Targeting Kinases that Phosphorylate the KSHV LANA Chromatin Binding Domain

靶向磷酸化 KSHV LANA 染色质结合域的激酶

基本信息

批准号：
8495960
负责人：
S DIANE HAYWARD
金额：
$ 16.56万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8495960
关键词：
Acquired Immunodeficiency Syndrome Affect Alanine Apoptosis Automobile Driving B-Lymphocytes Binding Biological Assay Cell Culture Techniques Cell Cycle Arrest Cell Proliferation Cell Survival Cells Chromatin Clinic Clinical Protocols Clinical Trials Collection Data Disease Endothelial Cells Enzymes Evaluation Experimental Designs Funding Genome Glutamic Acid Glycogen Synthase Kinase 3 Goals HIV-1 Histones Human Human Herpesvirus 8 Immune system Immunocompromised Host In Vitro Incidence Individual Infection Intervention Investigation Kaposi Sarcoma Maintenance Malignant - descriptor Malignant Neoplasms Measures Mediating Multicentric Angiofollicular Lymphoid Hyperplasia Mus Mutation Nature Organ Transplantation Outcome PIM1 gene Phosphorylation Phosphotransferases Physiologic pulse Play Post-Translational Protein Processing Property Proteins Recombinants Role Series Serine Serine/Threonine Phosphorylation Therapeutic Agents Therapeutic Intervention Threonine United States National Institutes of Health Viral Genome Virus Xenograft Model Yeasts base cell growth inhibitor/antagonist kinase inhibitor neoplastic cell novel strategies novel therapeutics pre-clinical prevent primary effusion lymphoma protein function research study tumor

项目摘要

DESCRIPTION (provided by applicant): KSHV is associated with the endothelial malignancy Kaposi sarcoma (KS) and the B cell cancers primary effusion lymphoma (PEL) and multicentric Castleman disease. The incidence of these cancers is increased in individuals who are immunocompromised such as those undergoing organ transplantation and those who are infected with human immunodeficiency virus 1 (HIV-1) and have AIDS. Treatments for KSHV associated malignancies remain sub-optimal and new treatment approaches are needed. The Kaposi sarcoma associated herpesvirus (KSHV) encoded LANA protein is expressed in all KSHV infected cells and KSHV associated malignancies. LANA is essential for replication and retention of KSHV genomes in infected cells and also provides cell growth and cell survival functions. The critical roles played by LANA in driving KSHV associated cell proliferation make LANA an attractive target for strategies to treat KSHV associated disease. LANA tethers KSHV genomes to cell chromatin through a chromatin binding domain. This function is essential for the maintenance of the viral genomes in infected cells. Post-translational modification of proteins by phosphorylation is a mechanism by which protein function is frequently regulated. We noted that the LANA chromatin binding domain contains serine and threonine residues that could be subject to phosphorylation. Investigation revealed that, while mutation of these residues to alanine prevented chromatin binding, conversion to phosphomimetic glutamic acid residues restored chromatin binding. Based on this evidence for a role of phosphorylation in LANA function, we undertook a screen to identify kinases that phosphorylate LANA. This screen started with 289 human kinases that were purified in active form from recombinant yeast. In a series of assays we subsequently identified four kinases from this collection that phosphorylated the critical serine and threonine residues in the LANA chromatin binding domain. Preliminary experiments using an inhibitor to one of these kinases revealed that kinase inhibition affected both LANA interaction with chromatin and the levels of LANA present in the cell. The promising nature of these observations has led us to propose a more in depth analysis of the outcome of inhibition of the four identified kinases in KSHV infected PEL and endothelial cell cultures. The goal of this application is to determine the optimal strategy for inhibitor mediated intervention in KSHV disease by examining the effects of inhibitors of the four identified kinases and, where available, of non-specific inhibitors of these kinases that are already in use in the clinic and could potentially be more rapidly transitioned into pre-clinical and clinical protocols for KSHV associated disease. We will examine the effect of kinase inhibition on KSHV genome maintenance, KSHV infected PEL and endothelial cell growth and viability and induction of markers of cell cycle arrest and apoptosis. We will also determine the mechanism of inhibitor induced LANA instability.

描述（由申请人提供）： KSHV与内皮恶性肿瘤Kaposi Sarcoma（KS）和B细胞癌一级积液淋巴瘤（PEL）和多中心Castleman病有关。这些癌症的发病率增加了免疫功能低下的个体，例如接受器官移植的人以及感染人类免疫缺陷病毒1（HIV-1）并具有艾滋病的人。 KSHV相关的恶性肿瘤的治疗仍然是最佳的，需要新的治疗方法。 Kaposi肉瘤相关的疱疹病毒（KSHV）编码的LANA蛋白在所有KSHV感染的细胞和KSHV相关的恶性肿瘤中表达。 LANA对于在感染细胞中的KSHV基因组复制和保留至关重要，还提供细胞生长和细胞存活功能。扮演的关键角色 LANA在驱动KSHV相关的细胞增殖方面使LANA成为治疗KSHV相关疾病的策略的吸引人目标。 Lana通过染色质结合结构域将KSHV基因组染成细胞染色质。该功能对于维持感染细胞中的病毒基因组至关重要。通过磷酸化对蛋白质的翻译后修饰是一种经常调节蛋白质功能的机制。我们注意到，LANA染色质结合结构域含有可能受到磷酸化的丝氨酸和苏氨酸残基。研究表明，尽管这些残基与丙氨酸的突变阻止了染色质结合，但转化为磷酸谷氨酸残基恢复了染色质结合。基于这个证据表明磷酸化在LANA功能中的作用，我们进行了筛选以鉴定磷酸化LANA的激酶。该屏幕始于289种人类激酶，这些激酶从重组酵母中以活性形式纯化。在一系列测定中，我们随后从该集合中鉴定出四个激酶，这些激酶磷酸化了LANA染色质结合结构域中的临界丝氨酸和苏氨酸残基。使用抑制剂对这些激酶之一的初步实验表明，激酶抑制作用都影响了LANA与染色质的相互作用和细胞中存在的LANA水平。这些观察结果的有希望的性质使我们对KSHV感染的PEL和内皮细胞培养物中抑制四种鉴定激酶的结果进行了更深入的分析。目标的目标应用是通过检查四种已鉴定的激酶的抑制剂以及可用的抑制剂的影响来确定抑制剂介导的KSHV疾病干预的最佳策略这些激酶的非特异性抑制剂已在临床中已经使用，并且可能会更快地转变为KSHV相关疾病的临床前和临床方案。我们将研究激酶抑制对KSHV基因组维持，KSHV感染的PEL和内皮细胞生长以及生存能力以及细胞周期停滞和凋亡标记的诱导的影响。我们还将确定抑制剂诱导的LANA不稳定性的机制。