Biology subjects

Holland, E.

Publications and source records attributed to Holland, E..

10 recordsLinked to original sources

bioRxiv2026

FFPE-CUTAC: A Single Assay, Multiple Layers

Background: Archival biobanks of formalin-fixed paraffin-embedded (FFPE) specimens represent a vast and underused resource for retrospective molecular studies linked to clinical follow-up. However, fixation-induced chemical modification and nucleic acid fragmentation limit scalable genomic and transcriptomic profiling. FFPE-CUTAC (Cleavage Under Targeted Accessible Chromatin) is an RNA polymerase II-targeted DNA assay specifically designed to profile regulatory activity in FFPE tissue. Here, we assess its archival robustness, information content, compatibility with RNA-seq, and ability to recover copy-number alterations. Results: In meningioma and breast cancer cohorts, we benchmarked FFPE-CUTAC against matched fresh-frozen RNA-seq, FFPE RNA-seq and FFPE whole-genome sequencing. For FFPE specimens spanning up to 25 years, FFPE-CUTAC showed no systematic collection-year-associated decline in library yield or signal-quality metrics. In contrast, FFPE RNA-seq is more affected by the FFPE tissue degradation and specimen age on read composition, gene-body coverage, and gene detection. By measuring RNA polymerase II occupancy on chromatin rather than mature RNA abundance, FFPE-CUTAC is less constrained by transcript half-life, polyadenylation or transcript-capture design. FFPE-CUTAC extends beyond gene regions to non-coding regulatory elements, while the shared gene-level signals supported its integration with existing RNA-seq cohorts for large-scale, long-term clinical association study. FFPE-CUTAC libraries also retain DNA dosage information and can be used to recover chromosome-arm gain, loss and intact states with 96% concordance to matched whole-genome sequencing. Conclusions: By converting routinely preserved pathology sections into integrated regulatory and copy-number profiles, FFPE-CUTAC provides a practical foundation for constructing clinically annotated disease maps to support molecular stratification, prognostic modeling and treatment-association studies.

cancer biology↗

bioRxiv2025

PTEN deficiency linked to chromosome 10q loss leads to aggressive NF2mutant meningioma biology

While most meningiomas are benign and can be surgically removed, a subset behaves aggressively, recurs quickly, and can ultimately be fatal. Recent work has focused on defining this aggressive group. To better characterize this clinically distinct, high-risk group, we analyzed bulk RNA sequencing (RNA-seq) from human meningiomas using a dimension-reduced reference landscape. We identified an NF2 mutant subtype enriched for chromosome 10q loss and low PTEN expression, both of which were strongly associated with shorter time to recurrence. To directly test causality, we engineered several genetically accurate, immunocompetent in vitro and in vivo mouse models with Pten knockdown or knockout. Across models, Pten loss markedly accelerated YAP1-driven tumorigenesis. Projection of Pten-deficient mouse tumors onto bulk and single-cell RNA-seq datasets from human meningiomas revealed that mouse tumors faithfully recapitulate the transcriptional programs of aggressive NF2 mutant meningiomas and robustly predicted clinical outcomes. Collectively, these findings demonstrate that PTEN loss cooperates with YAP1 activation to drive aggressive NF2 mutant meningioma biology, and that the high fidelity of this model to human disease establishes it as a robust platform for mechanistic investigation and therapeutic testing. More broadly, this work highlights the power of cross-species integration in both validating preclinical models and enhancing the translational relevance and utility of dimension-reduced reference landscapes across diverse disease types. One Sentence SummaryPTEN loss causally drives aggressive NF2 mutant meningiomas, and cross-species integration provides a novel, high-fidelity system to validate preclinical models to human disease.

cancer biology↗

bioRxiv2025

Gain-of-function mutant p53 regulates long-noncoding RNA LINC00643 to modulate HIF1α in glioblastoma

Gain-of-function mutations of p53 (GOF-MUT-p53) act as oncogenes by regulating gene transcription. We screened for the genome-wide transcriptional targets of GOF-MUT-p53 in glioblastoma (GBM) and found that a significant subset of them were long non-coding RNAs (lncRNA). Among these, LINC00643 was strongly repressed by GOF-MUT-p53 but not wild-type p53. LINC00643 was downregulated in GBM and low-grade glioma and correlated with patient survival. LINC00643 and its conserved third exon (Exon3) suppressed GBM cell proliferation, migration, invasion, stem cell self-renewal, and in vivo tumor growth Mechanistically, ChIRP-seq identified HIF1 as a key LINC00643 interactor. Under hypoxia, LINC00643 repressed HIF1 expression and its target genes by interacting with the HIF1 enhancer. Knockdown of GOF-MUT-p53 upregulated LINC00643 and reduces HIF1, revealing a regulatory axis. These findings show extensive regulation of lncRNAs by GOF-MUT-p53 and uncover a novel mechanism by which GOF-MUT-p53 drives GBM through repression of LINC00643 and dysregulation of the HIF1 pathway.

cancer biology↗

bioRxiv2025

Systems Pharmacology Screening Reveals Mertk and other Targetable Kinases in Supratentorial Ependymomas

Ependymomas (EPN) are rare central nervous system tumors that account for approximately 10% of intracranial tumors in children and 4% in adults. Despite their clinical and molecular heterogeneity, spanning supratentorial, posterior fossa, and spinal subtypes, treatment remains limited to surgery and radiotherapy, with chemotherapy offering minimal benefit. Here, we performed transcriptomic analysis of 370 human ependymoma samples and identified two distinct molecular subgroups: EPN-E1 and EPN-E2. The EPN-E1 cluster is enriched for supratentorial tumors harboring ZFTA-RELA fusions (ZFTA-RELAfus), which occur in over 70% of cases and are associated with poor prognosis. To identify targeted therapies for this aggressive subtype, we validated a ZFTA-RELAfus mouse model that recapitulates the human EPN-E1 transcriptome and used it for target discovery. Through Kinome Regularization, a machine learning-driven polypharmacology approach, we identified MERTK as a critical regulator of tumor cell viability. Genetic depletion or pharmacologic inhibition of Mertk reduced cell growth ex vivo, and treatment with a clinical-grade MERTK inhibitor significantly suppressed tumor proliferation in vivo. Both human EPN-E1 tumors and ZFTA-RELAfus mouse tumors exhibited elevated expression of MERTK and its ligand GAS6, and MERTK inhibition led to suppression of pro-survival signaling pathways including MEK/ERK and PI3K/AKT. Notably, over 80% of genes upregulated in ZFTA-RELAfus tumors were downregulated following MERTK inhibition, indicating a strong dependency on this pathway for tumor maintenance. These findings define a signaling vulnerability in ZFTA-RELA-driven ependymomas and support the clinical development of MERTK-targeted therapies for patients with the high-risk EPN-E1 subtype.

cancer biology↗

bioRxiv2024

DNAJC13 localization to endosomes is opposed by its J domain and its disordered C-terminal tail

Endosomes are a central sorting hub for membrane cargos. DNAJC13/RME-8 plays a critical role in endosomal trafficking by regulating the endosomal recycling or degradative pathways. DNAJC13 localizes to endosomes through its N-terminal Plekstrin Homology (PH)-like domain, which directly binds endosomal phosphoinositol-3-phosphate (PI(3)P). However, little is known about how DNAJC13 localization is regulated. Here, we show that two regions within DNAJC13, its J domain and disordered C-terminal tail, act as negative regulators of its PH-like domain. Using a structure-function approach combined with quantitative proteomics, we mapped these control points to a conserved YLT motif in the C-terminal tail as well as the catalytic HPD triad in its J domain. Mutation of either motif enhanced DNAJC13 endosomal localization in cells and increased binding to PI(3)P in vitro. Further, these effects required the N-terminal PH-like domain. We show that, similar to other PI(3)P binding domains, the N-terminal PH-like domain binds PI(3)P weakly in isolation and requires oligomerization for efficient PI(3)P binding and endosomal localization. Together, these results demonstrate that interaction between DNAJC13 and PI(3)P serves as a molecular control point for regulating DNAJC13 localization to endosomes. Significance StatementO_LIDNAJC13 controls endosomal sorting by regulating proteins which mediate the endosomal recycling and degradative subdomains. C_LIO_LIHere we show that subcellular localization of DNAJC13 is regulated through the coordinated action of three of its domains: the PH-like domain which has low affinity for PI(3)P, the J domain, and a YLT motif in its disordered C-terminus. C_LIO_LIThis study defines a novel mechanism by which DNAJC13 function is regulated. C_LI

cell biology↗

bioRxiv2024

Transcriptomic landscape identifies two unrecognized ependymoma subtypes and novel pathways in medulloblastoma

Medulloblastoma and ependymoma are common pediatric central nervous system tumors with significant molecular and clinical heterogeneity. We collected bulk RNA sequencing data from 888 medulloblastoma and 370 ependymoma tumors to establish a comprehensive reference landscape. Following rigorous batch effect correction, normalization, and dimensionality reduction, we constructed a unified landscape to explore gene expression, signaling pathways, RNA fusions, and copy number variations. Our analysis revealed distinct clustering patterns, including two primary ependymoma compartments, EPN-E1 and EPN-E2, each with specific RNA fusions and molecular signatures. In medulloblastoma, we observed precise stratification of Group 3/4 tumors by subtype and in SHH tumors by patient age. This landscape serves as a vital resource for identifying biomarkers, refining diagnoses, and enables the mapping of new patients bulk RNA-seq data onto the reference framework to predict biology and outcome from nearest neighbor analysis facilitate accurate disease subtype identification. The landscape is accessible via Oncoscape, an interactive platform, empowering global exploration and application. One Sentence SummaryA landscape built using only Transcriptomic analysis for medulloblastoma and ependymoma reveals novel insights about subtype-specific biology.

bioinformatics↗

bioRxiv2024

Microenvironment T-Type calcium channels regulate neuronal and glial processes to promote glioblastoma growth

BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor. The aim of this study was to elucidate the role of microenvironment and intrinsic T-type calcium channels (Cav3) in regulating tumor growth and progression. MethodsWe grafted syngeneic GBM cells into Cav3.2 knockout mice to assess the role of microenvironment T-Type calcium channels on GBM tumor growth. We performed single-cell RNA-seq (scRNA-seq) of tumors from WT and Cav3.2 KO mice to elucidate the regulation of tumors by the microenvironment. We used neurons from WT and Cav3.2 KO mice in co-culture with GBM stem cells (GSC) to assess the effects of Cav3.2 on neuron/GSC synaptic connections and tumor cell growth. ResultsCav3.2 KO in the microenvironment led to significant reduction of GBM growth and prolongation of animal survival. scRNA-seq showed that microenvironment Cav3.2 regulates neuronal and glial biological processes. Microenvironment Cav3.2 downregulated numerous genes associated with regulating the OPC cell state in GBM tumors such as SOX10 and Olig2. Neuronal Cav3.2 promoted neuron/GSC synaptic connections and GSC growth. Treatment of GSCs with the Cav3 blocker mibefradil downregulated genes associated with neuronal processes. The Cav3 blocker drug mibefradil synergized with temozolomide (TMZ) and radiation to reduce in vivo tumor growth and prolong animal survival. ConclusionsTogether these data reveal a role for microenvironment Cav3 in promoting GBM tumor progression through regulating neuronal and glial processes particularly associated with the OPC-cell state. Targeting both intrinsic and microenvironment Cav3 with the inhibitor mibefradil significantly enhanced the anti-GBM effects of TMZ and radiation. Key PointsO_LIMicroenvironment Cav3.2 promotes GBM progression C_LIO_LIMicroenvironment Cav3.2 promotes neuronal and glial processes C_LIO_LIPharmacological targeting of intrinsic and microenvironment Cav3 synergizes with TMZ/radiation C_LI Importance of the StudyIn this study, we demonstrate for the first time that microenvironment Cav3.2 contributes to GBM progression and growth by regulating neuronal and glial processes. Our findings highlight the importance of T-type calcium channels in the microenvironment as well as the tumor and provides preclinically relevant data for the use of mibefradil to inhibit GBM growth in combination with standard of care therapies.

cancer biology↗

bioRxiv2023

Functional genomic analysis of adult and pediatric brain tumor isolates

BackgroundAdult and pediatric tumors display stark differences in their mutation spectra and chromosome alterations. Here, we attempted to identify common and unique gene dependencies and their associated biomarkers among adult and pediatric tumor isolates using functional genetic lethal screens and computational modeling. MethodsWe performed CRISRP-Cas9 lethality screens in two adult glioblastoma (GBM) tumor isolates and five pediatric brain tumor isolates representing atypical teratoid rhabdoid tumors (ATRT), diffuse intrinsic pontine glioma, GBM, and medulloblastoma. We then integrated the screen results with machine learning-based gene-dependency models generated from data from >900 cancer cell lines. ResultsWe found that >50% of candidate dependencies of 280 identified were shared between adult GBM tumors and individual pediatric tumor isolates. 68% of screen hits were found as nodes in our network models, along with shared and tumor-specific predictors of gene dependencies. We investigated network predictors associated with ADAR, EFR3A, FGFR1 (pediatric-specific), and SMARCC2 (ATRT-specific) gene dependency among our tumor isolates. ConclusionsThe results suggest that, despite harboring disparate genomic signatures, adult and pediatric tumor isolates share a preponderance of genetic dependences. Further, combining data from primary brain tumor lethality screens with large cancer cell line datasets produced valuable insights into biomarkers of gene dependency, even for rare cancers. Importance of the StudyOur results demonstrate that large cancer cell lines data sets can be computationally mined to identify known and novel gene dependency relationships in adult and pediatric human brain tumor isolates. Gene dependency networks and lethality screen results represent a key resource for neuro-oncology and cancer research communities. We also highlight some of the challenges and limitations of this approach.

cancer biology↗

bioRxiv2022

Olverembatinib inhibits SARS-CoV-2-Omicron variant-mediated cytokine release

The Omicron variant has become dominant in the U.S. and around the world. This variant is found to be 2-fold more infectious than the Delta variant, posing a significant threat of severe cases and death. We and others have recently reported that the N-terminus domain (NTD) of the SARS-CoV-2 of various variants is responsible for inducing cytokine release in human PBMCs. Here, we demonstrate that the NTD of the Omicron variant remains highly effective at inducing cytokine release in human PBMCs. Furthermore, we show that Ponatinib and a novel compound, Olverembatinib, are potent Omicron NTD-mediated cytokine release inhibitors. Target profiling revealed that Olverembatinib blocks most of the previously identified kinases responsible for cytokine release. Together, we propose that Ponatinib and Olverembatinib may represent an attractive therapeutic option for treating moderate to severe COVID-19 cases. HIGHLIGHTSO_LIThe N-terminus domain (NTD) of the SARS-CoV-2 Omicron variant strongly induces multiple inflammatory molecules in PBMCs, unaffected by the mutations observed in the NTD. C_LIO_LIThe cytokine release mediated by the Omicron variant is comparable to the Delta variant. C_LIO_LIOlverembatinib, a clinical-stage multi-kinase inhibitor, potently inhibits Omicron NTD-mediated cytokine release. C_LIO_LIOlverembatinib could relieve severe symptoms associated with COVID-19 Omicron and Delta variants. C_LI

immunology↗

bioRxiv2019

Beyond Conventional Models: Recreating the Initiation, Evolution, and Genome of GBM

BackgroundImagining ways to prevent or treat glioblastoma (GBM) have been hindered by a lack of understanding of its pathogenesis. Although PDGF-AA overexpression may be an early event, critical details of the core biology are lacking. Existing PDGF-driven models replicate its microscopic appearance but not the genomic architecture characteristic of the human disease. Here we report a new model of GBM that overcomes this barrier to authenticity. MethodsUsing a method developed to study neural stem cells, we investigated the effects of PDGF-AA on subventricular zone (SVZ) cells, the putative cell of origin of GBM. We micro-dissected SVZ tissue from p53-null and wild-type adult mice, established primary cultures in media supplemented with PDGF-AA, and assessed cell viability, proliferation, genome stability, and tumour forming potential. ResultsCounterintuitive to its canonical role as a growth factor, we observed immediate and massive death of SVZ cells in PDGF-AA. Wild-type cells did not survive in PDGF-AA. However, a small fraction of null cells evaded apoptosis, displayed attenuated proliferation, gradually accumulated whole chromosome gains and losses, and, signalled by sudden rapid proliferation and growth factor independence, became tumorigenic in immune-competent syngeneic mice. Transformed cells had an OPC-like profile, were resistant to PDGFR- inhibition, and harboured highly abnormal karyotypes similar to those seen in human GBMs. ConclusionThis model associates genome instability in SVZ cells with chronic exposure to PDGF-AA; it is the first model to replicate the genomic landscape of GBM and first in which the earliest phases of GBM can be directly observed. IMPORTANCE OF STUDYWe have developed a mouse model in which the initiation, evolution and genomic landscape of GBM can be thoroughly studied thus paving the way for ideas about how this deadly brain cancer might be prevented, interrupted at an occult stage, or treated with very different therapies.

cancer biology↗