Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition.

The mechanistic target of rapamycin (mTOR) is a kinase whose activation is associated with poor prognosis in pre-B cell acute lymphoblastic leukemia (B-ALL). These and other findings have prompted diverse strategies for targeting mTOR signaling in B-ALL and other B-cell malignancies. In cellular models of Philadelphia Chromosome-positive (Ph+) B-ALL, mTOR kinase inhibitors (TOR-KIs) that inhibit both mTOR-complex-1 (mTORC1) and mTOR-complex-2 (mTORC2) enhance the cytotoxicity of tyrosine kinase inhibitors (TKIs) such as dasatinib. However, TOR-KIs have not shown substantial efficacy at tolerated doses in blood cancer clinical trials. Selective inhibition of mTORC1 or downstream effectors provides alternative strategies that may improve selectivity towards leukemia cells. Of particular interest is the eukaryotic initiation factor 4F (eIF4F) complex that mediates cap-dependent translation. Here we use novel chemical and genetic approaches to show that selective targeting of either mTORC1 kinase activity or components of the eIF4F complex sensitizes murine BCR-ABL-dependent pre-B leukemia cells to dasatinib. SBI-756, a small molecule inhibitor of eIF4F assembly, sensitizes human Ph+ and Ph-like B-ALL cells to dasatinib cytotoxicity without affecting survival of T lymphocytes or natural killer cells. These findings support the further evaluation of eIF4F-targeted molecules in combination therapies with TKIs in B-ALL and other blood cancers.

Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

Many roads from mTOR to eIF4F.

The process of cell growth depends on a complex co-ordinated programme of macromolecular synthesis that can be tuned to environmental constraints. In eukaryotes, the mTOR [mammalian (or mechanistic) target of rapamycin] signalling pathway is a master regulator of this process, in part by regulating mRNA translation through control of the eIF4F (eukaryotic initiation factor 4F) initiation complex. The present review discusses the role of this relationship in mTOR-regulated gene expression, and its contribution to phenotypes associated with deregulated mTOR signalling, such as cancer.

Targeting synthetic lethal interactions between Myc and the eIF4F complex impedes tumorigenesis.

The energetically demanding process of translation is linked to multiple signaling events through mTOR-mediated regulation of eukaryotic initiation factor (eIF)4F complex assembly. Disrupting mTOR constraints on eIF4F activity can be oncogenic and alter chemotherapy response, making eIF4F an attractive antineoplastic target. Here, we combine a newly developed inducible RNAi platform and pharmacological targeting of eIF4F activity to define a critical role for endogenous eIF4F in Myc-dependent tumor initiation. We find elevated Myc levels are associated with deregulated eIF4F activity in the prelymphomatous stage of the Eµ-Myc lymphoma model. Inhibition of eIF4F is synthetic lethal with elevated Myc in premalignant pre-B/B cells resulting in reduced numbers of cycling pre-B/B cells and delayed tumor onset. At the organismal level, eIF4F suppression affected a subset of normal regenerating cells, but this was well tolerated and rapidly and completely reversible. Therefore, eIF4F is a key Myc client that represents a tumor-specific vulnerability.

The cap-translation inhibitor 4EGI-1 induces apoptosis in multiple myeloma through Noxa induction.

BACKGROUND: Cancer cells are frequently addicted to deregulated oncogenic protein translation. The small molecule 4EG-I selectively inhibits the cap-dependent translation of mRNAs. As multiple myeloma is an incurable disease that requires new therapeutic approaches, we investigated whether targeting the translation initiation pathway could be a target for myeloma therapy. METHODS: Six myeloma cell lines and primary samples were included in this study. The 4EGI-1 effect was determined by AnnexinV staining and caspase activation. Modification of Bcl-2 protein expression was analysed, and the significance of modified proteins was analysed by knock-down experiments. RESULTS: We demonstrated that 4EGI-1 impaired the assembly of the eIF4F complex and decreased the expression of the eIF4E-regulated proteins in myeloma cells. Furthermore, we showed that 4EGI-1 induced strong apoptosis in five out of six myeloma cell lines. Apoptosis is associated with the activation of the intrinsic mitochondrial pathway. The 4EGI-1 triggered Noxa induction only in cells undergoing apoptosis through endoplasmic reticulum (ER) stress. Furthermore, Noxa silencing prevented myeloma cells from 4EGI-1-induced apoptosis. Finally, Noxa induction led to a disruption of Mcl-1/Bim complexes in parallel to the generation of 'Mcl-1-free Noxa'. CONCLUSION: Our results suggested that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for multiple myeloma therapy.

Eukaryotic initiation factor 4F: a vulnerability of tumor cells.

Protein synthesis is a complex, tightly regulated process in eukaryotic cells and its deregulation is a hallmark of many cancers. Translational control occurs primarily at the rate-limiting initiation step, where ribosomal subunits are recruited to template mRNAs through the concerted action of several eukaryotic initiation factors (eIFs). One factor that interacts with both the mRNA and ribosomes, and appears limiting for translation is eIF4F, a complex composed of the cap-binding protein, eIF4E; the scaffold protein, eIF4G; and the ATP-dependent DEAD-box helicase, eIF4A. eIF4E appears to play an important role in tumor initiation and progression since its overexpression can cooperate with oncogenes to accelerate transformation in cell lines and animal models, and its levels are elevated in many human cancers. This, therefore, represents a vulnerability for transformed cells, and presents an opportunity for therapeutic intervention. In this review, we discuss approaches for targeting eIF4F activity.

Functional overlap between eIF4G isoforms in Saccharomyces cerevisiae.

Initiation factor eIF4G is a key regulator of eukaryotic protein synthesis, recognizing proteins bound at both ends of an mRNA to help recruit messages to the small (40S) ribosomal subunit. Notably, the genomes of a wide variety of eukaryotes encode multiple distinct variants of eIF4G. We found that deletion of eIF4G1, but not eIF4G2, impairs growth and global translation initiation rates in budding yeast under standard laboratory conditions. Not all mRNAs are equally sensitive to loss of eIF4G1; genes that encode messages with longer poly(A) tails are preferentially affected. However, eIF4G1-deletion strains contain significantly lower levels of total eIF4G, relative to eIF4G2-delete or wild type strains. Homogenic strains, which encode two copies of either eIF4G1 or eIF4G2 under native promoter control, express a single isoform at levels similar to the total amount of eIF4G in a wild type cell and have a similar capacity to support normal translation initiation rates. Polysome microarray analysis of these strains and the wild type parent showed that translationally active mRNAs are similar. These results suggest that total eIF4G levels, but not isoform-specific functions, determine mRNA-specific translational efficiency.

Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma.

BACKGROUND: Insulin-like growth factor (IGF)-I signalling stimulates proliferation, survival, and invasion in malignant mesothelioma and other tumour types. Studies have found that tumourigenesis is linked to dysregulation of cap-dependent protein translation. METHODS: The effect of IGF stimulation on cap-mediated translation activation in mesothelioma cell lines was studied using binding assays to a synthetic 7-methyl GTP-cap analogue. In addition, cap-mediated translation was genetically repressed in these cells with a dominant active motive of 4E-BP1. RESULTS: In most mesothelioma cell lines, IGF-I stimulation resulted in a hyperphosphorylation-mediated inactivation of 4E-BP1 compared with that in normal mesothelial cells. An inhibitor of Akt diminished IGF-I-mediated phosphorylation of 4E-BP1, whereas inhibiting MAPK signalling had no such effect. IGF-I stimulation resulted in the activation of the cap-mediated translation complex as indicated by an increased eIF4G/eIF4E ratio in cap-affinity assays. Akt inhibition reversed the eIF4G/eIF4E ratio. Mesothelioma cells transfected with an activated 4E-BP1 protein (4E-BP1(A37/A46)) were resistant to IGF-I-mediated growth, motility, and colony formation. In a murine xenograft model, mesothelioma cells expressing the dominant active 4E-BP1(A37/A46) repressor protein showed abrogated tumourigenicity compared with control tumours. CONCLUSION: IGF-I signalling in mesothelioma cells drives cell proliferation, motility, and tumourigenesis through its ability to activate cap-mediated protein translation complex through PI3K/Akt/mTOR signalling.

c-Myc and eIF4F are components of a feedforward loop that links transcription and translation.

The Myc/Max/Mad family of transcription factors and the eukaryotic initiation factor 4F (eIF4F) complex play fundamental roles in regulating cell growth, proliferation, differentiation, and oncogenic transformation. eIF4F is involved in the recruitment of ribosomes to mRNAs and is thought to generally be the rate-limiting phase of translation. Here, we show that c-Myc directly activates transcription of the three subunits of eIF4F (eIF4E, eIF4AI, and eIF4GI). These transcriptional effects are mediated through canonical E-boxes (5'CACGTG3') present in the promoters of these genes. In addition, the c-Myc antagonist Mad1 down-regulates the expression of eIF4F subunits. We also show that MycER activation stimulates protein synthesis at the level of translation initiation. Increased eIF4F levels result in stimulation of c-Myc mRNA translation specifically, as assessed by quantitative reverse transcription-PCR. We use a murine model of lymphomagenesis to show the expression of eIF4F subunits is also up-regulated by c-Myc in vivo. Our results suggest the presence of a feedforward loop involving c-Myc and eIF4F that serves to link transcription and translation and that could contribute to the effects of c-Myc on cell proliferation and neoplastic growth.

MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F.

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell-free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

The translation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity, and memory in the hippocampus.

Long-lasting synaptic plasticity and memory requires mRNA translation, yet little is known as to how this process is regulated. To explore the role that the translation repressor 4E-BP2 plays in hippocampal long-term potentiation (LTP) and learning and memory, we examined 4E-BP2 knock-out mice. Interestingly, genetic elimination of 4E-BP2 converted early-phase LTP to late-phase LTP (L-LTP) in the Schaffer collateral pathway, likely as a result of increased eIF4F complex formation and translation initiation. A critical limit for activity-induced translation was revealed in the 4E-BP2 knock-out mice because L-LTP elicited by traditional stimulation paradigms was obstructed. Moreover, the 4E-BP2 knock-out mice also exhibited impaired spatial learning and memory and conditioned fear-associative memory deficits. These results suggest a crucial role for proper regulation of the eIF4F complex by 4E-BP2 during LTP and learning and memory in the mouse hippocampus.

Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells.

Common human malignancies acquire derangements of the translation initiation complex, eIF4F, but their functional significance is unknown. Hypophosphorylated 4E-BP proteins negatively regulate eIF4F assembly by sequestering its mRNA cap binding component eIF4E, whereas hyperphosphorylation abrogates this function. We found that breast carcinoma cells harbor increases in the eIF4F constituent eIF4GI and hyperphosphorylation of 4E-BP1 which are two alterations that activate eIF4F assembly. Ectopic expression of eIF4E in human mammary epithelial cells enabled clonal expansion and anchorage-independent growth. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppressed their tumorigenicity, whereas loss of these 4E-BP1 phosphorylation site mutants accompanied spontaneous reversion to a malignant phenotype. Thus, eIF4F activation is an essential component of the malignant phenotype in breast carcinoma.

New paradigms for nutrient control of genome translation.

Organisms respond adaptively to changing environmental conditions by altering genome expression. Translational control of gene expression by nutrients and metabolites is one of the most robust biologic mechanisms that enable rapid alterations in the cellular proteome in response to changes in the local environment. Recently, investigators have identified new mechanisms for nutrient control of translation in mammals; evidence supports the conservation of translational control mechanisms in eukaryotes that heretofore had only been demonstrated in prokaryotes.