Abstract EIF1AX (eukaryotic translation initiation factor 1A) is a component of the translation pre-initiation complex (PIC). Recurrent EIF1AX mutations, first reported in uveal melanomas, are found in ∼1% of papillary thyroid cancers in a mutually exclusive manner with other oncogenic driver events (BRAF, RAS, and oncogenic fusions). By contrast, they are enriched in advanced thyroid cancers (9% of anaplastic and 11% poorly-differentiated thyroid cancers), and are strongly associated with RAS mutations (p<0.0001). EIF1AX mutations cluster in the N-terminal (NTT) or C-terminal tails (CTT). EIF1AX NTT missense mutations in thyroid cancer occur within the first 15 amino acids, whereas the CTT mutation disrupts a splice acceptor site at exon 6 (A113splice). A113splice is the most prevalent defect and is private to this disease, and results in two differentially spliced mRNAs: (1) Cryptic splice variant: by use of a cryptic acceptor site in exon 6 that leads to a 132 AA protein that excludes 12 AA; (2) Truncated splice variant: by retaining intron 5, leading to a 115 AA truncated protein. EIF1AX mutants retain the ability to recruit the ternary complex, as shown by co-IP with EIF2 after ectopic expression of NTT or A113splice EIF1AX mutants in HEK293T cells, or after CRISPR-mediated knock-in of A113splice into Cal62 RAS-mutant thyroid cancer cells. The EIF1AX mutants had greater affinity to EIF5 compared to WT, consistent with a more stable PIC. As translation initiation is a rate-limiting step, the altered affinity of EIF1AX mutants to PIC components could impact the rate of protein synthesis. We tested this by L-azidohomoalanine labeling, which showed contrasting roles of the two A113-generated splice variants expressed at endogenous levels: i.e. the cryptic splice variant increased protein synthetic rate, whereas the truncated splice variant strongly inhibited protein translation. Despite inhibiting translation, the truncated splice variant showed a paradoxical increase in 4EBP1 phosphorylation. Upon A113splice knock-in, where both variants are expressed, translation is increased, which we hypothesize results from the combined effects of 4EBP1 phosphorylation, caused by relief of negative feedback events upstream in the pathway, with increased PIC assembly caused by the cryptic splice variant. We are currently determining whether the altered rate of protein synthesis is global or selective. Of note, cells expressing the cryptic, but not the truncated splice form, showed a transforming phenotype as assessed by soft agar colony formation. As EIF1AX mutations co-occur with RAS mutations in advanced thyroid cancers, it is likely that RAS-induced PI3K-AKT/mTOR signaling may provide a further cooperative benefit and play a key role in disease progression, and in generating specific tumor cell dependencies. Citation Format: Gnana P. Krishnamoorthy, Inigo Landa, Jeffrey A. Knauf, James Nagarajah, Gunnar Rätsch, Hans-Guido Wendel, James A. Fagin. Functional characterization of EIF1AX mutations in thyroid cancer predicts for gain of function by increasing translational rate with concomitant derepression of upstream inputs from mTOR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 892.