Recurrent Glioblastoma Multiforme Research Articles

Advanced tumor electric fields therapy: A review of innovative research and development and prospect of application in glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive malignant tumor with a high mortality rate and is the most prevalent primary intracranial tumor that remains incurable. The current standard treatment, which involves surgery along with concurrent radiotherapy and chemotherapy, only yields a survival time of 14-16 months. However, the introduction of tumor electric fields therapy (TEFT) has provided a glimmer of hope for patients with newly diagnosed and recurrent GBM, as it has been shown to extend the median survival time to 20 months. The combination of TEFT and other advanced therapies is a promising trend in the field of GBM, facilitated by advancements in medical technology. In this review, we provide a concise overview of the mechanism and efficacy of TEFT. In addition, we mainly discussed the innovation of TEFT and our proposed blueprint for TEFT implementation. Tumor electric fields therapy is an effective and highly promising treatment modality for GBM. The full therapeutic potential of TEFT can be exploited by combined with other innovative technologies and treatments.

383 Epigenetic Potentiation of Anti-tumor Immune Responses Using Viral Mimicry for Gliomas

INTRODUCTION: Human endogenous retroviruses (HERVs) account for approximately 8% of our genome and are evolutionary relics of a germline retroviral infection. Although normally silenced by a master epigenetic regulator, np220, HERVs may be re-expressed in cellular states with marked epigenetic dysregulation and induce innate anti-viral inflammatory responses. This viral mimicry immune response sensitizes tumors to immunotherapy by eliciting double-stranded RNAs (dsRNA)-mediated interferon signaling cascades. Hence, viral mimicry could serve as a predictor of clinical responses to immunotherapy. METHODS: We conducted a preclinical study on the role of np220 in GBM and mediating viral mimicry responses. RESULTS: Using a tailored bulk transcriptional dataset from 71 newly diagnosed high-grade GBMs, we demonstrated significantly negative correlations between np220 expression and 48 HERV superfamilies. Additionally, using a transcriptional deconvolution pipeline of low-and high-grade gliomas, we demonstrated that np220 is negatively correlated with innate antiviral immune response signatures (TLR3) (RTLR3 = -0.300, pTLR3 = 0.00006). Our in vitro studies have shown that NP220 knockdown induces intracellular HERV-mediated dsRNA-signaling cascades via RIG-1 and TLR3. NP220 knockdown exponentially increases PD-L1 expression (p-value: <0.001), and, increases cytoplasmic accumulation of cellular endogenous dsRNA in established human and mouse glioma cell lines which may induce the interferon-mediated response. This response was recapitulated in recurrent GBM samples with radiographic responses to checkpoint inhibition (anti-PD1) with widely increased expression of dsRNA, PD-L1 and perivascular CD8 cell infiltration, suggesting dsRNA-signaling may mediate response to immunotherapy. Furthermore, we showed np220 is linked to reduced immune cell infiltration (CD8, Dendritic Cells) in the glioma microenvironment through deconvolution of RNA-seq data from TCGA (RCD8 = -.202, RDC = -0.198) (pCD8 = 8.79E-06, pDC = 1.34E-05). CONCLUSIONS: Our findings suggest that np220 could serve as a target to potentiate glioma ICI for immune response.

Adjuvant re-irradiation vs. no early re-irradiation of resected recurrent glioblastoma: pooled comparative cohort analysis from two tertiary centers

BackgroundThe optimal management strategy for recurrent glioblastoma (rGBM) remains uncertain, and the impact of re-irradiation (Re-RT) on overall survival (OS) is still a matter of debate. This study included patients who achieved gross total resection (GTR) after a second surgery after recurrence, following the GlioCave criteria.MethodsInclusion criteria include being 18 years or older, having histologically confirmed locally recurrent IDHwt or IDH unknown GBM, achieving MRI-proven GTR after the second surgery, having a Karnofsky performance status of at least 60% after the second surgery, having a minimum interval of 6 months between the first radiotherapy and the second surgery, and a maximum of 8 weeks from second surgery to the start of Re-RT.ResultsA total of 44 patients have met the inclusion criteria. The median OS after the second surgery was 14 months. All patients underwent standard treatment after initial diagnosis, including maximum safe resection, adjuvant radiochemotherapy and adjuvant chemotherapy. Re-RT did not significantly impact OS. However, MGMT promoter methylation status and a longer interval (> 12 months) between treatments were associated with better OS. Multivariate analysis revealed the MGMT status as the only significant predictor of OS.ConclusionFactors such as MGMT promoter methylation status and treatment interval play crucial roles in determining patient outcomes after second surgery. Personalized treatment strategies should consider these factors to optimize the management of rGBM. Prospective research is needed to define the value of re-RT after second surgery and to inform decision making in this situation.

Abstract 4638: Integrated proteogenomic characterization of longitudinal glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor, with no curative treatment options and median patient survival time of almost one year. Despite multi-modal therapy including surgery, irradiation and chemotherapy, all patients experience tumor progression and recurrence. Our group proposed and validated the first single cell guided functional classification of primary GBM in four tumor-intrinsic cell states which informed clinical outcome and delivered therapeutic options. However, recurrent GBM remains therapeutically unresolved due in part to the diffusely invasive nature and in part to marked cellular heterogeneity of the tumor.The evolutionary trajectory of glioblastoma after therapy is a multifaceted biological process that extends beyond discrete genetic alterations alone. Here, we profiled by multi-omics platforms the largest dataset of matched primary and recurrent GBM including 123 longitudinal glioblastoma pairs, temporally separated by standard-of-care treatment. Genomics, proteomics, and phosphoproteomics all independently captured the loss of proliferative-progenitor characteristics and a significant upregulation of specialized neuronal and synaptic signaling programs in recurrent GBM. Proteomic and phosphoproteomic analyses revealed that the molecular transition from proliferative to neuronal state at recurrence is marked by coherent post-translational activation of the WNT/PCP signaling pathway and the BRAF protein kinase. Multi-omic analysis of Patient-Derived Xenograft (PDX) models mimicked the patterns of evolutionary trajectory, consisting of marked activation of neuronal signaling programs in recurrent patients. Inhibition of the BRAF kinase with small molecule inhibitors impaired both neuronal transition and migration capability of recurrent glioblastoma cells, which are the phenotypic hallmarks of glioblastoma progression after therapy. Accordingly, combinatorial treatment of temozolomide with BRAF inhibitor, vemurafenib, significantly prolonged the survival of mouse PDX models. This work provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance and highlights new therapeutic opportunities to effectively counter them in the clinic. Citation Format: Simona Migliozzi, Kyung-Hee Kim, Harim Koo, Jun-Hee Hong, Seung Min Park, Hyung Joon Kwon, Luciano Garofano, Young Taek Oh, Fulvio D'Angelo, Chan Il Kim, Anna-Luisa Di Stefano, Franck Bielle, Jinlong Yin, Marc Sanson, Do-Hyun Nam, Jason K. Sa, Anna Lasorella, Jong Bae Park, Antonio Iavarone. Integrated proteogenomic characterization of longitudinal glioblastoma [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 4638.

Abstract 2781: GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer, with incredibly high recurrence rates and virtually no long-term survival. Despite our growing understanding of GBM at the molecular level, we still don’t fully comprehend the molecular mechanisms driving recurrence in GBM. Current knowledge indicates that Glioma Stem Cells (GSCs) drive a resistant cell phenotype in GBM. Understanding the molecular events coordinated by GSCs leading to therapy-resistance can provide key advancements on how we treat recurrent GBM (rGBM). Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. The cross talk between cancer cells and the immune system is now classified as a hallmark of cancer. The current dogma is that cancer cells, including GSCs, influence recruitment of immune-suppressive cell subsets to tumor tissue, however how subsets of GSCs mimic this immune-suppressive effect within the tumor microenvironment remains to be elucidated. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3− cell subpopulation with high expression of TGFβ1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with regulatory T cell (Tregs) and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with TGFβ type II receptor (TGFBR2) expression in clinical GBM. Mechanistically, we show that blocking TGFBR2 or XBP1 signaling, key intermediary of this process, counteracts the immune-suppressive phenotype of rGBM cells by restoring CD4 and CD8 tumor killing capacity and reversing exhaustion in co-culture experiments. siRNA-mediated knock-down of TGFBR2 in rGBM cells also decreased CD206 expression and reduced release of anti-inflammatory chemokines in THP1 cells. By combining miRNA-based network analysis and advanced nanoparticle formulation for miRNA delivery we show that miR-16/124-3p effectively target all nodes of this immunosuppressive axis successfully blocking the immunosuppressive nature of rGBM cells. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM [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 2781.

Abstract 387: Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to its current standard-of-care regimen, which includes surgical resection followed by adjuvant ionizing radiation and temozolomide. Therapy resistance can be attributed to the abundance of genomic alterations in GBM tumors that evolve over time, which contribute to intra-and inter-tumoral heterogeneities that render targeted therapies as inadequate treatment options. Thus, there is an urgent unmet need for effective therapies targeting primary and recurrent GBM. Here, we investigated the impact of ionizing radiation on the transcriptome of patient derived GBM tumor spheres to identify key alterations and biological pathways involved in therapy resistance. Materials and methods: We acquired four independent human GBM tumor sphere cell lines, which were derived from patients and cultured in serum-free media. Tumor spheres were treated with either a sham radiation or a single dose of 10Gy. Using poly-A enrichment whole transcriptome sequencing, we evaluated transcriptional alterations occurring in GBM tumors spheres at 96h post-radiation. To do so, we performed differential expression analysis and gene set enrichment analysis (GSEA) to identify top differentially expressed genes and significant changes in biological phenomena. Additionally, we evaluated the cell viability response of these GBM tumor spheres to radiation. Results and discussion: PCA analysis of the four human GBM tumor sphere cell lines demonstrates that cell type has a substantial impact on variation among samples compared to non-irradiated and irradiated treatment conditions. Upon analysis of genes in common that are either upregulated or downregulated followed by GSEA, we identified enrichment of genes associated with inflammatory responses and ferroptosis repression (e.g., NUPR1, PTGS1, AOX1), and depletion of genes involved in fatty acid metabolism (e.g., INSIG1, PLA2G3, ACAT2). Furthermore, the GBM tumor sphere cell viability responses allowed us to stratify our models into radiosensitive and radioresistant subgroups. Conclusion: Radiosensitive human GBM tumor spheres exhibit transcriptional alterations in genes linked to inflammation and fatty acid metabolism to a greater extent compared to radioresistant tumor spheres. Our study characterizes the radiation response of patient derived GBM models, which is to be leveraged for combating therapeutic resistance. Citation Format: Arianna Richelle Izawa-Ishiguro, Subhiksha Nandakumar, Nicholas Carbone, Annalisa V. Ferrotta, Raashed Raziuddin, Kristen C. Vogt, Daniel A. Heller, Ingo K. Mellinghoff. Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme [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 387.

Abstract 5496: Multi-omic spatial analysis of the tumor microenvironment in gliomas

Abstract Introduction: The diverse tumor environment of high-grade glioma, which remains refractory to treatment, demands new innovative, multi-omic approaches to characterize tumor heterogeneity and expression profiles associated with progression and therapeutic response. Approach: We have applied an integrated, multi-omic approach using paired spatial in situ sequencing (Xenium) and protein profiling (PhenoCycler® Fusion) of glioma tissue to identify distinct glioma cellular phenotypes, activation states and metabolic pathways at true single cell resolution. Our multi-omic approach used hundreds of phenotypic RNA target probes and >50 antibodies in an unbiased single-cell analysis of primary, recurrent, and IDH1 mutant/wild type tumors with a range of heterogeneity. Summary: We were able to distinguish differentially regulated genes and pathways between aggregated primary and recurrent GBM, including cell cycle pathways being upregulated in primary vs recurrent GBM glial cell clusters and a down regulation of ERBB4 signaling in vascular cell clusters in primary GBMs. Our data also suggests that SOX11, known to be involved in tumorigenesis, is >2-fold upregulated in annotated tumor cells in primary compared to recurrent tumors. We were also able to quantitatively localize the expression of Epidermal Growth Factor Receptor, variant III (EGFRvIII) to specific tumor-cell sub-types, which is important given its association with therapeutic resistance. Further, we have been able to establish the cellular neighborhoods and cell-cell and receptor-ligand relationships within the tumors. Our spatial protein analysis identified distinct tumor and immune phenotypes with varying abundance of myeloid and lymphoid populations in IDH1 wt and mt tumors. Moreover, spatial proximity analyses and cellular neighborhood analyses revealed differences in higher order organizational landscapes that may contribute to the differential outcomes across wt and mt tumor subtypes. Conclusion: Multiomic spatial analysis enables deeper characterization of the glioma cellular and functional landscape to broaden our understanding of the key TME features that contribute to disease pathogenesis and prognoses. Our study provides an analytical framework to combine RNA and protein-based spatial data for a holistic investigation into a variety of glioma subtypes and aid in the identification of novel biomarkers, spatial neighborhoods, and functional states that drive glioma progression. Citation Format: Dmytro Klymyshyn, Vaibhav Jain, Lauren Whaley, Emily Hocke, Bassem Ben Cheikh, Alan Smith, Karen Abramson, Nadine Nelson, Diane Satterfield, Elizabeth Thomas, Giselle Lopez, Seetha Hariharan, Michael Brown, Niyati Jhaveri, Roger McLendon, David Ashley, Matthew Waitkus, Simon Gregory. Multi-omic spatial analysis of the tumor microenvironment in gliomas [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 5496.

Abstract 5238: uPAR as a novel immunotherapeutic target in recurrent glioblastoma

Abstract Glioblastoma (GBM) is the common malignant brain tumor in adults, accounting for approximately 15% of all CNS tumors, and 48.6% of malignant brain tumors, with a median survival of approximately 15 months, and minimal clinical progress having been made in the past two decades. GBM is characterized by extensive inter- and intra-tumoral heterogeneity as well as an extremely immunosuppressive tumor microenvironment. Following Standard-of-Care (SoC) surgical resection and chemoradiotherapy at primary diagnosis, few therapeutic avenues exist at recurrence, owing in part due to a lack of clinically relevant targets. Data from our multi-institutional target pipeline shows that the extracellular urokinase plasminogen activator receptor (uPAR) is significantly upregulated at recurrence on putative GBM brain tumor initiating cells (BTICs), which are believed to drive de novo tumor formation, tumor recurrence, and therapeutic resistance. uPAR plays an important role in the plasminogen activation system, and in the context of cancer, has been implicated in numerous pro-tumorigenic processes such as invasion, proliferation, epithelial-to-mesenchymal transition, and therapy resistance. We used CRISPR-Cas9 to genetically delete uPAR from our in-house patient-derived GBM cell lines, and found that in vitro, knockout of uPAR expression in recurrent GBM cells significantly reduces proliferation and sphere formation capacity, two stem-like traits associated with BTICs. Additionally, we found that uPAR knockout significantly reduced the frequency of BTICs in a given population. Additionally, uPAR knockout cells display increased sensitivity to standard-of-care chemoradiotherapy, implicating uPAR expression in therapy resistance, as seen in recurrent disease. We orthotopically injected immunocompromised mice to generate patient-derived xenograft models of GBM, and found that knockout of uPAR significantly increases survival, and decreases tumor burden. Further, we generated novel anti-uPAR single domain antibodies (sdAbs) and found that they specifically bind uPAR at low nanomolar concentrations in vitro. Using these sdAbs, we created an anti-uPAR CAR T which showed potent cytotoxicity in vitro, and drastically reduced tumor burden and extended survival in vivo. From this work we believe uPAR to be a clinically relevant target in recurrent GBM, and further investigation into therapeutic strategies to target uPAR-positive GBMs should be investigated further. Citation Format: William T. Maich, Muhammad Vaseem Shaikh, Anish Puri, Alisha Anand, Chirayu Chokshi, Sabra Salim, Neil Savage, Chitra Venugopal, Martin Rossotti, Thomas Kislinger, Kevin Henry, Sheila Singh. uPAR as a novel immunotherapeutic target in recurrent glioblastoma [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 5238.

Abstract 1151: Reconstruction of the spatial ecosystem of glioblastoma reveals relationships between tumor cell states and microenvironment

Abstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor, with no curative treatment options. Multiple studies have characterized at single cell resolution the GBM as being composed of transcriptional cell states interconnected with components in the tumor immune microenvironment (TME). Our group proposed and validated the first single cell guided functional classification of GBM in four tumor-intrinsic cell states which informed clinical outcome and delivered therapeutic options. However, single cell technologies are unable to unravel the spatial relationships among the cell states of GBM and between GBM cell states and TME. Spatially resolved transcriptomic technologies are emerging as powerful tools to reconstruct the spatial architecture of a tissue. We performed spatial transcriptomics of multicellular regions of interest (ROI) in 6 primary IDH wild-type GBM and 2 recurrent GBM with both CosMx Spatial Molecular Imager, which analyzes 1,000 RNA probes and 64 proteins at single cell resolution, and GeoMx Digital Spatial Profiler which profiles the whole transcriptome (~18,000 genes) at ROI resolution. The development of computational tools aimed to integrate spatial proximity and CosMx derived single-cell transcriptomics revealed spatial segregation of the tumor cell clones and cellular states and highlighted recurrent patterns of cell states, distinct TME cell types associated with coherent histopathological features across multiple samples. The development of a spatial informed intercellular communication algorithm and the reconstruction of ligand-receptor-target networks will allow the discovery of tumor cell states-TME cross-talks and the biological signaling regulated by these interactions that are driving the heterogeneity of GBM and therefore potentially therapeutically targetable. Analysis of matched regions of interest profiled by GeoMx and spatial proteomics with CosMx further cross-validated the spatial ecosystem of glioblastoma as reconstructed at single-cell resolution. Our studies established a scalable approach to resolve the transcriptional heterogeneity of GBM and reconstruct the architecture of GBM cell states and tumor microenvironment. Citation Format: Bruno Adabbo, Simona Migliozzi, Luciano Garofano, Fulvio D'Angelo, Pedro Davila, Sakir H. Gultekin, Daniel Bilbao Cortes, Benjamin Currall, Sion L. Williams, Marc Sanson, Franck Bielle, Anna Luisa Di Stefano, Michele Ceccarelli, Anna Lasorella, Antonio Iavarone. Reconstruction of the spatial ecosystem of glioblastoma reveals relationships between tumor cell states and microenvironment [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 1151.

Abstract 458: Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer. Despite aggressive therapy consisting of surgery followed by radio/chemotherapy GBM recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. We have recently developed PBAE nanoparticle formulations optimized for in vivo miRNA delivery to brain tumor stem cells, or GSCs. Carefully selecting one miRNA to target several mRNAs dysregulated within a pathway can allow the targeting of multiple nodes using a single agent, mimicking combinatorial therapies. To test this hypothesis, we performed bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens combined with gene set enrichment analysis (GSEA) and identified TGFBR2 signaling as a targetable pathway in rGBM. We show that SMAD2 signaling, a downstream effector of TGFBR2, is enriched in rGBM and that patients with elevated TGFBR2 transcripts respond poorly to current standard of care. Utilizing patient-derived xenograft lines from rGBM, we show that blocking TGFBR2 signaling using siRNAs or ITD1, a selective TGFBR2 inhibitor, decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM cells to temozolomide (TMZ) in-vitro. In vivo, we show that ITD1 is modestly effective in subcutaneous and ineffective in orthotopic rGBM models. Not surprisingly, these tumors exhibited classical growth patterns associated with acquisition of resistance. To overcome this obstacle, we performed a miRNA-based network analysis in rGBM and identified miR-590-3p as a putative tumor suppressor that targets SMAD2 signaling at multiple nodes in rGBM. In-vitro, transgenic miR-590-3p robustly decreased the expression of 37 putative oncogenes simultaneously concurrent with decreasing stem cell capacity, cell viability and re-sensitizing rGBM PDX cells to TMZ. In vivo delivery of miR-590-3p to established PDX-derived rGBM orthotopic tumors using novel PBAE nanocarriers robustly decreased tumor sizes and increased tumor necrosis. These results show that the promiscuous nature of miRNAs, in combination with cutting-edge nucleic acid delivery vehicles, can be leveraged as advanced targeted therapeutics for rGBM bypasses the resistance that is developed to standard of care and available small molecules. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes [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 458.

Abstract 5611: Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications

Abstract Glioblastoma (GBM), a highly aggressive brain cancer, poses a formidable challenge marked by its resistance to conventional therapies. F-actin, a crucial cytoskeletal protein, influences DNA repair and impacts genomic stability. F-actin dynamics also shape the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Altered F-actin organization contributes to GBM resistance to conventional therapies. Understanding F-actin's multifaceted role is vital for developing targeted strategies to overcome treatment resistance and enhance outcomes for GBM patients. This study goes deeper into the intricate interplay between the F-actin cytoskeleton and the acquisition of resistance in GBM cells. Our primary goal was to explore the effects of pharmacological or genetic F-actin depolymerization on the reversal of acquired resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM cells, particularly focusing on the underlying mechanisms involving DNA repair, the glioma stem cell (GSC) phenotype, and therapeutic implications. To establish resistant sublines, GBM U87-MG cells underwent cycles of IR and TMZ exposure, revealing a significant increase in IC50 values for TMZ and ID50 values for IR through viability assays. Subsequent characterization of these sublines growth was in both adherent and spheroid cultures uncovering a distinct sphere-forming capacity. Notably, alterations in DNA repair and the Rho GTPase pathway were observed, with evident disorganized F-actin polymerization in resistant cells, especially at the periphery of spheroids. Elevated expression of stemness markers (Nestin, CD133, and CD44) highlighted a GSC-like profile in the resistant cells, a characteristic further accentuated in spheroid cultures. To assess the relevance of the actin cytoskeleton in maintaining the resistant phenotype, cells were treated with Rho-GTPase inhibitor (C3) or actin polymerization inhibitor (Cytochalasin D). Both treatments resulted in reduced TMZ IC50 and IR ID50 values, partially attributed to the negative modulation of DNA repair capacity - observed by alkaline comet and host-cell reactivation assays. Additionally, F-actin disassembly was found to impact the expression of stemness markers, further reinforcing the connection between cytoskeletal dynamics and the GSC phenotype. Overall, our data strongly suggest that F-actin cytoskeleton dynamics plays a pivotal role in the development and maintenance of resistance in GBM. Targeting these cytoskeletal components sees a promising pharmacological strategy for resensitizing recurrent and resistant GBM tumors, not only by diminishing DNA repair capabilities but also by influencing the GSC-like phenotype, thus opening new avenues for therapeutic interventions. Citation Format: Yuli Thamires Magalhães, Fábio Luís Forti. Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications [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 5611.

Abstract 7100: Quantitative proteomics reveal the mechanism of TTFields therapy for glioblastoma

Abstract Glioblastoma (GBM) is a highly malignant brain tumor, and conventional treatments such as surgical resection and chemotherapy have limited effectiveness, with a high rate of recurrence and poor prognosis for patients. In 2011, the FDA approved a novel therapy for recurrent GBM known as Tumor Treating Fields (TTFields). TTFields use alternating intermediate-frequency (200 kHz) and low-intensity (1-3 V/cm) electric fields to interfere with tumor cells, inducing apoptosis and inhibiting tumor growth. This study aimed to explore the molecular mechanisms of TTFields therapy at the systems biology level using quantitative proteomics technology. We analyzed U87 cells before and after TTFields treatment using quantitative proteomics and identified a total of 7679 proteins. Among them, 804 proteins were significantly upregulated after TTFields treatment, and 1141 proteins were significantly downregulated. Pathway enrichment analysis of differentially expressed proteins showed that upregulated proteins were mainly associated with cell proliferation, cell cycle, and transcription, possibly representing compensatory responses to the TTFields treatment. Further analysis revealed a significant upregulation of the tumor proliferation key protein PARP1, which was validated by protein immunoblotting experiments. Based on this analysis, we hypothesized that combining TTFields with Olaparib (a PARP1 inhibitor) might enhance the sensitivity of TTFields. Subsequently, we performed cell proliferation experiments to evaluate the concomitant application strategy, and the results demonstrated that Olaparib in combination with TTFields treatment significantly reduced the survival rate of U87 cells compared to single-drug treatment. In conclusion, using quantitative proteomics, this study revealed the biological processes underlying the inhibitory effects of TTFields on GBM proliferation. Under TTFields intervention, the expression of PARP1 was upregulated, and the use of a PARP1 inhibitor in combination significantly increased the sensitivity of TTFields. Therefore, this study not only elucidated the molecular mechanisms of TTFields therapy for GBM treatment at the systems biology level, but also provided support for further enhancing the efficacy of TTFields. Citation Format: Qi Mei, Yanmei Gao, Rong Li, Chenxu Wang, Guangyuan Hu, He Huang. Quantitative proteomics reveal the mechanism of TTFields therapy for glioblastoma [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 7100.

Abstract 2917: Purifying cancer-associated fibroblasts (CAFs) from glioblastoma and defining their role in therapeutic resistance

Abstract Glioblastoma (GBM), a challenging brain tumor, necessitates the development of new therapies due to poor patient prognoses, with a median survival of 14-16 months. Standard of care, as established almost 20 years ago, remains to be surgical resection followed by chemotherapy and radiation and till date there is no defined standard of care for recurrent GBM, highlighting the urgent need for the development of novel therapies. Recent studies have implicated the tumor microenvironment in these poor prognoses. While cancer associated fibroblasts (CAFs) are a key cellular component of the microenvironment of many systemic tumors, their presence in GBM had been doubted until we recently developed intriguing data identifying cells in GBM that express fibroblast markers, have fibroblast-like morphology, and, most importantly, transcriptomically resemble CAFs from systemic cancers rather than other stromal cells that express similar surface markers such as pericytes (Jain et al. 2023). Similar to other solid cancers, GBM CAFs increase tumor growth and drive immunosuppression. The role of CAFs in regulating these processes could have significant implications in response to standard of care therapy as well as novel immunotherapies. While our initial CAF isolation had just 50% purity, we have developed a novel flow-sorting algorithm for CAF enrichment driven by our bioinformatic data. Through this protocol, we have successfully established, the first of its kind, a biobank of patient derived GBM CAFs with greater than 90% purity. These CAFs are now being utilized to investigate their contribution in conferring resistance to chemotherapy (Temozolomide) and radiotherapy. The CAFs are also being assessed for their role in suppressing T cell function and resistance to immunotherapies. In parallel, the origin of GBM CAFs and their development trajectory is being analyzed to find molecular markers to specifically target CAFs in the GBM microenvironment. The work put together will provide insight on mechanisms employed by CAFs in altering the GBM microenvironment and a basis for developing novel combination approaches, alleviating therapeutic resistance caused due to the presence of CAFs in the GBM microenvironment. The results of this study have the potential to significantly attenuate therapeutic resistance in GBM and provide much needed treatment options and improvement in survival, for what remains one of the most dismal diagnoses in the clinic. Citation Format: Ankita Sati, Aakriti Mathur, Meeki Lad, Atul Saha, Manish K. Aghi. Purifying cancer-associated fibroblasts (CAFs) from glioblastoma and defining their role in therapeutic resistance [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 2917.

Abstract A050: Ferritin, associated with worse overall survival in recurrent GBM, is reduced by combination of HDACi, ONC206 and radiation

Abstract Background: GBM is the most common primary brain malignancy with dismal prognosis. Current standard of care is chemoradiation&surgery. The main issue with GBM is the resistance & recurrence. GBM can have high Ferritin levels associated with disease progression & immunosuppression in the GBM TME. Ferritin light chain (FTL), a key protein in iron metabolism, is mutated in GBM leading to high ferritin levels (both light& heavy chain) & is associated with poor survival of (GBM) patients in part due to inhibition of ferroptosis. Panobinostat is a histone deacetylase inhibitor with antineoplastic & antiangiogenic effects in glioma. Imipridone ONC206 is under Phase I clinical development in treatment of adult & pediatric patients with newly diagnosed and/or recurrent primary brain tumors (NCT04732065 and NCT04541082).We investigated the novel combination of irradiation, Panobinostat and ONC206 with regard to GBM cell line sensitivity&ferritin levels. Materials & methods: We investigated cell viability and drug synergies of ONC206 plus Panobinostat with or without radiation in 4 human GBM cell lines at 72 hours. Changes in Ferritin level treatment in U87 & U251 cells were measured using cytokine profiling at 24 h time point. Information regarding recurrent cases of adult GBM from TCGA &GlioVis were used for survival data. Results: We previously have shown the synergy between Panobinostat, Irradiation and ONC206 (2023 AACR meeting). Treatment of U87 cells with Panobinostat and ONC206 at their IC50s +/- 4 Gy radiation at 24h time point led to reduction in secreted ferritin levels (Panobinostat alone: 26.3% decrease; ONC206 alone: 14.7% decrease; RT 4Gy alone 14.7% decrease; triple therapy: 32.2% decrease). In panobinostat treated U251 cells there was a 10% increase in ferritin at 24 h. ONC206 resulted in a reduction by 34.71%, 4 Gy RT caused a slight increase of 4% in ferritin amount while the triple showed 49.96% reduction in ferritin levels. mRNA expression of FTL gene (Ferritin Light Chain) is significantly higher in GBM tumors compared to normal brain tissue. From TCGA 17 cases of recurrent gliomas with FTL mutation were available with median survival of less than a year. In data from GlioVis of recurrent GBM cases 263 patients had high FTL with median survival of 12.6 months compared to 262 patients with low FTL with median survival of 14.9 months. Conclusions: GBM with high ferritin correlates with poor patient survival. FTL is associated with cell proliferation &angiogenesis of GBM cells & is protective from ferroptosis in glioma cells. ferritin may be a therapeutic target in recurrent & therapy resistant cases of GBM and ferroptosis modulating drugs &can be a novel treatment option. ONC206 & Panobinostat with irradiation is a novel combination & can be considered for temozolomide or radiation-resistant & recurrent cases. More studies are needed to investigate the mechanism & impact of ferritin in disease progression, therapy resistance& ferroptosis targeting therapeutics. We are currently exploring these directions including in vivo studies. Citation Format: Vida Tajiknia, Wafik El Deiry, Lanlan Zhou, William Macdonald, Praveen Srinivasan. Ferritin, associated with worse overall survival in recurrent GBM, is reduced by combination of HDACi, ONC206 and radiation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A050.

Abstract B001: Glioblastoma evolution and resistance is mediated by a shift in cell state and tumor microenvironmental interactions

Abstract Glioblastoma (GBM) is a lethal disease characterized by inevitable recurrence. Recently, single cell sequencing studies of primary GBM have defined various tumor cell states, specifically the neural progenitor-like (NPC-like), oligodendrocyte progenitor (OPC-like), astrocyte-like(AC-like), and mesenchymal(MES-like) states. Yet, the role these cell states play in treatment resistance and recurrence is not well defined. Furthermore, the spatial tumor microenvironmental (TME) context of tumor cells clearly plays a role in treatment response. Here we utilize in vivo recurrence models, and single cell and spatial transcriptomic (ST) analyses of primary and recurrent GBM to determine the pathways and spatial TME interactions mediating recurrence. Experimental Design: To define the evolution of the tumor landscape from primary to recurrence, we performed single cell RNA sequencing from stereotactically resected primary GBM (178K cells from 26 patients) and at recurrence (170K cells from 12 patients). ST analyses were also completed, as well as in vitro and in vivo mechanistic studies. Results: We identified a notable shift in the proportion of cell states in response to treatment from primary to recurrence with a significant decrease in AC-like cells (72% vs 55%, p<2e-16) and increase in MES-like cells (15% vs 34%, p<2e-16). This suggests that AC-like cells may be more treatment sensitive compared to other cell states, especially MES-like cells. We then worked to define potential TME interactions of these cell states to define drivers of recurrence. ST analyses revealed NPC-like cells in the infiltrative edge, AC-like cells in the tumor core, and MES-like cells in the hypoxic zones. We performed pseudotime trajectory analysis which identified that NPC-like cells divided into 2 daughter lineages: 1) AC-like cells which form the bulk of primary tumors and 2) MES-like cells that make up a large proportion of the recurrence. We then found that THY1, a cell surface receptor, was highly expressed in NPC-like cells in the primary tumors. While at recurrence, there was a remarkable increase in THY1 expression coincident with a switch to a predominantly MES-like state. Furthermore, ST analyses revealed upregulation of stem-like pathways in THY1 regions, as well as co-localization of MES-like THY1 cells with monocytes in hypoxic zones(p<0.001), suggesting an important role for TME interactions in mediating recurrence. To assess the biological implications of THY1, subsequent in vitro and in vivo studies revealed that THY1 knockout significantly decreased cell proliferation and tumor volume, and improved treatment response suggesting an important role for THY1 in driving treatment resistance and inevitable recurrence. Conclusions: Utilizing mechanistic and bioinformatic analyses, we show that a small population of NPC and MES-like THY1+ cells in primary GBM is likely to survive chemoradiation and mediate recurrence. Defining the interactions of these cell states in their TME and the mechanistic underpinnings of THY1 interactions may be critical to identifying new treatments. Citation Format: Sunita Shankar, Visweswaran Ravikumar, Arushi Tripathy, Sunjong Ji, Jackson Loper, Zainab Hassan, Ziad Fehmi, Yacoub Haydin, Dah-Luen Huang, Sagnik Bhadury, Reva Kulkarni, Jeffrey Regier, Daniel Wahl, Arvind Rao, Alnawaz Rehemtulla, Wajd N. Al-Holou. Glioblastoma evolution and resistance is mediated by a shift in cell state and tumor microenvironmental interactions [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B001.

Tumor-infiltrating CD8+ sub-populations in primary and recurrent glioblastoma: An in-silico study

BackgroundGlioblastoma multiforme (GBM) remains an incurable primary brain tumor. CD8+ tumor-infiltrating lymphocytes (TILs) can target malignant cells; however, their anti-tumoral immune responses mostly do not lead to GBM rejection in GBM patients. We profiled the sub-populations of tumor-infiltrating CD8+ T-cells, i.e., naïve, cytotoxic, and exhausted cells, in primary and recurrent GBM tissues and provided a blueprint for future precision-based GBM immunotherapy. MethodWe re-analyzed the raw data of single-cell RNA sequencing on the cells residing in the GBM microenvironment and leveraged tumor bulk RNA analyses to study the significance of CD8+ TILs sub-populations in primary and recurrent GBM. We investigated cell-cell interaction between exhausted CD8+ TILs and other immune cells residing in the primary and recurrent GBM microenvironments and profiled the expression changes following CD8+ TILs' transition from primary GBM to recurrent GBM. ResultsExhausted CD8+ TILs are the majority of CD8+ TILs sub-populations in primary and recurrent GBM, and cytotoxic CD8+ TILs display decreased expression of inhibitory immune checkpoint (IC) molecules in the primary and recurrent GBM. In the primary and recurrent GBM microenvironment, exhausted CD8+ TILs interact most with tumor-infiltrating dendritic cells. ConclusionThis study demonstrates the profiles of CD8+ TILs sub-populations in primary and recurrent GBM and provides a proof-of-concept for future precision-based GBM immunotherapy.

Brain Re-Irradiation Or Chemotherapy: a phase II randomised trial of re-irradiation and chemotherapy in patients with recurrent glioblastoma (BRIOChe) – protocol for a multi-centre open-label randomised trial

IntroductionGlioblastoma (GBM) is the most common adult primary malignant brain tumour. The condition is incurable and, despite aggressive treatment at first presentation, almost all tumours recur after a median of...

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

Practice Patterns for Managing Recurrent Glioblastoma Multiforme

Abstract Introduction Glioblastoma multiforme (GBM) is a devastating form of cancer with a poor prognosis despite available treatments. Managing recurrent GBM remains challenging and lacks guidelines. This study aims to provide practice patterns for managing recurrent GBMs in India. Methods A panel of experts was assembled to develop practice patterns using the Delphi technique. Their responses were analyzed anonymously to ensure impartiality and generate recommendations. The statements were intended to be nonbinding and focused on promoting best practices in the field, without legal or regulatory authority. Results A total of 23 experts participated in the study, providing their opinions on various aspects of managing recurrent GBM. Consensus was achieved on individualized and multidisciplinary management as the preferred approach. Surgery in combination with other treatments was found to impact survival in patients older than 65 years, with re-surgery and adjuvant radiation and chemotherapy being the preferred options. Gadolinium-enhanced magnetic resonance imaging (MRI) brain with spectroscopy and diffusion-weighted imaging was favored. Molecular profiling was considered significant, with O6-methylguanine DNA methyltransferase methylation being most relevant. Surgery was recommended for recurrent GBMs, primarily based on Karnofsky's performance score (KPS). Surgical adjuncts such as neuronavigation and intraoperative MRI were considered valuable. Radiation therapy, specifically stereotactic radiosurgery, was recommended for selected cases, while opinions on re-chemotherapy were divided. Palliative care was deemed important. Conclusion This study presents practice patterns for managing recurrent GBM in India, providing standardized recommendations for practice. By implementing these, clinicians can make informed decisions, leading to improved patient outcomes and reduced variability in the management of recurrent GBM.

Theranostic Uses of the Heme Pathway in Neuro-Oncology: Protoporphyrin IX (PpIX) and Its Journey from Photodynamic Therapy (PDT) through Photodynamic Diagnosis (PDD) to Sonodynamic Therapy (SDT).

ALA PDT, first approved as a topical therapy to treat precancerous skin lesions in 1999, targets the heme pathway selectively in cancers. When provided with excess ALA, the fluorescent photosensitizer PpIX accumulates primarily in cancer tissue, and ALA PDD is used to identify bladder and brain cancers as a visual aid for surgical resection. ALA PDT has shown promising anecdotal clinical results in recurrent glioblastoma multiforme. ALA SDT represents a noninvasive way to activate ALA PDT and has the potential to achieve clinical success in the treatment of both intracranial and extracranial cancers. This review describes the creation and evolution of ALA PDT, from the treatment of skin cancers to PDD and PDT of malignant brain tumors and, most recently, into a noninvasive form of PDT, ALA SDT. Current clinical trials of ALA SDT for recurrent glioblastoma and high-grade gliomas in adults, and the first pediatric ALA SDT clinical trial for a lethal brainstem cancer, diffuse intrinsic pontine glioma (DIPG), are also described.