cells, such as mastocytosis and allergies.

Gain-of-function microRNA screens identify miR-193a regulating proliferation and apoptosis in epithelial ovarian cancer cells.

MicroRNAs (miRNAs) are a small class of non­coding RNAs that negatively regulate gene expression, and are considered as new therapeutic targets for treating cancer. In this study, we performed a gain-of-function screen using miRNA mimic library (319 miRNA species) to identify those affecting cell proliferation in human epithelial ovarian cancer cells (A2780). We discovered a number of miRNAs that increased or decreased the cell viability of A2780 cells. Pro-proliferative and anti-proliferative miRNAs include oncogenic miR-372 and miR-373, and tumor suppressive miR-124a, miR-7, miR-192 and miR-193a, respectively. We found that overexpression of miR-124a, miR-192, miR-193a and miR­193b inhibited BrdU incorporation in A2780 cells, indicating that these miRNAs affected the cell cycle. Overexpression of miR­193a and miR-193b induced an activation of caspase 3/7, and resulted in apoptotic cell death in A2780 cells. A genome­wide gene expression analysis with miR-193a-transfected A2780 cells led to identification of ARHGAP19, CCND1, ERBB4, KRAS and MCL1 as potential miR-193a targets. We demonstrated that miR-193a decreased the amount of MCL1 protein by binding 3'UTR of its mRNA. Our study suggests the potential of miRNA screens to discover miRNAs as therapeutic tools to treat ovarian cancer.

Induction of apoptosis of leukemic cells by TRUE gene silencing using small guide RNAs targeting the WT1 mRNA.

TRUE gene silencing is a technology to eliminate specific cellular RNAs by using tRNase Z(L) and small guide RNA (sgRNA). Here we investigated how WT1-mRNA-targeting sgRNAs affect leukemic cells. We showed that sgRNA can be easily taken up by cells without any transfection reagents, and that the naked sgRNAs targeting the WT1 mRNA can reduce its mRNA levels and WT1 protein amounts in the WT1-expressing leukemic cells. Concomitantly, these sgRNAs efficiently induced apoptosis in these cells but not in WT1-nonexpressing cells. We also demonstrated that the reduction in the WT1 mRNA level is caused by its cleavage by tRNase Z(L).

Integration of pre-normalized microarray data using quantile correction.

An enormous amount of microarray data has been collected and accumulated in public repositories. Although some of the depositions include raw and processed data, significant parts of them include processed data only. If we need to combine multiple datasets for specific purposes, the data should be adjusted prior to use to remove bias between the datasets. We focused on a GeneChip platform and a pre-processing method, RMA, and examined simple quantile correction as the post-processing method for integration. Integration of the data pre-processed by RMA was evaluated using artificial spike-in datasets and real microarray datasets of atopic dermatitis and lung cancer. Studies using the spike-in datasets show that the quantile correction for data integration reduces the data quality at some extent but it should be acceptable level. Studies using the real datasets show that the quantile correction significantly reduces the bias. These results show that the quantile correction is useful for integration of multiple datasets processed by RMA, and encourage effective use of public microarray data.

Functional screening identifies a microRNA, miR-491 that induces apoptosis by targeting Bcl-X(L) in colorectal cancer cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain-of-function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD-1). We discovered a number of miRNAs that increased or decreased cell viability in DLD-1. They included known oncogenic miRNAs such as miR-372 and miR-373, and tumor suppressive miRNAs such as miR-124a, but also some for which this information was novel. Among them, miR-491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl-X(L) was a direct target of miR-491, and its silencing contributed to miR-491-induced apoptosis. Moreover, treatment of miR-491 suppressed in vivo tumor growth of DLD-1 in nude mice. Our study provides a new regulation of Bcl-X(L) by miR-491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl-X(L).