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

免疫检查点分子PD-1、CTLA-4和PD-L1的抑制最近在许多癌症中显示出巨大的临床前景。我们有证据表明，诱导DNA损伤会触发PD-L1的表达并激活抗原呈递分子。最近的临床报告表明，DNA修复基因的突变是免疫检查点抑制剂反应的主要遗传决定因素。我们的目标是建立DNA修复损失对这些抑制剂敏感性的精确影响。AIM 1.阐明DNA损伤如何诱导PDL-1表达我们的初步数据表明，在DNA修复缺陷和DNA损伤诱导后，PD-L1的表达在肿瘤细胞中显著上调。DNA损伤的诱导可以激活STING途径，导致导致I型IFN激活的转录应答，这反过来又显示出诱导PD-L1表达。我们将阐明DNA损伤是否可以激活STING并最终引发I型IFN途径上调PD-L1。AIM 2。确定DNA损伤后特异性新表位表达的意义体细胞突变可以产生新表位，其可以作为促进有效的抗肿瘤T细胞应答的新抗原。我们假设特异性肿瘤新抗原可能占主导地位，并预测免疫检查点阻断的治疗益处。为了确定潜在的表位特征，我们将在DNA损伤之前和之后，对我们的DNA修复缺陷细胞模型中的MHC I类呈递肽进行串联质谱分析。平行蛋白质组学将用于估计源蛋白质丰度和可能的营业额。使用这些参数，我们将使用生物信息学和系统建模工具，在我们的DNA修复缺陷模型中识别常见的新表位。我们将结合联合收割机预测算法与高通量肽结合试验，以确定从低，中，高丰度蛋白质的高，中，低亲和力肽组。将进行测定以确定命中肽是否可以刺激T细胞效应器应答（通过细胞毒性和细胞因子分泌例如IFN来测量）。Rotation project：我们的初步数据表明，在DNA修复缺陷和DNA损伤诱导时，免疫检查点分子PD-L1的表达在肿瘤细胞中显著上调。然而，这种上调的确切机制尚不清楚。先前的研究表明，DNA双链断裂、氧化性DNA损伤或DNA损伤反应激酶ATM的缺失诱导DNA损伤，可以引发I型干扰素（IFN）途径。在DNA损伤时，DNA在细胞质中积累，其激活STING途径，导致转录应答，导致I型IFN激活。已显示IFN-γ直接诱导PD-L1表达。因此，我们假设PD-L1表达被STING依赖性I型IFN信号转导引起的DNA损伤上调。为了充分阐明这一假设，我们将通过进行qRT-PCR测定，比较DNA损伤诱导前后一系列I型IFN基因（IFNB 1、IFNAR 1、MX 1、IFNL 1）在我们的DNA修复缺陷和熟练细胞系组中的表达。如果这些IFN靶基因上调，我们将在DNA损伤条件下使用抗IFNAR 1阻断IFN信号传导并测量PD-L1表达。为了确定PD-L1对DNA损伤的调节是否依赖于STING途径的激活，我们将使用siRNA靶向STING途径组分，即STING，TBK 1和IRF 3。该轮转项目将有助于确定DNA损伤后DNA修复缺陷细胞中PD-L1免疫逃避的细胞机制。技能优先级对齐：高级治疗学和定量生物学

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 expressionOur 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 damageSomatic 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