Differential Regulation of PI3KC2beta by Ras Protooncogenes In Acute Myeloid Leukemia

急性髓系白血病 Ras 原癌基因对 PI3KC2beta 的差异调节

• 批准号: 9115909
• 负责人: Russell Spencer Smith
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115909
• 关键词: Acute Myelocytic Leukemia; Alleles; American Cancer Society; Anchorage-Independent Growth; Apoptosis; Binding; Blood Cells; Bone Marrow; Cell Line; Cell division; Cell membrane; Cells; Cessation of life; Characteristics; Chronic; Complex; Data; Dimensions; Dominant-Negative Mutation; Endosomes; Epithelial Cells; Family; Fluorescent Probes; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Hematopoietic Neoplasms; Human; Imagery; In Vitro; Lipids; MEKs; Malignant Neoplasms; Mediating; Molecular; Mutate; Mutation; Myeloid Progenitor Cells; Neuroblastoma; Nucleotides; Oncogenic; PIK3CG gene; Pathway interactions; Phosphatidylinositols; Phosphotransferases; Play; Property; Protein Family; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogenes; Publishing; Regulation; Reporting; Research; Resistance; Role; Scaffolding Protein; Signal Transduction; Survival Rate; Testing; Therapeutic; Transfection; Tumor Suppressor Proteins; United States; Vesicle; Work; base; driving force; gain of function mutation; in vivo; inhibitor/antagonist; insight; leukemia; member; neuroblastoma cell; outcome forecast; overexpression; phosphoinositide-3,4-bisphosphate; prevent; ras Proteins; research study; tumor; tumor growth; tumorigenesis; tumorigenic

 DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is the most prevalent form of leukemia, resulting from oncogenic transformation of the myeloid progenitor cells. Mutation of Ras proteins to a continuously activated state predominates in cancer, including AML. Chronic Ras activation increases cell division, a hallmark of cancer. Ras GTPases cycle from an active GTP-loaded state to an inactive GDP-loaded state. We have discovered that the nucleotide-free transition-state of Ras formed during the exchange of GTP for GDP can negatively regulate protein targets, adding a new dimension to Ras signaling. There are three members of the Ras sub- family; KRas, HRas and NRas, each mutated in a distinct subset of cancers. Activated KRas is a common driving force in cancer. A second class of proteins, called PI3K's, is known for activating the AKT pathway that prevents pre-programed cell death, another hallmark of cancer. This project focuses on a class II PI3K called PI3KC2β, which is overexpressed in several cancers, including AML and neuroblastoma. It has been shown that inhibition of PI3KC2β leads decreased proliferation in AML cell lines. We have found that HRas, in its nucleotide free state, interacts with PI3KC2β inhibiting its activation in vitro. In contrat, my preliminary data shows that activated KRas (GTP-KRas) interacts with PI3KC2β, suggesting that the KRas:PI3KC2β complex is active. We have previously performed in vitro GST pulldowns to show that HRas, in the absence of nucleotide preferentially interacts with PI3KC2β and that this interaction is bimolecular. I will use this approach to test whether GTP-loaded KRas interacts with PI3KC2β. My first aim will be to define the sequence of HRas and KRas, which provides for such nucleotide-dependent interaction with PI3KC2β. The nucleotide free HRas:PI3KC2β interaction occurs on intracellular vesicles whereas activated KRas interacts with PI3KC2β on the plasma membrane. In this project I will switch the regions required for Ras localization between HRas and KRas to determine the requirements for PI3KC2β localization. My second aim will determine the activation state of PI3KC2β in cells during both of these interactions, along with the ability of HRas vs KRas to sequester PI3KC2β. These experiments will be achieved by transfecting fluorescent probes specific for PI3KC2β's lipid product PI(3,4)P2 (an AKT activator) into cells. This allows visualization of various lipid products using these genetically encoded probes and their co-localization with fluorescently tagged PI3KC2β. The co-transfection of HRas and/or KRas will determine the effects of each Ras isoform on PI3KC2β activity. My final aim utilizes AML cell lines where KRas is either wildtype or activated and determines the effects of modulating wild type HRas levels on proliferation and resistance to apoptosis and whether activated KRas negates this effect. Conversely, I will silence HRas (therefore preventing PI3KC2β inhibition by HRas) on the tumorigenic characteristics of AML lines. This research provides additional evidence that Ras can negatively regulate protein targets and that different Ras isoforms signal differently through the same target.

 描述（由申请人提供）：急性髓性白血病（AML）是最常见的白血病形式，由髓性祖细胞的致癌转化引起。Ras蛋白突变为持续活化状态在癌症中占主导地位，包括AML。慢性Ras激活增加细胞分裂，这是癌症的标志。Ras GTP酶从激活的GTP加载状态循环到非激活的GDP加载状态。我们已经发现，在GTP与GDP的交换过程中形成的Ras的无核苷酸过渡态可以负调节蛋白质靶点，为Ras信号转导增加了一个新的维度。Ras亚家族有三个成员; KRas、HRas和NRas，每个在不同的癌症亚组中突变。激活的KRas是癌症的常见驱动力。第二类蛋白质，称为PI 3 K，已知用于激活AKT通路，防止预编程细胞死亡，这是癌症的另一个标志。该项目的重点是一种称为PI 3 KC 2 β的II类PI 3 K，它在几种癌症中过表达，包括AML和神经母细胞瘤。已经显示，抑制PI 3 KC 2 β导致AML细胞系中增殖降低。 我们已经发现，HRas在其核苷酸游离状态下与PI 3 KC 2 β相互作用，抑制其在体外的活化。相反，我的初步数据显示活化的KRas（GTP-KRas）与PI 3 KC 2 β相互作用，表明KRas：PI 3 KC 2 β复合物是活性的。我们之前已经进行了体外GST下拉，以表明在不存在核苷酸的情况下，HRas优先与PI 3 KC 2 β相互作用，并且这种相互作用是双分子的。我将使用这种方法来测试GTP负载的KRas是否与PI 3 KC 2 β相互作用。我的第一个目标将是定义HRas和KRas的序列，它提供了与PI 3 KC 2 β的这种核苷酸依赖性相互作用。无核苷酸的HRas：PI 3 KC 2 β相互作用发生在细胞内囊泡上，而激活的KRas与质膜上的PI 3 KC 2 β相互作用。在本项目中，我将在HRas和KRas之间切换Ras定位所需的区域，以确定PI 3 KC 2 β定位的要求。我的第二个目标是确定在这两种相互作用期间细胞中PI 3 KC 2 β的活化状态，沿着HRas与KRas螯合PI 3 KC 2 β的能力。这些实验将通过将对PI 3 KC 2 β的脂质产物PI（3，4）P2（AKT激活剂）特异性的荧光探针导入细胞中来实现。这允许使用这些遗传编码的探针及其与荧光标记的PI 3 KC 2 β的共定位来可视化各种脂质产物。HRas和/或KRas的共转染将确定每种Ras同种型对PI 3 KC 2 β活性的影响。我的最终目的是利用AML细胞系，其中KRas是野生型或激活的，并确定调节野生型HRas水平对增殖和抗凋亡的影响，以及激活的KRas是否会抵消这种影响。相反，我将沉默HRas（因此阻止HRas对PI 3 KC 2 β的抑制）对AML细胞系的致瘤性特征。这项研究提供了额外的证据，表明Ras可以负调节蛋白质靶点，并且不同的Ras亚型通过相同的靶点发出不同的信号。