A Phase 0 Trial of Hydroxychloroquine in Patients with Stage III and IV Resectabl

描述（由申请人提供）：自噬使肿瘤能够在休眠状态下长时间存活代谢应激，因此，该途径是创建新抗癌疗法的新兴靶点。羟氯喹（HCQ）是一种阻断自噬的药物，提高溶酶体内pH值并抑制该途径的最后一步，从而损害自噬蛋白降解。这导致自噬体积累，潜在地干扰依赖于自噬的肿瘤细胞的存活，并使它们转向凋亡和/或坏死细胞死亡。在黑色素瘤细胞系中的初步结果表明，用氯喹（CQ）（一种在实验室中容易使用的与HCQ类似的药物）治疗增加了自噬体积累，并且从用不同浓度的CQ治疗中注意到增加的细胞死亡。此外，CQ在哺乳动物肿瘤同种异体移植模型中诱导自噬体积累。鉴于自噬途径在黑色素瘤中可能是活跃的，我们假设HCQ的施用可以在体内调节人类黑色素瘤肿瘤组织样品中的自噬过程。为了检验这一假设，建议在计划进行姑息性或根治性切除的III期或IV期黑色素瘤患者中进行HCQ的0期试验。具体而言，我们将寻找HCQ抑制自噬的迹象，比较治疗前与治疗后肿瘤标本中在电子显微镜（EM）上观察到的每个细胞的自噬囊泡的平均数量。将对治疗前和治疗后皮肤样本和PBMC进行类似的分析，以探索这些样本作为治疗效果替代标志物的潜力。虽然EM上的自噬体积累是用于检测自噬抑制的可靠方法，但其昂贵、繁琐并且需要技术人员来执行，因此不能广泛应用于临床样本以检测自噬抑制。因此，作为本试验的一部分，我们将研究几种蛋白质，这些蛋白质在实验室模型和人类肿瘤组织的一些初步研究中检测自噬的抑制作用，希望这些更简单的测定将有助于人类样本中的自噬检测。将被检查的标志物包括Beclin 1，一种重要的自噬调节因子，和LC 3，它被切割成LC 3-I和LC 3- II，后者在自噬过程中易位到自噬体膜。此外，我们的小组还发现，自噬缺陷肿瘤在应激下积累p62，一种多聚泛素结合蛋白，将聚集体递送到溶酶体进行降解，以及几种伴侣蛋白，包括葡萄糖相关蛋白70（GRp 170）。预期在具有功能性自噬的黑色素瘤中用HCQ治疗将导致EM上自噬体形成的增加以及通过免疫组织化学和Western印迹的LC 3-II、p62和GRp 170水平的增加。还将通过使用治疗前和治疗后活化的半胱天冬酶-3、TUNEL、Ki-67和有丝分裂率测定来探索HCQ的潜在促凋亡和/或抗增殖作用。该提案的目标是首次在人类肿瘤中调节自噬，使用这些实验的结果来指导未来临床试验中自噬标志物的开发。 公共卫生相关性：我们建议在患有黑色素瘤的人类中进行初步试验（O期试验），以观察施用羟氯喹（HCQ）（一种FDA批准的药物，几乎没有副作用）是否可以阻断自噬过程，这是几种不同类型肿瘤细胞的生存模式。我们将使用一种称为电子显微镜的方法来研究HCQ是否抑制黑色素瘤肿瘤组织以及血液和皮肤样本中的自噬，但由于这种方法昂贵且费力，我们还将检查四种蛋白质-Beclin 1，LC 3，p62和GRp 170-它们检测细胞系，动物模型，以及一些保存的人体组织样本，以证明这些更简单的测试也可以检测人类自噬的抑制。我们的希望是，我们将能够利用这些结果来更好地理解和检测自噬，从而更容易开发针对癌症中这一途径的未来药物。

DESCRIPTION (provided by applicant): Autophagy enables tumors to survive metabolic stress in a dormant state for prolonged periods of time and as such, this pathway is an emerging target for the creation of new anticancer therapies. Hydroxychloroquine (HCQ) is a drug that blocks autophagy, raising intralysosomal pH and inhibiting the final step of this pathway, impairing autophagic protein degradation. This results in autophagosome accumulation, potentially disturbing the survival of tumor cells reliant on autophagy and shunting them toward apoptotic and/or necrotic cell death. Preliminary results in melanoma cell lines indicate that treatment with chloroquine (CQ), a similar drug to HCQ used readily in the laboratory, increases autophagosome accumulation, and increased cell death was noted from treatment with CQ at varying concentrations. Additionally, CQ induced autophagosome accumulation in a mammalian tumor allograft model. Given that the autophagy pathway may be active in melanoma, we hypothesize that administration of HCQ can modulate the process of autophagy in melanoma tumor tissue samples in humans in vivo. To test this hypothesis, a phase 0 trial of HCQ in patients with stage III or IV melanoma planned for palliative or curative resection is proposed. Specifically, we will look for signs of autophagy inhibition by HCQ, comparing the mean number of autophagic vesicles per cell seen on electron microscopy (EM) in pre- versus post- treatment tumor specimens. Pre- and post- treatment skin samples and PBMCs will similarly be assayed to explore the potential of these samples as surrogate markers for treatment effect. While autophagosome accumulation on EM is a reliable method for detection of autophagy inhibition, it is expensive, tedious and requires a skilled individual to perform and hence, is not widely applicable to clinical specimens to detect autophagy inhibition. Thus, as part of this trial we will examine several proteins shown to detect inhibition of autophagy in laboratory models and in some preliminary studies of human tumor tissue, with the hope that these simpler assays will facilitate autophagy detection in human samples. Markers that will be examined include Beclin1, an essential autophagy regulator, and LC3, which is cleaved into LC3-I and LC3- II, with the latter translocated to the autophagosome membrane during autophagy. In addition, our group has discovered that autophagy deficient tumors under stress accumulate p62, a polyubiquitin binding protein that delivers aggregates to the lysosome for degradation, as well as several chaperone proteins including glucose related protein 70 (GRp170). It is expected that treatment with HCQ in melanomas with functional autophagy will result in increased autophagosome formation on EM and increased levels of LC3-II, p62 and GRp170 by immunohistochemistry and Western blot. Potential proapoptotic and/or ant proliferative effects of HCQ will also be explored through the use of pre- and post-treatment activated caspase-3, TUNEL, Ki-67 and mitotic rate assays. The goal of this proposal is to modulate autophagy for the first time in human tumors, using results from these experiments to guide development of markers of autophagy in future clinical trials. PUBLIC HEALTH RELEVANCE: We propose a preliminary trial in humans with melanoma (a Phase O trial) to see if administering hydroxychloroquine (HCQ), a FDA approved drug with little to no side effects, can block the process of autophagy, a survival mode for several different types of tumor cells. We will use a method called electron microscopy to study if HCQ is inhibiting autophagy in melanoma tumor tissue and blood and skin samples, but since this method is expensive and laborious we will also examine four proteins -- Beclin1, LC3, p62 and GRp170 -- which detect autophagy inhibition in cell lines, animal models, and some preserved human tissue samples to demonstrate that these simpler tests may also detect inhibition of autophagy in human beings. Our hope is that we will be able to use these results to better understand and detect autophagy, making it easier to develop future drugs against this pathway in cancer.