Abstract 5835: Genome-Wide CRISPR gene knockout screens combined with selpercatinib identifies potent combination therapies for RET driven medullary thyroid carcinoma
Authors: Rahmy Abdelrahman , Fallatah Arwa , Milewski David , Tian Meijie , Song Young , Kim Yong , Chou Hsien-Chao , Wen Xinyu , Wang Chaoyu , Wei Jun , Hawley Robert , Glod John , Khan Javed 
Published: March 2024

Abstract
Background: Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor driven primarily by activating mutations in the RET proto-oncogene. It accounts for approximately 13% of thyroid cancer-related deaths. Total thyroidectomy is the main treatment option for MTC patients; however, its efficacy is limited due to the high prevalence of metastatic disease at diagnosis. Vandetanib, cabozantinib, and selpercatinib are RET signaling inhibitors (RETi), that have been approved for treating advanced MTC. Despite their effectiveness, many patients relapse or have refractory disease due to eventual resistance to these RETis.
Methods: In our study, we employed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) gene knockout (KO) in MTC cell lines treated with selpercatinib or vehicle to investigate the genetic mechanisms of resistance or sensitivity to RETi. Our overarching goal was to identify potential synergistic or additive drug combinations that may prevent or treat resistant disease.
Results: Our research identified key genes and pathways confirming the sensitivity or resistance of MTC cells to RETi. Notably, apoptotic genes along with RAS and mTOR signaling pathways were found to play a significant role in resistance development. Knocking out negative regulators of these pathways (such as NF1 and TSC2) conferred growth advantages to cells treated with RETi. Alternatively, knocking out BCL2, an antiapoptotic protein, led to increased lethality. These findings suggested that targeting RAS, mTOR, or antiapoptotic pathways in conjunction with RETi may be synergistic or have additive activity. To validate the CRISPR screening, we selectively knocked down NF1 in MTC cells and found it led to a nearly two-fold increase in IC50 for selpercatinib. Furthermore, in-vitro and in-vivo testing confirmed significant synergistic activity when using the combination of trametinib, an MEK inhibitor (MEKi; downstream of the RAS pathway), with selpercatinib.
Conclusions and Future Directions: We have identified a potent synergistic combination of MEKi with RETi as a novel synergistic drug combination to treat MTC, which may also prevent resistance to RETi. These results will be translated to patients through clinical trials conduced at the NCI.
Citation Format: Abdelrahman Rahmy, Arwa Fallatah, David Milewski, Meijie Tian, Young Song, Yong Kim, Hsien-Chao Chou, Xinyu Wen, Chaoyu Wang, Jun Wei, Robert Hawley, John Glod, Javed Khan. Genome-Wide CRISPR gene knockout screens combined with selpercatinib identifies potent combination therapies for RET driven medullary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5835.