CARM1-mediated regulation of YAP1 as a therapeutic target in lung cancer

非小细胞肺癌(NSCLC)是癌症相关死亡的主要原因，可发生在吸烟者中 以及不吸烟的人。非小细胞肺癌和新近发现的河马肿瘤抑制通路的成分发生改变 研究表明，该通路可受K-RAS和EGFR上游信号事件的调节。 我们发现YAP1可以诱导SOX2和EMT标志，促进NSCLC的生长和转移。 我们发现非规范的IKB激酶TBK1(Tank Binding Kinase 1)可以与YAP1发生物理相互作用 并使其磷酸化；此外，在常氧条件下，TBK1的耗尽导致YAP1水平显著升高 条件，但仅在K-RAS突变，而不是EGFR突变的肺腺癌细胞中。这是专门针对 TBK1，因为耗尽密切相关的IKKE激酶不会提高YAP1的水平。我们发现归纳法 YAP1的发生在蛋白质稳定性水平上，这是由YAP1的精氨酸残基甲基化引起的 CARM1(PRMT4)。CARM1在多种癌症中过表达，高水平的CARM1与 非小细胞肺癌患者存活率低。根据这些结果，我们推测Tbk1对YAP1的调节 CARM1是一种显著促进非小细胞肺癌生长和转移的新机制 癌症。本申请中提出的研究将使用各种不同的 体外和体内分析，包括共培养研究和同基因移植模型结合 精氨酸甲基化的YAP1蛋白基因调控的全球分析。YAP1与YAP1的物理相互作用 TBK1、PRMT5和CARM1将在三种不同的人类肺癌TMA中进行评估，这些TMA来自低级别和 含有各种K-RAS突变的高级别肿瘤；这样的分析将阐明 YAP1与这些调控分子的物理相互作用影响这些细胞的生长和进展 肿瘤。将对原发肿瘤样本进行芯片-再芯片、芯片-序列和RNA-序列分析 以确定精氨酸甲基化的YAP1下游靶点。YAP1一直是 被证明具有显著的免疫抑制作用；通过抑制YAP1 CARM1有望增强免疫检查点抑制剂的疗效。将进行深入的研究 在同基因小鼠模型上进行，以评估CARM1抑制剂单独或联合使用如何影响 T细胞的抗肿瘤活性。此外，CARM1抑制剂正在进行血液学临床试验 恶性肿瘤增加了它们的用途最终可能扩展到K-RAS突变肺癌的可能性。我们 建议测试CARM1抑制剂EZM2302单独或与TBK1联合使用的抗癌效果 氨来昔诺、K-RAS G12C抑制剂AGM510或MEK抑制剂曲美替尼或PLK1/K-RAS。 鉴于CARM1和YAP1在包括胰腺癌在内的各种癌症中的致癌作用已确定 和肺，阐明这些新的调节模式的YAP1功能将导致识别 可能被靶向抗击K-RAS突变肺腺癌的脆弱性。

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality and can occur in smokers as well as non-smokers. Components of the Hippo tumor suppressive pathway are altered in NSCLC and recent studies have shown that this pathway can be modulated by upstream signaling events from K-Ras and EGFR. We had found that YAP1 could induce Sox2 and EMT markers, promoting the growth and metastasis of NSCLC. We find that the non-canonical IkB kinase TBK1 (Tank Binding Kinase 1) could physically interact with YAP1 and phosphorylate it; further, depletion of TBK1 led to a marked elevation of YAP1 levels under normoxic conditions, but only in K-Ras mutant, but not EGFR-mutant, lung adenocarcinoma cells. This was specific to TBK1, since depletion of the closely related IKKe kinase did not elevate YAP1 levels. We find that the induction of YAP1 occurs at the level of protein stability, brought about by the methylation of arginine residues of YAP1 by CARM1 (PRMT4). CARM1 is overexpressed in a variety of cancers and high levels of CARM1 correlates with poor survival in NSCLC patients. Based on these results, we hypothesize that the regulation of YAP1 by TBK1 and CARM1 is a novel mechanism which significantly promotes the growth and metastasis of non-small cell lung cancer. Studies proposed in this application will characterize this regulation mechanistically using a variety of in vitro and in vivo analysis, including co-culture studies and syngeneic transplantation models combined with global analysis of gene regulation by arginine-methylated YAP1 protein. The physical interaction of YAP1 with TBK1, PRMT5 and CARM1 will be assessed in three different human lung cancer TMAs from low grade and high grade tumors that harbor various K-Ras mutations; such an analysis will shed light on whether the levels and physical interaction of YAP1 with these regulatory molecules affect the growth and progression of these tumors. ChiP-re-ChIP, ChIP-Seq and RNA-Seq analysis will be conducted on primary tumor samples and adjacent normal tissue to identify the downstream targets of arginine-methylated YAP1. YAP1 has been demonstrated to have significant immunosuppressive effects; downregulating YAP1 through the inhibition of CARM1 can be expected to enhance the efficacy of immune checkpoint inhibitors. In depth studies will be conducted on syngeneic mouse models to assess how CARM1 inhibitors alone or in combination, affects the anti-tumor activity of T cells. Further, the fact that CARM1 inhibitors are in clinical trials for hematological malignancies raise the possibility that their utility can eventually be extended to K-Ras mutant lung cancers. We propose to test the anti-cancer efficacy of the CARM1 inhibitor EZM2302 alone or in combination with the TBK1 inhibitor Amlexanox, the K-Ras G12C inhibitor AGM510 or the MEK inhibitor Trametinib or the PLK1/K-Ras. Given the established oncogenic role of CARM1 and YAP1 in various cancers including those of the pancreas and the lung, an elucidation of these novel regulatory modes of YAP1 function would lead to the identification of vulnerabilities that could potentially be targeted to combat K-Ras mutant lung adenocarcinomas.