Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo^® and Caspase-3 activity assays, we generated dose–response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

The tumor microenvironment in glioblastoma (GB) is considered to be “cold”, i.e., the fraction of cytotoxic T cells, for instance, is low. Instead, macrophages are the major immune cell population in GB, which stem either from tissue response (resident microglia) or recruitment of macrophages from the periphery, thereby undergoing tumor-dependent “imprinting” mechanisms by which macrophages can adapt a tumor-supportive phenotype. In this regard, it is important to describe the nature of macrophages associated with GB, in particular under therapy conditions using the gold standard chemotherapy drug temozolomide (TMZ). Here, we explored the suitability of combining information from in vivo magnetic resonance spectroscopic (MRS) approaches (metabolomics) with in vitro molecular analyses to assess therapy response and characterize macrophage populations in mouse GB using an isogenic GL261 model. For macrophage profiling, expression levels of matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were determined, since their gene products affect macrophage–tumor cell communication by extensive cleavage of immunomodulatory membrane proteins, such as PD-L1. In tumor mice with an overall therapy response, expression of genes encoding the proteases ADAM8, ADAM10, and ADAM17 was increased and might contribute to the immunosuppressive phenotype of GB and immune cells. In tumors responding to therapy, expression levels of ADAM8 were upregulated by TMZ, and higher levels of PD-L1 were correlated significantly. Using a CRISPR/Cas9 knockout of ADAM8 in GL261 cells, we demonstrated that soluble PD-L1 (sPD-L1) is only generated in the presence of ADAM8. Moreover, primary macrophages from WT and ADAM8-deficient mice showed ADAM8-dependent release of sPD-L1, independent of the macrophage polarization state. Since ADAM8 expression is induced in responding tumors and PD-L1 shedding is likely to decrease the anti-tumor activities of T-cells, we conclude that immunotherapy resistance is caused, at least in part, by the increased presence of proteases, such as ADAM8.

Current standard adjuvant therapy of glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to therapy resistance. Thus, novel therapeutic approaches are highly demanded. We tested the therapeutic efficacy of the second-generation XPO1 inhibitor Eltanexor using assays for cell viability and apoptosis in GBM cell lines and GBM stem-like cells. For most GBM-derived cells, IC₅₀ concentrations for Eltanexor were below 100 nM. In correlation with reduced cell viability, apoptosis rates were significantly increased. GBM stem-like cells presented a combinatorial effect of Eltanexor with TMZ on cell viability. Furthermore, pretreatment of GBM cell lines with Eltanexor significantly enhanced radiosensitivity in vitro. To explore the mechanism of apoptosis induction by Eltanexor, TP53-dependent genes were analyzed at the mRNA and protein level. Eltanexor caused induction of TP53-related genes, TP53i3, PUMA, CDKN1A, and PML on both mRNA and protein level. Immunofluorescence of GBM cell lines treated with Eltanexor revealed a strong accumulation of CDKN1A, and, to a lesser extent, of p53 and Tp53i3 in cell nuclei as a plausible mechanism for Eltanexor-induced apoptosis. From these data, we conclude that monotherapy with Eltanexor effectively induces apoptosis in GBM cells and can be combined with current adjuvant therapies to provide a more effective therapy of GBM.

Glioblastoma (GB) is the most common primary malignant brain tumor in adults. Gboxin, a novel compound that targets oxidative phosphorylation via complex V inhibition, has shown promise in preclinical models of GB. We examined the efficacy of the pharmacokinetically optimized S-Gboxin under conditions replicating the GB microenvironment, including nutrient deprivation and hypoxia. We assessed cytotoxicity and growth-inhibitory effects of S-Gboxin in human GB cell lines, primary GB cultures, as well as immortalized and primary human astrocytes under different nutrient and oxygen deprivation scenarios. Oxygen consumption, cell migration, activation of the integrated stress response (ISR) as well as the relevance of the AMP-activated protein kinase (AMPK) were evaluated as variables under S-Gboxin treatment. S-Gboxin demonstrated cytotoxicity at low micromolar concentrations, with cell death enhanced under nutrient deprivation and hypoxia. S-Gboxin reduced cellular oxygen consumption and uncoupled mitochondria. Cytotoxicity was increased when mitochondrial fuels were the primary energy source. Additionally, S-Gboxin treatment resulted in elevated lactate production and glucose consumption. While the ISR marker ATF4 was induced by S-Gboxin in a dose-dependent manner, ISR inhibition with ISRIB did not affect its cytotoxicity. Conversely, S-Gboxin treatment combined with AMPK inhibition resulted in enhanced tumor cell death. Collectively, these findings demonstrate that S-Gboxin selectively targets cancer-specific metabolic vulnerabilities in GB cells. The synergistic action with AMPK inhibition suggests that this pathway contributes to maintain energy homeostasis in the presence of the drug. Therefore, S-Gboxin is a promising compound for GB therapy, especially in a combinatory approach with AMPK inhibition or other metabolic targeted therapies.

Glioblastoma resists therapy through interconnected survival programs. Combined XPO1, Bcl‐2, and Mcl‐1 inhibition (Eltanexor, Venetoclax, A1210477; EVA) enhances the efficacy of chemotherapy, yet resistance persists. We hypothesized that PI3K/AKT/mTOR (PAM) signaling drives adaptive escape. TCGA/GTEx analyses and immunohistochemistry indicated PAM pathway activation in glioblastoma, with AKT significantly overexpressed, whereas PIK3CA showed a clear trend towards higher expression, although it did not reach statistical significance. qPCR analysis showed that PAM‐component expression was highest in patient‐derived glioblastoma stem‐like cells (GSCs). In U87, U251, and GSCs, the dual PI3K/mTOR inhibitor Dactolisib combined with EVA and 5‐Azacytidine reduced viability and induced apoptosis more effectively than any of the Rapamycin combinations, and the difference in apoptosis was most pronounced in U87 and GSCs. Dactolisib monotherapy shifted the cell cycle towards G1 with a reduced S‐phase fraction. In CAM xenografts, [ ¹⁸ F]FDG‐PET/CT showed decreased tracer uptake, and Caspase‐3 activation with PARP cleavage was detected only under EVA + Dactolisib treatment. Taken together, our results suggest that dual PI3K/mTOR inhibition with Dactolisib disables a critical adaptive survival axis, warranting further preclinical evaluation.