Improving radiation response by targeting O2 metabolism via the PI3K/mTOR pathway

 描述(申请人提供)：放射治疗通常用于治疗实体肿瘤，包括头颈部鳞状细胞癌(HNSCC)；然而，许多患者仍然局部失败。因此，我们需要找到新的方法来提高其有效性。我们一直在研究PI3K/mTOR途径的抑制剂。在初步研究中，我们发现PI3K/mTOR双重抑制剂NVP-BEZ235和PI3K抑制剂NVP-BKM120能够在体外使细胞辐射增敏并诱导自噬，我们推测这是一种细胞保护反应，而不是一种细胞死亡模式。在目标1中，我们将使用BKM120在体外验证这一假说，并在裸鼠的侧翼和原位肿瘤中进行体内验证。我们将同时使用遗传方法(敲除关键的自噬基因)和药理学方法(抑制自噬、Spautin1和氯喹的化学物质)。我们也有初步数据表明，多种抑制PI3K/mTOR信号通路的药物，包括上述2种药物以及GDC-0980、GDC-0068和RAD001，在体外都能降低氧耗率(OCR)。我们还表明，BEZ235可以减少体内肿瘤的缺氧，因此，我们提出了一个新的模型，通过该模型，肿瘤内的氧合可能被调节，以增加辐射后的细胞杀伤。在目标2中，我们将探讨这些药物降低OCR的机制(S)。我们有两个假设，第一个假设是它们增加了丙酮酸脱氢酶E1亚单位Ser293的磷酸化，该亚单位是线粒体呼吸的关键守门人。PDHE1的磷酸化抑制其功能，从而减少丙酮酸进入柠檬酸循环，从而降低OCR.我们的第二个假设是，抑制mTOR的药物下调了参与细胞呼吸的线粒体蛋白的表达。在目标3中，我们将研究通过抑制PI3K/mTOR减少O2消耗是否导致体内辐射敏感性的增加。我们的方法之一是使用药物GDC-0980，它不会影响固有的(体外)放射敏感性，但会降低OCR。因此，如果这种药物导致体内辐射反应增加，它很可能是通过影响氧合作用来实现的。在目标3中，我们还将继续筛选FDA批准的426个化合物库中的药物，以寻找其他降低OCR的药物。然后，我们将测试它们在体内对肿瘤缺氧的影响(根据OCR降低的程度)，并确定它们是否与PI3K/mTOR抑制剂在减少缺氧方面具有相加作用。这些目标的成功实现将为目前在临床上测试的PI3K/mTOR抑制剂与放射治疗HNSCC结合使用奠定基础，并为影响肿瘤细胞氧代谢的转化药物创造新的线索。

 DESCRIPTION (provided by applicant): Radiation therapy is commonly used to treat solid tumors including head and neck squamous cell cancer (HNSCC); however, many patients still fail locally. Therefore, we need to find new ways to increase its effectiveness. We have been investigating inhibitors of the PI3K/mTOR pathway. In preliminary studies we found that NVP-BEZ235, a dual PI3K/mTOR inhibitor, and NVP-BKM120, a PI3K inhibitor, radiosensitize cells in vitro and induce autophagy, which we hypothesize is a cytoprotective response rather than a mode of cell death. In Aim 1 we will test this hypothesis in vitro using BKM120 and also in vivo with flank and orthotopic tumors in nude mice. We will use both genetic approaches (knocking out key autophagy genes) and pharmacologic approaches (chemicals that inhibit autophagy, Spautin1 and chloroquine). We also have preliminary data that multiple drugs that inhibit PI3K/mTOR signaling including, the 2 above and GDC-0980, GDC-0068, and RAD001, decrease O2 consumption rate (OCR) in vitro. We have also shown that BEZ235 decreases tumor hypoxia in vivo; thereby, leading us to propose a new model by which oxygenation within tumors may be modulated to increase cell killing after radiation. In Aim 2 we will investigate the mechanism(s) by which these drugs decrease OCR. We have 2 hypotheses, the first of which is that they increase Ser293 phosphorylation of the E1 subunit of pyruvate dehydrogenase (PDH), which is a critical gatekeeper of mitochondrial respiration. Phosphorylation of PDH E1 inhibits its function, hence reduces entry of pyruvate into the citric acid cycle and consequently decreases OCR. Our second hypothesis is that drugs that inhibit mTOR downregulate the expression of mitochondrial proteins that are involved in cellular respiration. In Aim 3 we will investigate whether the decrease in O2 consumption by PI3K/mTOR inhibition leads to increased radiation sensitivity in vivo. One of the ways we will do this is by using the drug GDC-0980, which does not affect intrinsic (in vitro) radiosensitivity but does reduce OCR. Hence, if this drug leads to increased radiation response in vivo, it is likely through effects on oxygenation. In Aim 3 we will also continue our screen of a 426 chemical compound library of FDA- approved agents to search for other agents that decrease OCR. We will then test the top candidates (in terms of degree of reduction of OCR) for their effects on tumor hypoxia in vivo and determine whether they have an additive effect with PI3K/mTOR inhibitors on decreasing hypoxia. Successful completion of these aims will set the stage for PI3K/mTOR inhibitors currently being tested in the clinic to be used in combination with radiotherapy for HNSCC and generate new leads for translational drugs that impact upon tumor cell oxygen metabolism.