Differential Regulation of PI3KC2beta by Ras Protooncogenes In Acute Myeloid Leukemia

 描述(申请人提供)：急性髓系白血病(AML)是最常见的白血病形式，由髓系祖细胞的致癌转化引起。RAS蛋白突变为持续激活状态在包括急性髓系白血病在内的癌症中占主导地位。慢性RAS激活会增加细胞分裂，这是癌症的一个标志。RAS GTP酶从激活的GTP负载状态循环到非激活的GDP负载状态。我们发现，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向调节蛋白质靶标，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，每个成员都在不同的癌症亚群中发生突变。激活的KRAS是癌症的常见驱动力。第二类蛋白质被称为PI3K‘s，它激活AKT途径，防止预先编程的细胞死亡，这是癌症的另一个标志。该项目专注于一种名为PI3KC2β的II类PI3K，它在几种癌症中过表达，包括急性髓细胞白血病和神经母细胞瘤。已有研究表明，抑制PI3KC2β可导致急性髓系白血病细胞系增殖下降。我们发现，HRAS在无核苷酸状态下，在体外与PI3KC2β相互作用，抑制其激活。相反，我的初步数据显示，激活的KRAS(GTP-KRAS)与PI3KC2β相互作用，表明KRAS：PI3KC2β复合体是活跃的。我们之前已经在体外进行了GST下拉实验，以表明在没有核苷酸的情况下，HRA优先与PI3KC2β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加载gtp的KRAS是否与PI3KC2β交互。我的第一个目标将是定义HRAS和KRAS的序列，它提供了与PI3KC2β的这种核苷酸依赖的相互作用。无核苷酸的HRAS：PI3KC2β相互作用发生在细胞内的小泡上，而激活的KRAS与质膜上的PI3KC2β相互作用。在这个项目中，我将在HRA和KRAS之间切换RAS本地化所需的区域，以确定PI3KC2β本地化的要求。我的第二个目标是确定在这两个相互作用过程中细胞中PI3KC2β的激活状态，以及HRAS和KRAS隔离PI3KC2β的能力。这些实验将通过将针对PI3KC2β的S脂质产物PI(3，4)P2(AKT激活剂)的荧光探针导入细胞来完成。这使得使用这些基因编码的探针以及它们与荧光标记的PI3KC2β的共同定位可以可视化各种脂肪产品。HRAS和/或KRAS的共转染将决定每种RAS亚型对PI3KC2β活性的影响。我的最终目标是利用KRAS野生型或激活的AML细胞系，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是否抵消这一影响。相反，我将沉默HRA(因此阻止HRA抑制PI3KC2HRA)对急性髓细胞白血病细胞系的致瘤特性的影响。这项研究提供了额外的证据，证明RAS可以负向调节蛋白质靶标，不同的RAS亚型通过相同的靶标发出不同的信号。

 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.