Inhibition Of eIF4E Research Articles

Abstract PO5-27-09: Pharmacological eIF4E inhibition suppresses anti-tumor activity in ER+ breast cancer

Abstract Elevated levels of eukaryotic initiation factor 4E (eIF4E) are found in a broad range of cancers, including breast, and have been associated with aggressive, drug resistant tumors. Furthermore, elevated eIF4E activity is sufficient to cause transformation in various cancers, while inhibition suppresses tumor growth. eIF4E, the main regulator and rate limiting factor of protein synthesis, is the downstream integrator of several important oncogenic signaling pathways (PI3K/AKT/mTOR, Ras/Raf/MEK, and Myc). Activation of eIF4E results in increased protein levels of key proliferation and metabolism proteins such as Cyclin D1 (CCND1) and Ornithine Decarboxylase 1 (ODC1), as well as an overall increase in cellular synthesis machinery, causing cellular growth and proliferation. Here, we describe the development and characterization of novel, potent, and selective eIF4E inhibitors. Compounds from this chemical series have nanomolar activity in multiple biochemical and biophysical m7G cap-competition assays as well as potent inhibition of translation in cellular and biochemical assays. In cells, these compounds rapidly and reversibly decrease protein levels of several oncogenes, including CCND1, leading to a G1 cell cycle arrest. We demonstrate that eIF4E inhibitors cause growth inhibition in a variety of breast cancer cell lines, including ER+ breast cancer. These inhibitors also inhibit growth of breast cancer lines with acquired resistance to Palbociclib with the same potency as the parental cell lines. Additionally, eIF4E inhibition shows an increased sensitivity in resistant cell lines when combined with standard of care (SOC), including Palbociclib. Select analogs from the series demonstrate favorable ADMET/PK properties with good oral bioavailability and low safety risk. Lastly, these compounds have demonstrated near complete tumor growth inhibition in non-breast cancer in vivo models. Ongoing experiments will address in vivo efficacy in ER+ breast cancer in both monotherapy and in combination with SOC. Citation Format: Matthew Friedersdorf, Devon Blake, Sarah Thompson, Krista Marran, Jessica Sorrentino. Pharmacological eIF4E inhibition suppresses anti-tumor activity in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-09.

Abstract 682: Development of novel eIF4E inhibitors to potently and selectively suppress tumor growth across multiple indications

Abstract Despite considerable progress in treating cancer, drug resistance remains the primary hurdle to achieving cures in patients. Tumors often develop resistance through reactivation of the drug-targeted pathway, or activation of alternative parallel pathways, thereby maintaining the original downstream signaling effects. One of the furthest downstream effectors and critical nodes for many oncogenic signaling pathways is eukaryotic translation initiation factor 4E (eIF4E); the main regulator and rate limiting factor for protein synthesis. eIF4E is reactivated by many resistance mechanisms to promote translation of multiple pro-oncogenic factors including Cyclin D1/3, making it an attractive target to potentiate the anti-cancer activity of targeted therapies and to overcome drug resistance. Here, we present the development of novel, potent, and selective eIF4E inhibitors for use in both treatment-naïve and resistance settings across multiple tumor indications. To capitalize on the potential of targeting eIF4E, we developed a series of compounds with unique properties that maintain anti-tumor efficacy while minimizing toxicity. Our novel, selective, and potent oral eIF4E inhibitors (RBX-eIF4Ei) elicit a reversible, dose-dependent cell cycle arrest. Unlike targeting eIF4A, eIF4E inhibition selectively regulates translation of cancer-dependent pathway proteins instead of global protein synthesis, thus reducing on-target toxicity and increasing tolerability. Cellular profiling demonstrates RBX-eIF4Ei are highly selective, nanomolar inhibitors across many tumor types including, NSCLC, CRC, breast, and melanoma. Additionally, RBX-eIF4Ei demonstrate consistent efficacy across both sensitive and resistant cell lines, including intrinsic and acquired resistance models. Combining RBX-eIF4Ei with standard of care targeted therapies produces additive responses in both settings, suggesting its potential anti-neoplastic benefits post progression. In vivo, daily oral monotherapy treatment with RBX-EIF4Ei causes significant tumor growth inhibition across a variety of indications including BRAFmut CRC, BRAFmut melanoma and ER+ breast cancer, with minimal signs of toxicity. Intratumoral concentration of RBX-eIF4Ei correlate with significant tumor cell growth inhibition, as well as reductions in eIF4E target proteins, ODC1 and Cyclin D1. Combination studies with standard of care in both the treatment naïve and resistant settings are ongoing. Collectively, these data support the addition of RBX-eIF4Ei to standard of care in both the naïve and treatment resistance settings across a variety of indications including NSCLC, breast, CRC, and melanoma. IND enabling studies are planned, marking a significant step toward advancing these promising eIF4E inhibitors into clinical development. Citation Format: Matthew B. Friedersdorf, Devon R. Blake, Sarah E. Thompson, Roheeth Pavana, Ramamurty V. Changalvala, Andrew S. Truong, Krista Marran, Abha Bias, Jessica A. Sorrentino. Development of novel eIF4E inhibitors to potently and selectively suppress tumor growth across multiple indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 682.

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.

Regulation of Eukaryotic Translation Initiation Factor 4E as a Potential Anticancer Strategy.

Eukaryotic translation initiation factors (eIFs) are highly expressed in cancer cells, especially eIF4E, the central regulatory node driving cancer cell growth and a potential target for anticancer drugs. eIF4E-targeting strategies primarily focus on inhibiting eIF4E synthesis, interfering with eIF4E/eIF4G interactions, and targeting eIF4E phosphorylation and peptide inhibitors. Although some small-molecule inhibitors are in clinical trials, no eIF4E inhibitors are available for clinical use. We provide an overview of the regulatory mechanisms of eIF4E and summarize the progress in developing and discovering eIF4E inhibitor strategies. We propose that interference with eIF4E/eIF4G interactions will provide a new perspective for the design of eIF4E inhibitors and may be a preferred strategy.

Dual targeting of protein translation and nuclear protein export results in enhanced antimyeloma effects.

Selinexor (KPT-330) is a small molecule inhibitor of XPO1, which mediates the transport of tumor suppressor proteins, oncogene messenger RNAs, and other proteins involved in governing cell growthfrom the cell nucleus to the cytoplasm. It is overexpressed in many cancer types. Because eukaryotic translation initiator factor 4E (eIF4E) plays a critical role in protein translation in cancer cells in multiple myeloma (MM), we evaluated the effectiveness of combined inhibition of protein translation and nuclear export in MM. Selinexor, an inhibitor of nuclear protein export, dose-dependently decreased eIF4E, IKZF1, and c-MYC protein levels. Using a doxycycline-inducible-pLKO-Tet-On vector, knockdown of eIF4E significantly enhanced the antiproliferative effects of selinexor, sensitized resistant MM cells to selinexor, and increased apoptosis in MM cells. Immunofluorescent analysis of MM cells showed that the combined treatment increased the localization of residual eIF4E to the nucleus compared with selinexor-only treatment. The overexpression of eIF4E at least partially rescued the effects of selinexor in MM cells by reducing G1 cell cycle arrest and increasing the selinexor-IC50 10-fold. Moreover, the combination of selinexor with pharmacologic inhibitors of protein translation showed synergistic anti-MM effects. These results suggest a synergistic anti-MM effect of selinexor combined with eIF4E inhibitors invitro. Our work provides a better understanding of the potential mechanism of resistance to selinexor and a rationale for combining selinexor with eIF4E inhibitors for the treatment of MM.

Identification of a First-in-Class Small-Molecule Inhibitor of the EIF4E-RBM38 Complex That Enhances Wild-type TP53 Protein Translation for Tumor Growth Suppression.

EIF4E, an mRNA cap-binding protein, is necessary for cap-dependent translation. Overexpression of EIF4E is known to promote cancer development by preferentially translating a group of oncogenic mRNAs. Thus, 4EGI-1, a disruptor of EIF4E-EIF4G1 interaction, was developed to inhibit oncoprotein expression for cancer therapy. Interestingly, RBM38, an RNA-binding protein, interacts with EIF4E on TP53 mRNA, prevents EIF4E from binding to TP53 mRNA cap, and inhibits TP53 expression. Thus, Pep8, an eight amino acid peptide derived from RBM38, was developed to disrupt the EIF4E-RBM38 complex, leading to increased TP53 expression and decreased tumor cell growth. Herein, we have developed a first-in-class small-molecule compound 094, which interacts with EIF4E via the same pocket as does Pep8, dissociates RBM38 from EIF4E, and enhances TP53 translation in RBM38- and EIF4E-dependent manners. Structure-activity relationship studies identified that both the fluorobenzene and ethyl benzamide are necessary for compound 094 to interact with EIF4E. Furthermore, we showed that compound 094 is capable of suppressing three-dimensional tumor spheroid growth in RBM38- and TP53-dependent manners. In addition, we found that compound 094 cooperates with the chemotherapeutic agent doxorubicin and EIF4E inhibitor 4EGI-1 to suppress tumor cell growth. Collectively, we showed that two distinct approaches can be used together to target EIF4E for cancer therapy by enhancing wild-type TP53 expression (094) and by suppressing oncoprotein expression (4EGI-1).

Oligomeric β-Amyloid Suppresses Hippocampal γ-Oscillations through Activation of the mTOR/S6K1 Pathway.

Neuronal synchronization at gamma frequency (30-100 Hz: γ) is impaired in early-stage Alzheimer's disease (AD) patients and AD models. Oligomeric Aβ1-42 caused a concentration-dependent reduction of γ-oscillation strength and regularity while increasing its frequency. The mTOR1 inhibitor rapamycin prevented the Aβ1-42-induced suppression of γ-oscillations, whereas the mTOR activator leucine mimicked the Aβ1-42-induced suppression. Activation of the downstream kinase S6K1, but not inhibition of eIF4E, was required for the Aβ1-42-induced suppression. The involvement of the mTOR/S6K1 signaling in the Aβ1-42-induced suppression was confirmed in Aβ-overexpressing APP/PS1 mice, where inhibiting mTOR or S6K1 restored degraded γ-oscillations. To assess the network changes that may underlie the mTOR/S6K1 mediated γ-oscillation impairment in AD, we tested the effect of Aβ1-42 on IPSCs and EPSCs recorded in pyramidal neurons. Aβ1-42 reduced EPSC amplitude and frequency and IPSC frequency, which could be prevented by inhibiting mTOR or S6K1. These experiments indicate that in early AD, oligomer Aβ1-42 impairs γ-oscillations by reducing inhibitory interneuron activity by activating the mTOR/S6K1 signaling pathway, which may contribute to early cognitive decline and provides new therapeutic targets.

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.

Metformin suppresses progression of muscle aging via activation of the AMP kinase-mediated pathways in Drosophila adults.

Metformin, a medicine used for the treatment of type 2 diabetes, was previously reported to suppress age-dependent hyperproliferation of intestinal stem cells in Drosophila. Here, we aimed to investigate its anti-aging effects on other tissues, such as adult muscle and elucidate the mechanisms underlying the anti-ageing effect. To evaluate the anti-muscle ageing effect of Metformin, we visualized ubiquitinated protein aggregates accumulated in adult muscle as the flies age by immunostaining and measured the total pixel size of the aggregates. We altered gene expression in the muscle by induction of dsRNA against the relevant mRNAs or mRNAs encoding the constitutively active mutant proteins using the Gal4/UAS system. We determined the mRNA levels by quantitative Real Time-Polymerase Chain Reaction (QRT-PCR). Continuous metformin feeding significantly extended the lifespan of Drosophila adults. Furthermore, the feeding suppressed the aging-dependent accumulation of ubiquitinated aggregates in adult muscle. To delineate the mechanism through which metformin influences the muscle aging phenotype, we induced the constitutively active AMPK specifically in the muscles and found that the activation of the AMPK-mediated pathway was sufficient for the anti-aging effect of Metformin. Furthermore, the AMPK-mediated downregulation of Tor-mediated pathways, subsequent induction of an eIF-4E inhibitor were involved in the effect. These genetic data suggested that the metformin effect is related to the partial suppression of protein synthesis in ribosomes. Furthermore, metformin stimulated autophagy induction in adult muscles. Our results suggest that metformin can be regarded as an anti-aging compound in Drosophila muscle. The stimulation of autophagy was also involved in the anti-aging effect, which delayed the progression of muscle aging in Drosophila adults.

MiR-34b-3p Inhibition of eIF4E Causes Post-stroke Depression in Adult Mice.

Post-stroke depression (PSD) is a serious and common complication of stroke, which seriously affects the rehabilitation of stroke patients. To date, the pathogenesis of PSD is unclear and effective treatments remain unavailable. Here, we established a mouse model of PSD through photothrombosis-induced focal ischemia. By using a combination of brain imaging, transcriptome sequencing, and bioinformatics analysis, we found that the hippocampus of PSD mice had a significantly lower metabolic level than other brain regions. RNA sequencing revealed a significant reduction of miR34b-3p, which was expressed in hippocampal neurons and inhibited the translation of eukaryotic translation initiation factor 4E (eIF4E). Furthermore, silencing eIF4E inactivated microglia, inhibited neuroinflammation, and abolished the depression-like behaviors in PSD mice. Together, our data demonstrated that insufficient miR34b-3p after stroke cannot inhibit eIF4E translation, which causes PSD by the activation of microglia in the hippocampus. Therefore, miR34b-3p and eIF4E may serve as potential therapeutic targets for the treatment of PSD.

EGPI-1, a novel eIF4E/eIF4G interaction inhibitor, inhibits lung cancer cell growth and angiogenesis through Ras/MNK/ERK/eIF4E signaling pathway

eIF4E plays an important role in regulating tumor growth and angiogenesis, and eIF4E is highly expressed in a variety of lung cancer cell lines. siRNA eIF4E can significantly inhibit the proliferation of lung cancer cells, indicating that inhibition of eIF4E may become a novel anti-tumor target. In the previous study, we synthesized a series of small molecule compounds with the potential to inhibit eIF4E. Among them, the compound EGPI-1 significantly inhibited the proliferation of a variety of lung cancer cells such as A549, NCI-H460, NCI-H1650 and 95D without inhibiting the proliferation of HUVEC cells. Further studies found that EGPI-1 interfered with the eIF4E/eIF4G interaction and inhibited the phosphorylation of eIF4E in NCI-H460 cells. The results of flow cytometry showed that EGPI-1 induced apoptosis and G0/G1 cycle arrest in NCI-H460 cell. Interestingly, we also found that EGPI-1 induced autophagy and DNA damage in NCI-H460 cells. The mechanism results showed that EGPI-1 inhibited the Ras/MNK/ERK/eIF4E signaling pathway. Moreover, EGPI-1 inhibited tube formation of HUVECs, as well as inhibited the neovascularization of CAM, proving the anti-angiogenesis activity of EGPI-1. The NCI-H460 xenograft studies showed that EGPI-1 inhibited tumor growth and angiogenesis in vivo by regulating Ras/MNK/ERK/eIF4E pathway. Our studies proved that eIF4E was a novel target for regulating tumor growth, and the eIF4E/eIF4G interaction inhibitor EGPI-1 was promising to develop into a novel anti-lung cancer drug.

Targeting of MNK/eIF4E overcomes chemoresistance in cervical cancer.

Eukaryotic translation initiation factor 4E (eIF4E) is activated in cancers in response to stress. This is regulated by MAP kinase interacting serine/threonine kinase (MNK) in cancerous but not normal cells. Chemoresistance causes treatment failure in advanced cervical cancer. In this study, we addressed chemotherapy effects on eIF4E for cervical cancer and reversal effects by MNK inhibitor cercosporamide for chemo-resistance mitigation. Cell assays and mouse tumour models were used to determine the efficacy of cercosporamide. Western blotting was applied to understand the affected cell signaling after cercosporamide treatment. Cercosporamide spared normal cervical epithelial cells. On cervical cancer cell lines, it showed inhibition of cell growth and migration, and induced apoptosis. Cercosporamide was effective on chemoresistant cancer cells and augmented the efficiency of doxorubicin and cisplatin both in vitro and in vivo. Cercosporamide suppressed eIF4E signaling. Of note, chemotherapy increased p-eIF4E. Cercosporamide abolished chemotherapy-induced eIF4E activation. The higher level of p-eIF4E in cancer cells compared with normal cervical epithelial cells explains the preferential toxicity of cercosporamide. This work demonstrates the ability of cercosporamide to overcome chemoresistance and highlight preferential inhibition of eIF4E via MNK inhibition in cervical cancer.

Reduced eIF4E function impairs B-cell leukemia without altering normal B-lymphocyte function

SummaryThe cap-binding protein eukaryotic initiation factor 4E (eIF4E) promotes translation of mRNAs associated with proliferation and survival and is an attractive target for cancer therapeutics. Here, we used Eif4e germline and conditional knockout models to assess the impact of reduced Eif4e gene dosage on B-cell leukemogenesis compared to effects on normal pre-B and mature B-cell function. Using a BCR-ABL-driven pre-B-cell leukemia model, we find that loss of one allele of Eif4e impairs transformation and reduces fitness in competition assays in vitro and in vivo. In contrast, reduced Eif4e gene dosage had no significant effect on development of pre-B and mature B cells or on survival or proliferation of non-transformed B lineage cells. These results demonstrate that inhibition of eIF4E could be a new therapeutic tool for pre-B-cell leukemia while preserving development and function of normal B cells.

A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders.

The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.

Abstract B33: Targeting PI3K/mTOR signaling with potent, selective and orally-available small-molecule inhibitors of eIF4E

Abstract Aberrant protein translation plays a role in the pathogenesis of multiple solid tumors and hematologic malignancies. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for several signaling pathways, such as PI3K/mTOR and MAPK, which are intimately involved in tumor cell growth and survival. As such, eIF4E has generated intense interest as a target for anticancer drug discovery. We have designed a series of potent, selective, and orally available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind free eIF4E, eIF4E-4EBP and eIF4E-eIF4F complexes within tumor cells. Ribosomal profiling of eIF4E inhibitor-treated tumor cells has identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also include a larger set of unique translationally regulated target mRNAs that are enriched for 5'-TOP, PRTE and CERT sequence elements in their 5'-untranslated regions. eIF4E inhibition results in potent antiproliferative activity and induction of apoptosis in a subset of tumor cell lines. Consistent with this observation, our eIF4E inhibitors show some similarities, yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiologic assays. Finally, significant antitumor efficacy was observed with eIF4E inhibition in both solid tumor and hematologic xenografts in vivo. Taken together, these results highlight the potential of targeting eIF4E as a novel and differentiated therapeutic strategy to treat cancer. Citation Format: Gregory S. Parker, Ivy N.J. Hung, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urklalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Gary G. Chiang, Kevin R. Webster. Targeting PI3K/mTOR signaling with potent, selective and orally-available small-molecule inhibitors of eIF4E [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B33.

5′-Cap‒Dependent Translation as a Potent Therapeutic Target for Lethal Human Squamous Cell Carcinoma

Skin squamous cell carcinomas (SCCs) are a major cause of death in patients who have undergone or will undergo organ transplantation. Moreover, these neoplasms cause significant disease and economic burden and diminish patients' life quality. However, no effective treatment or intervention strategies are available. In this study, we investigated the pathologic role of 5'-cap translation, which is regulated by the formation of a ternary initiation factor complex involving eIF4E, eIF4G, and eIF4A1. We detected increased expression of phosphorylated eIF4E, eIF4G, and eIF4A1 in human and murine skin SCCs. The increase in these ternary initiation factor complex proteins was associated with enhanced eIF4E translation targets cyclin D1 and c-Myc. Conversely, small interfering RNA-mediated depletion of eIF4E in human SCC cells (A431 and SCC-13) reduced eIF4G and proteins that regulate the cell cycle and proliferation. Notably, inhibition of Raf/MAPK/extracellular signal-regulated kinase signaling decreased eIF4E and phosphorylated eIF4E accumulation and significantly diminished cell-cycle gene expression and tumor volume of A431-derived xenograft tumors. Furthermore, disrupting the eIF4E with an allosteric inhibitor of eIF4E and eIF4G binding, 4EGI-1, decreased the eIF4E/eIF4G expression and reduced the proliferation. Finally, combined inhibition of the Raf/MAPK/extracellular signal-regulated kinase axis and eIF4E impaired 5'-cap‒dependent translation and abrogated tumor cell proliferation. These data demonstrate that 5'-cap‒dependent translation is a potential therapeutic target for abrogating lethal skin SCCs in patients who have undergone or will undergo organ transplantation.

Salubrinal in Combination With 4E1RCat Synergistically Impairs Melanoma Development by Disrupting the Protein Synthetic Machinery.

Increased protein synthesis is a key process in melanoma, which is regulated by the ALDH18A1 gene encoding pyrroline-5-carboxylate synthase (P5CS). P5CS is involved in proline biosynthesis and targeting ALDH18A1 has previously been shown to inhibit melanoma development by decreasing intracellular proline levels to increase the phosphorylation of eIF2α mediated by GCN2, which then impairs mRNA translation. Since there are no current inhibitors of P5CS, decreased eIF2α phosphorylation in melanoma was targeted using salubrinal (a specific inhibitor of eIF2α phosphatase enzymes). While salubrinal alone was ineffective, the combined use of salubrinal and 4E1RCat (a dual inhibitor of eIF4E:4E-BP1 and eIF4E:eIF4G interaction to prevent assembly of the eIF4F complex and inhibit cap-dependent translation) was found to be effective at decreasing protein synthesis, protein translation, and cell cycle progression to synergistically decrease melanoma cell viability and inhibited xenograft melanoma tumor development. The combination of these agents synergistically decreased melanoma cell viability while having minimal effect on normal cells. This is the first report demonstrating that it is possible to inhibit melanoma viability by targeting eIF2α signaling using salubrinal and 4E1RCat to disrupt assembly of the eIF4F complex.

Discovery of Lysine-Targeted eIF4E Inhibitors through Covalent Docking.

Eukaryotic translation initiation factor 4E (eIF4E) binds the m7GTP cap structure at the 5'-end of mRNAs, stimulating the translation of proteins implicated in cancer cell growth and metastasis. eIF4E is a notoriously challenging target, and most of the reported inhibitors are negatively charged guanine analogues with negligible cell permeability. To overcome these challenges, we envisioned a covalent targeting strategy. As there are no cysteines near the eIF4E cap binding site, we developed a covalent docking approach focused on lysine. Taking advantage of a "make-on-demand" virtual library, we used covalent docking to identify arylsulfonyl fluorides that target a noncatalytic lysine (Lys162) in eIF4E. Guided by cocrystal structures, we elaborated arylsulfonyl fluoride 2 to 12, which to our knowledge is the first covalent eIF4E inhibitor with cellular activity. In addition to providing a new tool for acutely inactivating eIF4E in cells, our computational approach may offer a general strategy for developing selective lysine-targeted covalent ligands.

Effect of eIF4E on Autophagy of CD138+ Cells in Multiple Myeloma

To investigate the effect of eukaryotic translation initiation factor 4E(eIF4E) on the autophagy of CD138+ plasma cells in multiple myeloma(MM). Multiple myeloma CD138+ plasma cells were treated with eIF4E inhibitor 4EGI, the changes of autophagy-related factors LC3-II and Beclin1 were detected by fluorescent quantitative PCR and Western blot, the changes of cell proliferation inhibition were detected by MTT assay, and cell apoptosis was detected by flow cytometry. Quantitative fluorescence PCR showed that after treatment of myeloma cells with 4EGI, the expression levels of LC3-II and Beclin1 mRNA gradually increased with the enhancomer of 4EGI concentration and the prolongation of action time, and the differences were statistically significant (48 h: LC3-Ⅱ,r=0.942, Beclin1,r=0.952; 80 μg/ml: LC3-Ⅱ,r=0.966, Beclin1,r=0.998）; Western blot showed that with the enhancement of 4EGI concentration, the expression of LC3-II and Beclin1 protein gradually increased（LC3-Ⅱ,r=0.923, Beclin1,r=0.977）; CCK-8 showed that the inhibition rate of cells gradually increased （r=0.996）; the apoptotic rate of 4EGI-treated groups (23.23±4.47, 7.59±1.67, 2.03±0.19) was significantly different from that of control group (0.03±0.04) (P<0.05). The inhibition of eIF4E can activate the autophagy of CD138+ plasma cells in multiple myeloma and induce the death of myeloma cells.

Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy.

Brain somatic mutations confer genomic diversity in the human brain and cause neurodevelopmental disorders. Recently, brain somatic activating mutations in MTOR have been identified as a major etiology of intractable epilepsy in patients with cortical malformations. However, the molecular genetic mechanism of how brain somatic mutations in MTOR cause intractable epilepsy has remained elusive. In this study, translational profiling of intractable epilepsy mouse models with brain somatic mutations and genome-edited cells revealed a novel translational dysregulation mechanism and mTOR activation-sensitive targets mediated by human MTOR mutations that lead to intractable epilepsy with cortical malformation. These mTOR targets were found to be regulated by novel mTOR-responsive 5'-UTR motifs, distinct from known mTOR inhibition-sensitive targets regulated by 5' terminal oligopyrimidine motifs. Novel mTOR target genes were validated in patient brain tissues, and the mTOR downstream effector eIF4E was identified as a new therapeutic target in intractable epilepsy via pharmacological or genetic inhibition. We show that metformin, an FDA-approved eIF4E inhibitor, suppresses intractable epilepsy. Altogether, the present study describes translational dysregulation resulting from brain somatic mutations in MTOR, as well as the pathogenesis and potential therapeutic targets of intractable epilepsy.