Defining the role of persistent DNA bridges in tumor-intrinsic immune activation in hereditary breast and ovarian cancer

项目摘要 染色体分离错误，如持久的DNA桥，加速了基因组的不稳定性，这是遗传学的一个标志。 癌这些毒性DNA损伤在遗传性乳腺癌和卵巢癌（HBOC）中发生频率较高 具有DNA修复缺陷的细胞，从而有缺陷的基因组维持。PARP抑制剂（PARPi）导致 在BRCA突变型HBOC中靶向肿瘤细胞死亡，并已被建议进一步增加 培养的BRCA突变细胞中的DNA桥。尽管PARPi具有治疗前景，但BRCA缺陷型癌症 对这种疗法产生了后天的抵抗力。最近的研究表明，免疫疗法可以预防或延缓 HBOC肿瘤中的PARPi抗性;然而，PARPi与免疫疗法协同作用的机制 仍然定义不清。我的初步数据显示PARPi治疗会导致持久的DNA涂层 与BRCA突变细胞中的先天免疫DNA传感器cGAS连接，这与I型增加一致 干扰素信号传导我假设PARPi诱导的持久性DNA桥是 PARPi的细胞毒性和与免疫疗法的协同作用，因此需要严格的进一步研究，以确定 最大化PARPi治疗在DNA修复缺陷肿瘤中的治疗潜力的策略。的 该提案的总体目标是，从而确定PARPI诱导持续性的机制， DNA桥导致HBOC中的肿瘤内在免疫激活。拟议的研究将调查 这一目标有两个具体目标。目标1将定义PARPi诱导的结构和核完整性 通过使用转座酶介导的荧光成像、免疫荧光、活细胞 成像和相关光学和电子显微镜。这些因素如何影响cGAS-STING激活将是 使用免疫印迹、RT-qPCR和酶免疫测定进行评估。目标2将建立持久的DNA 桥和庞大的后期桥作为反映DNA修复缺陷的潜在预测生物标志物， 染色体分离失败和随后的cGAS-STING激活，在PARPi奥拉帕尼治疗的BRCA- 突变型乳腺癌我将利用苏木精和伊红（H&E）染色样本的数字图像， 免疫组织化学染色的组织微阵列的去识别，配对的临床样本之前和期间 奥拉帕尼治疗，其将在正在进行的磷酸-STING染色的背景下进行询问。定义 染色体不稳定性驱动的免疫激活和肿瘤对PARPi的反应之间的相互作用将是第一个 改善HBOC治疗的一步。该提案将以支持性、协作性和 跨学科的环境，这将使我能够进行基础和转化癌症生物学研究。 这项培训将提高我的实验设计，严格的数据分析和分子生物学技术技能。 生物学、高分辨率显微镜和细胞病理学。我的培训计划也提供了充足的机会， 提高口头表达，写作，教学和指导技能，为我作为一个成功的职业生涯做好准备。 学术癌症生物学家领导一个研究小组，指导和培训下一代科学家。

PROJECT SUMMARY Chromosome segregation errors such as persistent DNA bridges accelerate genome instability, a hallmark of cancer. These toxic DNA lesions occur at a higher frequency in hereditary breast and ovarian cancer (HBOC) cells with DNA repair deficiencies, and thus defective genome maintenance. PARP inhibitors (PARPi) cause targeted tumor cell death in BRCA-mutant HBOC and have been suggested to further increase the frequency of DNA bridges in cultured BRCA-mutant cells. Despite the therapeutic promise of PARPi, BRCA-deficient cancers develop acquired resistance to this therapy. Recent studies show that immunotherapies can prevent or delay PARPi resistance in HBOC tumors; however, the mechanisms by which PARPi synergizes with immunotherapies remain poorly defined. My preliminary data shows that PARPi treatment leads to coating of persistent DNA bridges with the innate immune DNA sensor cGAS in BRCA-mutant cells, which coincides with increased type I interferon signaling. I hypothesize that PARPi-induced persistent DNA bridges are central to the mechanism of PARPi cytotoxicity and synergy with immunotherapies, therefore requiring rigorous further study to identify strategies that maximize the therapeutic potential of PARPi treatment in DNA repair-deficient tumors. The overall objective of this proposal is to thus define the mechanisms by which PARPi-induced persistent DNA bridges lead to tumor-intrinsic immune activation in HBOC. The proposed research will investigate this objective in two specific aims. Aim 1 will define the structure and nuclear integrity of PARPi-induced persistent bridges by use of transposase-mediated fluorescence imaging, immunofluorescence, live-cell imaging, and correlative light and electron microscopy. How these factors affect cGAS-STING activation will be assessed using immunoblotting, RT-qPCR, and enzyme immunoassays. Aim 2 will establish persistent DNA bridges and bulky anaphase bridges as potential predictive biomarkers that reflect DNA repair defects, chromosome segregation failure, and subsequent cGAS-STING activation in PARPi olaparib-treated BRCA- mutant breast cancer. I will leverage digital images of hematoxylin and eosin (H&E)-stained samples and perform immunohistochemistry staining on a tissue microarray of de-identified, paired clinical samples before and during olaparib treatment, which will be interrogated in the context of ongoing phospho-STING staining. Defining the interplay between chromosomal instability-driven immune activation and tumor response to PARPi will be a first step towards improved HBOC treatment. This proposal will be completed in a supportive, collaborative, and interdisciplinary environment that will allow me to perform fundamental and translational cancer biology research. This training will advance my experimental design, rigorous data analysis, and technical skills in molecular biology, high-resolution microscopy, and cytopathology. My training plan also provides ample opportunities to improve oral presentation, writing, teaching, and mentorship skills to prepare me for a successful career as an academic cancer biologist leading a research group to mentor and train the next generation of scientists.