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

确定持久性 DNA 桥在遗传性乳腺癌和卵巢癌肿瘤内在免疫激活中的作用

基本信息

批准号：
10606942
负责人：
Ece Kocak
金额：
$ 4.67万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10606942
关键词：
Acceleration Affect Anaphase Antibodies Archives Automobile Driving BRCA deficient BRCA1 gene Basic Science Biological Assay Biology Biopsy Breast Cancer Patient Cancer Biology Cancer cell line Cell Cycle Cell Death Cells Chromatids Chromosomal Instability Chromosome Segregation Clinical Cultured Cells Cytopathology Cytoplasm DNA DNA Repair DNA Repair Disorder DNA biosynthesis DNA lesion Data Data Analyses Defect Development Double Strand Break Repair Educational process of instructing Electron Microscopy Enzyme Immunoassay Enzyme-Linked Immunosorbent Assay Exhibits Experimental Designs Failure Frequencies Functional disorder Genome Genomic Instability Hematoxylin and Eosin Staining Method Hereditary Breast and Ovarian Cancer Syndrome Imaging Techniques Immune Immunofluorescence Immunologic Immunohistochemistry Immunooncology Immunotherapy Innate Immune Response Interferon Type I Knowledge Label Lead Maintenance Malignant Neoplasms Malignant neoplasm of ovary Mediating Mentors Mentorship Microscopy Mitotic Molecular Molecular Biology Mutagens Nuclear Nuclear Envelope Oral Paraffin Embedding Pathology Pathway interactions Poly(ADP-ribose) Polymerase Inhibitor Preparation Prevalence Production Proteins Research Resistance Resolution Role Rupture Sampling Scientist Signal Transduction Somatic Cell Stains Stimulator of Interferon Genes Stretching Structure Surface Technical Expertise Testing Therapeutic Tissue Microarray Tissues Training Translational Research Transposase Western Blotting Work Writing XCL1 gene cancer cell cancer therapy career collaborative environment cytokine cytotoxicity digital imaging fluorescence imaging immune activation immunoregulation improved in situ imaging innate immune mechanisms insight light microscopy live cell imaging mRNA Expression malignant breast neoplasm mutant neoplastic cell next generation novel novel marker potential biomarker predictive marker prevent recruit repaired replication stress response segregation sensor skills supportive environment synergism telomere therapy resistant tool tumor

项目摘要

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.

项目概要染色体分离错误，例如持久性 DNA 桥，会加速基因组不稳定，这是癌症。这些有毒 DNA 损伤在遗传性乳腺癌和卵巢癌 (HBOC) 中发生的频率较高 DNA修复缺陷的细胞，因此基因组维护有缺陷。 PARP 抑制剂 (PARPi) 引起 BRCA 突变 HBOC 中的靶向肿瘤细胞死亡，并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 缺陷型癌症对该疗法产生获得性耐药性。最近的研究表明，免疫疗法可以预防或延迟 HBOC 肿瘤中的 PARPi 耐药性；然而，PARPi 与免疫疗法协同作用的机制仍然不明确。我的初步数据显示 PARPi 处理导致持久性 DNA 涂层与 BRCA 突变细胞中的先天免疫 DNA 传感器 cGAS 桥接，这与 I 型增加一致干扰素信号传导。我假设 PARPi 诱导的持久 DNA 桥是该机制的核心 PARPi 细胞毒性以及与免疫疗法的协同作用，因此需要严格的进一步研究来确定最大化 PARPi 治疗 DNA 修复缺陷肿瘤的治疗潜力的策略。这该提案的总体目标是定义 PARPi 诱导的持续性机制 DNA 桥导致 HBOC 中肿瘤内在免疫激活。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、活细胞建立持久桥成像、相关光学和电子显微镜。这些因素如何影响 cGAS-STING 激活使用免疫印迹、RT-qPCR 和酶免疫分析进行评估。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， PARPi 奥拉帕尼治疗的 BRCA 中染色体分离失败以及随后的 cGAS-STING 激活突变乳腺癌。我将利用苏木精和曙红 (H&E) 染色样品的数字图像并执行在之前和期间对去识别的配对临床样本的组织微阵列进行免疫组织化学染色 olaparib 治疗，将在正在进行的磷酸 STING 染色的背景下进行询问。定义染色体不稳定驱动的免疫激活与肿瘤对 PARPi 反应之间的相互作用将是第一个朝着改善 HBOC 治疗迈出了一步。该提案将在支持、协作和跨学科环境将使我能够进行基础和转化癌症生物学研究。这次培训将提高我的实验设计、严格的数据分析和分子技术技能生物学、高分辨率显微镜和细胞病理学。我的培训计划还提供了充足的机会提高口头表达、写作、教学和指导技能，为我作为一名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。