Augmenting Anti-Tumor Immunity Using Radiation in the Setting of DNA Repair Defects

尽管PARP抑制剂被批准用于治疗晚期BRCA突变乳腺癌，但进展 无银存活时间仍然很短，只有7个月。这是一个很大的问题，因为一旦PARP 抑制剂失败，几乎没有全身性治疗选择，并且疾病无法治愈，导致需要 替代治疗策略。增强对肿瘤的免疫应答的一种策略是放射治疗（RT）， 其通过释放抗原和免疫刺激物刺激先天性和适应性免疫应答， 保守党调解员。然而，对于RT引发免疫刺激的物理机制知之甚少。 这些信号以及它们如何在DNA损伤和DNA修复缺陷的背景下被利用。我们 建议研究免疫刺激信号如何在RT诱导的DNA损伤的背景下被调制- 年龄和DNA修复缺陷，特别是BRCA 1突变。我们的初步数据表明BRCA 1突变 与质子RT组合，一种与质子RT相比诱导相对更多的成簇DNA损伤的RT形式。 光子RT，增强更高水平的微核-一种涉及循环免疫刺激的前体 GMP-AMP合酶（cGAS）和干扰素（IFN）基因刺激物（STING）途径。我们假设 DNA修复缺陷与高LET RT结合通过cGAS-1增强免疫应答。 STING途径。为了测试这一点，我们建议：1。确定高LET RT（质子）与低LET RT （光子）通过cGAS-STING途径影响具有BRCA 1缺陷的细胞的免疫原性。在 为此，我们将确定质子是否增强cGAS-STING传感途径介导的抗肿瘤免疫。 BRCA 1突变体和PARP中的免疫信号传导相对于光子抑制肿瘤。2.在BRCA 1中确定- 缺陷鼠乳腺肿瘤模型，无论是高LET RT（质子）还是低LET RT（光子）， 影响免疫原性，单独和在PARP和PDL 1抑制剂的情况下。我们将使用同源基因 具有和不具有功能性BRCA 1的小鼠肿瘤模型，以评价质子相对于单独光子的作用 以及PARP和PD 1抑制剂。我们将通过以下方法评估cGAS-STING途径的差异活化： 评估微核水平和STING、TBK 1、IRF 3和IFN-I的活化，并通过定量炎症反应， 细胞因子和RT后肿瘤浸润免疫细胞成分的改变。 该项目将产生创新的临床前数据，将RT的质量和cGAS-STING的激活直接联系起来 介导的抗肿瘤免疫信号传导。我们的研究有可能定义高LET RT作为一种方法， 增强侵袭性BRCA 1突变肿瘤患者的免疫应答，可能还包括其他肿瘤患者 DNA修复缺陷我们提出的工作是创新的，因为它的目的是确定集群的影响， 在抗肿瘤免疫重建的背景下，BRCA 1突变肿瘤上的高LET RT产生的DNA损伤 sponse，这些发现可能会为利用RT的独特物理特性作为工具带来新的机会 来激活免疫系统对抗癌症

Despite the approval of PARP inhibitors for the treatment of advanced BRCA-mutated breast cancer, progres- sion-free survival times are still quite short at only 7 months. This is a significant problem because once PARP inhibitors fail, few systemic treatment options remain and the disease cannot be cured, leading to the need for alternative treatment strategies. One strategy to enhance the immune response to tumors is radiotherapy (RT), which stimulates innate and adaptive immune responses through the release of antigens and immunostimula- tory mediators. However, relatively little is known about the physical mechanisms of RT that elicit immunostim- ulatory signals and how they can be harnessed in the context of DNA damage and DNA repair defects. We propose to investigate how immunostimulatory signals are modulated in the context of RT-induced DNA dam- age and DNA repair defects, particularly BRCA1 mutation. Our preliminary data indicate that BRCA1 mutation in combination with proton RT, a form of RT that induces relatively more clustered DNA lesions compared to photon RT, potentiates higher levels of micronuclei–a precursor of immunostimulation involving the cycling GMP-AMP synthase (cGAS) and stimulator of interferon (IFN) genes (STING) pathways. We hypothesize that DNA repair deficiency in combination with high-LET RT enhances an immune response through the cGAS- STING pathway. To test this we propose to: 1. Determine whether high-LET RT (protons) vs. low-LET RT (photons) influence the immunogenicity of cells with BRCA1 defects via the cGAS-STING pathway. In this aim, we will determine whether protons enhance cGAS-STING sensing pathway mediated anti-tumor im- mune signaling in BRCA1-mutant and PARP inhibited tumors relative to photons. 2. Determine in a BRCA1- defective murine mammary tumor model whether-high LET RT (protons) vs. low-LET RT (photons) in- fluence immunogenicity, alone and in the setting of PARP and PDL1 inhibitors. We will use syngeneic mouse tumor models with and without functional BRCA1 to evaluate the effect of protons vs. photons alone and with PARP and PD1 inhibitors. We will evaluate the differential activation of the cGAS-STING pathway by assessing micronuclei levels and activation of STING, TBK1, IRF3 and IFN-I and by quantifying inflammatory cytokines and alterations in tumor infiltrating immune cell components after RT. Successful completion of this project will generate innovative preclinical data to directly link the quality of RT and activation of cGAS-STING mediated anti-tumor immune signaling. Our research has the potential to define high LET RT as a way to aug- ment the immune response for patients with aggressive, BRCA1-mutated tumors and possibly for other tumors with DNA repair deficiency. Our proposed work is innovative in that it aims to define the effects of clustered DNA lesions generated by high LET RT on BRCA1 mutated tumors in the context of anti-tumor immune re- sponse, findings which may lead to new opportunities for harnessing unique physical properties of RT as a tool to activate the immune system to combat cancer.