Melanoma Resistance to Apoptosis: Mechanisms and Therapeutic Potential

 描述（由申请人提供）： 发现转移性黑色素瘤的新疗法仍然是一个紧迫的问题，因为缺乏野生型BRAF黑色素瘤的治疗选择，频繁复发和（某些药物）有限的反应，即使是最近令人兴奋的FDA批准的药物。该项目的长期目标是开发替代治疗方法，这将导致恶性黑色素瘤患者更持久的临床反应和更好的患者结局。具体来说，该提案旨在发现针对黑色素瘤用于抵抗细胞凋亡的抗凋亡防御和途径的治疗方法。 癌症起始细胞（CIC）与癌细胞对治疗的抗性有关，导致复发。因此，确定消除大部分黑素瘤细胞（去体积）以及黑素瘤的CIC群体（也称为黑素瘤起始细胞，MIC）的治疗策略至关重要。 BCL-2家族在调节细胞凋亡中是重要的。促凋亡和抗凋亡BCL-2家族成员之间的相互作用可以控制内源性凋亡的启动。ABT-737是抗凋亡蛋白BCL-2/BCL-XL/BCL-W的有效小分子抑制剂。我们已经确定了两种有前途的药物，合成类维生素A衍生物，芬维A胺（4-HPR）和γ分泌酶抑制剂（GSI-I），与ABT-737协同抑制多种抗凋亡BCL-2家族成员。当在体外测试时，在患者来源的异种移植物（PDX）模型中维持的黑素瘤细胞系和肿瘤细胞中，我们发现ABT-737与任一种药物伴侣组合不仅使黑素瘤细胞减体积，而且还杀死具有突变型或野生型BRAF的黑素瘤的CIC。这两种组合也极大地限制了肿瘤的自我更新。 基于miRNA的疗法为许多疾病提供了令人兴奋的新治疗机会。我们发现，通过靶向抗凋亡蛋白，死亡结构域沉默物（SODD），miR-26 a的替代诱导了黑色素瘤亚组的细胞死亡。因此，我们建议进一步证明两种靶向黑色素瘤抗凋亡防御的方法的有效性，并研究所涉及的机制：一次抑制多个抗凋亡BCL-2家族成员（目的1和2）和基于miRNA的靶向SODD的方法（目的3）。目标1：检查ABT-737与4-HPR或GSI组合在减瘤黑色素瘤和靶向MIC中确定疗效的机制。我们将使用CRISPR基因组编辑技术定义这些药物组合消除MIC的作用机制，并进一步检查组合如何诱导MCl-1降解。{Aim 2：研究ABT-737与4-HPR或GSI联合治疗的体内疗效和机制。我们将通过使用具有敲除细胞系的常规异种移植物模型、黑素瘤PDX模型、黑素瘤转移模型和低细胞数植入异种移植物模型来评估MIC引发的肿瘤形成来确定效果并检查所涉及的机制。{Aim第三章：研究miR-26 a替代在减瘤黑色素瘤和靶向MIC中的作用，并确定候选化合物作为miR-26 a治疗的潜在伙伴。我们将测试miR-26 a在如目标1中靶向MIC中的作用，并使用凋亡化合物文库和MAPK抑制剂文库筛选潜在的伴侣化合物。总之，这一提议扩展了我们令人兴奋的初步研究，这些研究已经确定了克服黑色素瘤细胞凋亡抗性的药物组合。我们将进一步测试这些有希望的组合对减瘤黑色素瘤的作用，消除MIC群体并限制其自我更新。我们还将确定单独或联合使用基于miRNA的原始疗法的有效性。结果可能会确定新的治疗方法来治疗黑色素瘤，在杀死MIC的独特效果。

 DESCRIPTION (provided by applicant): Discovering new therapies for metastatic melanoma is still a pressing issue, given the lack of treatment options for wild type BRAF melanoma, frequent relapses and (with some drugs) limited responders, even with the recent exciting FDA approved drugs. The long-term goal of this project is to develop alternative treatments, which will lead to longer lasting clinical responses and better patient outcomes for patients with malignant melanomas. Specifically, this proposal aims to discover treatments that target anti-apoptotic defenses and pathways used by melanoma to resist apoptosis. Cancer initiating cells (CICs) are implicated in cancer cells' resistance to treatment, resulting in relapse. It is therefore crucial to identify treatment strateies that eliminate the bulk of melanoma cells (de-bulk) as well as the CIC populations of the melanoma (also known as Melanoma Initiating Cells, MICs). The BCL-2 family is important in regulating apoptosis. The interactions among the pro- and anti-apoptotic BCL-2 family members can control the initiation of intrinsic apoptosis. ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins BCL-2/BCL-XL/BCL-W. We have identified two promising agents, the synthetic retinoid derivative, fenretinide (4-HPR) and a Gamma Secretase Inhibitor (GSI-I), that synergize with ABT-737 to inhibit multiple anti-apoptotic BCL-2 family members. When tested in vitro, in melanoma cell lines and tumor cells maintained in the patient derived xenograft (PDX) model, we found that ABT-737 combined with either drug partner not only de-bulked the melanoma cells, but also killed the CICs, for melanomas with either mutant or wild-type BRAF. Both combinations also dramatically limited tumor self-renewal. MiRNA-based therapies provide an exciting, novel therapeutic opportunity for many diseases. We show that replacement of miR-26a induced cell death in a subset of melanomas, through targeting the anti-apoptotic protein, silencer of death domain (SODD). Therefore, we propose to further demonstrate the efficacy of the two approaches of targeting anti-apoptotic defenses in melanoma and investigate the mechanisms involved: inhibiting multiple anti-apoptotic BCL-2 family members at once (Aims 1 and 2) and a miRNA-based approach to target SODD (Aim 3). Aim 1: Examine the mechanism(s) determining efficacy for the combinations of ABT-737 with 4-HPR or GSI in de-bulking melanoma and targeting MICs. We will define the mechanism of action for these drug combinations to eliminate MICs using CRISPR genomic editing technology, and further examine how the combinations induced MCl-1 degradation. {Aim 2: Investigate the efficacy and mechanisms of the combination treatments of ABT-737 with 4-HPR or GSI in vivo. We will determine the effects and examine the mechanisms involved by using a conventional xenograft models with knockout cell lines, a melanoma PDX model, a melanoma metastasis model, and a low-cell-number implantation xenograft model for assessing MIC-initiated tumor formation.} {Aim 3: Study the effects of miR-26a replacement in de-bulking melanoma and targeting MICs, and identify the candidate compounds as potential partners with miR-26a treatment. We will test the effects of miR-26a in targeting MICs as in Aim 1, and screen for potential partner compounds with an apoptosis compound library and a MAPK inhibitor library.} In summary, this proposal extends our exciting preliminary studies that have identified drug combinations which overcome melanoma resistance to apoptosis. We will further test these promising combinations with effects on de-bulking melanoma, and eliminating the MIC populations and limiting their self-renewal. We will also determine the effectiveness of an original miRNA-based therapy alone or in combination. Results are likely to identify new therapeutic approaches for treating melanoma, with unique effectiveness in killing MICs.