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

项目摘要 黑色素瘤是最致命的皮肤癌亚型，具有高频率的BRAF突变。超过90%的 BRAF突变为V600 E，导致丝裂原活化蛋白激酶（MAPK）过度活化 通路转移性黑色素瘤患者的治疗选择和对目前治疗的反应很少。 靶向BRAF和/或MEK 1/2激酶的策略由于耐药性的出现而仅仅是暂时的。 我们的实验室已经确定Cu是MAPK通路中的一个新的脆弱性，可以利用它来诱导 BRAFV 600 E黑色素瘤中的抗肿瘤活性。激酶活性需要Cu与MEK 1/2结合， 在BRAFV 600 E转移性黑色素瘤的鼠模型中，降低Cu水平减少了肿瘤发生。当Cu 传统上被认为是酶促反应的结构或催化辅因子，新发现的调节 激酶活性为信号通路带来了另一层调节，可以进一步研究， 可能成为治疗策略的目标。有趣的是，铜螯合剂，四硫代钼酸盐（TTM）， 与BRAF和MEK 1/2抑制剂协同作用，并在抑制剂抗性黑素瘤细胞中显示出有效性。 然而，与BRAF和MEK 1/2抑制剂类似，对Cu螯合的反应是细胞抑制性的和短期的， 因此，阐明其他铜依赖性激酶可能会解开TTM的其他机制，这些机制可以推动这一进程。 药物是晚期BRAF驱动的黑色素瘤的更有效的治疗选择。在研究Cu 依赖性激酶，我们确定了大肿瘤抑制因子1/2（LATS 1/2）作为铜螯合的目标。LATS 1/2 在Hippo途径中参与介导细胞生长的整合激酶。抑制LATS 1/2上调 癌基因Yes相关蛋白（雅普）。相反，LATS 1/2被磷酸化并被上游激活。 能够使LATS 1/2磷酸化雅普，阻止其核定位的激酶。增强型雅普核 在包括BRAFV 600 E突变型黑色素瘤在内的癌症中观察到了这种累积，在这些癌症中，它有活性启动一种新的免疫反应。 促进生长的转录程序。除了磷酸化，LATS 1/2激酶的其它调节剂 活动不明。我们发现，铜螯合抑制LATS 1/2激酶活性在体外，LATS 1/2结合， Cu.然而，目前尚不清楚Cu是否是必需的LATS 1/2激酶活性在体内。当Cu螯合 抑制致癌MAPK信号，我假设铜是LATS 1/2激酶活性所必需的， LATS 1/2的伴随抑制将抑制Hippo通路，从而限制Cu的功效。 螯合疗法在目标1中，我将定义LATS 1/2中的特异性Cu结合位点，以评估其贡献。 BRAFV 600 E突变黑素瘤细胞系中Cu至LATS 1/2激酶活性的变化。在目标2中，我将使用遗传和 在黑色素瘤的鼠模型中共同靶向雅普和Cu以评估本发明的功效的药理学手段 在BRAFV 600 E突变体环境中进行联合治疗，并进一步测试该方案与标准治疗方案的组合。 BRAF抑制剂vemurafenib。总的来说，本研究将提高我们对铜调节激酶的理解 并将提供对增强Cu螯合作为BRAF驱动的黑素瘤的持久治疗选择的见解。

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.