Protein eIF4G Research Articles

Abstract IA17: Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma

Abstract mTORC1 signaling is elevated in most lymphoid malignancies and frequently is associated with poor prognosis. Targeting mTORC1 with rapalogs or mTOR kinase inhibitors (TOR-KIs) are two strategies that both have limitations. Rapalogs are weak inducers of apoptosis, mainly due to incomplete mTORC1 inhibition, resulting in reduced phosphorylation of some substrates such as S6K1, but not 4E-BP1. TOR-KIs inhibit mTORC1 more completely, but also inhibit mTORC2, which likely contributes to dose-limiting toxicities in TOR-KI clinical trials. We have evaluated alternative strategies to target mTORC1 and downstream survival pathways in B-cell leukemia and lymphoma. In one approach, we have used novel bi-steric inhibitors of mTORC1, generated using our RevBlocksTM modular synthesis platform. These inhibitors engage both the FRB- and ATP-binding sites of mTOR and exhibit potent and selective inhibition of mTORC1. We found that RM-001, a proprietary bi-steric compound ~13-fold-selective for mTORC1 over mTORC2 in vitro, enhances the efficacy of the BCR-ABL1 inhibitor dasatinib in Philadelphia chromosome-positive pre-B cell acute lymphoblastic leukemia (Ph+ B-ALL) cell lines, causing profound and sustained phosphorylation inhibition of 4E-BP1 and S6, cell growth inhibition, and cell cycle arrest. Once-weekly dosing with RM-001 (10 mg/kg, IP) in a Ph+ B-ALL mouse model was well tolerated and effectively reduced leukemic burden as a single agent. A corresponding inhibition of phosphorylation of 4E-BP1 and S6 was observed in bone marrow. A key downstream effector pathway of mTORC1 is formation of the eIF4F translation initiation complex, which consists of eIF4E, eIF4G, and eIF4A. To assess targeting eIF4F as an alternative approach, we evaluated the function of eIF4F in both tumors and normal cells of the B lymphocyte lineage. In both B-ALL and diffuse large B-cell lymphoma (DLBCL) cells, disruption of eIF4F with a constitutively active 4E-BP1 mutant phenocopied the effect of TOR-KIs and bi-steric inhibitors. Notably, reduced gene dosage of Eif4e (eIF4E+/-) impaired the initiation and maintenance of B-cell transformation without affecting normal B-cell development and function. Most existing chemical inhibitors of eIF4F target either the cap-binding protein eIF4E or the RNA helicase eIF4A. We tested a novel eIF4G-binding molecule, SBI-756, shown previously to overcome resistance to BRAF inhibitors in melanoma models. SBI-756 (250 – 500 nM) disrupted eIF4F formation and caused marked cytotoxic effects in B-ALL, DLBCL, and normal B lymphocytes, while sparing T cells and NK cells. SBI-756 also synergized with targeted agents including dasatinib in Ph+ B-ALL and venetoclax in DLBCL. The combination of SBI-756 with venetoclax was more effective than single agents in a DLBCL xenograft model. These findings establish proof of concept for molecules that selectively bind to the scaffolding protein eIF4G. Collectively, these data highlight distinct strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma. Citation Format: Lee-or Herzog, Bianca J. Lee, Thanh-Trang Vo, Honyin Chiu, Sharmila Mallya, Amos Fung, Mallika Singh, James Aggen, Nidhi Tibrewal, Jacqueline A.M. Smith, David Wildes, Ze'ev Ronai, Davide Ruggero, David A. Fruman. Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA17.

Abstract 6507: Inhibition of the translation initiation factor eIF4A1 enhances tumor cell radiosensitivity

Abstract The cellular radioresponse is regulated by constitutively expressed proteins and by new gene expression. We have previously shown that radiation induces changes in gene expression primarily via translational control. Regulation of gene translation largely occurs at the initiation stage, and many factors that regulate initiation impact the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4F is comprised of the cap-binding protein eIF4E, the RNA helicase eIF4A, and the scaffold protein eIF4G. Because eIF4A is considered to regulate the translation of mRNAs involved in cell survival and proliferation, we investigated it as a target for tumor cell radiosensitization. Knockdown of eIF4A1 in the U251 glioblastoma and the HeLa cervical carcinoma cell lines resulted in a decrease in translational efficiency as determined by polysome profiling and an increase in cellular radiosensitivity as determined by clonogenic survival analysis. There was also an increase in γH2AX foci remaining at 24h after irradiation, consistent with an inhibition of DNA double strand break repair. These results suggest that eIF4A serves as a determinant of cellular radiosensitivity. As an additional approach to inhibiting eIF4A, we utilized the rocaglate hydroxamate (-)-SDS-1-021, which prevents eIF4A inclusion into eIF4F. Treatment of U251, HeLa and PSN1 pancreatic carcinoma cells with 10 nM (-)-SDS-1-021 resulted in decreased translational efficiency, which was accompanied by a decrease in protein synthesis. Treatment of the tumor cell lines with (-)-SDS-1-021 immediately before radiation resulted in an increase in radiosensitivity as well as an increase in γH2AX foci remaining at 24h, similar to eIF4A1 knockdown. Importantly, whereas treatment of the normal human fibroblast cell line MRC9 with (-)-SDS-1-021 also inhibited translation and protein synthesis, there was no effect on radiosensitivity. To gain a better understanding of the mechanism of radiosensitization by (-)-SDS-1-021, RNAseq was performed using polysome-bound mRNAs isolated from U251 and MRC9 cells. Cells were treated with 10 nM (-)-SDS-1-021, immediately irradiated (2Gy), and then harvested six hours post-irradiation. In U251 cells treated with 2Gy alone, 192 genes were translationally upregulated. The upregulation of 160 of these genes was inhibited by (-)-SDS-1-021 in the combination treatment group. Similarly, in MRC9 cells, 322 genes were translationally upregulated in response to radiation, and the upregulation of 173 of these was inhibited in the combination treatment group. By Ingenuity Pathway Analysis, the radiation-induced, (-)-SDS-1-021-sensitive genes in U251 were significantly enriched for proteins involved in DNA replication, recombination, and repair, which is in contrast to the MRC9 (-)-SDS-1-021 genes. Altogether, these findings suggest that eIF4A1 is a viable target for tumor radiosensitization. Citation Format: Stacey L. Lehman, Theresa Wechsler, Kayla R. Schwartz, Amy Wahba, Lauren E. Brown, John A. Porco, Jerry Pelletier, Kevin Camphausen, Philip J. Tofilon. Inhibition of the translation initiation factor eIF4A1 enhances tumor cell radiosensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6507.

Should I Stay or Should I Go: eIF3 Remains Ribosome Associated and Is Required for Elongation

Wagner etal. (2020), Bohlen etal. (2020), and Lin etal. (2020) use Sel-TCP-seq or selective ribosome profiling to gain insights into mRNA translation initiation, highlighting distinctions between yeast and higher eukaryotes and a role for eIF3 in elongation.

Genome Editing of eIF4E1 in Tomato Confers Resistance to Pepper Mottle Virus.

Many of the recessive virus-resistance genes in plants encode eukaryotic translation initiation factors (eIFs), including eIF4E, eIF4G, and related proteins. Notably, eIF4E and its isoform eIF(iso)4E are pivotal for viral infection and act as recessive resistance genes against various potyviruses in a wide range of plants. In this study, we used Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis to test whether novel sequence-specific mutations at eIF4E1 in Solanum lycopersicum (tomato) cv. Micro-Tom could confer enhanced resistance to potyviruses. This approach produced heritable homozygous mutations in the transgene-free E1 generation. Sequence analysis of eIF4E1 from E0 transgenic plants expressing Cas9 and eIF4E-sgRNA transcripts identified chimeric deletions ranging from 11 to 43 bp. Genotype analysis of the eIF4E1-edited lines in E0, E1, and E2 transgenic tomato plants showed that the mutations were transmitted to subsequent generations. When homozygous mutant lines were tested for resistance to potyviruses, they exhibited no resistance to tobacco etch virus (TEV). Notably, however, several mutant lines showed no accumulation of viral particles upon infection with pepper mottle virus (PepMoV). These results indicate that site-specific mutation of tomato eIF4E1 successfully conferred enhanced resistance to PepMoV. Thus, this study demonstrates the feasibility of the use of CRISPR/Cas9 approach to accelerate breeding for trait improvement in tomato plants.

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergooncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Translation initiation factors GleIF4E2 and GleIF4A can interact directly with the components of the pre-initiation complex to facilitate translation initiation in Giardia lamblia

Translation initiation factor eIF4F is essential for cap-dependent translation initiation in eukaryotes. eIF4F is a trimeric complex consisting of a scaffold protein eIF4G, cap-binding protein eIF4E and DEAD-box RNA helicase eIF4A. eIF4F binds to the 5’ cap structure of the mRNA through eIF4E and facilitates the binding of the preinitiation complex (PIC) via protein-protein interactions of eIF4G with eIF3 in mammals or with eIF5 in yeast. Initiation factor eIF4A is known to unwind the secondary structures of the 5’UTRs encountered by the PIC during its initial binding to the mRNA and while scanning for the initiation codon. In Giardia, homologs for eIF4E (GleIF4E2) and eIF4A (GleIF4A) have been identified but not for eIF4G. To address how PIC is recruited to the 5’ end of mRNA in the absence of eIF4G homolog, we have used yeast two-hybrid assays to identify potential interactions of GleIF4E2 with the components of the PIC. The results show that GleIF4E2 can interact with the β subunit of the initiation factor GleIF2, a component of the PIC. ZDOCK modeling of the GleIF4E2-GleIF2β complex revealed that the dorsal side of GleIF4E2 is likely involved in binding to GleIF2β, which mimics the interaction of mammalian eIF4E with eIF4G, and with eIF4E binding proteins. These results suggest that GleIF4E2 can facilitate the recruitment of the PIC to the 5’end of the mRNA by binding directly to the components of the PIC. The role of GleIF4A in translation initiation in Giardia is not clearly understood as the short 5’ UTRs of the mRNA are unlikely to form secondary structures. Interestingly, Pateamine A, a specific inhibitor of human eIF4A, inhibited the growth of Giardia in a dose-dependent manner, suggesting that the activity of GleIF4A is probably required for translation. Using yeast two-hybrid assays, we have identified a novel interaction of GleIF4A with i subunit of the initiation factor GleIF3 (GleIF3i), another component of the PIC. These results indicate that the GleIF4A can also interact directly with the components of the PIC. ZDOCK modeling of the GleIF3i-GleIF4A complex suggests that GleIF3i could serve as a stimulator of GleIF4A activity.

Mitosis-related phosphorylation of the eukaryotic translation suppressor 4E-BP1 and its interaction with eukaryotic translation initiation factor 4E (eIF4E)

Eukaryotic translation initiation factor 4E (eIF4E)–binding protein 1 (4E-BP1) inhibits cap-dependent translation in eukaryotes by competing with eIF4G for an interaction with eIF4E. Phosphorylation at Ser-83 of 4E-BP1 occurs during mitosis through the activity of cyclin-dependent kinase 1 (CDK1)/cyclin B rather than through canonical mTOR kinase activity. Here, we investigated the interaction of eIF4E with 4E-BP1 or eIF4G during interphase and mitosis. We observed that 4E-BP1 and eIF4G bind eIF4E at similar levels during interphase and mitosis. The most highly phosphorylated mitotic 4E-BP1 isoform (δ) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-γ, phosphorylated at Thr-70, Ser-83, and Ser-101, bound to eIF4E during mitosis. Two-dimensional gel electrophoretic analysis corroborated the identity of the phosphorylation marks on the eIF4E-bound 4E-BP1 isoforms and uncovered a population of phosphorylated 4E-BP1 molecules lacking Thr-37/Thr-46–priming phosphorylation. Moreover, proximity ligation assays for phospho-4E-BP1 and eIF4E revealed different in situ interactions during interphase and mitosis. The eIF4E:eIF4G interaction was not inhibited but rather increased in mitotic cells, consistent with active translation initiation during mitosis. Phosphodefective substitution of 4E-BP1 at Ser-83 did not change global translation or individual mRNA translation profiles as measured by single-cell nascent protein synthesis and eIF4G RNA immunoprecipitation sequencing. Mitotic 5′-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis.

Abstract 4391: Disruption of the translation initiation complex by eIF4A mutant inhibits prostate tumor growth in vivo

Abstract Translation initiation in cancer cells is the rate-limiting step of oncogenic mRNAs translation/protein synthesis and is primarily mediated by the eukaryotic translation initiation factor 4 F (eIF4F) complex that consists of three subunits: eIF4E, which binds the cap of mRNA; eIF4A, an RNA helicase implicated in unwinding mRNA structure; and a scaffold protein eIF4G. Our prior crystal structure determination of human eIF4A in complex with Heat 1 domain of eIF4GI (722-999) revealed that eIF4G interacts with both N- and C-terminal domains of human eIF4A (eIF4A1-NTD and eIF4A1-CTD). Utilizing structural information gained from this complex, we generated a series of eIF4A mutants (eIF4A1-NTD: E41K, R45D, Y48A and eIF4A1-CTD: T271A, R324D, D296A/T298A) in order to identify the crucial site(s) of interaction with eIF4G Heat 1 domain. Using in vitro binding studies, we demonstrated that amongst various mutants tested, eIF4A-CTD (eIF4A1R324D) mutant disrupted the eIF4A/eIF4G Heat 1 complex formation, mirroring the binding residue E809K mutant of eIF4G that also interrupted eIF4A/eIF4G Heat 1 interaction. Given that eIF4G has an additional eIF4A-binding site named Heat 2 domain, we further investigated whether eIF4A1R324D mutant is able to disrupt binding to full-length eIF4G protein in vivo. Surprisingly, overexpression of Flag-tagged eIF4A1R324D in prostate cancer cell line blocked interaction with exogenous eIF4G as well as endogenous eIF4G. Taken together, these results strongly suggest that eIF4A1R324 residue plays a crucial role in mediating eIF4A/eIF4G complex formation in vivo. Furthermore, overexpression of eIF4A1R324D mutant significantly blocked oncogene c-myc and Bcl-2 mRNA translation thereby inhibiting prostate tumor growth in vivo. To conclude, our studies provide a previously unexplored strategy to identify small-molecule compounds disrupting eIF4A-eIF4G complex by targeting the pocket spanning eIF4AR324 and its binding site residues on eIF4G for cancer therapy. Citation Format: Puja Singh, Hanyong Chen, Hanxuan Li, Young-In Chi, Bin Liu, Junxuan Lu, Wei Li, Zigang Dong, Yibin Deng. Disruption of the translation initiation complex by eIF4A mutant inhibits prostate tumor growth in vivo [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 4391.

Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome.

The 4G family of eukaryotic mRNA translation initiation factors is composed of three members (eIF4GI, eIF4GII, and DAP5). Their specific roles in translation initiation are under intense investigations, but how their respective intracellular amounts are controlled remains poorly understood. Here we show that eIF4GI and eIF4GII exhibit much shorter half-lives than that of DAP5. Both eIF4GI and eIF4GII proteins, but not DAP5, contain computer-predicted PEST motifs in their N-termini conserved across the animal kingdom. They are both sensitive to degradation by the proteasome. Under normal conditions, eIF4GI and eIF4GII are protected from proteasomal destruction through binding to the detoxifying enzyme NQO1 [NAD(P)H:quinone oxidoreductase]. However, when cells are exposed to oxidative stress both eIF4GI and eIF4GII, but not DAP5, are degraded by the proteasome in an N-terminal-dependent manner, and cell viability is more compromised upon silencing of DAP5. These findings indicate that the three eIF4G proteins are differentially regulated by the proteasome and that persistent DAP5 plays a role in cell survival upon oxidative stress.

Translational Control of Canonical and Non-Canonical Translation Initiation Factors at the Sea Urchin Egg to Embryo Transition.

Sea urchin early development is a powerful model to study translational regulation under physiological conditions. Fertilization triggers an activation of the translation machinery responsible for the increase of protein synthesis necessary for the completion of the first embryonic cell cycles. The cap-binding protein eIF4E, the helicase eIF4A and the large scaffolding protein eIF4G are assembled upon fertilization to form an initiation complex on mRNAs involved in cap-dependent translation initiation. The presence of these proteins in unfertilized and fertilized eggs has already been demonstrated, however data concerning the translational status of translation factors are still scarce. Using polysome fractionation, we analyzed the impact of fertilization on the recruitment of mRNAs encoding initiation factors. Strikingly, whereas the mRNAs coding eIF4E, eIF4A, and eIF4G were not recruited into polysomes at 1 h post-fertilization, mRNAs for eIF4B and for non-canonical initiation factors such as DAP5, eIF4E2, eIF4E3, or hnRNP Q, are recruited and are differentially sensitive to the activation state of the mechanistic target of rapamycin (mTOR) pathway. We discuss our results suggesting alternative translation initiation in the context of the early development of sea urchins.

The Role of the Eukaryotic Translation Initiation Factor 4E (eIF4E) in Neuropsychiatric Disorders.

Protein synthesis in eukaryotic cells is a complex, multi-step and tightly regulated process. Translation initiation, the rate limiting step in protein synthesis, is dependent on the activity of eukaryotic translation Initiation Factor 4E (eIF4E). eIF4E is the cap-binding protein which, in synergy with proteins such as the helicase eIF4A and the scaffolding protein eIF4G, binds to mRNA, allowing the recruitment of ribosomes and translation initiation. The function of eIF4E is tightly regulated in cells under normal physiological conditions and can be controlled by post-translational modifications, such as phosphorylation, and by the binding of inhibitory proteins, including eIF4E binding proteins (4E-BPs) and CYFIP1. Recent studies have highlighted the importance of eIF4E in normal or aberrant function of the nervous system. In this mini-review, we will highlight the role of eIF4E function and regulation in the pathophysiology of neurodevelopmental and neuropsychiatric disorders.

Abstract 1886: A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy

Abstract The purpose of this project is to investigate the feasibility of using small molecules targeting cap-dependent translation to sensitize B cell lymphoma cells to killing by BCL-2 inhibitors (venetoclax/ ABT-199). We hypothesized that diffuse large B-cell lymphoma (DLBCL) cell survival requires cap-dependent translation, which is facilitated by eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interaction and is promoted by mTOR complex 1 (mTORC1). We used the compound SBI-756, a novel inhibitor targeting the scaffolding protein eIF4G (1). SBI-756 is more potent and selective than previously reported compounds targeting the eIF4E cap-binding protein, or compounds targeting eIF4A helicase. By treating DLBCL cells (germinal center B-cell subtype) with SBI-756 (sub μM concentrations), we discovered profound synergistic induction of apoptosis when combined with venetoclax. Cell viability was reduced even more compared to mTOR kinase inhibitor (TOR-KI) treatment combination. Moreover, SBI-756 was found to reduce the viability (IC50 = 653nM) of VAL lymphoma cells lacking 4E-BP1, while VAL were insensitive to TOR-KI treatment. By using Proximity Ligation Assay (PLA) we showed that SBI-756 treatment prevents eIF4E-eIF4G association in intact lymphoma cells. Dual luciferase assays validated this and indicated a dose-dependent reduction in cap-dependent translation following SBI-756 treatment. Also, Western blot analyses confirmed that SBI-756 treatment did not change mTOR substrates' phosphorylation, indicating that the SBI-756 effect is specific to preventing the eIF4E-eIF4G interaction. Furthermore, SBI-756 treatment reduced polysome formation with a corresponding increase in abundance of monosomes. Lastly, induced expression of 4E-BP1 mutant, which irreversibly binds eIF4E, sensitized DLBCL cells to venetoclax treatment to a similar extent as TOR-KI treatment. Ongoing studies will investigate: (1) The molecular mechanism by which translation inhibition by SBI-756 synergizes with venetoclax to prime DLBCL cells toward apoptosis, and (2) pharmacokinetic and pharmacodynamic effects of venetoclax and SBI-756 treatment on DLBCL progression in vivo. Hence, this project highlights a novel combination for treatment of aggressive lymphomas, and establishes its efficacy using preclinical models. Reference: 1. Feng Y., et al. SBI-0640756 attenuates the growth of clinically unresponsive melanomas by disrupting the eIF4F translation initiation complex. Cancer Res 2015;75(24):OF1-8. Citation Format: Lee-or Herzog, Nancy Nguyen, Honyin Chiu, Sharmila Mallya, Ze'ev A. Ronai, David A. Fruman. A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1886.

AN EIF4G-RECRUITING APTAMER INCREASES THE FUNCTIONALITY OF IN VITRO TRANSCRIBED MRNA

Background: As a versatile and safe vector, in vitro transcribed messenger RNA (ivt mRNA) is currently being intensively evaluated as an active pharmaceutical ingredient. Its therapeutic uses encompass vaccination, cell reprogramming, genome engineering, gene complementation and the expression of protein drugs (e.g., growth factors or antibodies). 
 Objectives: The therapeutic efficacy of ivt mRNA correlates with the efficacy of its translation. Untranslated regions (UTRs) from stable mRNA, such as globin mRNA, and optimized 5’ cap structures have been used to improve the functionality of ivt mRNA. However, the recruitment of the eukaryotic initiation factor 4E (eIF4E) protein to the 5’ end of transfected ivt mRNA remains a ratelimiting parameter for translation. 
 Method: We added aptamer sequences that bind the eiF4G protein to the 5’ UTR of ivt mRNA. 
 Results: One of the tested aptamer sequences produced a several fold increase in ivt mRNA expression (specifically, an increase of threefold to over tenfold depending on mRNA sequence and cell type). 
 Conclusion: This simple modification of the 5’ UTR of ivt mRNA may represent an efficacious and general method for improving the therapeutic index of all new mRNA-based therapeutic products.

Epstein-Barr Virus Protein EB2 Stimulates Translation Initiation of mRNAs through Direct Interactions with both Poly(A)-Binding Protein and Eukaryotic Initiation Factor 4G.

Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, which was previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (CBC and eukaryotic initiation factor 4F [eIF4F], respectively) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given messenger ribonucleoparticle (mRNP). Interestingly, such an effect can be obtained only if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrate the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm.IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNAs of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein, EB2 in the case of Epstein-Barr virus, is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 first is recruited to the mRNA cap structure in the nucleus and then interacts with the proteins eIF4G and PABP to enhance the initiation step of translation.

Viral and cellular mRNA-specific activators harness PABP and eIF4G to promote translation initiation downstream of cap binding

Regulation of mRNA translation is a major control point for gene expression and is critical for life. Of central importance is the complex between cap-bound eukaryotic initiation factor 4E (eIF4E), eIF4G, and poly(A) tail-binding protein (PABP) that circularizes mRNAs, promoting translation and stability. This complex is often targeted to regulate overall translation rates, and also by mRNA-specific translational repressors. However, the mechanisms of mRNA-specific translational activation by RNA-binding proteins remain poorly understood. Here, we address this deficit, focusing on a herpes simplex virus-1 protein, ICP27. We reveal a direct interaction with PABP that is sufficient to promote PABP recruitment and necessary for ICP27-mediated activation. PABP binds several translation factors but is primarily considered to activate translation initiation as part of the PABP-eIF4G-eIF4E complex that stimulates the initial cap-binding step. Importantly, we find that ICP27-PABP forms a complex with, and requires the activity of, eIF4G. Surprisingly, ICP27-PABP-eIF4G complexes act independently of the effects of PABP-eIF4G on cap binding to promote small ribosomal subunit recruitment. Moreover, we find that a cellular mRNA-specific regulator, Deleted in Azoospermia-like (Dazl), also employs the PABP-eIF4G interaction in a similar manner. We propose a mechanism whereby diverse RNA-binding proteins directly recruit PABP, in a non-poly(A) tail-dependent manner, to stimulate the small subunit recruitment step. This strategy may be particularly relevant to biological conditions associated with hypoadenylated mRNAs (e.g., germ cells/neurons) and/or limiting cytoplasmic PABP (e.g., viral infection, cell stress). This mechanism adds significant insight into our knowledge of mRNA-specific translational activation and the function of the PABP-eIF4G complex in translation initiation.

Downregulation of eIF4G by microRNA-503 enhances drug sensitivity of MCF-7/ADR cells through suppressing the expression of ABC transport proteins.

Overexpression of adenosine triphosphate-binding cassette (ABC) transport protein is emerging as a critical contributor to anticancer drug resistance. The eukaryotic translation initiation factor (eIF) 4F complex, the key modulator of mRNA translation, is regulated by the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathway in anticancer drug-resistant tumors. The present study demonstrated the roles of ABC translation protein alterations in the acquisition of the Adriamycin (ADM)-resistant phenotype of MCF-7 human breast cells. Quantitative polymerase chain reaction and western blot analysis were applied to examine the differences in mRNA and protein levels, respectively. It was found that the expression of the ABC sub-family B member 1, ABC sub-family C member 1 and ABC sub-family G member 2 transport proteins were upregulated in MCF-7/ADR cells. An MTT assay was used to detect the cell viability, from the results MCF-7/ADR cells were less sensitive to ADM, tamoxifen (TAM) and taxol (TAX) treatment compared with MCF-7 cells. We predicted that the 3′-untranslated region of eukaryotic translation initiation factor 4-γ 1 (eIF4G) contains a potential miRNA binding site for microRNA (miR)-503 through using computational programs. These binding sites were confirmed by luciferase reporter assays. eIF4G mRNA degradation was accelerated in cells transfected with miR-503 mimics. Furthermore, it was demonstrated that eIF4G and ABC translation proteins were significantly downregulated in MCF-7/ADR cells after transfection with miR-503. It was found that miR-503 mimics could sensitize the cells to treatment with ADM, TAM and TAX. These findings demonstrated for the first time that eIF4G acted as a key factor in MCF-7/ADR cells, and may be an efficient agent for preventing and reversing multi-drug resistance in breast cancer.

Coupling between the DEAD-box RNA helicases Ded1p and eIF4A.

Eukaryotic translation initiation involves two conserved DEAD-box RNA helicases, eIF4A and Ded1p. Here we show that S. cerevisiae eIF4A and Ded1p directly interact with each other and simultaneously with the scaffolding protein eIF4G. We delineate a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as a modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. Our results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F, the complex comprising eIF4G, eIF4A, and eIF4E.

CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A.

SUMMARYTargeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition.

NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII.

Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap-binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut-off host-cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot-and-mouth disease virus (FMDV) leader proteinase (Lb(pro)), human rhinovirus 2 (HRV2) 2A proteinase (2A(pro)) and coxsackievirus B4 (CVB4) 2A(pro) with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed (13)C/(15) N sequential backbone assignment of human eIF4GII residues 551-745 and examined their binding to murine eIF4E. eIF4GII551-745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain-like Lb(pro) only forms a stable complex with eIF4GII(551-745) in the presence of eIF4E, with KD values in the low nanomolar range; Lb(pro) contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin-like 2A(pro) from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K(D) values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut-off.

Abstract B45: Vertical inhibition of the PI3K pathway potently sensitizes diffuse large B cell lymphoma to BCL-2 antagonism

Abstract The PI3K/AKT/mTOR axis is one of the most commonly mutated pathways in cancer, where aberrant activation promotes cell growth, proliferation, and survival. However, selective inhibitors targeting PI3K/AKT/mTOR have been hindered by their inability to effectively induce cell death in certain cancers. With the introduction of inhibitors that directly inhibit key pro-survival factors such as BCL-2 and BCL-XL (ABT-263 and ABT-199), the potential to achieve cancer cell death using combinations of targeted inhibitors has become a realizable goal. Here we show that selective inhibition of key components in the PI3K pathway (PI3K, AKT, or mTOR) potently sensitized a panel of DLBCL cell lines to ABT-263-induced apoptosis. While the degree of sensitization varied according to which PI3K pathway component was targeted, dual inhibition of both PI3K and mTOR consistently elicited the most potent sensitization across several cell lines. Previous work in other cancer types has linked the potency of this combination to the capacity of PI3K pathway inhibitors to reduce MCL-1 in an mTORC1-dependent manner. However, we found that this was not the case for DLBCL. For example, ABT-263 resistance induced by over-expression of MCL-1 was overcome when cells were co-treated with a dual-PI3K/mTOR inhibitor NVP-BEZ235 despite maintained expression of MCL-1. Instead, inhibition of the PI3K pathway led to a general increase in mitochondrial priming as measured by BH3 profiling. This occurred through distinct effects from both AKT and mTORC1 on the abundance of multiple BCL-2 family proteins at the mitochondria including reductions in the pro-survival proteins BCL-2, MCL-1, and BCL-XL as well as increases in Bim and Bad. In addition to these direct effects on BCL-2 family proteins, we found that inhibition of the PI3K pathway reduced expression of cancer-relevant proteins c-Myc and eIF4G as determined by reverse phase protein array. Thus, this project highlights the broad effects of dual-PI3K/mTOR inhibitors that sensitize DLBCL to apoptosis, and suggests that inhibition of the PI3K pathway has distinct effects on cell survival signaling among different cancer cell types. Citation Format: Jong-Hoon Scott Lee, Sarah Tang, Veronica Ortiz, David A. Fruman. Vertical inhibition of the PI3K pathway potently sensitizes diffuse large B cell lymphoma to BCL-2 antagonism. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B45.