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Translocator protein 18 kDa (TSPO) acts as an immune resistance gene involved in the T-cell mediated immune control of glioblastoma

ABSTRACT: Glioblastoma is the most malignant and common type of primary brain tumor with a median survival of less than 21 months. It is particularly resistant to current immunotherapies. Thus, yet unknown mechanisms of immune resistance may be active in glioblastoma. Translocator protein 18 kDa (TSPO) expression is upregulated in glioblastoma and correlates with malignancy and poor prognosis but also with increased immune infiltration. An immunomodulatory role of TSPO in glioblastoma has not been reported. Here, we studied the role of TSPO in the regulation of immune resistance of human glioblastoma cells. We used public bulk and single-cell gene expression data to identify cell types, genes and signalling pathways correlating with TSPO expression. To validate inflammatory triggers and the role of TSPO experimentally, we co-cultured tumor-infiltrating T cells and cytotoxic T cell lines with autologous and antigen-loaded allogeneic, primary brain tumor initiating cells (BTICs). TSPO proficient and deficient BTIC lines and clones were generated through genetic manipulation. Inflammatory factors triggering TSPO expression were determined by ELISA and validated experimentally using blocking antibodies and recombinant proteins. The impact of TSPO on glioblastoma cell resistance against cytotoxic T cells was studied by cytotoxicity assays and by activity analysis of components of the apoptotic cascade. Individual death-inducing pathways suppressed by TSPO were determined using recombinant proteins and TSPO-regulated genes associated with apoptosis resistance were identified by gene expression analysis in TSPO proficient and deficient BTICs and subsequent functional analyses. TSPO expression in glioblastoma cells correlated with cytotoxic T cell infiltration, expression of TNFR and IFNGR, the activity of their downstream pathways, expression of TRAIL receptors and with PD-L1. Coculture of BTICs with tumor-reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO protected BTICs selectively against TRAIL-induced apoptosis by regulating both intrinsic and extrinsic apoptosis pathways. TSPO regulated the expression of multiple genes associated with apoptosis-resistance of tumors. Among them, we studied the peptidase inhibitors PI3 and SLPI, which sensitized BTICs towards TRAIL-induced apoptosis. Our data revealed that TSPO expression in glioblastoma cells is induced through T cell derived cytokines IFNγ and TNFα. TSPO expression protects glioblastoma cells against cytotoxic T cell attack through the death inducing ligand TRAIL. Our data provided an indication that therapeutic targeting TSPO may be a suitable approach to sensitize glioblastoma to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL-resistance.

ORGANISM(S): Homo sapiens

PROVIDER: GSE210654 | GEO | 2023/05/17

REPOSITORIES: GEO

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Project description:Background: TRAIL (TNF-related apoptosis inducing ligand) exhibits selective proapoptotic activity in multiple tumor types, while sparing normal cells. This selectivity makes TRAIL an attractive therapeutic candidate for cancer patients. However, despite encouraging activity in preclinical models, clinical trials with TRAIL mimetics/death receptor agonists demonstrated insufficient activity, largely due to emerging resistance to these agents. Herein, we investigated the cytotoxic activity of a novel, TRAIL-based chimeric protein AD-051.4 combining TRAIL and VEGFA-derived peptide sequences, in hematological malignancies. We characterize key molecular mechanisms leading to resistance and propose rational pharmacological combinations sensitizing cells to AD-051.4. Methods: Sensitivity of DLBCL, classical Hodgkin lymphoma, (cHL), Burkitt lymphoma (BL) and acute myeloid leukemia (AML) to AD-051.4 was assessed in vitro with MTS assay and apoptosis tests (Annexin V/PI staining). Markers of apoptosis were assessed using immunoblotting, flow cytometry, or fluorogenic caspase cleavage assays. Resistant cell lines were obtained by incubation with increasing doses of AD-O51. Transcriptomic analyses were performed by RNA sequencing. Sensitizing effects of selected pathway modulators (BCL2, dynamin and HDAC inhibitors) were assessed using MTS/apoptosis assays. Results: AD-051.4 exhibited low-nanomolar cytotoxic activity in DLBCL cells, but not in other lymphoid or AML cell lines. AD-051.4 induced death-receptor (DR) mediated, caspase-dependent apoptosis in sensitive DLBCL cells, but not in primary resistant cells. The presence of DRs and caspase 8 in cancer cells was crucial for AD-O51.4-driven apoptosis. To understand the potential mechanisms of resistance in an unbiased way, we engineered AD-051.4-resistant cells and evaluated resistance-associated transcriptomic changes. Resistant cells exhibited changes in the expression of multiple genes and pathways associated with apoptosis, endocytosis and HDAC-dependent epigenetic reprogramming, suggesting potential therapeutic strategies of sensitization to AD-051.4. In subsequent analyses, we demonstrated that HDAC inhibitors, BCL2 inhibitors and endocytosis/dynamin inhibitors sensitized primary resistant DLBCL cells to AD-051.4 Conclusions: Taken together, we identified rational pharmacologic strategies sensitizing cells to AD-051.4, including BCL2, histone deacetylase inhibitors and dynamin modulators. Since AD-051.4 exhibits favorable pharmacokinetics and an acceptable safety profile, further clinical development is expected. Identification of resistance mechanisms in a clinical setting might indicate a personalized pharmacological approach to override the resistance.

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Translocator protein 18 kDa (TSPO) acts as an immune resistance gene involved in the T-cell mediated immune control of glioblastoma

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