Investigating paradoxical YAP activation as an emergent limitation to Cu chelation therapy in BRAF V600E-driven melanoma

研究矛盾的 YAP 激活作为 BRAF V600E 驱动的黑色素瘤中铜螯合疗法的紧急限制

基本信息

批准号：
9982040
负责人：
Tiffany Tsang
金额：
$ 2.23万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9982040
关键词：
Ablation Affinity Chromatography Amino Acids Antineoplastic Agents BRAF gene Binding Binding Sites Biochemical Reaction Biological Assay CRISPR/Cas technology Cell Line Cell Nucleus Chelating Agents Chelation Therapy Combined Modality Therapy Copper Cytostatics Data Development Drug resistance Effectiveness Embryo Exhibits Fibroblasts Foxes Frequencies Gene Expression Genetic Genetic Transcription Genetically Engineered Mouse Growth Hyperactive behavior Immunofluorescence Immunologic Immunohistochemistry In Vitro Knock-out MAP2K1 gene Malignant Neoplasms Measures Mediating Melanoma Cell Metastatic Melanoma Micronutrients Mitogen-Activated Protein Kinases Molecular Monophenol Monooxygenase Mus Mutagenesis Mutation Nuclear Nuclear Protein Oncogenic Pathway interactions Patients Pharmaceutical Preparations Pharmacology Phosphorylation Phosphotransferases Process Protein Inhibition Proteins Regulation Resistance Role SCID Beige Mouse Signal Pathway Signal Transduction Signaling Molecule Skin Cancer Structure Testing Therapeutic Transcription Coactivator Treatment Efficacy Tumor Burden Tumor Suppressor Proteins Tumor Tissue Verteporfin Western Blotting Xenograft Model base cell growth chelation cofactor cohort improved in vitro activity in vivo inhibitor/antagonist insight melanoma mouse model mutant novel pre-clinical prevent programs protein activation protein expression response standard of care tetrathiomolybdate tumor tumor growth tumorigenesis tumorigenic upstream kinase

项目摘要

PROJECT SUMMARY Melanoma is the most lethal subtype of skin cancer with a high frequency of BRAF mutations. Over 90% of BRAF mutations are V600E, which results in hyperactivation of the mitogen-activated protein kinase (MAPK) pathway. Patients with metastatic melanoma have minimal therapeutic options and responses to current strategies targeting BRAF and/or MEK1/2 kinases are only transient due to the emergence of drug resistance. Our lab has identified Cu as a novel vulnerability within the MAPK pathway that can be leveraged to induce antineoplastic activity in BRAFV600E melanomas. Cu binding to MEK1/2 is required for kinase activity, and lowering Cu levels reduced tumorigenesis in a murine model of BRAFV600E metastatic melanoma. While Cu is traditionally thought of as a structural or catalytic cofactor for enzymatic reactions, a newfound role in modulating kinase activity has brought another layer of regulation to signaling pathways that can be further studied and potentially targeted in therapeutic strategies. Interestingly, a Cu chelator, tetrathiomolybdate (TTM), both synergized with inhibitors of BRAF and MEK1/2 and showed effectiveness in inhibitor resistant melanoma cells. However, similar to BRAF and MEK1/2 inhibitors, the response to Cu chelation is cytostatic and short-term and thus, elucidation of additional Cu-dependent kinases may unlock other mechanisms of TTM that can propel this drug to be a more efficacious therapeutic option for late-stage BRAF-driven melanoma. In studying Cu dependent kinases, we identified large tumor suppressor 1/2 (LATS1/2) as targets of Cu chelation. LATS1/2 are integral kinases involved in mediating cell growth within the Hippo pathway. Inhibition of LATS1/2 upregulate oncogenic Yes- associated protein (YAP). Conversely, LATS1/2 are phosphorylated and activated by upstream kinases enabling LATS1/2 to phosphorylate YAP, preventing its nuclear localization. Enhanced YAP nuclear accumulation is observed in cancers including BRAFV600E-mutant melanoma, where it is active to initiate a growth-promoting transcriptional program. Aside from phosphorylation, additional modulators of LATS1/2 kinase activity are unknown. We found that Cu chelation inhibits LATS1/2 kinase activity in vitro and that LATS1/2 bind Cu. However, it is not clear whether Cu is necessary for LATS1/2 kinase activity in vivo. While Cu chelation inhibits oncogenic MAPK signaling, I hypothesize that Cu is needed for LATS1/2 kinase activity and that concomitant inhibition of LATS1/2 will dampen the Hippo pathway, consequently limiting the efficacy of Cu chelation therapy. In Aim 1, I will define the specific Cu binding sites within LATS1/2 to assess the contribution of Cu to LATS1/2 kinase activity in BRAFV600E mutant melanoma cell lines. In Aim 2, I will use genetic and pharmacologic means to co-target YAP and Cu in murine models of melanoma to assess the efficacy of the combination therapy in a BRAFV600E mutant setting and further test this regime in combination with a standard of care BRAF inhibitor vemurafenib. Overall, this study will improve our understanding of Cu regulation of kinases and will provide insight into enhancing Cu chelation as a durable therapeutic option for BRAF-driven melanomas.

项目概要黑色素瘤是最致命的皮肤癌亚型，BRAF 突变频率很高。 90%以上 BRAF 突变为 V600E，导致丝裂原激活蛋白激酶 (MAPK) 过度激活途径。转移性黑色素瘤患者的治疗选择和对当前治疗的反应很少由于耐药性的出现，针对 BRAF 和/或 MEK1/2 激酶的策略只是暂时的。我们的实验室已将 Cu 确定为 MAPK 途径中的一个新漏洞，可用于诱导 BRAFV600E 黑色素瘤的抗肿瘤活性。 Cu 与 MEK1/2 结合是激酶活性所必需的，并且降低铜水平可减少 BRAFV600E 转移性黑色素瘤小鼠模型中的肿瘤发生。虽然铜是传统上被认为是酶促反应的结构或催化辅助因子，是调节中的新发现的作用激酶活性给信号通路带来了另一层调节，可以进一步研究和治疗策略的潜在目标。有趣的是，铜螯合剂四硫代钼酸盐 (TTM) 与 BRAF 和 MEK1/2 抑制剂协同作用，并在抑制剂耐药性黑色素瘤细胞中显示出有效性。然而，与 BRAF 和 MEK1/2 抑制剂类似，对 Cu 螯合的反应是细胞抑制性的、短期且持续的。因此，阐明其他 Cu 依赖性激酶可能会解开 TTM 的其他机制，从而推动这一过程该药物成为晚期 BRAF 驱动的黑色素瘤更有效的治疗选择。在研究铜依赖性激酶，我们确定大肿瘤抑制因子 1/2 (LATS1/2) 作为 Cu 螯合的靶标。 LATS1/2 是参与 Hippo 途径内介导细胞生长的整合激酶。抑制 LATS1/2 上调致癌 Yes 相关蛋白 (YAP)。相反，LATS1/2 被上游磷酸化并激活激酶使 LATS1/2 能够磷酸化 YAP，从而阻止其核定位。增强型YAP核在包括 BRAFV600E 突变型黑色素瘤在内的癌症中观察到积累，在这些癌症中，它会主动启动促进生长的转录程序。除了磷酸化之外，LATS1/2 激酶的其他调节剂活动未知。我们发现 Cu 螯合在体外抑制 LATS1/2 激酶活性，并且 LATS1/2 结合铜。然而，尚不清楚 Cu 是否是体内 LATS1/2 激酶活性所必需的。当Cu螯合时抑制致癌 MAPK 信号传导，我假设 LATS1/2 激酶活性需要 Cu，并且同时抑制 LATS1/2 将抑制 Hippo 通路，从而限制 Cu 的功效螯合疗法。在目标 1 中，我将定义 LATS1/2 内的特定 Cu 结合位点以评估贡献 BRAFV600E 突变黑色素瘤细胞系中 Cu 对 LATS1/2 激酶活性的影响。在目标 2 中，我将使用遗传和在黑色素瘤小鼠模型中共同靶向 YAP 和 Cu 的药理学方法，以评估 YAP 和 Cu 的功效在 BRAFV600E 突变环境中进行联合治疗，并与标准组合进一步测试该方案护理 BRAF 抑制剂维莫非尼。总的来说，这项研究将提高我们对铜对激酶调节的理解并将提供有关增强铜螯合作为 BRAF 驱动的黑色素瘤的持久治疗选择的见解。