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

• 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
• 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

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.

项目摘要 染色体隔离错误，例如持续的DNA桥加速基因组不稳定性，这是一个标志 癌症。这些有毒的DNA病变在遗传性乳腺癌和卵巢癌（HBOC）中发生较高的频率。 具有DNA修复缺陷的细胞，因此维持基因组缺陷。 PARP抑制剂（PARPI）原因 BRCA突变HBOC的靶向肿瘤细胞死亡，并已被提议进一步增加 培养的BRCA突变细胞中的DNA桥。尽管有parpi的治疗诺言，但BRCA缺陷癌症 发展对这种疗法的抗药性。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；但是，PARPI与免疫疗法协同作用的机制 保持较差。我的初步数据表明，PARPI治疗导致持续DNA的涂层 BRCA突变细胞中与先天免疫DNA传感器CGA的桥梁，这与I型增加相吻合 干扰素信号传导。我假设parpi引起的持续DNA桥是机制的核心 PARPI细胞毒性和与免疫疗法的协同作用，因此需要进行严格的进一步研究以识别 在DNA修复缺陷型肿瘤中最大化PARPI治疗的治疗潜力的策略。这 该提案的总体目的是为了定义parpi诱导的持久性的机制 DNA桥导致HBOC中的肿瘤内部免疫激活。拟议的研究将调查 这个目标是两个具体目标。 AIM 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，活细胞的持续桥梁 成像以及相关的光和电子显微镜。这些因素如何影响CGAS插入激活 使用免疫印迹，RT-QPCR和酶免疫测定评估。 AIM 2将建立持久的DNA 桥梁和笨重的后期桥作为反映DNA修复缺陷的潜在预测生物标志物， 染色体隔离失败，随后在Parpi Olaparib处理的BRCA中激活CGA 突变乳腺癌。我将利用苏木精和曙红（H＆E）染色的样品的数字图像并执行 在组织微阵列上进行的免疫组织化学染色，以在 Olaparib治疗，将在持续的磷酸化染色的背景下进行讯问。定义 染色体不稳定性驱动的免疫激活与对PARPI的肿瘤反应之间的相互作用将是第一个 迈向改善HBOC治疗。该建议将以支持，协作和 跨学科环境将使我能够进行基本和转化的癌症生物学研究。 该培训将推进我的实验设计，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足够的机会 提高口头陈述，写作，教学和指导能力，以使我为成功的职业做好准备 学术癌症生物学家领导研究小组指导和培训下一代科学家。