Abstract LB431: Direct comparison of focal adhesion kinase (FAK) therapeutic modalities in cancer: Kinase, FAT, and PROTAC

Abstract Introduction: Focal Adhesion Kinase (FAK) is a multifunctional non-receptor tyrosine kinase and scaffolding protein that has emerged as a potential therapeutic target for the treatment of cancer due to its upregulation and role in cancer cell survival. However, it remains unknown which cancer types are most susceptible to FAK modulation or the best pharmacological strategy to target FAK. FAK is endogenously regulated by an alternative transcript termed FAK-related non-kinase (FRNK), which inhibits FAK through the Focal Adhesion Targeting (FAT) domain. Current FAK-kinase inhibitors that block the catalytic site have shown limited efficacy in multiple Phase I/II clinical trials. FAK-degrading PROTACs have also shown inadequate induction of antiproliferative effects despite successful protein degradation. However, we have shown promising results with our novel FAK-FAT inhibitors, which target the scaffolding function of FAK to induce anoikis and inhibit proliferation in cancer cells. A direct comparison of these different FAK modulators in various cancer types has yet to be performed, creating a significant gap in understanding the best approach for targeted FAK inhibition for the treatment of cancer. Methods: The Broad Institute’s Cancer Dependency Map (DepMap) was utilized to select a cancer cell line cohort based on dependency on FAK for survival. Four cancer types, including breast, brain, lung, and skin cancers were selected based on the correlation of FAK expression for survival and previous literature evidence for dependency on FAK. Three FAK-dependent and one FAK-independent cell lines per cancer type were selected for a total of 16 cancer cell lines. Genetic adenoviral constructs that block the FAK-FAT domain (Ad-FRNK) and knockdown FAK levels (shRNA) were utilized to investigate the role of FAK in cancer cell survival. These constructs were used as they recapitulate the pharmacological strategy of our FAK-FAT inhibitor UA-2012 and FAK degrading PROTACs, respectively. Results: Cell viability was significantly decreased in 10/13 FAK-dependent cell lines after transduction with either adFRNK or FAK targeting shRNAs, six of which showed a statically significant decrease in cell viability upon transduction with adFRNK compared to shRNAs. No cell lines showed a greater response to FAK shRNAs compared to adFRNK. We then evaluated the ability of the FAK kinase inhibitor Defactinib, FAK degrading PROTAC BI-3663, and our novel FAK-FAT domain inhibitor UA-2012 to induce cancer cell death. Overall, FAK-FAT domain inhibition with UA-2012 resulted in the most potent reduction in viability of FAK-dependent cells, particularly in skin and breast cancer cell lines. Treatment with Defactinib resulted in low IC50 values regardless of FAK, and PROTAC BI-3663 displayed minimal effects with high IC50 values often above 200 µM despite successful protein degradation. Conclusion: This data suggests that the FAK-FAT domain may be the major modulator of FAK-mediated survival in breast and skin cancer cells. Citation Format: Lauren Reyes, Ruchi Khatiwala, Timothy Marlowe. Direct comparison of focal adhesion kinase (FAK) therapeutic modalities in cancer: Kinase, FAT, and PROTAC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (7Suppl): Abstract nr LB431.