Melanoma Resistance to Apoptosis: Mechanisms and Therapeutic Potential

恶性黑色素瘤的一个基本特征是对凋亡的抵抗，这在很大程度上决定了 黑色素瘤对治疗的抵抗。促凋亡和抗凋亡成员之间的复杂相互作用 BCL-2家族是细胞凋亡的中枢控制点，也是一个很有前途的药物靶点。BH3模拟ABT-737是一种 有效的小分子抗凋亡蛋白Bcl2/Bclxl/Bclw的抑制剂，并显示出对 作为单一药物或联合治疗的癌症治疗。 我们发现ABT-737单独对黑色素瘤细胞的细胞毒性很小，并且进行了基因敲除实验 用RNAi技术证明抗凋亡蛋白Mcl-1，而不是Bcl2或Bclxl是主要的 黑色素瘤对ABT-737治疗耐药的介体。此外，ABT-737表现出较强的增效作用。 与蛋白酶体抑制剂(MG-132或Bortezomib)或紫杉醇联合使用时的致命性，以及 机制研究表明，蛋白酶体抑制剂和紫杉醇均中和Mcl-1‘S的作用。 这些令人兴奋的结果表明，中和Mcl-1功能的药物是优秀的候选药物 与ABT-737联合治疗黑色素瘤，并需要临床前模型的验证 并进一步探索靶向Mcl-1与ABT-737联合应用的其他药物的治疗潜力。 还提出了癌症启动细胞(或癌症干细胞)，其优选地启动癌症， 可能是癌症抵抗治疗的原因，而消除这些细胞可能对 开发任何长期成功的癌症治疗方法。我们将这些细胞称为类癌细胞。我们 建议研究丰富的黑色素瘤起始细胞是否对现有细胞更具抵抗力 化疗药物比未分选的黑色素瘤细胞，并检查是否组合 减少多种抗细胞凋亡防御的药物，如ABT-737加Bortezomib或紫杉醇将 克服黑色素瘤起始细胞的这种抵抗力。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。 具体目标1：直接测试富集型黑色素瘤起始细胞对现有和 新的体外治疗方法。I)确定浓缩的黑色素瘤起始细胞是否对 常规化疗药物，如替莫唑胺。Ii)确定ABT- 737与Bortezomib或紫杉醇一起使用，可以克服浓缩性黑色素瘤起始细胞的耐药性。 具体目标2：验证ABT-737与Bortezomib或 紫杉醇治疗黑色素瘤的两种新的异种移植模型：靶向人的模型 体内黑色素瘤起始细胞和黑色素瘤患者样本的直接异种移植模型。 具体目标3：研究BH3模拟ABT-737与其他药物联合治疗的可能性 抑制Mcl-1的化合物。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种抗细胞凋亡的Bcl-2来开发新的联合治疗药物 成员，并验证这些治疗组合在杀死黑色素瘤方面的疗效，最重要的是， 黑色素瘤启动细胞群。我们相信，这里提出的研究将很快导致未来 临床研究和ABT-737(或类似药物)在治疗人类黑色素瘤患者中的临床应用。 重要的是，这种方法不应该受到个体不同信令签名的限制 黑色素瘤。这项提议是由经验丰富的黑色素瘤基础科学家合作提出的，这是一种皮肤干细胞 专家，黑色素瘤肿瘤学家，人类癌症异种移植模型专家，这个团队 将在开发基于逆转细胞凋亡抵抗的黑色素瘤治疗方面取得重要进展。

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. Specific 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. Specific 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. Specific 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.