Targeting Kinases that Phosphorylate the KSHV LANA Chromatin Binding Domain

描述（由申请人提供）： KSHV与内皮恶性肿瘤卡波西肉瘤（KS）、B细胞癌、原发性渗出性淋巴瘤（PEL）和多中心Castleman病相关。这些癌症的发病率在免疫功能低下的个体中增加，例如接受器官移植的个体和感染人类免疫缺陷病毒1（HIV-1）并患有AIDS的个体。KSHV相关恶性肿瘤的治疗仍然是次优的，需要新的治疗方法。 卡波西肉瘤相关疱疹病毒（KSHV）编码的拉娜蛋白在所有KSHV感染的细胞和KSHV相关恶性肿瘤中表达。拉娜对于KSHV基因组在感染细胞中的复制和保留是必需的，并且还提供细胞生长和细胞存活功能。扮演的关键角色 通过拉娜驱动KSHV相关细胞增殖，使得拉娜成为治疗KSHV相关疾病策略的有吸引力的靶标。拉娜通过染色质结合结构域将KSHV基因组连接到细胞染色质。 这种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，拉娜染色质结合结构域含有丝氨酸和苏氨酸残基，可以进行磷酸化。研究表明，虽然这些残基突变为丙氨酸阻止染色质结合，但转化为磷酸模拟谷氨酸残基恢复染色质结合。基于磷酸化在拉娜功能中的作用的证据，我们进行了筛选以鉴定磷酸化拉娜的激酶。该筛选从289种人激酶开始，所述人激酶以活性形式从重组酵母纯化。在一系列试验中，我们随后从该集合中鉴定出四种激酶，其磷酸化拉娜染色质结合结构域中的关键丝氨酸和苏氨酸残基。使用这些激酶之一的抑制剂的初步实验显示，激酶抑制影响拉娜与染色质的相互作用和细胞中存在的拉娜的水平。这些观察结果的有前途的性质，使我们提出了一个更深入的分析结果的抑制KSHV感染的PEL和内皮细胞培养的四个确定的激酶。这个目标 本发明的应用是通过检测四种鉴定的激酶的抑制剂的作用来确定抑制剂介导的干预KSHV疾病的最佳策略，并且，如果可用， 这些激酶的非特异性抑制剂已经在临床中使用，并且可能更快地过渡到KSHV相关疾病的临床前和临床方案中。我们将研究激酶抑制对KSHV基因组维持、KSHV感染的PEL和内皮细胞生长和活力以及诱导细胞周期停滞和凋亡标志物的影响。我们还将确定抑制剂诱导的拉娜不稳定性的机制。

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.