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，因为缺乏针对野生型BRAF黑色素瘤的治疗选择，频繁的复发，以及(对于一些药物)有限的反应，即使最近FDA批准了令人兴奋的药物。该项目的长期目标是开发替代疗法，这将为恶性黑色素瘤患者带来更持久的临床反应和更好的患者结局。具体地说，这项提案旨在发现针对黑色素瘤抗凋亡防御和抗凋亡途径的治疗方法。肿瘤起始细胞(CICs)参与了癌细胞对治疗的抵抗，导致复发。因此，确定消除大部分黑色素瘤细胞(去块状)以及黑色素瘤的CIC群体(也称为黑色素瘤起始细胞，MICS)的治疗策略至关重要。Bcl2家族在细胞凋亡的调控中起着重要作用。促凋亡和抗凋亡的bcl2家族成员之间的相互作用可以控制内源性细胞凋亡的启动。ABT-737是BCL-2/BCL-XL/BCL-W抗凋亡蛋白的有效小分子抑制剂。我们已经确定了两种有希望的药物，合成的维甲酸衍生物芬维甲素(4-HPR)和伽玛分泌酶抑制剂(GSI-I)，它们与ABT-737协同抑制多个抗凋亡的bcl2家族成员。当在体外测试时，在患者来源的异种移植(PDX)模型中维持的黑色素瘤细胞系和肿瘤细胞中，我们发现ABT-737与任何一种药物伙伴联合使用不仅可以减轻黑色素瘤细胞的体积，还可以杀死CICs，用于治疗带有突变型或野生型BRAF的黑色素瘤。这两种联合疗法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、新颖的治疗机会。我们发现，miR-26a的替换通过靶向抗凋亡蛋白死亡结构域沉默蛋白(SODD)诱导了黑色素瘤的细胞死亡。因此，我们建议进一步证明这两种靶向黑色素瘤抗凋亡防御的方法的有效性，并研究其中涉及的机制：同时抑制多个抗凋亡的bcl2家族成员(目标1和2)和基于miRNA的靶向SODD的方法(目标3)。目的1：探讨ABT737与4-hpr或GSI联合应用抑制黑色素瘤和靶向MIC的作用机制(S)。我们将利用CRISPR基因组编辑技术确定这些药物组合消除MICs的作用机制，并进一步研究这些药物组合如何诱导MCL-1降解。目的2：探讨ABT-737联合4-HPR或GSI体内治疗的疗效及机制。我们将通过使用传统的基因敲除细胞异种移植模型、黑色素瘤PDX模型、黑色素瘤转移模型和低细胞数植入异种移植模型来评估MIC启动的肿瘤形成，以确定其作用和机制。}{目标3：研究miR-26a替换在消除黑色素瘤和靶向MIC方面的作用，并确定候选化合物为miR-26a治疗的潜在合作伙伴。我们将测试miR-26a在靶向MICs方面的效果，就像目标1中的那样，并用凋亡化合物库和MAPK抑制剂库筛选潜在的合作伙伴化合物。}总之，这项建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对细胞凋亡的耐药性的药物组合。我们将进一步测试这些有希望的组合在消除黑色素瘤、消除MIC群体和限制其自我更新方面的效果。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。结果可能确定治疗黑色素瘤的新治疗方法，在杀死MICs方面具有独特的效果。

 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.