Assays for Screening Histone Modifications in Cancer

描述（由申请人提供）：为了更好地了解组蛋白变异及其翻译后修饰在慢性淋巴细胞白血病（CLL）的发生、进展和治疗中的作用，我们打算开发并应用基于高质量精度的质谱分析来分析CLL特异性组蛋白亚型的模式，并进行详细的分子表征，以确定不同遗传亚型CLL中存在的特异性组蛋白亚型及其对患者治疗和预后的影响。我们将通过分析组蛋白修饰的状态来实现这些目标，并描述组蛋白修饰的变化，这些变化是由染色质重塑试剂治疗和与特定基因突变相关的变化引起的。我们将重点关注与体外和体内CLL细胞诱导凋亡和分化相关的过程。基本原理是定义由这些治疗引起的染色质变化，并确定染色质预测治疗风险和结果的潜力。本提案中开发和验证的技术将使基础科学家能够检测和理解组蛋白亚型中观察到的特定变化，并使临床转化科学家能够有效地预测结果并应用修饰CLL患者组蛋白的治疗方法。此应用程序的目标是：目标1。目的：结合多维液相色谱法和高质量精度质谱法对原发性恶性造血肿瘤细胞中的组蛋白进行分析，并对组蛋白异构体进行表征。这些技术将被应用于CLL基因亚型的恶性细胞。我们假设这些技术将有助于组蛋白变异和翻译后修饰的表征，并允许靶向目前可用的免疫技术无法识别的特定cll特异性位点；目标2。获得CLL患者的组蛋白谱，并确定这些谱与用于预测该疾病早期进展的其他生物标志物的临床相关性。我们假设基于LC-MS的组蛋白分析的应用将导致新的标记物，这将有助于根据风险对CLL患者进行分层；和Aim 3。利用高质量精度液相色谱串联质谱技术鉴定、绘制和表征CLL亚型恶性细胞中组蛋白翻译后修饰位点。我们将在CLL患者黄匹吡醇治疗诱导H1释放后，确定细胞核和细胞质提取物中存在的组蛋白H1磷酸化亚型。我们假设蛋白质组学将允许鉴定选择组蛋白修饰，这些修饰将作为黄吡醇治疗CLL患者的机制终点和患者预后的标记物。

DESCRIPTION (provided by applicant): To better understand the role of histone variants and their post-translational modifications on the development, progression and treatment of chronic lymphocytic leukemia (CLL), we intend to develop and apply high mass accuracy mass spectrometry based assays to profile CLL-specific patterns of histone isoforms and conduct detailed molecular characterization to identify the specific histone isoforms present in different genetic subtypes of CLL and their impact on patient treatment and outcome. We will carry out these goals by profiling the state of histone modifications and characterize the changes in histone modifications that are induced by treatment with chromatin remodeling reagents and changes that are associated with specific genetic mutations. We will focus on processes that correlate with induction of apoptosis and differentiation in vitro and in vivo in CLL cells. The rationale is to define the changes in chromatin induced by these therapies and determine chromatin's potential to predict risk and outcome for the therapy. The techniques developed and validated in this proposal will allow basic scientists to detect and understand specific changes observed in histone isoforms and clinical-translational scientists to effectively predict outcome and apply therapies that modify histones in patients with CLL. The aims of this application are: Aim 1. To combine multidimensional liquid chromatography with high-mass accuracy mass spectrometry to profile histone proteins and characterize histone protein isoforms in primary malignant hematopoietic tumor cells. These techniques will be applied to profile malignant cells from genetic subtypes of CLL. We hypothesize these techniques will facilitate the characterization of histone variants and post-translational modifications and permit the targeting of specific CLL-specific sites not identifiable with currently available immunological techniques; Aim 2. Obtain the histone profiles of CLL patients and determine the clinical relevance of these profiles relative to other biomarkers used to predict early progression of this disease. We hypothesize that the application of LC-MS based histone profiling will lead to new markers that will facilitate the stratification of CLL patients according to risk; and Aim 3. To identify, map and characterize sites of histone post-translational modifications in malignant cells from CLL subtypes by high- mass accuracy liquid chromatography tandem mass spectrometry. We will determine the histone H1 phosphorylation isoforms present in nuclear and cytosolic extracts due to H1 release induced by flavopiridol therapy in CLL patients. We hypothesize that proteomics will allow the identification of select histone modifications that will serve as markers for mechanistic endpoints and patient outcome in flavopiridol therapy of CLL patients. PUBLIC HEALTH RELEVANCE: Core histones play a vital role in the regulation of cellular processes that involve access to chromosomal DNA and subsequent gene transcription. These processes are misregulated in a large proportion of cancers including chronic lymphocytic leukemia (CLL). To better understand the role of histone variants and their post- translational modifications on the development, progression and treatment of CLL, we intend to develop and apply high mass accuracy mass spectrometry based assays to profile CLL-specific patterns of histone isoforms and conduct detailed molecular characterization to identify the specific histone isoforms present in different genetic subtypes of CLL and their impact on patient treatment and outcome.