Glioblastoma Growth Research Articles

Co-treatment of human GBM cell lines with imipridone ONC206 and tumor treating fields: Effect on on cell death and p-AKT.

e14033 Background: Glioblastoma (GBM) is the most common primary brain malignancy with dismal prognosis. Tumor Treating Fields (TTFields) therapy is available for the treatment of both newly diagnosed and recurrent glioblastoma and represents a new category of treatment modalities in oncologic therapy. Despite decades of study, few FDA approved modalities are available for GBM and provide limited survival improvements for patients. AKT functions as a key serine/threonine kinase in the RTK/PTEN/PI3K pathway, and extensive genomic analysis of GBM has revealed that this pathway is mutated in a significant majority of GBM cases. Activation of this pathway ultimately leads to the activation of AKT, and phospho-AKT levels are elevated in the majority of GBM tumor samples and cell lines. Studies have shown that increased p-AKT levels contribute to uncontrolled growth, resistance to apoptosis, and enhanced invasive capabilities of glioma cells, which are characteristics of tumor progression in GBM. AKT serves as a critical hub in this pathway, enabling the amplification of growth signals, thereby making the inhibition of AKT an appealing and promising therapeutic target for treating GBM. Imipridone ONC206 is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). Here we show inhibition of p-AKT as well as upregulation of cleaved PARP following cotreatment of ONC206 and TTFields. Methods: We investigated the effect of ONC206 at IC50s plus TTFields in LN229, U138, U251 and T98G human GBM cell lines at different time points. Effect of treatment on cPARP, BCL-2, CHOP, caspase-10, and p-AKT were investigated using western blot. Results: Treatment with ONC206 at the IC50 in T98G cells with TTFields at 24h showed synergistic upregulation of cPARP as well as inhibition of BCL-2. At 96hour time point, inhibition of p-AKT was observed following all treatments (ONC206, TTFields, and co-application compared to control. In U251 cells, following treatment of ONC206 and TTFields, inhibition of BCL-2 was observed at 24h time point. Upregulation of CHOP and Casapse-10 at 96h time point was seen, as well as inhibition of p-AKT. Spheroid models showed structure disruption and growth arrest following combination treatment. At 72 h time point co treatment with ONC206 and TTFields resulted in synergistic upregulation of cleaved PARP. Conclusions: Here we showed cotreatment with TTFields and ONC206 results in inhibition of p-AKT, the key node in GBM growth pathway. This is the first report of cleaved PARP upregulation following TTFields and ONC206 in Human GBM cell line. Further studies to investigate the in vivo efficacy of TTField treatment in GBM with ONC206 are warranted. More studies are needed to understand the exact mechanism of synergy.

Dual-Targeted Novel Temozolomide Nanocapsules Encapsulating siPKM2 Inhibit Aerobic Glycolysis to Sensitize Glioblastoma to Chemotherapy.

Chemotherapy of glioblastoma (GBM) has not yielded success due to inefficient blood-brain barrier (BBB) penetration and poor glioma tissue accumulation. Aerobic glycolysis, as the main mode of energy supply for GBM, safeguards the rapid growth of GBM while affecting the efficacy of radiotherapy and chemotherapy. Therefore, to effectively inhibit aerobic glycolysis, increase drug delivery efficiency and sensitivity, a novel temozolomide (TMZ) nanocapsule (ApoE-MT/siPKM2 NC) is successfully designed and prepared for the combined delivery of pyruvate kinase M2 siRNA (siPKM2) and TMZ. This drug delivery platform uses siPKM2 as the inner core and methacrylate-TMZ (MT) as the shell component to achieve inhibition of glioma energy metabolism while enhancing the killing effect of TMZ. By modifying apolipoprotein E (ApoE), dual targeting of the BBB and GBM is achieved in a "two birds with one stone" style. The glutathione (GSH) responsive crosslinker containing disulfide bonds ensures "directional blasting" cleavage of the nanocapsules to release MT and siPKM2 in the high GSH environment of glioma cells. In addition, in vivo experiments verify that ApoE-MT/siPKM2 NC has good targeting ability and prolongs the survival of tumor-bearing nude mice. In summary, this drug delivery system provides a new strategy for metabolic therapy sensitization chemotherapy.

Characteristics of the Antitumor Effect of Doxorubicin and Pegylated Hyaluronidase on Models of Rat Brain Tumors.

Hyaluronidase increases tissue permeability and diffusion of the extracellular fluid by cleaving hyaluronan, the primary component of the extracellular matrix. Hyaluronidase pegylation (Hyal-PEG) decreases its clearance and enhances biodistribution. The pro- and anticancer activity of Hyal-PEG and a combination of Hyal-PEG with doxorubicin were studied in vitro (morphological analysis of rat glioblastoma 101.8 spheroids) and in vivo (by the survival time of rats after intracerebral transplantation of the tumor and morphological analysis). In the presence of doxorubicin and Hyal-PEG in the culture medium in vitro, spheroids lost their ability to adhere to the substrate and disintegrate into individual cells. Intracerebral transplantation of the tumor tissue with Hyal-PEG did not accelerate glioblastoma growth. The mean survival time for animals receiving transplantation of the tumor alone and in combination with Hyal-PEG was 13 and 20 days, respectively. In one rat with transplanted tumor and Hyal-PEG, this parameter increased by 53%. The survival time of rats receiving systemic therapy with doxorubicin and Hyal-PEG significantly increased (p=0.003). Antitumor effect of therapeutic doses of doxorubicin combined with Hyal-PEG was demonstrated on the model of rat glioblastoma 101.8 in vitro. Hyal-PEG inhibited adhesion of tumor cells, but did not cause their death. Transplantation of Hyal-PEG-treated tumor did not reduce animal survival time. Systemic administration of therapeutic doses of doxorubicin with Hyal-PEG increased survival time of rats with glioblastoma 101.8.

Therapeutic efficacy of a novel self-assembled immunostimulatory siRNA combining apoptosis promotion with RIG-I activation in gliomas

BackgroundCurrent cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5’-C; antisense strand, 3’-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy.MethodsIsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5’-C; antisense strand, 3’-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-β and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection.ResultsIsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-β and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors.ConclusionsThis study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5’-C; antisense strand, 3’-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.

Aurora kinase A inhibition plus Tumor Treating Fields suppress glioma cell proliferation in a cilium-independent manner

Tumor Treating Fields (TTFields) extend the survival of glioblastoma (GBM) patients by interfering with a broad range of tumor cellular processes. Among these, TTFields disrupt primary cilia stability on GBM cells. Here we asked if concomitant treatment of TTFields with other agents that interfere with GBM ciliogenesis further suppress GBM cell proliferation in vitro. Aurora kinase A (AURKA) promotes both cilia disassembly and GBM growth. Inhibitors of AURKA, such as Alisertib, inhibit cilia disassembly and increase ciliary frequency in various cell types. However, we found that Alisertib treatment significantly reduced GBM cilia frequency in gliomaspheres across multiple patient derived cell lines, and in patient biopsies treated ex vivo. This effect appeared glioma cell-specific as it did not reduce normal neuronal or glial cilia frequencies. Alisertib-mediated depletion of glioma cilia appears specific to AURKA and not AURKB inhibition, and attributable in part to autophagy pathway activation. Treatment of two different GBM patient-derived cell lines with TTFields and Alisertib resulted in a significant reduction in cell proliferation compared to either treatment alone. However, this effect was not cilia-dependent as the combined treatment reduced proliferation in cilia-depleted cell lines lacking, ARL13B, or U87MG cells which are naturally devoid of ARL13B+ cilia. Thus, Alisertib-mediated effects on glioma cilia may be a useful biomarker of drug efficacy within tumor tissue. Considering Alisertib can cross the blood brain barrier and inhibit intracranial growth, our data warrant future studies to explore whether concomitant Alisertib and TTFields exposure prolongs survival of brain tumor-bearing animals in vivo.

Two Mixed-Ligand Co(II) Complexes as Luminescent Materials and Loaded with Temozolomide-gel Particles in Nursing Against Glioma.

By employing a mixed-ligand strategy, we synthesized two new coordination polymers (CPs) featuring Co(II): {Co(H2L)(bib)]·2H2O}n (1) and {Co(L)(bib)2]·2H2O}n (2), where H4L represents 5-(3,5-dicarboxybenzyloxy) isophthalic acid, and bib denotes 1,4-bis(1-imidazolyl)benzene. These CPs were obtained through the reaction of H4L, a flexible carboxylic acid ligand, with Co(NO3)2·6H2O in various solvent mixtures, along with the N-donor co-ligand bib. Complexes 1 and 2 are formed through distinct coordination modes, resulting in their distinct structural features and excellent fluorescent properties. Based on ligand-centered fluorescence emission and the blue shift (CP 1) along with red shift (CP 2) characteristics, both complexes show promise for applications in fields such as blue fluorescence sensing materials and luminescent materials. After successfully synthesizing two CPs, CP 1 was chosen as the carrier for loading temozolomide (TMZ). Subsequently, leveraging the unique advantages of hydrogels, we developed a novel metal gel formulation loaded with TMZ. The inhibitory effect of this formulation on the growth of glioblastoma was evaluated. Our results demonstrate a significant suppression of glioblastoma cell proliferation by this system, providing an effective avenue for glioblastoma treatment.

PD-1/PD-L1 axis is involved in the interaction between microglial polarization and glioma

The tumor microenvironment plays a vital role in glioblastoma growth and invasion. PD-1 and PD-L1 modulate the immunity in the brain tumor microenvironment. However, the underlying mechanisms remain unclear. In the present study, in vivo and in vitro experiments were conducted to reveal the effects of PD-1/PD-L1 on the crosstalk between microglia and glioma. Results showed that glioma cells secreted PD-L1 to the peritumoral areas, particularly microglia containing highly expressed PD-1. In the early stages of glioma, microglia mainly polarized into the pro-inflammatory subtype (M1). Subsequently, the secreted PD-L1 accumulated and bound to PD-1 on microglia, facilitating their polarization toward the microglial anti-inflammatory (M2) subtype primarily via the STAT3 signaling pathway. The role of PD-1/PD-L1 in M2 polarization of microglia was partially due to PD-1/PD-L1 depletion or application of BMS-1166, a novel inhibitor of PD-1/PD-L1. Consistently, co-culturing with microglia promoted glioma cell growth and invasion, and blocking PD-1/PD-L1 significantly suppressed these processes. Our findings reveal that the PD-1/PD-L1 axis engages in the microglial M2 polarization in the glioma microenvironment and promotes tumor growth and invasion.

Corrigendum: miR-182 integrates apoptosis, growth, and differentiation programs in glioblastoma.

Corrigendum: miR-182 integrates apoptosis, growth, and differentiation programs in glioblastoma.

Skull-Derived Myeloid Cells Augment T Cell Responses and Suppress Glioblastoma Growth

INTRODUCTION: Recent studies have implicated skull marrow as a functional myeloid reservoir in CNS pathology. However, its contribution to malignancy remains unexplored. METHODS: Fluorescently labeled calvarial flaps were transplanted onto syngeneic glioblastoma-bearing mice, followed by flow cytometric profiling of tumor-infiltrative leukocytes. To assay the functional relevance of SDMCs, tumor-bearing C57BL/6 mice were treated with preemptive calvarial ablation (13 Gy irradiation), or conversely intracalvarial AMD3100 to augment myeloid egress from marrow niches. Tumor burden was monitored via survival and bioluminescent imaging. RESULTS: 72 h following CMFDA-stained calvarial transplantation into tumor-bearing Balb/C mice, 40.3% of tumor-infiltrative myeloid cells were fluorescent and thus skull-derived; transplantation in an alternative longitudinal model (GFP+ flaps, C57BL/6) validated that SDMCs were robust and viable at 3 weeks post-operatively. As MHCII+ cells were disproportionately enriched among SDMCs (87.9% vs 67.3%, p = 0.012), we interrogated the immunogenicity of these cells through targeted ablation. Skull-ablated mice had reduced infiltration by antigen-presenting cells than controls (cDCs: 1.1% vs 4.5%, p = 0.002; macrophages: 14.4% vs 36.6%, p < 0.001) and 6-fold greater necrosis (67.7% vs 13.5% apoptotic, p < 0.001), suggesting an impaired ability to mount a controlled, adaptive immune response. The stimulatory role of SDMCs was further demonstrated via intracalvarial AMD3100 administration, which through its promotion of myeloid precursor egress (0.26% vs 0.02%, p < 0.001) and cDC2 infiltration (10.4% vs 3.0%, p = 0.003) yielded robust CD4+ T cell activation and effector memory differentiation. These differences were clinically meaningful, as skull irradiation accelerated tumor growth and attenuated survival compared to controls, while AMD3100 treatment accomplished the opposite (median OS: 17d vs 22d, p < 0.001; 26.5d vs 22d, p = 0.006). CONCLUSIONS: The preferential polarization of SDMCs into immunostimulatory APCs warrants the continued exploration of local marrow-targeting agents (e.g., AMD3100) as therapeutic modalities.

1236 Skull-Derived Myeloid Cells Augment T Cell Responses and Suppress Glioblastoma Growth

INTRODUCTION: Recent studies have implicated skull marrow as a functional myeloid reservoir in CNS pathology. However, its contribution to malignancy remains unexplored. METHODS: Fluorescently labeled calvarial flaps were transplanted onto syngeneic glioblastoma-bearing mice, followed by flow cytometric profiling of tumor-infiltrative leukocytes. To assay SDMC function, tumor-bearing C57BL/6 mice were treated with preemptive calvarial ablation (13 Gy irradiation), or conversely intracalvarial AMD3100 to augment myeloid egress from marrow niches. Tumor burden was monitored via survival and bioluminescent imaging. RESULTS: 72 h following CMFDA-stained calvarial transplantation into tumor-bearing Balb/C mice, 40.3% of tumor-infiltrative myeloid cells were fluorescent and thus skull-derived; transplantation in an alternative longitudinal model (GFP+ flaps, C57BL/6) validated that SDMCs were robust and viable at 3 weeks post-operatively. As MHCII+ cells were disproportionately enriched among SDMCs (87.9% vs 67.3%, p = 0.012), we interrogated the immunogenicity of these cells through targeted ablation. Indeed, skull-ablated mice had reduced aggregate infiltration by antigen-presenting cells than controls (cDCs: 1.1% vs 4.5%, p = 0.002; macrophages: 14.4% vs 36.6%, p < 0.001) and 6-fold greater necrosis (67.7% vs 13.5% apoptotic, p < 0.001), suggesting an impaired ability to mount a controlled, adaptive immune response. The stimulatory role of SDMCs was further demonstrated via intracalvarial AMD3100 administration, which through its promotion of myeloid precursor egress (0.26% vs 0.02%, p < 0.001) and cDC2 infiltration (10.4% vs 3.0%, p = 0.003) yielded robust CD4+ T cell activation and effector memory differentiation. These differences were clinically meaningful, as skull irradiation accelerated tumor growth and attenuated survival compared to controls, while the opposite was observed with AMD3100 treatment (median OS: 17d vs 22d, p < 0.001; 26.5 d vs 22 d, p = 0.006). CONCLUSIONS: The preferential polarization of SDMCs into immunostimulatory APCs warrants the continued exploration of local marrow-targeting agents (e.g., AMD3100) as therapeutic modalities.

A fractional mathematical model approach on glioblastoma growth: tumor visibility timing and patient survival

In this paper, we introduce a mathematical model given by
 \begin{equation}
 { }^c \mathfrak{D}_t^\alpha u = \nabla \cdot \mathrm{D} \nabla u + \rho f(u) \quad \text{in } \Omega,
 \end{equation}
 where $f(u)=\frac{1}{1-u/\mathrm{K}}, \, u/\mathrm{K} \neq 1, \, \mathrm{K} > 0$, to enhance established mathematical methodologies for better understanding glioblastoma dynamics at the macroscopic scale. The tumor growth model exhibits an innovative structure even within the conventional framework, including a proliferation term, $f(u)$, presented in a different form compared to existing macroscopic glioblastoma models. Moreover, it represents a further refined model by incorporating a calibration criterion based on the integration of a fractional derivative, $\alpha$, which differs from the existing models for glioblastoma. Throughout this study, we initially discuss the modeling dynamics of the tumor growth model. Given the frequent recurrence observed in glioblastoma cases, we then track tumor mass formation and provide predictions for tumor visibility timing on medical imaging to elucidate the recurrence periods. Furthermore, we investigate the correlation between tumor growth speed and survival duration to uncover the relationship between these two variables through an experimental approach. To conduct these patient-specific analyses, we employ glioblastoma patient data and present the results via numerical simulations. In conclusion, the findings on tumor visibility timing align with empirical observations, and the investigations into patient survival further corroborate the well-established inter-patient variability for glioblastoma cases.

Metformin Reduces Viability and Inhibits the Immunoinflammatory Profile of Human Glioblastoma Multiforme Cells

Glioblastoma (GBM) is the predominant primary malignant brain tumor. Metformin, a well-known antidiabetic medication, has emerged as a potential therapeutic candidate in the treatment of GBM. We have herein investigated two aspects of the effect of MTF on GBM cells: the effect of MTF on GBM cell viability, as previous studies have shown that MTF can selectively affect human GBM tumors; and the immunomodulatory effect of MTF on GBM, as there is evidence that inflammation is associated with GBM growth and progression. The human GBM cell line (U87) was exposed to various doses of MTF (1 mM, 20 mM, and 50 mM), followed by examination of cell viability and inflammatory mediator secretion at various time points. We observed that MTF treatment exerted a dose-response effect on glioblastoma multiforme cell viability. It also had an immunomodulatory effect on GBM cells. Our study identified several mechanisms that led to the overall inhibitory effect of MTF on human GBM. Further inquiry is necessary to gain a better understanding of how these in vitro findings would translate into successful in vivo approaches.

Abstract 7229: In situ trans-differentiation therapy effectively terminates the growth and recurrence of glioblastoma

Abstract Background: Glioblastoma (GBM) is the most common and malignant brain tumor in adults, and remains almost invariably lethal due to its aggressive and invasive nature. The standard first-line treatment for GBM is surgical resection, postoperative radiotherapy and chemotherapy with temozolomide (TMZ), but it exhibits short-term effects due to a high recurrence rate of GBM is its (up to almost one hundred percent). This long-existing clinical challenge has demanded development of novel and effective therapeutic strategies. In situ cell trans-differentiation therapy which direct reprograms tumor cells into terminally differentiated benign cells represents a novel therapeutic strategy for tumors. We further explored the application of in situ trans-differentiation therapy for glioblastoma. Results: In the present study, we have conducted a large-scale screen for transcription factors which can reprogram glioblastoma cells into terminally differentiated postmitotic cells. Over-expression of a combo of selected trans-differentiation factors that possess the highest trans-differentiation rate effectively reprogramed human glioma cells into neuronal-like cells with characteristic neuronal morphology and expression of neuronal genes in culture, as a result substantially reducing glioblastoma cell proliferation and tumor development after orthotopic transplantation. With replicable recombinant oncolytic virus vectors, product candidate can specifically replicate in glioma cells, therefore transcription factors can be specifically delivered to all tumor cells rather than normal cells. In either cell line-derived or patient-derived tumor tissue xenograft models, intra-tumoral injection product candidate inhibited glioblastoma cells growth at an exceptionally high efficiency. Remarkably product candidate combination with TMZ resulted in a complete regression of tumor and no recurrence was observed in animal model cases. Conclusion: In summary, our findings have established a novel therapeutic approach for treating glioblastoma by terminating sustained proliferation of glioma cells via transcription factors-induced in situ cell trans-differentiation and supported further clinic investigations in patients. Citation Format: Huihui Zhao, Zhaozhong Li, Xiaohui Zhang, Congjian Zhao, Zhao Zhu, Yueguang Liu. In situ trans-differentiation therapy effectively terminates the growth and recurrence of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7229.

Juglone induces ferroptosis in glioblastoma cells by inhibiting the Nrf2-GPX4 axis through the phosphorylation of p38MAPK

BackgroundFerroptosis, a non-apoptotic form of cell death induced by accumulation of free iron ions and lipid peroxidation, its importance for cancer treatment is gradually being recognized. Research on the anti-cancer mechanism of juglone is accumulating. However, the specific mechanism by which it directs glioblastoma (GBM) to death is unknown.MethodsWe used in vitro and in vivo experiments to explore the anti-GBM effect generated by juglone through the ferroptosis pathway.ResultsJuglone mainly causes cell death by inducing ferroptosis. Mechanistically, juglone can significantly activate the phosphorylation of p38MAPK. According to transcriptome sequencing and protein interaction analysis, the Nrf2-GPX4 signaling pathway is identified as the primary pathway through which juglone mediates ferroptosis. In vitro and in vivo experiments further verified that juglone induces the ferroptosis of GBM by activating the phosphorylation of p38MAPK and negatively regulating the Nrf2-GPX4 signaling pathway.ConclusionJuglone induces ferroptosis and inhibits the growth of GBM by targeting the Nrf2/Gpx4 signaling pathway and thus holds promise as a novel ferroptosis inducer or anti-GBM drug.

Abstract 3079: AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma growth

Abstract Cancer cells undergo cellular adaptation through metabolic reprogrammin to sustain survival and rapid growth under various stress conditions. However, it remains unknown how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment. We used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify regulatory mechanisms of S/G deprivation induced metabolic flexibility, we employed high throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens. The high throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP+ ratio, and proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and poor prognosis of GBM patients. Our findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells and highlight the potential to target SSP for treating human GBM. Citation Format: Hye Jin Yun, Jong-Ho Lee. AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma growth [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 3079.

Abstract 7596: (TTFields) and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines

Abstract Background: Glioblastoma (GBM) is the most common primary brain malignancy with dismal prognosis. Tumor Treating Fields (TTFields) therapy is available for the treatment of both newly diagnosed and recurrent glioblastoma and represents a new category of treatment modalities in oncologic therapy. Despite decades of study, few FDA approved modalities are available for GBM and provide limited survival improvements for patients. AKT functions as a key serine/threonine kinase in the RTK/PTEN/PI3K pathway, and extensive genomic analysis of GBM has revealed that this pathway is mutated in a significant majority of GBM cases. Activation of this pathway ultimately leads to the activation of AKT, and p-AKT levels are elevated in the majority of GBM tumor samples and cell lines. Studies have shown that increased p-AKT levels contribute to uncontrolled growth, resistance to apoptosis, and enhanced invasive capabilities of glioma cells, which are characteristics of tumor progression in GBM. AKT serves as a critical hub in this pathway, enabling the amplification of growth signals, thereby making the inhibition of AKT an appealing and promising therapeutic target for treating GBM. Imipridone ONC206 is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). Here we show inhibition of p-AKT as well as upregulation of CHOP and caspase-10 following cotreatment of ONC206 and TTFields. Materials & Methods: We investigated the effect of ONC206 (at IC50s) plus TTFields in U251 and T98G human GBM cell lines at different time points. Effect of treatment on cPARP, BCL-2, CHOP, caspase-10, and p-AKT were investigated using western blot. Neurospheres were generated using mentioned cell lines and used for investigating effect of co-treatment on 3D structure. Results: Treatment with ONC206 at the IC50 in T98G cells with TTFields at 24h showed synergistic upregulation of cPARP as well as inhibition of BCL-2. At 96hour time point, inhibition of p-AKT was observed following all treatments (ONC206, TTFields, and co-application compared to control. In U251 cells, following treatment of ONC206 and TTFields, inhibition of BCL-2 was observed at 24h time point at all treatment conditions. Upregulation of CHOP and Casapse-10 at 96h time point was seen, as well as inhibition of p-AKT. Spheroid models showed structure disruption and growth arrest following combination treatment. Conclusion: Here we showed cotreatment results in inhibition of p-AKT, the key node in GBM growth pathway. Also, the upregulation of CHOP and caspase-10 that has not been discussed before can be the key to focus on novel therapeutic targets for GBM More studies are needed to elaborate the exact mechanism of synergy but these new findings can be used to develop novel treatment combination Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian Pinho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. (TTFields) and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines [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 7596.

OGDH and Bcl-xL loss causes synthetic lethality in glioblastoma.

Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the α-ketoglutarate dehydrogenase (OGDH) gene, which encodes an enzyme that is part of the tricarboxylic acid (TCA) cycle, as essential for GBM growth. Moreover, by combining transcriptome and metabolite screening analyses, we discovered that loss of function of OGDH by the clinically validated drug compound CPI-613 was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3 mimetic, ABT263) in patient-derived xenografts as well neurosphere GBM cultures. CPI-613-mediated energy deprivation drove an integrated stress response with an upregulation of the BH3-only domain protein, Noxa, in an ATF4-dependent manner, as demonstrated by genetic loss-of-function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with disruption of the TCA cycle might be a treatment strategy for GBM.

Synthesis of Novel Acetyl-11-keto-β-boswellic Acid Derivatives as Potential Anti-GBM Agents.

Acetyl-11-keto-β-boswellic acid (AKBA) is known to inhibit the growth of glioblastoma (GBM) cells and subcutaneous GBM. A series of acetyl-11-keto-β-boswellic acid (AKBA) derivatives containing the oxime-ester functionality or amide side chains were synthesized, and their anti-GBM activities were evaluated. Some of these compounds exhibited significant inhibitory activity against cell proliferation in U87 and U251 GBM cell lines, with IC50 values in the micromolar concentration range. Cellular thermal shift analysis showed that A-01 and A-10 improved the thermal stability of FOXM1, indicating that these highly active compounds may directly bind to FOXM1 in cells. Docking studies of the two most active compounds, A-01 and A-10, revealed key interactions between these compounds and the active site of FOXM1, in which the amide moiety at the C-24 position was essential for improving the activity. These results suggested that A-10 is a suitable lead molecule for the development of FOXM1 inhibitors. Thus, the rational design of AKBA derivatives with amide side chains holds significant potential for discovering of a new class of triterpenoids capable of inhibiting GBM cell proliferation.

Zeaxanthin impairs angiogenesis and tumor growth of glioblastoma: An in vitro and in vivo study

ObjectivesTo investigate the therapeutic effects of Zeaxanthin (Zea), one of the oxidized xanthophyll carotenoids belonging to the isoprenoids, on inhibiting the angiogenesis and tumor growth of glioblastoma (GBM) via an in vitro and in vivo study. MethodsThe effects of Zea on the proliferation, adhesion, migration and invasion of human GBM cell lines were detected by cell proliferation assay, cell adhesion assay and Transwell assay. The effect of Zea on angiogenesis was detected by rat aortic ring assay and human umbilical vein endothelial cells (HUVEC) in vitro tube formation assay. The effects of Zea on PARP, Caspase 3 and VEGFR2 phosphorylation as well as VEGFR2's downstream signaling pathway were detected by Western blot. The in vivo human GBM xenograft mouse model was employed to study the therapeutic efficacy of Zea. ResultsZea impaired the proliferation, adhesion, migration and invasion of U87 and U251 cells as well as HUVECs. Rat aortic ring experiments displayed Zea significantly inhibited angiogenesis during VEGF-induced microvascular germination. In vitro and in vivo vascular experiments verified that Zea inhibited VEGF-induced HUVEC proliferation and capillary-like tube formation. Additionally, Zea induced GBM cells apoptosis via increasing the expression of cleaved PARP and Caspase 3. In HUVECs and U251 GBM cells, Zea down-regulated VEGF-induced activation of the VEGFR2 kinase pathway. Meanwhile the expression of p-AKT, p-ERK, p-STAT3 and FAK were all attenuated in U251 cells. Moreover, the effects of Zea on GBM cells proliferation could be blocked by VEGFR2 kinase inhibitor SU5408. These results suggest that Zea may hinder GBM angiogenesis and tumor growth through down-regulating a cascade of oncogenic signaling pathways, both through the inhibition of angiogenesis and the anti-tumor mechanism of a direct cytotoxic effect. Besides, Zea inhibits GBM angiogenesis and tumor growth exemplified through a xenograft mouse model in vivo. ConclusionZea impairs angiogenesis and tumor growth of GBM both in vitro and in vivo. It can be declared that Zea is a potential valuable anticancer candidate for the future treatment strategy of GBM.

Cannabinoids in the treatment of glioblastoma.

Glioblastoma (GBM) is the most prevalent primary malignant tumor of the nervous system. While the treatment of other neoplasms is increasingly more efficacious the median survival rate of GBM patients remains low and equals about 14 months. Due to this fact, there are intensive efforts to find drugs that would help combat GBM. Nowadays cannabinoids are becoming more and more important in the field of cancer and not only because of their properties of antiemetic drugs during chemotherapy. These compounds may have a direct cytotoxic effect on cancer cells. Studies indicate GBM has disturbances in the endocannabinoid system-changes in cannabinoid metabolism as well as in the cannabinoid receptor expression. The GBM cells show expression of cannabinoid receptors 1 and 2 (CB1R and CB2R), which mediate various actions of cannabinoids. Through these receptors, cannabinoids inhibit the proliferation and invasion of GBM cells, along with changing their morphology. Cannabinoids also induce an intrinsic pathway of apoptosis in the tumor. Hence the use of cannabinoids in the treatment of GBM may be beneficial to the patients. So far, studies focusing on using cannabinoids in GBM therapy are mainly preclinical and involve cell lines and mice. The results are promising and show cannabinoids inhibit GBM growth. Several clinical studies are also being carried out. The preliminary results show good tolerance of cannabinoids and prolonged survival after administration of these drugs. In this review, we describe the impact of cannabinoids on GBM and glioma cells in vitro and in animal studies. We also provide overview of clinical trials on using cannabinoids in the treatment of GBM.