Uncovering ATR signaling networks underlying chemotherapeutic synergies

揭示化疗协同作用背后的 ATR 信号网络

• 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
• 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

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.

项目总结/摘要 DNA损伤传感器激酶ATR是人类细胞生存所必需的，在细胞耐受中起着至关重要的作用 DNA复制压力1 -3。癌细胞通常具有由于癌基因的复制应激水平升高的特征。 作用，因此易受ATR抑制剂的影响，使ATR成为抗癌治疗的主要靶标4 -7。 虽然ATR抑制剂已被证明与复制应激诱导因子组合特别有效，但其在治疗中的作用是非常重要的。 虽然这些药物协同作用的分子机制仍然知之甚少，主要是由于 ATR信令网络的绝对复杂性5，8-11。为了揭示这些信号事件的基础， 治疗协同作用，我将利用质谱进行公正和有针对性的磷酸蛋白质组学 在用不同化疗剂处理的癌细胞系中筛选显示与ATR协同作用 抑制剂的我在HCT 116癌细胞中进行的试验性无偏筛选已经开始识别和量化ATR 响应于化学疗法CPT和奥拉帕尼的信号传导，允许每个响应被条形码化以用于直接识别。 对比比较先导条形码揭示了每种药物诱导的ATR信号传导的显著差异， 这也提出了关于这种ATR信号是否在对这些耐药的癌症之间进一步变化的问题。 化疗8，12.此外，在研究CPT诱导的ATR信号转导时，我们发现了一组非- 典型的信号事件是独立的DNA切除，一个过程所需的激活 典型ATR信号1，13.这些非经典信号底物包括参与溶解的蛋白质 在CPT处理后丰度增加并有助于基因组的DNA：RNA杂合体 不稳定性14 -16.面对这些数据，我假设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信号传导通过调节而有助于CPT抗性 DNA：RNA杂交过程。我的目标是扩展CPT和奥拉帕尼诱导ATR的信号条形码 通过放大磷酸化蛋白质组学筛选， 发现了该文库将使得能够跨跨细胞的ATR信号传导应答的高通量靶向条形码化。 对CPT、奥拉帕尼和ATR抑制剂联合治疗具有差异性耐药的癌症，允许相关性 癌症特异性ATR信号的化疗耐药性。我还打算探讨小说的作用 利用遗传、生化和细胞研究癌症对CPT耐药性中的非切除依赖性ATR信号传导事件 生物技术总体而言，结果将描述药物和癌症特异性ATR反应，并建立 ATR信号传导的新模式对化疗结果的贡献。生成的知识将作为 ATR抑制剂化疗协同作用机制的全系统解剖框架 和耐药性，可能揭示药物靶点和更有选择性的联合治疗的机会。