High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/β complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR–15a-16-1–KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

The eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for the viability of the cells whose proposed function is to prevent the stalling of the ribosomes during translation elongation. eIF5A activity requires a unique and functionally essential post-translational modification, the change of a lysine to hypusine. eIF5A is recognized as a promoter of cell proliferation, but it has also been suggested to induce apoptosis. To date, the precise molecular mechanism through which eIF5A affects these processes remains elusive. In the present study, we explored whether eIF5A is involved in controlling the stress-induced expression of the key cellular regulator p53. Our results show that treatment of HCT-116 colon cancer cells with the deoxyhypusine (DHS) inhibitor N1-guanyl-1,7-diamineheptane (GC7) caused both inhibition of eIF5A hypusination and a significant reduction of p53 expression in UV-treated cells, and that eIF5A controls p53 expression at the level of protein synthesis. Furthermore, we show that treatment with GC7 followed by UV-induced stress counteracts the pro-apoptotic process triggered by p53 up-regulation. More in general, the importance of eIF5A in the cellular stress response is illustrated by the finding that exposure to UV light promotes the binding of eIF5A to the ribosomes, whereas UV treatment complemented by the presence of GC7 inhibits such binding, allowing a decrease of de novo synthesis of p53 protein.

The translation initiation factor eIF4E binds the mRNA m⁷G-cap to initiate recruitment and binding of eIF4F components such as eIF4G. eIF4E exhibits oncogenic activity in in vitro and in vivo assays and high eIF4E expression in cancers is associated with poor prognosis and resistance to chemotherapeutics and targeted agents. Targeting eIF4E has long been considered a promising anticancer strategy but this target has remained undruggable using conventional screening approaches. In an accompanying submission we describe a fragment-based screening approach that discovered a non-canonical binding site on eIF4E with unknown functional relevance. Lead compounds with potency of 100nM were generated and provided tools for probing the function of the non-canonical site. Here we have used the tool compounds in combination with a targeted protein degradation (dTAG) to explore the function of this non-canonical site. H1299 NSCLC clones expressing an eIF4E-FKBP12^F36V fusion protein but lacking endogenous eIF4E were established. Treatment of clones with a heterobifunctional dTAG^V-1 molecule, that selectively binds to FKBP12^F36V and recruits the VHL E3 ligase, induced rapid degradation of the eIF4E-FKBP12^F36V protein leading to inhibition of cell growth and reduced expression of the MCL1 protein biomarker. We then expressed wild-type or eIF4E mutants and determined their ability to rescue the phenotype associated with eIF4E-loss following treatment with dTAG^V-1. Expression of wild-type eIF4E rescued the phenotype associated with eIF4E-loss. The W73F mutation, that is reported to block canonical eIF4G binding, disrupted binding to eIF4G in cell lysate assays, but surprisingly the W73F mutant retained the ability to rescue the eIF4E degradation phenotype. Similar to our lead compound, mutations that impacted on the non-canonical pocket (L85R and L134R) disrupted the eIF4E:eIF4G interaction. However, L85R remained able to rescue the cellular and molecular responses, in contrast to the L134R mutant that could not rescue eIF4E loss. Interestingly, a W73F/L85R double mutant had a synergistic effect with a loss of function. Despite disrupting eIF4E:eIF4G binding and protein synthesis in lysates, treatment of H1299 cells with our lead compound did not inhibit proliferation. In contrast, compound treatment of the W73F mutant recapitulated the effects of combining W73F with the L85R non-canonical pocket mutation. In summary, targeting the non-canonical pocket was sufficient to disrupt the eIF4E:eIF4G binding in lysate assays. However, the differential impact of compounds or mutation on eIF4G binding in cells suggest that the eIF4E:eIF4G interaction in intact cells may be more complex than that predicted by the in vitro models. This observation may also explain the challenges associated with developing cellularly potent inhibitors of the eIF4E:eIF4G interaction. Our approach demonstrates the power of coupling fragment-based screening with target-degradation and genetic rescue approaches to find and explore novel functional pockets on difficult to drug proteins. Citation Format: Swee Y Sharp, Marianna Martella, Christopher I Milton, George Ward, Mladen Vinkovic, Caroline Richardson, Andrew Woodhead, Paul A Clarke. Coupling fragment-based screening with targeted protein degradation and genetic rescue to identify and explore the function of a non-canonical pocket on eIF4E [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A143.

Initiation of translation is considered the main rate-limiting step of protein synthesis and requires the recognition of 5’ m7G-cap on mature mRNAs and the formation of eukaryotic translation initiation factor 4F (eIF4F) multi-protein mRNA cap-binding complex. Formation of this complex requires the interaction of eIF4E and the scaffold protein eIF4G, and RNA helicase eIF4A. This eIF4F complex along with eIF3 mediate the recruitment of the 40S ribosomal particle to the 5′ cap of mRNA. Activation of eIF4E is a regulatory hub of many major oncogenic pathways, thus, targeting eIF4E has emerged as a potential therapeutic strategy in cancer. Here we have used a targeted protein degradation approach coupled with genetic rescue to explore the molecular and cellular dependency of cancer cells on eIF4E. Stable H1299 human NSCLC clones expressing FKBP12F36V-tagged eIF4E but lacking endogenous eIF4E were established. Treatment of multiple N- or C-tagged-eIF4E clones with dTAGv-1, an FKBP12F36V selective heterobifunctional molecule that recruits VHL, induced rapid degradation of eIF4E to undetectable levels by 6hr exposure. This also resulted in reduced expression of MCL1, a previously reported biomarker of eIF4E activity. Longer exposures to dTAGv-1 resulted in a cytostasis that was not associated with cell death. A diastereomer negative control of dTAGv-1 that cannot recruit VHL did not elicit loss of eIF4E or the downstream events associated with its loss. Global analysis of protein synthesis initiation by RIBOseq and proteome profiling following dTAGv-1 treatment out to 32hr exposure demonstrated surprisingly few alterations in protein expression despite the significant effect on cancer cell growth. We also expressed wild-type or eIF4E mutants predicted to disrupt key functions and determined their ability to rescue molecular or cellular phenotype associate with eIF4E-loss following dTAGv-1 treatment. Expression of wild-type eIF4E completely rescued cell growth and MCL1 expression. A W56A mutant predicted to disrupt mRNA-cap binding was unable to rescue eIF4E loss. In contrast, expression of W73F or S290A mutants (predicted to disrupt eIF4G binding or exhibit reduced eIF4E activity, respectively) were able to rescue the loss of eIF4E. In summary, our rescue experiments show that mRNA-cap binding by eIF4E is required and that eIF4E:eIF4G interaction in cells may be more complex than predicted. This may also explain the challenges associated with developing selective and cellularly potent inhibitors of the eIF4E:eIF4G interaction and that targeting mRNA-cap binding may be a more effective strategy. We predicted that removing eIF4E would impact on the global synthesis of many proteins. However, our data demonstrate that targeting eIF4E leads to limited effects on protein synthesis that remain sufficient to inhibit cancer cell growth. Further experiments are underway to understand which proteins drive this dependency on eIF4E. Citation Format: Swee Y. Sharp, Marianna Martella, Christopher I. Milton, George Ward, Caroline Richardson, Andrew Woodhead, Paul A. Clarke. Exploring the role of eIF4E in cancer cells with targeted protein degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3722.