Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5′-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.

Glioblastoma (GBM) is the most common primary brain malignancy in adults, with a 100% recurrence rate and 21-month median survival. Our lab and others have shown that GBM contains a subpopulation of glioma stem cells (GSCs) that expand during chemotherapy and may contribute to therapeutic resistance and recurrence in GBM. To investigate the mechanism behind this expansion, we applied gene set expression analysis (GSEA) to patient-derived xenograft (PDX) cells in response to temozolomide (TMZ), the most commonly used chemotherapy against GBM. Results showed significant enrichment of cancer stem cell and cell cycle pathways (False Discovery Rate (FDR) < 0.25). The ligand of numb protein 1 (LNX1), a known regulator of Notch signaling by targeting negative regulator Numb, is strongly upregulated after TMZ therapy (p < 0.0001) and is negatively correlated with survival of GBM patients. LNX1 is also upregulated after TMZ therapy in multiple PDX lines with concomitant downregulations in Numb and upregulations in intracellular Notch1 (NICD). Overexpression of LNX1 results in Notch1 signaling activation and increased GSC populations. In contrast, knocking down LNX1 reverses these changes, causing a significant downregulation of NICD, reduction in stemness after TMZ therapy, and resulting in more prolonged median survival in a mouse model. Based on this, we propose that during anti-GBM chemotherapy, LNX1-regulated Notch1 signaling promotes stemness and contributes to therapeutic resistance.

This year nearly 20,000 lives will be lost to Glioblastoma (GBM), a treatment-resistant primary brain cancer. In this study, we identified a molecular circuit driven by epigenetic regulation that regulates the expression of ciliary protein ALR13B. We also demonstrated that ARL13B subsequently interacts with purine biosynthetic enzyme IMPDH2. Removal of ARL13B enhanced TMZ-induced DNA damage by reducing de-novo purine biosynthesis and forcing GBM cells to rely on the purine salvage pathway. Furthermore, targeting can be achieved by using an FDA-approved drug, Mycophenolate Moefitil. Our results suggest a clinical evaluation of MMF in combination with TMZ treatment in glioma patients.

Extensive molecular profiling has revealed profound heterogeneity in glioblastoma (GBM), yet how cellular lineages organize over time to govern tumor propagation and therapeutic response remains poorly understood. Existing single-cell approaches define transcriptional states but provide limited insight into how clonal dynamics shape functional tumor behavior. Here, we integrate high-complexity combinatorial DNA barcoding with single-cell transcriptomics in direct-from-patient IDH1-wild-type GBM, enabling lineage-resolved mapping of progenitor organization in a human microenvironmental context. Across 235,155 malignant cells from nine tumors, clonal relationships form reproducible lineage tracks in which distinct progenitor populations give rise to specific differentiated cell types, revealing that tumor growth is sustained by multiple non-redundant progenitors rather than a single dominant population. These progenitors exhibit distinct propensities for self-renewal, fate restriction, and cross-compartment interactions, collectively accounting for the full spectrum of tumor states. Using this lineage-resolved framework, we identify complementary drug targets in distinct progenitor compartments and demonstrate that hierarchy-informed combination therapies disrupt progenitor-progenitor interactions and reshape lineage output. These findings move beyond descriptive heterogeneity to define functional logic underlying GBM propagation and establish a generalizable framework for rational, cell type-specific combinatorial therapies.

A major obstacle to identifying effective therapies for the aggressive brain tumor glioblastoma is the lack of human-specific, immunocompetent models that reflect the human tumor microenvironment. To address this, we developed the immune-Human Organoid Tumor Transplantation (iHOTT) model. This is an autologous co-culture platform that integrates patient-derived tumor cells and matched peripheral blood mononuclear cells (PBMCs) within human cortical organoids, enabling the study of the patient-specific immune response to the tumor and tumor-immune interactions. This platform preserves tumor and immune populations, immune signaling, and cell-cell interactions observed in patient tumors. Treatment of iHOTT with pembrolizumab, a checkpoint inhibitor, mirrored cell type shifts and cell interactions observed in patients. TCR sequencing further revealed pembrolizumab-driven expansion of stem-like CD4-T-cell clonotypes exhibiting patient-specific repertoires. These findings establish iHOTT as a physiologically relevant platform for exploring autologous tumor–immune interactions and underscore the critical need for antigen-targeted strategies to enhance immunotherapy in glioblastoma.

Benign-appearing intracranial cysts may become symptomatic due to mass effect and require surgical treatment. Mechanisms underlying cyst formation and growth remain poorly understood. This study identified 16 patients who underwent surgical treatment for benign intracranial cysts. Cyst wall pathologic samples (n = 8) were characterized as arachnoid or glioependymal cysts using H&E and immunofluorescence staining. Five samples (62.5%) were found to be glioependymal while three (37.5%) were arachnoid cysts. Cyst fluid examined in 4 cases resembled cerebrospinal fluid (CSF) and showed no significant differences in composition between pathological subtypes. Single-cell sequencing and RNA expression profile comparisons between glioependymal and arachnoid cysts revealed distinct cellular profiles. Analyses of the innermost cell layer (ependymal cells versus arachnoid cells) suggested differing mechanisms of pathogenesis. Glioependymal cysts harbored upregulated expression of sodium transporters and aquaporin channels, which suggests possible CSF-like fluid production whereas arachnoid cysts may be associated with mechanical fluid accumulation. Thus, intracranial cysts represent multiple unique pathologic entities with differing cell types and RNA expression profiles. Further study into mechanisms of glioependymal cyst formation may allow for targeted interventions to reduce fluid production and avoid surgery.