Primary Glioblastoma Samples Research Articles

A novel protein SPECC1-415aa encoded by N6-methyladenosine modified circSPECC1 regulates the sensitivity of glioblastoma to TMZ

BackgroundCircular RNAs (circRNAs) can influence a variety of biological functions and act as a significant role in the progression and recurrence of glioblastoma (GBM). However, few coding circRNAs have been discovered in cancer, and their role in GBM is still unknown. The aim of this study was to identify coding circRNAs and explore their potential roles in the progression and recurrence of GBM.MethodsCircSPECC1 was screened via circRNAs microarray of primary and recurrent GBM samples. To ascertain the characteristics and coding ability of circSPECC1, we conducted a number of experiments. Afterward, through in vivo and in vitro experiments, we investigated the biological functions of circSPECC1 and its encoded novel protein (SPECC1-415aa) in GBM, as well as their effects on TMZ sensitivity.ResultsBy analyzing primary and recurrent GBM samples via circRNAs microarray, circSPECC1 was found to be a downregulated circRNA with coding potential in recurrent GBM compared with primary GBM. CircSPECC1 suppressed the proliferation, migration, invasion, and colony formation abilities of GBM cells by encoding a new protein known as SPECC1-415aa. CircSPECC1 restored TMZ sensitivity in TMZ-resistant GBM cells by encoding the new protein SPECC1-415aa. The m6A reader protein IGF2BP1 can bind to circSPECC1 to promote its expression and stability. Mechanistically, SPECC1-415aa can bind to ANXA2 and competitively inhibit the binding of ANXA2 to EGFR, thus resulting in the inhibition of the phosphorylation of EGFR (Tyr845) and its downstream pathway protein AKT (Ser473). In vivo experiments showed that the overexpression of circSPECC1 could combine with TMZ to treat TMZ-resistant GBM, thereby restoring the sensitivity of TMZ-resistant GBM to TMZ.ConclusionsCircSPECC1 was downregulated in recurrent GBM compared with primary GBM. The m6A reader protein IGF2BP1 could promote the expression and stability of circSPECC1. The sequence of SPECC1-415aa, which is encoded by circSPECC1, can inhibit the binding of ANXA2 to EGFR by competitively binding to ANXA2 and inhibiting the phosphorylation of EGFR and AKT, thereby restoring the sensitivity of TMZ-resistant GBM cells to TMZ.

Racial Disparities in Glioblastoma Genomic Alterations: A Comprehensive Analysis of a Multi-Institution Cohort of 2390 Patients

Although molecular biomarkers have significantly advanced precision oncology in glioblastoma, the prevalence of these biomarkers by race remains underexplored. This study aims to characterize the genomic alterations in glioblastoma across Asian, Black, and White patients, offering insights into racial disparities that may influence treatment outcomes and disease progression. Analyzing data from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange database V13.0, this study examined 2390 primary glioblastoma samples from unique patients. Genomic alterations in 566 cancer-related genes were assessed using targeted next-generation sequencing panels from 3 large cancer institutes. The patient cohort included 112 Asians, 67 Blacks, and 2211 Whites. Statistical significance of associations between genomic alterations and race was evaluated using the chi-squared test, with the Benjamini-Hochberg method applied to control for multiple testing adjustments. Significant racial differences were observed in the frequency of genomic alterations. Asians exhibited a higher frequency of TP53 alterations (52.68%, P < 0.001), Blacks showed more frequent alterations in NRAS (7.46%, P < 0.001), MTOR (10.45%, P= 0.039), and TET2 (8.96%, P=0.039), and Whites had a higher occurrence of PTEN alterations (48.67%, P= 0.045). Additionally, Black patientshad an elevated rate of RET deletions (14.29%, P<0.001). This study identifies significant racial disparities in the alteration frequencies of 6 key glioblastoma genes: NRAS, TP53, MTOR, TET2, PTEN, and RET. These findings underscore the need for racial considerations in glioblastoma treatment strategies and highlight potential avenues for targeted therapeutic interventions. Further research is needed to explore the clinical implications of these genomic disparities.

Immunotherapy drives mesenchymal tumor cell state shift and TME immune response in glioblastoma patients.

Glioblastoma is a highly aggressive type of brain tumor for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected patients, and there is an urgent need to identify mechanisms of treatment resistance to design novel therapy strategies. Here we investigated the phenotypic and transcriptional dynamics at single-cell resolution during nivolumab immune checkpoint treatment of glioblastoma patients. We present the integrative paired single-cell RNA-seq analysis of 76 tumor samples from patients in a clinical trial of the PD-1 inhibitor nivolumab and untreated patients. We identify a distinct aggressive phenotypic signature in both tumor cells and the tumor microenvironment in response to nivolumab. Moreover, nivolumab-treatment was associated with an increased transition to mesenchymal stem-like tumor cells, and an increase in TAMs and exhausted and proliferative T cells. We verify and extend our findings in large external glioblastoma dataset (n = 298), develop a latent immune signature and find 18% of primary glioblastoma samples to be latent immune, associated with mesenchymal tumor cell state and TME immune response. Finally, we show that latent immune glioblastoma patients are associated with shorter overall survival following immune checkpoint treatment (P = .0041). We find a resistance mechanism signature in one fifth of glioblastoma patients associated with a tumor-cell transition to a more aggressive mesenchymal-like state, increase in TAMs and proliferative and exhausted T cells in response to immunotherapy. These patients may instead benefit from neuro-oncology therapies targeting mesenchymal tumor cells.

Abstract 5676: Spatial 3D genome organization reveals intratumor heterogeneity in primary glioblastoma samples

Abstract Glioblastoma (GBM) represents the most prevalent malignant primary brain tumor with highly unfavorable prognosis. Currently, most genomic studies are conducted at a single site of a tumor, which do not reflect the complete genetic or epigenetic information across the whole tumor. Furthermore, the intra-tumoral heterogeneity (ITH) of 3D genome organization has also not been studied yet. To address these gaps, we performed Hi-C experiments in 21 samples obtained from 9 GBM patients, with 15 of them being spatially mapped based on their 3D coordinates from the same patients. We identified extensive inter-tumoral and intra-tumoral heterogeneity in genome compartmentalization and chromatin interactions. Notably, in a patient with 9 spatially mapped samples from both temporal and frontal regions, we accumulated over 6 billion reads and defined high-resolution region-specific chromatin interactions, regulatory networks, and key regulators within the same patient. We detected structural variation (SV) and enhancer hijacking across all the samples, and identified recurrent events that affect known cancer-related genes such as CDKN2A/B. Finally, we introduce the concept of 'enhancer amputation', defined as the loss of enhancers due to SVs which lead to decreased expression of their original target genes. To our knowledge, this study represents the first large-scale exploration of the 3D genome in primary GBM patients and the first investigation of 3D genome organization in multiple regions of the same tumor. Our findings provide unprecedented insights into the ITH of GBM at the 3D genomic level, opening new avenues for understanding and potentially targeting this devastating disease. Citation Format: Qixuan Wang, Juan Wang, Radhika Mathur, Mark W. Youngblood, Qiushi Jin, Ye Hou, Lena A. Stasiak, Yu Luan, Joseph F. Costello, Feng Yue. Spatial 3D genome organization reveals intratumor heterogeneity in primary glioblastoma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5676.

GRIK1 promotes glioblastoma malignancy and is a novel prognostic factor of poor prognosis.

Primary tumors of the central nervous system (CNS) are classified into over 100 different histological types. The most common type of glioma is derived from astrocytes, and the most invasive glioblastoma (WHO IV) accounts for over 57% of these tumors. Glioblastoma (GBM) is the most common and fatal tumor of the CNS, with strong growth and invasion capabilities, which makes complete surgical resection almost impossible. Despite various treatment methods such as surgery, radiotherapy, and chemotherapy, glioma is still an incurable disease, and the median survival time of patients with GBM is shorter than 15 months. Thus, molecular mechanisms of GBM characteristic invasive growth need to be clarified to improve the poor prognosis. Glutamate ionotropic receptor kainate type subunit 1 (GRIK1) is essential for brain function and is involved in many mental and neurological diseases. However, GRIK1's pathogenic roles and mechanisms in GBM are still unknown. Single-nuclear RNA sequencing of primary and recurrent GBM samples revealed that GRIK1 expression was noticeably higher in the recurrent samples. Moreover, immunohistochemical staining of an array of GBM samples showed that high levels of GRIK1 correlated with poor prognosis of GBM, consistent with The Cancer Genome Atlas database. Knockdown of GRIK1 retarded GBM cells growth, migration, and invasion. Taken together, these findings show that GRIK1 is a unique and important component in the development of GBM and may be considered as a biomarker for the diagnosis and therapy in individuals with GBM.

CNSC-25. NEURAL SIGNATURE OF GBM EXHIBITS SYNAPTOGENIC AND OPC-LIKE FEATURES AND INDEPENDENTLY PREDICTS PATIENT SURVIVAL

Abstract Interactions of neural cells with glioma cells drive tumor growth. In this study, we identified an epigenetically-defined, malignant neural signature of IDH-wildtype glioblastoma (GBM) that significantly influenced patients’ outcome. We used DNA methylation-based reference signatures of neural cells to deconvolve 1.058 primary patient GBM samples. Samples were classified based on the neural reference signature and annotated as low or high neural GBM. A high neural GBM was linked to hypomethylation of CpG sites associated with invasiveness, neuron-to-glioma-interactions, and transsynaptic signaling. Through spatially resolved, single cell transcriptomic and proteomic profiling, we discovered a correlation between the high neural signature and the upregulation of early development programs in OPC and NPC-like cells. These traits were accompanied by diminished immune infiltration and immunological response, further highlighting the distinctive neural-like nature of GBM. High neural GBM demonstrated increased connectivity as evidenced by magnetoencephalography and resting state magnet resonance imaging. Spatiotemporal tumor sampling showed a homogeneous and stable neural signature in newly diagnosed and recurrent GBM. In newly diagnosed GBM, a high neural signature correlated with decreased survival in 306 patients (median OS 15.0 versus 20.0 months, p&amp;lt; 0.001). Patient-derived xenograft models corroborated the aggressive nature of high neural GBM, showing a significantly reduced median OS of 58 days compared to 73 days (p&amp;lt; 0.01). These high neural GBM xenografts also exhibited an elevated tumor burden and an increase in neuron-glioma synapse formation. Consideration of the extent of resection showed a dependency on the neural subgroup as high neural GBM had an increased benefit from a complete contrast enhancing tumor removal when compared to low neural GBM. As a preoperative biomarker, the concentration of BDNF was increased in serum of high neural GBM patients (p &amp;lt; 0.01). Overall, we present an epigenetically-defined malignant neural signature in GBM that is associated with an unfavorable patient survival.

A three-stage eccDNA based molecular profiling significantly improves the identification, prognosis assessment and recurrence prediction accuracy in patients with glioma

Glioblastoma (GBM) progression is influenced by intratumoral heterogeneity. Emerging evidence has emphasized the pivotal role of extrachromosomal circular DNA (eccDNA) in accelerating tumor heterogeneity, particularly in GBM. However, the eccDNA landscape of GBM has not yet been elucidated. In this study, we first identified the eccDNA profiles in GBM and adjacent tissues using circle- and RNA-sequencing data from the same samples. A three-stage model was established based on eccDNA-carried genes that exhibited consistent upregulation and downregulation trends at the mRNA level. Combinations of machine learning algorithms and stacked ensemble models were used to improve the performance and robustness of the three-stage model. In stage 1, a total of 113 combinations of machine learning algorithms were constructed and validated in multiple external cohorts to accurately distinguish between low-grade glioma (LGG) and GBM in patients with glioma. The model with the highest area under the curve (AUC) across all cohorts was selected for interpretability analysis. In stage 2, a total of 101 combinations of machine learning algorithms were established and validated for prognostic prediction in patients with glioma. This prognostic model performed well in multiple glioma cohorts. Recurrent GBM is invariably associated with aggressive and refractory disease. Therefore, accurate prediction of recurrence risk is crucial for developing individualized treatment strategies, monitoring patient status, and improving clinical management. In stage 3, a large-scale GBM cohort (including primary and recurrent GBM samples) was used to fit the GBM recurrence prediction model. Multiple machine learning and stacked ensemble models were fitted to select the model with the best performance. Finally, a web tool was developed to facilitate the clinical application of the three-stage model.

CHRM3 is a novel prognostic factor of poor prognosis and promotes glioblastoma progression via activation of oncogenic invasive growth factors.

Glioblastoma (GBM) is the most aggressive cancer of the brain and has a high mortality rate due to the lack of effective treatment strategy. Clarification of molecular mechanisms of GBM's characteristic invasive growth is urgently needed to improve the poor prognosis. Single-nuclear sequencing of primary and recurrent GBM samples revealed that levels of M3 muscarinic acetylcholine receptor (CHRM3) were significantly higher in the recurrent samples than in the primary samples. Moreover, immunohistochemical staining of an array of GBM samples showed that high levels of CHRM3 correlated with poor prognosis, consistent with The Cancer Genome Atlas database. Knockdown of CHRM3 inhibited GBM cell growth and invasion. An assay of orthotopic GBM animal model in vivo indicated that inhibition of CHRM3 significantly suppressed GBM progression with prolonged survival time. Transcriptome analysis revealed that CHRM3 knockdown significantly reduced an array of classic factors involved in cancer invasive growth, including MMP1/MMP3/MMP10/MMP12 and CXCL1/CXCL5/CXCL8. Taken together, CHRM3 is a novel and vital factor of GBM progression via regulation of multiple oncogenic genes and may serve as a new biomarker for prognosis and therapy of GBM patients.

TMIC-71. SPATIAL ANALYSIS OF GENETIC HETEROGENEITY AND TUMOR MICROENVIRONMENT INTERACTIONS IN GLIOBLASTOMA

Abstract Genetic variation can be considered a driver of somatic evolution in glioblastoma (GBM); however, the tumor microenvironment (TME) is a key contributor to the selection process and evolutionary potential of cancer cells. Although diverse ecosystems exist in GBM which impact tumor cell fitness and characterize an aggressive pathophysiology, little is known about how genetically distinct subpopulations of GBM cells interact with the surrounding TME. Therefore, we sought to investigate how the varying clonal genotypes of GBM cells are influenced by different types of selective pressures within the TME. Our imaging-based strategy is centered around Specific-to-Allele PCR Florescence In Situ Hybridization (STAR-FISH), an in situ PCR based probe hybridization method for detection of point mutations and copy number variations at the single-cell level. Using STAR-FISH to illuminate the spatial distribution of clones with mutations in the TERT promoter, we integrated multicolor immunofluorescent staining, DNA-FISH, reflectance imaging, and computational cellular analysis methods to achieve highly-multiplexed single cell spatial profiling of intact GBM specimens. With this strategy we are able to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix organization, nuclear morphology, mutational status, and several oncogenic amplifications on the same slide and location. We analyzed 20 matched primary and recurrent GBM samples, imaging several distinct regions of the same tumor. Our initial results have revealed interesting and potentially novel distributions of subclones residing within specific microenvironments. These results include TERT mutation positive clones enriched in areas possessing more structured extracellular matrix, as well as unique arrangements of infiltrating immune cells in the angiogenic tumor niche. This research underscores the importance of the TME in selecting for certain phenotypes that characterize somatic evolution, highlighting novel genetic and spatial dependencies which sustain heterogeneity in GBM.

Epigenetic Suppression of the IL-7 Pathway in Progressive Glioblastoma.

Background: Immune evasion in glioblastoma (GBM) shields cancer cells from cytotoxic immune response. Methods: We investigated CpG methylation in promoters, genes, and pathways in 22 pairs of formalin-fixed paraffin-embedded sequential (FFPE) GBM using restricted resolution bisulfite sequencing (RRBS) and bioinformatic analyses. Results: Gene ontology revealed hypermethylation in elements of the innate and adaptive immune system when recurrent GBM samples (GBMrec) were compared to control (CG) and primary GBM samples (GBMprim). Higher methylation levels of the IL-7 signaling pathway and response to IL-7 were found in GBMrec suggesting a progressive blockade of the IL-7 driven T cell response in sequential GBM. Analyses of the Cancer Genome Atlas array-based data confirmed hypermethylation of the IL-7 pathway in recurrent compared with primary GBM. We also quantified DNA CpG methylation in promoter and gene regions of the IL-7 ligand and IL-7 α-receptor subunit in individual samples of a large RRBS-based sequential cohort of GBM in a Viennese database and found significantly higher methylation levels in the IL-7 receptor α-subunit in GBMrec compared with GBMprim. Conclusions: This study revealed the progressive suppression of the IL-7 receptor-mediated pathway as a means of immune evasion by GBM and thereby highlighted it as a new treatment target.

Abstract LB024: Cancer stem cells are enriched in human recurrent glioblastoma

Abstract A number of studies have described an essential role of cancer stem cells (CSC) in glioblastoma (GBM) progression and recurrence based on isolation by candidate markers. However, a systematic evaluation of the proposed CSC in paired human primary and recurrent glioblastoma has been lacking and whether they represent a unified cell type with conserved features over tumor evolution is unresolved. In this study, we used human patient derived xenografts (PDX) to investigate the status of these cells in patients following therapeutics and recurrence. Firstly, to ensure the fidelity of tumor heterogeneity in serially passaged orthotopic GBM PDX, we collected samples at different passages and performed whole tumor single cell RNA sequencing analysis. The results illustrate the preservation of all unsupervised transcriptional cell clusters through multiple passages. Furthermore, treatment of tumor bearing mice with temozolomide, the standard of care chemotherapeutic drug for glioblastoma patients, increased apoptosis and decreased tumor cell burden but spared CSC. We then turned to paired human primary and recurrent GBM samples to inquire cancer stem cell marker expression and cell distribution. In all three pairs of PDX studied, cancer stem cell marker F3 serves as a reliable means to isolate sphere formation cells and is augmented in the recurrent samples. Inspection of whole tumors with single cell RNA sequencing nails down the enrichment of CSC in recurrent tumors, thus emphasizing the importance to eradicate these cells in future therapeutics. Citation Format: Xuanhua Peter Xie, Mungunsarnai Ganbold, Alicia Pedraza, Luis Parada. Cancer stem cells are enriched in human recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB024.

Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy.

Tumor recurrence is the main challenge in glioblastoma (GBM) treatment. Gold standard therapy temozolomide (TMZ) is known to induce upregulation of IL8/CXCL2/CXCR2 signaling that promotes tumor progression and angiogenesis. Our aim was to verify the alterations on this signaling pathway in human GBM recurrence and to investigate the impact of TMZ in particular. Furthermore, a combi-therapy of TMZ and CXCR2 antagonization was established to assess the efficacy and tolerability. First, we analyzed 76 matched primary and recurrent GBM samples with regard to various histological aspects with a focus on the role of TMZ treatment and the assessment of predictors of overall survival (OS). Second, the combi-therapy with TMZ and CXCR2-antagonization was evaluated in a syngeneic mouse tumor model with in-depth immunohistological investigations and subsequent gene expression analyses. We observed a significantly decreased infiltration of tumor-associated microglia/macrophages (TAM) in recurrent tumors, while a high TAM infiltration in primary tumors was associated with a reduced OS. Additionally, more patients expressed IL8 in recurrent tumors and TMZ therapy maintained CXCL2 expression. In mice, enhanced anti-tumoral effects were observed after combi-therapy. In conclusion, high TAM infiltration predicts a survival disadvantage, supporting findings of the tumor-promoting phenotype of TAMs. Furthermore, the combination therapy seemed to be promising to overcome CXCR2-mediated resistance.

PML Differentially Regulates Growth and Invasion in Brain Cancer.

Glioblastoma is the most malignant brain tumor among adults. Despite multimodality treatment, it remains incurable, mainly because of its extensive heterogeneity and infiltration in the brain parenchyma. Recent evidence indicates dysregulation of the expression of the Promyelocytic Leukemia Protein (PML) in primary Glioblastoma samples. PML is implicated in various ways in cancer biology. In the brain, PML participates in the physiological migration of the neural progenitor cells, which have been hypothesized to serve as the cell of origin of Glioblastoma. The role of PML in Glioblastoma progression has recently gained attention due to its controversial effects in overall Glioblastoma evolution. In this work, we studied the role of PML in Glioblastoma pathophysiology using the U87MG cell line. We genetically modified the cells to conditionally overexpress the PML isoform IV and we focused on its dual role in tumor growth and invasive capacity. Furthermore, we targeted a PML action mediator, the Enhancer of Zeste Homolog 2 (EZH2), via the inhibitory drug DZNeP. We present a combined in vitro–in silico approach, that utilizes both 2D and 3D cultures and cancer-predictive computational algorithms, in order to differentiate and interpret the observed biological results. Our overall findings indicate that PML regulates growth and invasion through distinct cellular mechanisms. In particular, PML overexpression suppresses cell proliferation, while it maintains the invasive capacity of the U87MG Glioblastoma cells and, upon inhibition of the PML-EZH2 pathway, the invasion is drastically eliminated. Our in silico simulations suggest that the underlying mechanism of PML-driven Glioblastoma physiology regulates invasion by differential modulation of the cell-to-cell adhesive and diffusive capacity of the cells. Elucidating further the role of PML in Glioblastoma biology could set PML as a potential molecular biomarker of the tumor progression and its mediated pathway as a therapeutic target, aiming at inhibiting cell growth and potentially clonal evolution regarding their proliferative and/or invasive phenotype within the heterogeneous tumor mass.

Tumor cell and immune cell profiles in primary human glioblastoma: Impact on patient outcome.

The distribution and role of tumor‐infiltrating leucocytes in glioblastoma (GBM) remain largely unknown. Here, we investigated the cellular composition of 55 primary (adult) GBM samples by flow cytometry and correlated the tumor immune profile with patient features at diagnosis and outcome. GBM single‐cell suspensions were stained at diagnosis (n = 44) and recurrence following radiotherapy and chemotherapy (n = 11) with a panel of 8‐color monoclonal antibody combinations for the identification and enumeration of (GFAP+CD45−) tumor and normal astrocytic cells, infiltrating myeloid cells —i.e. microglial and blood‐derived tumor‐associated macrophages (TAM), M1‐like, and M2‐like TAM, neutrophils. and myeloid‐derived suppressor cells (MDSC)— and tumor‐infiltrating lymphocytes (TIL) —i.e. CD3+T‐cells and their TCD4+, TCD8+, TCD4−CD8−, and (CD25+CD127lo) regulatory (T‐regs) subsets, (CD19+CD20+) B‐cells, and (CD16+) NK‐cells—. Overall, GBM samples consisted of a major population (mean ± 1SD) of tumor and normal astrocytic cells (73% ± 16%) together with a significant but variable fraction of immune cells (24% ± 18%). Within myeloid cells, TAM predominated (13% ± 12%) including both microglial cells (10% ± 11%) and blood‐derived macrophages (3% ± 5%), in addition to a smaller proportion of neutrophils (5% ± 9%) and MDSC (4% ± 8%). Lymphocytes were less represented and mostly included TCD4+ (0.5% ± 0.7%) and TCD8+ cells (0.6% ± 0.7%), together with lower numbers of TCD4−CD8− T‐cells (0.2% ± 0.4%), T‐regs (0.1% ± 0.2%), B‐lymphocytes (0.1% ± 0.2%) and NK‐cells (0.05% ± 0.05%). Overall, three distinct immune profiles were identified: cases with a minor fraction of leucocytes, tumors with a predominance of TAM and neutrophils, and cases with mixed infiltration by TAM, neutrophils, and T‐lymphocytes. Untreated GBM patients with mixed myeloid and lymphoid immune infiltrates showed a significantly shorter patient overall survival versus the other two groups, in the absence of gains of the EGFR gene (p = 0.02). Here we show that immune cell infiltrates are systematically present in GBM, with highly variable levels and immune profiles. Patients with mixed myeloid and T‐lymphoid infiltrates showed a worse outcome.

MSI1 Promotes the Expression of the GBM Stem Cell Marker CD44 by Impairing miRNA-Dependent Degradation.

Simple SummaryGlioblastoma (GBM) is the most lethal brain tumor with a median survival rate of approximately 14 months. GBM patients commonly suffer from tumor recurrence, indicating that populations of chemo/radio-resistant stem cell-like tumor cells survive treatments. Here we reveal that the neuronal stem cell marker Musashi1 (MSI1) is highly expressed in primary GBM and recurrences. We identify a novel regulatory role of MSI1 in GBM-derived cell lines and patient-derived tumorspheres, the enhancement of stemness marker expression, here demonstrated for CD44. Furthermore, we provide a rationale for MSI1-centered therapeutic targeting strategies to improve treatment options of this chemo/radio-resistant malignancy.The stem cell marker Musashi1 (MSI1) is highly expressed during neurogenesis and in glioblastoma (GBM). MSI1 promotes self-renewal and impairs differentiation in cancer and non-malignant progenitor cells. However, a comprehensive understanding of its role in promoting GBM-driving networks remains to be deciphered. We demonstrate that MSI1 is highly expressed in GBM recurrences, an oncologist’s major defiance. For the first time, we provide evidence that MSI1 promotes the expression of stem cell markers like CD44, co-expressed with MSI1 within recurrence-promoting cells at the migrating front of primary GBM samples. With GBM cell models of pediatric and adult origin, including isolated primary tumorspheres, we show that MSI1 promotes stem cell-like characteristics. Importantly, it impairs CD44 downregulation in a 3′UTR- and miRNA-dependent manner by controlling mRNA turnover. This regulation is disturbed by the previously reported MSI1 inhibitor luteolin, providing further evidence for a therapeutic target potential of MSI1 in GBM treatment.

A novel methylation signature predicts radiotherapy sensitivity in glioma

Glioblastoma (GBM) is the most common and malignant cancer of the central nervous system, and radiotherapy is widely applied in GBM treatment; however, the sensitivity to radiotherapy varies in different patients. To solve this clinical dilemma, a radiosensitivity prediction signature was constructed in the present study based on genomic methylation. In total, 1044 primary GBM samples with clinical and methylation microarray data were involved in this study. LASSO-COX, GSVA, Kaplan–Meier survival curve analysis, and COX regression were performed for the construction and verification of predictive models. The R programming language was used as the main tool for statistical analysis and graphical work. Via the integration analysis of methylation and the survival data of primary GBM, a novel prognostic and radiosensitivity prediction signature was constructed. This signature was found to be stable in prognosis prediction in the TCGA and CGGA databases. The possible mechanism was also explored, and it was found that this signature is closely related to DNA repair functions. Most importantly, this signature could predict whether GBM patients could benefit from radiotherapy. In summary, a radiosensitivity prediction signature for GBM patients based on five methylated probes was constructed, and presents great potential for clinical application.

High-throughput microRNA profile in adult and pediatric primary glioblastomas: the role of miR-10b-5p and miR-630 in the tumor aggressiveness

Glioblastoma (GBM) is the most common primary malignant neoplasm of the central nervous system and, despite the standard therapy; the patients' prognoses remain dismal. The miRNA expression profiles have been associated with patient prognosis, suggesting that they may be helpful for tumor diagnosis and classification as well as predictive of tumor response to treatment. We described the microRNA expression profile of 29 primary GBM samples (9 pediatric GBMs) and 11 non-neoplastic white matter samples as controls (WM) by microarray analysis and we performed functional in vitro assays on these 2 most differentially expressed miRNAs. Hierarchical clustering analysis showed 3 distinct miRNA profiles, two of them in the GBM samples and a group consisting only of cerebral white matter. When adult and pediatric GBMs were compared to WM, 37 human miRNAs were found to be differentially expressed, with miR-10b-5p being the most overexpressed and miR-630 the most underexpressed. The overexpression of miR-630 was associated with reduced cell proliferation and invasion in the U87 GBM cell line, whereas the inhibition of miR-10b-5p reduced cell proliferation and colony formation in the U251 GBM cell line, suggesting that these miRNAs may act as tumor-suppressive and oncogenic miRNAs, respectively. The present study highlights the distinct epigenetic profiling of adult and pediatric GBMs and underscores the biological importance of mir-10b-5p and miR-630 for the pathobiology of these lethal tumors.

Abstract 6646: Comprehensive cyclic immunofluorescent analysis reveals new target candidates for CAR T cell based immunotherapy of glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a highly malignant, non-curative tumor of the primary central nervous system with dismal prognosis. Treatment options are limited and conventional therapies, including surgery, radiotherapy, chemotherapy have not resulted in major improvements in the survival of patients. The advent of chimeric antigen receptor (CAR) T cell technology has opened new perspectives in the fight against cancer. Therefore, cell surface proteins that are suitable as targets for CAR T cells need to be identified. When considering a certain protein as a target for immunotherapy, two aspects have to be taken into account: Coverage of tumor cells as a factor for efficacy and the expression in healthy tissues under physiological conditions. The latter represents a potential threat, due to on-target/off-tumor toxicity. The novel MACSima™ Imaging Platform enables high-content, cyclic fluorescence-based imaging of individual biological samples in a fully automated fashion. We applied this method to subclassify different glioblastoma samples, to identify new glioblastoma marker and to analyze the toxicity profile of these markers. A selection of 96 markers, identified through flow cytometric analysis of cell surface expression on primary glioblastoma-derived xenografts, was used for characterization of diverse primary glioblastoma samples using the MACSima™ Imaging Platform. Cryosections were fixed using acetone, and each specimen was exposed to the 96 fluorochrome-conjugated antibodies by cycles of i) staining with antibody, ii) image acquisition, and iii) erasure of the fluorescence signal. Previously published markers, such as PDGFRα, p53, synaptophysin, CD44, nestin, podoplanin, GFAP, and EGFR were used to subclassify the glioblastoma into oligodendrocyte precursor (OPC), differentiated oligodendrocyte (DOC), astrocytic and mesenchymal (AsMes) or mixed subtype, according to Motomura et al.1 Analysis of well-established glioblastoma markers already used in CAR T cell-based clinical trials, such as IL-13Rα22, EGFRvIII3, or ErbB24 revealed a broad inter- and intratumor diversity of expression. Segmentation and correlation analysis enabled the selective detection of tumor cells and the identification of new glioblastoma specific markers, which might be candidates for future CAR T cell based therapies. To analyze the risk of “on-target/off-tumor” toxicity, differential expression of these markers on tumor tissue versus healthy tissue was determined. A higher expression level was detected on tumor tissue in comparison to healthy tissue in case of some candidates, underlining the capability of these markers for future CAR T cell-based therapies. Citation Format: Sandy Reiß, Stefan Tomiuk, Jutta Kollet, Jan Drewes, Melanie Jungblut, Andreas Bosio. Comprehensive cyclic immunofluorescent analysis reveals new target candidates for CAR T cell based immunotherapy of glioblastoma multiforme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6646.

Long Noncoding RNA SChLAP1 Forms a Growth-Promoting Complex with HNRNPL in Human Glioblastoma through Stabilization of ACTN4 and Activation of NF-κB Signaling.

Long noncoding RNAs (lncRNA) have essential roles in diverse cellular processes, both in normal and diseased cell types, and thus have emerged as potential therapeutic targets. A specific member of this family, the SWI/SNF complex antagonist associated with prostate cancer 1 (SChLAP1), has been shown to promote aggressive prostate cancer growth by antagonizing the SWI/SNF complex and therefore serves as a biomarker for poor prognosis. Here, we investigated whether SChLAP1 plays a potential role in the development of human glioblastoma (GBM). RNA-ISH and IHC were performed on a tissue microarray to assess expression of SChLAP1 and associated proteins in human gliomas. Proteins complexed with SChLAP1 were identified using RNA pull-down and mass spectrometry. Lentiviral constructs were used for functional analysis in vitro and in vivo. SChLAP1 was increased in primary GBM samples and cell lines, and knockdown of the lncRNA suppressed growth. SChLAP1 was found to bind heterogeneous nuclear ribonucleoprotein L (HNRNPL), which stabilized the lncRNA and led to an enhanced interaction with the protein actinin alpha 4 (ACTN4). ACTN4 was also highly expressed in primary GBM samples and was associated with poorer overall survival in glioma patients. The SChLAP1-HNRNPL complex led to stabilization of ACTN4 through suppression of proteasomal degradation, which resulted in increased nuclear localization of the p65 subunit of NF-κB and activation of NF-κB signaling, a pathway associated with cancer development. Our results implicated SChLAP1 as a driver of GBM growth as well as a potential therapeutic target in treatment of the disease.

OS6.2 Loss of CYP46A1 directs altered cholesterol homeostasis and opens therapeutic opportunities for glioblastoma

Abstract BACKGROUND Dysregulated cholesterol metabolism is a hallmark of many cancers, including glioblastoma (GBM), but its role in disease progression is not well understood. Here, we identified cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for elimination of cholesterol through conversion of cholesterol to 24(S)-hydroxycholesterol (24OHC), as one of the most dramatically dysregulated cholesterol metabolism genes in GBM. MATERIAL AND METHODS Molecular and clinical data was obtained from publicly genomic databases. Immunohistochemistry was applied to assess protein levels of CYP46A1 in primary GBM samples. Lentiviral constructs expressing CYP46A1 were transduced into LN229, LN18 and primary GBM GSCs for functional assays carried out in vitro and in vivo in an orthotopic xenograft model. RNA-seq was performed to identify downstream targets of 24OHC. RESULTS CYP46A1 was significantly decreased in GBM samples compared to normal brain tissue. Reduced CYP46A1 expression was associated with increasing tumour grade and poor prognosis in GBM patients. Ectopic expression of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. Treatment of GBM cells with 24OHC suppressed tumour growth through regulation of LXR and SREBP signalling. Efavirenz (EFV), an activator of CYP46A1 with BBB penetration, inhibited GBM growth in vivo. CONCLUSION Our findings demonstrate that CYP46A1 is a critical regulator of cellular cholesterol in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target.