TOR kinase inhibitors for leukemia therapy: mechanisms of action and resistance

描述(申请人提供)：大多数癌细胞表现出磷脂酰肌醇3-激酶(PI3K)及其下游酶AKT和TOR的激活。靶向PI3K/AKT/TOR网络是一种很有前途的癌症治疗策略，但目前尚不清楚哪种靶向模式将提供最佳的疗效和耐受性平衡。部分抑制TOR的化合物(如雷帕霉素)或同时抑制PI3K和TOR的化合物(如PI-103)在疗效和/或耐受性方面存在显著限制。该领域的一项重大突破是发现了高效、选择性的小分子竞争性TOR抑制剂。这些化合物被称为“活性部位TOR抑制剂”，当TORC1或TORC2这两个不同的多蛋白复合体中存在TOR酶时，它们完全抑制TOR酶。与变构抑制剂雷帕霉素相比，活性部位的TOR抑制剂在关键信号通路上具有更广泛的作用，在抑制小鼠和人白血病细胞存活方面更有效。值得注意的是，尽管这些化合物具有更强的抗白血病效果，但它们的免疫抑制作用不如雷帕霉素。活性部位的TOR抑制剂在白血病模型中似乎与选择性较低的PI3K/TOR抑制剂一样有效，但在小鼠中的耐受性要好得多。几种活性部位的TOR抑制剂正在进行早期临床试验。这项建议的总体目标是完善和扩大我们对活性部位TOR抑制剂的理解，最终目的是改善癌症患者的健康。我们将重点放在B细胞恶性肿瘤上，因为活性部位TOR抑制剂在B细胞白血病模型中有显著的效果，但其作用机制仍不清楚。该提案有两个具体目标。首先，我们将建立活性部位TOR抑制剂触发白血病细胞死亡的机制。在这一目标中，我们将使用遗传学方法来检验TORC1和TORC2都在白血病细胞中介导生存信号的假设。这将使用可诱导的Cre介导的基因敲除系统和shRNA介导的基因敲除系统来完成。然后，我们将测试TORC1和TORC2底物在维持白血病细胞存活方面的作用。其次，我们将确定细胞对活性部位TOR抑制剂的耐药机制。与任何靶向分子方法一样，不同类型的癌细胞对活性部位的TOR抑制剂表现出不同的敏感性。药物开发中的一个新主题是需要确定靶向药物的有效组合。使用对TOR抑制没有发生凋亡反应的细胞系，我们将使用候选和全局方法来确定可用药的耐药机制。然后，我们将在体外和体内测试联合策略。识别耐药机制和应用适当的药物组合将拓宽活性部位TOR抑制剂的潜在应用。

DESCRIPTION (provided by applicant): Most cancer cells display activation of phosphoinositide 3-kinase (PI3K) and the downstream enzymes AKT and TOR. Targeting the PI3K/AKT/TOR network is a promising strategy for cancer therapeutics, yet it is not clear which target profile will provide the best balance of efficacy and tolerability. Compounds that partially inhibit TOR (such as rapamycin) or compounds that inhibit both PI3K and TOR (such as PI-103) have significant limitations in efficacy and/or tolerability. A major breakthrough in this field has been the identification of novel compounds that are highly potent, selective, small molecule competitive inhibitors of TOR. Termed "active-site TOR inhibitors", these compounds fully inhibit the TOR enzyme when it is present in TORC1 or TORC2, two distinct multiprotein complexes. Compared to rapamycin, an allosteric inhibitor, active-site TOR inhibitors have a broader effect on key signaling pathways and are more effective at suppressing survival of murine and human leukemia cells. Remarkably, these compounds are less immunosuppressive than rapamycin despite their greater anti-leukemic efficacy. Active-site TOR inhibitors appear equally effective in leukemia models as the less selective PI3K/TOR inhibitors, yet are considerably better tolerated in mice. Several active-site TOR inhibitors are in early stage clinical trials. The overall objective of this proposal is to refine and extend our understanding of active-site TOR inhibitors, with the ultimate goal of improving the health of cancer patients. We will focus on B cell malignancies, since active-site TOR inhibitors have dramatic effects in B cell leukemia models yet the mechanism of action remains poorly understood. The proposal has two specific aims. First, we will establish the mechanisms by which active-site TOR inhibitors trigger leukemia cell death. In this aim we will use genetic approaches to test the hypothesis that both TORC1 and TORC2 mediate survival signaling in leukemia cells. This will be accomplished using inducible Cre-mediated knockout systems and shRNA-mediated knockdown. We will then test the roles of TORC1 and TORC2 substrates in maintaining survival in leukemia cells. Second, we will define mechanisms of cellular resistance to active-site TOR inhibitors. As with any targeted molecular approach, subtypes of cancer cells display differing sensitivity to active-site TOR inhibitors. An emerging theme in drug development is the need to identify effective combinations of targeted agents. Using cell lines that do not undergo apoptosis in response to TOR inhibition, we will use candidate and global approaches to identify druggable mechanisms of resistance. We will then test combination strategies in vitro and in vivo. Identifying mechanisms of resistance and applying appropriate drug combinations will broaden the potential application of active-site TOR inhibitors.