Biology subjects

Magri, A.

Publications and source records attributed to Magri, A..

9 recordsLinked to original sources

bioRxiv2023

VDAC1 selective molecules promote patients-derived cancer organoids death through mitochondrial-dependent metabolic interference

In the continuous pursuit of advanced cancer therapeutics, our research unveils the potential to selectively target Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1), a pivotal component in cellular metabolism and apoptosis. VDAC1s role in metabolic rewiring and its subsequent prominence in many cancer types offer a unique intervention point. The incorporation of a systematic, in silico to in vitro methodology identified novel VA (VDAC-Antagonist) molecules with the capability to selectively bind to VDAC1, displaying a substantial specificity towards cancer cells while sparing healthy ones. This research first led to the revelation of a specialized VDAC1 pocket, which accommodates the binding of these VA molecules, thereby instigating a selective displacement of NADH. The coenzyme is a critical metabolic substrate, and its displacement ensues in notable mitochondrial distress and a reduction in cell proliferation, specifically in cancer cells. Furthermore, meticulous analysis using organoids derived from intrahepatic cholangiocarcinoma patients (iCCA) demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules, correlating with the findings from commercial cell lines. Interestingly, VA molecules significantly reduced cell viability and demonstrated a lower impact on healthy cells than conventional treatments like gemcitabine. This differential impact is possibly due to the elevated expression of VDAC1 in various cancer cell lines, rendering them more susceptible to metabolic disruptions induced by VA molecules. This endeavor uncovers a multifaceted approach to cancer treatment, involving meticulous targeting of metabolic gatekeepers like VDAC1 using novel entities, thereby paving the way for developing more selective and refined cancer therapeutics. The identified VA molecules, albeit in the nascent stages, represent promising candidates for further optimization and development, potentially revolutionizing treatment modalities in cancer therapy through precise metabolic interventions.

biophysics↗

bioRxiv2023

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. However, our knowledge of the host pathways that define susceptibility to RSV infection and disease severity is limited. Hypoxia inducible factors (HIFs) define cellular metabolic processes in response to low oxygen and are recognised to play a key role in regulating inflammatory responses in the lower respiratory tract. We demonstrate a role for hypoxia inducible factors (HIFs) to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors significantly reduce expression of the RSV entry receptor nucleolin and inhibit viral driven cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for treating this respiratory pathogen.

molecular biology↗

bioRxiv2023

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5 stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bio-informatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIF expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen. Author SummaryOxygen levels influence HBV replication and hypoxia inducible factors (HIFs) activate HBV transcription. Long-read sequencing and mapping the HBV transcriptome showed an increased abundance of all viral RNAs under hypoxic conditions that was dependent on N6-methyladenosine modifications. Investigating the oxygen-dependent expression of RNA demethylases identified ALKBH5 as a hypoxic activated gene and silencing its expression showed a key role in regulating HBV and host gene expression under hypoxic conditions.

molecular biology↗

bioRxiv2023

CTCF regulates hepatitis B virus cccDNA chromatin topology

Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcripts, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessibility/sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.

microbiology↗

bioRxiv2023

ERK1/2-dependent TSPO overactivation associates with the loss of mitophagy and mitochondrial respiration in ALS

Mitochondrial dysfunction and the loss of mitophagy, aimed at recycling irreversibly damaged organelles, contribute to the onset of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting spinal cord motor neurons. In this work, we showed that the reduction of mitochondrial respiration, exactly oxygen flows linked to ATP production and maximal capacity, correlates with the appearance of the most common ALS motor symptoms in a transgenic mice model expressing SOD1 G93A mutant. This is the result of the equal inhibition in the respiration linked to complex I and II of the electron transport chain, but not their protein levels. Since the overall mitochondrial mass was unvaried, we investigated the expression of the Translocator Protein (TSPO), a small mitochondrial protein whose overexpression was recently linked to the loss of mitophagy in a model of Parkinsons disease. Here we clearly showed that levels of TSPO are significantly increased in ALS mice. Mechanistically, this increase is linked to the overactivation of ERK1/2 pathway and correlates with a decrease in the expression of the mitophagy-related marker, Atg12, indicating the occurrence of impairments in the activation of mitophagy. Overall, our work sets out TSPO as a key regulator of mitochondrial homeostasis in ALS.

neuroscience↗

bioRxiv2022

Circadian regulation of human immunodeficiency virus type 1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks curative therapies or vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organisms response to its environment and invading pathogens. Recent reports of diurnal variation in peripheral viral load in HIV-1 infected subjects highlights the need for mechanistic studies. Here, we demonstrate rhythmic HIV-1 replication in circadian-synchronised model systems and show the circadian transcription factors, BMAL1 and REV-ERB, bind conserved motifs in the HIV-1 promoter. REV-ERB competes with ROR for binding to the same regulatory motif, and we uncover a role for ROR inhibitors to perturb rhythmic HIV-1 replication and host gene expression. We demonstrate that many HIV-1 host factors are circadian regulated and likely to define rhythmic viral replication. In summary, this study increases our understanding of the mechanisms underlying the circadian regulation of HIV that can inform HIV therapy and management.

microbiology↗

bioRxiv2020

Viral genome wide association study identifies novel hepatitis C virus polymorphisms associated with sofosbuvir treatment failure

Chronic hepatitis C virus (HCV) infection is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. With the recent development of direct acting antivirals (DAA), treatment of chronically infected patients has become highly effective although a subset of patients do not respond to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies. We used pre-treatment whole genome sequencing of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We found that three common polymorphisms present in HCV NS2 and NS3 proteins (not direct targets of sofosbuvir) were associated with reduced treatment response. These polymorphisms were enriched in post-treatment HCV sequences of patients unresponsive to treatment; they were also associated with lower reductions in viral load in the first week of therapy. The finding of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of more systematic genome-wide analyses of HCV in uncovering indirect but clinically relevant mechanisms of antiviral resistance.

microbiology↗

bioRxiv2020

Hypoxic gene expression in chronic hepatitis B infected patients is not observed in state-of-art in vitro and mouse infection models

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

microbiology↗

bioRxiv2019

Evolving neoantigen profiles in colorectal cancers with DNA repair defects

BackgroundNeoantigens that arise as a consequence of tumour-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumour types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.\n\nMethodsWe performed Whole Exome Sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures and predicted neoantigens.\n\nResultsThe majority of CRC models showed remarkably stable mutational and neoantigen profiles, however those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures, and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly-evolving CRC.\n\nConclusionsThese results indicate that CRC carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

cancer biology↗