PROJECT SUMMARY/ABSTRACT Mutations in RAS proteins, in particular K-ras, are common in human cancer, and the signaling pathways emanating from this oncoprotein have been examined in detail. Despite intensive efforts to translate this knowledge into improved cancer care, K-ras mutant tumors remain a formidable scientific and clinical challenge. In this proposal, we focus on individual KRAS mutations that are commonly found in colorectal cancer (CRC), because these mutations are associated with different sensitivities to targeted inhibitors and thus may engage distinct signaling programs and require individualized therapeutic strategies. To access and exploit these distinct programs, we employ an innovative mass spectrometry-based technique that enables the majority of the human kinome to be analyzed simultaneously, providing the means to explore the K-ras induced kinome in an unbiased fashion. Our unique approach combines mass spectrometry and protein kinase-capture beads to monitor activated protein kinases from tumor cells, providing the ‘big picture’ of tumor kinase activity. Using this technology, in Aim 1 we will measure the basal activity of the kinome in (i) a model isogenic CRC system that represents common CRC associated KRAS mutant alleles and (ii) a panel of well characterized human KRAS mutant CRC cell lines, before and after KRAS knockdown, to identify kinases whose activity is dependent on expression of the various mutant forms of this oncogene. In Aim 2, we will explore the resiliency of the CRC KRAS kinome(s) to MEK inhibition to identify and target compensatory kinases that promote resistance. In Aim 3, we will use this information to carry out a targeted synthetic lethal screen in vivo, testing the effectiveness of inhibiting these activated protein kinases in KRAS dependent CRC xenografts and in CRC knock-in mouse models driven by a common KRAS exon 2 (G12D) or an exon 4 (A146T) mutation. In this way, we hope to identify individualized kinase inhibitor combinations that are effective in treating these common types of K-ras- driven CRC.