mTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3^IBTK-mediated non-degradative ubiquitination of eIF4A1

Reviewer #1 (Public Review):

In this study, the authors examined the role of IBTK, a substrate-binding adaptor of the CRL3 ubiquitin ligase complex, in modulating the activity of the eiF4F translation initiation complex. They find that IBTK mediates the non-degradative ubiquitination of eiF4A1, promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and tumor cell growth. Correspondingly, phosphorylation of IBTK by mTORC1/ S6K1 increases eIF4A1 ubiquitination and sustains oncogenic translation.

Strengths:

This study utilizes multiple biochemical, proteomic, functional, and cell biology assays to substantiate their results. Importantly, the work nominates IBTK as a unique substrate of mTORC1, and further validates eiF4A1 ( a crucial subunit of the ei44F complex) as a promising therapeutic target in cancer. Since IBTK interacts broadly with multiple members of the translational initial complex - it will be interesting to examine its role in eiF2alpha-mediated ER stress as well as eiF3-mediated translation. Additionally, since IBTK exerts pro-survival effects in multiple cell types, it will be of relevance to characterize the role of IBTK in mediating increased mTORC1 mediated translation in other tumor types, thus potentially impacting their treatment with eiF4F inhibitors.

Limitations/Weaknesses:

The findings are mostly well supported by data, but some areas need clarification and could potentially be enhanced with further experiments:

1. Since eiF4A1 appears to function downstream of IBTK1, can the effects of IBTK1 KO/KD in reducing puromycin incorporation (in Fig 3A), cap-dependent luciferase reporter activity (Fig 3G), reduced oncogene expression ( Fig 4A) or 2D growth/ invasion assays (Fig 4) be overcome or bypassed by overexpressing eiF4A1? These could potentially be tested in future studies.

2. The decrease in nascent protein synthesis in puromycin incorporation assays in Figure 3A suggest that the effects of IBTK KO are comparable to and additive with silvesterol. It would be of interest to examine whether silvesterol decreases nascent protein synthesis or increases stress granules in the IBTK KO cells stably expressing IBTK as well.

3. The data presented in Figure 5 regarding the role of mTORC1 in IBTK-mediated eiF4A1 ubiquitination needs further clarification on several points:

- It is not clear if the experiments in Figure 5F with Phos-tag gels are using the FLAG-IBTK deletion mutant or the peptide containing the mTOR sites as it is mentioned on line 517, page 19 "To do so, we generated an IBTK deletion mutant (900-1150 aa) spanning the potential mTORC1-regulated phosphorylation sites" This needs further clarification.

-It may be of benefit to repeat the Phos tag experiments with full-length FLAG-IBTK and/or endogenous IBTK with molecular weight markers indicating the size of migrated bands.

-Additionally, torin or Lambda phosphatase treatment may be used to confirm the specificity of the band in separate experiments.

-Phos-tag gels with the IBTK CRISPR KO line would also help confirm that the non-phosphorylated band is indeed IBTK.

-It is unclear why the lower, phosphorylated bands seem to be increasing (rather than decreasing) with AA starvation/ Rapa in Fig 5H.

Reviewer #2 (Public Review):

Summary:

This study by Sun et al. identifies a novel role for IBTK in promoting cancer protein translation, through regulation of the translational helicase eIF4A1. Using a multifaceted approach, the authors demonstrate that IBTK interacts with and ubiquitinates eIF4A1 in a non-degradative manner, enhancing its activation downstream of mTORC1/S6K1 signaling. This represents a significant advance in elucidating the complex layers of dysregulated translational control in cancer.

Strengths:

A major strength of this work is the convincing biochemical evidence for a direct regulatory relationship between IBTK and eIF4A1. The authors utilize affinity purification and proximity labeling methods to comprehensively map the IBTK interactome, identifying eIF4A1 as a top hit. Importantly, they validate this interaction and the specificity for eIF4A1 over other eIF4 isoforms by co-immunoprecipitation in multiple cell lines. Building on this, they demonstrate that IBTK catalyzes non-degradative ubiquitination of eIF4A1 both in cells and in vitro through the E3 ligase activity of the CRL3-IBTK complex. Mapping IBTK phosphorylation sites and showing mTORC1/S6K1-dependent regulation provides mechanistic insight. The reduction in global translation and eIF4A1-dependent oncoproteins upon IBTK loss, along with clinical data linking IBTK to poor prognosis, support the functional importance.

Weaknesses:

While these data compellingly establish IBTK as a binding partner and modifier of eIF4A1, a remaining weakness is the lack of direct measurements showing IBTK regulates eIF4A1 helicase activity and translation of target mRNAs. While the effects of IBTK knockout/overexpression on bulk protein synthesis are shown, the expression of multiple eIF4A1 target oncogenes remains unchanged.

Summary:

Overall, this study significantly advances our understanding of how aberrant mTORC1/S6K1 signaling promotes cancer pathogenic translation via IBTK and eIF4A1. The proteomic, biochemical, and phosphorylation mapping approaches established here provide a blueprint for interrogating IBTK function. These data should galvanize future efforts to target the mTORC1/S6K1-IBTK-eIF4A1 axis as an avenue for cancer therapy, particularly in combination with eIF4A inhibitors.