Stevens, Victoria L.

Abstract High-grade gliomas represent the most common type of primary adult malignant brain tumor. IDH1/2 mutations are linked to lower-grade gliomas and better patient outcomes; however, molecular profiling has revealed additional alterations that drive aggression. CDKN2A/B loss in IDH-mutant astrocytoma is recognized as a key marker of grade 4 tumor biology, yet there remain no effective targeted therapies in aggressive gliomas. Here, we analyzed a cohort of patient-derived IDH-mutant astrocytoma with whole-exome and RNA sequencing data and identified several candidate genes and pathways concurrently regulated with CDKN2A/B loss, revealing an enhanced signature related to vascular processes. We sought to elucidate the angiogenic mechanisms and functional outcomes associated with high-grade behavior following CDKN2A/B loss in IDH-mutant astrocytoma to inform future therapeutic strategies. We used grade 4 IDH-mutant astrocytoma patient-derived xenograft (PDX) and established cell lines harboring homozygous CDKN2A/B deletion. We re-expressed the cell cycle regulators p14, p15, and p16 using a Tet-inducible lentiviral system and confirmed reduced proliferation compared to parental lines. Re-expression of p14, p15, or p16 also resulted in decreased glioma cell migration and invasion. Intriguingly, re-expressing these regulators reduced VEGF secretion. HUVEC cells cultured with conditioned media (CM) from the re-expression glioma lines decreased canonical activation of PI3K/Akt and Stat3 signaling and a diminished capacity to promote endothelial migration, invasion, and tube formation. Furthermore, in vivo Matrigel plug assays using CM from the re-expression lines showed a decreased ability to recruit endothelial cells, pericytes, and form vascular organized structures. We identified the WT1 transcription factor as an upstream regulator of angiogenesis. WT1 expression was inversely correlated with CDKN2A/B expression in patient data, and knockdown of WT1 in astrocytoma cells reduced VEGF secretion and impaired ability to promote endothelial migration. Taken together, our findings reveal a novel role for WT1 in VEGF-driven angiogenesis in IDH-mutant astrocytoma with CDKN2A/B homozygous deletion.

Abstract BACKGROUND Standard of care surgery for high grade glioma (HGG) employs Magnetic Resonance Imaging to identify the contrast enhancing (CE) core for resection, leaving a non-enhancing (NE) invasive rim of tumor which contributes to recurrence. Multi-parametric MRI (mpMRI) has recently been used to map regional biological heterogeneity of neuronal and immune signatures in HGG, and continues to offer a promising strategy for non-invasive surveillance of tumor progression. Here, we present a graph network analysis relating mpMRI to biological signatures in the NE tumor rim and show the utility of communities of imaging features for discriminating tumor phenotypes. METHODS 53 mpMRI features across 101 spatially-localized RNA-sequenced biopsy samples from 64 patients with IDH-wt HGG were represented as nodes within a Neo4j graph network. Modularity optimization was used to identify communities of densely connected nodes. Communities of nodes representing high proportions of NE samples (one-tailed test) were kept for further analysis. Gene set enrichment analysis (GSEA) on samples within selected imaging communities was analyzed for biological process enrichment using clusterProfiler. Single sample GSEA (ssGSEA) using ssGSEA2 was used to annotate neuronal and immune cell states across samples within selected communities. mpMRI feature selection within each community identified the most relevant imaging features for isolating samples classified into neuronal and immune cell states. RESULTS 11 imaging communities gathered a high proportion of NE samples. Three NE communities showed significant enrichment of neuronal signatures, while one showed significant enrichment of immune pathways. One neuronal-predominant community showed unique upregulation of 14 pathways related to neuronal migration/astrocyte projection. CONCLUSION Network imaging communities can identify clusters of NE glioma samples showing contrasting neuronal and immune signatures, including distinct upregulation of migration and projection pathways. Mapping these imaging feature communities to specific biological processes implicated in glioma progression may aid treatment planning and prognostication for glioma patients.

Abstract BACKGROUND High-grade glioma (HGG) is the most common and aggressive form of primary brain cancer. Standard of care surgery employs Magnetic Resonance Imaging to identify the contrast enhancing (CE) core for resection, leaving a non-enhancing (NE) invasive rim of tumor which contributes to recurrence. Recent multi-omics analyses identified predominant neuronal and immune signatures within NE tumor. Here, we apply a graph network to integrate regionally heterogeneous data from a unique dataset of spatially-localized CE and NE biopsies from a large cohort of patients with HGG. METHODS RNA-sequencing of 1159 differentially expressed genes across 159 IDH-wt biopsies (74 patients) from NE/CE regions were represented as nodes in a Neo4j network. Candidate genes for network random walks occurred exclusively within NE communities detected by modularity optimization and localized by cell-type using single-cell RNA-sequencing (scRNAseq). CSF2RA and RBFOX1 were selected as sources (walk length = 5; 10,000 walks/source node). Frequently occurring genes (one-tailed test) in walks were ranked by correlation with CSF2RA/RBFOX1 expression in NE samples and analyzed by gene set enrichment analysis for biological processes (clusterProfiler). Random walk genes were visualized (DoHeatmap) against composite NE/CE scRNAseq profiles. RESULTS CSF2RA and RBFOX1 were upregulated in NE samples (logFC = 0.789, 1.85). Upregulation of CSF2RA, an immune receptor, in the Glycolytic/Plurimetabolic and RBFOX1, a neurodevelopmental regulator, in the Neuronal glioma subtypes were observed in NE samples (ANOVA p < 0.05). CSF2RA walk genes (n=46) enriched metabolic/proliferative signatures, while RBFOX1 walk genes (n=201) showed neurodevelopmental upregulation. scRNAseq localized CSF2RA to monocytes/macrophages while RBFOX1 was expressed in distinct astrocyte/neuroepithelial populations. CONCLUSION CSF2RA and RBFOX1’s gene networks modulate immune and neurodevelopmental processes influencing glioma progression in NE tumor. Understanding the region-specific roles of these networks may offer strategies for targeting cells in the invasive glioma rim.

Abstract BACKGROUND High-grade glioma (HGG) is the most common and aggressive form of primary brain cancer with poor prognosis. Standard of care surgery employs Magnetic Resonance Imaging to identify the contrast enhancing (CE) core for resection, typically leaving behind a non-enhancing (NE) invasive rim of tumor which invariably contributes to recurrence. Recent multi-omics analyses identified predominant neuronal and immune signatures within NE tumor, which could provide key insights for combatting recurrence. We present the novel application of graph network analyses to integrate multi-omic data from a unique dataset of multi-regional spatially-localized CE and NE biopsy samples from a large cohort of patients with HGG. MATERIAL AND METHODS 159 IDH-wildtype tumor biopsy samples from NE and CE regions of 74 glioma patients were profiled by RNA sequencing. Differentially expressed genes (DEGs) across NE and CE samples (n = 1159) were determined using the EdgeR package, and represented as nodes within a Neo4j graph network. Samples were hierarchically clustered for each gene (method = median), and edges were generated between gene nodes sharing ≥ 1 sample. Modularity optimization was used to identify communities of densely connected nodes. Genes occurring exclusively within NE communities were localized by cell type using external single-cell RNA sequencing datasets and identified as source node candidates for NE network random walks. These were performed with CSF2RA nodes as source (walk length = 5; 10,000 walks/source node). Random walks traversing samples with significantly elevated CSF2RA expression (one-tailed test) were kept for analysis. Genes occurring at high frequency (one-tailed test) within random walks were ranked by correlation with CSF2RA expression in NE samples and analyzed by gene set enrichment analysis (GSEA) for biological processes using clusterProfiler. RESULTS CSF2RA, an immune signaling receptor gene, was upregulated (logFC = 0.789) in NE samples and uniquely occurred within NE communities. We show its differential expression within the immune-enriched Glycolytic/Plurimetabolic (GPM) glioma subtype and confirm its enrichment in myeloid populations across single cell HGG datasets. NE network random walks identified 46 DEGs associated with CSF2RA. GSEA revealed upregulation of proliferative and metabolic signatures and downregulation of signaling pathways within CSF2RA’s network. CONCLUSION CSF2RA’s genetic network in NE tumor represents a potential mechanism for immunologic facilitation of glioma progression within this region. Given the expression of CSF2 ligand by glioma cells, which plays a crucial role in recruiting myeloid cells during peripheral cancer progression, modulating the expression of key genes in the CSF2RA network may be a promising approach for perturbing the crosstalk signals between immune and tumor cells in the NE region.