Inhibition Of eIF4E Research Articles

Abstract 1302: Targeting hormone receptor-dependent cancers with potent, selective and orally-available small molecule inhibitors of eIF4E

Abstract The PI3K/mTOR pathway is commonly dysregulated in many hormone receptor-dependent tumors and plays a key role in promoting tumor growth and mediating drug resistance. In particular, PI3K and mTORC1/2 inhibitors have been intensively studied in the treatment of hormone receptor-dependent cancers and have shown benefit in some clinical settings. However, issues such as dose-limiting toxicities and emergent resistance limit the broader utility of these inhibitors. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for both the PI3K/mTOR and MAPK signaling pathways. 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 to eIF4E either as its free form or with eIF4E-4EBP and eIF4F complexes within tumor cells and downregulate hormone receptor-dependent signaling. Ribosomal profiling of eIF4E inhibitor-treated tumor cells identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also a unique set of translationally regulated target mRNAs. 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 physiological assays. Finally, significant anti-tumor efficacy was observed with eIF4E inhibition in vitro and in vivo. Taken together, these results highlight the potential for targeting eIF4E as a novel therapeutic strategy to treat hormone-receptor dependent cancers. Citation Format: Gary G. Chiang, Gregory S. Parker, Ivy N. Hung, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Cody Parker, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urkalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Kevin R. Webster. Targeting hormone receptor-dependent cancers with potent, selective and orally-available small molecule inhibitors of eIF4E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1302.

Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

BackgroundThe most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery. A broad range of translation initiation factors are also commonly found to be either amplified or mis-regulated in tumours, including eIF4E (elongation initiation factor 4E). eIF4E is a subunit of the eIF4F protein initiation complex and required for its recruitment. Here we measure the formation of the eIF4F complex through interactions of eIF4E and eIF4G subunits, and the effect of oncogenic signalling pathways on complex formation.ResultsWe developed a protein fragment complementation (PCA) assay that can accurately measure the status of the eIF4E-eIF4G interaction in cells and quantify the signalling flux through the RAS/ERK and PI3K/AKT pathways regulating eIF4F assembly. Complex disruption induced by inhibition of either pathway was shown to be a function of the phosphorylation status of 4EBP1, a key mediator of eIF4F assembly that interacts directly with eIF4E, confirming 4EBP1’s ability to integrate multiple signals affecting cap-dependent translation. Maximal measured disruption of the eIF4F complex occurred under combined mTORC1 and mTORC2 inhibition, whilst combined inhibition of both RAS/ERK and PI3K/AKT pathways in parallel resulted in greater inhibition of eIF4F formation than individually. v-Myc-mediated resistance to dual mTORC/PI3K inhibition was also principally demonstrated to depend on the lack of competent 4EBP1 available in the cell to bind eIF4E.ConclusionsWe show that 4EBP1 is a critical regulator of the mitogen responsive RAS/ERK and PI3K/AKT pathways and a key transducer of resistance mechanisms that affect small molecule inhibition of these pathways, principally by attenuating their effects on cap-dependent translation. These findings highlight the importance of highly efficacious direct inhibitors of eIF4E and eIF4F assembly, which could potentially target a wide spectrum of tumours containing differing mutations that effect these pathways and which confer chemo-resistance.

Genetic and pharmacological inhibition of eIF4E effectively targets esophageal cancer cells and augments 5-FU's efficacy.

Aberrant activation of eIF4E is critically involved in the progression and chemoresistance of various cancers. Elevated expression of eIF4E has also been documented in human cancerous esophageal tissues. However, the role of eIF4E in esophageal cancer is unclear. We analysed the levels of eIF4E expression and eIF4E function in a number of normal and cancerous esophageal cancer cell lines, and studied its underlying mechanism. We observed that eIF4E expression varies in different esophageal cancer cell lines but was significantly elevated in all tested esophageal cell lines as compared to the control cell lines. We demonstrated that eIF4E inhibition via genetic and pharmacological approaches inhibits cancer cell growth and survival. This inhibition also augments 5-flurouracil's (5-FU's) efficacy as demonstrated with both the in vitro esophageal cancer culture system and our in vivo xenograft mouse model. Of note, the sensitivity of esophageal cancer cells to ribavirin or eIF4E knockdown correlates well with the expression levels of eIF4E, demonstrating that esophageal cells with higher eIF4E expression are more sensitive to eIF4E inhibition. We further confirmed that the mechanism of action of ribavirin on esophageal cancer cells was through suppressing the Akt/mTOR/eIF4E and eIF4E-regulated pathways. To our knowledge, our work is the first to demonstrate the multiple roles of eIF4E in esophageal cancer. eIF4E was shown to promote cancer cell growth and survival, and protected the cells from chemotherapy. Our work also demonstrated that ribavirin is an attractive candidate for the treatment of esophageal cancer.

Inhibition of eukaryotic translation initiation factor 4E is effective against chemo-resistance in colon and cervical cancer

Although cancer patients initially respond well to chemotherapy, they eventually develop resistance and relapse. In this work, we demonstrate that eIF4E-targeting therapy is a potential sensitizing strategy for overcoming chemoresistance and progression in cancer. We show that ribavirin, an anti-viral drug and pharmacological eIF4E inhibitor, effectively inhibits proliferation and decreases viability of paclitaxel-resistant cervical cancer and 5-FU-resistant colon cancer cells while is less toxic to human fibroblast cells. Importantly, oral administration of ribavirin significantly inhibits paclitaxel-resistant colon and 5-FU-resistant cervical cancer growth in xenograft mouse cancer model without causing significant toxicity in mice. Consistently, combination of ribavirin with paclitaxel or 5-FU sensitizes colon and cervical cancer cells to chemotherapeutic agents treatment in vitro and in vivo. We further confirm that the mechanism of the action of ribavirin in chemoresistant cancer cells is through suppressing eIF4E function. In addition, specific eIF4E knockdown via two independent siRNA mimics the effects of ribavirin in chemoresistant colon and cervical cancer cells. Cell cycle analysis indicate that ribavirin enhances the anti-proliferative effect of 5-FU by additionally arresting cells at G2/M phase via increasing cyclin B1, p-histone H3(Ser10) and Mad2 levels. Our work demonstrates that eIF4E inhibition using inhibitor or siRNA, either as single agent or in combination, could sensitize chemoresistant cancer cells to paclitaxel or 5-FU treatment and thereby improving the efficacy of chemodrug. Our findings demonstrate the therapeutic value of inhibiting eIF4E, particularly in chemoresistant cancers. Our findings also suggest ribavirin as a promising sensitizing drug for cancer treatment.

Synergistic effects of inhibiting the MNK-eIF4E and PI3K/AKT/ mTOR pathways on cell migration in MDA-MB-231 cells.

The study of eukaryotic initiation factor 4E (eIF4E) is a key focus in cancer research due to its role in controlling the translation of tumour-associated proteins, that drive an aggressive migratory phenotype. eIF4E is a limiting component of the eIF4F complex which is a critical determinant for the translation of mRNAs. Mitogen-activated protein kinase interacting protein kinases (MNK1/2) phosphorylate eIF4E on Ser209, promoting the expression of oncogenic proteins, whereas mTORC1 phosphorylates and de-activates the eIF4E inhibitor, 4E-BP1, to release translational repression. Here we show that inhibiting these pathways simultaneously effectively slows the rate of cell migration in breast cancer cells. However, a molecular hybridisation approach using novel, cleavable dual MNK1/2 and PI3K/mTOR inhibiting hybrid agents was less effective at slowing cell migration.

Two distinct nodes of translational inhibition in the Integrated Stress Response.

The Integrated Stress Response (ISR) refers to a signaling pathway initiated by stress-activated eIF2α kinases. Once activated, the pathway causes attenuation of global mRNA translation while also paradoxically inducing stress response gene expression. A detailed analysis of this pathway has helped us better understand how stressed cells coordinate gene expression at translational and transcriptional levels. The translational attenuation associated with this pathway has been largely attributed to the phosphorylation of the translational initiation factor eIF2α. However, independent studies are now pointing to a second translational regulation step involving a downstream ISR target, 4E-BP, in the inhibition of eIF4E and specifically cap-dependent translation. The activation of 4E-BP is consistent with previous reports implicating the roles of 4E-BP resistant, Internal Ribosome Entry Site (IRES) dependent translation in ISR active cells. In this review, we provide an overview of the translation inhibition mechanisms engaged by the ISR and how they impact the translation of stress response genes.

Inhibition of eIF4E cooperates with chemotherapy and immunotherapy in renal cell carcinoma

Although overexpression of the eukaryotic translation initiation factor 4E (eIF4E) is detected in patients with renal cell carcinoma (RCC) and associated with poor prognosis, the possible roles of eIF4E in RCC have not been revealed. The effects of eIF4E inhibition on cell growth, migration, survival, chemo-/immunotherapy and eIF4E pathways via pharmacological inhibitor and genetic siRNA knockdown were analyzed in RCC cells. In this work, we demonstrate that eIF4E is critically involved in multiple biological functions of RCC. We firstly inhibited eIF4E activity by ribavirin in two cell lines (Caki-1 and ACHN) representing RCC metastasis models. We demonstrated that ribavirin inhibited proliferation and migration and induced apoptosis in RCC in a dose-dependent manner. We further confirmed that the inhibitory effects of ribavirin were attributed to its ability in inhibiting eIF4E-regulated protein translation and activity. eIF4E inhibition using siRNA knockdown mimicked ribavirin's effector in RCC cells. Importantly, eIF4E inhibition by both ribavirin and siRNA knockdown significantly sensitized RCC response to chemo- and immunotherapeutic agents in vitro as well as in vivo. Our findings clearly demonstrate the roles of eIF4E in RCC growth, survival, metastasis and resistance. Ribavirin is an antiviral drug, and its clinical efficacy is currently being investigated in the treatment of various cancers. Our findings support and provide a preclinical evidence for clinical trial for the combination of ribavirin with chemo-/immunotherapy in RCC.

Abstract 4898: eIF4E reshapes the surface of migrating AML cells through regulating Hyaluronic Acid synthesis & CD44 expression

Abstract Formation of cell surface protrusions has long been associated with tumor cell migration, metastasis & bone marrow homing & retention. It was shown that increased expression of endogenous or exogenous hyaluronic acid synthesizing enzyme 3 (HAS3) induces growth of microvillus-like cell surface protrusions. Some of the biological signals triggered by HA are dependent on its recognition by the cell surface receptor CD44. In our studies we observe that overexpression of the eukaryotic translation initiation factor 4E (eIF4E) increases formation of cell surface protrusions resembling those produced by HAS3 upregulation. eIF4E regulates mRNA export & translation through binding to the m7G cap of mRNA in the nucleus & the cytoplasm, respectively. Strikingly, analysis of our two complimentary screens (immunoprecipitation of mRNAs bound to eIF4E in the nucleus, and mRNA export assay with ribavirin, an established inhibitor of eIF4E) that we carried out to identify candidate eIF4E mRNA export targets, showed that nearly all the enzymes involved in HA synthesis as well as CD44. Here, we hypothesize that eIF4E regulates HA synthesis & CD44 overexpression to reshape the surface of cancer cells producing HA-rich microvillus-like pseudopods. Following validation of the positive hits, we used immunofluorescence staining with biotinylated HA binding protein (HABP) to determine the effect of eIF4E expression on protrusion formation. Our data revealed that eIF4E overexpression increased formation of HA-rich cell surface protrusions & was correlated with increased invasiveness of eIF4E overexpressing cells in in vitro matrigel assay, compared to respective controls. Colocalization studies using confocal microscopy indicated that protrusions positively stained for CD44. Further, pharmacological inhibition with ribavirin and genetic knockdown of eIF4E (or HAS3/CD44 in the context of eIF4E overexpression) abrogated protrusion formation and decreased the invasion capacity of eIF4E overexpressing cells. Together, these data indicate that eIF4E modulates protrusion formation & tumor invasion through coordinately regulating the export of RNAs in the CD44-HA regulon. To establish the clinical relevance of our findings in AML, we examined primary AML specimens with high eIF4E levels for expression of HA synthesizing enzymes, CD44 & we directly measured HA using confocal methods. This comparison showed that these pathways are dysregulated in AML patients & that their inhibition correlates with response to eIF4E inhibition by ribavirin. While HA has long been considered as a component of the tumor microenvironment required for the migration of cancer cells though interaction with surface CD44, our results provide a novel role for eIF4E in the motility & bone marrow homing. These findings offer a rationale for potentially inhibiting tumor cell metastasis through the combinatorial inhibition of eIF4E and CD44 or HAS3. Citation Format: Hiba Zahreddine, Biljana Kraljacic-Culjkovic, Valbona Cali, Mark Lauer, Lucy Skrabanek, Nabilah Khan, Ronald Midura, Leandro Cerchietti, Vincent Hascall, Craig Jordan. eIF4E reshapes the surface of migrating AML cells through regulating Hyaluronic Acid synthesis & CD44 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4898. doi:10.1158/1538-7445.AM2017-4898

Antiviral Drug Ribavirin Targets Thyroid Cancer Cells by Inhibiting the eIF4E-β-Catenin Axis

BackgroundAlthough eukaryotic translation initiation factor 4E (eIF4E) is important in cancer development and progression, its role in thyroid cancer is not well understood. Ribavirin, an anti-viral drug, has been identified as an eIF4E inhibitor. Herein, we investigated the effects of ribavirin on thyroid cancer and its molecular mechanisms of action. Materials and MethodsThe effects of ribavirin on thyroid cancer was investigated using in vitro cellular assays and in vivo xenograft mouse model. The mechanism of its action on eIF4E-β-catenin axis was examined using genetic and biochemical approaches. ResultsWe show that ribavirin inhibited proliferation and induced apoptosis in the thyroid cancer cell lines 8505C and FTC-133. Ribavirin inhibited thyroid cancer growth in a xenograft mouse model. Ribavirin also sensitized thyroid cancer’s response to paclitaxel. Mechanistically, ribavirin suppressed eIF4E phosphorylation and overexpression of its wildtype and phosphor-mimetic form (S209D) but not of the non-phosphorylatable form (S209A), which rescued the inhibitory effects of ribavirin in thyroid cancer cells. We further demonstrated that ribavirin suppressed phosphorylation and activities of β-catenin and its subsequent gene transcriptional expression. β-Catenin overexpression rescued the effects of ribavirin in thyroid cancer cells. Importantly, we show that eIF4E regulated β-catenin and that the regulation depended on phosphorylation at S209. The in vivo inhibitory effects of ribavirin on phosphorylation of eIF4E and β-catenin were also observed in thyroid tumor. ConclusionsOur data clearly demonstrate that ribavirin acts on thyroid cancer cells by inhibiting eIF4E/β-catenin signaling. Our findings suggest that ribavirin has the potential to be repurposed for thyroid cancer treatment and also highlight the therapeutic value of inhibiting eIF4E-β-catenin in thyroid cancer.

Abstract IA23: eIF4E as anti-cancer target

Abstract The interaction of the cap-binding protein eIF4E with eIF4G is central for recruiting the small ribosomal subunit to the 5' end of mRNA. This interaction is regulated by the competitive interaction with the 4E-binding proteins (4E-BPs). Genome analyses have shown that mRNAs coding for proto-oncogenes, transcription factors, translation factors, growth factors and their receptors have statistically long 5' UTRs with large negative free energies estimated with mfold. Translation of these genes thus, depends heavily on an efficient translation-initiation machinery. This is in contrast to 5' TOP mRNAs and highly expressed genes with expression controlled at the transcription level that have small negative free estimated with mfold. Consistently, overexpression of 4E-BP1 has been shown to suppress tumor growth in mouse xenografts. In a high-throughput screen of compound libraries we discovered a small molecule that mimics the action of 4E-BP1 by displacing eIF4G from eIF4E. Surprisingly, the compound does not displace 4E-BP1 but rather stabilizes its binding both in vitro and in vivo. To understand this effect we have solved crystal structures of complexes between eIF4E and several inhibitor analogs. In addition, we determined the structure of eIF4E in complex with a long fragment of 4E-BP1. These data revealed the dual mechanism of the compound displacing the tumor promoter eIF4G while stabilizing the tumor suppressor 4E-BP1. Furthermore, we investigated the activities of 4EGI-1 aggressive breast cancer cells that have properties of cancer stem cells and find that the compound specifically suppresses the aggressive properties of these cancer cells. In addition, we discovered that eIF4E is transcriptionally upregulated in hypoxia as it promoter contains hypoxia response elements utilized by Hif1a. Finally, we tested 4EGI-1 against a panel of ~1000 cancer cell lines and tested the global impact on Jurkat cells in a proteomics approach. Citation Format: Gerhard Wagner, Evangelos Papadopoulos, Tingfang Yi, Melissa Leger-Abraham, Naotaka Sekiyama. eIF4E as anti-cancer target. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA23.

Ribavirin, an eIF4E Inhibitor, As a Potential Anti-Lymphoma Therapeutic - Preclinical and Early Clinical Data

Ribavirin, an antiviral drug used to treat infections including respiratory syncytial virus (RSV), inhibits the eukaryotic translation initiation factor 4E (eIF4E). EIF4E exports key mRNA transcripts from the nucleus and is a critical factor for translation of mRNAs into protein. Prospective trials of patients treated with ribavirin indicate that the drug has clinical activity and expected molecular effects of eIF4E inhibition in AML including relocalization of eIF4E to cytoplasm and decrease in eIF4E levels (Assouline Blood 2009 and Assouline Haematologica 2015). We demonstrated in pre-clinical models including a PDX triple hit lymphoma that eIF4E is also implicated in the pathogenesis of lymphomas (Culjkovic-Kraljacic Blood 2016).To elucidate the mechanism of action of ribavirin in DLBCL, we conducted eIF4E-immunoprecipitation followed by RNA-sequencing (RIP-seq) in OCI-Ly1 cells to identify RNAs that bind to eIF4E. We integrated this data with the RNA-sequencing of ribavirin-treated OCI-Ly1 cells (vs. vehicle) to further characterize RNAs that are more likely to be decreased by ribavirin. We performed pathway analysis with this data and found several lymphomagenic eIF4E transcripts to be significantly reduced by ribavirin treatment including the BCR, epigenomic regulators, interleukin signaling (IL-6, IL10), DNA damage response elements, and components of the splicing machinery. This suggests that ribavirin interferes with critical pathways in proliferating DLBCL cells and may be active in lymphoma patients.After observing a patient with an aggressive, refractory transformed lymphoma (CLL to HL) demonstrate an objective response on imaging following administration of ribavirin for RSV in absence of concurrent chemotherapy, we retrospectively analyzed (with IRB approval) outcomes of lymphoma patients undergoing autologous or allogeneic SCT who received ribavirin for antiviral indications. We searched our institutional electronic record system and SCT database for lymphoma patients meeting prospectively defined criteria as receiving ribavirin within 6 months prior to or any time after SCT. Ten patients were identified including 5 DLBCL (1 transformed from CLL and another from FL), 2 HL (1 transformed from CLL), 2 FL, and 1 MCL. All were male and median age at lymphoma diagnosis was 54 years (range 35-64). Median number of treatments received prior to SCT was 4 (2-8). Four were deemed to have inadequate response (3 with PD and 1 with insufficient PR) after salvage therapy and were treated with bendamustine prior to proceeding to SCT (3 with an investigational high dose regimen). Responses to therapy immediately prior to SCT included 4 CR, 5 PR, and 1 SD. Six underwent allo SCT and 4 auto SCT.All patients received ribavirin for RSV (4 inhalational, 6 oral) with a median length of treatment of 10 days (5-15). Median interval between SCT and ribavirin was 5 months (-1 to 23). Nine of 10 patients are currently alive with no evidence of lymphoma with a median OS of 17.8 months (4.6-85.5) and median PFS of 11.1 months (2.4-63.8).These retrospective data from patients with refractory lymphomas treated with ribavirin as antiviral therapy (just prior to or soon following SCT) demonstrate lymphoma-related outcomes superior to those expected based on disease risk profiles (9 of 10 with ongoing CRs). We recognize the limitations of this analysis, as well as potential selection biases and other possible explanations for these findings. However, our observations, in conjunction with preclinical data on eIF4E inhibition, raise the intriguing possibility that ribavirin may have clinically meaningful anti-lymphoma activity. Further assessment of larger numbers of patients, and rationally designed prospective clinical studies of ribavirin are justified and planned. [Display omitted] DisclosuresMartin:Acerta: Consultancy; Novartis: Consultancy; Gilead: Consultancy, Other: travel, accommodations, expenses; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: travel, accommodations, expenses; Teva: Research Funding.

Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation

Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5′-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N′-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5′-deoxy-5′-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5′-deoxy-5′-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5′-deoxy-5′-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents.

A Novel Role for the 4E-BP/eIF4E Axis in B Cell Antibody Class Switching

Abstract During an immune response, activated B cells give rise to antibody secreting plasma cells to fight infection. B cells undergo a process known as antibody class switch recombination (CSR) to produce different classes of antibodies with varying effector functions. The mammalian target of Rapamycin (mTOR) signaling pathway is activated during this process and inhibition of mTOR suppresses both proliferation and differentiation into antibody secreting cells. mTOR is a kinase that is found in two distinct complexes, mTORC1 and mTORC2 and B cells deficient in mTOR have impaired survival, proliferation and differentiation. It has been shown that antibody class switch recombination requires B cell division, however it is unclear if there is another regulatory mechanism through mTOR that is independent of proliferation. The mTORC1 inhibitor rapamycin, at either low concentrations or when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Mechanistic investigation of the mTORC1 substrate 4E-BP, an inhibitor of eIF4E and cap-dependent translation, showed that blocking the 4E-BP/eIF4E axis can decrease antibody class switching independently of proliferation. Genetic deletion of 4E-BP1 and 4E-BP2 isoforms partially rescues from the effects of rapamycin on class switching to IgG1. These results uncover a novel role for mTORC1 and the 4E-BP/eIF4E axis in B cell antibody class switching, suggesting that cap-dependent translation regulates key steps in B cell differentiation. Further study of this pathway may provide mechanistic insight into antibody-mediated autoimmune diseases such as lupus and arthritis.

EIF4E-phosphorylation-mediated Sox2 upregulation promotes pancreatic tumor cell repopulation after irradiation

Pancreatic cancer is a devastating disease characterized by treatment resistance and high recurrence rate. Repopulation of surviving tumor cells undergoing radiotherapy is one of the most common reasons for recurrence. Our previous studies have discovered a novel mechanism for repopulation after irradiation that activation of caspase-3 in irradiated tumor cells activates PKCδ/p38 axis to transmit proliferation signals promoting repopulation of surviving tumor cells. Here we found Sox2 expression is up-regulated in irradiated pancreatic cancer cells, which played a major role in tumor cell repopulation after irradiation. Over-expression of Sox2 strongly enhanced the growth-stimulating effect of irradiated dying tumor cells on living tumor cells through a paracrine modality. Furthermore, we identified activated eIF4E, which is phosphorylated by MNK1, as a regulator of Sox2 expression after irradiation, and pharmacologic inhibition of eIF4E with CGP57380 and Ribavirin significantly weakened Sox2-mediated tumor cell repopulation. Finally, we showed the activation of caspase 3/PKCδ/p38/MNK1 signal pathway in irradiated pancreatic tumor cells. Together, we showed a novel pathway regulating Sox2 expression and Sox2 may be a promising target to reduce recurrence due to repopulation of surviving tumor cells after radiotherapy.

Corrigendum] Knockdown of eukaryotic translation initiation factor 4E suppresses cell growth and invasion, and induces apoptosis and cell cycle arrest in a human lung adenocarcinoma cell line.

Mol Med Rep 12:[Related article:] 7971–7978, 2015; DOI: 10.3892/mmr.2015.4468 Following the publication of this article, an interested reader drew to our attention an anomaly associated with the presentation of Fig. 3A. The images selected for the '72 h Blank' and the '72 h Mock' panels were inadvertently selected from the same original photograph. This error arose during the editing process of our paper with a professional English Editing Service, and our failure to notice the mismatching of the images during the final proofreading of the manuscript prior to submission. A corrected version of Fig. 3 is presented here (right), which includes the corrected images for the 72 h Blank' and the '72 h Mock' panels. This error did not affect the overall conclusions reported in the present study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused.

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas

AbstractAggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

Translational control of nociception via 4E-binding protein 1.

Activation of the mechanistic/mammalian target of rapamycin (mTOR) kinase in models of acute and chronic pain is strongly implicated in mediating enhanced translation and hyperalgesia. However, the molecular mechanisms by which mTOR regulates nociception remain unclear. Here we show that deletion of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a major mTOR downstream effector, which represses eIF4E activity and cap-dependent translation, leads to mechanical, but not thermal pain hypersensitivity. Mice lacking 4E-BP1 exhibit enhanced spinal cord expression of neuroligin 1, a cell-adhesion postsynaptic protein regulating excitatory synapse function, and show increased excitatory synaptic input into spinal neurons, and a lowered threshold for induction of synaptic potentiation. Pharmacological inhibition of eIF4E or genetic reduction of neuroligin 1 levels normalizes the increased excitatory synaptic activity and reverses mechanical hypersensitivity. Thus, translational control by 4E-BP1 downstream of mTOR effects the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, and thereby contributes to enhanced mechanical nociception.

Knockdown of eukaryotic translation initiation factor4E suppresses cell growth and invasion, and induces apoptosis and cell cycle arrest in a human lung adenocarcinoma cell line.

Eukaryotic translation initiation factor 4E (eIF4E) was shown to be upregulated in malignant human tumors. To assess the effect of downregulation of eIF4E on the proliferation and invasiveness of a human lung adenocarcinoma cell line, a short hairpin (sh)RNA targeting eIF4E was constructed and transfected into A549 human lung adenocarcinoma cells. The expression of eIF4E was determined by reverse transcription-quantitative polymerase chain reaction and western blotting. Cell viability was assessed using a Cell Counting kit-8, and apoptosis levels and cell cycle distribution were assessed by flow cytometry. Invasiveness was assessed using Transwell chambers. Transfection of the A549 cells with eIF4E targeting shRNA reduced the mRNA and protein expression levels of eIF4E by >70% 48 and 72 h following transfection, and eIF4E targeting shRNA-transfected cells were significantly less viable compared with the cells transfected with scrambled shRNA. The rate of apoptosis was also significantly increased, significantly more cells were in the G0/G1 phase and fewer were in the S phase, indicating cell cycle arrest. The fraction of transfected cells migrating across Transwell inserts were also reduced. In conclusion, inhibition of eIF4E suppressed cell growth and invasion, induced apoptosis and cell cycle arrest, suggesting that eIF4E may be a potential therapeutic target in lung adenocarcinoma.

Gating by tryptophan 73 exposes a cryptic pocket at the protein-binding interface of the oncogenic eIF4E protein.

Targeting protein-protein interacting sites for potential therapeutic applications is a challenge in the development of inhibitors, and this becomes more difficult when these interfaces are relatively planar, as in the eukaryotic translation initiation factor 4E (eIF4E) protein. eIF4E is an oncogene that is overexpressed in numerous forms of cancer, making it a prime target as a therapeutic molecule. We report here the presence of a cryptic pocket at the protein-binding interface of eIF4E, which opens transiently during molecular dynamics simulations of the protein in solvent water and is observed to be stable when solvent water is mixed with benzene molecules. This pocket can also be seen in the ensemble of structures available from the solution-state conformations of eIF4E. The accessibility of the pocket is gated by the side-chain transitions of an evolutionarily conserved tryptophan residue. It is found to be feasible for accommodating clusters of benzene molecules, which signify the plasticity and ligandability of the pocket. We also observe that the newly formed cavity provides a favorable binding environment for interaction of a well-recognized small molecule inhibitor of eIF4E. The occurrence of this transiently accessible cavity highlights the existence of a more pronounced binding groove in a region that has traditionally been considered to be planar. Together, the data suggest that an alternate binding cavity exists on eIF4E and could be exploited for the rational design and development of a new class of lead compounds against the protein.

Signalling to eIF4E in cancer.

Translational control plays a critical role in the regulation of gene expression in eukaryotes and affects many essential cellular processes, including proliferation, apoptosis and differentiation. Under most circumstances, translational control occurs at the initiation step at which the ribosome is recruited to the mRNA. The eukaryotic translation initiation factor 4E (eIF4E), as part of the eIF4F complex, interacts first with the mRNA and facilitates the recruitment of the 40S ribosomal subunit. The activity of eIF4E is regulated at many levels, most profoundly by two major signalling pathways: PI3K (phosphoinositide 3-kinase)/Akt (also known and Protein Kinase B, PKB)/mTOR (mechanistic/mammalian target of rapamycin) and Ras (rat sarcoma)/MAPK (mitogen-activated protein kinase)/Mnk (MAPK-interacting kinases). mTOR directly phosphorylates the 4E-BPs (eIF4E-binding proteins), which are inhibitors of eIF4E, to relieve translational suppression, whereas Mnk phosphorylates eIF4E to stimulate translation. Hyperactivation of these pathways occurs in the majority of cancers, which results in increased eIF4E activity. Thus, translational control via eIF4E acts as a convergence point for hyperactive signalling pathways to promote tumorigenesis. Consequently, recent works have aimed to target these pathways and ultimately the translational machinery for cancer therapy.