Translation initiation in cancer cells is the rate-limiting step of oncogenic mRNAs translation/protein synthesis and is primarily mediated by the eukaryotic translation initiation factor 4 F (eIF4F) complex that consists of three subunits: eIF4E, which binds the cap of mRNA; eIF4A, an RNA helicase implicated in unwinding mRNA structure; and a scaffold protein eIF4G. Our prior crystal structure determination of human eIF4A in complex with Heat 1 domain of eIF4GI (722-999) revealed that eIF4G interacts with both N- and C-terminal domains of human eIF4A (eIF4A1-NTD and eIF4A1-CTD). Utilizing structural information gained from this complex, we generated a series of eIF4A mutants (eIF4A1-NTD: E41K, R45D, Y48A and eIF4A1-CTD: T271A, R324D, D296A/T298A) in order to identify the crucial site(s) of interaction with eIF4G Heat 1 domain. Using in vitro binding studies, we demonstrated that amongst various mutants tested, eIF4A-CTD (eIF4A1R324D) mutant disrupted the eIF4A/eIF4G Heat 1 complex formation, mirroring the binding residue E809K mutant of eIF4G that also interrupted eIF4A/eIF4G Heat 1 interaction. Given that eIF4G has an additional eIF4A-binding site named Heat 2 domain, we further investigated whether eIF4A1R324D mutant is able to disrupt binding to full-length eIF4G protein in vivo. Surprisingly, overexpression of Flag-tagged eIF4A1R324D in prostate cancer cell line blocked interaction with exogenous eIF4G as well as endogenous eIF4G. Taken together, these results strongly suggest that eIF4A1R324 residue plays a crucial role in mediating eIF4A/eIF4G complex formation in vivo. Furthermore, overexpression of eIF4A1R324D mutant significantly blocked oncogene c-myc and Bcl-2 mRNA translation thereby inhibiting prostate tumor growth in vivo. To conclude, our studies provide a previously unexplored strategy to identify small-molecule compounds disrupting eIF4A-eIF4G complex by targeting the pocket spanning eIF4AR324 and its binding site residues on eIF4G for cancer therapy.

Citation Format: Puja Singh, Hanyong Chen, Hanxuan Li, Young-In Chi, Bin Liu, Junxuan Lu, Wei Li, Zigang Dong, Yibin Deng. Disruption of the translation initiation complex by eIF4A mutant inhibits prostate tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4391.