Target In Glioblastoma Research Articles

Targeted therapy for glioblastoma utilizing hyaluronic acid-engineered liposomes for adriamycin delivery.

Glioblastoma (GBM) is a malignant tumor with highly heterogeneous and invasive characteristics leading to a poor prognosis. The CD44 molecule, which is highly expressed in GBM, has emerged as a highly sought-after biological marker. Therapeutic strategies targeting the cell membrane protein CD44 have emerged, demonstrating novel therapeutic potential. In this study, we constructed a nanodrug system (HA-Liposome@Dox) based on hyaluronic acid-engineered liposomes delivering adriamycin to target GBM. The system efficiently encapsulated Dox inside the liposomes through a hydrophilic-hydrophobic interaction mechanism, and the resulting HA-Liposome@Dox exhibited excellent loading efficacy, attributed to its uniform particle size distribution and negatively charged surface. Further evaluation revealed that HA-Liposome@Dox possessed excellent stability and safety and could promote the effective uptake of drug particles by CD44-overexpressing tumor cells, thus exerting a more potent cell-killing effect. Notably, in the treatment of GBM, HA-Liposome@Dox demonstrated significantly greater tumor growth inhibition compared to free Dox and prolonged the survival of tumor-bearing mice. Taken together, the present study not only verified the feasibility of HA-Liposome@Dox as an effective therapeutic tool against GBM and other CD44-positively expressing tumors, but also opened a promising new avenue for the clinical treatment of this type of refractory malignancies.&#xD.

Macrophage membrane-camouflaged pure-drug nanomedicine for synergistic chemo- and interstitial photodynamic therapy against glioblastoma.

Glioblastoma (GBM) persists as a highly fatal malignancy, with current clinical treatments showing minimal progress over years. Interstitial photodynamic therapy (iPDT) holds promise due to its minimally invasive nature and low toxicity but is impeded by poor photosensitizer penetration and inadequate GBM targeting. Here, we developed a biomimetic pure-drug nanomedicine (MM@CT), which co-assembles the photosensitizer chlorin e6 (Ce6) and the first-line chemotherapeutic drug (temozolomide, TMZ) for GBM, then camouflaged with macrophage membranes. This design eliminates the need for traditional excipients, ensuring formulation safety and achieving exceptionally high drug loading with 73.2 %. By leveraging the biomimetic properties of macrophage membranes, MM@CT evades clearance by the mononuclear phagocyte system and can overcome blood circulatory barriers to target intracranial GBM tumors due to its inherent tumor-homing ability. Consequently, this targeted strategy enables precise delivery of TMZ to the tumor site while significantly enhancing Ce6 accumulation within the tumor tissue. Upon intra-tumoral irradiation using an optical fiber, activated Ce6 synergizes with TMZ to exert both cytotoxic effects from chemotherapy and unique advantages from iPDT simultaneously attacking GBM tumors in a dual manner. In subcutaneous and intracranial GBM mouse models, MM@CT exhibits remarkable anti-tumor efficacy with minimal systemic toxicity, emerging as a promising GBM treatment strategy. STATEMENT OF SIGNIFICANCE: Glioblastoma (GBM) remains a formidable brain cancer, posing significant therapeutic challenges due to the presence of the blood-brain barrier (BBB) and tumor heterogeneity. To overcome these obstacles, we have developed MM@CT, a biomimetic nanomedicine with exceptional drug loading efficiency of 73.2 %. MM@CT incorporates the photosensitizer Ce6 and chemotherapy agent TMZ, encapsulated within nanoparticles and camouflaged with macrophage membranes. This innovative design enables efficient BBB penetration, precise tumor targeting, and synergistic application of chemotherapy and photodynamic therapy. Encouragingly, preclinical evaluations have demonstrated substantial antitumor activity with minimal systemic toxicity, positioning MM@CT as a promising therapeutic strategy for GBM.

CYBC1 Drives Glioblastoma Progression via ROS and NF-κB Pathways.

This study aims to investigate the role of Cytochrome b-245 chaperone 1 (CYBC1) in glioblastoma (GBM) progression, focusing on its involvement in reactive oxygen species (ROS) production and associated signaling pathways. Understanding the molecular mechanisms driven by CYBC1 could provide new therapeutic targets and prognostic markers for GBM. Publicly available datasets were analyzed to assess CYBC1 expression in GBM and its correlation with patient survival. GBM cell lines were genetically manipulated using the CRISPR/Cas9 system to deplete CYBC1. The effects of CYBC1 deficiency on cell proliferation, migration, invasion, and cell cycle dynamics were experimentally evaluated. Additionally, the impact of CYBC1 on the expression of NOXA1, a subunit of NADPH oxidase, and downstream signaling pathways such as NF-κB was explored. CYBC1 expression was significantly elevated in GBM tissues and correlated with poor patient survival. CYBC1 deficiency in GBM cells resulted in reduced cell viability, migration, and invasion. Mechanistically, CYBC1 positively regulated NOXA1 expression, which in turn enhanced ROS production and activated the ERK·AKT/NF-κB pathways. The suppression of CYBC1 led to decreased ROS levels, reduced phosphorylation of NF-κB, and downregulation of genes involved in epithelial-mesenchymal transition. CYBC1 is implicated in GBM progression by regulating NOXA1-mediated ROS production and activating the ERK·AKT/NF-κB pathways. This study suggests that CYBC1 could serve as a potential therapeutic target and prognostic marker in GBM, warranting further investigation into its molecular mechanisms and therapeutic potential.

Bright NIR-II emissive cyanine dye-loaded lipoprotein-mimicking nanoparticles for fluorescence imaging-guided and targeted NIR-II photothermal therapy of subcutaneous glioblastoma

Cyanine dye-containing nanoparticles have widely been used in “all-in-one” NIR fluorescence imaging (FI)-guided photothermal therapy (PTT) because of their intrinsically large extinction coefficient and available physical and chemical modulation methods to tune absorption and emission wavelengths. The combination of good brightness and excellent tumor-targeting capacity is the key to realize efficient NIR-II FI-guided PTT. In this study, by covalently decorating NIR-II absorptive cyanine dyes with bulky AIE motify, we demonstrate how steric hindrance suppresses π-π stacking-induced fluorescence quenching and contributes to the good brightness of NIR-II FI of subcutaneous glioblastoma. The resulting cyanine dye (C12-TPAE) is 5 times brighter than the original cyanine dye in the formulated liposomal nanoparticles and C12-TPAE-AL has a high photothermal conversion efficiency of 62.4%, with good colloidal and light stability. Importantly, the ApoE peptide is absorbed on the liposomal surface, yielding lipoprotein-mimicking nanoparticles, which achieve active targeting of glioblastoma and efficient FI-guided PTT without tumor recurrence without any side effects on normal organs (heart, kidneys, liver, spleen, or lung). This research highlights a facile design route for bright NIR-II emissive and NIR-II photothermal cyanine dyes and indicates that cyanine dye-containing biomimetic theranostic nanoplatforms are promising candidates for future precision therapy.

The Neuroprotective Role of A2A Adenosine Purinoceptor Modulation as a Strategy Against Glioblastoma.

Glioblastoma (GBM) is a highly lethal type of cancer, frequently presenting an unfavorable prognosis. The current treatment options for this neoplasia are still limited, highlighting the need for further research evaluating new drugs to treat GBM or to serve as an adjuvant to improve the efficiency of currently used therapies. In this sense, the inhibition of A2A receptors in the brain has presented a neuroprotective role for several diseases, such as neurodegenerative conditions, and it has been suggested as a possible pharmacological target in some types of cancer; thus, it also can be underscored as a potential target in GBM. Recently, Istradefylline (IST) was approved by the FDA for treating Parkinson's disease, representing a safe drug that acts through the inhibition of the A2A receptor, and it has also been suggested as an antineoplastic drug. Therefore, this work aims to explore the effects of A2A receptor inhibition as a therapy for GBM and assess the feasibility of this blockage occurring through the effects of IST.

TUBB2B regulates epithelial-mesenchymal transition via interaction with Vimentin to promote glioma migration and invasion

BackgroundEpithelial-mesenchymal transition (EMT) plays a crucial role in the migration and invasion capabilities of glioblastoma (GBM) cells. Several studies have established tubulin as a significant regulator of the EMT process. Tubulin beta 2B class IIb (TUBB2B), a critical component of microtubules, has been linked to the prognosis of various tumors. However, the specific biological function and mechanism of TUBB2B in GBM remain unclear.MethodsIn vitro experiments demonstrated that TUBB2B knockdown inhibited the migration and invasion of GBM cells, while its overexpression enhanced these capabilities. Western blot, immunofluorescence (IF) and co-immunoprecipitation (Co-IP) assays revealed that TUBB2B interacts with Vimentin. Molecular docking and residue mutation scanning indicated that TUBB2B interacts with Vimentin at the R391/K392/A393/F394 sites. In vivo experiments using nude mice confirmed that TUBB2B knockdown inhibited GBM cell invasion and migration.ResultsTUBB2B was upregulated in GBM tissue samples compared with normal tissues. The sites of TUBB2B(R391/K392/A393/F394) physically interacts with Vimentin to induce EMT, which promotes migration and invasion.ConclusionTUBB2B may regulate EMT and promote the migration and invasion of GBM cells through its interaction with Vimentin, highlighting TUBB2B as a potential therapeutic target for GBM.

Lipidomics-driven drug discovery and delivery strategies in glioblastoma.

With few viable treatment options, glioblastoma (GBM) is still one of the most aggressive and deadly types of brain cancer. Recent developments in lipidomics have demonstrated the potential of lipid metabolism as a therapeutic target in GBM. The thorough examination of lipids in biological systems, or lipidomics, is essential to comprehending the changed lipid profiles found in GBM, which are linked to the tumor's ability to grow, survive, and resist treatment. The use of lipidomics in drug delivery and discovery is examined in this study, focusing on how it may be used to find new biomarkers, create multi-target directed ligands, and improve drug delivery systems. We also cover the use of FDA-approved medications, clinical trials that use lipid-targeted medicines, and the integration of lipidomics with other omics technologies. This study emphasizes lipidomics as a possible tool in developing more effective treatment methods for GBM by exploring various lipid-centric techniques.

Characterizing and targeting glioblastoma neuron-tumor networks with retrograde tracing.

Glioblastomas are invasive brain tumors with high therapeutic resistance. Neuron-to-glioma synapses have been shown to promote glioblastoma progression. However, a characterization of tumor-connected neurons has been hampered by a lack of technologies. Here, we adapted retrograde tracing using rabies viruses to investigate and manipulate neuron-tumor networks. Glioblastoma rapidly integrated into neural circuits across the brain, engaging in widespread functional communication, with cholinergic neurons driving glioblastoma invasion. We uncovered patient-specific and tumor-cell-state-dependent differences in synaptogenic gene expression associated with neuron-tumor connectivity and subsequent invasiveness. Importantly, radiotherapy enhanced neuron-tumor connectivity by increased neuronal activity. In turn, simultaneous neuronal activity inhibition and radiotherapy showed increased therapeutic effects, indicative of a role for neuron-to-glioma synapses in contributing to therapeutic resistance. Lastly, rabies-mediated genetic ablation of tumor-connected neurons halted glioblastoma progression, offering a viral strategy to tackle glioblastoma. Together, this study provides a framework to comprehensively characterize neuron-tumor networks and target glioblastoma.

Hypoglycemic Agents Increase Regulatory Factor X1 to Inhibit Cancer Cell Behaviour in Human Glioblastoma Cells.

Glioblastoma multiforme is a deadly brain tumour in humans. We have shown that regulatory factor X1 (RFX1), a transcription factor, inhibits the proliferation, migration and invasion of human glioblastoma cells. This study was designed to identify the existing medications that could increase RFX1 in human glioblastoma cells and to determine whether these medications could inhibit the cancer cell behaviours. A bioinformatics approach was used to identify the medications that increased RFX1. The effects of these medications on human glioblastoma cell proliferation, migration and invasion were assayed under cell culture and mouse brain xenograft conditions. Pioglitazone, rosiglitazone and WY-14643 increased RFX1 based on bioinformatics prediction and Western blotting data. These hypoglycemic agents reduced the proliferation, migration and invasion of human glioblastoma cell cultures. These agents reduced metalloproteinase 2 (MMP2) activity in the culture medium. Silencing RFX1 attenuated hypoglycemic agent-induced inhibition of cancer cell behaviours and MMP2 activity. Pioglitazone reduced the xenograft tumour volume and migration distance of U87 human glioblastoma cells in the mouse brain. RFX1-siRNA attenuated these effects. Our results provide additional evidence for RFX1 as a therapeutic target for human glioblastoma and suggest that pioglitazone, rosiglitazone and WY-14643 inhibit cancer cell behaviour of human glioblastoma cells via upregulating RFX1.

RAB32 promotes glioma cell progression by activating the JAK/STAT3 signaling pathway.

This study aimed to investigate the role of RAB32 in glioblastomas and its molecular mechanisms that regulate gliomas. The expression and prognostic value of RAB32 were evaluated using western blotting and the Gene Expression Profiling Interactive, Chinese Glioma Genome Atlas, and The Cancer Genome Atlas databases. Lentivirus containing sh-RAB32 or OE-RAB32 was used to manipulate RAB32 expression in glioma cells. The effects of RAB32 on cell proliferation, migration, and invasion were determined by western blotting, cell counting kit-8, plate cloning, wound healing, and transwell assays. Gene set enrichment analysis was used to screen for associations between the JAK/STAT3 signaling pathway and RAB32. The role of this pathway was verified using JAK/STAT3 inhibitors. RAB32 expression was significantly upregulated in patients with glioma and in glioma cell lines. The expression level was positively correlated with the glioma grade and served as an independent prognostic factor. In vitro experiments revealed that RAB32 knockdown inhibited glioblastoma cell proliferation, migration, and invasion, while the opposite effects were observed with overexpression and could be inhibited by the JAK/STAT3 inhibitor BP-1-102. RAB32 promotes malignant progression of glioblastoma cells through the JAK/STAT signaling pathway, providing new possibilities for therapeutic targets for glioblastoma.

KLF14 inhibits tumor progression via FOSL1 in glioma

BackgroundGlioma, the most frequent central nervous system malignancy, is often promoted by the overexpression of Fos-like antigen 1 (FOSL1). However, the regulation of FOSL1 remains unexplored. The present study aimed to investigate the regulatory mechanism of FOSL1 to identify potential therapeutic targets for glioblastoma. MethodsThis study's initial investigation utilized dual-luciferase reporter gene assays and quantitative polymerase chain reaction (qPCR) assays to establish that Kruppel-like factor 14 (KLF14) inhibits the transcription of FOSL1. Subsequent immunohistochemistry and western blotting (WB) assays on glioma tissues confirmed a negative association between FOSL1 and KLF14. This study generated KLF14 knockdown cells and double knockdown cells of KLF14 and FOSL1 and further assessed cell growth through various experimental methods. The impact of KLF14 on tumor cell migration via FOSL1 was determined using qPCR and WB assays. A xenograft tumor model was utilized to verify tumor growth suppression by KLF14. ResultsThe present study demonstrated that KLF14 restrains FOSL1 transcription and is inversely correlated with FOSL1 in glioma tissues. KLF14 overexpression was found to counteract FOSL1's effect on cell migration and epithelial-to-mesenchymal transition in glioma cells, which coincided with decreased Snail2 and cluster of differentiation 44 (CD44) expressions. Further, KLF14 overexpression was shown to hinder tumor progression in vivo. ConclusionThis study highlights that FOSL1 is negatively regulated by KLF14 in glioblastoma and suggests that KLF14 overexpression can mitigate tumor growth by inhibiting FOSL1, thus identifying KLF14 as a novel molecular target for treating glioblastoma. Further research into the interplay and regulatory dynamics between KLF14 and FOSL1 under varying stress conditions can enhance the precision of glioblastoma treatment.

Unraveling the role of LncRNAs in glioblastoma progression: insights into signaling pathways and therapeutic potential.

Glioblastoma (GBM) is one of the most aggressive types of brain cancer, characterized by its poor prognosis and low survival rate despite current treatment modalities. Because GBM is lethal, clarifying the pathogenesis's underlying mechanisms is important, which are still poorly understood. Recent discoveries in the fields of molecular genetics and cancer biology have demonstrated the critical role that non-coding RNAs (ncRNAs), especially long non-coding RNAs (lncRNAs), play in the molecular pathophysiology of GBM growth. LncRNAs are transcripts longer than 200 nucleotides that do not encode proteins. They are significant epigenetic modulators that control gene e expression at several levels. Their dysregulation and interactions with important signaling pathways play a major role in the malignancy and development of GBM. The increasing role of lncRNAs in GBM pathogenesis is thoroughly examined in this review, with particular attention given to their regulation mechanisms in key signaling pathways such as PI3K/AKT, Wnt/β-catenin, and p53. It also looks into lncRNAs' potential as new biomarkers and treatment targets for GBM. In addition, the study discusses the difficulties in delivering lncRNA-based medicines across the blood-brain barrier and identifies areas that need more research to advance lncRNA-oriented treatments for this deadly cancer.

Metabolically-Driven Active Targeting of Magnetic Nanoparticles Functionalized with Glucuronic Acid to Glioblastoma: Application to MRI-Tracked Magnetic Hyperthermia Therapy.

Glioblastoma continues to pose a major global health challenge due to its incurable nature. The need for new strategies to combat this devastating tumor is therefore paramount. Nanotechnology offers unique opportunities to develop innovative and more effective therapeutic approaches. However, most nanosystems developed to treat glioblastomas, especially those based on metallic nanoparticles (NPs), have proven unsuccessful due to their inability to efficiently target these tumors, which are particularly inaccessible due to the restrictions imposed by the blood-brain tumor barrier (BBTB). Here, an innovative strategy is presented to efficiently target metallic NPs to glioblastomas through glucose transporters (GLUT) overexpressed on the endothelial cells of glioblastoma microvasculature, particularly GLUT1. Specifically, Iron Oxide Nanoparticles (IONPs) are functionalized with glucuronic acid to promote GLUT-mediated transcytosis which is drastically boosted by inducing mild hypoglycemia. This metabolically-driven active targeting strategy has yielded unprecedented efficacy in targeting metallic NPs to glioblastomas. Moreover, these IONPs, designed to act as magnetic hyperthermia (MH) mediators, are used to conduct a proof-of-concept preclinical study on MRI-tracked MH therapy following intravenous administration, resulting in significant tumor growth delay. These findings demonstrate unparalleled efficiency in glioblastoma targeting and lay the ground for developing alternative therapeutic strategies to combat glioblastoma.

Microglia induce an interferon-stimulated gene expression profile in glioblastoma and increase glioblastoma resistance to temozolomide.

Glioblastoma is the most malignant primary brain tumour. Even with standard treatment comprising surgery followed by radiation and concomitant temozolomide (TMZ) chemotherapy, glioblastoma remains incurable. Almost all patients with glioblastoma relapse owing to various intrinsic and extrinsic resistance mechanisms of the tumour cells. Glioblastomas are densely infiltrated with tumour-associated microglia and macrophages (TAMs). These immune cells affect the tumour cells in experimental studies and are associated with poor patient survival in clinical studies. The aim of the study was to investigate the impact of microglia on glioblastoma chemo-resistance. We co-cultured patient-derived glioblastoma spheroids with microglia at different TMZ concentrations and analysed cell death. In addition, we used RNA sequencing to explore differentially expressed genes after co-culture. Immunostaining was used for validation. Co-culture experiments showed that microglia significantly increased TMZ resistance in glioblastoma cells. RNA sequencing revealed upregulation of a clear interferon-stimulated gene (ISG) expression signature in the glioblastoma cells after co-culture with microglia, including genes such as IFI6, IFI27, BST2, MX1 and STAT1. This ISG expression signature is linked to STAT1 signalling, which was confirmed by immunostaining. The ISG expression profile observed in glioblastoma cells with enhanced TMZ resistance corresponded to the interferon-related DNA damage resistance signature (IRDS) described in different solid cancers. Here, we show that the IRDS signature, linked to chemo-resistance in other cancers, can be induced in glioblastoma by microglia. ISG genes and the microglia inducing the ISG expression could be promising novel therapeutic targets in glioblastoma.

CSIG-23. CYTOMEGALOVIRUS-INFECTED HUMAN GLIOBLASTOMA CELL LINES SHOW AN AGGRESSIVE PHENOTYPE ASSOCIATED WITH UPREGULATION OF IL-6/STAT3 AND AKT SIGNALING

Abstract BACKGROUND Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) pathogenesis by affecting stemness, angiogenesis and immune pathways. Clinical trials targeting CMV in GBM patients have shown some early promise, and CMV seropositivity has been associated with poorer outcomes. However, the underlying mechanisms remain unclear. Here we investigated the effects of CMV infection on critical signaling pathways in GBM (IL6/STAT3, and Akt signaling) based on the hypothesis that CMV may contribute to poorer outcomes in GBM by increasing oncogenic signaling. MATERIALS AND METHODS Human U251 and LN229 GBM cell lines were infected with mCherry expressing human CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. We performed basic phenotypic studies in vitro and investigated the status of IL6/STAT3, and Akt signaling by Western blotting. RESULTS Both LN229 and U251 cell lines were readily infectable by CMV. Infection of U251 cells was over 90% and in LN229 cells was approximately 60%. Interestingly expression of CMV was sustained over many passages in these cell lines. Infection was also verified by the presence of the CMV pp65 protein assessed by Western blotting. Measurement of cell proliferation indicated faster growth after CMV infection. Interrogation of key pro-oncogenic signaling pathways in GBM revealed a robust upregulation of IL6, as well as phospho(Tyr705)-STAT3 and phospho(Thr308)-Akt. This elevation of pro-oncogenic signaling pathways was observed in both cell lines compared with controls. CONCLUSIONS Here we show for the first time that infection of human GBM cell lines with CMV results in a more aggressive phenotype associated with upregulation of IL6/STAT3 signaling as well as a significant increase in phospho-Akt levels. We are currently studying sensitivity to standard therapies and additional molecular changes in these cells. These data support the hypothesis that CMV causes more aggressive disease and that CMV is a relevant therapeutic target in GBM.

CSIG-34. TYROSINE PHOSPHORYLATION OF NON-CANONICAL NF-ΚB P52 PROMOTES GLIOMA GROWTH AND CHEMORESISTANCE IN GBM

Abstract p52 is a nuclear factor-κB (NF-κB) transcription factor that comprises the non-canonical NF-κB pathway. The impact and mechanism of non-canonical NF-κB signaling remains understudied in glioblastoma (GBM). Subunit post-translational modification is a known mechanism of NF-κB activation, however phosphorylation of p52 has never been demonstrated. Elevated tyrosine kinase activity is a known-feature of GBM and portends worse survival. Further, p52 has been shown to upregulate factors that likely promote invasion of GBM, thereby reducing survival. Activation of p52 may serve as a link between increased kinase activity and poor survival in GBM. p52-knockout cell lines were generated using the CRISPR-Cas9 system and assessed for growth and survival differences in vitro and in syngeneic murine glioma models. Public glioma datasets and an institutional cohort were surveyed to corroborate p52-dependent survival differences in GBM. To determine whether p52 is post-translationally modified, we performed tandem mass spectrometry on p52 in GBM cells. Interacting factors were also analyzed and tested for site-specific modification of p52 via co-IP studies. We then generated PTM-null, knock-in glioma cell lines using CRISPR-CAS9 editing to demonstrate the functional significance of p52 PTMs, which were assessed using assays of relative cell growth, stemness, chemosensitivity and survival. Finally, we performed unbiased analysis of the p52 PTM-dependent transcriptome via RNA-seq. p52 loss significantly reduced glioma growth rate in vitro and improves murine survival. Retrospective analysis of GBM in patients suggests that p52 levels are negatively prognostic. Mass spectrometry identified multiple p52 PTMs with tyrosine phosphorylation sites as top hits. Identified phospho-tyrosine residues specifically interact with SRC-family kinases. PTM site editing significantly reduces glioma cell growth and stemness while enhancing chemosensitivity, reflecting broad transcriptome level changes. In sum, p52 activity enhances GBM growth and chemoresistance, which is partially modulated by tyrosine-specific phosphorylation. p52 warrants investigation as a therapeutic target in GBM, possibly in combination with kinase inhibitors and standard alkylating chemotherapy.

STEM-17. THE UROKINASE RECEPTOR AS A NOVEL IMMUNOTHERAPEUTIC TARGET IN BRAIN CANCERS

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. GBM is characterized by extensive inter- and intra-tumoral heterogeneity and an extremely immunosuppressive tumor microenvironment. Following Standard-of-Care surgical resection and chemoradiotherapy, patients inevitably relapse, at which point few therapeutic avenues exist, owing in part due to a lack of clinically relevant targets. Data from our target identification pipeline shows the 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, 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, and therapy resistance. We used CRISPR-Cas9 to genetically delete uPAR from our in-house patient-derived GBM cell lines, and found that knockout of uPAR in recurrent GBM cells significantly reduces various functional characteristics of BTICs in vitro. In our patient-derived mouse model of GBM, we found that knockout of uPAR significantly increases survival, and decreases tumor burden. Further, we generated novel anti-uPAR single domain antibodies (sdAbs) which specifically and efficiently bind uPAR at low nanomolar concentrations in vitro. We developed anti-uPAR CAR Ts using the binder sequence of the sdAbs, which showed potent cytotoxicity in vitro, and drastically reduced tumor burden and extended survival in vivo. Further, we identified uPAR as being highly expressed on brain metastases from multiple origins (lung-to-brain, melanoma-to-brain, etc.), and demonstrate that anti-uPAR CAR Ts effectively kill brain metastases in vitro, and significantly extend survival using in vivo brain metastasis models. From this work we believe uPAR to be a clinically relevant target in both recurrent GBM and brain metastases, and investigation into therapeutic strategies targeting uPAR-positive brain cancers should be explored further.

TMOD-03.BRAFV600E MUTATION INDUCES TWIST1 UPREGULATION ASSOCIATED WITH MIGRATION AND CEREBRAL FLUID DISSEMINATION IN GLIOBLASTOMA

Abstract INTRODUCTION BRAFV600E-mutant glioblastoma (GBM) with epithelioid features frequently show cerebrospinal fluid dissemination (CSFD), which results in poor prognoses. BRAF mutation is worth investigating since BRAF or downstream MEK inhibitors show dramatic response for BRAFV600E-mutant GBM including CSFD. This suggests BRAF mutation could play a role for inducing the clinical manifestation of CSFD found in newly diagnosed and recurrent BRAFV600E-mutant GBM. Because, BRAFV600E-mutant GBM often recur with CSFD, elucidating the mechanism by which BRAFV600E-mutant GBM exhibits CSFD is important to improve prognosis. (Objective) To elucidate the molecular mechanism of CSFD involved in BRAFV600E-mutant GBM. METHODS We introduced the heterozygous BRAFV600E mutation in human induced pluripotent stem cells (iPSC) in the context of GBM genetic alterations, CDKN2A/2B, and PTEN deletion, TERT promoter mutation and with EGFRvIII overexpression. We implanted these iPSC-derived neural progenitor cells into immunodeficient mouse brains to establish a BRAFV600E-mutant GBM model. At the same time, we created a model with the same genetic background without BRAFV600E. We compared their gene expression, pathology, and migration patterns to identify BRAFV600E specific characteristics. RESULTS RNA sequencing revealed BRAFV600E-mutant tumors upregulated migration, and downregulated cell-to-cell adhesion genes. TWIST1 was one of the most upregulated genes, and was validated by qPCR. Concomitant with TWIST1 expression we detected downregulation of E-cadherin. The BRAFV600E GBM model histologically showed a cluster-like invasion phenotype, while non-BRAF tumors showed homogenous invasion with typical GBM pathological findings. Immunohistochemical staining showed relatively high TWIST1 expression in cluster-like tumor cells of BRAFV600E tumors. Upon re-engraftment of primary tumor-derived cells, secondary BRAFV600E tumors presented leptomeningeal dissemination. TWIST1 knockdown resulted in a decrease in the migratory ability of our BRAFV600E model in vitro, and is under investigation in vivo. CONCLUSION BRAV600E mutation induces TWIST1 upregulation, and might facilitate migration and CSFD. This work nominates TWIST1 and its regulated genes as targets for BRAFV600E-mutant GBM with CSFD.

EXTH-16. UNBIASED DISCOVERY OF SYNERGISTIC VULNERABILITIES AGAINST GLIOBLASTOMA: AN INNOVATIVEIN VIVO CRISPR PROFILING PLATFORM TO UNLEASH THE POWER OF TUMOR-TARGETED CYTOKINE THERAPY

Abstract BACKGROUND Glioblastoma poses a formidable challenge due to its intricate intra- and intertumoral heterogeneities and its associated immunosuppressive milieu. The standards of care have limited activity, and no major advances have been made in decades. One promising avenue is tumor-targeted cytokine-based therapies, particularly L19-TNF, which is undergoing clinical trials in Europe and the US for patients with newly diagnosed and recurrent glioblastoma. However, there is still an opportunity to find safer and more tolerable combination partners to improve the efficacy of L19-TNF immunotherapy. To address this challenge, we employed a cutting-edge in vivo CRISPR screening approach to identify synergistic vulnerabilities with a focus on druggable targets. METHODS We systematically investigated the dependencies of tumor growth and key signaling pathways under the pressure of L19-TNF immunotherapy in the CT-2A orthotopic murine glioma model. We used an innovative in vivo CRISPR screening approach that enables timed sgRNA activation to mitigate off-target effects and overcome orthotopic tumor cell implantation challenges, such as the bottleneck of cell engraftment. RESULTS We first conducted a genome-wide in vivo screen and identified 400 top-scoring candidate genes, which we subsequently tested again in a targeted in vivo screen to refine our drug selection process. Through this, we uncovered novel and previously unexplored druggable targets for glioblastoma. Out of these, we validated four promising target-specific drugs in preclinical glioblastoma models in downstream in vivo experiments. In addition, we investigated modes of action and resistance. CONCLUSION Our research marks a significant advancement in pursuing novel treatment strategies for glioblastoma. We have unveiled a range of synergistic combinations and paved the way for the rational design of combinatory treatment approaches to maximize the therapeutic efficacy of L19-TNF immunotherapy, laying the groundwork for future clinical translation.

IMMU-10. IDENTIFICATION OF A NEW THERAPEUTIC ANTIGEN FOR GLIOBLASTOMA USING A MONOCLONAL ANTIBODY LIBRARY FROM PATIENT-DERIVED TUMOR SPHERES

Abstract Glioblastoma (GBM) has a poor prognosis despite intensive treatment by surgery, radiation, and chemotherapy, thus new treatment strategies are urgently needed. Various innovative cancer therapies targeting cancer-specific cell surface antigens, such as chimeric antigen receptor T cell therapy, are developed and more cell surface antigens that is highly specific for GBM cells are needed. Although extensive transcriptome analyses or proteomic analysis of GBM cells were performed, few transcripts specific for GBM cells have been identified. However, GBM cell-specific antigen epitopes formed by post-translational modifications of proteins may have been missed by transcriptome or proteomic analysis, and could still be discovered by thoroughly searching for cancer-specific monoclonal antibodies. In this study, we aimed to identify GBM-specific antigens using a monoclonal antibody library from patient-derived tumor spheres, create CAR-T cells against the antigen, and check antitumor efficiency of the CAR-T cells. Approximately 25,000 monoclonal antibody-producing hybridomas were establishment using BALB/c mice and patient derived tumor spheres. Among them, a antibodies that bind to plural glioblastomas and not to plural normal brain cells were selected and we identified the antigen as Prostaglandin F2 receptor negative regulator (PTGFRN) by the expression cloning method. PTGFRN witch is expressed in several cancers regulate migration, angiogenesis, and cell polarity and decreases the radiosensitivity of GBM cells. We created CAR-T cells against the antigen and co-cultured it with GBM cells, which significantly increased the production of IL-2 and INF-γ. Furthermore, the CAR-T cells injected intracranial in an orthotopic xenograft model with patient-derived GBM cells, significantly reduced tumor growth. PTGFRN CAR-T-cell therapy may be a potential novel therapeutic target for GBM.