Biology subjects

Dirks, P. B.

Publications and source records attributed to Dirks, P. B..

18 recordsLinked to original sources

bioRxiv2026

Metabolic rewiring induces a viral mimicry-mediated quiescence in glioblastoma

Glioblastoma is a highly aggressive brain tumour driven by therapy-resistant glioblastoma stem cells (GSCs) that switch between dormant and proliferative states through poorly defined programs. In this study, we identify SAT1, encoding the rate-limiting polyamine-catabolic enzyme SSAT1, as enriched in quiescent GSCs, linking metabolic rewiring to dormancy. Pharmacologic induction of SSAT1 with the polyamine analogue N1,N11-Diethylnorspermine (DENSpm) triggers a chemoprotective, reversible growth arrest in patient-derived GSCs, independent of stemness, differentiation or senescence. SAT1-induced dormancy coincides with reduced eIF5A hypusination and global protein translation, and a marked rise in double-stranded RNA that activates type I interferon signalling. Knocking down the mitochondrial antiviral-signalling protein, essential for antiviral innate immunity, partially reverses this growth arrest, establishing viral mimicry as functionally required for dormancy. Single-cell and cohort analyses across GSCs and neural stem cells demonstrate that susceptibility to metabolically induced quiescence is set by an intrinsic inflammatory tone and lysosomal dependency, defining a context-dependent metabolic-immune axis which can be targeted to prevent GBM progression.

cancer biology↗

bioRxiv2026

Convergence on BRAF and MAPK Signaling in Glioma Development in a P53-ENU model

Pediatric high-grade gliomas (HGGs) are aggressive and lethal brain tumors that account for 15- 20% of all pediatric central nervous system (CNS) malignancies and remain largely incurable. These tumors, despite having mutational targets that activate MAPK signaling, are frequently resistant to targeted therapies in their malignant states, but often show responses when they are in lower grade form. These findings suggest a need to identify and intercept early tumorigenic events that arise in earlier tumor developmental stages. To investigate the molecular events in glioma progression, we developed and characterized a NestinCre/+;Trp53fl/fl mouse model combined with in utero exposure to the classic chemical mutagen N-ethyl-N-nitrosourea (ENU). This model mimics the context of genetic predisposition paired with an environmental genotoxic insult, and mice with loss of TRP53 in early neural precursors who are exposed to ENU at embryonic day 13.5 reproducibly develop HGGs postnatally that retain features of the human tumors. By sampling discrete lesions at premalignant, early, and late stages, we observed progressive increases in genetic complexity, stemness features, and immune signatures across tumor evolution. Notably, a recurrent Braf mutation emerged in a majority of early lesions and persisted in advanced tumors, consistent with the occurrence of BRAFV600E mutations in human gliomas that arise in children and undergo transition from lower grade to higher grade stages. Additional components of the RAS-RAF-MAPK signaling cascade, including Kras, Nras and Nf1 were found to be mutated at the late tumor stage, indicating convergent activation of this pathway in this model. Cell lines derived from early lesions responded to BRAF inhibitors, but cells from endpoint tumors were less responsive. Together, this model reveals aspects of the molecular and cellular evolution of glioma development in vivo, and identifies RAS-MAPK signaling as a critical molecular bottleneck selected for the ENU-induced mutations. This genetic-environmental model may be valuable to understanding key determinants of glioma initiation and progression, and for evaluating new therapies that limit MAPK signaling.

cancer biology↗

bioRxiv2026

A Chemical-Genetic Interaction Matrix Reveals Drug Mechanism and Genetic Architecture

To probe drug mechanism of action (MOA) and interrogate the genetic architecture of human cells, we carried out isogenic genome-wide CRISPR/Cas9 knockout screens against 310 diverse drugs, bioactive compounds, and stress conditions. Stringent statistical correction for gene knockout fitness defects yielded a large-scale matrix of >12,000 high confidence chemical-genetic interactions (CGIs). This dataset revealed many previously unappreciated off-target effects for well-characterized compounds and novel MOAs for uncharacterized compounds. The CGI matrix uncovered dense genetic modules that yielded new biological insights into phospholipidosis, mitotic regulation, metabolism, the DNA damage response, and mTOR signaling. The dataset allowed identification of multi-drug sensitization and resistance mechanisms, inference of gene function, elaboration of cross-process connectivity, evaluation of the cell type specificity of CGIs, prediction of chemical synergism, and extensive annotation of understudied genes. This resource provides a map of the genetic landscape in human cells and a framework to help guide drug discovery.

systems biology↗

bioRxiv2025

Identification of Injury-Response State Determinants in Glioblastoma Stem Cells

Glioblastoma (GBM) is a common and highly lethal type of primary brain tumor in adults. Therapeutic failure is partly attributed to a fraction of Glioblastoma Stem Cells (GSCs) that show high levels of heterogeneity and plasticity. GSCs exist in a transcriptional gradient between two states: Developmental (Dev) and Injury Response (IR) in which IR-GSCs exhibit more invasive behaviors. While previous studies have identified fitness genes in GSCs, the genes required to establish and maintain the Dev and IR states remain poorly defined. To identify the regulators of the IR GSC state, we performed a phenotypic genome-wide CRISPR-Cas9 knockout (KO) screen in patient-derived GSCs based on cell surface expression of the IR marker CD44. Notably, we found that perturbations of the histone acetyltransferase EP300 in IR GSCs led to decreased CD44 cell surface expression, significant downregulation of gene expression signatures associated with the IR state, and to decreased self-renewal and invasion. Furthermore, genetic targeting of Ep300 in a mouse GBM model delayed tumor initiation and/or progression. Collectively, our results establish EP300 as a regulator of the IR state in GSCs and provide a mechanistic basis for its therapeutic targeting in GBM. SignificanceA genome-wide phenotypic CRISPR-Cas9 screen in a patient-derived Glioblastoma Stem Cell line identified the genes required to maintain the Injury-Response cellular state, with a focus on the histone acetyl transferase gene EP300. This study suggests how therapeutic targeting of cellular state could reduce the aggressiveness of GBM tumors.

cancer biology↗

bioRxiv2024

Targeting synthetic lethality between non-homologous end joining and radiation in very high-risk SHH medulloblastoma

Specific and biologically informed treatments for medulloblastoma, especially the highly lethal TP53 mutant SHH subgroup, remain elusive, where radiotherapy is the primary treatment option. Applying genome-wide CRISPR-Cas9 screening in combination with lethal doses of radiotherapy, we identified the main driver of radiation resistance in SHH medulloblastoma is loss of p53. A negative selection CRISPR-Cas9 screen across multiple models of Trp53-deficient SHH medulloblastoma revealed a strong synthetic lethal interaction between components of the non-homologous end-joining pathway and radiation, particularly DNA protein kinase (DNA-PK) and its binding partners. Both genetic and pharmacological perturbation of DNA-PK enhanced radiosensitivity in TP53-deficient SHH medulloblastoma, leading to cell death. In vivo treatment of somatic and germline TP53-mutant SHH medulloblastoma models with peposertib, a small-molecule inhibitor of DNA-PK, significantly improved survival when combined with radiotherapy, strongly supporting further clinical investigation.

cancer biology↗

bioRxiv2024

Glioblastoma stem cells show transcriptionally correlated spatial organization

Glioblastoma (GBM) is an aggressive brain cancer with a poor survival rate. Despite hundreds of clinical trials, there is no effective targeted therapy. Glioblastoma stem cells (GSCs) are an important GBM model system. In culture, these cells form spatial structures that share morphological aspects with their source tumors. We collected 17,000 phase contrast images of 15 patient-derived GSC lines growing to confluence. We find that GSCs grow in characteristic multicellular patterns depending on their transcriptional state. Interpretable computer vision algorithms identified specific image features that predict transcriptional state across multiple cell confluency levels. This relationship will be useful in developing GSC screens where image features can be used to identify how GSC biology changes in response to perturbations simply by imaging cultured cells on plates.

cancer biology↗

bioRxiv2024

PRMT5 is required for full-length HTT expression by repressing multiple proximal intronic polyadenylation sites

Expansion of the CAG trinucleotide repeat tract in exon 1 of the Huntingtin (HTT) gene above a threshold of [~]36 repeats causes Huntingtons disease (HD) through the expression of a polyglutamine-expanded form of the HTT protein. This mutation triggers wide-ranging cellular and biochemical pathologies leading to cognitive, motor, and psychiatric symptoms in HD patients. As accurate splicing is required to produce the full-length HTT protein of [~]348 kDa, targeting HTT splicing with small molecule drugs is a compelling approach to lower HTT protein levels to treat HD, and splice modulators are being tested in the clinic. Here, we identify PRMT5 as a novel regulator of HTT mRNA splicing and alternative polyadenylation. PRMT5 inhibition disrupts the splicing of HTT introns 9 and 10, leading to activation of multiple proximal intronic polyadenylation sites within these introns and promoting premature termination, cleavage and polyadenylation (PCPA) of the HTT mRNA, thus lowering total HTT protein levels. We also detected increasing levels of these truncated, intron-containing HTT transcripts across a series of neuronal differentiation samples which correlated with lower PRMT5 expression. Notably, PRMT5 inhibition in glioblastoma (GBM) stem cells potently induced neuronal differentiation. We posit that PRMT5-mediated regulation of intronic polyadenylation, premature termination and cleavage of the HTT mRNA modulates HTT expression and plays an important role during embryonic development and neuronal differentiation.

neuroscience↗

bioRxiv2023

Pan-cancer analysis of the ion permeome reveals functional regulators of glioblastoma aggression

Ion channels, transporters, and other ion-permeating proteins, collectively comprising the ion permeome (IP), are common drug targets. However, their roles in cancer are understudied. Our integrative pan-cancer analysis shows that IP genes display highly-elevated expression patterns in subsets of cancer samples significantly more often than expected transcriptome-wide. To enable target identification, we identified 410 survival-associated IP genes in 29 cancer types using a machine learning approach. Notably, GJB2 and SCN9A show prominent expression in neoplastic cells and associate with poor prognosis in glioblastoma (GBM), the most common and aggressive brain cancer. GJB2 or SCN9A knockdown in patient-derived GBM cells induces transcriptome-wide changes involving neural projection and proliferation pathways, impairs cell viability and tumor sphere formation, mitigates tunneling nanotube formation, and extends the survival of GBM-bearing mice. Thus, aberrant activation of IP genes appears as a pan-cancer feature of tumor heterogeneity that can be exploited for mechanistic insights and therapy development.

cancer biology↗

bioRxiv2023

Directed differentiation of human hindbrain neuroepithelial stem cells recapitulates cerebellar granule neurogenesis

Cerebellar granule neurons (CGNs) are the most abundant neurons in the human brain and modulate cerebellar output to the motor cortex. Dysregulation of CGN development underlies movement disorders and medulloblastomas. It is suspected that these disorders arise in progenitor states of the CGN lineage, for which human models are lacking. Here, we have differentiated human hindbrain neuroepithelial stem (hbNES) cells to CGNs in vitro using soluble growth factors, recapitulating key progenitor states in the lineage. We show that hbNES cells are not lineage committed and retain rhombomere 1 (r1) regional identity. Upon differentiation, hbNES cells first transit through a rhombic lip (RL) progenitor state at day 7, demonstrating human specific sub-ventricular cell identities. This RL state is followed by an ATOH1+ CGN progenitor state at day 14. By the end of a 56-day differentiation procedure, we obtain mature neurons expressing CGN markers GABAAa6 and vGLUT2. These neurons generate spontaneous and evoked action potentials. A small fraction of endpoint neurons were unipolar brush cells (UBC). We noted maintenance of a RL population throughout differentiation, as is consistent with human development. We show that sonic hedgehog (SHH) promotes {gamma}-aminobutyric acid (GABA)-ergic lineage specification and is a positive regulator of CGN progenitor proliferation. Interestingly, we observed that functional neuronal maturation is impaired by either elevated or absent SHH signaling. Impaired maturation under high SHH levels represents the potential of our system to model cerebellar tumorigenesis. Further, our data suggest a potential pro-differentiation role of SHH within a certain concentration range. Our work is, to our knowledge, the first detailed temporal characterization of the complete human CGN lineage in vitro. Our system recapitulates developmentally relevant progenitor states and is a new tool to model this specific cerebellar lineage, and how it may be disrupted to cause human disease.

developmental biology↗

bioRxiv2022

In vivo functional genomics identifies essentiality of potassium homeostasis in medulloblastoma

The identification of cancer maintenance genes--driver genes essential to tumor survival--is fundamental for developing effective cancer therapy. Transposon-based insertional mutagenesis screens can identify cancer driver genes broadly but not discriminate maintenance from progression or initiation drivers, which contribute to cancer phenotypes and tumorigenesis, respectively. We engineered a nested, double-jumping transposon system to first dysregulate gene expression during tumorigenesis and then restore gene expression following tumor induction, allowing for genome-wide screening of maintenance essentiality in vivo. In a mouse model of medulloblastoma, the most common pediatric malignancy, insertion and remobilization of this nested transposon uncovers potassium channel genes as recurrent maintenance drivers. In human medulloblastoma, KCNB2 is the most overexpressed potassium channel across Group 3, Group 4, and SHH subgroups, and Kcnb2 knockout in mice diminishes the replicative potential of medulloblastoma-propagating cells to mitigate tumor growth. Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives proliferative expansion of medulloblastoma-propagating cells. Thus, our novel transposon system reveals potassium homeostasis as essential to tumor maintenance through biomechanical modulation of membrane signaling.

cancer biology↗

bioRxiv2021

Molecular atlas of the human brain vasculature at the single-cell level

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

neuroscience↗

bioRxiv2021

A comparison of data integration methods for single-cell RNA sequencing of cancer samples

Tumours are routinely profiled with single-cell RNA sequencing (scRNA-seq) to characterize their diverse cellular ecosystems of malignant, immune, and stromal cell types. When combining data from multiple samples or studies, batch-specific technical variation can confound biological signals. However, scRNA-seq batch integration methods are often not designed for, or benchmarked, on datasets containing cancer cells. Here, we compare 5 data integration tools applied to 171,206 cells from 5 tumour scRNA-seq datasets. Based on our results, STACAS and fastMNN are the most suitable methods for integrating tumour datasets, demonstrating robust batch effect correction while preserving relevant biological variability in the malignant compartment. This comparison provides a framework for evaluating how well single-cell integration methods correct for technical variability while preserving biological heterogeneity of malignant and non-malignant cell populations.

bioinformatics↗

bioRxiv2021

The Mediator tail module cooperates with proneural factors to determine fate identity in Glioblastoma.

Glioblastoma stem cells (GSCs) exhibit latent neuronal lineage differentiation potential governed by the proneural transcription factor Achaete-scute homolog 1 (ASCL1) and harnessing and promoting terminal neuronal differentiation has been proposed as a novel therapeutic strategy. Here, using a genome-wide CRISPR suppressor screen we identified genes required for ASCL1-mediated neuronal differentiation. This approach revealed a specialized function of the Mediator complex tail module and of its subunits MED24 and MED25 for this process in GSCs, human fetal neural stem cells and pluripotent stem cells. We show that upon induction of neuronal differentiation MED25 is recruited to genomic loci co-occupied by ASCL1 to regulate neurogenic gene expression programs. MED24 and MED25 are sufficient to induce neuronal differentiation in GSC cultures and to mediate neuronal differentiation in multiple contexts. Collectively our data expand our understanding of the mechanisms underlying directed terminal neuronal differentiation in brain tumor stem cells and point to a unique function of the Mediator tail in neuronal reprogramming.

cell biology↗

bioRxiv2020

The white matter is a pro-differentiative microenvironment for glioblastoma

Glioblastomas are hierarchically organised tumours driven by glioma stem cells that retain partial differentiation potential. Glioma stem cells are maintained in specialised microenvironments, but how they undergo lineage progression outside of these niches remains unclear. Here we identify the white matter as a differentiative niche for glioblastomas with oligodendrocyte lineage competency. Tumour cells in contact with white matter acquire pre-oligodendrocyte-like fate, resulting in decreased proliferation and invasion. Differentiation is a response to white matter injury, which is caused by tumour infiltration itself in a tumoursuppressive feedback loop. Mechanistically, tumour cell differentiation is driven by selective white matter upregulation of SOX10, a master regulator of normal oligodendrogenesis. SOX10 overexpression or treatment with myelination-promoting agents that upregulate endogenous SOX10, mimic this response, leading to white matter-independent pre-oligodendrocyte-like differentiation and tumour suppression in vivo. Thus, glioblastoma recapitulates an injury response and exploiting this latent programme may offer treatment opportunities for a subset of patients.

cancer biology↗

bioRxiv2019

Comprehensive analysis of lncRNAs reveals candidate prognostic biomarkers in multiple cancer types

Long non-coding RNAs (lncRNAs) are increasingly recognized as functional units in cancer pathways and powerful molecular biomarkers, however most lncRNAs remain uncharacterized. Here we performed a systematic discovery of prognostic lncRNAs in 9,326 patient tumors of 29 types using a proportional-hazards elastic net machine-learning framework. lncRNAs showed highly tissue-specific transcript abundance patterns. We identified 179 prognostic lncRNAs whose abundance correlated with patient risk and improved the performance of common clinical variables and molecular tumor subtypes. Pathway analysis revealed a large diversity of the high-risk tumors stratified by lncRNAs and suggested their functional associations. In lower-grade gliomas, discrete activation of HOXA10-AS indicated poor patient prognosis, neurodevelopmental pathway activation and a transcriptomic similarity to glioblastomas. HOXA10-AS knockdown in patient-derived glioblastoma cells caused decreased cell proliferation and deregulation of glioma driver genes and proliferation pathways. Our study underlines the pan-cancer potential of the non-coding transcriptome for developing molecular biomarkers and innovative therapeutic strategies.

cancer biology↗

bioRxiv2018

High-resolution structural genomics reveals new therapeutic vulnerabilities in glioblastoma

We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating the highest-resolution 3D genome maps to-date for this cancer. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including individual physical interactions between regulatory regions and their target genes. Residing in structurally conserved regions in GSCs was CD276, a gene known to play a role in immuno-modulation. We show that, unexpectedly, CD276 is part of a stemness network in GSCs and can be targeted with an antibody-drug conjugate to curb self-renewal, a key stemness property. Our results demonstrate that integrated structural genomics datasets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.\n\nSIGNIFICANCEIn adult GBM, GSCs act as therapy-resistant reservoirs to nucleate tumor recurrence. New therapeutic approaches that target these cell populations hold the potential of significantly improving patient care and overall prognosis for this always-lethal cancer. Our work describes new links between 3D genome architecture and stemness properties in GSCs. In particular, through integration of multiple genomics and structural genomics datasets, we found an unexpected connection between immune-related genes and self-renewal programs in GBM. Among these, we show that targeting CD276 with knockdown strategies or specific antibody-drug conjugates achieve suppression of self-renewal. Strategies to target CD276+ cells are currently in clinical trials for solid tumors. Our results indicate that CD276-targeting agents could be deployed in GBM to specifically target GSC populations.\n\nHIGHLIGHTSO_LIWe generated high (sub-5 kb) resolution Hi-C maps for stem-like cells from GBM patients.\nC_LIO_LIIntegration of Hi-C and genomics datasets dissects mechanisms of gene regulation.\nC_LIO_LI3D genomes poise immune-related genes, including CD276, for expression.\nC_LIO_LITargeting CD276 curbs self-renewal properties of GBM cells.\nC_LI

cancer biology↗

bioRxiv2018

Subclonal architecture, evolutionary trajectories and patterns of inheritance of germline variants in pediatric glioblastoma

Pediatric glioblastoma (pGBM) is a lethal cancer with no effective therapies. Intratumoral genetic heterogeneity and mode of tumor evolution have not been systematically addressed for this cancer. Whole-genome sequencing of germline-tumor pairs showed that pGBM is characterized by intratumoral genetic heterogeneity and consequent subclonal architecture. We found that pGBM undergoes extreme evolutionary trajectories, with primary and recurrent tumors having different subclonal compositions. Analysis of variant allele frequencies supported a model of tumor growth involving slow-cycling cancer stem cells that give rise to fast-proliferating progenitor-like cells and to non-dividing cells. pGBM patients germlines had subclonal structural variants, some of which underwent dynamic frequency fluctuations during tumor evolution. By sequencing germlines of mother-father-patient trios, we found that inheritance of deleterious germline variants from healthy parents cooperate with de novo germline and somatic events to the tumorigenic process. Our studies therefore challenge the current notion that pGBM is a relatively homogeneous molecular entity.

cancer biology↗

bioRxiv2018

The functional genomic circuitry of human glioblastoma stem cells

SummarySuccessful glioblastoma (GBM) therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells to interrogate function of the coding genome, we identify diverse actionable pathways responsible for growth that reveal the gene-essential circuitry of GBM stemness. In particular, we describe the Sox developmental transcription factor family; H3K79 methylation by DOT1L; and ufmylation stress responsiveness programs as essential for GBM stemness. Additionally, we find mechanisms of temozolomide resistance and sensitivity that could lead to combination strategies with this standard of care treatment. By reaching beyond static genome analysis of bulk tumors, with a genome wide functional approach, we dive deep into a broad range of biological processes to provide new understanding of GBM growth and treatment resistance.\n\nSignificanceGlioblastoma (GBM) remains an incurable disease despite an increasingly thorough depth of knowledge of the genomic and epigenomic alterations of bulk tumors. Evidence from multiple approaches support that GBM reflects an aberrant developmental hierarchy, with GBM stem cells (GSCs), fueling tumor growth and invasion. The properties of this tumor subpopulation may also in part explain treatment resistance and disease recurrence. Unfortunately, we still have a limited knowledge of the molecular circuitry of these cells and progress has been slow as we have not been able, until recently, to interrogate function at the genome-wide scale. Here, using parallel genome-wide CRISPR-Cas9 screens, we identify the essential genes for GSC growth. Further, by screening in the presence of low and high dose temozolomide, we identify mechanisms of drug resistance and sensitivity. These functional screens in patient derived cells reveal new aspects of GBM biology and identify a diversity of actionable targets such as genes governing stem cell traits, epigenome regulation and the response to stress stimuli.

cancer biology↗