Genomic and biochemical studies indicate the central role of ERK signaling as the key driver of colorectal cancer (CRC) proliferation. Yet selective RAF/MEK/ERK pathway inhibitors have been ineffective in the treatment of CRC thus far. We hypothesize that profound ERK inhibition is required to suppress the growth of ERK activated tumors and that high basal receptor-driven RAS activation in CRC causes rapid adaptive and acquired resistance to ERK inhibition that is not overcome with current inhibitors due to their narrow therapeutic index. Additionally, augmenting this high basal receptor-driven RAS activation could provide sufficient ERK activation to stimulate tumor growth, providing a selective pressure for genomic alterations that amplify upstream signaling in CRC. In this proposal, we will define the spectrum of genomic alterations in CRC that amplify ERK signaling and could be targeted with ERK inhibition and will functionally and biochemically characterize novel alterations in the RAF and MEK proteins. We will devise strategies that profoundly inhibit ERK by co-targeting ERK activation and receptor re- activation and by using novel combinations and dosing schedules that expand the therapeutic index of ERK inhibition. We have shown that high receptor tyrosine kinase (RTK) signaling in CRC has consequences for response to targeted therapies as (1) response rates to RAF plus RTK (i.e., EGFR) inhibitors is better than for RAF inhibitors alone in patients with BRAF V600E CRC, (2) wild-type RAS amplification, a recurrent mechanism of resistance to RAF inhibitor combinations in CRC never detected in the large number of analyzed RAF inhibitor-resistant melanomas, increases activated RAS in an RTK-dependent manner, and (3) low activity BRAF mutants in CRC can act as oncogenes by increasing signal transduction from activated RAS and amplifying EGFR signaling. This proposal will now apply an understanding of these specific lineage properties to define the molecular profiles that depend on ERK signaling and devise effective strategies to inhibit ERK signaling in CRC in the clinic. We believe these studies will guide the development of new treatments for CRC and improve the outcomes of patients with these tumors.