Mediator Kinases and AML Cell Proliferation

 描述(由申请人提供)：这个合作的，多PI项目试图获得一个有效的癌基因CDK8及其高度同源的同源基因CDK19如何控制AML细胞生长的详细了解。我们发现，抑制CDK8和CDK19的活性可以选择性地杀死培养中的急性髓系白血病(AML)细胞和AML小鼠模型中的AML细胞。CDK8和CDK19作为介体相关蛋白，有望广泛调控基因表达。(Mediator是一个多亚单位复合体，调节RNA聚合酶II转录本的全基因组表达。)然而，值得注意的是，多种细胞类型的全球基因表达谱表明，抑制CDK8和CDK19激酶活性对转录的影响有限。这些结果表明，CDK8和CDK19(即转录外)在AML细胞生长调控中起着不同的作用。该项目的一个关键因素是能够快速和选择性地抑制细胞中CDK8和CDK19的活性。在过去的四年里，我们严格检测了天然产物皮质抑素A(CA)在体外、细胞和体内选择性抑制介体激酶的能力。正如该提案(以及即将发表的一篇论文)中详细描述的那样，CA是CDK8和CDK19的一种异常有效和选择性的抑制剂(例如，即使在IC50剂量100倍的情况下也是选择性的)。有了这种首创的试剂，我们可以解决一些关于介体激酶的生物学和癌症相关角色的基本和长期存在的问题。重要的是，这些问题将在治疗相关的急性髓细胞白血病细胞系MOLM-14中得到解决，最近发现该细胞系需要介体激酶功能才能增殖和存活。理解生物学的第一个关键步骤 任何一种激酶的作用都是识别它的底物。已知的介体蛋白激酶底物很少；其中，CDK8的磷酸化(CDK19底物未知)在某些情况下会触发蛋白质降解。因此，在目标1A中，我们建议完成定量蛋白质组学实验(SILAC)，包括磷酸蛋白质组学和全蛋白质组分析，以确定介体蛋白激酶底物，并确定介体蛋白激酶抑制如何影响MOLM-14蛋白质组。另一个与介体蛋白激酶相关的主要问题是它们如何/是否影响细胞代谢。代谢变化对肿瘤发生和细胞分化至关重要，已知CDK8调节这些过程中的每一个。此外，CDK8与模式生物的新陈代谢调节和营养感知有着古老的联系，这表明人类CDK8(或许还有CDK19)可能作为代谢调节因子发挥更广泛的功能。为了开始认真解决这个问题，我们将完成全球性的非靶向代谢组学实验(与新陈代谢公司合作)。在CA处理的MOLM-14细胞和未处理的MOLM-14细胞中(Aim 1B)。后续研究将开始建立在我们在目标1A和目标1B的结果的基础上；然而，我们预计这个探索性的R21项目将产生许多关键发现，远远超出两年的时间框架。

 DESCRIPTION (provided by applicant): This collaborative, multi-PI project seeks to gain a detailed understanding of how a potent oncogene, CDK8, and its highly homologous paralog, CDK19, control AML cell growth. We discovered that inhibition of CDK8 and CDK19 kinase activity selectively kills acute myeloid leukemia (AML) cells in culture and in mouse models of AML. As Mediator-associated kinases, CDK8 and CDK19 are poised to broadly regulate gene expression. (Mediator is a multi-subunit complex that regulates genome-wide expression of RNA polymerase II transcripts.) Remarkably, however, global gene expression profiling in multiple cell types indicate that inhibition of CDK8 and CDK19 kinase activity has limited effects on transcription. These results suggest distinct roles for the "Mediator kinases" CDK8 and CDK19 (i.e. beyond transcription) in the regulation of AML cell growth. A key element of this project is the ability to rapidly and selectively inhibit CDK8 and CDK19 kinase activity in cells. Over the past four years, we have rigorously examined the natural product cortistatin A (CA) for its ability to selectively inhibit Mediator kinases in vitro, in cells, and in vivo. As detailed inthe proposal (and in a forthcoming publication), CA is an unusually potent and selective inhibitor of CDK8 and CDK19 (e.g. selective even at 100-times the IC50 dose). With this first-of-its-kind reagent, we can address some fundamental and long-standing questions about the biological and cancer-relevant roles of Mediator kinases. Importantly, these questions will be addressed in the therapeutically relevant AML cell line MOLM- 14, recently shown to require Mediator kinase function for proliferation and survival. A first critical step toward understanding the biological role of any kinase is to identify its substrates. Few Mediator kinase substrates are known; among these, phosphorylation by CDK8 (no CDK19 substrates are known) triggers protein degradation in some cases. Therefore, in Aim 1A we propose to complete quantitative proteomics experiments (SILAC), including phosphoproteomics and whole proteome analyses, to identify Mediator kinase substrates and determine how Mediator kinase inhibition affects the MOLM-14 proteome. Another major unanswered question related to Mediator kinases is how/whether they impact cell metabolism. Metabolic changes are fundamentally important for oncogenesis and cell differentiation, and CDK8 is known to regulate each of these processes. Moreover, CDK8 has ancient links to metabolic regulation and nutrient sensing in model organisms, suggesting human CDK8 (and perhaps CDK19) may function more broadly as a metabolic regulator. To begin to seriously address this question, we will complete global, untargeted metabolomics experiments (in collaboration with Metabolon, Inc.) in CA-treated vs. untreated MOLM-14 cells (Aim 1B). Follow-up studies will begin to build upon our results in Aim 1A and Aim 1B; however, we anticipate that many key discoveries will result from this exploratory R21 project that will extend well beyond the 2-year time frame.