Mediator Kinases and AML Cell Proliferation

 描述（由申请人提供）：这个合作的多PI项目旨在详细了解有效的癌基因CDK 8及其高度同源的parkingCDK 19如何控制AML细胞生长。我们发现，抑制CDK 8和CDK 19激酶活性可以选择性地杀死培养物和小鼠AML模型中的急性髓性白血病（AML）细胞。作为介体相关激酶，CDK 8和CDK 19有望广泛调节基因表达。（介体是调节RNA聚合酶II转录物的全基因组表达的多亚基复合物。然而，值得注意的是，多种细胞类型中的全局基因表达谱表明，抑制CDK 8和CDK 19激酶活性对转录的影响有限。这些结果表明，“介体激酶”CDK 8和CDK 19（即转录之外）在AML细胞生长的调节中具有不同的作用。 该项目的一个关键要素是能够快速和选择性地抑制细胞中的CDK 8和CDK 19激酶活性。在过去的四年中，我们严格检查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。正如提案（以及即将出版的出版物）中所详述的，CA是CDK 8和CDK 19的异常强效和选择性抑制剂（例如，即使在IC 50剂量的100倍下也具有选择性）。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作用的基本和长期问题。重要的是，这些问题将在治疗相关的AML细胞系MOLM- 14中得到解决，该细胞系最近显示需要介体激酶功能来增殖和存活。 理解生物学的第一个关键步骤 任何激酶的作用都是鉴定其底物。已知的介体激酶底物很少;其中，CDK 8的磷酸化（没有已知的CDK 19底物）在某些情况下触发蛋白质降解。因此，在目标1A中，我们建议完成定量蛋白质组学实验（SILAC），包括磷酸化蛋白质组学和全蛋白质组分析，以鉴定介体激酶底物并确定介体激酶抑制如何影响MOLM-14蛋白质组。 与介体激酶相关的另一个主要未回答的问题是它们如何/是否影响细胞代谢。代谢变化对于肿瘤发生和细胞分化至关重要，已知CDK 8调节这些过程中的每一个。此外，CDK 8与模式生物中的代谢调节和营养感测有着古老的联系，这表明人类CDK 8（可能还有CDK 19）可能更广泛地发挥代谢调节剂的作用。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Metabolon，Inc.合作）。在CA处理的与未处理的MOLM-14细胞中（Aim 1B）。后续研究将开始建立在我们的目标1A和目标1B的结果;然而，我们预计许多关键发现将来自这个探索性的R21项目，该项目将远远超出2年的时间范围。

 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.