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抗性，这在很大程度上决定了黑色素瘤对治疗的抗性。Bcl-2家族的促凋亡和抗凋亡成员的复杂相互作用是凋亡的中心控制点和有前途的药物靶点。BH 3模拟物ABT-737是抗凋亡蛋白Bcl-2/Bcl-XL/Bcl-w的有效小分子抑制剂，并且其作为单一药剂或组合疗法显示出用于癌症治疗的前景。我们已经发现，单独的ABT-737在黑素瘤细胞中诱导的细胞毒性很小，并且利用RNAi技术的敲低实验证明，抗凋亡蛋白Mcl-1而不是Bcl-2或Bcl-XL是黑素瘤对ABT-737治疗的抗性的主要介导物。此外，ABT-737与蛋白酶体抑制剂（MG-132或硼替佐米）或Paclitaxel联合使用时显示出强烈的协同致死作用，机制研究表明，蛋白酶体抑制剂和Paclitaxel均能中和Mcl-1的功能。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杰出候选者，需要在临床前模型中进行验证，并进一步探索与ABT-737联合靶向Mcl-1的其他药物的治疗潜力。还已经提出，优选引发癌症的癌症引发细胞（或癌症干细胞）可能是癌症对疗法的抗性的原因，并且消除这些细胞对于开发具有长期成功的任何癌症疗法可能是必不可少的。我们将这些细胞称为癌模仿细胞。我们建议研究富集的黑色素瘤起始细胞是否比黑色素瘤中未分选的细胞对现有化疗药物更具抗性，并检查减少多种抗凋亡防御的药物组合，如ABT-737加硼替佐米或紫杉醇是否会克服黑色素瘤起始细胞的这种抗性。这些研究将是了解黑色素瘤起始细胞是否对治疗更具抗性的重要组成部分。目的1：在体外直接检测富集的黑色素瘤起始细胞对现有和新疗法的反应。i）确定富集的黑素瘤起始细胞是否对常规化疗剂如替莫唑胺更具抗性。ii）确定ABT- 737与硼替佐米或紫杉醇的组合是否克服富集的黑素瘤起始细胞中的抗性。目标二：使用两种新的异种移植模型验证ABT-737与Bortezelatin或Paclitazone联合治疗黑素瘤的疗效：靶向体内人黑素瘤起始细胞的模型和用于黑素瘤患者样本的直接异种移植模型。目的3：研究BH 3模拟物ABT-737与其他抑制Mcl-1的化合物的联合治疗潜力。i）通过microRNA下调黑素瘤中Mcl-1的新方法。ii）已知抑制Mcl-1的其他药物。该项目提出通过靶向多个抗凋亡Bcl-2成员来开发新的组合疗法，并验证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中的功效。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应用。重要的是，这种方法不应受到单个黑色素瘤的不同信号特征的限制。该提案是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的合作，该团队将在开发基于逆转细胞凋亡抗性的黑色素瘤治疗方面取得重要进展。 公共卫生相关性： 恶性黑色素瘤在很大程度上对现有疗法无反应，预后非常差，过去30年来，这种疾病的医学治疗进展甚微。此外，黑色素瘤的发病率在美国（包括VA人群）迅速增加，美国兵役期间的阳光照射也与黑色素瘤发病率增加有关。此外，黑色素瘤在年轻女性中也迅速增加，这在VA和现役军队中越来越重要。因此，缺乏有效的治疗恶性黑色素瘤是一个紧迫的问题，在医学和退伍军人事务部病人护理使命。该提案使用最先进的研究模型来验证我们开发的新治疗策略。本文提出的研究结果可能会迅速导致临床试验，并立即适用于黑色素瘤患者的临床护理，利用已经在其他癌症临床试验中的新药物组合。

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.