GBM Xenografts Research Articles

EXTH-14. PRECLINICAL EVALUATION OF NOVEL CDK4/6 INHIBITOR GLR2007 IN GLIOBLASTOMA MODELS

Abstract Glioblastoma multiforme (GBM) is characterized by a high frequency of cyclin-dependent kinase (CDK)4 and CDK6 pathway dysregulation. None of the approved CDK4/6 inhibitors are indicated for GBM. Poor blood–brain barrier (BBB) penetration is a potential factor limiting treatment efficacy in GBM. GLR2007 is an investigational CDK4/6 inhibitor with the potential for improved penetration across the BBB. These preclinical studies investigated the anti-tumor efficacy in GBM xenograft models and central nervous system distribution of GLR2007. In vivo evaluation of the anti-tumor efficacy of GLR2007 versus vehicle, abemaciclib, and/or palbociclib was performed in two BALB/c nude mouse GBM xenograft models. Efficacy was expressed as tumor growth inhibition (TGI, change in mean tumor volume from baseline as a percentage of change in vehicle group) or increased survival time. Following 21 days of treatment, TGI in BN2289 subcutaneous xenografts was 39.4% and 56.4% for 25 mg/kg and 50 mg/kg GLR2007 groups, respectively, 34.0% for 25 mg/kg palbociclib, and 24.9% for 25 mg/kg abemaciclib (p < 0.001 in all groups vs vehicle control). In U87-luc orthotopic xenografts, compared with vehicle controls, median survival time was 50.0% (p = 0.0009) longer in the 12.5 mg/kg GLR2007 group, similar to the 150 mg/kg abemaciclib group (54.4%, p = 0.0002), and was 182.6% (p < 0.0001) longer in mice treated with 50 mg/kg GLR2007. Quantitative whole-body autoradiography was used to determine the distribution of [14C]GLR2007 in Sprague Dawley rats following a single oral dose. Total radioactivity levels in the brain exceeded those in plasma by 2.3–4.5-fold from 2–6 h after dosing. These preclinical studies suggest the potential of GLR2007 for the treatment of GBM, supported by evidence that GLR2007 showed numerically greater anti-tumor efficacy than approved CDK4/6 inhibitors palbociclib and abemaciclib in GBM xenograft models, and evidence of substantial central nervous system distribution.

Targeting UDP-α-d-glucose 6-dehydrogenase alters the CNS tumor immune microenvironment and inhibits glioblastoma growth

Glioblastoma (GBM, WHO grade IV glioma) is the most common and lethal malignant brain tumor in adults with a dismal prognosis. The extracellular matrix (ECM) supports GBM progression by promoting tumor cell proliferation, migration, and immune escape. Uridine diphosphate (UDP)-glucose 6-dehydrogenase (UGDH) is the rate-limiting enzyme that catalyzes the biosynthesis of glycosaminoglycans that are the principal component of the CNS ECM. We investigated how targeting UGDH in GBM influences the GBM immune microenvironment, including tumor-associated microglia/macrophages (TAMs) and T cells. TAMs are the main immune effector cells in GBM and can directly target tumor cells if properly activated. In co-cultures of GBM cells and human primary macrophages, UGDH knockdown in GBM cells promoted macrophage phagocytosis and M1-like polarization. In orthotropic human GBM xenografts and syngeneic mouse glioma models, targeting UGDH decreased ECM deposition, increased TAM phagocytosis marker expression, reduced M2-like TAMs and inhibited tumor growth. UGDH knockdown in GBM cells also promoted cytotoxic T cell infiltration and activation in orthotopic syngeneic mouse glioma models. The potent and in-human-use small molecule GAG synthesis inhibitor 4-methylumbelliferone (4-MU) was found to inhibit GBM cell proliferation and migration in vitro, mimic the macrophage and T-cell responses to UGDH knockdown in vitro and in vivo and inhibit growth of orthotopic murine GBM. Our study shows that UGDH supports GBM growth through multiple mechanisms and supports the development of ECM-based therapeutic strategies to simultaneously target tumor cells and their microenvironment.

OS06.3A LSD1-directed therapy curtails glioblastoma tumorigenicity by limiting the adaptation of tumor initiating cells to stressful environments

Abstract BACKGROUND Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, therapy resistance and poor blood-brain-barrier penetration hinder the amelioration of the standard-of-care. Included in the GBM mass are the tumor initiating cells (TICs), representing the driver of GBM growth and relapse in virtue of their stem-like traits and therapy-resistance. Being constantly exposed to environmental stress, including nutrients deficiency, hypoxia and therapeutic insults, all GBM cells -and TICs in particular- have to be highly adaptive in order to survive. Thus, their ability to cope with stress could be targeted to curtail TICs maintenance and the whole GBM aggressiveness. The key of TICs adaptation relies, among the others, on their epigenetic plasticity, hence encouraging epigenetic drugs testing. MATERIAL AND METHODS By exploiting patient-derived GBM TICs and orthotopic xenograft models, we tested the antitumorigenic features of a novel, selective, orally bioavailable and brain-penetrant Lysine-specific histone demethylase 1 inhibitor (LSD1i). We confirmed the specificity of its effects by LSD1 genetic targeting. A combination of RNA-seq, Chromatin Immunoprecipitation(ChIP)-seq, Mass Spectrometry and reverse genetic experiments unraveled LSD1 molecular players in GBM TICs. RESULTS We identified LSD1 as a druggable target in human GBM: LSD1i treatment, mirrored by LSD1 genetic targeting, impairs growth, viability, stem-like traits and in vivo tumorigenicity of GBM TICs. Mechanistically, LSD1 is crucial for the expression of the activating transcription factor 4 (ATF4), which coordinates the integrated stress response (ISR) to manage stressful stimuli as nutrient deprivation and endoplasmic reticulum stress. By mimicking these stress cues in vitro, we found that LSD1i triggers a delayed but unabated ATF4 translation which provokes an over-lasting ISR, eventually culminating in GBM TICs apoptosis. Lastly, LSD1 demethylase activity is dispensable for ATF4 induction. Rather, LSD1i exerts its anti-tumorigenic potential by interfering with LSD1 scaffolding function in GBM TICs. CONCLUSION LSD1-directed therapy is likely a promising strategy to hinder GBM. By sensitizing GBM TICs to stress, LSD1i endangers the GBM TICs pool. The effectiveness of LSD1i administration in different patient-derived GBM TICs and xenografts, regardless of their molecular profile, places a strong rationale toward the clinical translation of this approach for GBM management. FUNDING Italian association for Cancer Research (AIRC) and Italian Ministry of Health

Downregulated CLIP3 induces radioresistance by enhancing stemness and glycolytic flux in glioblastoma

BackgroundGlioblastoma Multiforme (GBM) is a malignant primary brain tumor in which the standard treatment, ionizing radiation (IR), achieves a median survival of about 15 months. GBM harbors glioblastoma stem-like cells (GSCs), which play a crucial role in therapeutic resistance and recurrence.MethodsPatient-derived GSCs, GBM cell lines, intracranial GBM xenografts, and GBM sections were used to measure mRNA and protein expression and determine the related molecular mechanisms by qRT-PCR, immunoblot, immunoprecipitation, immunofluorescence, OCR, ECAR, live-cell imaging, and immunohistochemistry. Orthotopic GBM xenograft models were applied to investigate tumor inhibitory effects of glimepiride combined with radiotherapy.ResultsWe report that GSCs that survive standard treatment radiation upregulate Speedy/RINGO cell cycle regulator family member A (Spy1) and downregulate CAP-Gly domain containing linker protein 3 (CLIP3, also known as CLIPR-59). We discovered that Spy1 activation and CLIP3 inhibition coordinately shift GBM cell glucose metabolism to favor glycolysis via two cellular processes: transcriptional regulation of CLIP3 and facilitating Glucose transporter 3 (GLUT3) trafficking to cellular membranes in GBM cells. Importantly, in combination with IR, glimepiride, an FDA-approved medication used to treat type 2 diabetes mellitus, disrupts GSCs maintenance and suppresses glycolytic activity by restoring CLIP3 function. In addition, combining radiotherapy and glimepiride significantly reduced GBM growth and improved survival in a GBM orthotopic xenograft mouse model.ConclusionsOur data suggest that radioresistant GBM cells exhibit enhanced stemness and glycolytic activity mediated by the Spy1-CLIP3 axis. Thus, glimepiride could be an attractive strategy for overcoming radioresistance and recurrence by rescuing CLIP3 expression.

Alkylaminophenol and GPR17 Agonist for Glioblastoma Therapy: A Combinational Approach for Enhanced Cell Death Activity.

Drug resistance and tumor heterogeneity limits the therapeutic efficacy in treating glioblastoma, an aggressive infiltrative type of brain tumor. GBM cells develops resistance against chemotherapeutic agent, temozolomide (TMZ), which leads to the failure in treatment strategies. This enduring challenge of GBM drug resistance could be rational by combinatorial targeted therapy. Here, we evaluated the combinatorial effect of phenolic compound (2-(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), GPR17 agonist 2-({5-[3-(Morpholine-4-sulfonyl)phenyl]-4-[4-(trifluoromethoxy)phenyl]-4H-1,2,4-triazol-3-yl}sulfanyl)-N-[4-(propan-2-yl)phenyl]acetamide (T0510.3657 or T0) with the frontline drug, TMZ, on the inhibition of GBM cells. Mesenchymal cell lines derived from patients’ tumors, MMK1 and JK2 were treated with the combination of THTMP + T0, THTMP + TMZ and T0 + TMZ. Cellular migration, invasion and clonogenicity assays were performed to check the migratory behavior and the ability to form colony of GBM cells. Mitochondrial membrane permeability (MMP) assay and intracellular calcium, [Ca2+]i, assay was done to comprehend the mechanism of apoptosis. Role of apoptosis-related signaling molecules was analyzed in the induction of programmed cell death. In vivo validation in the xenograft models further validates the preclinical efficacy of the combinatorial drug. GBM cells exert better synergistic effect when exposed to the cytotoxic concentration of THTMP + T0, than other combinations. It also inhibited tumor cell proliferation, migration, invasion, colony-forming ability and cell cycle progression in S phase, better than the other combinations. Moreover, the combination of THTMP + T0 profoundly increased the [Ca2+]i, reactive oxygen species in a time-dependent manner, thus affecting MMP and leading to apoptosis. The activation of intrinsic apoptotic pathway was regulated by the expression of Bcl-2, cleaved caspases-3, cytochrome c, HSP27, cIAP-1, cIAP-2, p53, and XIAP. The combinatorial drug showed promising anti-tumor efficacy in GBM xenograft model by reducing the tumor volume, suggesting it as an alternative drug to TMZ. Our findings indicate the coordinated administration of THTMP + T0 as an efficient therapy for inhibiting GBM cell proliferation.

Ultrasound-activated nano-TiO2 loaded with temozolomide paves the way for resection of chemoresistant glioblastoma multiforme

BackgroundGlioblastoma multiforme (GBM) is one of the most daunting issues to modern therapeutics, with a higher mortality rate post-diagnosis. Temozolomide (TMZ) is the only available treatment; however, the frequent resistance leaves the oncologists at a dead end. Therefore, new approaches to circumvent the GBM are highly desired. We have employed TiO2 nanosticks loaded with TMZ as nanomedicine for TMZ-resistant GBM resection in this contribution.ResultsThe ultrasonication triple-action effect could greatly facilitate tumor ablation by enhancing the TiO2 nanosticks traversing across BBB, releasing the TMZ payload from TiO2 nanosticks and reactive oxygen species (ROS) generation from TiO2 nanosticks within the GBM milieu. The tumor ablation was confirmed by MTT and Annexin(v)-PI assays, apoptotic proteins expression via western blot and ROS level detection in vitro, whereas tumor volume, weight, survival rate, and relative photon flux in the xenograft and orthoptic TMZ-resistant GBM murine models as in vivo.ConclusionWe found this nanomedicine-based ultrasound modality highly efficient in GBM treatment and is of future clinical application value due to the employment of already FDA-approved techniques and nanomedicine.

OTME-14. TGF-beta signaling in microtube formation of glioblastoma

Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas.By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs.Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3.Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.

ETMM-05. LACTIC ACID FACILITATES GLIOBLASTOMA GROWTH THROUGH MODULATION OF THE EPIGENOME

Glioblastoma (GBM) is the most common primary malignant brain tumor with an unfavorable prognosis. While GBMs utilize glucose, there are other carbon sources at their disposal. Lactate accumulates to a significant amount in the infiltrative margin of GBMs. In the current study, we demonstrated that lactate rescued patient-derived xenograft (PDX) GBM cells from nutrient deprivation mediated cell death and inhibition of growth. Transcriptome analysis, ATAC-seq and CHIP-seq. showed that lactic acid exposure entertained a signature of cell cycle progression and oxidative phosphorylation (OXPHOS) /tricarboxylic acid (TCA)-cycle. LC/MS analysis demonstrated that U-13C-Lactate elicited substantial labeling of TCA-cycle metabolites, acetyl-CoA and histone protein acetyl-residues in PDX derived GBM cells. Given that acetyl-CoA is pivotal for histone acetylation we observed a dose-dependent elevation of histone marks (e.g. H3K27ac), which was rescued by genetic and pharmacological inhibition of lactic acid-uptake, ATP-citrate lyase, p300 histone-acetyl-transferase and OXPHOS, resulting in reversal of lactate mediated protection from cell death. CHIP-seq. analysis demonstrated that lactic acid facilitated enhanced binding of H3K27ac to gene promoters and cis-regulatory elements. Consistently, ATAC-seq. analysis highlighted enhanced accessibility of the chromatin by lactic acid. In a combined tracer experiment (U-13C-glucose and 3-C13-lactate), we made the fundamental observation that lactic acid carbons were predominantly labeling the TCA cycle metabolites over glucose, implying a critical role of lactic acid in GBMs. Finally, pharmacological blockage of the TCA-cycle, using a clinically validated drug, extended overall survival in an orthotopic PDX model in mice without induction of toxicity, implying a critical role of lactic acid in GBMs and establishing lactic acid metabolism as a novel drug target for GBM.

An Upstream Open Reading Frame in Phosphatase and Tensin Homolog Encodes a Circuit Breaker of Lactate Metabolism

The metabolic role of micropeptides generated from untranslated regions remains unclear. Here we describe MP31, a micropeptide encoded by the upstream open reading frame (uORF) of phosphatase and tensin homolog (PTEN) acting as a "circuit breaker" that limits lactate-pyruvate conversion in mitochondria by competing with mitochondrial lactate dehydrogenase (mLDH) for nicotinamide adenine dinucleotide (NAD+). Knocking out the MP31 homolog in mice enhanced global lactate metabolism, manifesting as accelerated oxidative phosphorylation (OXPHOS) and increased lactate consumption and production. Conditional knockout (cKO) of MP31 homolog in mouse astrocytes initiated gliomagenesis and shortened the overall survival of the animals, establishing a tumor-suppressing role for MP31. Recombinant MP31 administered intraperitoneally penetrated the blood-brain barrier and inhibited mice GBM xenografts without neurological toxicity, suggesting the clinical implication and application of this micropeptide. Our findings reveal a novel mode of MP31-orchestrated lactate metabolism reprogramming in glioblastoma.

EXTH-19. EVALUATING THE ANTI-TUMOR EFFECT OF A NOVEL THERAPEUTIC AGENT, MAGMAS INHIBITOR, IN MALIGNANT GLIOMA

Abstract BACKGROUNDS Glioblastoma (GBM) is an aggressive infiltrative brain tumor, and has an extremely poor prognosis despite the use of multiple treatment modalities, including surgery, radiation, and chemotherapy. Meanwhile, mitochondrial changes represent a significant part of cancer cell biology since cancer cells must survive and adapt to challenging microenvironments, specifically in conditions where tumor growth makes oxygen and glucose scarce. As GBM is characterized by extensive hypoxia-induced phenotypic changes such as abnormal vascular proliferation and necrosis, regulation of mitochondrial function could be a novel approach for treating GBM that currently lacks effective therapies. Magmas (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. We previously demonstrated that a novel Magmas inhibitor, BT#9, significantly exerted anti-tumor effect in glioma in vitro, and may cross the blood brain barrier in vivo, indicating that Magmas inhibitor may be a new chemotherapeutic agent for the treatment of GBM. METHODS In this study, the antitumor effect of Magmas inhibitor BT#9 was tested in an orthotopic xenograft model of human GBM. The molecular mechanism of BT#9 was investigated using glioma cell lines. RESULTS The mice were tolerated to BT#9, and there was no statistical difference in the weight of animals between the control and MTD (Maximum-tolerated Dose, 50mg/kg) groups. The immunocompromised mice, intracranially implanted with human D-54 GBM xenografts, survived significantly longer than the controls (P< 0.5) when treated with BT#9 at MTD. In vitro study showed that the MAP kinase pathways are involved in BT#9-induced tumor suppression. DISCUSSION This is the first study on the role of Magmas in glioma in vivo. Our findings suggested that Magmas plays a key role in glioma survival and targeting Magmas by Magmas inhibitor has the potential to become a therapeutic strategy in glioma patients.

EPCO-16. LACTIC ACID IS AN EPIGENETIC METABOLITE THAT DRIVES GLIOBLASTOMA SURVIVAL AND GROWTH

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor with an unfavorable prognosis and a reprogrammed metabolism. While tumors utilize glucose, there are other carbon sources at their disposal. Originally considered as a waste product of glucose catabolism, lactate accumulates to a significant amount in tumor tissue. We launched our studies with the central hypothesis that lactate is metabolized by GBM cells to promote their survival via modulation of the epigenome. We showed that lactate rescued patient-derived xenograft (PDX) GBM cells from nutrient deprivation mediated cell death and inhibition of growth. Transcriptome analysis, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and CHIP-seq. showed that lactic acid exposure entertained a signature of cell cycle progression, oxidative phosphorylation (OXPHOS) and MYC target expression. LC/MS analysis demonstrated that U-13C-Lactate elicited substantial labeling of TCA-cycle metabolites, acetyl-CoA and histone protein acetyl-residues in PDX derived GBM cells. Given that acetyl-CoA is pivotal for histone acetylation we observed a dose-dependent elevation of histone marks (e.g. H3K27ac), which was rescued by genetic and pharmacological inhibition of lactic acid-uptake, ATP-citrate lyase, p300 histone-acetyl-transferase and OXPHOS, resulting in reversal of lactate mediated protection from cell death or facilitation of GBM growth. CHIP-seq. analysis demonstrated that lactic acid facilitated enhanced binding of H3K27ac to gene promoters and cis-regulatory elements (e.g. super-enhancers). Consistently, ATAC-seq. analysis highlighted enhanced accessibility of the chromatin by lactic acid. Finally, we assessed whether lactic acid is actively metabolized in vivo, utilizing an orthotopic PDX model of GBM. In a combined tracer experiment (U-13C-glucose and 3-C13-lactate), we made the fundamental observation that lactic acid carbons were predominantly labeling the TCA cycle metabolites over glucose, implying a critical role of lactic acid in GBMs and establishing lactic acid metabolism as a novel drug target for GBM that may be targeted with epigenetic drugs.

RBIO-03. HETEROGENEITY OF HUMAN PATIENT-DERIVED XENOGRAFTS GROWTH RATES RESPONSES TO THE RADIATED MICROENVIRONMENT

Abstract BACKGROUND Radiotherapy, combined with surgical resection and chemotherapy, remains a first-line treatment for infiltrative gliomas. However, these tumors are not surgically curable, and often recur, even within the prior radiation field, and may demonstrate a more aggressive phenotype. Importantly, high grade gliomas display diverse molecular phenotypes, and whether this genetic variability leads to divergent behaviour in the radiated tumor microenvironment is unknown. Herein, we characterize the effects of the irradiated brain microenvinroment on nine additional unique GBM cell lines to better understand the nuances of how tumor molecular phenotypes influence cellular dynamics. METHODS Female athymic nude mice were randomly divided into cranial radiation (15 Gy) and non-radiated groups. Mice then underwent intracranial implantation with one of the selected patient-derived xenograft (PDX) GBM cell lines (GBM 6, 10, 12, 39, 46, 76, 123, 164, 196; total n=8-15, per group, per line). Kaplan-Meyer (K-M) and log-rank tests were performed to compare the survival between irradiated and non-irradiated groups. RESULT Of nine previously untested human GBM lines, we found that five demonstrated shorter survival in the pre-radiated brain (GBM 6, 46, 76, 164, 196). However, two lines yielded prolonged survival in the pre-radiated brain (GBM 10, 12); GBM 39, 123 whose rate of growth was not impacted by the radiated brain. CONCLUSION These results highlight the likely critical impact of the irradiated microenvironment on tumor behaviour, yet illustrate that different tumors may exhibit opposing responses. Although further evaluation will be needed to understand mechanisms of divergent behavior, our data suggest the increased rate of growth in the radiated microenvironment may not apply to the fastest-growing tumor lines, which could instead demonstrate a paradoxical response.

STEM-20. A CANCER STEM CELL-SELECTIVE APOPTOSIS-INDUCING SMALL MOLECULE FOR THE TREATMENT OF GBM

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation and maintenance, drug resistance, and recurrence following surgery. New therapeutic strategies for the treatment of GBM have recently focused on targeting CSCs. Here we have used an unbiased large-scale screening approach to identify drug-like small molecules that induce apoptosis in GBM CSCs in a cell type-selective manner. METHODS A luciferase-based survival assay of patient-derived GBM CSC lines was established to perform a large-scale screen of ∼one million drug-like small molecules with the goal of identifying novel compounds that are selectively toxic to chemoresistant GBM CSCs. Compounds found to kill GBM CSC lines as compared to control cell types were further characterized. A caspase activation assay was used to evaluate the mechanism of induced cell death. A xenograft animal model using patient-derived GBM CSCs was employed to test the leading candidate for suppression of in vivo tumor formation. RESULTS We identified a small molecule, termed RIPGBM, from the cell-based chemical screen that induces apoptosis in primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of RIPGBM appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an intracranial GBM xenograft mouse model, RIPGBM was found to significantly suppress tumor formation. CONCLUSIONS Our chemical genetics-based approach has identified a small molecule drug candidate and a potential drug target that selectively targets cancer stem cells and provides an approach for the treatment of GBMs.

TAMI-46. TNFα SECRETED BY GLIOMA ASSOCIATED MACROPHAGES PROMOTES ENDOTHELIAL ACTIVATION AND RESISTANCE AGAINST ANTI-ANGIOGENIC THERAPY

Abstract Glioblastoma (GBM) remains a universal fatal disease and improving survival remains a challenge. One of the most prominent features of GBM is hyper-vascularization, characterized by abnormally dilated, distorted, and leaky vessels. Bone marrow (BM)-derived macrophages are recognized to be an important host cell population that are actively recruited to the tumor and referred to as glioma-associated macrophages (GAMs). GAMs are found to associate closely with blood vessels, and thought to provide a critical role in tumor neo-vascularization. However, the mechanisms by which GAMs regulate and promote endothelial cells (ECs) in the process of tumor vascularization and response to anti-angiogenic therapy (AATx) is not understood. To understand how GBM, GAMs and EC interacts, we designed and developed a novel GBMs-GAMs-EC co-culture system as well as a unique in vivo model in which the BM of NOD/SCID mice were reconstituted with red fluorescent protein (RFP)-BM cells to create a chimeric mouse and then using intracranial injection of GFP-U87 cells to create a GBM xenograft. Our study is the first to demonstrate that glioma cells-secreted IL8 and CCL2 which stimulate GAMs to secrete TNFα and promote EC activation. TNFα inhibition led to normalization of the tumor vasculature and significant improvement in survival in glioma mice model. More importantly, we validate these findings using clinical data showing that TNFα is a predictor of overall survival and response to AATx in GBM patients. We further demonstrated that increased macrophage recruitment and upregulation of TNFα in GAMs activating EC in response to bevacizumab treatment is one of the critical molecular mechanisms underlying the failure of AATx in GBM. Collectively, these results provide compelling evidence to further explore the clinical impact of inhibiting TNFα concurrent with AATx.

STEM-27. miR-10b-5p MODULATES 5hmC EXPRESSION AND THE STEM-LIKE PHENOTYPE IN GBM

Abstract Epigenetic alterations such as DNA methylation and dysregulation of non-coding RNAs (e.g. miRNAs) are found in all types of cancer and are thought to play important roles in tumorigenesis. GBM is characterized by small subsets of cells, referred to as glioma stem cells (GSCs), that display stem-like properties implicated in tumor initiation, therapeutic resistance, and recurrence. DNA methylation patterns are altered in GBM and GSCs and are thought to play critical roles in tumor initiation and propagation. DNA methylation is a reversible process catalyzed, in part, by the ten-eleven translocation (TET) family of enzymes. These enzymes function as deoxygenases that catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients. However, the mechanisms leading to the loss of 5hmC in glioma and the role this phenomenon plays in gliomagenesis remains poorly understood. We found that Sox2 expression decreases TET2 expression and its product 5hmC in GSCs and identified miR-10b-5p as a molecular intermediary of this process. We show that miR-10b-5p expression is high in GBM compared to non-tumor in clinical specimens and high levels of this miRNA correlate with poor patient outcome. Expression of transgenic miR-10b-5p enhanced sphere formation capacity of GSCs and the expression of stem cell markers and drivers. Additionally, using a combination of molecular and biochemical endpoints, we show that miR-10b-5p modifies 5hmC levels by regulating TET2 in GSCs. Finally, we show that repression of miR-10b-5p increases 5hmC levels and inhibits tumor propagation in GBM xenograft models. Taken together, these results present a new molecular mechanism that controls 5hmC and the tumor propagating capacity of GSCs and suggests that miR-10b-5p inhibition and other strategies for enhancing TET2 function can be developed to treat GBM.

TAMI-56. HYPOXIA-DRIVEN NECROSIS DRIVES DYNAMIC TUMOR PROGRESSION IN GLIOBLASTOMA

Abstract The onset of necrosis correlates with aggressive malignant progression in solid tumors. Whether necrosis arises from or promotes accelerated tumor proliferation and progression remains unknown, primarily due to inadequate models systems that fail to induce or capture these dynamic changes as they develop. In glioblastoma (GBM; WHO grade IV), the most malignant primary brain tumor, vascular pathology and central necrosis precedes rapid, radial expansion resulting in patient mortality. While genetic alterations in GBM have been highly characterized, biological adaptations arising from sustained hypoxia/anoxia require further mechanistic investigation. This sustained nutrient deprivation leads to necrosis which dramatically changes the tumor microenvironment (TME). To reveal the dynamic temporal and spatial changes promoting expansive progression, we are generating mouse models that more appropriately capture events found in human gliomas, accounting for unique microenvironmental stressors often lacking in GBM animal models, specifically central necrosis. Combining hypoxia-induced focal necrosis within high grade gliomas with intravital microscopy captures TME restructuring to understand its impact on glioma progression. Our studies use both genetically characterized patient-derived orthotopic GBM xenografts, alongside an immunocompetent RCAS/tv-a model, to determine how antagonizing these processes impacts disease progression and outcomes across multiple GBM subtypes. Complementary to this, we are investigating how increasing hypoxia and necrosis-driven signaling events promotes tumor-associated macrophages (TAMs) influx and reprogramming during glioma progression. Our preliminary data indicate substantial differences pre- and post-necrosis regarding TAM enrichment, immunosuppressive phenotypic conversion and their biological impact, however these mechanisms have not yet been elucidated. Our models capture glioma growth dynamics, reactive neuroinflammation and therapeutic resistance, facilitating innovative pre-clinical interventions to improve patient outcome.

EXTH-27. YAP/TAZ TRANSCRIPTIONAL CO-ACTIVATORS CREATE THERAPEUTIC VULNERABILITY TO VERTEPORFIN IN EGFR MUTANT GLIOBLASTOMA

Abstract Glioblastoma (GBM), neoplasms derived from glia and neuro-glial stem/progenitor cells, are the most common and lethal malignant primary brain tumors diagnosed in adults, with a median survival of 14 months with current treatments. GBM tumorigenicity is often driven by genetic aberrations in receptor tyrosine kinases (RTKs), such as EGFR, and the Pi-3 kinase (PI3K) signaling pathway. Using a Drosophila glioma model and human patient-derived GBM cells, we identified the YAP and TAZ transcription co-activators, effectors of the Hippo pathway that promote gene expression via TEAD co-factors, as key drivers of GBM tumorigenicity downstream of oncogenic EGFR signaling. YAP and TAZ are highly expressed in EGFR mutant human GBMs, and their knockdown in EGFR mutant GBM cells inhibits proliferation, blocks neuro-glial stem/progenitor cell fate, and elicits apoptosis. Our results indicate that YAP/TAZ-TEAD directly regulate transcription of SOX2, C-MYC, and EGFR itself to create a feedforward loop to drive survival and proliferation as well as stem/progenitor cell fate in human GBM cells. Moreover, the benzoporphyrin derivative verteporfin, a disruptor of YAP/TAZ-TEAD mediated transcription, specifically induced apoptosis of cultured patient-derived GBM cells, suppressed expression of YAP/TAZ transcriptional targets, including EGFR, and conferred significant survival benefit in an orthotopic xenograft GBM model. Our efforts led us to design and initiate a phase 0 clinical trial of Visudyne, an FDA-approved liposomal formulation of verteporfin, where we used intraoperative fluorescence to observe and verify verteporfin uptake into tumor cells in GBM tumors in human patients. Together, our data indicate that verteporfin is a promising therapeutic agent for EGFR amplified and mutant GBM. On the basis of these results, we have initiated as phase 1/2 clinical trial of Visudyne in patients with recurrent GBM characterized by EGFR amplification and/or mutation.

Abstract 3789: Therapeutic targeting of the YAP/TAZ pathway in glioblastoma with verteporfin

Abstract Glioblastoma (GBM) is a particularly lethal brain neoplasm accounting for half of malignant primary brain tumors diagnosed in adults and maintains a devastating diagnosis, with a median survival of 14 months despite standard clinical interventions of surgical, chemotherapeutic, and radiation treatments. GBM tumorigenicity is often driven by aberrations in receptor tyrosine kinases (RTKs) such as EGFR, and the Pi-3 kinase (PI3K) signaling pathway. Using a Drosophila glioma model and human patient-derived glioma cells, we identified YAP/TAZ, effectors of the Hippo pathway that promote expression of stemness factors and oncogenes via activation of the TEAD transcription factors, as drivers of glioma tumorigenicity. This led to the discovery that YAP/TAZ are highly expressed in human patient-derived glioma stem cells and that knockdown of these inhibits cell growth. We hypothesized that Verteporfin, a benzoporphyrin derivative approved by the FDA for the treatment of macular degeneration, may have therapeutic benefit for glioblastoma due to its published ability to inhibit interaction between YAP and TEAD transcription factors. Using in vitro culture-based assays and in vivo in GBM xenograft models, we observed Verteporfin specifically induces apoptosis in GBM cells, slows tumor growth, and durably suppressesexpression of known and novel TEAD transcriptional targets in tumor tissue. We validated several direct targets using chromatin immunoprecipitation, and are seeking to further identify the full transcriptional program controlled by YAP/TAZ-TEAD in GBM. Our efforts have led to a phase 0 clinical trial where we were able to observe Verteporfin uptake in GBM tumors in human patients. These data indicate that Verteporfin is a promising therapeutic target for GBM. Citation Format: Nathaniel H. Boyd, Krish Vigneswaran, Shoeb Lallani, Se-Yeong Oh, Andrew Boucher, Jeffrey Olson, Renee Read. Therapeutic targeting of the YAP/TAZ pathway in glioblastoma with verteporfin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3789.

Therapeutic targeting of CD38 enhances temozolomide activity in glioblastoma cells and modulates their tumor microenvironment by eliciting an enhanced CD8+ T cell response

Abstract Glioblastoma (GBM) is an aggressive primary malignant brain tumor with poor prognosis. Surgical resection followed by chemoradiotherapy (temozolomide [TMZ] + radiation) results in a median survival of only 12-15 months. Recently, immunotherapy with targeted antibodies, therapeutic vaccination and adoptive cell therapy offers a promising option for treating GBM. Herein we observed that CD38, a catalytic receptor and regulator of NAD homeostasis, is expressed on GBM cells and their microenvironment in primary patient-derived GBM tissues. Blacher et al have shown that blocking CD38 enzymatic functions results in reduced GBM cell growth. This evidence prompted us to investigate the antitumor and immune engaging effects of a clinical grade anti CD38 therapeutic monoclonal antibody (Daratumumab/DARA) in preclinical models of GBM. We demonstrated that DARA significantly enhanced the direct apoptotic effect of TMZ and induced ADCC/CDC of GBM cells, irrespective of the presence of microglia. In an in vitro 3D simulated GBM + T cell coculture system, DARA induced activation (CD69/GITR upregulation/decreased PD1) and enhanced proliferation of CD8+ T cells/cTL activity against GBM cells. DARA also resulted in increased IFNγ and decreased IL-10; suggesting an improved antitumor cytokine milieu and ensued in downregulated inflammatory cytokine associated JAK2/STAT3 signaling pathway. Finally, we tested TMZ+DARA in a human GBM xenograft in vivo model and noted significantly prolonged survival of mice [TMZ+DARA vs. vehicle: 98.13±1.88 vs. 63.63±3.77 days, p<0.001]. Altogether, these observations led us to investigate the clinical activity of DARA+ TMZ in GBM patients in a phase I/II clinical trial.

Metabolite Imaging at the Margin: Visualizing Metabolic Tumor Gradients Using Mass Spectrometry.

Glioblastoma multiforme (GBM) tumors are highly metabolic and vascularized, yet little has been reported regarding the spatial localization of metabolic activity within these tumors. A mass spectrometry imaging (MSI) study by Randall and colleagues in this issue provides provocative observations of metabolic gradients in xenograft GBM models. The intensity of acylcarnitines is dramatically increased at tumor margins, which interface with normal tissue, but not in tumor margins at the edge of the brain. A secondary examination of drug metabolites suggests that the observed metabolic gradients are pharmacologically relevant. These findings underscore previously undescribed spatial metabolic heterogeneity in GBM biology and opportunities for MSI investigations.See related article by Randall et al., p. 1258.