Meghan Kellett ¹ Vibha Sharma ¹ Madeline E Sherlock ^2,3 Umarani Pugazhenthi ¹ Madison Rose ¹ Molishree U Joshi ⁴ Monika Dzieciatkowska ⁵ Vu Nguyen ⁶ Philip Reigan ⁶ Kirk Hansen ⁵ Jeffrey S Kieft ^2,3 Rebecca E Schweppe ^1,7*

• ¹ Division of Endocrinology, Metabolism, and Diabetes, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States
• ² New York Structural Biology Center, New York, United States
• ³ Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States
• ⁴ Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, 80045, United States
• ⁵ Division of Structural Biology and Biophysics, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, 80045, United States
• ⁶ Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, United States
• ⁷ University of Colorado Anschutz Medical Campus, Aurora, United States

Advanced thyroid cancer, including Papillary (PTC) and Anaplastic Thyroid Cancer (ATC) are the leading causes of endocrine cancer deaths. Thus, there is a critical need to identify novel therapeutic targets to improve standard of care. Focal Adhesion Kinase (FAK) is overexpressed and phosphorylated in thyroid cancer and drives thyroid cancer growth, invasion, and metastasis.FAK is a nonreceptor tyrosine kinase that is autophosphorylated at tyrosine 397 (Y397) in response to integrin or growth factor receptor signaling, resulting in the recruitment of SRC proto-oncogene and downstream signaling pathways. FAK is predominately localized at the plasma membrane but has recently been shown to accumulate in the nucleus as well as the nucleolus to drive tumor growth. The nucleolus is a membraneless subnuclear organelle that is involved in ribosomal biogenesis through the transcription, processing, and assembly of ribosomal RNA (rRNA). The role of FAK in ribosome biogenesis is currently unknown. We have found that pY397 FAK is localized in the nucleolus of advanced thyroid cancer cells where it interacts with nucleolar proteins involved in ribosome biogenesis. Furthermore, we have found that pY397 is important for nucleolar accumulation of FAK and that nucleophosmin 1 (NPM1), the main structural component of the granular component of the nucleolus, is required for pY397 FAK nucleolar accumulation. Functionally, we identified that nuclear FAK and FAK kinase activity are necessary for anchorage independent growth. We discovered that depletion of FAK results in decreased protein synthesis with a specific decrease in the 60S ribosome subunit. Of the nucleolar FAK interacting proteins, the core snoRNP, NOP56, plays a key role in 60S subunit biogenesis. Accordingly, we have shown that pY397 FAK co-localizes with NOP56 and that knockdown of NOP56 phenocopies FAK depletion. Overall, these findings highlight a novel function for FAK to regulate ribosome biogenesis and suggest that nucleolar FAK represents a promising therapeutic target.