PROJECT ABSTRACTFocal Adhesion Kinase (FAK) is a multifunctional non-receptor tyrosine kinase and scaffolding protein that isoverexpressed in numerous solid tumors including melanoma while minimally expressed in normal tissue. FAKhas been widely investigated as a cancer drug target due its contribution in multiple aspects of tumor progressionincluding adhesion invasion proliferation survival metastasis angiogenesis and immune cell suppression.However development of FAK inhibitors has largely focused on inhibition of the kinase enzyme of FAK opposedto inhibition of key scaffolding interactions. Particularly limited efforts have been made at the discovery andbiological evaluation of inhibitors of the focal adhesion targeting (FAT) scaffolding domain of FAK the domainrequired for FAK localization to focal adhesions. During this period of support we have identified the firstdiscovered stapled peptide-based FAK inhibitor (UA-1907) that binds to and co-crystallizes with the FAT domainand competitively inhibits FAK-paxillin binding. We have identified a myristoylated derivative (UA-2012) withimproved cellular potency favorable drug-like properties and in vivo efficacy; and developed a bivalent peptide(UA-2023) with low nanomolar binding to FAT. However the mechanistic differences between these novel FATdomain peptides and traditional FAK-kinase inhibitors on perturbation of focal adhesion complexes and the FAKinteractome have yet to be elucidated. Furthermore we have preliminary data showing that FAT inhibitionprovides selective anti-cancer effects in NRAS mutant melanoma cells. Melanoma is the deadliest form of skincancer and there are no current effective targeted therapies against NRAS mutant melanoma which represents~30% of all patients. We hypothesize that FAK FAT domain inhibitors have distinct biological effects onthe focal adhesion complex in cancer cells compared to FAK kinase inhibitors; and cancer cells withalterations in NRAS signaling pathways have a molecular dependence on FAK FAT scaffolding forsurvival thus promoting selective anti-cancer efficacy. In specific aim 1 we will identify the uniquedifferences between FAK FAT scaffold inhibitors and FAK kinase inhibitors on modulation of the focal adhesioncomplex and the FAK interactome. In specific aim 2 we will define the molecular mechanisms ofsensitivity/resistance to FAK FAT inhibition in melanoma cells with activating mutations in NRAS and BRAFpathways. In specific aim 3 we will evaluate in vivo efficacy of novel FAK FAT domain inhibitors in mouse modelsof NRAS and BRAF driven cancer. Overall in this project we will utilize FAT stapled peptides to validate thatFAT domain targeting provides additional biological efficacy on the focal adhesion complex compared to FAK-kinase inhibitors and that NRAS mutant melanoma has a unique sensitivity to FAK FAT inhibition..