Arsenic Trioxide and Acute Myeloid Leukemia

描述(申请人提供)：三氧化二砷在急性髓细胞白血病(AML)的一种亚型--急性早幼粒细胞白血病(APL)中取得了显著的治疗成功，促使人们广泛努力将这种疗法推广到其他恶性肿瘤。我们和其他人确定，As_2O_3通过产生活性氧(ROS)诱导APL细胞凋亡。我们还发现，PML-RARA的降解不是As_2O_3促凋亡程序中的关键事件，但高ROS产生能力是关键事件。因此，我们推测，通过联合治疗提高ROS的生成能力，我们将能够有效地靶向非APL AML的细胞凋亡。膜上定位的NADPH氧化酶和线粒体是ROS产生的主要来源。AML细胞与多形核细胞有共同的谱系起源，多形核细胞使用NADPH氧化酶产生的ROS来杀灭细菌。我们发现，AML细胞经As_2O_3处理后，NADPH氧化酶的关键成分p47Phox的表达增加，但没有引起激活。我们的初步结果显示，治疗上可达到的As2O3浓度与Fenretinide(4-HPR)(一种批准用于临床试验的维甲酸)的组合，在导致AML细胞凋亡方面具有协同作用，这些AML细胞表达可检测到的(基础或As2O3诱导的)NADPH氧化酶成分，并延长携带AML的SCID/NOD小鼠的生存时间。在生理条件下，线粒体呼吸产生的ROS被谷胱甘肽酶、谷胱甘肽过氧化物酶、谷胱甘肽S转移酶？(商品及服务税？)谷胱甘肽还原酶(GR)。抑制这些酶的活性，再加上癌细胞线粒体呼吸功能障碍导致的ROS生成增加，应该会产生超出耐受的ROS水平，导致线粒体介导的AML细胞凋亡。事实上，抑制GPX的三氧化二砷与我们开发的利尿酸(一种临床上使用的利尿药)及其新的衍生物结合在一起，可以抑制GST吗？GR、GR协同诱导AML细胞凋亡。这种作用不依赖于NADPH氧化酶的表达。我们建议研究这些发现背后的机制及其在原发AML和活体(动物模型)中的作用。在目标1中，我们将通过神经酰胺依赖的途径来研究As_2O_3诱导NADPH氧化酶表达和4-HPR激活NADPH氧化酶的机制。在目标2中，我们将通过抑制ROS降解和减少抗凋亡蛋白Mcl-1来研究As_2O_3和Eacrynic及其衍生物协同诱导细胞凋亡的机制。在目标3中，我们将研究As_2O_3和4-HPR或As_2O_3和Eacrynic在原代AML样本中的凋亡效应。重要的是，我们将研究使用原代人类AML细胞中NADPH氧化酶/髓过氧化物酶或GST？/过氧化氢酶水平分别作为患者对As_2O_3/4-HPR或As_2O_3/Eacrynic治疗敏感或耐药的潜在预测标志物的可能性。在特定的目标4中，我们将测试联合治疗在人AML异种移植中的疗效。如果进一步的研究证实这些治疗组合确实有效地诱导AML细胞系和原代细胞的凋亡，并且它们在异种移植中具有治疗效果，而没有过多的毒性，因为每种被测试化合物目前的临床使用，那么将有可能将这些发现迅速转化为临床试验。公共卫生相关性：三氧化二砷是一种全球使用的药物，可使90%的急性早幼粒细胞白血病患者临床完全缓解，且无明显毒性。我们的治疗设计包括使用4HPR，一种已经在临床试验中的药物，它激活了产生ROS的膜系统，并使用了新的非利尿剂乙基丙烯酸衍生物，它阻断了减少ROS的酶，并通过几种机制显著增强了As_2O_3诱导的白血病细胞死亡。我们的目标是在难治性、继发性或老年急性髓系白血病患者的临床试验中联合使用As2O3和4种HPR或EA类似物。

DESCRIPTION (provided by applicant): The outstanding therapeutic success of As2O3 in a subtype of acute myelocytic leukemia (AML), the acute promyelocytic leukemia (APL), prompted widespread effort to extend this therapy to other malignancies. We and others determined that, through generation of reactive oxygen species (ROS), As2O3 induced apoptosis of APL cells. We also showed that degradation of PML-RARa was not a crucial event in the pro-apoptotic program of As2O3, but high ROS production ability was. We thus postulate that by enhancing the ROS-generating capacity through combination therapies we will be able to effectively target non-APL AML for apoptosis. Membrane localized NADPH oxidase and mitochondria are the main sources of ROS production. AML cells share lineage derivation with polymorphonuclear cells, which use NADPH oxidase generated ROS to kill bacteria. We showed that As2O3 treatment of AML cells increases the expression, without causing activation, of a crucial component of NADPH oxidase, p47phox. Our preliminary results show that a combination of therapeutically achievable concentration of As2O3 with fenretinide (4-HPR), a retinoid approved for clinical trials, synergizes in causing apoptosis in AML cells which express detectable (basal or As2O3-induced) NADPH oxidase components and extends survival of AML bearing SCID/NOD mice. Under physiological conditions ROS generated as a byproduct of mitochondrial respiration are detoxified by glutathione-enzymes, glutathione peroxidase (GPx), glutathione-s-transferase ? (GST?) and glutathione reductase (GR). Inhibition of the activities of these enzymes combined with increased ROS generation due to malfunction of the mitochondrial respiration in cancer cells, should generate levels of ROS that are beyond tolerable, causing mitochondria-mediated apoptosis of AML cells. Indeed, a combination of As2O3, which inhibits GPx, with ethacrynic acid (a clinically used diuretic drug) and its new derivative, which we have developed, and which inhibit GST? and GR, synergistically induce apoptosis in AML cells. This effect is independent of NADPH oxidase expression. We propose to study the mechanisms underlying these findings and their effect in primary AML and in vivo (animal models) in 4 specific aims. In aim 1 we will study the mechanism of As2O3 induced NADPH oxidase expression and its activation by 4-HPR through a ceramide dependent pathway. In aim 2 we will study the mechanism of the synergy of apoptosis induction by As2O3 and ethacrynic acid, and its derivative, through inhibition of ROS degradation and decrease of an antiapoptotic protein Mcl-1. In aim 3 we will study the apoptotic effects of As2O3 and 4-HPR or As2O3 and ethacrynic acid in primary AML samples. Importantly, we will investigate the possibility of using NADPH oxidase/myeloperoxidase or GST?/catalase levels in primary human AML cells as potential predictive markers of patient stratification to sensitive or resistant to As2O3/4-HPR or As2O3/ethacrynic acid therapies, respectively. In specific aim 4 we will test the efficacy of the combination treatments in xenografts of human AML. If further studies confirm that these treatment combinations are indeed effective in inducing apoptosis in AML cell lines and primary cells, and that they have therapeutic effect in xenografts, without excessive toxicity, because of the current clinical use of each of the tested compounds, it will be possible to rapidly translate these finding into clinical trials. PUBLIC HEALTH RELEVANCE: Arsenic trioxide (As2O3) is a drug used world-wide that induces complete clinical remission in 90% of APL patients without significant toxicity. Our therapeutic design includes the use of 4 HPR, a drug already in clinical trial, that activates a membrane system for the production of ROS and the use of new non-diuretic derivatives of ethyacrynic acid which block enzymes that diminish ROS and markedly enhanceAs2O3 induced leukemic cell death by several mechanisms. Our goal will be to utilize As2O3 in combination with 4 HPR or EA analogs in clinical trials in patients with refractory, secondary or in elderly patients with acute myelogenous leukemia.