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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

基本信息

批准号：
8835288
负责人：
Russell Spencer Smith
金额：
$ 4.31万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
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/8835288
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 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 public health relevance ras Proteins research study tumor tumor growth tumorigenesis tumorigenic

项目摘要

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.