eIF4E Expression Research Articles

RIPK1 inhibition mitigates neuroinflammation and rescues depressive-like behaviors in a mouse model of LPS-induced depression

BackgroundDepression is often linked to inflammation in the brain. Researchers have been exploring ways to reduce this inflammation to improve depression symptoms. One potential target is a protein called RIPK1, which is known to contribute to brain inflammation. However, it’s unclear how RIPK1 influences depression. Our study aims to determine whether RIPK1 inhibition could alleviate neuroinflammation-associated depression and elucidate its underlying mechanisms.MethodsTo investigate our research objectives, we established a neuroinflammation mouse model by administering LPS. Behavioral and biochemical assessments were conducted on these mice. The findings were subsequently validated through in vitro experiments.ResultsUsing LPS-induced depression models, we investigated RIPK1’s role, observing depressive-like behaviors accompanied by elevated cytokines, IBA-1, GFAP levels, and increased inflammatory signaling molecules and NO/H2O2. Remarkably, Necrostatin (Nec-1 S), a RIPK1 inhibitor, mitigated these changes. We further found altered expression and phosphorylation of eIF4E, PI3K/AKT/mTOR, and synaptic proteins in hippocampal tissues, BV2, and N2a cells post-LPS treatment, which Nec-1 S also ameliorated. Importantly, eIF4E inhibition reversed some of the beneficial effects of Nec-1 S, suggesting a complex interaction between RIPK1 and eIF4E in LPS-induced neuroinflammation. Moreover, citronellol, a RIPK1 agonist, significantly altered eIF4E phosphorylation, indicating RIPK1’s potential upstream regulatory role in eIF4E and its contribution to neuroinflammation-associated depression.ConclusionThese findings propose RIPK1 as a pivotal mediator in regulating neuroinflammation and neural plasticity, highlighting its significance as a potential therapeutic target for depression.

Hyperactivation of mTOR/eIF4E Signaling Pathway Promotes the Production of Tryptophan-To-Phenylalanine Substitutants in EBV-Positive Gastric Cancer.

Although messenger RNA translation is tightly regulated to preserve protein synthesis and cellular homeostasis, chronic exposure to interferon-γ (IFN-γ) in several cancers can lead to tryptophan (Trp) shortage via the indoleamine-2,3-dioxygenase (IDO)- kynurenine pathway and therefore promotes the production of aberrant peptides by ribosomal frameshifting and tryptophan-to-phenylalanine (W>F) codon reassignment events (substitutants) specifically at Trp codons. However, the effect of Trp depletion on the generation of aberrant peptides by ribosomal mistranslation in gastric cancer (GC) is still obscure. Here, it is shows that the abundant infiltrating lymphocytes in EBV-positive GC continuously secreted IFN-γ, upregulated IDO1 expression, leading to Trp shortage and the induction of W>F substitutants. Intriguingly, the production of W>F substitutants in EBV-positive GC is linked to antigen presentation and the activation of the mTOR/eIF4E signaling pathway. Inhibiting either the mTOR/eIF4E pathway or EIF4E expression counteracted the production and antigen presentation of W>F substitutants. Thus, the mTOR/eIF4E pathway exposed the vulnerability of gastric cancer by accelerating the production of aberrant peptides and boosting immune activation through W>F substitutant events. This work proposes that EBV-positive GC patients with mTOR/eIF4E hyperactivation may benefit from anti-tumor immunotherapy.

Cinnamaldehyde protects SH-SY5Y cells against advanced glycation end-products induced ectopic cell cycle re-entry

Accumulation of advanced glycation end-products (AGEs) in the brain contributes significantly to cognitive impairment in patients with diabetes by disrupting the post-mitotic state of neuronal cells, thereby triggering ectopic cell cycle re-entry (CCR) and subsequent neuronal apoptosis. Cinnamaldehyde (CINA), a potential mitigator of cognitive impairment due to its blood glucose-lowering properties, warrants exploration for its role in counteracting diabetes-related neurological damage. In this study, we examined the neuroprotective effect of CINA on AGE-damaged SH-SY5Y human neuroblastoma cells differentiated in vitro. We investigated the impact of CINA on AGE-induced neuronal CCR and apoptosis, finding that it substantially suppressed aberrant DNA replication, precluded cells from entering the mitotic preparatory phase, and diminished apoptosis. Additionally, CINA inhibited the expression of eIF4E without altering S6K1 phosphorylation. These findings indicate that CINA safeguards neuronal cells from AGE-related damage by preventing abnormal CCR, preserving the post-mitotic state of neuronal cells, and reducing AGE-induced apoptosis, potentially through the inhibition of eIF4E-controlled cell proliferation. Our results highlight the prospective utility of CINA in managing diabetic neuropathy.

EIF4E plays the role of a pathogenic gene in psoriasis, and the inhibition of eIF4E phosphorylation ameliorates psoriasis-like skin damage.

Psoriasis is a complex inflammatory skin disease with uncertain pathogenesis. eIF4E (eukaryotic translation initiation factor 4E) and its phosphorylation state p-eIF4E are highly expressed in psoriatic tissues. However, the role eIF4E played in psoriasis is still unclear. To investigate the function of eIF4E and p-eIF4E in psoriasis and to figure out whether eFT-508 (Tomivosertib, eIF4E phosphorylation inhibitor) can relieve the disease severity and become a promising candidate for the psoriasis treatment. We first verified the expression of eIF4E and p-eIF4E in psoriasis patients' lesional skin. Then, we demonstrated the effect of eIF4E and p-eIF4E on the abnormal proliferation and inflammatory state of keratinocytes by using eIF4E-specific small interfering RNA (si-eIF4E) and eFT-508. In this study, all cell experiments were performed under the psoriasis-model condition. Moreover, the external application of eFT-508 on imiquimod (IMQ)-induced psoriasis mice was performed to explore its potential clinical value. Results showed that eIF4E and p-eIF4E were significantly overexpressed in skin lesions of psoriasis patients. Knocking down eIF4E or adding eFT-508 can relieve the abnormal proliferation and the excessive inflammatory state of keratinocytes by reducing the expression of cyclin D1, IL-1β, CXCL10, IL23, Wnt 5a, NBS1 and p-AKT from mRNA or protein levels. Furthermore, these results were consistent with those obtained from the invitro experiments. Then, we conclude that eIF4E plays the role of the pathogenic gene in psoriasis, and eFT-508 may be a promising candidate for anti-prosoriasis drugs.

DIA-Based Proteomics Reveals the Protective Effect of Quercetin against Cyclophosphamide-Induced Testicular Toxicity in Mice

Cyclophosphamide (CP) is a common employed immunosuppressive and anti-neoplastic alkylating antineoplastic drug with documented male reproductive harm. Quercetin, a widely distributed anti-oxidant flavonoid, has been demonstrated to have a variety of biological roles, including tissue-protective properties. Here, administration of quercetin alleviated the CP-induced damage to the testicles, as evidenced by improvements in body weight, testis and epididymis index, serum testosterone concentration, the pathomorphological of testis, and reduced apoptosis in testicular cells. Moreover, Proteomic data indicated that all DEPs were classified into five categories, Cluster 1 (C1) and C4 were regulated by cyclophosphamide and quercetin. KEGG pathway analyses showed that mTOR signaling pathways and Proteasome were the most abundant pathways in C1 and C4, respectively. In the protein-protein interaction (PPI) network analysis, quercetin inhibits the expression of GSK3B, EIF4E, and RPS6KA2 proteins in CP-induced spermatogenesis failure in mice via the mTOR signaling pathway, protecting against spermatogonia injury. On the other hand, quercetin alleviates spermatogonia injury by stimulating the production of Rps23, Rpl8, Rps11, and Mrps17 proteins in CP-induced testis injury in mice via the pathways of Proteasome. Our results have showed that quercetin has a putative protective effect via modulating the pathways of mTOR and Proteasome in CP induced testis injury

Abstract A143: 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 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-FKBP12F36V fusion protein but lacking endogenous eIF4E were established. Treatment of clones with a heterobifunctional dTAGV-1 molecule, that selectively binds to FKBP12F36V and recruits the VHL E3 ligase, induced rapid degradation of the eIF4E-FKBP12F36V 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 dTAGV-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.

Eukaryotic Initiation Factor 4E (eIF4E) as a Target of Anti-Psoriatic Treatment

Eukaryotic translation initiation factor 4E (eIF4E) has been known to play a critical role in the regulation of gene expression and essential cellular processes such as proliferation, apoptosis and differentiation. We explored herein its role in the pathophysiology of psoriasis. The inhibition of eIF4E by siRNA or briciclib, an eIF4E small molecule inhibitor, downregulated the expression of eIF4E itself and its two complex partners eIF4A and G, as well as other eIFs (e.g. eIF1A, eIF2α, eIF3A, eIF3B, eIF5 and eIF6). This inhibition also abolished psoriatic inflammation in both the imiquimod and TGFß mouse model, as well as in a human 3D-psoriasis tissue model. Downregulation of eIF4E and the other eIFs by application of briciclib (particularly when given topically) was linked to the normalization of cellular proliferation, restoration of the inflammatory milieu and epidermal hyperplasia of psoriasis, and normalization of levels of pro-inflammatory cytokines (e.g. TNFα, IL-1b, IL-17 and IL-22), and as well as keratinocyte differentiation markers (e.g. KRT16 and FLG). These results demonstrate translational imbalance and underline the crucial role played by eIF4E and other eIFs in the pathophysiology of psoriasis. This work opens up avenues for the development of novel topical anti-psoriatic treatment strategies by targeting eIF4E.

Newly Synthesized Anticancer Purine Derivatives Inhibiting p-EIF4E Using Surface-Modified Lipid Nanovesicles.

Translation of mRNA is one of the processes adopted by cancer cells to maintain survival via phosphorylated (p)-eIF4E overexpression. Once p-eIF4E binds to the cap structure of mRNA, it advocates a nonstop translation process. In this regard, 15 new-based GMP analogs were synthesized to target eIF4E and restrain its binding to cap mRNA. The compounds were tested against three types of cancer cell lines: Caco-2, HepG-2, MCF-7, and normal kidney cells (Vero cells). Most of the compounds showed high potency against breast cancer cells (MCF-7), characterized by the highest cancer type for overexpression of p-eIF4E. Compound 4b was found to be the most active against three cell lines, colon (Caco-2), hepatic (HepG-2), and breast (MCF-7), with positive IC50 values of 31.40, 27.15, and 21.71 μM, respectively. Then, chitosan-coated niosomes loaded with compound 4b (Cs/4b-NSs) were developed (as kinetically enhanced molecules) to improve the anticancer effects further. The prepared Cs/4b-NSs showed pronounced cytotoxicity compared to the free 4b against Caco2, Hepg2, and MCF-7 with IC50 values of 16.15, 26.66, and 6.90 μM, respectively. Then, the expression of both the phosphorylated and nonphosphorylated western blot techniques was conducted on MCF-7 cells treated with the most active compounds (based on the obtained IC50 values) to determine the total protein expression of both eIF4E and p-eIF4e. Interestingly, the selected most active compounds displayed 35.8-40.7% inhibition of p-eIF4E expression when evaluated on MCF-7 compared to Ribavirin (positive control). CS/4b-NSs showed the best inhibition (40.7%). The findings of the present joint in silico molecular docking, simulation dynamic studies, and experimental investigation suggest the potential use of niosomal nanovesicles as a promising nanocarrier for the targeted delivery of the newly synthesized compound 4b to eukaryotic initiation factor 4E. These outcomes support the possible use of Cs/4b-NSs in targeted cancer therapy.

Inhibition of Eukaryotic Initiating Factor eIF4E Overcomes Abemaciclib Resistance in Gastric Cancer.

Aberrant activating mutations in cyclin-dependent kinases 4 and 6 (CDK4/6) are common in various cancers, including gastroesophageal malignancies. Although CDK4/6 inhibitors, such as abemaciclib and palbociclib, have been approved for breast cancer treatment, their effectiveness as a monotherapy remains limited for gastroesophageal tumors. The present study explored the underlying mechanism of abemaciclib resistance. Abemaciclib-resistant gastric cancer cell lines were generated, and the phospho-eukaryotic translation initiation factor 4E (p-eIF4E) and eIF4E expression was compared between resistant and parental cell lines. In order to analyze the role of eIF4E in cell resistance, siRNA knockdown was employed. The effectiveness of ribavirin alone and its combination with abemaciclib was evaluated in the gastric cancer xenograft mouse model. The upregulation of eIF4E was a common feature in gastric cancer cells exposed to prolonged abemaciclib treatment. Gastric cancer cells with increased eIF4E levels exhibited a better response to eIF4E inhibition, especially those that were resistant to abemaciclib. Ribavirin, which is an approved anti-viral drug, significantly improved the efficacy of abemaciclib, both in vitro and in vivo, by inhibiting eIF4E. Importantly, ribavirin effectively suppressed the abemaciclib-resistant gastric cancer growth in mice without causing toxicity. These findings suggest that targeting eIF4E can enhance the abemaciclib treatment for gastric cancer, proposing the potential combination therapy of CDK4/6 inhibitors with ribavirin for advanced gastric cancer.

Biological functions and research progress of eIF4E.

The eukaryotic translation initiation factor eIF4E can specifically bind to the cap structure of an mRNA 5' end, mainly regulating translation initiation and preferentially enhancing the translation of carcinogenesis related mRNAs. The expression of eIF4E is closely related to a variety of malignant tumors. In tumor cells, eIF4E activity is abnormally increased, which stimulates cell growth, metastasis and translation of related proteins. The main factors affecting eIF4E activity include intranuclear regulation, phosphorylation of 4EBPs, and phosphorylation and sumoylation of eIF4E. In this review, we summarize the biological functions and the research progress of eIF4E, the main influencing factors of eIF4E activity, and the recent progress of drugs targeting eIF4E, in the hope of providing new insights for the treatment of multiple malignancies and development of targeted drugs.

RIP3/MLKL regulates necroptosis via activating 4EBP1-eIF4E pathway.

Necroptosis is a cell death type mediated by receptor interacting protein 3 (RIP3)/mixed lineage kinase domain-like protein (MLKL). It has been reported that mammalian target of rapamycin plays a regulatory role in necroptosis. Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1)-eukaryotic initiation factor 4E (eIF4E) pathway is a key down streamer of mammalian target of rapamycin. However, whether 4EBP1-eIF4E pathway is involved in necroptosis is still unknown. This study aims to investigate the changes of 4EBP1-eIF4E pathway in necroptosis. TNF-α/SM-164/Z-VAD-FMK (TSZ), a necroptosis inducer, was used to induce necroptosis in murine fibroblastoid cell line L929. Cell necrosis was observed under an optical microscope. Then, TSZ was added to L929 cells with RIP3 and MLKL gene knockout. Propidium iodide (PI) staining was used to observe cell necrosis. Real-time fluorescence quantitative PCR and Western blotting were used to determine the mRNA and protein expression of 4EBP1 and eIF4E, respectively. After treating L929 cells with TSZ, the number of necrotic cells was increased, the mRNA and protein expression levels of 4EBP1 were significantly downregulated, and the ratio of phosphorylated 4EBP1 (p-4EBP1) to 4EBP1 was increased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly upregulated, and the ratio of phosphorylated eIF4E (p-eIF4E) to eIF4E was increased (both P<0.01). After knocking out RIP3 and MLKL in L929 cells, PI positive necrotic cells were significantly reduced, the mRNA and protein expression levels of 4EBP1 were significantly upregulated, and the ratio of p-4EBP1 to 4EBP1 was decreased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly downregulated, and the ratio of p-eIF4E to eIF4E was decreased (both P<0.01). 4EBP1-eIF4E pathway is activated in the RIP3/MLKL mediated-necroptosis.

Continuous dosing of AUM001 with cyclical gemcitabine/nab-paclitaxel to maintain the tumor growth kinetics in KPC xenograft model.

e15112 Background: PDAC is a highly desmoplastic and aggressive disease with limited therapeutic options and poor 5-year survival outcome. Gemcitabine (GEM) plus nab-paclitaxel (nPTX) is widely used as a first line treatment for PDAC but advancement for greater improvement on efficacy is still warranted. Dysregulated mRNA translation of pro-tumorigenic genes has contributed to the proliferative and metastatic nature of PDAC. Targeting oncogenic mRNA translation via MNK-coupled phosphorylation of eIF4E could serve as a target for inhibition to synergize chemotherapy. AUM001 is a novel MNK1/MNK2 inhibitor undergoing clinical trials in various solid tumors. We hypothesize that combination of AUM001 with GEM-nPTX would have synergistic effect in modulating the tumor growth kinetics and maintaining the tumor in a stable state. Methods: PDAC KPC cells were subcutaneously implanted into BL6 mice and randomized into five treatment groups once tumor volumes of 60-80mm3 were reached. Saline (control), GEM-nPTX were administered intraperitoneally on days 1, 4 and 8 while AUM001 was given orally on a daily basis for 8 days. The combination of GEM-nPTX and AUM001 were administered at similar doses and frequency. AUM001 was given for another 3 weeks in the continuous dosing group. Body weight and tumor volume were measured thrice weekly. Immunohistochemistry staining was performed on FFPE sections for Ki67, angiogenesis and stromal markers. Apoptosis was analyzed by Annexin V-propidium iodide flow cytometric assay. P &lt; 0.05 was considered as statistically significant. Results: GEM-nPTX and AUM001 (GPA) or GEM-nPTX (GP) alone resulted in approximately 30% reduction in relative tumor volume. The continuous dosing of AUM001 (GPAc) after the cessation of chemotherapy showed a marked 55% decline in relative tumor volume ( P= 0.0079) compared to GPA. The phosphorylation of EIF4E (target of MNK1/2) and Myc expression were significantly inhibited in GPAc vs GPA groups at end of observation (EOO) (P &lt; 0.05). VEGFA expression at EOO was found to be suppressed in GPAc compared to GPA group ( P= 0.0159) with no difference in MVD ( P&gt; 0.05). Greater fraction of apoptotic cells was detected in GPAc at EOO ( P= 0.055) than GPA ( P&gt; 0.999) when referenced to GP group. Conclusions: This study demonstrated that continuous dosing of AUM001 with cyclical GEM-nPTX is well-tolerated and confers good anti-tumor effect. Pharmacodynamic analysis also suggests that AUM001 may potentiate the reduction in pro-tumorigenic protein expression. This is the first study depicting the continuous dosing of AUM001 after cessation of chemotherapy could help to maintain the tumor state, facilitating its possible use in clinics as maintenance therapy in PDAC.

RIPK1-dependent necroptosis promotes vasculogenic mimicry formation via eIF4E in triple-negative breast cancer

Necroptosis is a caspase-independent form of programmed cell death. Receptor interacting protein kinase 1 (RIPK1) is a key molecule in the initiation of necroptosis and the formation of the necrotic complex. Vasculogenic mimicry (VM) provides a blood supply to tumor cells that is not dependent on endothelial cells. However, the relationship between necroptosis and VM in triple-negative breast cancer (TNBC) is not fully understood. In this study, we found that RIPK1-dependent necroptosis promoted VM formation in TNBC. Knockdown of RIPK1 significantly suppressed the number of necroptotic cells and VM formation. Moreover, RIPK1 activated the p-AKT/eIF4E signaling pathway during necroptosis in TNBC. eIF4E was blocked by knockdown of RIPK1 or AKT inhibitors. Furthermore, we found that eIF4E promoted VM formation by promoting epithelial-mesenchymal transition (EMT) and the expression and activity of MMP2. In addition to its critical role in necroptosis-mediated VM, eIF4E was essential for VM formation. Knockdown of eIF4E significantly suppressed VM formation during necroptosis. Finally, through clinical significance, the results found that eIF4E expression in TNBC was positively correlated with the mesenchymal marker vimentin, the VM marker MMP2, and the necroptosis markers MLKL and AKT. In conclusion, RIPK1-dependent necroptosis promotes VM formation in TNBC. Necroptosis promotes VM formation by activating RIPK1/p-AKT/eIF4E signaling in TNBC. eIF4E promotes EMT and MMP2 expression and activity, leading to VM formation. Our study provides a rationale for necroptosis-mediated VM and also providing a potential therapeutic target for TNBC.

Azacitidine Is Synergistically Lethal with XPO1 Inhibitor Selinexor in Acute Myeloid Leukemia by Targeting XPO1/eIF4E/c-MYC Signaling.

Acute myeloid leukemia (AML) is a high-mortality malignancy with poor outcomes. Azacitidine induces cell death and demonstrates treatment effectiveness against AML. Selinexor (KPT-330) exhibited significant benefits in combination with typical induction treatment for AML patients. Here, we explore the antitumor effect of KPT-330 combined with AZA in AML through CCK-8, flow cytometry, RT-qPCR, western blot, and RNA-seq. Our results showed that KPT-330 combined with AZA synergistically reduced cell proliferation and induced apoptosis in AML primary cells and cell lines. Compared to the control, the KPT-330 plus AZA down-regulates the expression of XPO1, eIF4E, and c-MYC in AML. Moreover, the knockdown of c-MYC could sensitize the synergy of the combination on suppression of cell proliferation and promotion of apoptosis in AML. Moreover, the expression of XPO1 and eIF4E was elevated in AML patient cohorts, respectively. XPO1 and elF4E overexpression was associated with poor prognosis. In summary, KPT-330 with AZA exerted synergistic effects by suppressing XPO1/eIF4E/c-MYC signaling, which provided preclinical evidence for further clinical application of the novel combination in AML.

Abstract 3722: Exploring the role of eIF4E in cancer cells with targeted protein degradation

Abstract 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.

Translation-dependent skin hyperplasia is promoted by type 1/17 inflammation in psoriasis

BackgroundPsoriasis vulgaris (PV) is a chronic skin inflammatory disease and characterized by aberrant epidermal hyperplasia. The molecule eukaryotic initiation factor (eIF) 4E controls translation initiation of certain protein synthesis and determines cell cycle or differentiation fate. ObjectiveTo determine the role of eIF4E in keratinocytes abnormal differentiation in the context of psoriasis. MethodsThe expression of eIF4E in psoriatic skin lesions and normal skin from human subjects was examined by western blot and immunohistochemistry. In a murine model of psoriasis-like dermatitis that is induced by topical imiquimod, 4EGI-1 was used to inhibit eIF4E activities. To measure murine skin eIF4E and keratinocytes differentiation, immunofluorescence and western blot assays were conducted. Normal human epidermal keratinocytes (NHEK) were isolated, cultured, and stimulated with cytokines including TNF-α, IFN-γ, and IL-17A, respectively. Immunofluorescence and western blot were performed to test eIF4E and effect of 4EGI-1 in a co-culture system. ResultsCompared with healthy controls, skin lesions from patients with PV exhibited a higher expression of eIF4E, which was positively correlated with the epidermal thickness. This expression pattern of eIF4E was replicated by the imiquimod-induced murine model. Skin hyperplasia and eIF4E activities in the murine model were attenuated by the administration of 4EGI-1. Both IFN-γ and IL-17A, rather than TNF-α, are sufficient to induce NHEK abnormal differentiation. This effect can be disrupted by 4EGI-1. ConclusioneIF4E plays a crucial role in keratinocytes abnormal differentiation driven by type 1/17 inflammation in the context of psoriasis. The initiation of abnormal translation provides an alternative treatment target for psoriasis.

Regulation of eIF4E guides a unique translational program to control erythroid maturation.

Translation control is essential in balancing hematopoietic precursors and differentiation; however, the mechanisms underlying this program are poorly understood. We found that the activity of the major cap-binding protein eIF4E is unexpectedly regulated in a dynamic manner throughout erythropoiesis that is uncoupled from global protein synthesis rates. Moreover, eIF4E activity directs erythroid maturation, and increased eIF4E expression maintains cells in an early erythroid state associated with a translation program driving the expression of PTPN6 and Igf2bp1. A cytosine-enriched motif in the 5' untranslated region is important for eIF4E-mediated translation specificity. Therefore, selective translation of key target genes necessary for the maintenance of early erythroid states by eIF4E highlights a unique mechanism used by hematopoietic precursors to rapidly elicit erythropoietic maturation upon need.

Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

Comprehensive multi-omics analysis of the m7G in pan-cancer from the perspective of predictive, preventive, and personalized medicine.

The N7-methylguanosine modification (m7G) of the 5' cap structure in the mRNA plays a crucial role in gene expression. However, the relation between m7G and tumor immune remains unclear. Hence, we intended to perform a pan-cancer analysis of m7G which can help explore the underlying mechanism and contribute to predictive, preventive, and personalized medicine (PPPM/ 3PM). The gene expression, genetic variation, clinical information, methylation, and digital pathological section from 33 cancer types were downloaded from the TCGA database. Immunohistochemistry (IHC) was used to validate the expression of the m7G regulator genes (m7RGs) hub-gene. The m7G score was calculated by single-sample gene-set enrichment analysis. The association of m7RGs with copy number variation, clinical features, immune-related genes, TMB, MSI, and tumor immune dysfunction and exclusion (TIDE) was comprehensively assessed. CellProfiler was used to extract pathological section characteristics. XGBoost and random forest were used to construct the m7G score prediction model. Single-cell transcriptome sequencing (scRNA-seq) was used to assess the activation state of the m7G in the tumor microenvironment. The m7RGs were highly expressed in tumors and most of the m7RGs are risk factors for prognosis. Moreover, the cellular pathway enrichment analysis suggested that m7G score was closely associated with invasion, cell cycle, DNA damage, and repair. In several cancers, m7G score was significantly negatively correlated with MSI and TMB and positively correlated with TIDE, suggesting an ICB marker potential. XGBoost-based pathomics model accurately predicts m7G scores with an area under the ROC curve (AUC) of 0.97. Analysis of scRNA-seq suggests that m7G differs significantly among cells of the tumor microenvironment. IHC confirmed high expression of EIF4E in breast cancer. The m7G prognostic model can accurately assess the prognosis of tumor patients with an AUC of 0.81, which was publicly hosted at https://pan-cancer-m7g.shinyapps.io/Panca-m7g/. The current study explored for the first time the m7G in pan-cancer and identified m7G as an innovative marker in predicting clinical outcomes and immunotherapeutic efficacy, with the potential for deeper integration with PPPM. Combining m7G within the framework of PPPM will provide a unique opportunity for clinical intelligence and new approaches. The online version contains supplementary material available at 10.1007/s13167-022-00305-1.

374 Eukaryotic translation initiation factor 4E (eIF4E) as a target of anti-psoriatic treatment

Eukaryotic translation initiation factor 4E (eIF4E) has been known to play a critical role in the regulation of gene expression in eukaryotes and to affect many essential cellular processes, including proliferation, apoptosis and differentiation. We and others found eIF4E and other eIF subunits upregulated in human psoriatic skin and thus hypothesized that they are involved in the pathophysiology of psoriasis and can serve as direct topical targets for anti-psoriatic treatment. We used specific eIF4E siRNA or briciclib an eIF4E inhibitor in HaCaT cells, a human 3D-psoriasis tissue model as well as the imiquimod and TGFß mouse model to downregulate the protein and mRNA expression of eIF4E itself and its two complex partners eIF4A and G, as well as other eIFs (e.g.