eIF4E Phosphorylation Research Articles

Phospho‐inositide‐dependent kinase 1 regulates signal dependent translation in megakaryocytes and platelets

BackgroundRegulated protein synthesis is essential for megakaryocyte (MK) and platelet functions, including platelet production and activation. PDK1 (phosphoinositide‐dependent kinase 1) regulates platelet functional responses and has been associated with circulating platelet counts. Whether PDK1 also directly regulates protein synthetic responses in MKs and platelets, and platelet production by MKs, remains unknown. ObjectiveTo determine if PDK1 regulates protein synthesis in MKs and platelets. MethodsPharmacologic PDK1 inhibitors (BX‐795) and mice where PDK1 was selectively ablated in MKs and platelets (PDK1−/−) were used. PDK1 signaling in MKs and platelets (human and murine) were assessed by immunoblots. Activation‐dependent translation initiation and protein synthesis in MKs and platelets was assessed by probing for dissociation of eIF4E from 4EBP1, and using m7‐GTP pulldowns and S35 methionine incorporation assays. Proplatelet formation by MKs, synthesis of Bcl‐3 and MARCKs protein, and clot retraction were employed for functional assays. ResultsInhibiting or ablating PDK1 in MKs and platelets abolished the phosphorylation of 4EBP1 and eIF4E by preventing activation of the PI3K and MAPK pathways. Inhibiting PDK1 also prevented dissociation of eIF4E from 4EBP1, decreased binding of eIF4E to m7GTP (required for translation initiation), and significantly reduced de novo protein synthesis. Inhibiting PDK1 reduced proplatelet formation by human MKs and blocked MARCKs protein synthesis. In both human and murine platelets, PDK1 controlled Bcl‐3 synthesis. Inhibition of PDK1 led to complete failure of clot retraction in vitro. ConclusionsPDK1 is a previously unidentified translational regulator in MKs and platelets, controlling protein synthetic responses, proplatelet formation, and clot retraction.

Eukaryotic Initiation Factor 4E phosphorylation acts a switch for its binding to 4E-BP1 and mRNA cap assembly

Translational regulation has invited considerable interest consequent of its circumstantial dysregulation during cancer genesis. eIF4E (Eukaryotic Initiation Factor 4E) has been identified as an important factor involved in tumor progression by way of instrumenting the convergence of oncogenic signals for up-regulation of Cap-dependent translation. In the backdrop of dramatic eIF4E over-expression in a large population of human cancers, we suggest that the tumorigenic property of eIF4E is strictly attributed to its phosphorylation state. We provide evidence that while phosphorylated eIF4E fails to be sequestered by 4E-BP1, its dephosphorylated form shows overwhelming binding with 4E-BP1 without any consideration to the state of 4E-BP1 phosphorylation to suggest that eIF4E-4EBP1 binding is governed by eIF4E phosphorylation instead of 4E-BP1. We also show that eIF4E engages in Cap-assembly formation preferably in a phosphorylation-dependent manner to suggest that eIF4E phosphorylation rather than 4E-BP1 regulates its availability for Cap-assembly.

Type I Interferons Act Directly on Nociceptors to Produce Pain Sensitization: Implications for Viral Infection-Induced Pain.

One of the first signs of viral infection is body-wide aches and pain. Although this type of pain usually subsides, at the extreme, viral infections can induce painful neuropathies that can last for decades. Neither of these types of pain sensitization is well understood. A key part of the response to viral infection is production of interferons (IFNs), which then activate their specific receptors (IFNRs) resulting in downstream activation of cellular signaling and a variety of physiological responses. We sought to understand how type I IFNs (IFN-α and IFN-β) might act directly on nociceptors in the dorsal root ganglion (DRG) to cause pain sensitization. We demonstrate that type I IFNRs are expressed in small/medium DRG neurons and that their activation produces neuronal hyper-excitability and mechanical pain in mice. Type I IFNs stimulate JAK/STAT signaling in DRG neurons but this does not apparently result in PKR-eIF2α activation that normally induces an anti-viral response by limiting mRNA translation. Rather, type I IFNs stimulate MNK-mediated eIF4E phosphorylation in DRG neurons to promote pain hypersensitivity. Endogenous release of type I IFNs with the double-stranded RNA mimetic poly(I:C) likewise produces pain hypersensitivity that is blunted in mice lacking MNK-eIF4E signaling. Our findings reveal mechanisms through which type I IFNs cause nociceptor sensitization with implications for understanding how viral infections promote pain and can lead to neuropathies.SIGNIFICANCE STATEMENT It is increasingly understood that pathogens interact with nociceptors to alert organisms to infection as well as to mount early host defenses. Although specific mechanisms have been discovered for diverse bacterial and fungal pathogens, mechanisms engaged by viruses have remained elusive. Here we show that type I interferons, one of the first mediators produced by viral infection, act directly on nociceptors to produce pain sensitization. Type I interferons act via a specific signaling pathway (MNK-eIF4E signaling), which is known to produce nociceptor sensitization in inflammatory and neuropathic pain conditions. Our work reveals a mechanism through which viral infections cause heightened pain sensitivity.

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.

Abstract A53: Phosphorylation of eIF4E promotes phenotype switching and MDSC-mediated immunosuppression in melanoma

Abstract Introduction: Melanoma is the deadliest form of skin cancer. Melanoma phenotype switching is characterized by reduced expression of melanocyte lineage transcription factor MITF and its downstream targets, leading to increased invasiveness of melanoma cells and resistance to both targeted therapy and immunotherapy, and worse prognosis. In melanoma, MAPK and PI3K pathways ultimately converge upon eukaryotic translation initiation factor 4E (eIF4E) to induce its phosphorylation (p-eIF4E), which is critical for the oncogenicity of eIF4E. Here, we investigate the role of p-eIF4E in melanoma progression and tumor immunity. Methods: We crossed p-eIF4E deficient (eIF4EKI) mice with an inducible melanoma mouse model. We monitored the primary tumor outgrowth, metastasis, and survival of the eIF4EKI mice versus eIF4EWT mice. Melanoma samples were isolated for further investigation. Results: Compared to the eIF4EWT mice, eIF4EKI mice exhibit significantly delayed tumor growth, reduced metastasis, and increased survival. Increased expression of MITF and downstream melanoma antigens were observed in eIF4EKI tumors, suggesting a p-eIF4E-mediated phenotype switching. Cytokine array analysis revealed a novel proinvasive cytokine signature in eIF4EWT melanoma primary culture, further supporting a role of phospho-eIF4E in phenotype switching. The cytokine profiling also revealed a pro-myeloid-derived suppressor cell (MDSC) cytokine signature in the eIF4EWT tumor, indicating a p-eIF4E-linked immunosuppression. In support of the immune suppressive cytokine signature associated with phospho-eIF4E expressing melanomas, immune phenotyping of eIF4EWT melanomas showed a significant increase in MDSCs and less cytotoxic T cells, compared to eIF4EKI melanomas. Finally, pharmacologic inhibition of p-eIF4E in combination with anti-PD-1 immunotherapy results in a synergistic delay in primary tumor outgrowth and reduced metastasis. Conclusions: Here we showed that phosphorylation of eIF4E promotes melanoma phenotype switching, leading to increased invasiveness and reduced expression of tumor-associated antigens. Further, by increasing the secretion of pro-MDSC cytokines, p-eIF4E permits an immunosuppressive microenvironment. Pharmacologic inhibition of p-eIF4E sensitizes melanoma to anti-PD-1 immunotherapy, potentially by increasing melanoma antigen expression and compromising MDSC-mediated immunosuppression. This study provides a novel therapeutic approach for the treatment of melanoma. Citation Format: Fan Huang, Christophe Gonçalves, Qianyu Guo, Joelle Rémy-Sarrazin, Audrey Emond, William Yang, Dany Plourde, Margarita Bartish, Jie Su, Yao Zhan, Marina G. Gimeno, Elie Khoury, Alexandre Benoit, David Dankort, Wilson H. Miller, Sonia V. del Rincón. Phosphorylation of eIF4E promotes phenotype switching and MDSC-mediated immunosuppression in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A53.

Stepwise Evolution of Fragment Hits against MAPK Interacting Kinases 1 and 2.

Dysregulation of translation initiation factor 4E (eIF4E) activity occurs in various cancers. Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) play a fundamental role in activation of eIF4E. Structure-activity relationship-driven expansion of a fragment hit led to discovery of dual MNK1 and MNK2 inhibitors based on a novel pyridine-benzamide scaffold. The compounds possess promising in vitro and in vivo pharmacokinetic profiles and show potent on target inhibition of eIF4E phosphorylation in cells.

EIF4E regulates STEAP1 expression in peritoneal metastasis.

Gastric cancer is the most prominent form of malignancy in China, and the high mortality associated with it is mostly due to peritoneal metastasis. We have previously elucidated that the RNA-binding protein poly r(C) binding protein 1 (PCBP1) and miR-3978 function as repressors of peritoneal metastasis, partially by downregulation of six-transmembrane epithelial antigen of the prostate 1 (STEAP1). We now show that STEAP1 is regulated at the level of cap-dependent translation initiation by phosphorylated eIF4E. Chemically inhibiting phosphorylation of eIF4E or genetic ablation of phosphorylated eIF4E inhibit translational upregulation of STEAP1 in the peritoneal metastasis mimicking cell line MKN45 in comparison to the normal mesothelial cell line HMrSV5. Thus phosphorylation of eIF4E is required for peritoneal metastasis of gastric cancer via translational control of STEAP1. Chemical inhibitors targeting phosphorylation of eIF4E or its interaction with the translation initiation complex thus might prove effective in treating patients with peritoneal metastasis.

Co-overexpression of AXL and c-ABL predicts a poor prognosis in esophageal adenocarcinoma and promotes cancer cell survival.

Background: Esophageal adenocarcinoma (EAC) is highly aggressive and characterized by poor prognosis. AXL expression has been linked to Barrett's tumorigenesis and resistance to chemotherapy, which is associated with c-ABL intracellular localization. However, the molecular and functional relationship between AXL and c-ABL and the clinical significance of the co-expression of these proteins in EAC remain unclear.Methods: We used immunohistochemical analysis (IHC) on tissue microarrays containing human EAC samples (n=53) and normal esophageal tissues (n=11) in combination with corresponding deidentified clinicopathological information to evaluate the expression and the prognostic significance of AXL and c-ABL in EAC. The data were statistically analyzed using Kruskal-Wallis, the chi-square, the Fisher's exact, and Pearson tests. The Kaplan-Meier method and Cox proportional hazards regression model were used to evaluate cancer patient survival. We used a serum deprivation EAC cell model to investigate the pro-survival function of AXL and c-ABL using cell viability, apoptosis, and lactate dehydrogenase activity assays. We performed in vitro assays, including Western blotting, quantitative real-time PCR, and translational chromatin immunoprecipitation (TrIP-Chip) to study the molecular relationship between AXL and c-ABL in EAC cells.Results: IHC analysis revealed that AXL and c-ABL were overexpressed in 55% and 66% of EAC samples, respectively, as compared to normal tissues. Co-overexpression of the two proteins was observed in 49% of EAC samples. The chi-square test indicated a significant association between AXL and c-ABL expression in the EAC samples (χ2 = 6.873, p = 0.032), and the expression of these proteins was significantly associated with EAC patient age (p < 0.001), tumor stage (p < 0.01), and lymph node status (p < 0.001). AXL and c-ABL protein expression data analysis exhibited an identical clinicopathological association profile. Additionally, we found a significant association between expression of AXL (χ2 = 16.7, p = 0.002) or c-ABL (χ2 = 13.4, p = 0.001) and survival of EAC patients. The Cox proportional hazards model and log rank test predicted a significant increase in mortality of patients with high expression of AXL [hazard ratio (HR): 2.86, 95% confidence interval (CI): 1.53 - 5.34, p = 0.003] or c-ABL [HR: 3.29, 95% CI: 1.35 - 8.03, p = 0.001] as compared to those patients with low expression of AXL or c-ABL proteins. Molecular investigations indicated that AXL positively regulates c-ABL protein expression through increased cap-dependent protein translation involving phosphorylation of EIF4E in EAC cells. Next, we investigated the functional relationship between AXL and c-ABL in EAC cells. We demonstrated that the pro-survival activity of AXL requires c-ABL expression in response to serum deprivation.Conclusion: This study highlights the importance of the co-overexpression of AXL and c-ABL proteins as a valuable prognostic biomarker and targeting these proteins could be an effective therapeutic approach in EAC or other solid tumors expressing high levels of AXL and c-ABL proteins.

Inhibition of Growth of TSC2-Null Cells by a PI3K/mTOR Inhibitor but Not by a Selective MNK1/2 Inhibitor.

Lymphangioleiomyomatosis (LAM) is a rare metastatic cystic lung disease due to a mutation in a TSC tumor suppressor, resulting in hyperactive mTOR growth pathways. Sirolimus (rapamycin), an allosteric mTORC1 inhibitor, is a therapeutic option for women with LAM but it only maintains lung volume during treatment and does not provide benefit for all LAM patients. The two major mTORC1 protein synthesis pathways are via S6K/S6 or 4E-BP/eIF4E activation. We aimed to investigate rapamycin in combination with compounds that target associated growth pathways, with the potential to be additive to rapamycin. In this study we demonstrated that rapamycin, at a clinically tolerable concentration (10 nM), inhibited the phosphorylation of S6, but not the critical eIF4E releasing Thr 37/46 phosphorylation sites of 4E-BP1 in TSC2-deficient LAM-derived cells. We also characterized the abundant protein expression of peIF4E within LAM lesions. A selective MNK1/2 inhibitor eFT508 inhibited the phosphorylation of eIF4E but did not reduce TSC2-null cell growth. In contrast, a PI3K/mTOR inhibitor omipalisib blocked the phosphorylation of Akt and both S6K/S6 and 4E-BP/eIF4E branches, and additively decreased the growth of TSC2-null cells with rapamycin. Omipalisib, or another inhibitor of both major mTORC1 growth pathways and pAkt, might provide therapeutic options for TSC2-deficient cancers including, but not limited to, LAM.

Inhibitory effects of SEL201 in acute myeloid leukemia.

MAPK interacting kinase (MNK), a downstream effector of mitogen-activated protein kinase (MAPK) pathways, activates eukaryotic translation initiation factor 4E (eIF4E) and plays a key role in the mRNA translation of mitogenic and antiapoptotic genes in acute myeloid leukemia (AML) cells. We examined the antileukemic properties of a novel MNK inhibitor, SEL201. Our studies provide evidence that SEL201 suppresses eIF4E phosphorylation on Ser209 in AML cell lines and in primary patient-derived AML cells. Such effects lead to growth inhibitory effects and leukemic cell apoptosis, as well as suppression of leukemic progenitor colony formation. Combination of SEL201 with 5’-azacytidine or rapamycin results in synergistic inhibition of AML cell growth. Collectively, these results suggest that SEL201 has significant antileukemic activity and further underscore the relevance of the MNK pathway in leukemogenesis.

EIF4E‑related miR‑320a and miR‑340‑5p inhibit endometrial carcinoma cell metastatic capability by preventing TGF‑β1‑induced epithelial‑mesenchymal transition.

Endometrial cancer (EC) is a common form of cancer in women. Metastasis is the main cause of EC treatment failure. Eukaryotic translation initiation factor 4E (eIF4E) is an oncogene that is overexpressed in a variety of malignancies and their distant metastases. The present study analyzed microarray data from the Oncomine database and revealed that high eIF4E expression was associated with poor prognosis and high pathological grade of EC. The expression of eIF4E was higher in EC tissues compared with in adjacent normal tissues. In addition, microRNA (miR)-320a and miR-340-5p expression levels were downregulated in EC tissues compared with those in adjacent normal tissues, which suggested that these microRNAs may serve as EC tumor suppressor genes. miR-320a and miR-340-5p could bind to the 3′-UTR of eIF4E mRNA, thus downregulating the expression of eIF4E and phosphorylated (p)-eIF4E in EC cells. Overexpression of miR-320a or miR-340-5p effectively suppressed HEC-1A cell migration and invasion. The downregulation of eIF4E and p-eIF4E following miR-320a or miR-340-5p transfection reduced the invasiveness and metastatic capability of EC cells in a manner associated with decreased expression of matrix metallopeptidase (MMP)-3 and MMP-9. In addition, one of the effects of transforming growth factor β1 (TGF-β1), which is to induce the phosphorylation of eIF4E, was suppressed by miR-320a and miR-340-5p overexpression. These two microRNAs also attenuated the features of TGF-β1-induced epithelial-mesenchymal transition (EMT). In conclusion, the results of the present study demonstrated that eIF4E was upregulated in EC, whereas miR-320a and miR-340-5p were downregulated in EC compared with adjacent normal tissues. In vitro, miR-320a and miR-340-5p inhibited the migratory capability of EC cells by downregulating MMP-3 and MMP-9 and prevented TGF-β1-induced EMT through p-eIF4E.

Deciphering the mechanistic effects of eIF4E phosphorylation on mRNA-cap recognition.

The mRNA cap-binding oncoprotein "eIF4E" is phosphorylated at residue S209 by Mnk kinases, and is closely associated with tumor development and progression. Despite being well-established, mechanistic details at the molecular level of mRNA recognition by eIF4E due to phosphorylation have not been clearly elucidated. We investigated this through molecular modeling and simulations of the S209 phosphorylated derivative of eIF4E and explored the associated implication on the binding of the different variants of mRNA-cap analogs. A key feature that emerges as a result of eIF4E phosphorylation is a salt-bridge network between the phosphorylated S209 (pS209) and a specific pair of lysine residues (K159 and K162) within the cap-binding interface on eIF4E. This interaction linkage stabilizes the otherwise dynamic C-terminal region of the protein, resulting in the attenuation of the overall plasticity and accessibility of the binding pocket. The pS209-K159 salt-bridge also results in an energetically less favorable environment for the bound mRNA-cap primarily due to electrostatic repulsion between the negative potentials from the phosphates in the cap and those appearing as a result of phosphorylation of S209. These observations collectively imply that the binding of the mRNA-cap will be adversely affected in the phosphorylated derivative of eIF4E. We propose a mechanistic model highlighting the role of eIF4E phosphorylation as a regulatory tool in modulating eIF4E: mRNA-cap recognition and its potential impact on translation initiation.

Eukaryotic initiation factor 4E is a novel effector of mTORC1 signaling pathway in cross talk with Mnk1.

Cellular signals that influence Cap-dependent translation have assumed significant relevance in the backdrop of their enforced dysregulation during oncogenesis. Eukaryotic initiation factor 4E(eIF4E), the mRNA cap-binding protein, has emerged as a key player to facilitate tumor progression through upregulated cap-dependent translation synchronized with enhanced cell division. We provide evidence that eIF4E phosphorylation is regulated by mTORC1 by virtue of its interaction with Raptor through a novel TPTPNPP motif and consequent phosphorylation invitro and in vivo in a Rapamycin-sensitive manner. While we show that phosphorylation pattern of eIF4E responds faithfully to Rapamycin inhibition, the prolonged exposure to Rapamycin rescues the loss of eIF4E phosphorylation through Mnk1 activation. We also present evidence that eIF4E interacts with the amino terminal domain of S6K1 in a phospho-dependent manner, and this interaction is instrumental in overriding Rapamycin inhibition of S6K1. The data endorses eIF4E as a regulatory subunit that modulates the functional attributes of mTOR effectors to synchronize cap-dependent translation with growth assertion.

Higher matrix stiffness as an independent initiator triggers epithelial-mesenchymal transition and facilitates HCC metastasis

BackgroundIncreased liver stiffness exerts a detrimental role in driving hepatocellular carcinoma (HCC) malignancy and progression, and indicates a high risk of unfavorable outcomes. However, it remains largely unknown how liver matrix stiffness as an independent cue triggers epithelial-mesenchymal transition (EMT) and facilitates HCC metastasis.MethodsBuffalo rat HCC models with different liver stiffness backgrounds and an in vitro Col I-coated cell culture system with tunable stiffness were used in the study to explore the effects of matrix stiffness on EMT occurrence and its underlying molecular mechanism. Clinical significance of liver stiffness and key molecules required for stiffness-induced EMT were validated in HCC cohorts with different liver stiffness.ResultsHCC xenografts grown in higher stiffness liver exhibited worse malignant phenotypes and higher lung metastasis rate, suggesting that higher liver stiffness promotes HCC invasion and metastasis. Cell tests in vitro showed that higher matrix stiffness was able to strikingly strengthen malignant phenotypes and independently induce EMT occurrence in HCC cells, and three signaling pathways converging on Snail expression participated in stiffness-mediated effect on EMT including integrin-mediated S100A11 membrane translocation, eIF4E phosphorylation, and TGF β1 autocrine. Additionally, the key molecules required for stiffness-induced EMT were highly expressed in tumor tissues of HCC patients with higher liver stiffness and correlated with poor tumor differentiation and higher recurrence.ConclusionsHigher matrix stiffness as an initiator triggers epithelial-mesenchymal transition (EMT) in HCC cells independently, and three signaling pathways converging on Snail expression contribute to this pathological process. This work highlights a significant role of biomechanical signal in triggering EMT and facilitating HCC invasion and metastasis.

Phosphorylation of eukaryotic initiation factor 4E is dispensable for skeletal muscle hypertrophy.

The eukaryotic initiation factor 4E (eIF4E) is a major mRNA cap-binding protein that has a central role in translation initiation. Ser209 is the single phosphorylation site within eIF4E and modulates its activity in response to MAPK pathway activation. It has been reported that phosphorylation of eIF4E at Ser209 promotes translation of key mRNAs, such as cyclin D1, that regulate ribosome biogenesis. We hypothesized that phosphorylation at Ser209 is required for skeletal muscle growth in response to a hypertrophic stimulus by promoting ribosome biogenesis. To test this hypothesis, wild-type (WT) and eIF4E knocked-in (KI) mice were subjected to synergist ablation to induce muscle hypertrophy of the plantaris muscle as the result of mechanical overload; in the KI mouse, Ser209 of eIF4E was replaced with a nonphosphorylatable alanine. Contrary to our hypothesis, we observed no difference in the magnitude of hypertrophy between WT and KI groups in response to 14 days of mechanical overload induced by synergist ablation. Similarly, the increases in cyclin D1 protein levels, ribosome biogenesis, and translational capacity did not differ between WT and KI groups. Based on these findings, we conclude that phosphorylation of eIF4E at Ser209 is dispensable for skeletal muscle hypertrophy in response to mechanical overload.

Discovery of N-Phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine Derivatives as Potent Mnk2 Inhibitors: Design, Synthesis, SAR Analysis, and Evaluation of in vitro Anti-leukaemic Activity.

Aberrant expression of eukaryotic translation initiation factor 4E (eIF4E) is common in many types of cancer including acute myeloid leukaemia (AML). Phosphorylation of eIF4E by MAPK-interacting kinases (Mnks) is essential for the eIF4E-mediated oncogenic activity. As such, the pharmacological inhibition of Mnks can be an effective strategy for the treatment of cancer. A series of N-phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine derivatives was designed and synthesised. The Mnk inhibitory activity of these derivatives as well as their anti-proliferative activity against MV4-11 AML cells was determined. These compounds were identified as potent Mnk2 inhibitors. Most of them demonstrated potent anti-proliferative activity against MV4-11 AML cells. The cellular mechanistic studies of the representative inhibitors revealed that they reduced the level of phosphorylated eIF4E and induced apoptosis by down-regulating the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and by cleaving poly(ADP-ribose)polymerase (PARP). The lead compound 7k possessed desirable pharmacokinetic properties and oral bioavailability. This work proposes that exploration of the structural diversity in the context of Nphenyl- 4-(1H-pyrrol-3-yl)pyrimidin-2-amine would offer potent and selective Mnk inhibitors.

A new mechanism for translational control in plants.

In unstressed conditions, the cap-binding protein, eIF4E, binds the 5' cap structure (m7G) on mRNA and recruits other translation initiation factors to bring the ribosome to the mRNA. Bruns etal. show that, in plants, phosphorylation of eIF4E by the master metabolic regulatory protein, sucrose nonfermenting-related kinase 1 (SnRK1), reduces translation globally. This negative regulation of translation inhibits geminivirus infection and is speculated to be a response to various abiotic stresses.

Abstract 4142: Physical and functional interactions between MNK and mTOR signaling regulate the activation and differentiation of T cells

Abstract The role of the PI3K/mTOR and MAPK signaling pathways in regulating T cell activation and differentiation is well established. It has been shown that mTOR acts as a sensor of the T cell metabolic state, coordinating diverse inputs to determine the balance between effector versus memory cell fates. Similarly, the MAPK interacting kinases, MNK1 and MNK2, are key downstream effector kinases that mediate post-transcriptional gene regulation of critical mediators of the T cell response, including immune checkpoint proteins and cytokines, via the phosphorylation of specific RNA binding proteins such as eIF4E and hnRNPA1. eFT508 is a potent and selective inhibitor of both MNK1 and MNK2, which enhances anti-tumor immune responses by decreasing the expression of immune response modulators including PD-1, PD-L1, LAG3, TIM3 and IL-10. Furthermore, eFT508 treatment boosts memory T cell populations while increasing the effectiveness of cytotoxic T lymphocytes. Phosphoproteomic analysis of the effects of eFT508 on early T cell activation identified novel eFT508 sensitive phosphopeptides, including a subset that overlaps with mTOR-dependent phosphorylation sites. In vitro biochemical analysis has shown that eFT508 is not a mTOR kinase inhibitor and that most of these sites are not direct MNK substrates. These results suggest inhibition of MNK by eFT508 can have significant effects on mTOR signaling. Consistent with these findings, we observe a physical association between MNK and mTOR that is disrupted by either eFT508 or the allosteric mTOR inhibitor rapamycin. Our phosphoproteomic analysis also identified novel MNK-dependent phosphorylation sites within the translation initiation factor eIF4G1 that regulate the ability of mTOR to recognize eIF4G1 as a substrate. In addition, phosphorylation of the mTOR target 4EBP1 is decreased upon treatment of T cells with eFT508, although 4EBP1 itself is not a direct substrate of MNK in vitro. These data are consistent with a role for MNK in regulating mRNA translation, not only through phosphorylation of eIF4E, but also by modulating the activity of mTOR toward specific substrates. Given the role of mTOR in controlling memory T cell differentiation, our results provide a potential mechanistic basis to explain the impact of MNK inhibition on T cell differentiation. Ongoing studies are further characterizing the consequences of eFT508 regulation on MNK/mTOR signaling and T cell differentiation. This work significantly expands our current understanding of eFT508’s mechanism of action as well as its ability to promote a prolonged anti-tumor immune response. Citation Format: Craig R. Stumpf, Vikas K. Goel, Rajesh K. Sharma, Gary G. Chiang, Peggy A. Thompson, Kevin R. Webster. Physical and functional interactions between MNK and mTOR signaling regulate the activation and differentiation of T cells [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 4142.

Impact of eIF4E phosphorylation at Ser209 via MNK2a on tumour recurrence after curative surgery in localized clear cell renal cell carcinoma.

Background: We investigated the roles of eIF4E phosphorylation (Ser209) in tumour recurrence after curative nephrectomy for localized clear cell renal cell carcinoma (ccRCC).Methods: Expression of eIF4E, p eIF4E and MNKs (MAPK interacting kinases), was evaluated in surgical specimens obtained from consecutive non metastatic ccRCC patients (n = 290) by immunohistochemistry (IHC), immunoblotting, and qRT PCR at the protein and mRNA levels. In human RCC cell lines, the effects of eIF4E phosphorylation were examined using immunoblotting, proliferation, migration and invasion assays with pharmacological inhibitors (CGP57380 or ETP45835) and specific small interfering (si) RNAs against MNK1/2(a/b).Results: In postoperative follow-up (median, 7.9 y), 40 patients experienced metastatic recurrence. In multivariate Cox analyses, higher IHC expression of p eIF4E in ccRCC significantly predicted a longer recurrence-free interval. eIF4E is phosphorylated mainly by MNK2a in tumour specimens and cell lines. In 786-O and A-498 cell lines, pharmacological inhibition of MNKs decreased p-eIF4E and increased vimentin and N cadherin but did not influence proliferation. Similarly, MNK2 or MNK2a inhibition with siRNA reduced p-eIF4E and enhanced vimentin translation, cell migration and invasion in the cell lines.Conclusions: MNK2a-induced eIF4E phosphorylation may suppress metastatic recurrence of ccRCC, partially due to vimentin downregulation at the translational level, consequently leading to inhibition of epithelial–mesenchymal transition.

Inhibiting MNK Selectively Targets Cervical Cancer via Suppressing eIF4E-Mediated β-Catenin Activation

Targeting β-catenin has been shown to have great potential therapeutic value in cervical cancer. Because β-catenin is also essential for normal cells, strategies to specifically target cancer will require identification of druggable factors capable of distinguishing β-catenin signaling pathways between cancer and normal cells. Expression of p-eIF4E and p-β-catenin was analyzed in malignant and normal cervical tissues and cells. The effects and its underlying mechanisms of targeting MNK and eukaryotic translation initiation factor 4E (eIF4E) were determined in cervical cancer and normal cells. Inhibiting MNK/eIF4E axis selectively targets cervical cancer without affecting normal cervical cells, via suppressing eIF4E-mediated β-catenin activation. We found that eIF4E phosphorylation was upregulated in cervical cancer cells and tissues but not normal cervical counterparts, and its phosphorylation at Ser 209 activates Wnt/β-catenin signaling, promotes growth and migration in cervical cancer, in an MNK-dependent manner. MNK inhibition via genetic small interfering RNA (siRNA) knockdown or pharmacologic inhibitor effectively decreased phosphorylation of eIF4E and β-catenin, leading to reduced β-catenin activity and transcript levels of Wnt target genes in cervical cancer cells. Consistently, we found that MNK kinase inhibitor is effective in inhibiting proliferation and migration, and inducing apoptosis in cervical cancer but not normal cervical cells. The combination of MNK kinase inhibitor with paclitaxel achieved greater efficacy in cervical cancer cells than paclitaxel alone. Our work identifies MNK-eIF4E axis as a specific and critical regulator of β-catenin activity in cervical cancer but not normal cervical cells, and suggests that targeting MNK is a useful therapeutic strategy in cervical cancer.