The role of the LZTR1 ubiquitin ligase in stem cells and cancer

 描述(申请人提供)：在人类癌症的遗传分析中，焦点基因拷贝数变异(CNV)和点突变通过精确定位候选驱动基因提供了精细的信息。最近，我们进行了一项大规模的分析，我们将体细胞点突变和焦点CNV信息整合在一个框架中，以提名与多形性胶质母细胞瘤(GBM)有关的新驱动基因，GBM是人类最具侵袭性的癌症类型之一。从这一分析中出现的排名最靠前的基因是LZTR1，它编码的是cullin-3(CUL3)泛素连接酶复合体的底物适配器，底物仍有待发现。在GBM中，LZTR1以功能丧失突变和局部缺失为靶点，因此作为一种新的肿瘤抑制基因，这一概念最近在其他肿瘤中得到证实。从机制的角度来看，我们发现LZTR1有意想不到的能力来破坏人类GBM中最具侵略性的细胞亚群--胶质瘤干细胞(GSCs)的自我更新和生长。这项建议的中心目标是鉴定和功能表征LZTR1泛素连接酶复合体的底物，并破译LZTR1如何在机制上阻止正常神经细胞中的肿瘤发展。我们的主要假设是，LZTR1-CUL3蛋白复合体通过调节一组特定底物的蛋白分解来抑制肿瘤生长。我们的初步数据已经确定并验证了一组具有公认线粒体活性的蛋白质作为新的LZTR1底物。再加上我们最近观察到的LZTR1定位于线粒体，这些结果是令人兴奋的新发现，将LZTR1的改变与癌症中线粒体功能的解除调节联系起来。在目标1中，LZTR1底物将通过一种新的基于质谱学的技术进行全面鉴定，该技术已经成功地识别了LZTR1令人兴奋的新底物候选。底物的功能验证将在直接从原发人类GBM产生的高度相关的细胞模型中进行，并将与在人类癌症中发现的LZTR1突变情况有关。在目标2中，我们将在神经系统有条件地敲除LZTR1基因的新的遗传小鼠模型中，确定LZTR1在大脑中的正常活性以及携带LZTR1失活突变的人GBM模型。我们还将使用睡美人插入突变系统来识别与LZTR1缺失相关的基因改变，以促进脑肿瘤的发生。

 DESCRIPTION (provided by applicant): In the genetic analysis of human cancer, focal gene copy number variation (CNV) and point mutations provide exquisite information on candidate driver genes by pinpointing their exact location. Recently, we conducted a large-scale analysis in which we integrated somatic point mutations and focal CNV information in a single framework to nominate new driver genes implicated in glioblastoma multiforme (GBM), one of the most aggressive types of human cancer. The top-ranking gene that emerged from this analysis is LZTR1, which codes for the substrate adaptor of a Cullin-3 (Cul3) ubiquitin ligase complex for which the substrates still await discovery. In GBM, LZTR1 is targeted by loss-of-function mutations and focal deletions, thus behaving as a new tumor suppressor gene, a notion recently confirmed in other tumors. From a mechanistic standpoint, we have discovered the unexpected capacity of LZTR1 to impair self-renewal and growth of the most aggressive cellular subpopulation in human GBM, the glioma stem cells (GSCs). The central objective of this proposal is to identify and functionally characterize the substrates of the LZTR1 ubiquitin ligase complex and decipher how mechanistically LZTR1 operates to prevent tumor development in normal neural cells. Our overarching hypothesis is that the LZTR1-Cul3 protein complex suppresses tumor growth through the regulated proteolysis of a particular set of substrates. Our preliminary data have already identified and validated a set of proteins with recognized mitochondrial activities as new LZTR1 substrates. Together with our recent observation that LZTR1 localizes at mitochondria, these results are exciting new findings that link alterations of LZTR1 to deregulation of mitochondrial functions in cancer. In Aim 1, the LZTR1 substrates will be comprehensively identified from a novel mass spectrometry-based technology that has already successfully recognized exciting, new substrate candidates of LZTR1. The functional validation of the substrates will be pursued in highly relevant cellular models directly generated from primary human GBM and will be related to the landscape of mutations of LZTR1 discovered in human cancer. In Aim 2, we will determine the normal activity of LZTR1 in the brain and model human GBM harboring inactivating mutations of LZTR1 in a new genetic mouse model in which the LZTR1 gene is conditionally knocked out in the nervous system. We will also use the Sleeping Beauty insertional mutagenesis system to identify the genetic alterations that cooperate with loss of LZTR1 for brain tumorigenesis.