Drug Resistance In Glioblastoma Research Articles

Rapid Biofabrication of an Advanced Microphysiological System Mimicking Phenotypical Heterogeneity and Drug Resistance in Glioblastoma.

Microphysiological systems (MPSs) reconstitute tissue interfaces and organ functions, presenting a promising alternative to animal models in drug development. However, traditional materials like polydimethylsiloxane (PDMS) often interfere by absorbing hydrophobic molecules, affecting drug testing accuracy. Additive manufacturing, including 3D bioprinting, offers viable solutions. GlioFlow3D, a novel microfluidic platform combining extrusion bioprinting and stereolithography (SLA) is introduced. GlioFlow3D integrates primary human cells and glioblastoma (GBM) lines in hydrogel-based microchannels mimicking vasculature, within an SLA resin framework using cost-effective materials. The study introduces a robust protocol to mitigate SLA resin cytotoxicity. Compared to PDMS, GlioFlow3D demonstrated lower small molecule absorption, which is relevant for accurate testing of small molecules like Temozolomide (TMZ). Computational modeling is used to optimize a pumpless setup simulating interstitial fluid flow dynamics in tissues. Co-culturing GBM with brain endothelial cells in GlioFlow3D showed enhanced CD133 expression and TMZ resistance near vascular interfaces, highlighting spatial drug resistance mechanisms. This PDMS-free platform promises advanced drug testing, improving preclinical research and personalized therapy by elucidating complex GBM drug resistance mechanisms influenced by the tissue microenvironment.

Acquired Temozolomide Resistance Instructs Patterns of Glioblastoma Behavior in Gelatin Hydrogels.

Acquired drug resistance in glioblastoma (GBM) presents a major clinical challenge and is a key factor contributing to abysmal prognosis, with less than 15 months median overall survival. Aggressive chemotherapy with the frontline therapeutic, temozolomide (TMZ), ultimately fails to kill residual highly invasive tumor cells after surgical resection and radiotherapy. Here, a 3D engineered model of acquired TMZ resistance is reported using two isogenically matched sets of GBM cell lines encapsulated in gelatin methacrylol hydrogels. Response of TMZ-resistant versus TMZ-sensitive GBM cell lines within the gelatin-based extracellular matrix platform is benchmarked and drug response at physiologically relevant TMZ concentrations is further validated. The changes in drug sensitivity, cell invasion, and matrix-remodeling cytokine production are shown as the result of acquired TMZ resistance. This platform lays the foundation for future investigations targeting key elements of the GBM tumor microenvironment to combat GBM's devastating impact by advancing the understanding of GBM progression and treatment response to guide the development of novel treatment strategies.

Targeting drug resistance in glioblastoma (Review).

Glioblastoma (GBM) is the most common malignancy of the central nervous system in adults. The current standard of care includes surgery, radiation therapy, temozolomide; and tumor‑treating fields leads to dismal overall survival. There are far limited treatments upon recurrence. Therapies to date are ineffective as a result of several factors, including the presence of the blood‑brain barrier, blood tumor barrier, glioma stem‑like cells and genetic heterogeneity in GBM. In the present review, the potential mechanisms that lead to treatment resistance in GBM and the measures which have been taken so far to attempt to overcome the resistance were discussed. The complex biology of GBM and lack of comprehensive understanding of the development of therapeutic resistance in GBM demands discovery of novel antigens that are targetable and provide effective therapeutic strategies.

Gene regulatory network topology governs resistance and treatment escape in glioma stem-like cells.

Poor prognosis and drug resistance in glioblastoma (GBM) can result from cellular heterogeneity and treatment-induced shifts in phenotypic states of tumor cells, including dedifferentiation into glioma stem-like cells (GSCs). This rare tumorigenic cell subpopulation resists temozolomide, undergoes proneural-to-mesenchymal transition (PMT) to evade therapy, and drives recurrence. Through inference of transcriptional regulatory networks (TRNs) of patient-derived GSCs (PD-GSCs) at single-cell resolution, we demonstrate how the topology of transcription factor interaction networks drives distinct trajectories of cell-state transitions in PD-GSCs resistant or susceptible to cytotoxic drug treatment. By experimentally testing predictions based on TRN simulations, we show that drug treatment drives surviving PD-GSCs along a trajectory of intermediate states, exposing vulnerability to potentiated killing by siRNA or a second drug targeting treatment-induced transcriptional programs governing nongenetic cell plasticity. Our findings demonstrate an approach to uncover TRN topology and use it to rationally predict combinatorial treatments that disrupt acquired resistance in GBM.

Emerging Insights into the PI3K/AKT/mTOR Signaling Pathway and Non-Coding RNA-mediated Drug Resistance in Glioblastoma.

Glioblastoma multiforme [GBM] is a highly aggressive grade IV central nervous system tumor with a dismal prognosis. Factors such as late detection, treatment limitations due to its aggressive nature, and, notably, drug resistance significantly affect clinical outcomes. Despite the effectiveness of Temozolomide [TMZ], a potent chemotherapy agent, the development of drug resistance remains a major challenge. Given the poor survival rates and chemoresistance, there is an urgent need for novel treatment strategies. Non-coding RNAs, particularly microRNAs [miRNAs], offer a promising approach to GBM diagnosis and treatment. These small non-coding RNAs play crucial roles in tumor progression, either suppressing or promoting oncogenic characteristics. The phosphoinositide-3 kinase [PI3K]/AKT/ mTOR pathway, which regulates essential biological processes like proliferation and survival, is a key target of miRNAs in cancer. Studies have underscored the significance of PI3K/AKT/mTOR signaling in drug resistance development and its interplay with non-coding RNAs as mediators of tumorigenesis. This review aims to outline the involvement of PI3K/AKT/mTOR signaling in miRNA modulation and strategies to overcome chemoresistance in GBM.

Glioblastoma stem cells deliver ABCB4 transcribed by ATF3 via exosomes conferring glioblastoma resistance to temozolomide

Glioblastoma stem cells (GSCs) play a key role in glioblastoma (GBM) resistance to temozolomide (TMZ) chemotherapy. With the increase in research on the tumour microenvironment, exosomes secreted by GSCs have become a new focus in GBM research. However, the molecular mechanism by which GSCs affect drug resistance in GBM cells via exosomes remains unclear. Using bioinformatics analysis, we identified the specific expression of ABCB4 in GSCs. Subsequently, we established GSC cell lines and used ultracentrifugation to extract secreted exosomes. We conducted in vitro and in vivo investigations to validate the promoting effect of ABCB4 and ABCB4-containing exosomes on TMZ resistance. Finally, to identify the transcription factors regulating the transcription of ABCB4, we performed luciferase assays and chromatin immunoprecipitation-quantitative PCR. Our results indicated that ABCB4 is highly expressed in GSCs. Moreover, high expression of ABCB4 promoted the resistance of GSCs to TMZ. Our study found that GSCs can also transmit their highly expressed ABCB4 to differentiated glioma cells (DGCs) through exosomes, leading to high expression of ABCB4 in these cells and promoting their resistance to TMZ. Mechanistic studies have shown that the overexpression of ABCB4 in GSCs is mediated by the transcription factor ATF3. In conclusion, our results indicate that GSCs can confer resistance to TMZ in GBM by transmitting ABCB4, which is transcribed by ATF3, through exosomes. This mechanism may lead to drug resistance and recurrence of GBM. These findings contribute to a deeper understanding of the mechanisms underlying drug resistance in GBM and provide novel insights into its treatment.

Abstract 6770: Acquired temozolomide resistance instructs patterns of glioblastoma cell motility and proteome expression within gelatin hydrogels

Abstract Acquired drug resistance in glioblastoma (GBM) presents a major clinical challenge and is one of the key factors contributing to the abysmal prognosis with less than 15 months median overall survival. The aggressive chemotherapy with the frontline therapeutic, temozolomide (TMZ), ultimately fails to kill residual, highly invasive tumor cells after surgical resection and radiotherapy. Here, we present a robust three-dimensional (3D) in vitro hydrogel model of acquired temozolomide (TMZ) resistance using isogenically-matched glioblastoma (GBM) cell lines. We demonstrate distinct drug response patterns between TMZ-sensitive and TMZ-resistant GBM cells, emphasizing our model's promise for future pre-clinical investigations aimed at developing novel therapies targeting drug-resistant GBM. Importantly, our model detects apoptosis-related protein changes in response to TMZ, validating its ability to study drug-induced molecular changes. Furthermore, our study delves into the migratory potential of GBM cells within the 3D extracellular matrix environment. Additionally, we uncover significant alterations in cytokine expression associated with matrix remodeling and angiogenesis, providing new avenues for therapeutic investigation. Although our findings are derived from a specific set of isogenically-matched cell lines, this model highlights the potential to explore personalized treatment strategies. Ongoing research in our lab is dedicated to gaining a more profound understanding of matrix remodeling and the migratory potential of TMZ-resistant GBM cells. Moreover, our future work will aim to expand our study to include a wider range of cell lines and patient-derived xenografts within our advanced microphysiological systems that include key elements of GBM tumor microenvironment such as perivascular niche. This endeavor seeks to establish connections between observed therapeutic behaviors and clinical outcomes, ultimately contributing to the development of more effective treatments for GBM patients. Citation Format: Viktoriia Kriuchkovskaia, Ela Eames, Rebecca Riggins, Brendan Harley. Acquired temozolomide resistance instructs patterns of glioblastoma cell motility and proteome expression within gelatin hydrogels [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 6770.

Identification of potential glioma drug resistance target proteins based on ultra-performance liquid chromatography-mass spectrometry differential proteomics.

In this study, to screen for candidate markers of temozolomide (TMZ) resistance in glioblastoma, we artificially established TMZ drug-resistant glioblastoma (GBM) cell lines, U251-TMZ and U87-TMZ. In the U251-TMZ and U87-TMZ cell lines, we screened and analyzed differentially expressed proteins using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) differential proteomics. Compared with the U251 and U87 control cell lines, 95 differential proteins were screened in the U251-TMZ and U87-TMZ cell lines, of which 28 proteins were upregulated and 67 proteins were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the co-upregulated proteins showed that most of the differentially expressed proteins were located in the cytoplasm and were significantly upregulated in the biological processes related to vesicular transport in the intimal system and inflammatory response mediated by myeloid leukocytes. Seven candidates were identified as potential GBM markers of TMZ resistance. Combined with existing research findings, our study supports that UAP1L1 and BCKDK are promising potential markers of TMZ resistance in GBM. This is important for further understanding the molecular mechanisms that drive the development and enhancement of TMZ resistance.

Fam20C Kinase as a Key Regulator of Bevacizumab Resistance in Mesenchymal Glioblastoma

AbstractA significant hurdle in treating glioblastoma (GBM) is addressing the development of drug resistance. In this study, the role of Family of Sequence Similarity 20, Member C (Fam20C) as a central player in bevacizumab resistant GBM mouse model is investigated. In vivo analyses confirm that Fam20C upregulation accelerates drug resistance and correlates with tumor progression. Proteomic analyses of conditioned media and cell lysates subsequent to Fam20C knockout (KO) in GBM cells reveal the regulatory role of Fam20C in both intracellular and extracellular aspects of epithelial‐mesenchymal transition (EMT) and genes associated with AKT signaling. Moreover, In vitro experiments demonstrate that Fam20C activates the AKT signaling pathway, promoting cell proliferation. Elevated levels of Fam20C are observed in human GBM, particularly in the mesenchymal subtype, which correlates with diminished survival rates and increased resistance to various drugs, including temozolomide (TMZ), bevacizumab, epidermal growth factor receptor (EGFR) inhibitors, and other antibody‐based drugs. Notably, even in cases of resistance to gefitinib and hepatocyte growth factor (HGF) antibodies, Fam20C expression is elevated. These findings highlight the pivotal role of Fam20C in driving drug resistance in GBM, suggesting it as a promising target for combination therapies aimed at surmounting this formidable resistance barrier.

Drug resistance in glioblastoma: from chemo- to immunotherapy.

As the most common and aggressive type of primary brain tumor in adults, glioblastoma is estimated to end over 10,000 lives each year in the United States alone. Stand treatment for glioblastoma, including surgery followed by radiotherapy and chemotherapy (i.e., Temozolomide), has been largely unchanged since early 2000. Cancer immunotherapy has significantly shifted the paradigm of cancer management in the past decade with various degrees of success in treating many hematopoietic cancers and some solid tumors, such as melanoma and non-small cell lung cancer (NSCLC). However, little progress has been made in the field of neuro-oncology, especially in the application of immunotherapy to glioblastoma treatment. In this review, we attempted to summarize the common drug resistance mechanisms in glioblastoma from Temozolomide to immunotherapy. Our intent is not to repeat the well-known difficulty in the area of neuro-oncology, such as the blood-brain barrier, but to provide some fresh insights into the molecular mechanisms responsible for resistance by summarizing some of the most recent literature. Through this review, we also hope to share some new ideas for improving the immunotherapy outcome of glioblastoma treatment.

P13.04.A REPRODUCIBLE CRISPR-SEQ PIPELINE FOR NGS FUNCTIONAL GENOMICS SCREENINGS FOR GLIOMA CELL LINES

Abstract BACKGROUND The CRISPR/Cas9 genome editing technology has revolutionized the field of functional genomics by enabling precise editing of DNA. Over the past few years, CRISPR technology has been widely employed to identify genetic dependencies in a broad spectrum of applications paving the way for the discovery and validation of relevant molecular targets, for example potential drug targetsGlioblastoma are incurable primary tumors of the central nervous system. Functional genomics could enable the discovery of novel therapeutic targets or even combination therapies. MATERIAL AND METHODS Here, we present a bioinformatics pipeline that combines functional screens and target editing analysis to identify potential vulnerabilities and drug targets in glioblastoma or other diseases. The pipeline consists of two workflows, one performing functional screening and one performing base editing analysis. This developed CRISPR-seq pipeline is reproducible and containerized, allowing high-throughput processing. All processes are modularized in Nextflow DSL2 (Domain Specific Language) and versioned which facilitates maintainability and customization, as well as the addition of new tools. A quality control report is also automatically generated, allowing the user an overview of their ran experiments. The pipeline also helps us to keep the data FAIR (Findable, Accessible, Interoperable and Reusable) and optimized, making it easier to share with other researchers and collaborate on future studies. RESULTS This pipeline was applied to functional screens using CRISPR/Cas9 genome-wide knockout or activation libraries to detect genetic vulnerabilities that might enhance drug treatment efficacy, e.g. for ataxia telangiectasia and Rad3 related (ATR) inhibition in experimental glioma.With this pipeline, we were able to identify several genes that are involved in drug resistance in glioblastoma, by starting from raw data (fastq files) to genes presenting vulnerabilities.The results obtained through our pipeline provided important information on the disease's molecular mechanisms, which were subsequently confirmed through wet lab validation. CONCLUSION In conclusion, the crisprseq software described in this abstract is a powerful tool for identifying potential genetic dependencies. The pipeline aims at analyzing this output data in a reproducible and containerized way using Nextflow and respecting nf-core community standards. By combining functional screens and base editing analysis with bioinformatics tools, this pipeline allows for the identification of essential genes and pathways, as well as the characterization of specific mutations that contribute to the development and progression of glioblastoma.

The Regulatory Roles of Mitochondrial Metabolism Dynamics and Mitochondria Calcium Uniporter (MCU) in Bevacizumab Resistance of GBM

AbstractAdapted oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle activations are essential tumor microenvironments for abnormal energy consumption to acquire malignancy and drug resistance during cancer development and progression. To elucidate the molecular mechanism related to the mitochondrial metabolic dynamics and drug resistance in glioblastoma (GBM), a longitudinal GBM orthotopic mouse model with acquired resistance to bevacizumab is established. The longitudinal proteomic analysis results show that OXPHOS, TCA, and calcium signaling gene sets are enriched in the bevacizumab pre‐resistance phase for preparing resistance phase. Then, the APEX system to GBM to biotinylate and purify proteins of the mitochondria matrix is applied. The organelle specific proteomic analysis shows that the pore‐forming subunits of the mitochondrial calcium uniporter protein (MCU) are essential for acquiring bevacizumab resistance. Additionally, a combination effect of hypoxia and the MCU‐specific inhibitor DS16570511 in vitro shows that cell growth and proliferation are reduced via inhibition of NF‐κB and CEBP/β signaling pathways. In conclusion, the hypoxic tumor microenvironment induced by bevacizumab treatment affects mitochondrial metabolic dynamics, and targeting MCU is a promising therapeutic option in combination with bevacizumab in recurrent GBM.

The antiferroptotic role of TRIM7: Molecular mechanism and synergistic effect with temozolomide

The antiferroptotic role of TRIM7: Molecular mechanism and synergistic effect with temozolomide

Effect of Polyunsaturated Fatty Acids on Temozolomide Drug-Sensitive and Drug-Resistant Glioblastoma Cells.

Glioblastomas (GBMs) are notoriously difficult to treat, and the development of multiple drug resistance (MDR) is common during the course of the disease. The polyunsaturated fatty acids (PUFAs) gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) have been reported to improve MDR in several tumors including breast, bladder, and leukaemia. However, the effects of PUFAs on GBM cell MDR are poorly understood. The present study investigated the effects of PUFAs on cellular responses to temozolomide (TMZ) in U87MG cells and the TMZ-resistant (TMZR) cells derived from U87MG. Cells were treated with PUFAs in the absence or presence of TMZ and dose-response, viable cell counting, gene expression, Western blotting, flow cytometry, gas chromatography-mass spectrometry (GCMS), and drug efflux studies were performed. The development of TMZ resistance caused an increase in ABC transporter ABCB1 and ABCC1 expression. GLA-, EPA-, and DHA-treated cells had altered fatty acid composition and accumulated lipid droplets in the cytoplasm. The most significant reduction in cell growth was seen for the U87MG and TMZR cells in the presence of EPA. GLA and EPA caused more significant effects on ABC transporter expression than DHA. GLA and EPA in combination with TMZ caused significant reductions in rhodamine 123 efflux from U87MG cells but not from TMZR cells. Overall, these findings support the notion that PUFAs can modulate ABC transporters in GBM cells.

Multiscale modeling of drug resistance in glioblastoma with gene mutations and angiogenesis

Drug resistance is a prominent impediment to the efficacy of targeted therapies across various cancer types, including glioblastoma (GBM). However, comprehending the intricate intracellular and extracellular mechanisms underlying drug resistance remains elusive. Empirical investigations have elucidated that genetic aberrations, such as gene mutations, along with microenvironmental adaptation, notably angiogenesis, act as pivotal drivers of tumor progression and drug resistance. Nonetheless, mathematical models frequently compartmentalize these factors in isolation. In this study, we present a multiscale agent-based model of GBM, encompassing cellular dynamics, intricate signaling pathways, gene mutations, angiogenesis, and therapeutic interventions. This integrative framework facilitates an exploration of the interplay between genetic mutations and the vascular microenvironment in shaping the dynamic evolution of tumors during treatment with tyrosine kinase inhibitor. Our simulations unveil that mutations influencing the migration and proliferation of tumor cells expedite the emergence of phenotype heterogeneity, thereby exacerbating tumor invasion under both treated and untreated conditions. Moreover, angiogenesis proximate to the tumor fosters a protumoral milieu, augmenting mutation-induced drug resistance by increasing the survival rate of tumor cells. Collectively, our findings underscore the dual roles of intrinsic genetic mutations and extrinsic microenvironmental adaptations in steering tumor growth and drug resistance. Finally, we substantiate our model predictions concerning the impact of gene mutations and angiogenesis on the responsiveness of targeted therapies by integrating single-cell RNA-seq, spatial transcriptomics, bulk RNA-seq, and clinical data from GBM patients. The multidimensional approach enhances our understanding of the complexities governing drug resistance in glioma and offers insights into potential therapeutic strategies.

Novel kinome profiling technology reveals drug treatment is patient and 2D/3D model dependent in glioblastoma.

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome™) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

Involvement of cell shape and lipid metabolism in glioblastoma resistance to temozolomide.

Temozolomide (TMZ) has been used as standard-of-care for glioblastoma multiforme (GBM), but the resistance to TMZ develops quickly and frequently. Thus, more studies are needed to elucidate the resistance mechanisms. In the current study, we investigated the relationship among the three important phenotypes, namely TMZ-resistance, cell shape and lipid metabolism, in GBM cells. We first observed the distinct difference in cell shapes between TMZ-sensitive (U87) and resistant (U87R) GBM cells. We then conducted NMR-based lipid metabolomics, which revealed a significant increase in cholesterol and fatty acid synthesis as well as lower lipid unsaturation in U87R cells. Consistent with the lipid changes, U87R cells exhibited significantly lower membrane fluidity. The transcriptomic analysis demonstrated that lipid synthesis pathways through SREBP were upregulated in U87R cells, which was confirmed at the protein level. Fatostatin, an SREBP inhibitor, and other lipid pathway inhibitors (C75, TOFA) exhibited similar or more potent inhibition on U87R cells compared to sensitive U87 cells. The lower lipid unsaturation ratio, membrane fluidity and higher fatostatin sensitivity were all recapitulated in patient-derived TMZ-resistant primary cells. The observed ternary relationship among cell shape, lipid composition, and TMZ-resistance may be applicable to other drug-resistance cases. SREBP and fatostatin are suggested as a promising target-therapeutic agent pair for drug-resistant glioblastoma.

Abstract 1661: Development of a patient-derived glioblastoma model in the transgenic Rag2 Null rat: In vivo imaging, histology, and combination therapy assessment

Abstract An important obstacle to translating promising drug treatments against glioblastoma (GBM) to the clinic is lack of resistant GBM animal models that more accurately parallel the clinical course of GBM drug resistance found in patients. In this project, a recurrent patient derived intracranial GBM tumor model (GB10) was developed for the first time in the novel Rag2 Null transgenic rat to establish a reproducible preclinical brain tumor model. Histologic characterization confirmed markers of high nuclear proliferation (30% Ki67) and presence of neovascularity (3% Von Willebrand Factor VIII) in all tumors. Serial in vivo imaging was performed to determine a clinically relevant late stage of tumor across all rats before initiation of treatment, accounting for heterogeneity of tumor size and growth rate characteristics. Overall survival was measured following oral administration of control, Temozolomide (TMZ), or combination therapy with TMZ and Idasanutlin. Eighteen Rag2 Null rats were intracranially implanted with GB10, a well characterized, recurrent cell line from the Mayo Clinic Brain Tumor Patient-Derived Xenograft National Resource. Bi-weekly high resolution, 2-dimensional T2 magnetic resonance imaging (2DT2 MRI) was performed on a Bruker 9.4 tesla scanner until tumor volume reached 30 µL. 14 days later, simultaneous positron emission tomography and magnetic resonance imaging (PET MRI) was performed on each rat with three dimensional T1 post-contrast (3DT1 CE), amino acid-based 18F-Flouroethyltyrosine (18F-FET), and amide proton transfer-chemical exchange saturation transfer (amide-CEST). Measured tumor parameters included volumetry (3DT1 CE), metabolic activity (18F-FET), and mobile phase intracellular protein content (amide-CEST), to confirm late-stage viable tumors of similar size (15.3 mm^2 +/-1.38 based on SEM, N=16) and metabolism (2:1 ratio of activity of tumor relative to contralateral control brain tissue). Two rats were removed: 1 died prematurely of infection and 1 had extra-axial tumor location. Oral treatment groups were as follows: Control (N= 5), TMZ (N=6) or TMZ + Idasanutlin (N=5). Kaplan Meier survival curves for the TMZ treated group (80.2+/-4.1 days) and combined TMZ + Idasanutlin group (93.8+/-6.2 days) were both significantly increased (P<0.001) compared to control (67.4+/-4.5 days). Survival for the TMZ + Idasanutlin treated group was also significantly increased (P<0.001) relative to TMZ treated group indicating efficacy of combination therapy. In conclusion, a robust, resistant, patient derived GBM animal model was developed using serial, high field, non-invasive, multimodality, molecular imaging along with histopathologic analysis. Feasibility of assessing treatment response was also demonstrated with both a standard of care and experimental combination therapy, in the late stage of GBM growth. Citation Format: Luke R. Jackson, Megan R. Masi, Hamideh zarrinmayeh, Sudip K. Das, Mark A. Green, Scott E. Snyder, Nian Wang, Karen Pollok, Gary D. Hutchins, Elizabeth R. Butch, Michael C. Veronesi. Development of a patient-derived glioblastoma model in the transgenic Rag2 Null rat: In vivo imaging, histology, and combination therapy assessment [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 1661.

Abstract 6255: CRISPRa-mediated induction of apoptosis in therapy-resistant glioblastoma cells via derepression of pro-apoptotic genes

Abstract Induction of apoptosis in cancer cells via de-repressing dormant pro-apoptotic genes might be a successful therapeutic strategy. In this study, we utilized several dCas9-mediated CRISPR activation (CRISPRa) systems to activate two key pro-apoptotic genes DR4 and XAF1 in glioblastoma cells. CRISPRa is used to activate endogenous genes via either recruiting transcription factors or epigenetic modifiers to regulatory regions in the genome. Previously conducted studies showed that VPR, Suntag, and SAM systems are the most effective transcription factor-based systems for activating several genes in a variety of cell lines. However, epigenetic modifier-based systems that involve the recruitment of TET demethylases have more permanent effects. Therefore, we hypothesized that combinations of these systems may induce stronger activation. We systematically tested various CRISPRa systems and their combinations to activate XAF1 and DR4, which are known to be epigenetically silenced in drug-resistant glioblastoma cells. Inducing the expression of these genes, we aimed to observe apoptosis in glioblastoma cells either at a basal state or upon treatment with the pro-apoptotic ligand TRAIL. By transcriptomic alteration and cell viability analyses, we demonstrated that the combinatorial system including dCas9-TET1 and MS2-p65-hsf1 was the most efficient one for the activation of DR4 and XAF1 genes in both 293T and glioblastoma cells. Another system in which multiple TET1 enzymes were recruited to the gene promoter together with MS2-p65-hsf1 was found to be effective similarly, suggesting that the promoter demethylation is critical for the activation of these genes. Activation of these genes with the most efficient systems caused apoptosis and also sensitized glioblastoma cells to TRAIL treatment. Our preliminary work on measuring methylation levels of several CpG sites in the promoter regions of DR4 is in line with our observations of cell-based phenotypes. Together, our study indicates that the combination of transcription factor-based and demethylation-based CRISPR activation systems can induce pro-apoptotic gene expression in cancer cells and lead cancer cells to undergo apoptosis. Future applications of this methodology might involve the development of cancer-specific pro-apoptotic therapies. [E.Y. and A.K. contributed equally to this work.] Citation Format: Ebru Yilmaz, Alisan Kayabolen, Semra Cemre Atalar, Buket Yigit, Burge Ulukan, Mujdat Zeybel, Tugba Bagci-Onder. CRISPRa-mediated induction of apoptosis in therapy-resistant glioblastoma cells via derepression of pro-apoptotic genes [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 6255.

Fundamental signaling pathways for glioblastoma drug resistance and developing robust organoid models for drug discovery

This article presents a review of the current literature on the molecular mechanisms of treatment resistance in glioblastoma. As mounting research continues to explore novel methods of treating glioblastoma, from using organoid models for drug screening to developing novel cellular therapies, it is critical to understand the fundamental molecular landscape that makes glioblastoma difficult to treat. This review explores the means of chemoresistance to the conventional chemotherapy temozolomide. Consideration of DNA repair pathways, p53-mediated apoptosis and autophagy, convergent proliferation pathways, and epigenetic mechanisms demonstrate avenues for the development of sophisticated drug targets and combination treatments. Ultimately, this article highlights each of these mechanisms and presents referential material for future endeavors in organoid-based drug screening.