The role of DNA repair mutations and DNA damage in the response to immune checkpoint blockade

Inhibition of the immune checkpoint molecules, PD-1, CTLA-4 and PD-L1 have recently shown great clinical promise in many cancers. We have evidence that inducing DNA damage triggers expression of PD-L1 and activates antigen presentation molecules. Recent clinical reports have shown that mutations in DNA repair genes are a major genetic determinant of response to immune checkpoint inhibitors. We aim to establish the precise impact of DNA repair loss on sensitivity to these inhibitors.
AIM 1. Elucidate how DNA damage induces PDL-1 expression
Our preliminary data suggest that upon DNA repair deficiency and DNA damage induction, expression of PD-L1 is significantly upregulated in tumour cells. Induction of DNA damage can activate the STING pathway, resulting in a transcriptional response leading to type I IFN activation, which in turn has been shown to induce PD-L1 expression. We will elucidate whether DNA damage can activate STING and ultimately prime the type I IFN pathway to upregulate PD-L1.
AIM 2. Determine the significance of specific neoepitope expression upon DNA damage
Somatic mutations can give rise to neoepitopes, which may serve as neoantigens promoting potent anti-tumour T cell responses. We hypothesise that specific tumour neoantigens may be dominant and predict therapeutic benefit to immune checkpoint blockade. To define potential epitope signatures, we will perform tandem mass spectrometric analysis for MHC class I-presented peptides in our DNA repair deficient cell models, before and after DNA damage. Parallel proteomics will be used to estimate source protein abundance and where possible turnover. Using these parameters, we will identify common neo-epitopes in our DNA repair deficient models, using bioinformatics and systems modelling tools. We will combine predictive algorithms with high-throughput peptide binding assays to identify sets of high, medium and low-affinity peptides from low, medium and high abundance proteins. Assays will be performed to determine whether hit peptides can stimulate a T-cell effector response (measured by cytotoxicity and cytokine secretion e.g IFN).

Rotation project:
Our preliminary data suggest that upon DNA repair deficiency and DNA damage induction, expression of the immune checkpoint molecule, PD-L1 is significantly upregulated in tumour cells. However, the precise mechanism underlying this upregulation is unknown. Previous studies have shown that induction of DNA damage by DNA double strand breaks, oxidative DNA damage or loss of the DNA damage response kinase ATM, can prime the Type I Interferon (IFN) pathway. Upon DNA damage, DNA accumulates in the cytoplasm, which activates the STING pathway, resulting in a transcriptional response leading to type I IFN activation. A direct induction of PD-L1 expression by IFN- has been shown. Therefore, we hypothesize that PD-L1 expression is upregulated by DNA damage due to STING-dependent type I IFN signaling. To fully elucidate this hypothesis, we will compare expression of a range of type I IFN genes (IFNB1, IFNAR1, MX1, IFNL1) in our panel of DNA repair deficient and proficient cell lines, before and after DNA damage induction by performing qRT-PCR assays. If these IFN target genes are upregulated, we will block IFN signaling using anti-IFNAR1 under DNA damage conditions and measure PD-L1 expression. To determine whether PD-L1 regulation upon DNA damage is dependent on the activation of the STING pathway, we will use siRNA to target STING pathway components, namely STING, TBK1 and IRF3. This rotation project will help determine the cellular mechanism of immune evasion by PD-L1 in DNA repair-deficient cells upon DNA damage.

Skills Priority Alignment: Advanced Therapeutics and Quantitative Biology