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

项目总结 黑色素瘤是最致命的皮肤癌亚型，具有高频率的BRAF突变。90%以上的 BRAF突变为V600E，导致丝裂原活化蛋白激酶(MAPK)过度激活 路径。转移性黑色素瘤患者的治疗选择和对电流的反应最小 针对BRAF和/或MEK1/2激酶的策略只是暂时的，因为出现了耐药性。 我们的实验室已经发现铜是MAPK通路中的一个新的漏洞，可以被用来诱导 BRAFV600E黑色素瘤的抗肿瘤活性。激酶活性需要铜与MEK1/2结合，并且 降低铜水平可降低BRAFV600E转移性黑色素瘤小鼠模型的肿瘤发生。而铜是 传统上被认为是酶反应的结构或催化辅因子，这是一种新发现的调节作用 激酶活性为信号通路带来了另一层调节，可以进一步研究和 在治疗策略中可能有针对性。有趣的是，一种铜络合剂四硫代钼酸(TTM)，两者 与BRAF和MEK1/2的抑制剂有协同作用，并在耐药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，对铜螯合的反应是细胞抑制和短期的 因此，阐明其他铜依赖的激酶可能会解锁TTM的其他机制，从而推动这一点 药物将成为治疗晚期BRAF驱动的黑色素瘤的更有效的选择。在铜的研究中 依赖的激酶，我们确定大的肿瘤抑制因子1/2(LATS1/2)是铜螯合的靶点。LATS1/2是 在河马途径中参与调节细胞生长的整合型激酶。LATS1/2上调表达的抑制作用 致癌是相关蛋白(YAP)。相反，LATS1/2被上游磷酸化和激活 使LATS1/2能够磷酸化YAP的激酶，阻止其核定位。增强型YAP核 在包括BRAFV600E突变黑色素瘤在内的癌症中观察到积聚，在那里它活跃地启动 促进生长的转录程序。除磷酸化外，LATS1/2激酶的其他调节剂 活动是未知的。我们发现铜的络合作用在体外抑制了LATS1/2的活性，并与LATS1/2结合 CU.然而，目前尚不清楚铜是否对体内LATS1/2激酶的活性是必需的。而铜的络合 抑制致癌MAPK信号，我假设LATS1/2激酶活性需要铜，并且 同时抑制LATS1/2将抑制河马通路，从而限制铜的作用 螯合疗法。在目标1中，我将定义LATS1/2中的特定铜结合位点，以评估其贡献 BRAFV600E突变黑色素瘤细胞系中铜对LATS1/2激酶活性的影响。在《目标2》中，我将使用基因和 在小鼠黑色素瘤模型中联合靶向YAP和铜的药理学方法评估 在BRAFV600E突变环境中进行联合治疗，并结合标准的 CARE BRAF抑制剂维莫拉非尼。总体而言，这项研究将提高我们对铜对激酶的调控的理解 并将为加强铜螯合作为BRAF驱动的黑色素瘤的持久治疗选择提供洞察力。

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.