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

非小细胞肺癌（NSCLC）是癌症相关死亡率的主要原因，可发生在吸烟者中 以及不吸烟者。Hippo肿瘤抑制途径的组分在NSCLC中发生改变， 研究表明，该途径可被来自K-Ras和EGFR的上游信号事件调节。 我们发现YAP 1可以诱导Sox 2和EMT标记，促进NSCLC的生长和转移。 我们发现非经典的IkB激酶TBK 1（Tank Binding Kinase 1）可以与YAP 1发生物理相互作用， 并使其磷酸化;此外，TBK 1的消耗导致在常氧条件下YAP 1水平显著升高。 条件下，但仅在K-Ras突变体，而不是EGFR突变体，肺腺癌细胞。这是专门针对 TBK 1，因为密切相关的IKKe激酶的消耗并没有提高YAP 1水平。我们发现， YAP 1的甲基化发生在蛋白质稳定性水平，这是由YAP 1的精氨酸残基的甲基化引起的， CARM1（PRMT4）。CARM 1在多种癌症中过表达，高水平的CARM 1与癌症相关。 NSCLC患者生存率低。基于这些结果，我们推测TBK 1对YAP 1的调节可能与其对细胞增殖的调控有关。 CARM 1是促进非小细胞肺癌生长和转移的新机制， 癌本申请中提出的研究将使用各种不同的方法来机械地表征这种调节。 体外和体内分析，包括共培养研究和同系移植模型， 通过精氨酸甲基化YAP 1蛋白进行的基因调控的全局分析。YAP 1与 TBK 1、PRMT 5和CARM 1将在三种不同的人肺癌TMA中进行评估， 携带各种K-Ras突变的高级别肿瘤;这样的分析将阐明是否存在K-Ras突变。 YAP 1与这些调节分子的物理相互作用影响这些细胞的生长和进展。 肿瘤的将对原发性肿瘤样品进行ChiP-re-ChIP、ChIP-Seq和RNA-Seq分析， 邻近的正常组织，以确定腺嘌呤甲基化YAP 1的下游靶点。YAP 1已经 证明具有显著的免疫抑制作用;通过抑制 CARM 1有望增强免疫检查点抑制剂的功效。深入研究将 在同系小鼠模型上进行，以评估CARM 1抑制剂单独或组合如何影响 T细胞的抗肿瘤活性。此外，CARM 1抑制剂在血液学临床试验中的事实， 恶性肿瘤增加了它们的效用最终可以扩展到K-Ras突变肺癌的可能性。我们 我们建议测试CARM 1抑制剂EZM 2302单独或与TBK 1组合的抗癌功效。 抑制剂氨来萨诺、K-Ras G12 C抑制剂AGM 510或MEK抑制剂曲美替尼或PLK 1/K-Ras。 考虑到CARM 1和YAP 1在各种癌症（包括胰腺癌）中的致癌作用， 和肺，阐明这些新的调控模式的YAP 1功能将导致识别， 这些弱点可能是针对打击K-Ras突变肺腺癌的目标。

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.