We are only beginning to scratch the surface of the complexity underlying the role of KRAS in cancer, and there is a pressing need for a deeper understanding of KRAS biology in order to develop effective targeted therapeutics. Intriguingly, the KRAS gene encodes two proteins, KRAS4A and KRAS4B, and there is profound evidence from in vivo mouse models that suggests that the often ignored and poorly understood Kras4A protein is the main player in the development of mutant Kras tumors. Specifically, mice with Kras4B but not Kras4A are highly resistant to carcinogen-induced lung tumors, and there is evidence that Kras4A may play a role in lung stem cells and tumor initiating cells. Furthermore, we have compelling preliminary evidence showing that the Kras4A proto-oncogene (WT Kras4A) interacts with mutant Kras, and that this interaction depends on the type of activating mutation in Kras and influences the selection of tumor initiating cells. Objectives: This study wil (1) define the role of Kras4A expressing cells in vivo in the normal lung and in cancer development; (2) define the signaling features unique to Kras4A and the biochemical interactions underlying the role of Kras4A in tumor cell selection; and (3) identify novel genetic loci which modulate these features of Kras4A in tumorigenesis, which will broaden our understanding of the genetic context in which Kras functions, and may reveal new pathways and genes that converge with KRAS function that can be pharmacologically targeted to treat cancer. Three specific aims are proposed to address these objectives: Aim 1: Determine the role of Kras4A in lung regeneration and tumorigenesis using in vivo models A novel mouse model coupling Cre-recombinase and Kras4A expression (Kras4ACreERT2) will be used to label Kras4A expressing cells in the lung, and to interrogate the role of these cells in lung regeneration and tumor initiation. Kras4A expressing cells will be traced after airway damage, and after tumor induction. Aim 2: Functionally analyze Kras4A signaling in vitro Kras4A signaling pathways will be determined by biochemical analyses of canonical Ras effector pathways and genome-wide expression array experiments in MEFs and epithelial cells with and without Kras4A. The biochemical interaction of WT Kras4A with different mutant oncoproteins of Kras will be explored by analysis of signaling and Kras dimerization, in collaboration with the lab of Dr. Xiaolin Nan. Aim 3: Identify genetic factors in mice that interact with Kras in modulating lung tumor initiation. Tissue already collected from 3 populations of backcrossed mice totaling nearly 400 animals will be used to map genetic factors that are involved in the selection of tumor cells harboring specific mutations at Kras, as well as genetic factors involved in the regulation o Kras splicing and Kras4A expression.