Expanding multiplexed kinase biosensor analysis to SWATH-MS (1 of 2)

描述(由申请人提供)：激酶抑制剂药物已经改变了化疗的面貌，预计仍将是白血病治疗的主要焦点。然而，它们的长期疗效是不同的，可以归因于它们在靶点上的活动在患者之间的差异，这种差异最早可以在开始治疗后一周观察到。我们计划验证一项技术，该技术能够在治疗开始时量化疗效，使医生和患者有机会立即监测个人的反应和成功的可能性，而不是依赖目前的观察和等待方法。在帕克实验室获得IMAT R21奖的支持下，我们已经建立了用于分析慢性粒细胞白血病(CML)中BCR-Abl激酶活性和抑制的概念验证。授予耶鲁大学土耳其实验室的IMAT奖也支持了一项合作工作，为开发其他激酶的底物进行概念验证工作，包括Src家族和JAK2激酶。在这项R33先进技术开发和验证应用中，我们将利用这两个结果来进一步开发我们的方法，以产生新的试剂和方案，用于检测完整的活细胞中特定的激酶或激酶家族的活性和抑制。我们还将在与印第安纳大学西蒙癌症中心血液恶性肿瘤组织库合作收集的更大的患者样本面板中验证这项技术的定量和多路复用能力。在目标1中，我们计划通过开发一种同位素编码策略来解决吞吐量和标准化问题，该策略将允许在不同的细胞(对照、多个患者样本)中并行执行同一生物传感器的分析，然后合并和同时分析。在目标2中，我们将扩展生物传感器和质谱分析工具箱，以包括与慢性粒细胞白血病耐药和/或白血病生物学其他方面重要的其他激酶和激酶家族。在目标3中，我们将验证这项技术在患者细胞中的应用，包括优化生物稳定性和递送。这一先进的发展将建立方法学的定量实用，并扩大这项技术的适用性，使之适用于仅与慢性粒细胞白血病相关的激酶和抑制剂。最终，这项工作可能会改变癌症的治疗，使医生和患者能够做出关于抑制剂治疗选择的明智决定，实时监测疗效，并及时发现治疗失败，以便在耐药性和复发成为问题之前进行干预。

DESCRIPTION (provided by applicant): Kinase inhibitor drugs have changed the face of chemotherapy and are projected to remain a major focus of leukemia treatment. However, their long-term efficacy is variable and can be attributed to differences in their activity on their targts from patient to patient that can be observed as early as one week after beginning treatment. We plan to validate a technology that enables quantification of efficacy at the very beginning of treatment, giving physicians and patients the opportunity to monitor an individual's response and likelihood of success immediately, rather than relying on the current watch-and-wait approach. We have established proof-of-concept for analyzing Bcr-Abl kinase activity and inhibition in chronic myelogenous leukemia (CML) with the support of an IMAT R21 award to the Parker laboratory. An IMAT award to the Turk laboratory at Yale University also supported work leading to a collaborative effort on proof-of-concept for developing substrates for other kinases, including the Src-family and JAK2 kinases. In this R33 advanced technology development and validation application, we will leverage both of these outcomes to further develop our approach to generate new reagents and protocols for detecting the activity and inhibition of specific kinases or kinase families in intact, live cells. We will also validate the quantitative and multiplexing capabilities of this technology in larger panels of patient samples, collected in collaboration with the Indiana University Simon Cancer Center Hematological Malignancies Tissue Bank. In Aim 1, we plan to address throughput and standardization by developing an isotope-coding strategy that will allow analyses of the same biosensor to be performed in different cells (controls, multiple patient samples) in parallel, followed by pooling and simultaneous analysis. In Aim 2, we will expand the biosensor and mass spectrometry analysis toolbox to include other kinases and kinase families that are important to CML drug resistance and/or other aspects of leukemia biology. In Aim 3, we will validate the application of this technology to patient cells, including optimization of biostability and delivery. This advanced development will establish the quantitative utility of the methodology, and expand the applicability of this technology to kinases and inhibitors relevant beyond CML alone. Ultimately, this work could transform cancer treatment by enabling physicians and patients to make informed decisions about inhibitor treatment choice, monitor efficacy in real time, and catch treatment failure in time to intervene before resistance and relapse become a problem.