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

抑制免疫检查点分子PD-1， CTLA-4和PD-L1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损伤触发PD-L1的表达并激活抗原递呈分子。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。我们的目标是确定DNA修复损失对这些抑制剂敏感性的精确影响。目的1。我们的初步数据表明，在DNA修复缺陷和DNA损伤诱导下，肿瘤细胞中PD-L1的表达显著上调。DNA损伤的诱导可以激活STING通路，导致I型IFN激活的转录反应，进而诱导PD-L1表达。我们将阐明DNA损伤是否可以激活STING并最终启动I型IFN通路上调PD-L1。目标2。确定特异性新表位表达对DNA损伤的意义体细胞突变可以产生新表位，它可能作为新抗原促进有效的抗肿瘤T细胞反应。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效果。为了确定潜在的表位特征，我们将对DNA修复缺陷细胞模型中DNA损伤前后的MHC i类肽进行串联质谱分析。平行蛋白质组学将用于估计源蛋白丰度和可能的周转。使用这些参数，我们将使用生物信息学和系统建模工具在DNA修复缺陷模型中识别常见的新表位。我们将结合预测算法和高通量肽结合分析，从低、中、高丰度蛋白质中识别高、中、低亲和肽。将进行检测以确定击中肽是否能刺激t细胞效应反应（通过细胞毒性和细胞因子分泌（如IFN）来测量）。轮换项目：我们的初步数据表明，在DNA修复缺陷和DNA损伤诱导下，肿瘤细胞中免疫检查点分子PD-L1的表达显著上调。然而，这种上调背后的确切机制尚不清楚。先前的研究表明，DNA双链断裂、DNA氧化损伤或DNA损伤反应激酶ATM的缺失诱导DNA损伤，可以启动I型干扰素（IFN）途径。当DNA损伤时，DNA在细胞质中积累，激活STING通路，导致转录反应，导致I型IFN激活。IFN-直接诱导PD-L1表达已被证实。因此，我们假设由于sting依赖性I型IFN信号导致的DNA损伤导致PD-L1表达上调。为了充分阐明这一假设，我们将通过进行qRT-PCR分析，比较DNA修复缺陷和熟练细胞系在DNA损伤诱导前后一系列I型IFN基因（IFNB1、IFNAR1、MX1、IFNL1）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ifnar1阻断IFN信号传导，并测量PD-L1的表达。为了确定PD-L1对DNA损伤的调节是否依赖于STING通路的激活，我们将使用siRNA靶向STING通路组分，即STING、TBK1和IRF3。这个轮换项目将有助于确定DNA修复缺陷细胞中PD-L1在DNA损伤时逃避免疫的细胞机制。技能优先排序：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