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

项目总结 PIP5K1A在大约5%的癌症中被扩增或突变，但它作为药物靶点的潜力并没有 到目前为止已经实现了。已知该酶可合成关键的调节脂质PI(4，5)P2。放大 因此，PIP5K1A破坏了PI(4，5)P2的动态平衡，但质膜的散布在下游发挥作用 PI(4，5)P2的突变使恶性疾病的相关功能(S)难以识别。我们的目标 在本R03中测定的是破坏PI(4，5)P2动态平衡和PIP5K1A扩增的程度 改变PI3K信号，因为PI3K被认为是肿瘤发生的中心途径，而PI(4，5)P2是其 底物。我们的中心假设是PI(4，5)P2稳态是PI3K信号强度的关键决定因素 健康和疾病。最近的证据表明，PIP5K酶通过与WITS结合而受到负调控 相关的PIP4K酶，不依赖于PIP4K的催化活性。我们自己未发表的研究表明， PIP4K蛋白在细胞中也是低亲和力的PI(4，5)P2结合蛋白，提示PIP4K蛋白是三段式的PI(4，5)P2 因此，PIP5K1A的过度表达有望上调PI(4，5)P2水平，但关键是， 这是以一种催化独立的方式进行的，因为过度表达的PIP5K隔离了可用的负电荷 调节器，PIP4K。PI(4，5)P2水平升高进而驱动PI3K信号转导增强。这项工作的基本原理是 通过确定PIP5K1A扩增破坏PI(4，5)P2动态平衡和增强 PI3K信号强度，我们将阐明开发PIP5K1A小分子抑制剂的途径。为此， 我们将解决以下具体目标：(1)确定PI3K信令可以调制的程度 通过实验操纵PI(4，5)P2的动态平衡。我们假设PI(4，5)P2动态平衡升高 通过对PIP5K-PIP4K-PI(4，5)P2的调控，PI3K信号会发生相应的变化 输出。我们将改变内源性PIP4K和PIP5K在293A细胞中的表达水平和定位，以及 使用直接测量(PIP3产生和Akt磷酸化)来确定PI3K信号。(2)确定如何 PIP5K1A在癌细胞中的表达变化激活了PI3K信号通路，并推动了细胞增殖。我们将测试 PIP5K1A在癌细胞中过表达可上调PI3K信号转导和增殖。我们 将在未转化细胞和二倍体细胞中过表达PIP5K1A，并在有 PIP5K1A扩增产物。PI3K信号将被确定为与目标1中的一样，以及增殖。在此之前 这个项目的完成，我们预计会发现PI(4，5)P2动态平衡升高会增强PI3K活性； 此外，我们将通过PIP5K1A扩增在癌细胞中表现出这种升高的动态平衡， 导致致癌PI3K信号和增殖增加。这一发现将具有重大意义，因为它将 确定PIP5K1A既是增强PI3K信号的新标记，也是一个有希望的新抑制靶点 肿瘤中的PI3K信号转导。我们相信我们的方法是创新的，因为它考虑了动态平衡 调节PI(4，5)P2合成的机制，而不仅仅是探索PIP5Ks的单独催化功能。

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 Our own unpublished work shows that PIP4K proteins are also low-affinity PI(4,5)P2 binding proteins in cells, suggesting a tripartite PI(4,5)P2 homeostat; consequently, PIP5K1A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.