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

 描述（由申请人提供）：放射治疗通常用于治疗实体瘤，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效力。我们一直在研究PI 3 K/mTOR通路的抑制剂。在初步研究中，我们发现NVP-BEZ 235（一种双重PI 3 K/mTOR抑制剂）和NVP-BKM 120（一种PI 3 K抑制剂）在体外使细胞放射增敏并诱导自噬，我们假设这是一种细胞保护反应而不是细胞死亡模式。在目标1中，我们将使用BKM 120在体外以及在裸鼠中用侧腹和原位肿瘤在体内检验这一假设。我们将使用遗传方法（敲除关键的自噬基因）和药理学方法（抑制自噬的化学物质，Spautin 1和氯喹）。我们也有初步数据表明，多种抑制PI 3 K/mTOR信号传导的药物，包括上述2种和GDC-0980、GDC-0068和RAD 001，在体外降低O2消耗率（OCR）。我们还表明，BEZ 235减少了体内肿瘤缺氧;因此，我们提出了一种新的模型，通过该模型可以调节肿瘤内的氧合，以增加辐射后的细胞杀伤。在目标2中，我们将研究这些药物降低OCR的机制。我们有两个假设，第一个是它们增加了丙酮酸脱氢酶（PDH）E1 β亚基的Ser 293磷酸化，这是线粒体呼吸的关键守门人。PDH E1 β的磷酸化抑制其功能，因此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PI 3 K/mTOR抑制减少O2消耗是否导致体内辐射敏感性增加。其中一种方法是使用药物GDC-0980，它不会影响内在（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，则可能是通过对氧合的影响。在目标3中，我们还将继续筛选FDA批准的426种化合物库，以寻找其他降低OCR的药物。然后，我们将测试最佳候选物（就OCR的降低程度而言）对体内肿瘤缺氧的影响，并确定它们是否与PI 3 K/mTOR抑制剂对减少缺氧具有累加效应。这些目标的成功完成将为目前在临床上测试的PI 3 K/mTOR抑制剂与HNSCC的放射治疗联合使用奠定基础，并为影响肿瘤细胞氧代谢的转化药物产生新的线索。

 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.