Characterization of aberrant splicing in pediatric central nervous system tumors reveals CLK1 as a candidate oncogenic dependency.

Pediatric brain cancer is the leading cause of disease-related mortality in children, and many aggressive tumors still lack effective treatment strategies. Despite extensive studies characterizing these tumor genomes, alternative transcriptional splicing patterns remain underexplored. Here, we systematically characterized aberrant alternative splicing across pediatric brain tumors, identifying pediatric high-grade gliomas (HGGs) among the most heterogeneous. Through integration with UniProt Knowledgebase annotations, we identified 12,145 splice events in 5,424 genes, leading to functional changes in protein activation, folding, and localization. We discovered that the master splicing factor and cell-cycle modulator, CDC-like kinase 1 (CLK1), is aberrantly spliced in HGGs to include exon 4, resulting in a gain of two phosphorylation sites and subsequent activation of CLK1. Inhibition of CLK1 with Cirtuvivint in the pediatric HGG KNS-42 cell line significantly decreased both cell viability and proliferation in a dose-dependent manner. Morpholino-mediated depletion of CLK1 exon 4 splicing reduced RNA expression, protein abundance, and cell viability. Notably, KNS-42 cells treated with the CLK1 exon 4 morpholino demonstrated differential expression 78 genes and differential splicing with loss or gain of a functional site in 193 genes annotated as oncogene or tumor suppressor genes (TSGs). These genes were enriched for cancer-associated pathways, with 20 identified as significant gene dependencies in pediatric HGGs. Our findings highlight a dependency of pediatric HGGs on CLK1 and its roles contributing to tumor splicing heterogeneity through transcriptional dysregulation of splicing factors and transcriptional modulation of oncogenes. Overall, aberrant splicing in HGGs and other pediatric brain tumors represents a potentially targetable oncogenic pathway contributing to tumor growth and maintenance.

The Open Pediatric Cancer Project.

Background: In 2019, the Open Pediatric Brain Tumor Atlas (OpenPBTA) was created as a global, collaborative open-science initiative to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Here, we extend the OpenPBTA to create the Open Pediatric Cancer (OpenPedCan) Project, a harmonized open-source multi-omic dataset from 6,112 pediatric cancer patients with 7,096 tumor events across more than 100 histologies. Combined with RNA-Seq from the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA), OpenPedCan contains nearly 48,000 total biospecimens (24,002 tumor and 23,893 normal specimens). Findings: We utilized Gabriella Miller Kids First (GMKF) workflows to harmonize WGS, WXS, RNA-seq, and Targeted Sequencing datasets to include somatic SNVs, InDels, CNVs, SVs, RNA expression, fusions, and splice variants. We integrated summarized CPTAC whole cell proteomics and phospho-proteomics data, miRNA-Seq data, and have developed a methylation array harmonization workflow to include m-values, beta-vales, and copy number calls. OpenPedCan contains reproducible, dockerized workflows in GitHub, CAVATICA, and Amazon Web Services (AWS) to deliver harmonized and processed data from over 60 scalable modules which can be leveraged both locally and on AWS. The processed data are released in a versioned manner and accessible through CAVATICA or AWS S3 download (from GitHub), and queryable through PedcBioPortal and the NCI's pediatric Molecular Targets Platform. Notably, we have expanded PBTA molecular subtyping to include methylation information to align with the WHO 2021 Central Nervous System Tumor classifications, allowing us to create research- grade integrated diagnoses for these tumors. Conclusions: OpenPedCan data and its reproducible analysis module framework are openly available and can be utilized and/or adapted by researchers to accelerate discovery, validation, and clinical translation.

Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.

TARGETED DOWN REGULATION OF CORE MITOCHONDRIAL GENES DURING SARS-COV-2 INFECTION.

Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been reported in COVID-19 patients, but the timing and organs affected vary among reports. Here, we reveal the dynamics of COVID-19 through transcription profiles in nasopharyngeal and autopsy samples from patients and infected rodent models. While mitochondrial bioenergetics is repressed in the viral nasopharyngeal portal of entry, it is up regulated in autopsy lung tissues from deceased patients. In most disease stages and organs, discrete OXPHOS functions are blocked by the virus, and this is countered by the host broadly up regulating unblocked OXPHOS functions. No such rebound is seen in autopsy heart, results in severe repression of genes across all OXPHOS modules. Hence, targeted enhancement of mitochondrial gene expression may mitigate the pathogenesis of COVID-19.

Role of miR-2392 in driving SARS-CoV-2 infection.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provide an exciting avenue toward antiviral therapeutics. From patient transcriptomic data, we determined that a circulating miRNA, miR-2392, is directly involved with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia, as well as promoting many symptoms associated with coronavirus disease 2019 (COVID-19) infection. We demonstrate that miR-2392 is present in the blood and urine of patients positive for COVID-19 but is not present in patients negative for COVID-19. These findings indicate the potential for developing a minimally invasive COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we design a miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters, and may potentially inhibit a COVID-19 disease state in humans.

Adenovirus Remodeling of the Host Proteome and Host Factors Associated with Viral Genomes.

Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. Adenovirus (AdV) encodes proteins from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteome changes induced by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, unbiased proteomics approach to analyze changes to the host proteome during infection with adenovirus serotype 5 (Ad5) virus. We used whole-cell proteomics to measure total protein abundances in the proteome during Ad5 infection. Since host antiviral proteins can antagonize viral infection by associating with viral genomes and inhibiting essential viral processes, we used Isolation of Proteins on Nascent DNA (iPOND) proteomics to identify proteins associated with viral genomes during infection with wild-type Ad5 or an E4 mutant virus. By integrating these proteomics data sets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. IMPORTANCE Viral infections induce myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, post-translational modifications, interactions, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We used an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner, suggesting ways that adenovirus counteracts potentially inhibitory host defenses. This study provides a global view of adenovirus-mediated proteome remodeling, which can serve as a model to investigate virus-host interactions of DNA viruses.

The Great Deceiver: miR-2392's Hidden Role in Driving SARS-CoV-2 Infection.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 positive patients, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we have developed a novel miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters and may potentially inhibit a COVID-19 disease state in humans.

The E3 ubiquitin ligase Cul4b promotes CD4+ T cell expansion by aiding the repair of damaged DNA.

The capacity for T cells to become activated and clonally expand during pathogen invasion is pivotal for protective immunity. Our understanding of how T cell receptor (TCR) signaling prepares cells for this rapid expansion remains limited. Here we provide evidence that the E3 ubiquitin ligase Cullin-4b (Cul4b) regulates this process. The abundance of total and neddylated Cul4b increased following TCR stimulation. Disruption of Cul4b resulted in impaired proliferation and survival of activated T cells. Additionally, Cul4b-deficient CD4+ T cells accumulated DNA damage. In T cells, Cul4b preferentially associated with the substrate receptor DCAF1, and Cul4b and DCAF1 were found to interact with proteins that promote the sensing or repair of damaged DNA. While Cul4b-deficient CD4+ T cells showed evidence of DNA damage sensing, downstream phosphorylation of SMC1A did not occur. These findings reveal an essential role for Cul4b in promoting the repair of damaged DNA to allow survival and expansion of activated T cells.