Levrero, M. | Search bioRxiv

bioRxiv2022

RNA helicase DDX5 mediates adaptive response to multi-kinase inhibitors in liver cancer

ObjectiveTo determine the role of RNA helicase DDX5 in sorafenib/multi-tyrosine kinase inhibitor (mTKI) response. Sorafenib and mTKIs downregulate DDX5 in vitro and preclinical hepatocellular carcinoma (HCC) models. In turn, sorafenib-mediated DDX5 downregulation activates Wnt/{beta}-catenin and non-canonical NF-{kappa}B signaling, resulting in ferroptosis escape and mTKI resistance. Design and ResultsMolecular, pharmacologic and bioinformatic approaches were employed in human HCC cell lines, preclinical HCC models, and HCCs from TCGA. Earlier studies linked sorafenib effectiveness to ferroptosis. Herein we demonstrate sorafenib/mTKIs downregulate DDX5 in vitro and in vivo. To understand the effect of DDX5 downregulation, we compared TCGA-derived HCCs expressing low vs. high DDX5 focusing on ferroptosis-related genes. Glutathione Peroxidase 4 (GPX4), a key ferroptosis regulator, was significantly overexpressed in DDX5LOW HCCs. Importantly, DDX5-knockdown (DDX5KD) HCC cell lines lacked lipid peroxidation by GPX4 inhibition, indicating DDX5 downregulation suppresses ferroptosis. RNAseq of wild type vs. DDX5KD cells untreated or treated with sorafenib, identified a unique set of genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps genes from Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, including NF-{kappa}B inducing Kinase required for non-canonical NF-{kappa}B activation. Pharmacologic inhibition of these pathways in combination with sorafenib reduced DDX5KD cell viability. Mechanistically, sorafenib-mediated NIK expression induced NRF2 transcription, while DDX5KD extended NRF2 half/life by stabilizing p62/SQSTM1, enhancing GPX4 expression and ferroptosis escape. ConclusionSorafenib/mTKI-mediated DDX5 downregulation results in adaptive mTKI resistance by enhancing NRF2 expression, leading to ferroptosis escape. We propose inhibition of the pathways leading to NRF2 expression will enhance the therapeutic effectiveness of sorafenib/mTKIs. O_LIWhat is already known on this topic - In advanced HCC, mTKIs/sorafenib, offer limited survival benefits due to resistance. Combination of VEGF (bevacizumab) and PD-L1 antibody (atezolizumab) has led to its adoption as first line treatment; however, mTKIs are still widely used in patients with contra-indications to bevacizumab; also, in atezolizumab/bevacizumab failure, mTKIs are the gold standard in second or later-line systemic therapy, emphasizing the importance of delineating the mechanism of mTKI resistance. C_LIO_LIWhat this study adds - We show mTKIs and sorafenib downregulate the RNA helicase DDX5. This downregulation of DDX5 by sorafenib enables activation of Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, leading to expression of genes that enable escape from ferroptotic cell death. C_LIO_LIHow this study might affect research, practice or policy The Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways activated by sorafenib/DDX5 downregulation can serve as druggable targets to enhance the anti-cancer effect of mTKIs by inducing ferroptosis, thereby opening-up new therapeutic directions against mTKI resistance. C_LI

cancer biology↗

bioRxiv2022

Performance of the cobas(R) HBV RNA Automated Investigational Assay for the Detection and Quantification of Circulating HBV RNA in Chronic HBV Patients

BackgroundThe amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment. ObjectivesWe evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas(R) 6800/8800 systems. Study DesignThe sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas(R) HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens. Resultscobas(R) HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL). ConclusionsThe cobas(R) 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.

microbiology↗