Targeting A20 to Activate Glioblastoma Immune Microenvironment and Improve the Response to Immunotherapy

• Applicant: Dr. Nirmeen Elmadany
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Immunology
• Term: from 2022 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 514016909

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor with a median survival of 12-15 months. GBM is a molecularly heterogenous tumor that inevitably resists conventional therapy via several mechanisms; including immune cell evasion. This suppression of adaptive immunity is to a large extend mediated by myeloid cells. GBM-associated myeloid cells (GAMMs) represent up to 50% of the GBM microenvironment. GAMMs comprise two cell populations; brain-resident microglia and monocyte-derived macrophages. Several studies demonstrated that GAMMs are characterized by immunosuppressive phenotypes, which actively promote tumor growth. Furthermore, GAMMs contribute to the recruitment of peripheral monocytes and T cells through cytokines and chemokines. Although GAMMs are in principle enabled to present antigens on major histocompatibility complex class II molecules, specifically within the GBM microenvironment, CD80/CD86 receptors bind check-point cytotoxic T-lymphocyte-associated Protein 4 (CTLA4) on T cells surface, thereby suppressing their activity. Moreover, GAMMs express programmed cell death protein ligand 1 (PD-L1) which bind to PD1 on T cells surface, another immune check-point protein, to further suppress T cell effector functions. A20 has been emerged as a key modulator of the immune system. A20 suppresses signaling pathways downstream of toll-like receptors (TLR). TLRs are one class of the pattern recognition receptors; expressed by myeloid cells like microglia and macrophages, to activate the innate immunity as a first line defense system. Analyses of publicly available gene expression datasets indicate that A20 is expressed by brain-resident microglia as well as GAMMs. Therefore, targeting A20 in GAMMs might overcome the immunosuppression, and activate T cells to effectively recognize and eliminate glioblastoma cells. In a targeted CRISPR screening to identify druggable targets that suppress macrophages/ microglia, A20 was identified as the top hit of genes as an immune-suppressor. In subsequent preliminary in vitro experiments, A20 was knocked-out in isolated primary mouse and human macrophages. Higher level of proinflammatory chemokines and cytokines was detected. Moreover, co-cultures of primary human macrophages and T cells could indeed activate T cells. In vivo validation of these findings is crucial. Using cutting-edge technologies; including 10X chromium system for transcriptomic analysis which will be integrated with spatial transcriptomics and proteomics, I aim at investigating the impact of A20-ablation in GAMMs on immune cell populations in GBM microenvironment, immune-checkpoint blockade response, glioblastoma cell growth, and mouse overall survival. For this purpose, I designed a mouse model to reproduce the preliminary data and further evolve the project in vivo. Moreover, I planned to use 3D organoid culture from human samples to predict the translation of the data in GBM patients.

• DFG Programme: WBP Position