Novel TNFα-based strategies to overcome immune evasion in diffuse intrinsic pontine glioma

• Applicant: Dr. Theophilos Tzaridis
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Hematology, Oncology ; Pediatric and Adolescent Medicine
• Term: from 2021 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 454298163

Diffuse intrinsic pontine glioma (DIPG) is a devastating primary brain tumour in desperate need of novel treatment strategies. One exciting new paradigm for treatment is immunotherapy. We recently found that DIPG cells with p53 mutations lack surface MHC-I, which makes them resistant to killing by cytotoxic T cells. MHC-I can be restored by treatment with Tumor Necrosis Factor α (TNFα), which enhances T-cell mediated killing and increases responsiveness to immune checkpoint inhibition. Notably, our previous studies used only one model of DIPG, raising questions about the generalisability of our findings. Moreover, only one checkpoint inhibitor (anti-PD-1) was tested in combination with TNFα, and 50% of the mice succumbed to their tumours, highlighting the importance of testing other checkpoint inhibitors and investigating the mechanisms of resistance to this therapy. To address these questions, we will first study the mechanisms of MHC-I regulation by p53 in DIPG cells and primary patient samples. Expression of MHC-I, as well as ERAP1 and TAP1 – key regulators of antigen processing and MHC-I localisation – will be assessed in p53-wild type and p53-deficient murine and human DIPG cell lines and patient samples. Next, sensitivity of human DIPG cells to T-cell mediated killing will be tested in vitro by co-culture with human T-cells. To identify the most effective checkpoint molecules to combine with TNFα, expression of checkpoint molecules will be studied in murine and human DIPG cells and in primary patient material. Based on these data, inhibitors of the most widely expressed checkpoint molecules will be tested, alone or in combination with TNFα, in two murine models of DIPG. Finally, to determine whether combinations of checkpoint inhibitors are more effective than single agents, TNFα will be combined with the two most promising checkpoint inhibitors identified in the previous experiments. Although we expect that combining TNFα with checkpoint inhibitors will lead to enhanced responses, DIPG is a highly aggressive tumour and resistance may develop as a result of changes in tumour cells themselves (e.g. via downregulation of MHC-I or tumour-associated antigens) or in their microenvironment (e.g. through impaired T-cell infiltration or upregulation of immunosuppressive signals). To understand the mechanisms of resistance, we will use single-cell RNA sequencing to compare gene expression in untreated mice and in mice that have developed resistance to TNFα + anti-PD-1 therapy. Moreover, we will perform digital spatial profiling of tumour sections from these mice to examine the spatial distribution of relevant immune cells and their transcriptome profiles relative to tumour cells. Overall, this project aims to identify biomarkers of non-responsiveness to T cell-based immunotherapy and novel ways to enhance the response to this therapeutic modality in DIPG, thereby improving both the quality and length of patients’ lives.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Robert Wechsler-Reya, Ph.D.