Inhibition Of eIF4E Research Articles

The Oncogene eIF4E Reprograms the Nuclear Pore Complex to Promote mRNA Export and Oncogenic Transformation

The eukaryotic translation initiation factor eIF4E is a potent oncogene that promotes the nuclear export and translation of specific transcripts. Here, we have discovered that eIF4E alters the cytoplasmic face of the nuclear pore complex (NPC), which leads to enhanced mRNA export of eIF4E target mRNAs. Specifically, eIF4E substantially reduces the major component of the cytoplasmic fibrils of the NPC, RanBP2, relocalizes an associated nucleoporin, Nup214, and elevates RanBP1 and the RNA export factors, Gle1 and DDX19. Genetic or pharmacological inhibition of eIF4E impedes these effects. RanBP2 overexpression specifically inhibits the eIF4E mRNA export pathway and impairs oncogenic transformation by eIF4E. The RanBP2 cytoplasmic fibrils most likely slow the release and/or recycling of critical export factors to the nucleus. eIF4E overcomes this inhibitory mechanism by indirectly reducing levels of RanBP2. More generally, these results suggest that reprogramming the NPC is a means by which oncogenes can harness the proliferative capacity of the cell.

Development of Biochemical Assays for the Identification of eIF4E-Specific Inhibitors

Control of mRNA translation plays a critical role in cell growth, proliferation, and differentiation and is tightly regulated by AKT and RAS oncogenic pathways. A key player in the regulation of this process is the mRNA 5′ cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E). eIF4E contributes to malignancy by selectively enabling the translation of a limited pool of mRNAs that generally encode key proteins involved in cell cycle progression, angiogenesis, and metastasis. Several data indicate that the inhibition of eIF4E in tumor cell lines and xenograft models impairs tumor growth and induces apoptosis; eIF4E, therefore, can be considered a valuable target for cancer therapy. Targeting the cap-binding pocket of eIF4E should represent a way to inhibit all the eIF4E cellular functions. We present here the development and validation of different biochemical assays based on fluorescence polarization and surface plasmon resonance techniques. These assays could support high-throughput screening, further refinement, and characterization of eIF4E inhibitors, as well as selectivity assessment against CBP80/CBP20, the other major cap-binding complex of eukaryotic cells, overall providing a robust roadmap for development of eIF4E-specific inhibitors.

Translation Initiation Factor eIF4E Is a Target for Tumor Cell Radiosensitization

A core component in the cellular response to radiation occurs at the level of translational control of gene expression. Because a critical element in translation control is the availability of the initiation factor eIF4E, which selectively enhances the cap-dependent translation of mRNAs, we investigated a regulatory role for eIF4E in cellular radiosensitivity. eIF4E silencing enhanced the radiosensitivity of tumor cell lines but not normal cells. Similarly, pharmacologic inhibition of eIF4E with ribavirin also enhanced tumor cell radiosensitivity. eIF4E attenuation did not affect cell-cycle phase distribution or radiation-induced apoptosis, but it delayed the dispersion of radiation-induced γH2AX foci and increased the frequency of radiation-induced mitotic catastrophe. Radiation did not affect 4E-BP1 phosphorylation or cap-complex formation but it increased eIF4E binding to more than 1,000 unique transcripts including many implicated in DNA replication, recombination, and repair. Taken together, our findings suggest that eIF4E represents a logical therapeutic target to increase tumor cell radiosensitivity.

Structure-Guided Design, Synthesis, and Evaluation of Guanine-Derived Inhibitors of the eIF4E mRNA–Cap Interaction

The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5' terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the compound has an IC(50) of 2.5 μM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E inhibitors with physicochemical properties suitable for cellular activity assessment.

Abstract 2798: Cytotoxic and cystostatic properties of rapamycin are due to differential effects on the phosphorylation of mTORC1 substrates S6 kinase and 4E-BP1

Abstract The mammalian target of rapamycin (mTOR) has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapy. Used clinically at nano-molar concentrations, rapamycin suppresses phosphorylation of mTOR complex 1 (mTORC1) substrate S6 kinase and has been considered a cytostatic drug. However, we have reported that higher (micro-molar) doses are required for complete cell cycle arrest. Such high doses suppress phosphorylation of another mTORC1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). In this state, eIF4E is sequestered and unable to facilitate mRNA translation. Dual ablation of S6 kinase and eIF4e resulted in a larger increase in G1 content than by ablation of S6 kinase alone. This confirmed the complete cytostatic effect of high-dose rapamycin. We also demonstrated that the differential dose effects of rapamycin correlate with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. Therefore, high-dose rapamycin ensures complete non-functionality of mTORC1. Interestingly, in the absence of serum, high dose rapamycin induces apoptosis and dual inhibition of S6 kinase and eIF4e does not prevent it. We have established that the differential cytostatic and cytotoxic effects of rapamycin are due to intact TGFb signaling. In serum, TGFb is activated upon low dose rapamycin (suppression of of S6 kinase phosphorylation). The lack of TGFb in the absence of serum allows cells to progress through the cell cycle; at high rapamycin doses, the inactivity of eIF4e results in cell death. This study reveals the mechanism for both cytostatic and cytotoxic properties of rapmaycin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2798. doi:1538-7445.AM2012-2798

Elevated expression of eukaryotic translation initiation factor 4E is associated with proliferation, invasion and acquired resistance to erlotinib in lung cancer

Eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting factor for cap-dependent translation initiation, which is known to regulate oncogenesis. Elevated eIF4E and its negative impact on prognosis in human non-small cell lung cancer (NSCLC) have been reported previously. However, its potential as a therapeutic target and role in regulation of sensitivity to EGFR inhibitors is an area of ongoing investigations. In this study, we detected increased levels of eIF4E in 16 human NSCLC cell lines compared with their normal bronchial epithelial cells. Consistently, human tissue array analysis showed that eIF4E expression was significantly higher in human NSCLC tissues than normal tissues. Inhibition of eIF4E using eIF4E siRNA inhibited the growth and invasion of NSCLC cells. These data suggest that eIF4E overexpression plays a crucial role in positive regulation of the growth and invasion of NSCLC cells. By proteomics, we found that eIF4E levels were elevated in erlotinib-resistant cell lines compared with the sensitive parental cell line. In agreement, assembly of the eIF4F cap complex and several oncogenic proteins regulated by the cap-dependent translation mechanism, were also increased in erlotinib-resistant cells. Thus, erlotinib-resistant cells exhibit elevated eIF4E expression and cap-dependent translation. Inhibition of eIF4F with different means (e.g., gene knockdown) downregulated c-Met expression and partially restored cell sensitivity to erlotinib, suggesting that elevated eIF4E contributes to development of erlotinib resistance, likely through positive regulation of c-Met expression. Taken together, we suggest that elevated eIF4E in NSCLC cells is associated with proliferation, invasion and acquired erlotinib resistance.

A Phase I Combination Study of Ribavirin and Low Dose Cytarabine Arabinoside (ara-C) in M4/M5 Acute Myeloid Leukemia (AML) and AML with High eIF4E,

Abstract 3606The conventional therapy for acute myeloid leukemia (AML), including cytarabine and an anthracycline with or without transplant, results in a durable remission in only a minority of AML patients. Furthermore, half of all newly diagnosed AML patients are over the age of 65, and many are poor candidates for high dose chemotherapy due to comorbidities and poor performance status. Thus, new effective treatment strategies are needed. Overexpression of the eukaryotic translation initiation factor 4E (eIF4E) occurs in greater than 30% of all cancers including M4 and M5 subtypes of AML. eIF4E is an oncogene that selectively regulates expression post-transcriptionally, both at the levels of mRNA translation and export, for genes that are critical for cell division, growth and angiogenesis. Novel strategies to target eIF4E are entering clinical development and have shown some promising activity in cancer. We have identified ribavirin as an inhibitor of eIF4E with anti-cancer activity in patients with AML. Ribavirin is a well-characterized and well-tolerated anti-viral drug. We have shown that it inhibits oncogenic transformation mediated by overexpression of eIF4E and reduces clonogenic potential of cancer cells with elevated levels of eIF4E. Our phase II proof-of-principle clinical trial examining the efficacy of ribavirin treatment in elderly or relapsed M4 and M5 AML patients was the first clinical study to target eIF4E in human malignancy, and demonstrated that ribavirin effectively targets eIF4E in patients, leading to clinical improvement. Unfortunately, all patients who achieved a response acquired resistance and relapsed shortly thereafter. We have found that ara-C and ribavirin have an additive effect on primary leukemia specimens in vitro. Hence, a Phase I trial was commenced combining low dose ara-C with ribavirin in AML patients. The primary objective of the trial was to determine the maximum tolerated dose and recommended phase II dose of ribavirin and low dose ara-C. Additional objectives were to determine safety and to examine molecular correlates and the pharmacokinetic/pharmacodynamic profile of the combination treatment. A total of 17 patients were enrolled on study and evaluated for safety. No dose limiting toxicities have been observed to date. The current dose being tested is 4400 mg/day of ribavirin and ara-C 20 mg subcutaneous twice a day for 10 days and dose escalation continues. We have observed that ribavirin plasma levels are substantially lower than obtained in the monotherapy trial. In addition, of the 12 patients who completed at least one cycle of therapy, we have seen one complete response (CR), 2 blasts responses, and one stable disease. The patient with CR had treatment-related AML due to breast cancer therapy and had relapsed shortly after an allogeneic stem cell transplantation. She achieved a CR after 5 cycles of low dose ara-C and ribavirin. After 6 cycles, the patient was put on maintenance ribavirin monotherapy and continues to be in remission after 16 cycles. Interestingly, in this patient, toxicities due to ara-C led to its dose reduction that correlated with a near doubling of plasma ribavirin levels prior to achieving remission, suggesting that ara-C may alter absorption of ribavirin. A full molecular response was observed in patients with clinical response and none in patients that did not respond, indicating that ribavirin levels were sufficient to affect eIF4E localization and levels. Further safety, pharmacokinetic, pharmacodynamic, and response data will be presented. Disclosures:Off Label Use: Use of investigational agent Ribavirin for the treatment of AML. Bergeron:Celgene: Research Funding; Merck: Research Funding; Roche: Honoraria.

Ribavirin Treatment Effects on Breast Cancers Overexpressing eIF4E, a Biomarker with Prognostic Specificity for Luminal B-Type Breast Cancer

We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer. Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis-free survival (DMFS), overall and within each intrinsic breast cancer subtype. At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating with reduced mRNA export and protein expression of important eIF4E targets. This effect was suppressed by knockdown of eIF4E. Although eIF4E expression is elevated in all breast cancer cell lines, variability in ribavirin responsiveness was observed, indicating that other factors contribute to an eIF4E-dependent phenotype. Assessment of the prognostic value of high eIF4E mRNA in patient tumors found that significant discrimination between good and poor outcome groups was observed only in luminal B cases, suggesting that a specific molecular profile may predict response to eIF4E-targeted therapy. Inhibition of eIF4E is a potential breast cancer therapeutic strategy that may be especially promising against specific molecular subtypes and in metastatic as well as primary tumors.

Abstract 1953: Elevated eukaryotic translation initiation factor 4E (eIF4E) is involved in erlotinib resistance

Abstract The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, erlotinib and gefitinib, are effective therapies for non-small cell lung cancer (NSCLC) with somatic mutations in EGFR. However, all patients eventually develop resistance (i.e., acquired resistance) to these agents. Thus, there is a great challenge and urgent need to understand the underlying mechanism so as to develop effective strategies to overcome the resistance. Eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting factor for cap-dependent translation initiation, a well known mechanism that regulates oncogenesis. In human cancers, increased eIF4E expression has been documented repeatedly to be associated with malignant progression. Accordingly therapeutic suppression of eIF4E expression (e.g., with eIF4E antisense oligonucleotides) effectively reduces tumor growth. However, the role of eIF4E in regulation of erlotinib resistance has not been studied. In this study, we detected increased levels of eIF4E in 16 human NSCLC cell lines compared with their normal bronchial epithelial cells. Consistently, human tissue array analysis showed that eIF4E expression was significantly higher in human NSCLC tissues than normal tissues. Inhibition of eIF4E using eIF4E siRNA substantially inhibited the growth of NSCLC cells. These data suggest that eIF4E overexpression plays a crucial role in supporting the growth of NSCLC cells. By SILAC proteomic and subsequent Western blot analysis, we found that eIF4E levels were substantially elevated in erlotinib-resistant cell line (i.e., HCC827/ER) compared with the sensitive parental cell line (i.e., HCC827). In agreement, assembly of the eIF4F cap complex and several oncogenic proteins such as HIF1α, c-Myc, and Mcl-1, which are regulated by the cap-dependent translation mechanism, were also increased in HCC827/ER cells. Thus, it appears that erlotinib-resistant cells exhibit elevated eIF4E expression and cap-dependent translation. Inhibition of eIF4E with either siRNA or the small molecule eIF4E inhibitor, 4EGI-1, in HCC828/ER cells, partially restored cell sensitivity to erlotinib, suggesting that elevated eIF4E in part contributes to development of erlotinib resistance. In addition, we detected increased levels of eIF4E mRNA in HCC827/ER cells, suggesting that elevated levels of elF4E protein are likely due to increased transcription. Our ongoing work is devoted to understand how eIF4E levels are elevated during the development of erlotinib resistance. (This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH/NCI RO1 CA118450 and PO1 CA116676, and Emory Winship Cancer Institute CCB seed grant; SY Sun, FR Khuri, SS Ramaligam and RK Owonikoko are Georgia Cancer Coalition Distinguished Cancer Scholars) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1953. doi:10.1158/1538-7445.AM2011-1953

Inhibition of eIF4E with ribavirin cooperates with common chemotherapies in primary acute myeloid leukemia specimens

Inhibition of eIF4E with ribavirin cooperates with common chemotherapies in primary acute myeloid leukemia specimens

Eradication of metastatic gastric ca of the antrum resistant to trastuzumab and cetuximab following immunochemogene treatment with SV-IV, a stealth nanoparticle formulation composed of clamp pna against mRNA of SGCA1/B1, docetaxel, and MUC1 chimeric mab

IntroductionThe unmet medical need for metastatic gastric Ca of the antrum is very high because of potent chemoresistance. We aim to circumvent these resistant factors.MethodsWe obtained surgically tumour cells from...

Stabilizing the eIF4G1 α-Helix Increases Its Binding Affinity with eIF4E: Implications for Peptidomimetic Design Strategies

Eukaryotic initiation factor (eIF)4E is overexpressed in many types of cancer such as breast, head and neck, and lung. A consequence of increased levels of eIF4E is the preferential translation of pro-tumorigenic proteins such as c-Myc, cyclin D1, and vascular endothelial growth factor. Inhibition of eIF4E is therefore a potential therapeutic target for human cancers. A novel peptide based on the eIF4E-binding peptide eIF4G1, where the α-helix was stabilized by the inclusion of α-helix inducers as shown by CD measurements, was synthesized. The helically stabilized peptide binds with an apparent Kd of 9.43±2.57 nM, which is ∼15.7-fold more potent than the template peptide from which it is designed. The helically stabilized peptide showed significant biological activity at a concentration of 400 μM, unlike the naturally occurring eIFG1 peptide when measured in cell-based cap-dependent translational reporter and WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assays. Fusion of the template peptide and the stabilized peptide to the cell-penetrating peptide TAT produced more active but equally potent inhibitors of cap-dependent translation in cell lines. They also equally disrupted cell metabolism as measured in a WST-1 assay. Propidium iodide staining revealed that the TAT-fused, helically stabilized peptide caused more cell death than the TAT-fused eIF4G1 template peptide with substantial decreases in the G1 and G2 cell populations. Annexin-staining experiments also indicated that the TAT-fused eIF4G1 derivative peptides caused cell death by apoptosis. The results presented should offer further insight into peptidomimetics development for eIF4E.

Infant Acute Lymphoblastic Leukemias Are Pan-Sensitive to Obatoclax Across molecular/Cytogenetic Subtypes, Especially MLL-ENL, and gene Expression Profiles Determine Obatoclax IC50: A Report on the Children's Oncology Group (COG) P9407 Trial

AbstractAbstract 2757 Introduction:The outcome of infants with acute lymphoblastic leukemia (ALL) remains poor because of the association of frequently occurring MLL translocations with drug resistance and vulnerability of the very young to treatment complications. The two most common MLL partner genes in infant ALL, AF4 (AFF1) and ENL (MLLT1), are associated with particularly poor survival. Better therapies are urgent. One candidate is obatoclax (GeminX Biotechnologies, Inc.), which targets interactions of pan-anti-apoptotic BCL-2 family proteins with BH3 proteins and is now in a Phase I trial for relapsed/refractory pediatric cancers (COG ADVL0816). Previously we showed potent single agent in vitro activity of obatoclax against MLL-rearranged infant ALL (Zhang ASH 2008). Here we evaluate correlations of obatoclax activity with MLL translocation status and gene expression profiles in a large number of cases of infant ALL to define molecular determinants of sensitivity. Methods:Bone marrow, peripheral blood or apheresis samples from the time of diagnosis in 54 infants (age 1–365 d, median 168 d; WBC count 15–1230×103/μL, median 445×103/μL) with ALL (n=52) or bilineal acute leukemia (n=2) were examined, 48 of which were from the COG P9407 trial. By molecular/cytogenetic classification, the cases were MLL-AF4+ (n= 28), MLL-ENL+ (n= 11), other MLL rearrangement positive (other MLL+) (n= 8) or MLL germline (MLL-) (n= 7). Single agent IC50 values from MTT assays after 72 h obatoclax exposures were determined in all cases (including 13 previously tested; Zhang ASH 2008) by plotting the surviving fractions. IC50s in the MLL-AF4+ group were compared to those in each of the other 3 molecular/cytogenetic groups by Wilcoxon's test. Gene expression profiling was performed on Affymetrix HG_U133 Plus2.0 arrays in 47 of the 48 COG P9407 cases. Spearman test was used to identify correlation between log2 expression levels for each probeset and IC50 values across subjects. A heatmap of significant probesets (p≤0.001) was generated by transforming expression levels to z-scores and ordering rows and columns by complete linkage hierarchical clustering. Ingenuity pathway analysis was applied to all probesets with p≤0.01 to identify pathways significantly correlated with IC50. Additional MTT assays were initiated to test sensitivity to agents targeting these pathways. Results:Even though most cases in all 4 groups were sensitive to obatoclax as indicated by IC50s within a clinically achievable range, MLL translocation status still had a significant effect on IC50. MLL-AF4+ cases were least sensitive and MLL-ENL+ cases were most sensitive to obatoclax. Respective IC50 ranges across all 54 cases were: MLL-AF4+, 26–918 nM; MLL-ENL+, 13–294 nM; other MLL+ 10–356 nM; MLL−, 31–488. Compared to MLL-AF4+, the IC50s in MLL-ENL+ cases were significantly lower (p=0.047), IC50s in other MLL+ cases were lower but the difference did not achieve significance (p=0.10), and IC50s in MLL- cases were not significantly different (p=0.64). In the 47 COG P9407 cases studied by MTT assay and gene expression profiling, 450 probesets defined a cluster of 16 cases with higher IC50s, which were predominantly MLL-AF4+ (68.7%). Ingenuity analysis identified significant correlations of the following canonical pathways with the IC50 in the same 47 cases: glycolysis/gluconeogenesis, mTOR signaling, regulation of eIF4 and p70S6K signaling, EIF2 signaling, and fructose and mannose metabolism. In preliminary analyses, cell lines with t(4;11) exhibited time and dose-dependant sensitivity to the eIF4e inhibitor ribavirin. Conclusions:In infant ALL, obatoclax has broad-spectrum activity and there is pan-sensitivity across MLL translocation subtypes and MLL− cases. Still specific MLL partner genes have a strong effect on obatoclax IC50 and there is exquisite sensitivity in MLL-ENL+ cases. This result is important because MLL-ENL is associated with particularly poor survival when conventional therapies are used. The association of differentially expressed genes in canonical cell signaling and metabolism pathways with differences in obatoclax sensitivity forms the basis to combine obatoclax with targeted agents directed at restoring these pathways to enhance responsiveness even further. Disclosures:Felix:None: Patent not licensed.

The Eukaryotic Translation Initiation Factor eIF4E, a Recently Identified Therapeutic Target In AML, Is a Direct Transcriptional Target of NFkB

AbstractAbstract 3146It has been well observed that NFκB activity is elevated in a wide range of hematopoietic malignancy including acute myeloid leukemia (AML). However, the downstream effectors that mediate the biological effects of NFκB are not well described. In the present study, we demonstrate that the eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NFκB. Elevated levels of eIF4E correlate with oncogenic transformation in in-vitro and in-vivo models and are associated with poor prognosis in several cancer types including AML. In a Phase II clinical trial in poor prognosis M4 and M5 AML patients, we demonstrated that the anti-viral drug, ribavirin, targeted eIF4E activity in patients correlating with clinical benefit including remissions and blast responses. Below, we describe the first steps in understanding the dysregulation of eIF4E expression in hematological malignancy.Examination of the human eIF4E promoter proximal region revealed the presence of putative κB sites proximal to the transcriptional start region. Given this, we hypothesized that eIF4E was an inducible target of NFκB. First, we tested whether eIF4E expression was modulated in response to NFκB stimulation by several agents including TNFα and PMA. We carried these studies out in multiple cell lines including BJAB, Jurkat and fibroblasts. We found that PMA or TNFα treatment led to substantial increases in eIF4E levels versus untreated controls. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated p65 loading on the eIF4E promoter. This binding correlated with a transcriptionally active complex. Importantly, eIF4E levels were not elevated upon stimulation in p65-/- fibroblasts whereas elevation was observed in control fibroblasts. Further, expression of a dominant negative form of the inhibitor of NFκB, IκB (IκB2NΔ4), substantially increased the expression of eIF4E. Luciferase reporter assays confirm the element is necessary and sufficient for activation of eIF4E. Importantly, this p65 binding element in the eIF4E gene is evolutionarily conserved in mammals. Finally, using EMSA, we observed that this eIF4E promoter element is in active NFκB complexes in M4 and M5 primary AML specimens.We examined the physiological relevance of these findings in primary M4/M5 AML specimens and AML cell lines. The use of certain demethylase inhibitors such as azacytidine have been reported to have undesirable off target effects, including the activation of NFκB. Consistent with these reports, we observe that treatment with 3 uM azacytidine leads to elevated p65 levels and furthermore, elevated eIF4E levels. Consistently, this led to the upregulation of several eIF4E target genes including myeloid cell leukemia protein Mcl-1 and anti-apoptotic proteins such as mdm2. The addition of the eIF4E inhibitor ribavirin led to reduction in these eIF4E targets with no change in eIF4E levels. Consistently, in primary colony assays, azacytidine (3 uM) alone did not substantially reduce colonies in M4/M5 AML specimens relative to untreated cells, whereas the addition of a combination of ribavirin and azacytidine reduced growth 2 to 10 fold. These findings strongly suggest a physiological link between NFκB and eIF4E and furthermore, have implications for the design of novel therapeutic modalities. For instance, ribavirin could substantially dampen unwanted proliferative effects of azacytidine; thus improving its efficacy.This report is the first direct link ever observed between the NFκB pathway and eIF4E and thus, between NFkB and protein translation & mRNA export. Remarkably, most of the genes regulated at the level of mRNA export or translation by eIF4E are targets at the transcriptional level by NFκB. These include VEGF, cyclin D1, mdm2 and Mcl-1. In this way, the NFκB pathway is powerfully positioned to amplify the effects of eIF4E on post-transcriptional gene expression by both promoting the expression of eIF4E and its downstream effectors. Thus, NFκB can amplify the proliferation and survival pathways governed by eIF4E at multiple levels. Disclosures:No relevant conflicts of interest to declare.

Acetaminophen Improves Protein Translational Signaling in Aged Skeletal Muscle

Age-related muscle atrophy is characterized by increased oxidative stress, diminished Akt enzymatic function, and reduced phosphorylation of the mammalian target of rapamycin (mTOR), which can be attenuated by chronic acetaminophen ingestion. Here we hypothesize that age-related impairments in Akt/mTOR function are associated with reduced protein translational signaling, and that these changes, if present, can be attenuated by acetaminophen treatment. Compared to 6- and 27-month old animals, the expression of the mTOR-complex proteins raptor and GβL and the phosphorylation of tuberin/TSC2 (Thr1462) were reduced in the soleus muscles of very aged rats (33 months old). These changes in Akt/mTOR pathway signaling proteins were in turn associated with decreased phosphorylation of S6 kinase p85S6K (Thr412) and eukaryotic translation initiation factor-4E (eIF4E) binding protein-1 (4EBP1, Thr37/46), reduced phosphorylation of S6 ribosomal protein (Ser235/236), and increased inhibition of eIF4E by binding to 4EBP1. Age-associated alterations in the Akt/mTOR pathway signaling and in the phosphorylation of the stress-responsive eIF2α protein were attenuated by chronic acetaminophen treatment (30 mg/kg body weight per day). Ex vivo incubation of adult muscles with hydrogen peroxide mimicked the age-related decreases seen in eIF4E and 4EBP1 phosphorylation, whereas the inclusion of acetaminophen in the muscle bath attenuated this effect. Aging is associated with impairments in the regulation of proteins thought to be important in controlling mRNA translation, and acetaminophen may be useful for the treatment of age-related muscle atrophy by reducing oxidative stress.

Knockdown of eIF4E suppresses cell growth and migration, enhances chemosensitivity and correlates with increase in Bax/Bcl-2 ratio in triple-negative breast cancer cells

Elevated activity of the eukaryotic translation initiation factor 4E (eIF4E) plays crucial roles in tumorigenesis and disease progression by disproportionately increasing translation of mRNAs coding proteins that play significant roles in all aspects of malignancy, providing that eIF4E as an attractive target for therapeutic intervention. In this study, we showed that inhibition of eIF4E by small interfering RNAs (siRNA) resulted in cell cycle arrest and suppression of colony formation in MDA-MB-231 triple-negative (TN) breast cancer cells. Migration transwell assay revealed that repression of eIF4E effectively inhibited motility of MDA-MB-231 cancer cells. Importantly, we showed that silencing of eIF4E sensitized MDA-MB-231 cells to chemotherapeutic drugs of cisplatin, adriamycin, paclitaxel and docetaxel as assessed by MTT assay. Moreover, Western blot assay showed that eIF4E siRNA increased Bax/Bcl-2 ratio in MDA-MB-231 cells. Taken together, we showed that knockdown of eIF4E suppressed cell growth and migration, enhanced chemosensitivity, suggesting a potential therapeutic target in TN breast carcinoma.

Autocrine Semaphorin3A stimulates eukaryotic initiation factor 4E-dependent RhoA translation in breast tumor cells

Translation of the small G protein RhoA in neurons is regulated by the eukaryotic translation initiation factor eIF4E. Here we show that this translation factor also regulates RhoA expression and activity in breast cancer cells. The introduction of eIF4E into breast tumor cells increased RhoA protein levels, while expression of an eIF4E siRNA reduced RhoA expression. Previous studies indicate that the axon repulsion factor Semaphorin3A (Sema3A) stimulates the eIF4E-dependent translation of RhoA in neurons, and breast tumor cells support autocrine Sema3A signaling. Accordingly, we next examined if autocrine Sema3A signaling drives eIF4E-dependent RhoA translation in breast cancer cells. The incubation of breast tumor cells with recombinant Sema3A rapidly increased eIF4E activity, RhoA protein levels, and RhoA activity. This Sema3A activity was blocked in tumor cells expressing an shRNA-specific for the Sema3A receptor, Neuropilin-1 (NP-1), as well as in cells incubated with an eIF4E inhibitor. Importantly, RhoA protein levels were reduced in Sema3A shRNA-expressing compared to control shRNA-expressing breast tumor cells, demonstrating that autocrine Sema3A increases RhoA expression in breast cancer. Considering that Sema3A suppresses axon extension by stimulating RhoA translation, we next examined if the Sema3A/RhoA axis impacts breast tumor cell migration. The incubation of control breast tumor cells, but not RhoA shRNA-expressing cells, with rSema3A significantly reduced their migration. Collectively, these studies indicate that Sema3A impedes breast tumor cell migration in part by stimulating RhoA. These findings identify common signaling pathways that regulate the navigation of neurons and breast cancer cells, thus suggesting novel targets for suppressing breast tumor cell migration.

Direct inhibition of eIF4E reduced cell growth in endometrial adenocarcinoma

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancer and is emerging as a potential therapeutic target. Yet data on the expression of eIF4E in endometrial cancer are lacking. Immunohistochemistry was used to evaluate the expression of eIF4E in 62 endometrial cancer surgical specimens. We subsequently evaluated whether inhibition of eIF4E by siRNA would have an impact on cell growth in endometrial cancer cell lines, using the MTT cell proliferation assay. According to a chosen cutoff value, 36 (58.1%) of 62 patient specimens scored as eIF4E positive. The positivity of eIF4E was significantly more frequent in tumors extending outside the uterus (stage III/IV vs. stage I/II, P=0.027). Lastly, downregulation of eIF4E by siRNA reduced the growth of HEC-1A cells significantly but had a somewhat weaker effect in Ishikawa cells (P<0.001). Expressions of eIF4E correlated with prognosis of endometrial adenocarcinomas. Our results suggest that eIF4E might be a promising therapeutic target in endometrial cancer.

Abstract 643: Therapeutically targeting eIF4E overexpressing breast cancers destined for early metastatic relapse

Abstract Pathways downstream of PI3K/AKT/mTOR that regulate protein translation, ribosome biogenesis, transcription, metabolism and autophagy are essential to the formation and progression of many human cancers as evidenced by the clinical approval of such selective mTOR inhibitors as temsirolimus (CCI-779, Wyeth) and everolimus (RAD-001, Novartis). However, clinical limitations to the effective use of mTOR inhibitors include the lack of validated tumor marker(s) predictive of mTOR inhibitor responsiveness, mTOR inhibitor release of S6 kinase-mediated negative feedback resulting in secondary activation of PI3K/AKT, and transcriptional upregulation of the downstream effector eIF4E. Using breast cancer cell lines (MCF7, MCF7/HER2, BT474, SKBr3, MDA231) showing varying sensitivity to RAD-001 (IC50 values for MTT viability @ 5 d from &amp;lt;0.2 nM to &amp;gt;20 nM), we observe marked suppression of phos-p70S6K and secondary phos-AKT activation, with variable suppression of phosSer65-4E-BP1 and downstream translation of eIF4E-dependent survival and proliferation proteins (e.g. cyclin D1). To circumvent these limitations of mTOR inhibitors, we are directly targeting eIF4E, as this m7G 5'cap-binding initiator of translation has been shown to be oncogenic and appears elevated in up to 30% of malignancies, with breast cancer ratios of eIF4E/4E-BP1&amp;gt;1 indicating that mTOR inhibition may not be able to reverse eIF4E-driven breast tumorigenesis. Moreover, initial clinical studies of two different eIF4E inhibitors, an investigational antisense oligonucleotide (LY2275796; Isis/Lilly) that reduces eIF4E levels and the approved antiviral ribavirin that physically mimics the m7G 5’ cap structure, indicate that selective in vivo inhibition of eIF4E can be achieved with little or no host toxicity. Our in vitro comparison of phenotypically diverse human breast cancer models indicates that RAD-001 and ribavirin differentially inhibit growth and suppress phos-AKT and cyclin D1 levels in a phenotype-dependent manner. To evaluate the potential of this eIF4E drug target as a prognostic biomarker, 621 node-negative and adjuvant naïve breast cancer cases with expression microarray data and distant metastasis-free survival (DMFS) clinical annotation were curated from public databases, and the normalized eIF4E transcript levels were dichotomized (high, low) for Kaplan-Meier outcome analyses. Increased eIF4E transcript levels are significantly associated with worse DMFS, but only for estrogen receptor (ER)-positive breast cancer cases (n = 400, log rank p = 0.04). These preclinical and clinical findings provide new rationale for pursuing the breast cancer therapeutic potential of targeting eIF4E, especially in eIF4E mRNA overexpressing ER-positive breast cancer cases most likely to experience metastatic relapse within 5 years of diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 643.

Abstract A6: Targeting fatty acid synthase abrogates PI3K signaling in ovarian cancer by dephosphorylation and ubiquitin-mediated proteasomal degradation of downstream effectors

Abstract Ovarian carcinoma is fourth leading cause of cancer death in women and accounts for highest mortality of all gynecological malignancies. The phosphatidylinositol-3 kinase (PI3K) cascade controls proliferation, differentiation, tumorigenesis, angiogenesis and apoptosis. Many ovarian carcinomas harbor aberrations within the PI3K pathway. Amplification of PI3K is observed in ∼40% of ovarian carcinomas and cell lines. The PI3K downstream target AKT phosphorylates mTOR, which is hyperactivated in many cancers. mTOR activates S6 via p70S6K, which is frequently activated in ovarian cancer. S6 protein being a component of the 40S ribosomal subunit is involved in translation control. mTOR also phosphorylates eukaryotic translation initiation factor 4E (eIF4E) inhibitor binding protein 1 (4EBP1). Phosphorylated 4EBP1 dissociates from eIF4E and activates cap-dependent mRNA translation. In addition, many ovarian carcinomas harbor aberrations of the ErbB receptors ErbB1 (EGFR; 55%) or ErbB2 (HER2/neu; 35%), respectively. Importantly, PI3K signaling plays crucial roles in transmitting ErbB-derived signals and stimulating cancer growth. Irrespectively, clinical studies yet reveal that monotherapies with ErbB1 or ErbB2 inhibitors or antibodies are largely inefficient in ovarian carcinomas. Therefore, additional molecular targeting strategies are urgently needed. Fatty acid synthase (FASN) being overexpressed in ∼80% of ovarian carcinomas is a marker for poor prognosis. It supports formation of lipid rafts in the plasma membranes, which accommodate transmembrane growth factor receptors incl. ErbB proteins. Thereby, FASN facilitates signal generation at the cell membranes. Most importantly, inhibition of FASN delays disease progression of ovarian carcinoma xenografts. Recently, we reported that the FASN inhibitor C75 downregulates ErbB1 and ErbB2 in ovarian cancer and sensitizes the cells against ErbB targeting drugs (Grunt et al., BBRC, 385, 454). We now demonstrate that C75 abrogates A2780 ovarian cancer cell growth. This correlates with silencing of PI3K downstream signaling as evidenced by reduced phosphorylation of AKT, mTOR, p70S6K and 4EBP1 in Western blot analyses, which is caused by both specific protein dephosphorylation/deactivation and by ubiquitin-mediated proteasomal degradation of these PI3K effector proteins. In contrast, specific phosphorylation/activation of the mitogen-activated protein kinase ERK1/2 is increased, although ERK1/2 steady-state levels are concurrently decreased by C75. In comparison, the PI3K inhibitor LY294002 blocks phosphorylation while concurrently upregulating steady-state levels of AKT, mTOR, p70S6K, and 4EBP1, and it activates ERK1/2. This suggests i) that PI3K/AKT normally cross-inhibits ERK1/2, which can be abrogated by silencing of PI3K/AKT, and ii) that PI3K, but not ERK1/2, signaling is crucial for growth arrest of ovarian cancer cells. Notably, our data demonstrate for the first time that C75-mediated silencing of PI3K signaling is caused by reduced phosphorylation and diminished protein stability due to increased ubiquitination and proteasomal degradation. Thus, C75 provides additive anticancer action, when compared to the PI3K inhibitor LY294002, which directly targets PI3K and downstream signaling, but does not stimulate effector protein degradation. In summary, FASN represents a promising anticancer drug target, which should be further developed for clinical use in ovarian carcinoma. Supp. ‘Med.-Wiss. Fonds Bürgerm. Wien’ Citation Information: Clin Cancer Res 2010;16(7 Suppl):A6