Laukhuff, Harrison

Abstract Introduction Leptomeningeal disease (LMD) is the seeding of tumor cells in the leptomeninges and the cerebrospinal fluid (CSF) and carries a poor prognosis of 2-4 months. CSF sampling is required to study LMD, but genomic analysis may not be able to identify non-genomic and post-translational modifications. Proteomics can address this gap but is limited by reproducibility and disease heterogeneity. Integrating proteomic with genomic data may prioritize driver genes that promote LMD. Methods 23 patients with breast cancer-associated LMD with matched CSF proteomic and genomic data were studied. Variant allele frequencies (VAFs) for genes TP53, NFE2L2, ERBB2 and PIK3CA were derived, and proteomic profiling was performed on matched CSF. Spearman correlations between driver-gene VAF and protein expression were computed, restricting to genes with VAF>0 in ≥ 3 samples, and Benjamini-Hochberg correction was applied across gene-protein pairs. Proteins significantly associated with VAF were analyzed using over representation analysis (ORA) and gene segment enrichment analysis (GSEA) with KEGG, Gene Ontology, and MSigDB. Results TP53 VAF was associated with upregulation of proteins involved in glycolysis and cell proliferation, with enrichment of hallmark glycolysis (FDR=2.6x10-8), mTORC1 signalling (FDR=8.5x10-8), and reactive oxygen species pathways (FDR=2.7x10-3), and downregulation of complement (FDR=6.8 x 10-6) and coagulation cascades (FDR=0.011). NFE2L2 VAF was associated with epithelial-mesenchymal transition by both GSEA (FDR=2.3x10-9) and ORA (FDR=4.6x10-3), TGF-β signalling (FDR=0.015) and glycoprotein metabolism (FDR=1.6x10-7). ERBB2 VAF was associated with an upregulation of cell-cycle regulation (FDR=6.4x10-10), mTORC1 pathway (FDR=3.2x10-4), and a downregulation of proteins involved in cell-cell adhesion (FDR=4.0x10-9), axon guidance (GSEA FDR=0.025), and developmental growth (GOBP FDR=3.4x10-4). Conclusion Distinct CSF proteomic signatures were associated with specific driver mutations, implicating metabolic and oxidative stress pathways (TP53), stromal remodelling (NFE2L2), and disruption of CNS structural pathways (ERBB2). Validation in prospective LMD cohorts is needed to confirm these mutation-specific signatures and inform diagnostic and therapeutic strategies.

Abstract Background Glioblastoma (GBM) is the most common malignant primary brain tumor and is associated with a poor prognosis. Repurposed posaconazole (PCZ) has demonstrated efficacy in vitro by targeting key metabolic pathways. The aim of this study was to determine the neuropharmacological profile of PCZ in patients with GBM through a Phase 0 first-in-human trial for this indication (NCT04825275). Methods We conducted an open-label, non-randomized, parallel-arm trial in participants with primary or recurrent GBM. Patients in the treatment arm received oral PCZ for 7-10 days presurgically to achieve steady-state concentrations. Microdialysis catheters were implanted to measure interstitial drug levels. Postoperative tissue and fluid samples were analyzed to evaluate PCZ pharmacokinetics (mass spectrometry) and pharmacodynamics, including vascular density, apoptosis, and metabolite concentrations (lactate and pyruvate) compared to controls. Results The trial was closed early due to slow accrual, enrolling 2 participants in the PCZ arm and three in the control arm. No drug-related safety issues were observed. Although PCZ was undetectable in microdialysate fluid, it accumulated in tumor tissue, with concentrations showing an association with CD31-measured endothelial content. PCZ-treated tumors exhibited trends toward lower BCL2 expression indicative of increased apoptosis, and significantly lower pyruvate levels in the tumor periphery compared to controls. Plasma lactate levels in treated patients normalized to control levels over 24 h. Conclusions Despite limited accrual, this trial provides the first clinical evidence that oral PCZ accumulates in human GBM tissue and shows preliminary signals of metabolic modulation. These findings provide biological rationale warranting further clinical investigation, potentially utilizing direct tissue sampling rather than microdialysis.

Abstract GB13 is a novel biologic therapy in the final stages of preclinical development for the treatment of glioblastoma (GBM) and H3 K27-altered diffuse midline glioma (DMG) that has received Orphan Drug Designation by the Food and Drug Administration for treatment of malignant gliomas. GB13 targets a specific tumor-restricted receptor of interleukin 13 called interleukin 13 receptor alpha 2 (IL13Rα2). IL13Rα2 is highly expressed in malignant gliomas and, importantly, is not expressed on non-cancerous cells of the brain. GB13 consists of an engineered proprietary mutant of IL13, which binds exclusively to IL13Rα2, and is fused to a highly cytotoxic exotoxin molecule. In vitro data demonstrate that GB13 potently kills IL13Rα2+ cancer cells while sparing non-cancerous cells. These characteristics make GB13 an attractive agent for safe and efficacious targeting of tumor cells expressing IL13Rα2 in upcoming clinical trials. In the current studies, we employed patient-derived GBM and DMG cell and animal models to provide key conclusions that support GB13 as an effective treatment option for gliomas in the clinic. First, we demonstrated that single acute infusions of GB13 by enhanced delivery (CED) is effective at decreasing orthotopic tumor volumes and increasing animal survival and that a long-term infusion can more significantly extend animal survival. Second, we determined that IL13Rα2 is requisite for GB13 activity as tumors that do not express the receptor are insensitive to the cytotoxic effects of GB13, demonstrating a precise and clear biomarker for clinical trial patient selection. Finally, we determined that GB13 works in concert with clinical standard-of-care radiation therapy to increase cell death, compared to radiation alone. Based on this work, we and our clinical collaborators are confident that the current work confirms GB13 has many important characteristics as an exciting therapy, which continues development towards a Phase I clinical trial for both GBM and pediatric DMG.