Multiplex FRET Imaging of Kinase-Epigenome Interregulations in Live Cancer Cells

活体癌细胞中激酶-表观基因组相互调控的多重FRET成像 激酶抑制剂已被应用于缓解胰腺癌的发展。然而，适应性表观遗传学 包括组蛋白调节在内的反应可以导致大规模基因表达的改变，这可以 最终导致癌细胞的异质性耐药反应和危及生命的肿瘤复发 疾病。在目前阶段，酪氨酸激酶活性如何动态地与 表观遗传组蛋白调节以确定癌细胞对药物治疗的反应。因此，调查 而操纵组蛋白调节和密码的调节在癌症治疗中具有重要意义 和药物筛查。在这个提案中，我们将利用定向进化和高内容筛选的力量 方法系统研制动态荧光共振能量转移(FRET)生物传感器 监测和定量单细胞中关键的组蛋白甲基化(H3K4、H3K9、H3K27)。我们还将 应用此方法优化粘着斑激酶(FAK)FRET生物传感器用于FAK的可视化 激活酶活性。与我们优化的现有Src FRET生物传感器一起，这些生物传感器将被 使用CRIPSR整合到胰腺癌细胞的基因组中，以最小化信号的异质性 跨越不同的单个细胞。我们将进一步加入新的FRET对，这些FRET对发出与 流行的FRET对(CFP和YFP)，用于同时监测同一活细胞中的两个不同信号，例如一个 组蛋白甲基化和一种激酶的激活。使用共同的分子信号作为跨不同 单个细胞，这些关键的分子事件将与相关的FRET成像方法一起映射 (CFIM)是我们实验室开发的，用于生成动态组-表观组相互作用的动态图景。 药理试剂将被用来研究它们对这些分子动态格局的影响 相互作用和适应性表观遗传反应。然后我们将把这些多重分子图谱关联到 在这些药理试剂下的癌症转归，并因此提供量化的多重指数 在单细胞水平上评估药物疗效，目标是将耐药性降至最低。三个具体目标是 据此提出：(1)开发和优化组蛋白甲基化和酪氨酸激酶FRET生物传感器；(2) 应用CRISPR将FRET生物传感器基因工程导入胰腺癌细胞系用于校准 在单细胞中的抑制效果；(3)组蛋白甲基化和激酶活性的多重成像 用于评估对激酶抑制的适应性表观遗传反应的PDAC细胞。鉴于其重要性和紧迫性 需要新的成像工具来研究癌细胞中的动态组-表观组连接，开发了FRET 生物传感器和成像系统应该为解开癌症的分子网络提供强有力的手段 生物学，并允许以最小的耐药性进行药物筛选的多路和高通量平台。因此， 该项目的成功将为癌症研究和研究领域贡献变革性的使能技术 药剂学，朝着根除胰腺癌的最终目标前进。

Multiplex FRET Imaging of Kinase-Epigenome Interregulations in Live Cancer Cells Kinase inhibitors have been applied to mitigate pancreatic cancer development. However, adaptive epigenetic responses including histone modulations can lead to the alteration of large scale gene expressions which can ultimately result in heterogeneous drug resistant responses of cancer cells and life-threatening relapse of diseases. At the current stage, it remains unclear how tyrosine kinase activities are dynamically coupled with epigenetic histone modulations to determine cancer cell responses upon drug treatment. Therefore, investigating and manipulating the regulation of histone modulations and codes have crucial implications in cancer treatment and drug screening. In this proposal, we will harness the power of directed evolution and high-content screening methods to systematically develop fluorescence resonance energy transfer (FRET) biosensors for the dynamic monitoring and quantification of crucial histone methylations (H3K4, H3K9, H3K27) in single cells. We will also apply this approach to optimize a focal adhesion kinase (FAK) FRET biosensor for the visualization of FAK kinase activity. Together with an existing Src FRET biosensor optimized by us, these biosensors will be incorporated into the genome of pancreatic cancer cells using CRIPSR to minimize the heterogeneity of signals across different individual cells. We will further incorporate new FRET pairs emitting colors distinct from the popular FRET pair (CFP and YFP) to simultaneously monitor two different signals in the same live cell, e.g. one histone methylation and one kinase activation. Using a common molecular signal as a reference across different individual cells, these crucial molecular events will be mapped together with correlative FRET imaging method (CFIM) developed in our labs to generate dynamic landscapes of kinome-epigenome interactions. Pharmacological reagents will be applied to study their impact on these dynamic landscape of molecular interactions and adaptive epigenetic responses. We will then correlate these multiplex molecular profiles to cancer outcomes under these pharmacological reagents, and hence provide quantified multiplex indices to evaluate drug efficacy at the single-cell level with the goal of minimizing drug resistance. Three specific aims are accordingly proposed: (1) Develop and optimize histone methylation and tyrosine kinase FRET biosensors; (2) Apply CRISPR to genetically engineer FRET biosensors into pancreatic cancer cell lines for the calibration of inhibitor efficacy in single cells; (3) Multiplex imaging of histone methylations and kinase activities in the same PDAC cells for assessing adaptive epigenetic responses upon kinase inhibition. Given the importance and critical needs of new imaging tools to investigate the kinome-epigenome connection in cancer cells, developed FRET biosensors and imaging system should provide powerful means to unravel the molecular network for cancer biology, and allow multiplex and high throughput platform for drug screening with minimal resistance. As such, the success of the project will contribute transformative enabling technologies to the field of cancer research and pharmaceutics, toward an ultimate goal of eradicating pancreatic cancers.