Biology subjects

McBrayer, S. K.

Publications and source records attributed to McBrayer, S. K..

9 recordsLinked to original sources

bioRxiv2026

KDM6 Enzymes are the Mechanistic Targets of Mutant IDH that Dictate Replication Stress Sensitivity

Cancer-associated isocitrate dehydrogenase (IDH) mutations sensitize gliomas to replication stress, although the underlying mechanisms are unclear. IDH-mutant enzymes synthesize (R)-2-hydroxyglutarate (R2HG), which broadly inhibits 2-oxoglutarate-dependent enzymes. We performed forward genetic screens targeting all 2-oxoglutarate-dependent enzymes and discovered that KDM6 histone demethylases play a vital role in protecting cells from replication stress. Genetic or R2HG-mediated repression of KDM6 catalytic activity sensitized glioma cells to disparate replication stress-inducing drugs, including Ataxia-telangiectasia and Rad3-related (ATR) and dihydroorotate dehydrogenase (DHODH) inhibitors. This liability is generalizable because KDM6A loss-of-function mutations commonly observed in urothelial carcinomas sensitized bladder cancer cells to DHODH inhibition, thereby phenocopying IDH mutations in glioma. To exploit these oncogene-induced replication stress vulnerabilities, we developed an effective, on-target, and well-tolerated DHODH inhibitor, GLIO-1, that is poised for clinical translation. Collectively, we reveal KDM6 activity as a fundamental determinant of replication stress sensitivity and nominate pan-cancer, mechanism-based biomarkers of ATR and DHODH inhibitor efficacy. STATEMENT OF SIGNIFICANCEWe discovered that the KDM6 enzymes are the mechanistic targets of R2HG that mediate mutant IDH-induced replication stress hypersensitivity. We report a promising new DHODH inhibitor, GLIO-1, and nominate KDM6 and IDH mutations as predictive biomarkers for the antitumor effects of GLIO-1 and other replication stress inducers.

cancer biology↗

bioRxiv2026

Pharmacological induction of mitochondrial stress counteracts therapy resistance in glioblastoma stem-like cells

Glioblastoma (GBM) stem-like cells (GSCs) contribute to therapeutic resistance and recurrence. We sought to define cellular processes underlying GSC resilience. We discovered that GSCs, unlike differentiated GBM cells (DGCs) or non-malignant neural cells, depend on mitochondrial function for survival. To target this vulnerability, we exploited doxycycline (DOXY), an antibiotic used in humans, to interfere with mitochondrial protein translation. DOXY induced cell death and inhibited sphere formation in GSCs, but not in DGCs or non-malignant cells, indicating a differentiation state-selective effect. Mechanistically, DOXY induced mitochondrial dysfunction and activated a stress-responsive apoptotic program involving HRI-mediated signaling. DOXY displayed antitumor efficacy in patient-derived GBM organoid and orthotopic xenograft models. Our study reveals that DOXY can selectively target undifferentiated glioma cells, informing a drug repurposing-based strategy.

cancer biology↗

bioRxiv2025

Mutant IDH silences GSX2 to reprogram neural progenitor cell fate and promote gliomagenesis

Isocitrate dehydrogenase (IDH) mutations arise early in gliomas and are associated with a defined neurodevelopmental cancer cell hierarchy. However, how mutant IDH contributes to this hierarchy and whether this interaction promotes gliomagenesis remain unclear. We captured the dynamics of IDH-mutant glioma initiation in genetically engineered mice through time-resolved, single-cell genomics. Mutant IDH activates and induces lineage switching of neural progenitor cells (NPCs). These actions expand oligodendrocyte precursor cells, the predominant cell-of-origin for these tumors, at the expense of interneurons. Lineage switching is mediated by promoter hypermethylation and silencing of Gsx2, a homeobox gene required for neurogenesis. Critically, Gsx2 ablation recapitulates NPC fate reprogramming by mutant IDH. We provide a new model of neural cell fate control by IDH oncogenes and insights into the developmental origins of glioma.

cancer biology↗

bioRxiv2025

Nitrogen metabolism profiling reveals cell state-specific pyrimidine synthesis pathway choice

Conventional stable isotope tracing assays track one or several metabolites. However, cells use an array of nutrients to sustain nitrogen metabolic pathways. This incongruency hampers a system level understanding of cellular nitrogen metabolism. Therefore, we created a platform to simultaneously trace 30 nitrogen isotope-labeled metabolites. This platform revealed that while primitive cells engage both de novo and salvage pyrimidine synthesis pathways, differentiated cells nearly exclusively salvage uridine despite expressing de novo pathway enzymes. This link between cell state and pyrimidine synthesis routes persisted in physiological contexts, including primary murine and human tissues and tumor xenografts. Mechanistically, we found that Ser1900 phosphorylation of CAD, the first enzyme of the de novo pathway, was enriched in primitive cells and that mimicking this modification in differentiated cells abrogated their preference for pyrimidine salvage. Collectively, we establish a method for nitrogen metabolism profiling and define a mechanism of cell state-specific pyrimidine synthesis pathway choice.

cell biology↗

bioRxiv2025

L-2-hydroxyglutarate impairs neuronal differentiation through epigenetic activation of MYC expression

High levels of L- and D-2-hydroxyglutarate, the reduced forms of -ketoglutarate (KG), are implicated in human neurodevelopmental disorders and cancer. Both enantiomers exert effects on epigenetics by modulating a family of KG-dependent dioxygenases involved in histone, DNA and RNA demethylation. L-2HG dehydrogenase (L2HGDH) converts L-2HG to KG. Its deficiency is a rare, autosomal recessive inborn error of metabolism (IEM) characterized by systemic elevations of L-2HG, progressive neurological disability and a high risk of malignancy in the brain. The mechanisms behind these aberrations are unknown. Here we used an isogenic, patient-derived induced pluripotent stem cell (iPSC) system to study the impact of L2HGDH deficiency on neural progenitor cell (NPC) function and neuronal differentiation. We demonstrate that L2HGDH deficiency causes accumulation of L-2HG, NPC hyperproliferation, increased clonogenicity, excessive growth, and defective neuronal differentiation in 2D cultures and cortical spheroids. Editing the L2HGDH locus to wild-type reverses these effects. Blocking L-2HG accumulation in NPCs with a glutaminase inhibitor also induces neuronal differentiation. L-2HG-dependent inhibition of KDM5 histone demethylases leads to widespread retention of H3K4me2 and H3K4me3, markers of active gene expression. These marks are prominently elevated at the MYC locus in L2HGDH-deficient cells, and consequently cells express high MYC both in 2D culture and in many distinct cell types within cortical spheroids. Although thousands of loci display altered histone methylation, genetically or pharmacologically normalizing MYC is sufficient to completely reverse defective neuronal differentiation. These data indicate that the primary metabolic disturbance in an iPSC IEM model activates the MYC oncogene, favoring stem cell self-renewal and suppressing lineage commitment to neurons.

genetics↗

bioRxiv2025

A transcriptional biosensor reveals mechanisms of α-ketoglutarate signaling to chromatin

Alpha-ketoglutarate (KG) is required for chromatin demethylation but mechanisms controlling KG abundance in the nucleus are poorly defined. Therefore, we designed a biosensor system to monitor this metabolite pool in human cells using an KG-responsive cyanobacterial transcription factor, NtcA. We then coupled this system with a genetic screen to identify genes that regulate KG in the nucleus, defining an inter-organelle pathway in which sequential mitochondrial activities of the GPT2 transaminase and SLC25A11 transporter supply nuclear KG. Using a mouse model of GPT2 deficiency, a human inborn error of metabolism, we found that this pathway controls chromatin methylation in the developing brain. Our work provides a tool to assess KG signaling to chromatin and a framework for leveraging forward genetics to study nuclear metabolite pools.

synthetic biology↗

bioRxiv2024

Matched three-dimensional organoids and two-dimensional cell lines of melanoma brain metastases mirror response to targeted molecular therapy

PurposeDespite significant advances in the treatment paradigm for patients with metastatic melanoma, melanoma brain metastasis (MBM) continues to represent a significant treatment challenge. The study of MBM is limited, in part, by shortcomings in existing preclinical models. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional cultures prepared from primary tissue samples with minimal processing that recapitulate genotypic and phenotypic features of parent tumors and are grown without artificial extracellular scaffolding. We aimed to develop the first matched patient-derived SXO and cell line models of MBM to investigate responses to targeted therapy. MethodsMBM SXOs were created by a novel protocol incorporating techniques for establishing glioma and cutaneous melanoma organoids. A BRAFV600K-mutant and BRAF-wildtype MBM sample were collected directly from the operating room for downstream experiments. Organoids were cultured in an optimized culture medium without an artificial extracellular scaffold. Concurrently, matched patient-derived cell lines were created. Drug screens were conducted to assess treatment response in SXOs and cell lines. ResultsOrganoid growth was observed within 3-4 weeks, and MBM SXOs retained histological features of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, and strong SOX10 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF and MEK inhibitors. ConclusionHere, we describe the creation of a scaffold-free organoid model of MBM. Further study using SXOs may improve the translational relevance of preclinical studies and enable the study of the metastatic melanoma tumor microenvironment.

cancer biology↗

bioRxiv2023

Human plasma-like medium facilitates metabolic tracing and enables upregulation of immune signaling pathways in glioblastoma explants

BackgroundIn vivo stable isotope tracing is useful for natively surveying glioma metabolism but can be difficult to implement. Stable isotope tracing is tractable using in vitro glioma models, but most models lack nutrient conditions and cell populations relevant to human gliomas. This limits our ability to study glioma metabolism in the presence of an intact tumor microenvironment (TME) and immune-metabolic crosstalk. MethodsWe optimized an in vitro stable isotope tracing approach for human glioma explants and glioma stem-like cell (GSC) lines that integrates human plasma-like medium (HPLM). We performed 15N2-glutamine tracing in GSC monocultures and human IDH-wildtype glioblastoma explants and developed an analytical framework to evaluate microenvironment-dependent metabolic features that distinguish them. We also conducted spatial transcriptomics to assess transcriptional correlates to metabolic activities. ResultsHPLM culture preserved glioma explant viability and stemness while unmasking metabolic and immune programs suppressed by conventional culture conditions. Stable isotope tracing in HPLM revealed TME-dependent and TME-independent features of tumor metabolism. Tissue explants recapitulated tumor cell-intrinsic metabolic activities, such as synthesis of immunomodulatory purines. Unlike GSC monocultures, tissue explants captured tumor cell- extrinsic activities associated with stromal cell metabolism, as exemplified by astrocytic GDP- mannose production in heterocellular explants. Finally, glioma explants displayed tumor subtype-specific metabolic reprogramming, including robust pyrimidine degradation in mesenchymal cells. ConclusionsWe present a tractable approach to assess glioma metabolism in vitro under physiological nutrient levels and in the presence of an intact TME. This platform opens new avenues to interrogate glioma metabolism and its interplay with the immune microenvironment. Importance of the StudyMetabolic reprogramming is a hallmark of tumor biology, but in vitro studies of glioma metabolism often fail to replicate the nutrient complexity and cellular heterogeneity of the TME. We developed a method to perform stable isotope tracing in glioma explants grown in HPLM to analyze metabolism in a nutrient context that reflects in vivo conditions. By comparing metabolic activities between glioma cell monocultures and explanted tumor tissues, our approach captures features of tumor metabolism that are driven by the microenvironment. We show that HPLM not only sustains cell fitness and identity in tumor explants but also evokes distinct metabolic patterns and immune activation signatures repressed by standard culture conditions. Our approach offers a tractable and scalable way to study tumor cell intrinsic and microenvironmental metabolism in faithful tissue culture glioma models, complementing powerful yet low-throughput in vivo stable isotope tracing approaches. Key PointsO_LIHPLM supports culture of glioma explants and stimulates metabolic and immune transcriptional responses. C_LIO_LIStable isotope tracing in glioma explants reveals contributions of tumor cells, stromal cells, and gene expression programs to tumor metabolism. C_LI

cancer biology↗

bioRxiv2021

Semi-automated computational assessment of cancer organoid viability using rapid live-cell microscopy

The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess cellular viability in tissue specimens are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed "Apex Imaging". We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.

bioinformatics↗