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

活癌细胞中激酶-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减轻胰腺癌的发展。然而，适应性表观遗传 包括组蛋白调节的反应可导致大规模基因表达的改变， 最终导致癌细胞的异质性耐药反应和危及生命的复发。 疾病在目前阶段，仍不清楚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药物治疗后的癌细胞应答。因此，调查 操纵组蛋白调节和编码的调节在癌症治疗中具有重要意义 和药物筛选。在这个提议中，我们将利用定向进化和高内容筛选的力量 方法系统地开发荧光共振能量转移（FRET）生物传感器的动态 监测和定量单细胞中的关键组蛋白甲基化（H3 K4、H3 K9、H3 K27）。我们还将 应用这种方法来优化粘着斑激酶（FAK）FRET生物传感器，用于FAK的可视化 激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 使用CRIPSR掺入胰腺癌细胞的基因组中以最小化信号的异质性 在不同的个体细胞中。我们将进一步纳入新的FRET对发射的颜色不同于 流行的FRET对（CFP和YFP），以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活化。使用一个共同的分子信号作为参考， 这些关键的分子事件将与相关的FRET成像方法一起绘制 （CNOM）在我们的实验室中开发，以生成激酶组-表观基因组相互作用的动态景观。 药理学试剂将被应用于研究它们对这些分子动态景观的影响 相互作用和适应性表观遗传反应。然后，我们将这些多重分子谱与 这些药理学试剂下的癌症结果，并因此提供量化的多重指标， 在单细胞水平上评估药物疗效，以最大限度地减少耐药性。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） 将CRISPR应用于基因工程FRET生物传感器到胰腺癌细胞系中，用于校准 （3）在同一细胞中组蛋白甲基化和激酶活性的多重成像; 用于评估激酶抑制后的适应性表观遗传应答的PDAC细胞。鉴于其重要性和关键性， 需要新的成像工具来研究癌细胞中的激酶组-表观基因组连接， 生物传感器和成像系统将为解开癌症分子网络提供强有力的手段 生物学，并允许多重和高通量平台，用于药物筛选，具有最小的耐药性。因此，在本发明中， 该项目的成功将为癌症研究领域提供变革性的技术， 药物，以达到根除胰腺癌的最终目标。

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.