Melanoma Resistance to Apoptosis: Mechanisms and Therapeutic Potential

描述(由申请人提供)： 恶性黑色素瘤的一个基本特征是对细胞凋亡的抵抗，这在很大程度上决定了黑色素瘤对治疗的抵抗。Bcl2家族促凋亡和抗凋亡成员的复杂相互作用是细胞凋亡的中心控制点，也是一个很有前途的药物靶点。BH3模拟ABT-737是一种有效的小分子抑制蛋白，可抑制抗细胞凋亡蛋白Bcl2/Bclxl/Bclw，无论是作为单一药物还是联合治疗，它都显示出很好的治疗癌症的前景。我们发现ABT-737单独对黑色素瘤细胞的细胞毒作用很小，RNAi技术的基因敲除实验表明，抗凋亡蛋白Mcl-1是黑色素瘤对ABT-737耐药的主要介质，而不是Bcl2或Bclxl。此外，ABT737与蛋白酶体抑制剂(MG-132或Bortezomib)或紫杉醇均显示出很强的协同致死性，机制研究表明蛋白酶体抑制剂和紫杉醇都中和了Mcl-1‘S的作用。这些令人兴奋的结果表明，中和Mcl-1功能的药物是与ABT-737联合治疗黑色素瘤的优秀候选药物，需要在临床前模型中进行验证，并进一步探索其他靶向Mcl-1与ABT-737联合治疗的药物的治疗潜力。也有人提出，癌症起始细胞(或癌症干细胞)可能是癌症抵抗治疗的原因，而消除这些细胞可能是开发任何长期成功的癌症治疗方法的关键。我们将这些细胞称为类癌细胞。我们建议研究在黑色素瘤中，浓缩的黑色素瘤启动细胞是否比未分选的细胞对现有化疗药物更具耐药性，并检查减少多种抗凋亡防御的药物组合，如ABT-737加Bortezomib或紫杉醇是否可以克服黑色素瘤启动细胞的这种耐药性。这些研究将是了解黑色素瘤启动细胞是否对治疗更具抵抗力的重要组成部分。目的1：在体外直接检测富集型黑色素瘤起始细胞对现有疗法和新疗法的反应。I)确定浓缩的黑色素瘤起始细胞是否对替莫唑胺等传统化疗药物更具抗药性。Ii)确定ABT-737与Bortezomib或紫杉醇的组合是否克服了浓缩黑色素瘤起始细胞的耐药性。目的：采用两种新的异种移植模型：体内靶向人黑色素瘤起始细胞模型和黑色素瘤患者样本直接异种移植模型，验证ABT-737联合波特佐米或紫杉醇治疗黑色素瘤的疗效。目的：探讨BH3类似物ABT-737与其他抑制Mcl-1的化合物联合应用的治疗潜力。I)通过microRNAs下调黑色素瘤中Mcl-1的新方法。Ii)其他已知可抑制Mcl-1的药物。该项目建议通过靶向多个抗凋亡的Bcl-2成员来开发新的联合治疗药物，并验证这些治疗组合在杀死黑色素瘤，最重要的是，黑色素瘤起始细胞群方面的效果。我们相信，这里提出的研究将很快导致未来的临床研究和ABT-737(或类似药物)在治疗人类黑色素瘤患者的临床使用。重要的是，这种方法不应该受到个体黑色素瘤不同信号特征的限制。这项建议是由经验丰富的黑色素瘤基础科学家、皮肤干细胞专家、黑色素瘤肿瘤学家和人类癌症异种移植模型专家合作提出的，该团队将在开发基于逆转细胞凋亡抵抗的黑色素瘤治疗方法方面取得重要进展。 公共卫生相关性： 恶性黑色素瘤在很大程度上对现有的治疗方法没有反应，预后非常差，在过去的30年里，这种疾病的医学治疗几乎没有什么进展。此外，在美国，黑色素瘤的发病率正在迅速增加，包括退伍军人中的黑色素瘤人群，在美国服兵役期间暴露在阳光下也与黑色素瘤发病率的增加有关。此外，年轻女性中的黑色素瘤也在迅速增加，退伍军人和现役军人中的黑色素瘤人数越来越多。因此，缺乏有效的恶性黑色素瘤治疗方法是医学和退伍军人管理局患者护理任务中的一个紧迫问题。这项建议使用最先进的研究模型来验证我们开发的新的治疗策略。这里提出的研究结果可能会很快导致临床试验，并立即适用于黑色素瘤患者的临床护理，利用已经在临床试验中的其他癌症的新药物组合。

DESCRIPTION (provided by applicant): A fundamental characteristic of malignant melanoma is resistance to apoptosis, which largely determines melanoma's resistance to therapy. The complex interaction of pro-apoptotic and anti-apoptotic members of the Bcl-2 family is a central control point of apoptosis and a promising drug target. The BH3 mimetic ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2/Bcl-XL/Bcl-w, and it exhibits promise for cancer treatment either as a single agent or in combination therapy. We have found that ABT-737 alone induced little cytotoxicity in melanoma cells, and knockdown experiments with RNAi techniques demonstrated that anti-apoptotic protein Mcl-1, but not Bcl-2 or Bcl-XL is the main mediator of melanoma resistance to ABT-737 treatment. In addition, ABT-737 displayed strong synergistic lethality when combined with either proteasome inhibitors (MG-132 or Bortezomib) or Paclitaxel, and mechanistic studies suggested that both proteasome inhibitors and Paclitaxel neutralize Mcl-1's functions. These exciting results indicate that drugs that neutralize Mcl-1 function are outstanding candidates for combination therapy with the ABT-737 for treating melanomas, and demand validation in preclinical models and further exploring the therapeutic potential of other drugs which target Mcl-1 in combination with ABT-737. It has also been proposed that cancer initiating cells (or cancer stem cells), which preferably initiate cancers, may be responsible for cancer resistance to therapy, and eliminating these cells may be essential for developing any cancer therapy with long term success. We will refer these cells as cancer imitating cells. We propose to investigate whether enriched melanoma initiating cells are more resistant to existing chemotherapeutic agents than non-sorted cells in melanoma, and to examine whether combinations of drugs that reduce multiple anti-apoptotic defenses such as ABT-737 plus Bortezomib or Paclitaxel will overcome this resistance of melanoma initiating cells. These studies will be an important component to understand whether melanoma initiating cells are more resistant to therapy. Aim 1: To directly test the responses of enriched melanoma initiating cells to existing and new therapies in vitro. i) To determine whether enriched melanoma initiating cells are more resistant to conventional chemotherapeutic agents such as Temozolomide. ii) To determine whether combinations of ABT- 737 with either Bortezomib or Paclitaxel overcome the resistance in enriched melanoma initiating cells. Aim 2: To verify the efficacy of the combinations of ABT-737 with either Bortezomib or Paclitaxel for treating melanoma using two novel xenotransplantation models: a model targeting human melanoma initiating cells in vivo, and a direct xenograft model for melanoma patient samples. Aim 3: To investigate the therapeutic potential of combining BH3 mimetic ABT-737 with other compounds which inhibit Mcl-1. i) A new approach to down-regulating Mcl-1 in melanomas by microRNAs. ii) Other drugs known to inhibit Mcl-1. This project proposes to develop novel combination therapeutics by targeting multiple anti-apoptotic Bcl-2 members, and to verify the efficacy of the treatment combinations in killing melanomas, and most importantly, melanoma initiating cell populations. We believe that the studies proposed here will quickly lead to future clinical studies and to the clinical use of ABT-737 (or similar agents) in treating human melanoma patients. Importantly, this approach should not be restricted by different signaling signatures of individual melanomas. This proposal is a collaboration of experienced melanoma basic scientists, a skin stem cell expert, a melanoma oncologist, and an expert in xenotransplantation models of human cancers, and this team will make important advances in developing melanoma treatments based on reversing resistance to apoptosis. PUBLIC HEALTH RELEVANCE: Malignant melanoma is largely unresponsive to existing therapies with a very poor prognosis, and there has been little progress over the past 30 years in medical treatment of this disease. In addition, the incidence of melanoma is increasing rapidly in the United States including in VA populations, and sun exposure during US military service has also been linked to increased melanoma incidence. Furthermore, melanoma is also rapidly increasing in young women, an increasingly significant population in the VA and in the active-duty military. Thus, the paucity of effective treatments for malignant melanoma is a pressing issue in medicine and in the VA patient care mission. This proposal uses state-of-the-art research models to validate the novel treatment strategy we have developed. The results from the studies proposed here will likely lead quickly to clinical trials and to immediate applicability to the clinical care of patients with melanoma, utilizing new combinations of drugs already in clinical trials for other cancers.