Development of deep proteomic sequencing platforms for molecular markers in DLBCL

描述（由申请人提供）：DLBCL弥漫性大B细胞淋巴瘤（DLBCL）是一种遗传和临床异质性疾病，包括最常见的非霍奇金淋巴瘤，开发DLBCL分子标记的深度蛋白质组学测序平台。迄今为止，揭示DBLCL的分子发病机制，确定治疗靶点，并根据特定的分子病变对患者进行分层的努力都依赖于基因组水平的分析，这些分析确定了不同的亚结构分类和致病机制，包括b细胞受体（BCR）依赖的生存信号。从这些研究中出现的潜在生物标志物有待进一步验证。定量蛋白质组学有能力在蛋白质/途径水平上揭示额外的、新的分子特征，否则这些特征可能由于转录后和翻译后机制而无法通过基于基因组的方法检测到。然而，使用定量蛋白质组学来分析分子特征并促进临床组织中生物标志物的发现一直受到关键障碍的阻碍，例如低丰度蛋白质的检测不足，过于复杂的样品工作流程通常与原发性肿瘤中可用的蛋白质数量不相容，以及低通量。我们已经开发出一种新型的非常高的峰值容量液相色谱系统，直接与质谱相结合，可以解决这些障碍，并实现广泛和定量的蛋白质组覆盖。在适当的时候，该平台将能够对足够大的临床队列进行分析，以提供统计学上的蛋白质生物标志物鉴定。在初步研究中，我们利用我们的深层蛋白质组测序平台的原型版本来询问原发性DLBCL肿瘤活检和患者衍生的DLBCL细胞系的蛋白质组。该分析揭示了之前未被认识到的营养/燃料利用以及相关的增殖和生存优势的途径水平异质性，这些异质性与功能性BCR信号的存在/缺失有关。FOA PA-12-220鼓励开发“定量检测与造血恶性肿瘤相关的新型生物标志物的特定技术”，我们建议测试、验证和改进我们的蛋白质组学平台，以系统地检测原发性DLBCL肿瘤中的蛋白质组（Aim 1）和磷蛋白质组（Aim 2）特征。我们的目标是识别和验证DLBCL分子亚群中差异富集的坐标通路。我们的研究计划将揭示假定的DLBCL的新分层标记物，并证明我们的深度蛋白测序LC-MS/MS平台将来可用于更大的NHL患者队列。

DESCRIPTION (provided by applicant): Development of deep proteomic sequencing platforms for molecular markers in DLBCL Diffuse large B cell lymphoma (DLBCL) is a genetically and clinically heterogeneous disease that comprises the most common non-Hodgkin's lymphoma. To date efforts to unearth the molecular pathogenesis of DBLCL, identify therapeutic targets, and stratify patients according to specific sets of molecular lesions have relied on genome-level analyses that identified distinct substructure classifications and pathogenic mechanisms, including B-cell receptor (BCR)-dependent survival signaling. The potential biomarkers that have emerged from these studies await further validation. Quantitative proteomics has the power to reveal additional, novel molecular signatures at the protein/pathway level that may otherwise escape detection through genome-based approaches due to post-transcriptional and post-translational mechanisms. However, use of quantitative proteomics to profile molecular signatures and facilitate biomarker discovery in clinical tissues has been hampered by critical barriers such as poor detection of low-abundance proteins, overly complex sample workflows that are often incompatible with the protein quantities available in primary tumors, and low throughput. We have developed a novel and very high peak capacity liquid chromatography system coupled directly to mass spectrometry that can address these barriers and achieve extensive and quantitative proteome coverage. In the fullness of time, this platform will enable analyses of sufficiently large clinical cohorts to provde statistically-powered identification of protein biomarkers. In preliminary studies, we utilized a prototype version of our deep proteome sequencing platform to interrogate the proteome of primary DLBCL tumor biopsies and patient derived DLBCL cell lines. This analysis revealed previously unappreciated pathway-level heterogeneity in nutrient/fuel utilization and associated proliferation and survival advantages that segregate with the presence/absence of functional BCR signaling. In response to FOA PA-12-220, which encourages development of "specific technologies for quantitative detection of novel biomarkers associated with hematopoietic malignancies," we propose to test, validate and improve our proteomics platform for systematic interrogation of both proteome (Aim 1) and phosphoproteome (Aim 2) signatures in primary DLBCL tumors. Our goal is to identify and validate coordinate pathways that are differentially enriched in molecular subsets of DLBCL. Our study plan will reveal putative new stratification markers for DLBCL and credential our deep protein sequencing LC-MS/MS platform for future use on much larger NHL patient cohorts.