Interrogation of MLL2 as a tumor suppressor gene in lung cancer

描述(申请人提供)：小细胞肺癌(SCLC)是最具侵袭性的肺癌。由于存活率很低，而且缺乏针对性的治疗，我们必须了解驱动小细胞肺癌的主要基因和途径。为了确定新的潜在的小细胞肺癌驱动因素，我们对人小细胞肺癌外显子和转录体进行了测序，发现组蛋白甲基转移酶MLL2经常发生失活突变。鳞癌基因组图谱研究显示，MLL2基因是鳞状肺癌中最常见的突变基因之一，提示MLL2可能是多种类型肺癌中的抑癌基因。MLL2在许多人类癌症中经常发生突变，但关于MLL2与癌症之间的联系的研究很少。我们的初步数据提供了MLL2是一种肿瘤抑制因子的功能证据。通过赋予组蛋白H3K4单甲基化，MLL2是一种新出现的转录增强子功能的关键调节因子，但这一作用在肿瘤发生中尚未被探索。在这项拨款提案中，我们将MLL2作为肺癌的关键肿瘤抑制因子进行研究。具体目的1：验证MLL2在肺癌中是肿瘤抑制因子的假说。我们之前已经使用了致敏的自体小鼠小鼠模型来证明SCLC突变的基因在小细胞肺癌的功能上是重要的。我们建议建立MLL2突变的SCLC和SQCC的新模型。这些模型不仅将揭示MLL2肿瘤抑制活性的潜在机制，而且将成为未来测试针对以下目标的新疗法的理想临床前工具 Mll2停用带来的漏洞。具体目标2：确定由MLL2调控的促进癌症的靶基因和途径。MLL2是一种甲基转移酶，它调节组蛋白H3K4的单甲基化，这是转录增强子的标志。我们假设转录增强子的MLL2调节控制对肺癌抑制重要的肿瘤抑制转录本的表达。我们将使用原发肿瘤和细胞培养研究来确定依赖于MLL2状态的转录变化。通路分析将确定已知的对小细胞肺癌重要的信号通路是否在MLL2干扰下发生改变，并将确定可能对小细胞肺癌重要的新通路。为了确定小细胞肺癌中直接受MLL2调控的增强子，ChIPseq分析将确定哪些差异表达的基因表现出直接的MLL2结合，并在MLL2失活时，组蛋白H3K4单甲基化减少和其他与活性增强子相关的标记。直接的MLL2靶基因的一个子集可能作为小细胞肺癌肿瘤抑制基因，并将被研究在小细胞肺癌中的功能作用。这些研究将阐明MLL2突变肺癌的分子基础，并将为临床前研究提供新的小鼠模型。由于目前尚无针对小细胞肺癌或小细胞肺癌的靶向治疗方法，因此使用严谨的小鼠模型来分析mll2在肺癌中的作用是至关重要的。

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.