MODELS FOR GENETIC ASSESSMENT OF TUMOR MAINTENANCE GENES IN PDAC

在超过95%的PDAC中发现了导致组成型活性K-RAS的突变，并且被认为是这种类型癌症的起始事件。尽管K-RAS突变在PDAC和其他癌症中的频率很高，但迄今为止开发针对癌蛋白的治疗方法的努力都失败了。这一失败导致人们将重点放在由K-RAS控制的效应子途径上，将其作为可能的治疗靶点（见项目2）。项目3基于抑制K-RAS突变细胞生长的替代策略，该策略植根于我们小组和其他人最近在识别所谓的“K-RAS合成致死基因”方面的成功。几 这些基因已经通过RNAi筛选和其它策略被鉴定为在K-RAS突变癌细胞中选择性需要的。然而，这些基因中没有一个作为潜在的肿瘤维持基因在体内被充分检查，并且没有一个在PDAC的背景下被验证。在项目3的目标1中，我们将使用RNAi和生物化学方法来研究抑制四个候选合成致死基因/途径的效果：STK 33，TBK 1，NF-{K}B和丙酮酸激酶基因的M2亚型 （PK-M2）在小鼠和人来源的PDAC衍生细胞系中的作用。在目标2中，我们将创建一种新的PDAC小鼠模型，该模型将允许在已建立的PDAC背景下评估这些和其他候选肿瘤维持基因。使用由两种位点特异性重组酶（Cre和Flp）控制的条件等位基因的组合，该模型将允许K-RAS的条件活化（具有或不具有p53中的伴随突变）以促进PDAC发展，随后是感兴趣基因的条件失活。该系统将首先用于测试抑制Stk 33、Tbk 1或PK-M2的后果 在已建立的，自体肿瘤中。在本项目和本PO 1中的其他项目中鉴定的其他潜在肿瘤维持基因将在未来进行检测。在目标3中，我们将使用目标1中开发的系统来进行更广泛的候选合成致死基因筛选，这些基因可能有助于PDAC疗法的开发。这些数据将用于优先考虑将在目标2中开发的体内系统中进一步测试的基因。

Mutations that result in constitutively active K-RAS are found in over 95% of PDACs and are believed to be an initiating event for this type of cancer. Despite the high frequency of K-RAS mutations in PDAC and other cancers, efforts to date to develop therapies directed against the oncoprotein have failed. This failure has led to a focus on the effector pathways controlled by K-RAS as possible therapeutic targets (see Project 2). Project 3 is based on an alternative strategy for inhibiting the growth of K-RAS-mutant cells, which is rooted in recent success of our group and others in identifying so-called "K-RAS synthetic lethal genes". Several such genes have been identified through RNAi screens and other strategies as being required selectively In K-RAS mutant cancer cells. However, none of these genes have been adequately examined in vivo as potential tumor maintenance genes and none have been validated in the context of P DAC. In Aim 1 of Project 3, we will use RNAi and biochemical methods to investigate the effects of inhibiting four candidate synthetic lethal genes/pathways¿STK33, TBK1, NF-{K}B, and the M2 isoform of the pyruvate kinase gene (PK-M2)¿in PDAC-derived cell lines of mouse and human origin. In Aim 2, we will create a novel mouse model of PDAC that will allow for the assessment of these and other candidate tumor maintenance genes in the context of established PDAC. Using a combination of conditional alleles controlled by two site-specific recombinases (Cre and Flp), this model will allow for conditional activation of K-RAS (with or without accompanying mutation In p53) to promote PDAC development, followed by conditional inactivation of the gene of interest. This system will be deployed first to test the consequences of inhibiting Stk33, Tbk1 or PK-M2 In established, autochthonous tumors. Additional potential tumor maintenance genes identified in this project and other projects in this PO1 will be tested In the future. In Aim 3, we will use the systems developed In Aim 1 to conduct a broader screen of candidate synthetic lethal genes that might be useful in the development of therapies for PDAC. These data will tie used to prioritize genes that will be further tested in the in vivo system developed in Aim 2.