Dissecting a novel role of FAK in modulating immunity during metastatic reactivation and progression - PROJECT SUMMARY Metastatic relapse is the dominant cause of death in cancer patients. In many cancers, including breast cancer and melanoma, metastatic relapse is caused by the reactivation of dormant tumor cells in a manner that permits immune evasion. However, the mechanisms governing dormancy and immune evasion in incipient metastasis are poorly understood. Here, we identify a critical and new role of focal adhesion kinase (FAK) in promoting metastatic reactivation and immune evasion by abrogating innate immune signaling. By integrating in vivo genetic perturbation screening with analysis of RNA-seq data, we find that loss of components of the Sting cascade, a central innate immunity pathway, leads to metastatic reactivation and progression. Given that intact immunogenic outputs of Sting restrict metastasis, we reasoned that negative regulators of this pathway enable immune escape and reactivation. Analysis of murine and human single-cell RNA-seq data across the dormancy– reactivation–metastasis spectrum reveals a strong inverse correlation between Sting-dependent immune outputs and FAK. Through a series of genetic validation experiments, we show that Fak is sufficient and necessary for ablating Sting-dependent immune outputs. In conditional mouse models, we show that Fak is required for metastatic reactivation by suppressing T cell function. Together, these results suggest a fundamentally new role of Fak in tumor immunobiology during metastatic reactivation. From a mechanistic standpoint, we find that Fak, via its FERM domain, binds to Irf3, the key transcription factor mediating immunogenic outputs downstream of Sting. This interaction blunts Irf3 phosphorylation, dimerization, and nuclear translocation, therefore suppressing Irf3-mediated IFN transcriptional regulation. We further show that Fak depletion using shRNA or a proteolysis- targeting chimera (PROTAC), but not inhibition of Fak kinase activity, restores Sting-Irf3-mediated IFN expression in vitro, and suppresses metastatic reactivation and progression in vivo. This proposal seeks to dissect the structural and mechanistic basis of Fak in regulating the dormancy–reactivation–metastasis spectrum, and inform and test novel therapeutic approaches. Building upon our preliminary findings, we propose in Aim 1 to determine the impact of Fak:Irf3 interaction on metastatic reactivation and progression. Through the use of cutting-edge approaches including CRISPR-dependent base editing, we will determine not only the precise residues required for mediating protein–protein interaction, but also the physiological relevance of disrupting Fak:Irf3 binding and its impact on metastatic reactivation and progression. In Aim 2, we will test whether Fak depletion sensitizes incipient and established metastases to immunotherapy. Successful completion of this work will aid in the development of first-in-class therapeutic approaches that reduce suffering in patients with metastatic relapse. With guidance from exceptional mentors and collaborators, and access to an outstanding scientific research environment at Columbia University Irving Medical Center, this project will prepare me for a successful career as a physician-scientist in the field of cellular biology.
