Uncovering ATR signaling networks underlying chemotherapeutic synergies

揭示化疗协同作用背后的 ATR 信号网络

基本信息

批准号：
10679717
负责人：
William Comstock
金额：
$ 4.42万
依托单位：
CORNELL 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 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10679717
关键词：
Address Bar Codes Biochemical Biological Camptothecin Cancer cell line Cell Survival Cells Chemicals Chemoresistance Chronic Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Colon Carcinoma Combined Modality Therapy DNA DNA Damage DNA Repair DNA biosynthesis Data Dissection Drug Synergism Drug Targeting Event Excision Exhibits Generations Genes Genetic Genomic Instability HCT116 Cells Human Hybrids Intelligence Knowledge Libraries Malignant Neoplasms Maps Mass Spectrum Analysis Molecular Molecular Profiling Monitor Mutate Oncogenes Outcome Pharmaceutical Preparations Pharmacotherapy Phosphorylation Phosphotransferases Play Poison Poly(ADP-ribose) Polymerase Inhibitor Predisposition Process Proteins RNA RNA Processing Regulation Replication-Associated Process Resistance Resolution Role Signal Induction Signal Transduction Source Structure Synthetic Peptide Libraries System Techniques Therapeutic Topoisomerase Work cancer cell cancer therapy cancer type cell type chemotherapeutic agent chemotherapy clinically relevant ds-DNA experimental study genotoxicity inhibitor insight novel nuclease overexpression phosphoproteomics refractory cancer replication stress response ribonuclease H1 scale up sensor stressor synergism therapy outcome

项目摘要

PROJECT SUMMARY/ABSTRACT The DNA damage sensor kinase ATR is essential for human cell viability and plays a crucial role in cell tolerance to DNA replication stress1–3. Cancer cells often feature elevated levels of replication stress due to oncogene action and are therefore susceptible to inhibitors of ATR, making ATR a prime target in anti-cancer therapies4–7. While ATR inhibitors have been shown to be particularly effective in combination with replication stress-inducing agents, the molecular mechanisms behind these drug synergies remain poorly understood, primarily due to the sheer complexity of the ATR signaling network5,8–11. To uncover the signaling events that underlie these therapeutic synergies, I will utilize mass spectrometry to perform both unbiased and targeted phosphoproteomic screens in cancer cells lines treated with different chemotherapeutic agents shown to synergize with ATR inhibitors. Pilot unbiased screens I conducted in HCT116 cancer cells have begun to identify and quantify ATR signaling in response to chemotherapies CPT and Olaparib, allowing each response to be barcoded for direct comparison. Comparing pilot barcodes revealed dramatic differences in ATR signaling induced by each drug, also raising questions regarding whether such ATR signaling varies further between cancers resistant to these chemotherapies8,12. Moreover, when investigating CPT-induced ATR signaling, we uncovered a set of non- canonical signaling events that are independent of DNA resection, a process required for the activation of canonical ATR signaling1,13. These non-canonical signaling substrates include proteins involved in the dissolution of R-loops: DNA:RNA hybrids that increase in abundance upon CPT treatment and contribute to genome instability14–16. Faced with these data, I hypothesize that ATR signaling varies between drug treatments and cancer cell types, and that non-canonical ATR signaling contributes to CPT resistance via regulation of DNA:RNA hybrid processing. I aim to expand signaling barcodes for CPT and Olaparib-induced ATR responses several fold via scaled up phosphoproteomic screens, curating a synthetic peptide library from events uncovered. This library will enable high-throughput targeted barcoding of ATR signaling responses across cancers differentially resistant to CPT, Olaparib, and ATR inhibitor combination therapy, allowing for correlation of chemotherapy resistance with cancer-specific ATR signaling. I also aim to investigate the role of novel resection-independent ATR signaling events in cancer resistance to CPT by using genetic, biochemical, and cell biological techniques. Overall, results will delineate drug- and cancer-specific ATR responses and establish the contribution of a new mode of ATR signaling to chemotherapy outcomes. Generated knowledge will serve as a framework for systems-wide dissection of the mechanisms underlying ATR inhibitor chemotherapeutic synergies and resistances, potentially revealing drug targets and opportunities for more selective combination therapies.

项目概要/摘要 DNA 损伤传感器激酶 ATR 对于人类细胞活力至关重要，并且在细胞耐受性中发挥着至关重要的作用 DNA 复制压力1-3 导致癌细胞通常因癌基因而出现复制压力水平升高。作用，因此对 ATR 抑制剂敏感，使 ATR 成为抗癌治疗的主要靶点4-7。虽然 ATR 抑制剂已被证明与复制应激诱导结合特别有效但这些药物协同作用背后的分子机制仍然知之甚少，这主要是由于 ATR 信号网络的复杂性5,8-11 揭示了这些信号背后的信号事件。治疗协同作用，我将利用质谱法进行无偏和靶向磷酸化蛋白质组学在用不同化疗药物治疗的癌细胞系中进行的筛选显示与 ATR 具有协同作用我在 HCT116 癌细胞中进行的试点无偏筛选已开始识别和量化 ATR。响应化疗 CPT 和 Olaparib 的信号传导，允许对每个响应进行条形码以直接比较试点条形码揭示了每种药物诱导的 ATR 信号的显着差异，还提出了这样的问题：这种 ATR 信号传导在对这些药物耐药的癌症之间是否存在进一步差异？此外，在研究 CPT 诱导的 ATR 信号传导时，我们发现了一组非- 独立于 DNA 切除的典型信号事件，DNA 切除是激活 DNA 所需的过程典型的 ATR 信号传导 1,13 这些非典型的信号传导底物包括参与溶解的蛋白质。 R 环：DNA:RNA 杂交体，在 CPT 处理后丰度增加并对基因组做出贡献面对这些数据，我发现 ATR 信号在药物治疗之间存在差异。和癌细胞类型，并且非经典 ATR 信号通过调节导致 CPT 耐药我的目标是扩展 CPT 和奥拉帕尼诱导的 ATR 的信号条形码。通过扩大磷酸蛋白质组筛选反应数倍，从事件中构建合成肽库该库将实现跨 ATR 信号响应的高通量靶向条形码。癌症对 CPT、奥拉帕尼和 ATR 抑制剂联合治疗有不同的耐药性，从而可以进行相关性分析我还旨在研究新型 ATR 信号传导对化疗耐药的影响。利用遗传、生化和细胞研究癌症对 CPT 耐药性中与切除无关的 ATR 信号传导事件总体而言，结果将描述药物和癌症特异性的 ATR 反应并建立所产生的知识将作为一种新的 ATR 信号传导模式对化疗结果的贡献。 ATR 抑制剂化疗协同作用机制的全系统剖析框架和耐药性，可能揭示药物靶点和更具选择性的联合疗法的机会。