Development of multiplex single cell phosphotyrosine profiling tools for B-cell malignancies

摘要 酪氨酸激酶的异常激活会导致细胞信号失控，从而导致癌细胞增殖。 侵袭和转移。靶向酪氨酸激酶抑制剂正在取得显著的成功 恶性血液病领域--兼论研究活化酪氨酸激酶的意义 人类疾病中的途径。了解哪些通路是活跃的，因此可能成为靶点 在治疗方面，需要对细胞蛋白的酪氨酸磷酸化进行全面的分析。目前， 酪氨酸磷酸蛋白质组学(pTyr组学)方法，包括质谱学和微阵列系统 由于技术复杂性，主要用作研究工具，而不是用于临床诊断。这个 缺乏可靠和方便的pTyr组学工具阻碍了与酪氨酸相关的生物标记物的发现 激酶途径和新的治疗策略的发展。相比之下，流式细胞术 整合在临床实验室中，作为单细胞水平的血细胞免疫表型鉴定的常规工具。 为了开发一种基于全球酪氨酸磷酸化状态的分子诊断工具，我们 利用哺乳动物细胞中主要的pTyr结合模块SH2结构域，开发了SH2图谱 作为检测癌细胞全球酪氨酸磷酸化状态变化的探针。最近，我们发现 一组慢性淋巴细胞白血病(CLL)患者在诊断时通过SH2特征分析显示高水平 在约5年的临床随访期间疾病进展的可能性。这一结果表明了使用 SH2作为一种预测性生物标志物，促使我们开发一种新的检测平台，通过SH2 可以在临床实验室中常规进行单细胞分辨率的分析。 在这里，我们建议开发结合两种新的流式细胞术的BCR途径聚焦的SH2谱分析方法 技术(SH2-Flow)。在目标1中，我们将准备一个通用的B细胞受体(BCR)探针库(BCR-SH2 面板)，其中荧光染料以共价和化学计量比偶联到SH2结构域，允许 多重结合分析。在目标2中，我们将建立一种人B细胞的多色流式细胞术分析 用该套SH2荧光探针对淋巴细胞进行检测。在目标3中，我们将开发一种基于SH2的结合试验 使用CyTOF质量细胞仪在单细胞分辨率下进行高度多重SH2谱分析。带来了一条路径- 通过补充现有基于抗体的方法，将聚焦的磷酸酪氨酸分析工具应用于常规临床实践 免疫表型将对临床研究产生重大影响，可能还会对患者护理产生影响。这个 新的检测方法可以用来发现基于信号的预测生物标记物，疾病的指示物 进展，以及评估靶向药物反应的工具。

Summary Aberrant activation of tyrosine kinases causes dysregulated cell signaling resulting in cancer cell proliferation, invasion, and metastasis. Targeted tyrosine kinase inhibitors are achieving the remarkable success in the field of hematologic malignancies, exemplifying the significance of studying activated tyrosine kinase pathways in human diseases. To understand what pathways are active and may thus be targeted therapeutically, comprehensive analysis of tyrosine phosphorylation of cellular proteins is needed. Currently, tyrosine phosphoproteomics (pTyr-omics) approaches including mass spectrometry and microarray systems are predominantly used as research tools rather than for clinical diagnosis due to technical complexities. The lack of robust and convenient pTyr-omics tools hampers the discovery of biomarkers relevant to tyrosine kinase pathways and the development of new therapeutic strategies. By contrast, flow cytometry has been integrated in clinical laboratories as a routine tool for immunophenotyping of blood cells at the single cell level. In an effort to develop a molecular diagnostic tool based on the global tyrosine phosphorylation state, we developed SH2 profiling in which SH2 domains, a major pTyr binding module in mammalian cells, are utilized as probes to detect changes in the global tyrosine phosphorylation state of cancer cells. Recently, we found that a group of chronic lymphocytic leukemia (CLL) cases selected by SH2 profiling at diagnosis showed high likelihood of disease progression during ~5 years of clinical follow-up. This result suggests the potential to use the SH2 profile as a predictive biomarker, motivating us to develop a new assay platform by which the SH2 profiling can be routinely performed in clinical laboratories with single-cell resolution. Here we propose to develop BCR pathway-focused SH2 profiling assays integrating two new flow cytometry technologies (SH2-flow). In Aim 1, we will prepare a universal B Cell Receptor (BCR) probe library (BCR-SH2 panel) in which fluorescent dyes are covalently and stoichiometrically coupled to SH2 domains, allowing for multiplexed binding assays. In Aim 2, we will develop a multi-color flow cytometry assay of human B lymphocytes using the set of fluorescent SH2 probes. In Aim 3, we will develop an SH2 binding assay based on CyTOF mass cytometer for highly multiplex SH2 profiling at a single cell resolution. Bringing a pathway- focused phosphotyrosine profiling tool to routine clinical practice by complementing existing antibody-based immunophenotyping would have a remarkable impact on clinical research and possibly on patient care. The new assays could be utilized for discovery of signaling-based predictive biomarkers, indicators for disease progression, and tools to assess targeted drug responses.