Biology subjects

Brown, M. A.

Publications and source records attributed to Brown, M. A..

At least 19 recordsLinked to original sources

bioRxiv2026

TCF4 and CtBP1 Repress LEF1 to Maintain a TCF4-centric Transcriptional Program in Colon Cancer.

Abstract The canonical WNT signaling pathway guides cell growth throughout the human lifespan. In adults, WNT plays an essential role in tissue homeostasis by maintaining tissue stem cells. In the intestine, the WNT transcription factor, TCF4, is necessary for stem cell maintenance. Aberrant activation of WNT signaling is generally accepted as an initiating event in colon cancer, leading to constitutive TCF4 activity. To investigate the role of this key transcription factor in global gene regulation, we abrogated TCF4 expression in colon cancer cells. Loss of TCF4 function resulted in the over-expression of the WNT transcription factor, LEF1. LEF1 was transcriptionally competent and over-compensated for TCF4 (TCF7L2) silencing in a WNT reporter assay. TCF4 enlisted the transcriptional repressor CtBP1, and bound the LEF1 promoter, indicative of direct repression. TCF4 and LEF1 drove different transcriptional programs, with TCF4 favoring MYC and cell cycle progression, while LEF1 favored immune-related gene signatures. TCF4 was the primary regulator of MYC expression, yet also the mediator of LEF1 repression, suggestive of competition centered on TCF4 transcriptional output despite the {beta}-catenin saturated colon cancer nucleus. We conclude that TCF4 has dual activator-repressor activity in colon cancer.

cell biology↗

bioRxiv2024

Olfactory bulb tracks breathing rhythms and place in freely behaving mice

Vertebrates sniff to control the odor samples that enter their nose. These samples can not only help identify odorous objects, but also locations and events. However, there is no receptor for place or time. Therefore, to take full advantage of olfactory information, an animals brain must contextualize odor-driven activity with information about when, where, and how they sniffed. To better understand contextual information in the olfactory system, we captured the breathing and movements of mice while recording from their olfactory bulb. In stimulus- and task-free experiments, mice structure their breathing into persistent rhythmic states which are synchronous with statelike structure in ongoing neuronal population activity. These population states reflect a strong dependence of individual neuron activity on variation in sniff frequency, which we display using "sniff fields" and quantify using generalized linear models. In addition, many olfactory bulb neurons have "place fields" that display significant dependence of firing on allocentric location, which were comparable with hippocampal neurons recorded under the same conditions. At the population level, a mouses location can be decoded from olfactory bulb with similar accuracy to hippocampus. Olfactory bulb place sensitivity cannot be explained by breathing rhythms or scent marks. Taken together, we show that the mouse olfactory bulb tracks breathing rhythms and self-location, which may help unite internal models of self and environment with olfactory information as soon as that information enters the brain.

neuroscience↗

bioRxiv2024

The splicing modulator CLK1 is a candidate oncogenic dependency in pediatric high-grade gliomas

BackgroundPediatric brain tumors are the leading cause of disease-related mortality in children, yet many aggressive tumors lack effective therapies. RNA splicing is a hallmark of cancer, but it has not yet been systematically studied in pediatric brain tumors. MethodsWe analyzed 729 pediatric brain tumors spanning histologies and molecular subtypes to quantify differential tumor splicing. We developed the Splicing Burden Index (SBI) to enable cross-sample comparisons and performed hierarchical clustering of highly variable splice events to define splicing-informed tumor groups. These were integrated with clinical outcomes, pathway activity, and proteogenomic data. Recurrent splice events were prioritized for predicted functional impact, and in vitro perturbation studies were performed targeting the splicing kinase CDC-like kinase 1 (CLK1). ResultsSBI revealed substantial inter- and intra-histology heterogeneity. Clusters were enriched for histologies and molecular subtypes, several of which were independently associated with survival beyond histology and clinical covariates. Spliceosome pathway activity varied across clusters and was associated with worse survival, yet was not correlated with SBI, indicating distinct dimensions of splicing dysregulation. Functional prioritization identified a recurrent in CLK1 exon 4, required for canonical kinase activity. CLK1 exon 4 inclusion followed an oncofetal pattern and showed context-dependent associations with outcome distinct from total CLK1 expression. Pharmacologic inhibition and exon 4-specific perturbation of CLK1 reduced tumor cell viability and disrupted cancer-relevant splicing and transcriptional programs. ConclusionsThis study systematically characterizes splicing in pediatric brain tumors, identifies splicing-informed subgroups, and prioritizes CLK1 exon 4 as an oncofetal tumor-specific event, motivating further preclinical exploration. Key Points[bullet] Splicing analysis of 729 pediatric CNS tumors identifies splicing-defined clusters. [bullet]CLK1 exon 4 inclusion is widespread and developmentally regulated. [bullet]Exon-level CLK1 regulation shows context-dependent links to prognosis in aggressive CNS tumors.

genomics↗

bioRxiv2024

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr({Phi}Stx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC({Phi}Stx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC({Phi}Stx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality. Significance StatementEngineered smart microbes that secrete therapeutics are emerging as treatment modalities, particularly for gut-based diseases. With the growing threat of multidrug-resistant infection, non-antibiotic treatments are urgently needed. The gastrointestinal pathogen enterohemorrhagic E coli (EHEC) can cause the potentially lethal hemolytic uremic syndrome, a toxin-driven disease. Given concerns that antibiotics increase toxin release, treatment is largely limited to supportive care. Here, we show that pre-treatment with a commensal E. coli (HS-PROT3EcT) engineered to secrete an antibody that blocks an essential EHEC virulence factor delays the establishment of an EHEC-like infection in mice. This study strongly suggests that smart microbes that deliver payloads that block colonization factors of gut pathogens can be developed as critically needed alternatives to antibiotics for fighting bacterial infections.

microbiology↗

bioRxiv2024

The Open Pediatric Cancer Project

BackgroundIn 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 present an extension of the OpenPBTA called 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). FindingsWe 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 NCIs 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. ConclusionsOpenPedCan 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.

genomics↗

bioRxiv2024

Playbook Workflow Builder: Interactive Construction of Bioinformatics Workflows from a Network of Microservices

Many biomedical research projects produce large-scale datasets that may serve as resources for the research community for hypothesis generation, facilitating diverse use cases. Towards the goal of developing infrastructure to support the findability, accessibility, interoperability, and reusability (FAIR) of biomedical digital objects and maximally extracting knowledge from data, complex queries that span across data and tools from multiple resources are currently not easily possible. By utilizing existing FAIR application programming interfaces (APIs) that serve knowledge from many repositories and bioinformatics tools, different types of complex queries and workflows can be created by using these APIs together. The Playbook Workflow Builder (PWB) is a web-based platform that facilitates interactive construction of workflows by enabling users to utilize an ever-growing network of input datasets, semantically annotated API endpoints, and data visualization tools contributed by an ecosystem. Via a user-friendly web-based user interface (UI), workflows can be constructed from contributed building-blocks without technical expertise. The output of each step of the workflows are provided in reports containing textual descriptions, as well as interactive and downloadable figures and tables. To demonstrate the ability of the PWB to generate meaningful hypotheses that draw knowledge from across multiple resources, we present several use cases. For example, one of these use cases sieves novel targets for individual cancer patients using data from the GTEx, LINCS, Metabolomics, GlyGen, and the ExRNA Communication Consortium (ERCC) Common Fund (CF) Data Coordination Centers (DCCs). The workflows created with the PWB can be published and repurposed to tackle similar use cases using different inputs. The PWB platform is available from: https://playbook-workflow-builder.cloud/.

bioinformatics↗

bioRxiv2023

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. HighlightsO_LIPlasma membrane enriched proteomics defines surfaceome of neuroblastoma C_LIO_LIMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancers C_LIO_LIDLK1 expression is driven by a super-enhancer C_LIO_LIDLK1 silencing in neuroblastoma cells results in cellular differentiation C_LIO_LIADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models C_LI

cancer biology↗

bioRxiv2023

AutoGVP: a dockerized workflow integrating ClinVar and InterVar germline sequence variant classification

SummaryWith the increasing rates of exome and whole genome sequencing, the ability to classify large sets of germline sequencing variants using up-to-date American College of Medical Genetics - Association for Molecular Pathology (ACMG-AMP) criteria is crucial. Here, we present Automated Germline Variant Pathogenicity (AutoGVP), a tool that integrates germline variant pathogenicity annotations from ClinVar and sequence variant classifications from a modified version of InterVar (PVS1 strength adjustments, removal of PP5/BP6). This tool facilitates large-scale, clinically-focused classification of germline sequence variants in a research setting. Availability and ImplementationAutoGVP is an open-source dockerized workflow implemented in R and freely available on GitHub at https://github.com/diskin-lab-chop/AutoGVP.

bioinformatics↗

bioRxiv2022

The Children's Brain Tumor Network (CBTN) - Accelerating Research in Pediatric Central Nervous System Tumors through Collaboration and Open Science

Pediatric brain tumors are the leading cause of cancer-related death in children in the United States and contribute a disproportionate number of potential years of life lost compared to adult cancers. Moreover, survivors frequently suffer long-term side effects, including secondary cancers. The Childrens Brain Tumor Network (CBTN) is a multi-institutional international clinical research consortium created to advance therapeutic development through the collection and rapid distribution of biospecimens and data via open-science research platforms for real-time access and use by the global research community. The CBTNs 32 member institutions utilize a shared regulatory governance architecture at the Childrens Hospital of Philadelphia to accelerate and maximize the use of biospecimens and data. As of August 2022, CBTN has enrolled over 4,700 subjects, over 1,500 parents, and collected over 65,000 biospecimen aliquots for research. Additionally, over 80 preclinical models have been developed from collected tumors. Multi-omic data for over 1,000 tumors and germline material is currently available with data generation for > 5,000 samples underway. To our knowledge, CBTN provides the largest open-access pediatric brain tumor multi-omic dataset annotated with longitudinal clinical and outcome data, imaging, associated biospecimens, child-parent genomic pedigrees, and in vivo and in vitro preclinical models. Empowered by NIH-supported platforms such as the Kids First Data Resource and the Childhood Cancer Data Initiative, the CBTN continues to expand the resources needed for scientists to accelerate translational impact for improved outcomes and quality of life for children with brain and spinal cord tumors.

cancer biology↗

bioRxiv2022

OpenPBTA: An Open Pediatric Brain Tumor Atlas

SummaryPediatric brain and spinal cancer are the leading disease-related cause of death in children, thus we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Childrens Brain Tumor Network and Pacific Pediatric Neuro-Oncology Consortium created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to create OpenPBTA, an open collaborative project which establishes over 40 scalable analysis modules to genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals that TP53 loss is a significant marker for poor overall survival in ependymomas and H3 K28-altered diffuse midline gliomas and further identifies universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas. OpenPBTA is a foundational analysis platform actively being applied to other pediatric cancers and inform molecular tumor board decision-making, making it an invaluable resource to the pediatric oncology community. In BriefThe OpenPBTA is a global, collaborative open-science initiative which brought together researchers and clinicians to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Shapiro, et. al create over 40 open-source, scalable modules to perform cancer genomics analyses and provide a richly-annotated somatic dataset across 58 brain tumor histologies. The OpenPBTA framework can be used as a model for large-scale data integration to inform basic research, therapeutic target identification, and clinical translation. HighlightsOpenPBTA collaborative analyses establish resource for 1,074 pediatric brain tumors NGS-based WHO-aligned integrated diagnoses generated for 641 of 1,074 tumors RNA-Seq analysis infers medulloblastoma subtypes, TP53 status, and telomerase activity OpenPBTA will accelerate therapeutic translation of genomic insights

genomics↗

bioRxiv2022

Direct laser writing of 3D electrodes on flexible substrates

This report describes a 3D microelectrode array integrated on a thin-film flexible cable for neural recording in small animals. The micro electrode array fabrication process integrates traditional silicon thin-film processing techniques and direct laser writing of 3D structures at micron resolution via two-photon lithography. While direct laser writing of 3D printed electrodes has been described before, this report is the first to provide a method for high-aspect-ratio laser-written structures integrated with microfabricated electrical traces. One prototype is a 16-channel array composed of 350 m long shanks spaced on a grid with 90 m pitch. Other devices shown here include biomimetic mosquito-needles that penetrate through the dura of birds and porous electrodes designed to promote tissue ingrowth or enhance charge injection capacity for neural stimulation. These devices are just a few examples of a new design space that will enable high-channel-count 3D electrode arrays with features definable at single micrometer resolution. Using a custom laser writer, the 3D printing process is rapid (1 mm3/min). This high-speed printing combined with standard wafer-scale processes will enable efficient device fabrication and new studies examining the relationship between electrode geometry and electrode performance. We anticipate highest impact in small animal models, nerve interfaces, retinal implants, and other applications requiring small, high density 3D electrodes.

bioengineering↗

bioRxiv2020

Germline ERBB3 mutation in familial non-small cell lung carcinoma: expanding the role of the ErbB family in oncogenesis.

BackgroundLung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. MethodsGermline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and sub-cellular localisation were assessed; and cellular proliferation, pAkt/Akt, and pERK levels were determined. ResultsA novel germline variant in ERBB3 (c.1946T>G: p.Iso649Arg), coding for receptor tyrosineprotein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80kD form (which enhances proliferation) compared to the full-length (180kD) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localisation pre- and post-EGF stimulation; however, EGFR levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared to wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 minutes post-stimulation compared to wtErbB3 transfection, demonstrating altered signalling pathway activation by mutErbB3. Cumulatively, these results support this mutation as tumorogenic. ConclusionsThis is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

genomics↗

bioRxiv2020

TCF7L2 Silencing Reprograms the 4D Nucleome of Colorectal Cancer Cells

Canonical Wnt signaling is crucial for intestinal homeostasis as the major Wnt signaling effector in the intestines, TCF4, is required for stem cell maintenance. The capability of TCF4 to maintain the stem cell phenotype is contingent upon {beta}-catenin, a potent transcriptional activator which interacts with histone acetyltransferases and chromatin remodeling complexes. In colorectal cancer, mutations result in high levels of nuclear {beta}-catenin causing aberrant cell growth. Here, we used RNAi to explore the influence of TCF4 on chromatin structure (Hi-C) and gene expression (RNA sequencing) across a 72-hour time series in colorectal cancer. We found that TCF4 reduction results in a disproportionate upregulation of gene expression genome-wide, including a powerful induction of SOX2. Hi-C analysis revealed a general increase in chromatin compaction across the entire time series, though this did not influence gene expression. Analysis of local chromosome organization demonstrated a TAD boundary loss which influenced the expression of a cluster of CEACAM genes on chromosome 19. Four-dimensional nucleome (4DN) analysis, which integrates structural (Hi-C) and functional (RNA sequencing) data, identified EMT and E2F as the two most deregulated pathways and LUM, TMPO, and AURKA as highly influential genes in these networks. Results from gene expression, chromatin structure, and centrality analyses were then integrated to generate a list of candidate transcription factors for reprogramming of colorectal cancer cells to a vulnerable state. The top ranked transcription factor in our analysis was c-JUN, an oncoprotein known to interact with TCF4 and {beta}-catenin.

cancer biology↗

bioRxiv2020

Sniff-synchronized, gradient-guided olfactory search by freely-moving mice

For many organisms, searching for relevant targets such as food or mates entails active, strategic sampling of the environment. Finding odorous targets may be the most ancient search problem that motile organisms evolved to solve. While chemosensory navigation has been well characterized in micro-organisms and invertebrates, spatial olfaction in vertebrates is poorly understood. We have established an olfactory search assay in which freely-moving mice navigate noisy concentration gradients of airborne odor. Mice solve this task using concentration gradient cues and do not require stereo olfaction for performance. During task performance, respiration and nose movement are synchronized with tens of milliseconds precision. This synchrony is present during trials and largely absent during inter-trial intervals, suggesting that sniff-synchronized nose movement is a strategic behavioral state rather than simply a constant accompaniment to fast breathing. To investigate the spatiotemporal structure of these active sensing movements, we used machine learning methods to parse motion trajectories into elementary movement motifs. Motifs fall into two clusters, which correspond to investigation and approach states. Investigation motifs lock precisely to sniffing, such that the individual motifs preferentially occur at specific phases of the sniff cycle. This work clarifies sensorimotor strategies for mouse olfactory search and guides ongoing work into the underlying neural mechanisms.

neuroscience↗

bioRxiv2019

annoFuse: an R Package to annotate and prioritize putative oncogenic RNA fusions

BackgroundGene fusion events are a significant source of somatic variation across adult and pediatric cancers and are some of the most clinically-effective therapeutic targets, yet low consensus of RNA-Seq fusion prediction algorithms makes therapeutic prioritization difficult. In addition, events such as polymerase read-throughs, mis-mapping due to gene homology, and fusions occurring in healthy normal tissue require informed filtering, making it difficult for researchers and clinicians to rapidly discern gene fusions that might be true underlying oncogenic drivers of a tumor and in some cases, appropriate targets for therapy. ResultsWe developed annoFuse, an R package, and shinyFuse, a companion web application, to annotate, prioritize, and explore biologically-relevant expressed gene fusions, downstream of fusion calling. We validated annoFuse using a random cohort of TCGA RNA-Seq samples (N = 160) and achieved a 96% sensitivity for retention of high-confidence fusions (N = 603). annoFuse uses FusionAnnotator annotations to filter non-oncogenic and/or artifactual fusions. Then, fusions are prioritized if previously reported in TCGA and/or fusions containing gene partners that are known oncogenes, tumor suppressor genes, COSMIC genes, and/or transcription factors. We applied annoFuse to fusion calls from pediatric brain tumor RNA-Seq samples (N = 1,028) provided as part of the Open Pediatric Brain Tumor Atlas (OpenPBTA) Project to determine recurrent fusions and recurrently-fused genes within different brain tumor histologies. annoFuse annotates protein domains using the PFAM database, assesses reciprocality, and annotates gene partners for kinase domain retention. As a standard function, reportFuse enables generation of a reproducible R Markdown report to summarize filtered fusions, visualize breakpoints and protein domains by transcript, and plot recurrent fusions within cohorts. Finally, we created shinyFuse for algorithm-agnostic interactive exploration and plotting of gene fusions. ConclusionsannoFuse provides standardized filtering and annotation for gene fusion calls from STARFusion and Arriba by merging, filtering, and prioritizing putative oncogenic fusions across large cancer datasets, as demonstrated here with data from the OpenPBTA project. We are expanding the package to be widely-applicable to other fusion algorithms and expect annoFuse to provide researchers a method for rapidly evaluating, prioritizing, and translating fusion findings in patient tumors.

bioinformatics↗

bioRxiv2019

A RUNX3 enhancer polymorphism associated with ankylosing spondylitis influences recruitment of Interferon Regulatory Factor 5 and factors of the Nucleosome Remodelling Deacetylase Complex in CD8+ T-cells

ObjectivesTo investigate the functional consequences of the single nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter strongly associated with ankylosing spondylitis (AS).\n\nMethodsThe effects of rs4648889 on transcription factor (TF) binding were tested by DNA pull-down and quantitative mass spectrometry. The results were validated by electrophoretic mobility gel shift assays (EMSA), Western blot (WB) analysis of the pulled-down eluates, and chromatin immuno-precipitation (ChIP)-qPCR.\n\nResultsSeveral TFs showed differential allelic binding to a 50bp DNA probe spanning rs4648889. Binding was increased to the AS-risk A allele for IKZF3 (aiolos) in nuclear extracts from CD8+ T-cells (3.7-fold, p<0.03) and several components of the NUcleosome Remodeling Deacetylase (NuRD) complex, including Chromodomain-Helicase-DNA-binding protein 4 (3.6-fold, p<0.05) and Retinoblastoma-Binding Protein 4 (4.1-fold, p<0.02). In contrast, binding of interferon regulatory factor (IRF) 5 was increased to the AS-protective G allele. These results were confirmed by EMSA, WB and ChIP-qPCR.\n\nConclusionsThe association of AS with rs4648889 most likely results from its influence on the binding of this enhancer-like region to TFs, including IRF5, IKZF3 and members of the NuRD complex. Further investigation of these factors and RUNX3-related pathways may reveal important new therapeutic possibilities in AS.

genetics↗

bioRxiv2019

Pediatric High Grade Glioma Resources From the Children’s Brain Tumor Tissue Consortium (CBTTC) and Pediatric Brain Tumor Atlas (PBTA)

BackgroundPediatric high grade glioma (pHGG) remains a fatal disease. Increased access to richly annotated biospecimens and patient derived tumor models will accelerate pHGG research and support translation of research discoveries. This work describes the pediatric high grade glioma set of the Childrens Brain Tumor Tissue Consortium (CBTTC) from the first release (October 2018) of the Pediatric Brain Tumor Atlas (PBTA).\n\nMethodspHGG tumors with associated clinical data and imaging were prospectively collected through the CBTTC and analyzed as the Pediatric Brain Tumor Atlas (PBTA) with processed genomic data deposited into PedcBioPortal for broad access and visualization. Matched tumor was cultured to create high grade glioma cell lines analyzed by targeted and WGS and RNA-seq. A tissue microarray (TMA) of primary pHGG tumors was also created.\n\nResultsThe pHGG set included 87 collection events (73 patients, 60% at diagnosis, median age of 9 yrs, 55% female, 46% hemispheric). Analysis of somatic mutations and copy number alterations of known glioma genes were of expected distribution (36% H3.3, 47% TP53, 24% ATRX and 7% BRAF V600E variants). A pHGG TMA (n=77), includes 36 (53%) patient tumors with matched sequencing. At least one established glioma cell line was generated from 23 patients (32%). Unique reagents include those derived from a H3.3 G34R glioma and from tumors with mismatch repair deficiency.\n\nConclusionThe CBTTC and PBTA have created an openly available integrated resource of over 2,000 tumors, including a rich set of pHGG primary tumors, corresponding cell lines and archival fixed tissue to advance translational research for pHGG.\n\nIMPORTANCE OF STUDYHigh-grade gliomas (HGG) remain the leading cause of cancer death in children. Since molecularly heterogeneous, preclinical studies of pediatric HGG will be most informative if able to compare across groups. Given their relatively rarity, there are few readily available biospecimens and cellular models to inform preclinical laboratory and genomic translational research. Therefore, the aim of this CBTTC study was to highlight the panel of pediatric HGG cases whose primary tumors have undergone extensive genomic analysis, have clinical data, available imaging and additional biospecimens, including tumor, nucleic acids, cell lines and FFPE tissue on a tissue microarray (TMA).

cancer biology↗

bioRxiv2019

Shotgun metagenomics reveals an enrichment of potentially cross-reactive bacterial epitopes in ankylosing spondylitis patients, as well as the effects of TNFi therapy and the host’s genotype upon microbiome composition

Diverse evidence including clinical, genetic and microbiome studies support a major role of the gut microbiome in the common immune-mediated arthropathy, ankylosing spondylitis (AS). To further investigate this we performed metagenomic analysis of a case-control cohort of 250 Han-Chinese subjects. Previous reports of gut dysbiosis in AS were re-confirmed and several notable bacterial species and functional categories were differentially abundant. TNF-inhibitor (TNFi) therapy at least partially restored the perturbed microbiome observed in untreated AS cases to that of healthy controls, including several important bacterial species that have been previously associated with AS and other related diseases. Enrichment of bacterial peptides homologous to HLA-B27-presented epitopes was observed in the stools of AS patients, suggesting that either HLA-B27 fails to clear these or that they are involved in driving HLA-B27-associated immune reactions. TNFi therapy of AS patients was also associated with a reduction of potentially arthritogenic bacterial peptides, relative to untreated patients. An AS-associated SNP in RUNX3 significantly influenced the microbiome in two independent cohorts, highlighting a host genotype (other than HLA-B27) potentially influencing AS via the microbiome. These findings emphasise the key role that the gut microbiome plays in driving the pathogenesis of AS.

microbiology↗