Solving the elusive mechanism of rapamycin action in lymphocytes

描述(申请人提供)：mTOR是一种普遍表达的丝氨酸/苏氨酸激酶。MTOR酶存在于两个细胞复合体TORC1和TORC2中。MTOR是基于它与药物雷帕霉素的相互作用而被发现和命名的。该化合物是一种变构抑制剂，可以部分阻断TORC1的活性，通常不抑制TORC2的功能。雷帕霉素治疗T和B淋巴细胞可以极大地阻止由抗原和其他有丝分裂原引发的增殖，而雷帕霉素(西罗莫斯；Rapamune(R))是FDA批准的一种免疫抑制剂。然而，雷帕霉素及其类似物对包括癌细胞在内的其他细胞系统的增殖影响较小。虽然雷帕霉素对淋巴细胞的影响已经研究了20年，但为什么这种化合物选择性地诱导淋巴细胞增殖，目前还不清楚。矛盾的是，一类新型的ATP竞争性mTOR激酶抑制剂(TOR-Kis)比雷帕霉素引起的免疫抑制更少，尽管对TORC1和TORC2都有更完全的抑制。该项目的目标是确定雷帕霉素选择性阻断正常淋巴细胞增殖的机制。基于初步数据，我们提出了两个假设，它们构成了不同的具体目标的基础。目的1验证雷帕霉素对TORC1的关键底物S6激酶有更全面和持久的抑制作用的假设。有两种S6蛋白，S6K1和S6K2，但它们在淋巴细胞活化和增殖中的作用尚未见报道。我们将使用一种新的小鼠模型，该模型将允许一种化学遗传方法来特异性地抑制正常T和B细胞中的S6K活性。我们将使用这个模型来比较S6K抑制的效果与雷帕霉素和TOR-Kis的效果。目的2验证雷帕霉素抑制mTOR不依赖于激酶的功能的假说。我们将产生一种新的小鼠品系，在该品系中，激酶死亡的mTOR可以以细胞特异性的方式表达。这将使我们能够测试这样的预测，即表达激酶死亡mTOR的淋巴细胞将保持对雷帕霉素仍然敏感的剩余增殖。我们还将开始一种候选方法来测试可能的激酶依赖的mTOR功能。 公共卫生相关性：一种名为雷帕霉素的药物被用来抑制免疫系统，防止器官移植的排斥反应。然而，与其他类型的细胞相比，为什么雷帕霉素选择性地阻断免疫细胞的激活尚不清楚。这项建议旨在解决这个长期存在的问题。

DESCRIPTION (provided by applicant): mTOR is a serine/threonine kinase that is expressed ubiquitously. The mTOR enzyme is present in two cellular complexes, TORC1 and TORC2. mTOR was discovered and named based on its interaction with the drug rapamycin. This compound is an allosteric inhibitor that partially blocks TORC1 activity and generally does not inhibit TORC2 function. Rapamycin treatment of T and B lymphocytes profoundly blocks proliferation triggered by antigen and other mitogens, and rapamycin (sirolimus; Rapamune(R)) is an FDA-approved immunosuppressant. However, rapamycin and analogs have lesser effects on proliferation in other cellular systems including cancer cells. Although the effects of rapamycin on lymphocytes have been studied for 20 years, it remains unclear why the compound induces a profound block of proliferation selectively in lymphocytes. Paradoxically, a novel class of ATP-competitive mTOR kinase inhibitors (TOR-KIs) causes less immunosuppression than rapamycin despite causing more complete inhibition of both TORC1 and TORC2. The goal of this project is to define mechanisms by which rapamycin selectively blocks proliferation of normal lymphocytes. Based on preliminary data, we propose two hypotheses that form the basis of separate specific aims. Aim 1 will test the hypothesis that rapamycin causes more complete and sustained inhibition of S6 kinases, key substrates of TORC1. There are two S6 kinases, S6K1 and S6K2, but their roles in lymphocyte activation and proliferation have not been reported. We will use a novel mouse model that will allow a chemical genetic approach to specifically inhibit S6K activity in normal T and B cells. We will use this model to compare the effects of S6K inhibition with the effects of rapamycin and TOR-KIs. Aim 2 will test the hypothesis that rapamycin inhibits kinase-independent functions of mTOR. We will generate a novel mouse strain in which kinase-dead mTOR can be expressed in a cell-specific manner. This will allow us to test the prediction that lymphocytes expressing kinase-dead mTOR will retain residual proliferation that remains sensitive to rapamycin. We will also begin a candidate approach to test possible kinase-independent mTOR functions. PUBLIC HEALTH RELEVANCE: A drug called rapamycin is used to suppress the immune system and prevent rejection of organ transplants. However, it is not known why rapamycin selectively blocks the activation of immune cells, compared to other cell types. This proposal seeks to solve this long-standing question.