PIP5K1A as a novel driver of PI3K signaling in health and disease

PIP5K1A 作为健康和疾病中 PI3K 信号传导的新型驱动因素

• 批准号: 10214915
• 负责人: Gerald R Hammond
• 金额: $ 15.65万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214915
• 关键词: Address; Affinity; Automobile Driving; Binding; Binding Proteins; Bladder; Breast; Cancer cell line; Cell Line; Cell Proliferation; Cell membrane; Cells; Coupling; Diploid Cells; Disease; Drug Targeting; Enzymes; Epidermal Growth Factor; Foundations; Future; Gap Junctions; Genetic; Genome; Goals; Health; Homeostasis; Inositol; Integral Membrane Protein; Knowledge; Lipids; Liver; Lung; Malignant - descriptor; Malignant Neoplasms; Measures; Membrane; Membrane Lipids; Mutate; Oncogenic; Output; Pathway interactions; Peripheral; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Production; Proteins; Signal Transduction; Structure; Testing; Therapeutic; Uterus; Work; cancer cell; drug discovery; inhibitor/antagonist; innovation; novel; novel marker; overexpression; phosphatidylinositol 4-phosphate; phosphoinositide-3,4,5-triphosphate; small molecule; small molecule inhibitor; stem; success; trafficking; tumor; tumorigenesis; unpublished works

PROJECT SUMMARY PIP5K1A is amplified or mutated in approximately 5% of cancers, yet its potential as a drug target has not been realized to date. The enzyme is known to synthesize the key regulatory lipid PI(4,5)P2. Amplification of PIP5K1A therefore disrupts PI(4,5)P2 homeostasis, but the diaspora of plasma membrane function downstream of PI(4,5)P2 has made it difficult to identify the related function(s) disrupted in malignant disease. Our objective in this R03 is to determine the extent to which disrupted PI(4,5)P2 homeostasis and PIP5K1A amplification changes PI3K signaling, since PI3K is known to be a central pathway in tumorigenesis and PI(4,5)P2 is its substrate. Our central hypothesis is that PI(4,5)P2 homeostasis is a key determinant of PI3K signal strength in health and disease. Recent evidence indicates that PIP5K enzymes are negatively regulated by binding to with the related PIP4K enzymes, independent of PIP4K catalytic activity. Our own unpublished work shows that PIP4K proteins are also low-affinity PI(4,5)P2 binding proteins in cells, suggesting a tripartite PI(4,5)P2 homeostat; consequently, PIP5K1A over-expression is expected to upregulate PI(4,5)P2 levels but crucially, to do so in a catalytically independent manner, since the over-expressed PIP5K sequesters the available negative regulator, PIP4K. Elevated PI(4,5)P2 levels then drive enhanced PI3K signaling. The rationale for this work is that by identifying the mechanism by which PIP5K1A amplification disrupts PI(4,5)P2 homeostasis and enhances PI3K signal strength, we will illuminate the path to developing small molecule inhibitors of PIP5K1A. To this end, we will address the following specific aims: (1) Determine the extent to which PI3K signaling can be modulated by experimental manipulation of PI(4,5)P2 homeostasis. We hypothesize that elevated PI(4,5)P2 homeostasis via modulation of PIP5K-PIP4K-PI(4,5)P2 interactions will produce corresponding changes in PI3K signaling output. We will change the expression level and localization of endogenous PIP4K and PIP5K in 293A cells, and determine PI3K signaling using direct measures (PIP3 production and Akt phosphorylation). (2) Identify how changes in PIP5K1A expression in cancer cell lines activates PI3K signaling and drive proliferation. We will test the hypothesis that PIP5K1A overexpression in cancer cells upregulates PI3K signaling and proliferation. We will over-express PIP5K1A in non-transformed and diploid cells, as well as reducing expression in cell lines with PIP5K1A amplification. PI3K signaling will be determined as in aim 1, along with proliferation. Upon the completion of this project, we expect to find that elevated PI(4,5)P2 homeostasis enhances PI3K activity; moreover, we will show that this elevated homeostasis manifests in cancer cells with PIP5K1A amplification, causing increased oncogenic PI3K signaling and proliferation. This finding will be significant, because it will identify PIP5K1A as both a novel marker of enhanced PI3K signaling, and as a promising new target to inhibit PI3K signaling in tumors. We believe our approach is innovative, because it considers the homeostatic mechanism regulating PI(4,5)P2 synthesis, rather than just exploring the isolated catalytic function of PIP5Ks.

项目摘要 PIP 5 K1 A在大约5%的癌症中被扩增或突变，但其作为药物靶点的潜力还没有被证实。 到目前为止已经实现。已知该酶合成关键调节脂质PI（4，5）P2。扩增 因此，PIP 5 K1 A破坏PI（4，5）P2稳态，但下游质膜功能的离散 PI（4，5）P2的功能异常使得难以鉴定恶性疾病中被破坏的相关功能。我们的目标 在该R 03中，确定破坏PI（4，5）P2稳态和PIP 5 K1 A扩增的程度 改变PI 3 K信号传导，因为已知PI 3 K是肿瘤发生中的中心途径，PI（4，5）P2是其 衬底我们的中心假设是PI（4，5）P2稳态是PI 3 K信号强度的关键决定因素， 健康和疾病。最近的证据表明，PIP 5 K酶通过与 相关的PIP 4K酶，独立于PIP 4K催化活性。我们未发表的研究表明， PIP 4K蛋白在细胞中也是低亲和力的PI（4，5）P2结合蛋白，表明三重PI（4，5）P2 因此，预期PIP 5 K1 A过表达上调PI（4，5）P2水平，但关键的是， 以催化独立的方式这样做，因为过表达的PIP 5 K隔离了可用的阴性表达， 调节器，PIP 4K。升高的PI（4，5）P2水平然后驱动增强的PI 3 K信号传导。这项工作的基本原理是 通过鉴定PIP 5 K1 A扩增破坏PI（4，5）P2稳态并增强PI（4，5）P2稳态的机制， PI 3 K信号强度，我们将阐明开发PIP 5 K1 A小分子抑制剂的途径。为此目的， 我们将致力于以下具体目标：（1）确定PI 3 K信号可以被调节的程度 通过PI（4，5）P2稳态的实验操作。我们假设PI（4，5）P2稳态升高 通过调节PIP 5 K-PIP 4K-PI（4，5）P2的相互作用，将在PI 3 K信号转导中产生相应的变化 输出.我们将改变内源性PIP 4K和PIP 5 K在293 A细胞中的表达水平和定位， 使用直接测量（PIP 3产生和Akt磷酸化）确定PI 3 K信号传导。(2)确定如何 癌细胞系中PIP 5 K1 A表达的变化激活PI 3 K信号传导并驱动增殖。我们将测试 癌细胞中PIP 5 K1 A过表达上调PI 3 K信号传导和增殖的假设。我们 将在非转化细胞和二倍体细胞中过表达PIP 5 K1 A，并减少细胞系中的表达 PIP 5 K1 A扩增。PI 3 K信号传导将如目的1中所述，沿着增殖来测定。于 本课题完成后，我们期望发现PI（4，5）P2稳态的升高可增强PI 3 K活性; 此外，我们将证明这种升高的稳态在具有PIP 5 K1 A扩增的癌细胞中表现出来， 导致致癌PI 3 K信号传导和增殖增加。这一发现意义重大，因为它将 将PIP 5 K1 A鉴定为增强PI 3 K信号传导的新标记物，并鉴定为抑制PI 3 K信号传导的有希望的新靶点， 肿瘤中的PI 3 K信号传导。我们相信我们的方法是创新的，因为它考虑了体内平衡， PI（4，5）P2合成的调控机制，而不仅仅是探索PIP 5 Ks的孤立催化功能。