Interrogation of MLL2 as a tumor suppressor gene in lung cancer

描述（由申请人提供）：小细胞肺癌（SCLC）是最具侵袭性的肺癌。由于生存率低和缺乏靶向治疗，我们了解驱动SCLC的主要基因和途径至关重要。为了鉴定新的潜在SCLC驱动因子，我们对人SCLC外显子组和转录组进行了测序，并在组蛋白甲基转移酶MLL2中发现了频繁的失活突变。鳞状细胞肺癌（SQCC）的癌症基因组图谱研究表明，MLL2是SQCC中最常见的突变基因之一，这表明MLL2可能是多种肺癌类型的肿瘤抑制因子。MLL2在许多人类癌症中经常发生突变，但很少有关于MLL2与癌症之间联系的研究。我们的初步数据提供了MLL2是肿瘤抑制因子的功能证据。通过赋予组蛋白H3K4单甲基化，MLL2新近成为转录增强子功能的关键调节因子，但这种作用尚未在肿瘤发生中探索。在这项资助提案中，我们将MLL2作为肺癌中的一个关键肿瘤抑制因子进行研究。具体目的1：检验MLL2是肺癌中的肿瘤抑制因子的假设。我们以前曾使用致敏的本地小鼠模型的SCLC显示，SCLC突变基因是功能上的重要SCLC。我们建议生成MLL2突变型SCLC和SQCC的新模型。该模型不仅将揭示MLL2肿瘤抑制活性的潜在机制，而且将是未来测试针对以下目标的新疗法的理想临床前工具： MLL2失活所带来的脆弱性。具体目标2：鉴定促进癌症的MLL2调控的靶基因和途径。MLL2是调节组蛋白H3K4单甲基化的甲基转移酶，组蛋白H3K4单甲基化是转录增强子的标志。我们推测MLL2对转录增强子的调节控制了对肺癌抑制很重要的肿瘤抑制转录本的表达。我们将使用原发性肿瘤和细胞培养研究来确定依赖于MLL2状态的转录变化。通路分析将确定对SCLC重要的已知信号通路是否在MLL2扰动后改变，并将鉴定可能对SCLC重要的新通路。为了鉴定SCLC中直接MLL2调节的增强子，ChIPseq分析将确定哪些差异表达的基因表现出直接MLL2结合，以及在MLL2失活后，降低的组蛋白H3K4单甲基化和与活性增强子相关的其他标记。直接MLL2靶基因的一个子集本身可以作为SCLC肿瘤抑制因子，并将检查其在SCLC中的功能作用。这些研究将阐明MLL2突变型肺癌的分子基础，并为临床前研究提供新的小鼠模型。由于SCLC或SQCC没有靶向治疗，因此我们使用严格的小鼠模型来剖析MLL2在肺癌中的作用至关重要。

DESCRIPTION (provided by applicant): Small cell lung carcinoma (SCLC) is the most aggressive form of lung cancer. With dismal survival rates and a lack of targeted therapies it is critical that we understand the major genes and pathways that drive SCLC. To identify new potential SCLC drivers, we have sequenced human SCLC exomes and transcriptomes and found frequent inactivating mutations in the histone methyltransferase MLL2. The Cancer Genome Atlas study of squamous lung carcinoma (SQCC) revealed that MLL2 is among the most frequently mutated genes in SQCC, suggesting that MLL2 may be a tumor suppressor across multiple lung cancer types. MLL2 is frequently mutated across many human cancers but there has been little study of the link between MLL2 and cancer. Our preliminary data provide functional evidence that MLL2 is a tumor suppressor. By conferring histone H3K4 monomethylation, MLL2 is newly emerging as a key regulator of transcriptional enhancer function but this role has not been explored in tumorigenesis. In this grant proposal, we interrogate MLL2 as a critical tumor suppressor in lung cancer. Specific Aim 1: To test the hypothesis that MLL2 is a tumor suppressor in lung cancer. We have previously used a sensitized autochthonous mouse model of SCLC to show that an SCLC-mutated gene is functionally important for SCLC. We propose to generate new models of MLL2-mutant SCLC and SQCC. The models will not only reveal mechanisms underlying MLL2 tumor suppressor activity but will be ideal preclinical tools for future testing of novel therapies directed towards vulnerabilities conferred by MLL2 inactivation. Specific Aim 2: To identify target genes and pathways regulated by MLL2 that promotes cancer. MLL2 is a methyltransferase that regulates histone H3K4 monomethylation, a mark of transcriptional enhancers. We hypothesize that MLL2 regulation of transcriptional enhancers controls the expression of tumor suppressive transcripts important for lung cancer suppression. We will use primary tumors and cell culture studies to identify transcriptional changes that depend on MLL2 status. Pathway analyses will determine whether known signaling pathways important for SCLC are altered upon MLL2 perturbation and will identify new pathways that may be important for SCLC. To identify direct MLL2-regulated enhancers in SCLC, ChIPseq analyses will determine which of the differentially expressed genes exhibit direct MLL2 binding and, upon MLL2 inactivation, decreased histone H3K4 monomethylation and other marks associated with active enhancers. A subset of direct MLL2 target genes may act themselves as SCLC tumor suppressors and will be examined for functional roles in SCLC. These studies will elucidate molecular underpinnings of MLL2-mutant lung cancer and will provide new mouse models for preclinical studies. As there are no targeted therapies for SCLC or SQCC, it is critical that we use rigorous mouse models to dissect the role of MLL2 in lung cancer.