Hyperphosphorylation Of 4E-BP1 Research Articles

Accessory cells precondition naive T cells and regulatory T cells for cytokine-mediated proliferation

Abstract Naïve T cells and regulatory T cells, when purified, do not proliferate to the common cytokine receptor γ-chain family cytokines interleukin (IL)-2, IL-7 or IL-15, despite their expression of cognate cytokine receptors. Cell-to-cell contact with dendritic cells (DCs) enabled proliferation of the T cell to these cytokines, independent of antigen recognition or T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in mice depleted of DCs. We propose calling this a “preconditioning effect”. Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2-target genes. Preconditioning was necessary to activate these two pathways, and induced weak Ca 2+mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation and prolonged S6 phosphorylation occurred, and T cells underwent proliferation. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells.

Accessory cells precondition naïve T cells and regulatory T cells for cytokine-mediated proliferation

Naïve Tcells and regulatory Tcells, when purified, do not proliferate to the γc-cytokines IL-2, IL-7, or IL-15, despite their expression of cognate cytokine receptors. Dendritic cells (DCs) enabled the Tcell proliferation to these cytokines, through cell-to-cell contact, but independent of Tcell receptor stimulation. This effect lasted after separation of Tcells from DCs, enabling enhanced proliferation of the Tcells in DC-depleted hosts. We propose calling this a "preconditioning effect". Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in Tcells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2 target genes. "Preconditioning" was necessary to activate these two pathways and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation, and prolonged S6 phosphorylation occurred. Collectively, accessory cells provide Tcell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of Tcells.

Umbralisib/TGR-1202 As a Novel Dual PI3K/CK1 Inhibitor Has a Unique Therapeutic Role in Silencing Oncogenes in Aggressive Lymphomas

Introduction: A number of oncogenes have been shown to promote the pathogenesis and correlate with poor survival in aggressive lymphoma. For example, deregulation of C-MYC is prevalent in diffuse large B cell lymphoma (DLBCL), chromosome translocations involving C-MYC and BCL2 define “double hit lymphoma” (DHL), and chromosome translocation involving CCND1 is pathognomonic for mantle cell lymphoma (MCL). Despite impressive progress being made in the treatment of these aggressive lymphomas, the treatment options for patients with relapsed aggressive lymphoma, especially DHL, remain inadequate. Interestingly, translation of C-MYC, BCL2, CCND1, and a number of other oncogenes is highly sensitive to inhibition of the eukaryotic translation initiation factor 4F (eIF4F) (Manier et al., 2017; Wolfe et al., 2014), which is comprised of the eIF4E, eIF4A, and eIF4G subunits. However, inhibitors of eIF4F are currently not available for the clinic. eIF4F is negatively regulated by the eIF4E binding protein 1 (4E-BP1), which in the hypo-phosphorylated form sequesters eIF4E (Haghighat et al., 1995). To allow for robust initiation of translation in cancer cells 4E-BP1 is hyper-phosphorylated by upstream signals, such as mechanistic target of rapamycin (mTOR). Our recent results indicate that casein kinase 1 epsilon (CK1ε) positively regulate translation via phosphorylating4E-BP1 (Deng et al., 2016). There may be additional poorly understood signals regulating 4E-BP1. Although no CK1ε inhibitor has been developed for the clinic, we recently discovered that the PI3Kδ inhibitor umbralisib/TGR-1202 acts additionally to inhibit CK1ε. Given that TGR-1202 has demonstrated a favorable safety profile in clinical trials and is capable of targeting both PI3Kδ and CK1ε, the drug may be particularly valuable for the silencing of oncogenes in aggressive lymphoma. Our hypothesis is the following: If hyper-phosphorylation of 4E-BP1 serves as a nexus for upregulating oncogene translation, then co-targeting signals phosphorylating 4E-BP1, such as CK1ε and mTOR, may potently and synergistically silence oncogenes in aggressive lymphomas.Materials and Methods: Cytotoxicity was studied in lymphoma cell lines and using Cell TiterGlo (Promega®). Screen of drug synergy was performed between pairs of TGR-1202, mTOR inhibitors, proteasome inhibitors, and CK1 inhibitors. The Bliss additivism model was used to determine the expected inhibition of cell growth and the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Expression of oncogenes was studied by Western blot and qPCR. 4E-BP1 was studied by Western blot and by 4E-BP1 cap-binding assay using m7GTP Sepharose beads. Translation was determined using a bicistronic reporter. Drug studies were validated by gene knockout of expected molecular targets such as CK1ε and PI3Kδ using CRISPR/Cas9. To further investigate the global effects of targeting translation initiation, we are conducting in parallel proteomic and RNA-Seq studies in 3 cell lines representing DLBCL, DHL, and MCL. Furthermore, the mechanism will be validated by rescuing experiments through over-expression of eIF4E, MYC, or CCND1. The efficacy of TGR-1202-containing regimens is being investigated in xenograft mouse models of MCL.Results: We investigated the combinatorial effect of TGR-1202 and the proteasome inhibitor carfilzomib. The combination of TGR-1202 and carfilzomib (TC) was highly synergistic in mantle cell lymphoma and double hit lymphoma cell lines (Figure 1A). TC synergistically inhibited phosphorylation of 4E-BP1 at the Ser65 in both MCL and DHL (Figure1B). The TC combination synergistically produced cleavage of poly-ADP ribose polymerase (PARP), indicating apoptosis as its mechanism (Figure 1C). TC reduced the protein expression levels of several oncogenes responsible for malignancies such as c-Myc, cyclin D1, and Bcl-2. In contrast, this combination had no significant effect on the mRNA level of c-Myc and cyclin D1 (Figure 1D).Conclusion: A novel therapeutic strategy can be developed by combining umbralisib/TGR-1202 and the proteasome inhibitor carfilzomib for the treatment of DLBCL, DHL, and MCL. The combination induces apoptosis by inhibiting the phosphorylation of 4E-BP1, leading to translational silencing and suppressed protein levels of c-Myc, cyclin D1, and Bcl-2. [Display omitted] DisclosuresAli:Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. O'Connor:Celgene: Honoraria, Research Funding; Trillium Therapeutics: Research Funding.

Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 knockout

TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein 1 (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been comprehensively investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR-associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.

Newcastle Disease virus infection activates PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways to benefit viral mRNA translation via interaction of the viral NP protein and host eIF4E.

Newcastle disease virus (NDV), a member of the Paramyxoviridae family, can activate PKR/eIF2α signaling cascade to shutoff host and facilitate viral mRNA translation during infection, however, the mechanism remains unclear. In this study, we revealed that NDV infection up-regulated host cap-dependent translation machinery by activating PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways. In addition, NDV infection induced p38 MAPK/Mnk1 signaling participated 4E-BP1 hyperphosphorylation for efficient viral protein synthesis when mTOR signaling is inhibited. Furthermore, NDV NP protein was found to be important for selective cap-dependent translation of viral mRNAs through binding to eIF4E during NDV infection. Taken together, NDV infection activated multiple signaling pathways for selective viral protein synthesis in infected cells, via interaction between viral NP protein and host translation machinery. Our results may help to design novel targets for therapeutic intervention against NDV infection and to understand the NDV anti-oncolytic mechanism.

Casein Kinase 1 Delta Is a Novel Regulator of mRNA Translation and Druggable Target in Aggressive Lymphomas

Introduction: As an oncoprotein and a transcription factor, C-MYC has been extensively validated as a key driver in many cancers such as Diffuse large B cell lymphoma (DLBCL). C-MYC has been intensely investigated as a therapeutic target in preclinical models. However, no drugs have been successfully developed to target c-Myc, and the c-Myc oncoprotein has been recognized as undruggable. Recent data from our lab and others suggest that the translation of C-MYC and some other oncogenes may be preferentially repressed using inhibitors of the translation apparatus. Translation can be divided into three steps, namely translational initiation, elongation and termination. Translational initiation involves various kinases that stimulate phosphorylation of the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BP) such as 4E-BP1. In its hypo-phosphorylated state 4E-BP1 sequesters eIF4E and acts as a "brake" for translational initiation. mTORC1 has been established as a key activator for hyper-phosphorylation of 4E-BP1. Hyper-phosphorylation of 4E-BP1 allows the release of eIF4E for the assembly of the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of 3 subunits, including the mRNA 5ʹ-cap-binding subunit eIF4E, the large scaffolding subunit eIF4G, and the RNA helicase subunit eIF4A. Subsequently, the 40S and 60S ribosomes are assembled on the AUG start codon of mRNA and translational elongation proceeds along the polysomes. Despite the extensive fund of knowledge in translation, there has been limited success in targeting translation as a therapeutic option for cancers. Omacetaxine mepesuccinate (homo-harringtonine) is approved for the treatment of chronic myeloid leukemia (CML), using the mechanism of competing for aminoacyl-tRNA thereby inhibiting translational elongation. No other translation inhibitors have been approved. Insights into novel regulators of translation will be key to successful development of drugs targeting translation for the treatment of cancers. Such drugs may be particularly useful for cancers driven by translationally regulated oncogenes such as C-MYC. Materials and Methods: Gene knockout of molecular targets such as CK1δ was done using CRISPR/Cas9. Assembly of eIF4F was determined by cap-binding assay using m7GTP Sepharose beads. Global and gene-specific translation was determined using (a) surface sensing of translation (SUnSET) assay and (b) polysome profile followed by Western blotting and qPCR. To further investigate the mode of the action and the effect on the translation, we are conducting an unbiased, proteome-wide experiment and RNA-seq studies in 2 cell lines representing DLBCL and MCL. We will determine the binding partners of CK1δ using co-immunoprecipitation followed by LC-MS. Finally, we determined in vitro the pharmacological activity of CK1δ inhibitors using the Cell Titer Glo assay and Annexin V assay; and their in vivo activity is being studied using mouse models of human lymphoma xenografts. Results: Knocking down CK1δ led to marked reduction in the phosphorylation of 4E-BP1 and translational initiation. CK1δ inhibitors phenocopied the effects of CK1δ knockdown on 4E-BP1 or eIF4F, and profoundly inhibited mRNA translation, as demonstrated using the polysome profiling and SUnSET assays. CK1δ inhibitors inhibited the translation of important oncogenes including C-MYC, CCND1, and MDM2 in lymphoma cells. CK1δ inhibitors potently induced cell death in a number of human lymphoma cell lines such as DLBCL and mantle cell lymphoma (MCL). We expect to complete and present the in vivo and omics studies by the time of the ASH meeting in December. Conclusion: Our results suggest that CK1δ is a key regulator of translation and a promising druggable target in lymphoma. CK1δ inhibitors, if successfully developed, will represent a new class of antineoplastic drugs with a completely new mechanism of action. Figure Disclosures Ali: VOR Biopharma: Patents & Royalties. O'Connor:Acetylon Pharma: Other: Travel expenses, Research Funding; Allos Therapeutics: Consultancy; Millenium: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Seattle Genetics, Inc.: Consultancy, Other: Travel expenses, Research Funding; Spectrum Pharma: Consultancy, Other: Travel expenses, Research Funding; Celgene: Research Funding; Novartis: Consultancy, Honoraria; Roche: Research Funding.

Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.

Silencing c-Myc Translation As a Therapeutic Strategy through Targeting PI3K Delta and CK1 Epsilon in Hematological Malignancies

Introduction: c-Myc is a master transcription factor and one of the most frequently altered genes across a vast array of human cancers including diffuse large B-cell lymphoma (DLBCL), and is thus an attractive therapeutic target . However, no direct inhibitor of c-Myc has been successfully developed for the treatment of any cancer. The c-Myc protein has a short half-life of less than 30 minutes , and the complex secondary structures in the 5' untranslated region (UTR) of MYC mRNA make its translation highly dependent on the eukaryotic translation initiation factor 4F (eIF4F) . eIF4F exists as a complex comprised of the eIF4E, eIF4A, and eIF4G subunits. eIF4E can be sequestered by 4E-BP1, which acts as a "brake" for initiation of mRNA translation . Hyper-phosphorylation of 4E-BP1, caused by upstream signals such as mTORC1, leads to release of eIF4E from 4E-BP1, assembly of the eIF4F complex, and robust mRNA translation. Surprisingly, neither FDA approved mTORC1 inhibitors nor the investigational mTORC1/mTORC2 inhibitor MLN0128 has demonstrated adequate activity in aggressive lymphoma. The therapeutic effects of mTOR inhibition in c-Myc driven aggressive lymphoma remain poorly understood.Recognizing phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating mTOR, we hypothesized that co-targeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. Since both PI3K and proteasome are proven drug targets in blood cancer, such co-targeting strategy may be expeditiously studied in clinical trials for c-Myc driven aggressive lymphoma.Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo (Promega®). The Bliss additivism model was used to determine the expected inhibition of cell growth and the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Expression of c-Myc was investigated at the translation and transcription levels, using a combination of Western blot, qPCR, and a bi-cistronic luciferase reporter we developed to study cap dependent translation. Gene expression profiling (GEP) studies were conducted using RNAseq, and analyzed by the Fisher t-test and running enrichment score (RES) between different treatment groups. Mechanisms of synergy were determined through interrogating the effects of small molecule inhibitors and shRNA targeting regulators of various regulators of 4E-BP1. Structural studies of TGR-1202 were performed by in silico docking, and validated by synthesis of novel analogs of TGR-1202. Activity of TGR-1202 on CK1 epsilon was studied by kinome profiling (Reaction Biology®), cell free kinase assay of CK1e (Promega®), and cell based assay of CK1e autophosphorylation.Results: We found that a novel PI3K delta isoform inhibitor TGR-1202, but not the approved PI3Kd inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells (Figure 1). TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of eIF4E-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc dependent transcription (Figure 2). Furthermore, the synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kd inhibitors, was active against casein kinase-1 (CK1) epsilon (Figure 3). Targeting CK1e using a selective chemical inhibitor or shRNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc.Conclusion: These results suggest that TGR-1202 is a first-in-class dual PI3Kd/CK1e inhibitor, which may in part explain the preliminary clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1e should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc. [Display omitted] [Display omitted] [Display omitted] DisclosuresLentzsch:BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria. O'Connor:Seattle Genetics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Spectrum: Research Funding; Spectrum: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding.

Abstract 2896: 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity

Abstract Taxane based chemotherapy is commonly used to treat a wide variety of solid tumors including breast, prostate, lung and ovarian. Taxanes bind to and stabilize microtubules inhibiting their function in interphase and mitosis, and leading to cancer cell apoptosis. Although taxanes are widely used clinically and their target, tubulin, is ubiquitously expressed, patient response is heterogeneous and the molecular mechanism of clinical drug resistance remains elusive. In addition to their antimitotic effects, taxanes affect several signaling pathways whose perturbation by microtubule stabilization may determine cell sensitivity or resistance. Our recent work has identified one such pathway by showing that Taxol treatment inhibits the translation of HIF-1α mRNA by inducing its sequestration to P-bodies where it is silenced. However, the signaling cascade that is initiated in response to microtubule damage leading to translation suppression of HIF-1α is not understood. Using polysome analysis we showed that Taxol treatment inhibited translation at the initiation step of protein synthesis, evidenced by the increase in the 80S peak. Post-translational modifications of various translation initiation factors are implicated in the regulation of translation initiation. Hence, we analyzed phosphorylation states of several initiation factors, including eIF4E, eIF2α, 4E-BP1, 4E-BP2 and rpS6. We didn't observe any change in the phosphorylation state of these initiation factors except for 4E-BP1. We observed that Taxol treatment led to 4E-BP1 hyper-phosphorylation, at a unique previously unrecognized, site. Using isogenic cell lines with acquired mutations at the taxane binding site we conclusively showed that microtubule disruption was required for the hyper-phosphorylation of 4E-BP1. Phosphorylation of 4E-BP1 is known to activate cap-dependent translation. Instead, our data using polysome profiling revealed that Taxol-induced 4E-BP1 hyper-phosphorylation was associated with translation suppression. We confirmed these results by performing the cap-binding assay, which demonstrated that Taxol treatment had no effect on cap-dependent translation. Taken together these results suggest a novel biological function for 4E-BP1 hyper-phosporylation. Further, treatment with the mTOR inhibitor, Torin-1, had no effect on Taxol-induced 4E-BP1 hyper-phosphorylation, suggesting mTOR pathway independence. Interestingly, treatment with the CDK inhibitor Roscovitine, prevented both Taxol-induced 4E-BP1 hyper-phosphorylation and Taxol-induced cell death. Although we do not yet know which of the three CDKs exerts the inhibitory effect on taxanes, our data suggest that there is a functional link between 4E-BP1 hyper-phosphorylation and taxane activity. We are currently investigating the role of CDK1, CDK2 and CDK5 in this pathway to decipher the novel biological function of 4E-BP1 hyper-phosphorylation. Citation Format: Prashant Khade, Paraskevi Giannakakou. 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2896.

Mitotic 4E-BP1 hyperphosphorylation and cap-dependent translation

Cap-dependent translation is a form of epigenetic regulation that is largely invisible to conventional gene expression analyses such as RNA seq and cDNA microarray. mTOR-dysregulated cap-dependent ...

Abstract 2121: Microtubule-regulated mRNA translation signaling and therapeutic implications in cancer

Abstract Drugs that affect microtubule dynamics and function, both those that stabilize microtubules (e.g. taxanes) and those that depolymerize them (e.g. vincas, Eribulin) are widely used in cancer chemotherapy. However, their mechanism of action at the molecular level and the signaling pathways regulated by microtubule dynamics are not fully understood, precluding chemotherapy customization to the individuals. Previously, we have reported that microtubule dynamics control the translation of HIF-1α mRNA. We showed that treatment with microtubule-targeting drugs (MTDs) shifts HIF-1α mRNA from actively translating polysomes to non-translating ribosomes resulting in inhibition of HIF-1α transcriptional activity. Using cell lines with acquired mutations at different MTD binding sites we conclusively showed that microtubule disruption was required for the inhibition of HIF translation. However, additional mRNAs susceptible to MTD treatment or the exact signaling pathway that senses microtubule disruption and leads to inhibition of translation are not known. Polysome RNA-Seq analyses of untreated and drug treated cells has identified additional mRNAs whose polysome association is shifted towards inactive translation similar to HIF-1α mRNA. We are using bioinformatics analyses to identify a common microtubule-susceptibility motif in addition to validation experiments at the protein and pathway level. The polysome analysis showed that protein synthesis is inhibited at the stage of translation initiation. In an effort to better understand the underlying mechanism of translation inhibition of HIF-1α mRNA and additional microtubule-dependent mRNAs, we assessed 4E-BP1 protein as its phosphorylation is a key regulator of cap dependent mRNA translation. We observed that taxanes or Eribulin treatment hyper-phosphorylates 4E-BP1 without affecting ribosomal protein S6 (rpS6) phosphorylation. Using the taxane-resistant breast cancer cell line MB-231/K20T which harbors an acquired mutation at the taxane binding site of β-tubulin we showed that Taxol treatment has no effect on 4E-BP1 hyper- phosphorylation, consistent with its lack of microtubule activity. In contrast, Eribulin, which has a distinct binding site on tubulin, is able to overcome taxol-resistance by fully engaging the microtubule-4E-BP1 axis. Interestingly, in renal cell carcinoma (RCC) cells, for which treatment with microtubule inhibitors has no clinical benefit, we didn't observe 4E-BP1 hyper-phosphorylation after taxane or Eribulin treatment, even though microtubules were affected. These results suggest that the microtubule-4E-BP1 axis is compromised in RCC and that there is a link between 4E-BP1 phosphorylation and MTD efficacy. Hence, we propose that identifying the link of 4E-BP1 dependent signaling with microtubules will have important therapeutic implications in MTD chemotherapy. Citation Format: Prashant Khade, Paraskevi Giannakakou. Microtubule-regulated mRNA translation signaling and therapeutic implications in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2121. doi:10.1158/1538-7445.AM2015-2121

Abstract 2129: Translational regulation of Id1 expression in glioma cells by the PI-3K pathway involves PPM1G-mediated dephosphorylation of 4E-BP1

Abstract Increasing evidence suggests that Id1, a helix-loop-helix transcriptional modulator that inhibits differentiation and is elevated in stem cells, is involved in the initiation and aggressive behavior of malignant glial neoplasms. Our own previous study also found that overexpression of Id1 increases the transforming potential of human glioma cells (Xu et al., Oncotarget 5:1241-52, 2014). Since a high percentage of glioblastomas show overactivity of the PI-3K pathway and a link between this signaling pathway and Id1 expression has not previously been established, we sought to determine whether PI-3K signaling regulates Id1 expression. We explored this question using multiple established glioma cell lines (including SF767, LN229, U87, U251, U118, SF539). We initially find higher basal Id1 expression in glioma cell lines with dysregulated PI-3K signaling. To determine the role of PI-3K signaling on Id1 expression, glioma cell lines with increased flux through this signaling pathway were further assessed. In each case, forced expression of wild-type PTEN or treatment with either the PI-3K inhibitor LY294002 or the AKT inhibitor MK2206 leads to lower Id1 expression at the protein but not mRNA level. Pulse-chase assay and polyribosome profile analysis of Id1 mRNA confirm that inhibition of the PI-3K pathway reduces Id1 translation but not protein stability. The PI-3K pathway is known to regulate protein translation through activation of TORC1 leading to hyper-phosphorylation of 4E-BP1, a form of this protein that can no longer bind and inhibit the translation initiation factor eIF4E. Interestingly, while inhibition of PI-3K and AKT reduces 4E-BP1 phosphorylation and expression of Id1 in all cases, inhibition of TORC1 with rapamycin did not consistently have a similar effect. These results suggest a PI-3K/AKT-dependent, mTOR-independent mechanism for increasing Id1 translation. In fact, we identify a potential mechanism for this regulation of Id1 protein expression involving the serine-threonine phosphatase PPM1G. PPM1G and 4E-BP1 are associated by co-immunoprecipitation indicating interactions in the cell. In addition, knockdown of PPM1G expression by siRNA increases both 4E-BP1 phosphorylation and Id1 expression. Furthermore, PPM1G is not only phosphorylated but this phosphorylation is regulated by PI-3K activity. Finally, we find that PI-3K-dependent phosphorylation of PPM1G reduces its activity using an in vitro phosphatase assay and speculate that this regulation of PPM1G directly increases 4E-BP1 phosphorylation and activation of Id1 translation. In conclusion, our findings provide the first evidence that the PI-3K/AKT signaling pathway translationally regulates Id1 expression through modulation of PPM1G activity, possibly by phosphorylation, altering the balance between hyper- and hypo-phosphorylated 4E-BP1. Citation Format: Kaiming Xu, Lanfang Wang, Hui-Kuo G. Shu. Translational regulation of Id1 expression in glioma cells by the PI-3K pathway involves PPM1G-mediated dephosphorylation of 4E-BP1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2129. doi:10.1158/1538-7445.AM2015-2129

Total 4EBP1 Is Elevated in Liver of Rats in Response to Low Sulfur Amino Acid Intake

Translation initiation is known to be regulated by the binding of eukaryotic initiation factor 4E (eIF4E) by binding proteins (4EBPs), and there is evidence that amino acid deprivation and other cellular stresses upregulate 4EBP1 expression. To pursue the question of whether diets limited in an essential amino acid lead to induction of 4EBP1 expression in vivo, diets that varied in methionine and cystine content were fed to rats for 7 days, and 4EBP1 mRNA and protein levels and 4EBP1 phosphorylation state were determined. Total 4EBP1 mRNA and protein abundance increased in liver of rats with severely deficient intakes of sulfur amino acids (0.23% or 0.11% methionine without cystine) but not in animals with a less restricted intake of sulfur amino acids (0.11% methionine plus 0.35% cystine) but a similarly restricted intake of total diet (53 to 62% of control). The amount of 4EBP1 binding activity (α + β forms) was elevated in liver of rats fed sulfur amino acid-deficient diets, whereas the hyperphosphorylation of 4EBP1 was not affected by dietary treatment. Results suggest that changes in total 4EBP1 expression should be considered when examining mechanisms that attenuate protein synthesis during amino acid deficiency states.

Abstract 4195: Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E-BP1

Abstract Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Deregulation of translation initiation leads to tumor development and progression. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Hyperphosphorylation of 4E-BP1, which leads to inactivation of 4E-BP1, has been linked to human cancers with increased malignancy. While it has been demonstrated previously that the phosphosphorylation of 4E-BP1 is controlled by mTOR in the mTOR complex 1 (mTORC1), the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1 through an shRNA library screen of Ser/Thr protein phosphatases. Co-immunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells, and purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 and SW480 cells results in an increase in the phosphorylation of 4E-BP1 at both Thr37/46 and Ser65 sites. This PPM1G-mediated dephosphorylation of 4E-BP1 is specific as the phosphorylation of upstream regulators of 4E-BP1 including Akt and mTOR is not altered in PPM1G knockdown cells. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mTOR inhibitor treatment is significantly slowed down in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when expression of PPM1G is depleted. As the result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of protein translation by negatively controlling the phosphorylation of 4E-BP1. Thus, PPM1G may serve as a tumor suppressor by directly counteracting mTOR to inhibit cell growth in cancer. 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 4195. doi:1538-7445.AM2012-4195

Translation Regulatory Protein 4E-Binding Protein (BP)-1 Phosphorylation Identifies Diffuse Large B Cell Lymphoma Patients with Low IPI Scores Who Experience Short Survival After Chemotherapy.

Abstract 1925Poster Board I-948Eukaryotic initiation factor 4E binding protein 1 (4E-BP1) regulates the function of eukaryotic initiation factor 4E (eIF-4E) which mediates nucleo-cytoplasmic transport and cap-dependent translation of specific transcripts. eIF-4E expression itself is elevated in several carcinomas and hematopoietic malignancies. Hypophosphorylated 4E-BP1 binds eIF-4E and inhibits cap-dependent translation. Phosphorylated 4E-BP1 dissociates from eIF-4E, thereby allowing translation of several cap-dependent transcripts responsible for cell cycling, survival and angiogenesis. Hyper-phosphorylation of 4E-BP1 may thus be associated with oncogenesis and the malignant phenotype; its influence on the response to chemotherapy and prognosis of diffuse large B cell lymphoma (DLBCL) is unknown. We examined diagnostic tissues from 85 patients with DLBCL for 4E-BP1 expression and phosphorylation, and correlated these with clinical outcomes. Samples were stained for total and phosphorylated 4E-BPI using immunohistochemistry (IHC). Slides were scored for percent cells positive for the two markers (0% to 100%) and the intensity of expression (0 to 3+) by hematopathologists (A.R. and H.M.) blinded to the clinical data. An Expression Score was calculated from their product ([% cells positive × intensity]/10), thus ranging from 0-30. In parallel, expression of bcl-2 was determined by IHC on the same samples. Patients had a median age of 75 years (range, 25-91). The majority received CHOP-based chemotherapy with/without rituximab. Total 4E-BP1 was expressed uniformly (by 97% of the cases). However, there was marked heterogeneity in the level of phosphorylation of 4E-BP1 (none [Expression Score = 0] in 35%, uniformly high [Expression Score = 30] in 34%, and variable [Expression Score = 1-29] in the remainder [31%]). We therefore examined if the level of phosphorylation of 4E-BP1 varied with clinical features at diagnosis, and whether it correlated with response to chemotherapy and survival. The level of expression or phosphorylation of 4E-BP1 did not correlate with the clinical stage, IPI score or bcl-2 expression at diagnosis, and did not influence the likelihood of achieving complete remission with CHOP-based chemotherapy. However, in patients with low IPI scores (IPI 0-2), the level of phosphorylation of 4E-BP1 correlated significantly with overall survival (OS). In these patients, the OS was significantly different (P = 0.031) in patients with low levels of phosphorylation of 4E-BP1 (Expression Score = 0-15) compared to those with higher levels of 4E-BP1 phosphorylation (Expression Score = 16-30). The median survival was >146.4 months vs 20.6 months, and the 5-year survival was 73% vs 31%, in patients with low and high levels of 4E-BP1 phosphorylation, respectively. The difference in survival between the two groups was not a consequence of differences in age or IPI score at diagnosis or inclusion of rituximab in the treatment regimen. The level of phosphorylation of 4E-BP1 did not have any further impact on the survival of patients with high IPI scores (IPI 4-5). We conclude that while the large majority of DLBCL express the translation regulatory protein 4E-BP1, the level of phosphorylation (indicative of biological activity) of this protein varies widely. Importantly, the level of phosphorylation of 4E-BP1 further stratifies DLBCL patients who would be expected to have a favorable prognosis based on low IPI scores, and identifies a subset of these patients who do poorly following standard CHOP-based chemotherapy. Since hyper-phosphorylation of 4E-BP1 increases the activity of eIF-4E, this study provides the rationale for trials incorporating novel targeted agents that inhibit eIF-4E into the therapeutic regimen, specifically in DLBCL patients with low IPI scores and high levels of 4E-BP1 phosphorylation. Disclosures:No relevant conflicts of interest to declare.

Inhibition of mammalian target of rapamycin (mTOR) signalling in C2C12 myoblasts prevents myogenic differentiation without affecting the hyperphosphorylation of 4E-BP1

Current accepted models suggest that hypophosphorylated 4E-binding protein (4E-BP1) binds to initiation factor 4E (eIF4E) to inhibit cap-dependent translation, a process readily reversed by its phosphorylation following activation of mammalian target of rapamycin (mTORC1) signalling. Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Here we show that myogenic differentiation is associated with increased rates of translation, the up-regulation of both 4E-BP1 mRNA and protein levels and enhanced levels of eIF4E/4E-BP1 complex. Paradoxically, treatment of C2C12 myoblasts with an inhibitor of mTOR signalling (RAD001) which inhibits translation, promotes the hyperphosphorylation of 4E-BP1 on novel sites and prevents the increase in 4E-BP1 levels. In contrast, eIF4E appears to be under translational control with a significant delay between induction of mRNA and subsequent protein expression.

Adenovirus-mediated eukaryotic initiation factor 4E binding protein-1 in combination with rapamycin inhibits tumor growth of pancreatic ductal adenocarcinoma in vivo

Over-expression of eIF4E indicates a poor prognosis in different tumors. In the present study, we investigated the frequency of eIF4E, 4E-BP1 and phosphorylated 4E-BP1 expression in PDAC cell lines, gastric carcinoma (GC) cell lines and human embryonic pancreatic cells, as well as gene therapy using translation repressor gene 4E-BP1 in combination with the mTOR inhibitor rapamycin. We also assessed the significance of eIF4E expression in 80 PDAC cases. Combination therapy of adenovirus vector-delivered 4E-BP1 gene and rapamycin was administered to determine their growth inhibition effect in vitro and in vivo in mice. Our study revealed that all PDAC cell lines, GC cell lines and human embryonic pancreas-derived cells expressed the 25-kDa eIF4E protein (MIAPaca-2 cells also expressed the 13-kDa protein 4E-BP1). The 80 PDAC specimens showed a heterogeneous pattern of eIF4E staining. No significant correlation between eIF4E expression and TNM classification was found. Adenovirus vectors Ad-4E-BP1 and Ad-GFP efficiently showed transgenic expression with hyperphosphorylation of 4E-BP1; however, insignificant growth inhibition of the PDAC and GC cell lines was observed. Combination therapy with rapamycin significantly inhibited proliferation and tumor growth in vitro as well as in vivo. Therefore, combination of Ad 4E-BP1 and rapamycin may be a more effective adjuvant therapy.

Expression of the cap-mediated translation pathway in non-Hodgkins lymphomas: diagnostic and therapeutic implications

8595 Background: The eukaryotic initiation factor (eIF) complex controls cap-mediated mRNA translation. This plays a key role in cancer by mediating expression of proteins critical for cell growth, transformation and tumorigenesis (e.g., cyclin D1, survivin, c-myc, Bcl-2 and VEGF). eIF4 complex (eIF4E, eIF4G and eIF4A) activity is inhibited by eIF4E binding protein-1 (4E-BP1) which thus has tumor suppressor activity. Hyper-phosphorylated 4E-BP1 is unable to bind eIF4E and promotes translation initiation and protein synthesis. Methods: We analyzed eIF complex expression in B-cell non-Hodgkins lymphomas (NHL) using immunohistochemistry (IHC) for total 4E-BP1 and Western immunoblotting (WB) for eIF4G (total), eIF4E and 4E-BP1 (total and phosphorylated). IHC was performed on 37 NHL (9 follicular [FL], 9 diffuse large cell [DLCL], 2 Burkitt’s, 5 mantle cell, 5 extranodal marginal zone/lymphoplasmacytoid, 7 small lymphocytic) and 8 reactive tissues (6 nodes, 2 tonsils). Staining intensity was graded from 0 to 3+. WB was performed on 6 NHL (2 FL, 4 DLCL) and 2 reactive tissues. Results: IHC showed a consistently high level (2–3+) of cytoplasmic 4E-BP1 expression in malignant cells in 36 of 37 NHL samples. In contrast, there was marked regional specificity of cytoplasmic expression in reactive tissues (3+ in the mantle and marginal zone cells but absent/minimal (0- 1+) in germinal centers and T cells). Nuclear expression was seen in 4 NHL samples but not in reactive tissues. Incipient/focal involvement by FL showed 3+ staining in neoplastic follicles. WB showed that phosphorylated:total 4E-BP1 ratio was increased (2-fold over reactive samples) in 3 of 4 DLCL, but not in FL. The levels of expression of eIF4G, total and phospho-eIF4E and total 4E-BP1 were comparable in reactive and NHL samples, as expected in whole tissue lysates. Conclusion: (1) 4E-BP1 detection by IHC offers a sensitive diagnostic tool for distinguishing reactive follicles from neoplastic B-cell proliferations. (2) 4E-BP1 hyper-phosphorylation may be a mechanism that can increase expression of growth and proliferation inducing proteins in aggressive NHL. (3) These findings provide the rationale for evaluating cap-dependent translation inhibition as a novel therapeutic strategy in NHL. No significant financial relationships to disclose.

Effect of branched-chain amino acids on muscle atrophy in cancer cachexia.

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.

Cell hydration and mTOR‐dependent signalling

Insulin- and amino acid-induced signalling by the mammalian target of rapamycin (mTOR) involves hyperphosphorylation of the p70 ribosomal S6 protein kinase (p70S6-kinase) and the eukaryotic initiation factor 4E (eIF4E) binding protein 4E-BP1 and contributes to regulation of protein metabolism. This review considers the impact of cell hydration on mTOR-dependent signalling. Although hypoosmotic hepatocyte swelling in some instances activates p70S6-kinase, the hypoosmolarity-induced proteolysis inhibition in perfused rat liver is insensitive to mTOR inhibition by rapamycin. Likewise, swelling-dependent proteolysis inhibition by insulin and swelling-independent proteolysis inhibition by leucine, a potent activator of p70S6-kinase and 4E-BP1 hyperphosphorylation, in perfused rat liver is insensitive to rapamycin, indicating that at least rapamycin-sensitive mTOR signalling is not involved. Hyperosmotic dehydration in different cell types produces inactivation of signalling components around mTOR, thereby attenuating insulin-induced glucose uptake, glycogen synthesis, and lipogenesis in adipocytes, and MAP-kinase phosphatase MKP-1 expression in hepatoma cells. Direct inactivation of mTOR, stimulation of the AMP-activated protein kinase, and the destabilization of individual proteins may impair mTOR signalling under dehydrating conditions. Further investigation of the crosstalk between the mTOR pathway(s) and hyperosmotic signalling will improve our understanding about the contribution of cell hydration changes in health and disease and will provide further rationale for fluid therapy of insulin-resistant states.