Assays for Screening Histone Modifications in Cancer

描述（申请人提供）：为了更好地了解组蛋白变体及其翻译后修饰在慢性淋巴细胞白血病（CLL）发生、发展和治疗中的作用，我们打算开发和应用基于高质量准确度质谱的测定来分析CLL，组蛋白异构体的特定模式，并进行详细的分子表征，以确定存在于不同基因组中的特定组蛋白异构体。CLL的亚型及其对患者治疗和结果的影响。我们将通过分析组蛋白修饰的状态来实现这些目标，并表征染色质重塑试剂治疗诱导的组蛋白修饰的变化以及与特定基因突变相关的变化。我们将集中在过程中，与诱导细胞凋亡和分化在体外和体内的CLL细胞。其基本原理是定义这些疗法诱导的染色质变化，并确定染色质预测治疗风险和结果的潜力。该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在组蛋白亚型中观察到的特定变化，并使临床翻译科学家能够有效地预测结果并应用修饰CLL患者组蛋白的疗法。本申请的目的是：目的1。将联合收割机多维液相色谱与高质量准确度质谱联用分析原发性恶性造血系统肿瘤细胞的组蛋白谱和组蛋白异构体。这些技术将应用于分析来自CLL遗传亚型的恶性细胞。我们假设这些技术将有助于组蛋白变体和翻译后修饰的表征，并允许靶向目前可用的免疫学技术无法识别的特定CLL特异性位点;目的2。获得CLL患者的组蛋白谱，并确定这些谱相对于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的组蛋白谱分析的应用将产生新的标志物，其将促进根据风险对CLL患者进行分层;以及目的3。通过高质量准确度液相色谱串联质谱法鉴定、定位和表征CLL亚型恶性细胞中组蛋白翻译后修饰的位点。我们将确定组蛋白H1磷酸化亚型存在于细胞核和胞质提取物由于H1释放flavopiridol治疗诱导CLL患者。我们假设，蛋白质组学将允许识别选择组蛋白修饰，这将作为CLL患者的flavopiridol治疗的机制终点和患者结局的标记。 公共卫生相关性：核心组蛋白在调节细胞过程中起着至关重要的作用，这些细胞过程涉及染色体DNA的进入和随后的基因转录。这些过程在包括慢性淋巴细胞白血病（CLL）在内的大部分癌症中被错误调节。为了更好地理解组蛋白变体及其翻译后修饰对CLL的发生、进展和治疗的作用，我们打算开发和应用基于高质量准确度质谱的测定法来分析CLL特异性组蛋白同种型模式，并进行详细的分子表征以鉴定存在于CLL的不同遗传亚型中的特异性组蛋白同种型及其对患者治疗和结果的影响。

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.