Phosphoproteomic signatures for early detection and stratification of AML

 描述(申请人提供)：急性髓系白血病仍然是一种致命的疾病，特别是对那些对强化化疗没有反应的患者。尽管下一代DNA测序技术产生了重要的新数据，但仍难以将特定的基因突变与驱动白血病细胞异常增殖的分子机制联系起来。此外，在核型正常的AML患者中，约50%的患者缺乏关键基因如Flt3或NPM1的突变，他们对诱导化疗具有抵抗力。与基因测序相比，磷酸蛋白质组学方法提供了一种直接和定量的方法来衡量介导白血病发生的信号分子，从而避免了从肿瘤中积累的大量无功能的“乘客”DNA损伤中破译“驱动因素”突变的需要。在这项研究计划的初步研究中，我们发现了信号通路支持AML细胞生长和存活的新机制。在相关工作中，我们开发了深度序列质谱仪，这是一个可扩展的平台，提供真正的全基因组蛋白质组定量。与FOA PA-12-220中描述的目标一致，我们将进一步改进和利用我们的深度SEQ平台，在高危AML患者的原发肿瘤中识别基于磷酸化的功能性生物标记物，以“开发和改进与血液系统恶性肿瘤相关的新型生物标志物的定量检测的特定技术”，重点放在“早期发现、预测进展和复发，尤其是在高危个体中”。我们将使用毛细管纳米免疫分析和靶向质谱学验证我们的磷蛋白质组数据，这两种分析格式适合于医疗保健临床应用。我们新的发现和验证策略将捕获与高危AML患者治疗失败相关的信号通路，并为将我们的磷酸蛋白质组数据转化为改进的诊断奠定基础。我们的研究计划有两个特定的目标：特定的目标1是在AML患者的诊断标本中建立化疗耐药和治疗失败的磷酸蛋白质组学特征。我们将使用深度SEQ质谱仪来识别与化疗耐药和治疗失败相关的磷酸化标记物。重要的是，我们已经与四个主要癌症中心的同事达成了合作协议，以获取关于遗传背景和临床结果的注释良好的初级AML患者样本。具体目标2是验证磷酸蛋白质组标志物，并评估其在急性髓细胞白血病患者独立队列中的预后表现。我们将在一个更大的、独立的患者队列中验证我们的磷蛋白生物标记物，以确定预后表现。为了促进最终转化为临床应用，我们将使用毛细管纳米免疫分析和靶向质谱学的补充技术进行这些验证研究。

 DESCRIPTION (provided by applicant): Acute myeloid leukemia remains a lethal disease, especially for patients who do not respond to intensive chemotherapy. Despite significant new data resulting from next-generation DNA sequencing techniques, it remains difficult to link specific genetic mutations to the molecular mechanisms that drive aberrant proliferation of leukemic cells. Moreover, some 50% of AML patients who display a normal karyotype and who lack mutations in key genes such as FLT3 or NPM1 are resistant to induction chemotherapy. In contrast to gene sequencing, phosphoproteomic approaches provide a direct and quantitative measure of the signaling molecules that mediate leukemogenesis, circumventing the need to decipher 'driver' mutations from the very large number of non-functional 'passenger' DNA lesions that accumulate in tumors. In preliminary studies for this research proposal, we discovered novel mechanisms by which signaling pathways support AML cell growth and survival. In related work, we have developed DEEP SEQ mass spectrometry, a scalable platform that provides true, genome-wide proteome quantification. Consistent with the objectives described in FOA PA-12-220, which encourages "development and improvement of specific technologies for quantitative detection of novel biomarkers associated with hematopoietic malignancies," with a focus on "early detection, prediction of progression, and recurrence of hematopoietic malignancies, especially in high-risk individuals," we will further improve and utilize our DEEP SEQ platform to identify phosphorylation-based, functional biomarkers in primary tumors of high-risk AML patients. We will validate our phosphoproteomic data using capillary nanoimmunoassays and targeted mass spectrometry, analytical formats suitable for point-of-care clinical applications. Our novel discovery and validation strategy will capture the signaling pathways associated with therapy failure in high-risk AML patients, and lay the foundation for translation of our phosphoproteomic data into improved diagnostics. Our study plan has two specific aims: Specific Aim 1 is to establish phosphoproteomic signatures of chemoresistance and therapy failure in diagnostic specimens from patients with AML. We will use DEEP SEQ mass spectrometry to identify phosphorylation markers associated with chemoresistance and therapy failure. Importantly, we have secured collaborative agreements with colleagues at four major cancer centers to access primary AML patient samples that are well-annotated with respect to genetic background and clinical outcomes. Specific Aim 2 is to validate phosphoproteomic markers and access their prognostic performance in independent cohorts of AML patients. We will validate our phosphoproteomic biomarkers across a larger, independent patient cohort to determine prognostic performance. To facilitate eventual translation to clinical application we will perform these validation studies using the complementary techniques of capillary nanoimmunoassays and targeted mass spectrometry.