Novel Roles of p53 & ROS in Therapy of Refractory CLL

描述（由申请人提供）：慢性淋巴细胞白血病（CLL）是美国和欧洲最常见的成人白血病。最近我们对CLL生物学的理解和新疗法的发展，如基于氟达拉滨的方案，已经导致治疗结果的改善。然而，许多CLL患者，特别是由于染色体17p缺失或p53突变导致p53缺失的患者，对目前的治疗药物具有很高的耐药性，预后非常差。缺乏p53的CLL细胞治疗反应差的潜在机制尚不清楚，治疗这类CLL患者的有效疗法仍有待开发。目前的研究计划旨在回答这一领域的一些关键问题，并开发治疗难治性CLL的新治疗策略。我们之前的研究表明，来自晚期疾病患者的CLL细胞表现出线粒体DNA （mtDNA）突变和功能障碍，活性氧（ROS）的产生升高，对氟达拉滨的敏感性降低。此外，p53通过与线粒体pol 3的相互作用，在维持线粒体遗传完整性方面发挥着重要作用。重要的是，我们观察到ROS增加的癌细胞对天然化合物2-苯乙酯异硫氰酸酯（PEITC）诱导的进一步ROS应激高度敏感。我们的初步研究显示，PEITC对难治性CLL细胞有很好的活性，这导致了开发这种化合物用于CLL临床治疗的重大努力。基于这些观察结果，我们假设p53缺失的CLL细胞容易发生线粒体突变和功能障碍，导致凋亡反应减少和对常规治疗药物的耐药性，但对PEITC仍然敏感，PEITC通过ros介导的机制杀死耐药CLL细胞。这个研究项目将调查3个具体目标。(1)我们将利用来自不同p53状态CLL患者的原代白血病细胞比较其mtDNA突变率，并使用一种新型CLL培养系统来测试p53缺失在线粒体突变和药物对药物凋亡反应的衰减中的作用。(2)研究PEITC等ros应激剂有效杀伤缺乏p53且对常规药物耐药的原代CLL细胞的能力和机制。(3)通过携带或不携带p53的人CLL细胞的小鼠异种移植模型，检测PEITC单独或联合其他药物的体内治疗活性，评估PEITC和其他ros应激剂是否能诱导CLL缓解并提高生存率。本研究将揭示p53在影响CLL细胞线粒体遗传稳定性、功能和药物敏感性中的新作用，为通过ros介导的机制有效杀伤难治性CLL细胞提供新策略。由于PEITC是一种在蔬菜中发现的天然化合物，对正常细胞的毒性较低，因此在临床上应用于难治性CLL治疗是可行的，具有重要意义。公共卫生相关性：慢性淋巴细胞白血病（CLL）是西方世界最常见的成人白血病，在发病率和死亡率方面都造成了重大的健康负担。由于染色体17p缺失或p53基因突变导致p53缺失的CLL细胞对目前的治疗药物具有耐药性，这些患者的临床预后较差。本研究项目的主要目的是研究p53缺失导致线粒体功能障碍和耐药的潜在机制，并测试新的药物和治疗策略，以有效杀死缺乏p53的耐药CLL细胞。本研究将揭示p53在影响CLL细胞线粒体功能和药物敏感性中的重要作用，并将为常规药物难治性CLL患者的有效治疗寻找新的药物。这项研究将对这种最常见的成人白血病的临床治疗产生直接影响。

DESCRIPTION (provided by applicant): Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States and Europe. Recent progress in our understanding of CLL biology and the development of new therapy such as fludarabine-based regimens has led to improvements of the therapeutic outcomes. However, many CLL patients, particularly those with loss of p53 due to chromosome 17p deletion or p53 mutations, are highly resistant to the current therapeutic agents and have very poor prognosis. The underlying mechanisms responsible for the poor therapeutic responses of CLL cells lacking p53 are poorly understood, and effective therapies to treat this population of CLL patients remain to be developed. The current research proposal aims to answer some of the key questions in this area, and to develop novel therapeutic strategies for treating refractory CLL. Our previous studies showed that CLL cells from patients in advanced disease stages exhibit mitochondrial DNA (mtDNA) mutations and dysfunction, elevated generation of reactive oxygen species (ROS), and decreased sensitivity to fludarabine. Furthermore, p53 plays an important role in maintaining mitochondrial genetic integrity through its interaction with mitochondrial pol 3. Importantly, we observed that cancer cells with increased ROS are highly sensitive to further ROS stress induced by a natural compound 2-phenethyl isothiocyanate (PEITC). Our preliminary studies have revealed promising activity of PEITC against refractory CLL cells, leading to major efforts to develop this compound for clinical treatment of CLL. Based on these observations, we hypothesize that CLL cells with p53 deletion are prone to develop mitochondrial mutations and dysfunction leading to a decrease in apoptotic response and resistance to conventional therapeutic agents, but remain sensitive to PEITC which kills drug-resistant CLL cells through ROS-mediated mechanism. This research project will investigate 3 specific aims. (1) We will use primary leukemia cells from CLL patients with different p53 status compare their mtDNA mutation rates, and use a novel CLL culture system to test the role p53 deletion in mitochondrial mutations and attenuation of drug apoptotic response to therapeutic agents. (2) Investigate the ability and mechanisms of PEITC and other ROS-stressing agents to effectively kill primary CLL cells that lack p53 and are resistant to conventional drugs. (3) Test the in vivo therapeutic activity of PEITC, alone or in combination with other drugs, using mouse xenograft models bearing human CLL cells with or without p53 to evaluate if PEITC and other ROS-stressing agents can induce CLL remission and improve survival. This study will reveal the novel roles of p53 in affecting mitochondrial genetic stability and function and drug sensitivity in CLL cells, and provide new strategies to effectively kill refractory CLL cells through ROS-mediated mechanism. Because PEITC is a natural compound found in vegetables with low toxicity to normal cells, its applications for clinical treatment of refractory CLL is feasible and highly significant. PUBLIC HEALTH RELEVANCE: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world and causes a significant health burden both in morbidity and mortality. CLL cells with p53 loss due to chromosome 17p deletion or p53 gene mutation are resistant to current therapeutic agents and these patients have poor clinical outcomes. The main objectives of this research project are to investigate the underlying mechanisms by which the loss p53 leads to mitochondrial dysfunction and drug resistance, and to test novel agents and therapeutic strategies to effectively kill the drug-resistant CLL cells lacking p53. This study will reveal the important role of p53 in affecting mitochondrial function and drug sensitivity in CLL cells, and will identify new therapeutic agents for effective treatment of CLL patients who are refractory to conventional drugs. This research will have directly implications in clinical treatment of this most common adult leukemia.