eIF4F Translation Initiation Complex Research Articles

Eukaryotic translation initiation factor EIF4G1 p.Ser637Cys mutation in a family with Parkinson's disease with antecedent essential tremor.

Essential tremor (ET) and Parkinson's disease (PD) are common chronic movement disorders that can cause a substantial degree of disability. However, the etiology underlying these two conditions remains poorly understood. In the present study, Whole-exome sequencing of peripheral blood samples from the proband and Sanger sequencing of the other 18 family members, and pedigree analysis of four generations of 29 individuals with both ET and PD in a nonconsanguineous Chinese family were performed. Specifically, family members who had available medical information, including historical documentation and physical examination records, were included. A novel c.1909A>T (p.Ser637Cys) missense mutation was identified in the eukaryotic translation initiation factor 4γ1 (EIF4G1) gene as the candidate likely responsible for both conditions. In total, 9 family members exhibited tremor of the bilateral upper limbs and/or head starting from ages of ≥40 years, 3 of whom began showing evidence of PD in their 70s. Eukaryotic initiation factor 4 (eIF4)G1, a component of the translation initiation complex eIF4F, serves as a scaffold protein that interacts with many initiation factors and then binds to the 40S ribosomal subunit. The EIF4G1 (p.Ser637Cys) might inhibit the recruitment of the mRNA to the ribosome. In conclusion, the results from the present study suggested that EIF4G1 may be responsible for the hereditary PD with 'antecedent ET' reported in the family assessed.

MRNA Translation Inhibition Targets Bioenergetic Homeostasis in AML Cells in Vitro and In Vivo and Synergizes with Cytarabine and Venetoclax

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. Recently, inhibition of BCL-2 with venetoclax in combination with hypomethylating agents has emerged as an attractive strategy for high-risk AML cases. Another promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1) (Oki et al, Nature Comm, 2021). However, clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. To curtail these drawbacks, we adopted a strategy of inhibiting an important effector of the mTORC1 signaling pathway controlling mRNA translation - the eukaryotic initiation factor 4A (eIF4A), subunit of the translation initiation complex eIF4F. Recent evidence suggests that translational programs mediated by the mTORC1/4E-BP/eIF4F axis can support resistance to therapy in various cancer models driven by oncogenic kinases, in part by allowing cellular metabolic plasticity (Hulea L et al, Cell Metab. 2018). In fact, metabolism, and specifically mitochondrial oxidative phosphorylation, has emerged as a central dependency of AML cells, sustaining resistance to therapy and recurrence. Using the MOLM-14 human AML cell line to model therapy-resistant disease, we previously demonstrated the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, both in vitro and in vivo (Fooks et al, J Exp Clin Cancer Res 2022). eIF4Ai affected cellular metabolism, by reducing mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Concomitantly, eIF4Ai decreased intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mitochondrial ROS. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Importantly, CR-1-31-B acted synergistically in vitro in combination with cytarabine or venetoclax. Recently, we have expanded our characterization of the eIF4Ai/venetoclax combination in MOML-14 cells, and showed that venetoclax potentiates the CR-1-31-B-induced inhibition of cellular respiration, glycolysis and ATP production (Seahorse). The combination provokes a robust apoptotic response with different temporal dynamics to single treatment, as measured using the Incucyte platform. In vivo, we have validated the strong anti-leukemic response induced by CR-1-31-B in a MOLM-14 transplantation model, after only 7 days of treatment, and showed that venetoclax slightly potentiates this effect. In addition, eIF4A inhibition reduces the levels of several metabolic proteins (GLS1, IDH1), which supports our previous observations of decreased levels of TCA cycle metabolites. We have confirmed the metabolic and pro-apoptotic effect of eIF4Ai in a second cellular model of AML, U937. Consistent with previous results, in U937 cells CR-1-31-B induces apoptosis at low nM concentrations, reduces cellular bioenergetics, ATP production, as well as levels of pro-proliferative (cyclin D3, CDK4) and anti-apoptotic (BCL-2, MCL-1) proteins. Our collective studies highlight (i) the importance of the crosstalk between mRNA translation and metabolic regulation and (ii) that direct inhibition of translation represents an appealing therapeutic strategy for clinical cases of therapy resistance that are dependent on the mTORC1/eIF4F axis. This is of interest as several translation inhibitors are currently tested in phase 1/2 clinical trials in solid malignancies. # co-corresponding authors

VNLG-152R and its deuterated analogs potently inhibit/repress triple/quadruple negative breast cancer of diverse racial origins in vitro and in vivo by upregulating E3 Ligase Synoviolin 1 (SYVN1) and inducing proteasomal degradation of MNK1/2.

Triple-negative breast cancer (TNBC) and its recently identified subtype, quadruple negative breast cancer (QNBC), collectively account for approximately 13% of reported breast cancer cases in the United States. These aggressive forms of breast cancer are associated with poor prognoses, limited treatment options, and lower overall survival rates. In previous studies, our research demonstrated that VNLG-152R exhibits inhibitory effects on TNBC cells both in vitro and in vivo and the deuterated analogs were more potent inhibitors of TNBC cells in vitro. Building upon these findings, our current study delves into the molecular mechanisms underlying this inhibitory action. Through transcriptome and proteome analyses, we discovered that VNLG-152R upregulates the expression of E3 ligase Synoviolin 1 (SYVN1), also called 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) in TNBC cells. Moreover, we provide genetic and pharmacological evidence to demonstrate that SYVN1 mediates the ubiquitination and subsequent proteasomal degradation of MNK1/2, the only known kinases responsible for phosphorylating eIF4E. Phosphorylation of eIF4E being a rate-limiting step in the formation of the eIF4F translation initiation complex, the degradation of MNK1/2 by VNLG-152R and its analogs impedes dysregulated translation in TNBC cells, resulting in the inhibition of tumor growth. Importantly, our findings were validated in vivo using TNBC xenograft models derived from MDA-MB-231, MDA-MB-468, and MDA-MB-453 cell lines, representing different racial origins and genetic backgrounds. These xenograft models, which encompass TNBCs with varying androgen receptor (AR) expression levels, were effectively inhibited by oral administration of VNLG-152R and its deuterated analogs in NRG mice. Importantly, in direct comparison, our compounds are more effective than enzalutamide and docetaxel in achieving tumor growth inhibition/repression in the AR+ MDA-MD-453 xenograft model in mice. Collectively, our study sheds light on the involvement of SYVN1 E3 ligase in the VNLG-152R-induced degradation of MNK1/2 and the therapeutic potential of VNLG-152R and its more potent deuterated analogs as promising agents for the treatment of TNBC across diverse patient populations.

CDKAL1 Drives the Maintenance of Cancer Stem-Like Cells by Assembling the eIF4F Translation Initiation Complex.

Cancer stem-like cells (CSCs) have a unique translation mode, but little is understood about the process of elongation, especially the contribution of tRNA modifications to the maintenance of CSCs properties. Here, it is reported that, contrary to the initial aim, a tRNA-modifying methylthiotransferase CDKAL1 promotes CSC-factor SALL2 synthesis by assembling the eIF4F translation initiation complex. CDKAL1 expression is upregulated in patients with worse prognoses and is essential for maintaining CSCs in rhabdomyosarcoma (RMS) and common cancers. Translatome analysis reveals that a group of mRNAs whose translation is CDKAL1-dependent contains cytosine-rich sequences in the 5' untranslated region (5'UTR). Mechanistically, CDKAL1 promotes the translation of such mRNAs by organizing the eIF4F translation initiation complex. This complex formation does not require the enzyme activity of CDKAL1 but requires only the NH2 -terminus domain of CDKAL1. Furthermore, sites in CDKAL1 essential for forming the eIF4F complex are identified and discovered candidate inhibitors of CDKAL1-dependent translation.

Spatial patterns of the cap-binding complex eIF4F in human melanoma cells

As a central node of protein synthesis, the cap-binding complex, eukaryotic translation initiation factor 4 F (eIF4F), is involved in cell homeostasis, development and tumorigenesis. A large body of literature exists on the regulation and function of eIF4F in cancer cells, however the intracellular localization patterns of this complex are largely unknown. Since different subsets of mRNAs are translated in distinct subcellular compartments, understanding the distribution of translation initiation factors in the cell is of major interest. Here, we developed an in situ detection method for eIF4F at the single cell level. By using an image-based spot feature analysis pipeline as well as supervised machine learning, we identify five distinct spatial patterns of the eIF4F translation initiation complex in human melanoma cells. The quantity of eIF4F complex per cell correlated with the global mRNA translation activity, and its variation is dynamically regulated by cell state or extracellular stimuli. In contrast, the spatial patterns of eIF4F complexes at the single cell level could distinguish melanoma cells harboring different oncogenic driver mutations. This suggests that different tumorigenic contexts differentially regulate the subcellular localization of mRNA translation, with specific localization of eIF4F potentially associated with melanoma cell chemoresistance.

Targeting a Highly Deregulated eIF4F Translation Initiation Complex Sensitises IM-Resistant Cells to Tyrosine Kinase Inhibitors and Effectively Suppresses BCR-ABL1 Protein Expression

Background We previously reported that Abelson helper integration site-1 (AHI-1) is highly deregulated in chronic myeloid leukemia (CML) leukemic stem cells (LSCs) and mediates tyrosine kinase inhibitor (TKI) resistance. AHI-1 physically interacts with BCR-ABL1 and its SH3 domain interacts with BCR-ABL1 substrates, like dynamin-2, which regulate leukemic properties of CML LSCs. However, the molecular and biological roles of AHI-1 and its interacting partners in mediating TKI resistance remain largely unknown. Objective To investigate the molecular functions of AHI-1 and its SH3 domain in regulation of TKI resistance, using advanced antibody microarray analysis. Methods A high content antibody microarray was performed in BCR-ABL1+ cells co-transduced with wild-type (WT) AHI-1 or the deletion of AHI-1 SH3 domain (SH3Δ) with or without imatinib (IM). Changes in antibody signals for protein expression or phosphorylation were determined using limma and pathway enrichment analysis by g:Profiler. eIF4G1 genetic inhibition by lentiviral-mediated shRNA or pharmacological inhibition by SBI-756, was performed in TKI-resistant cells to assay translation initiation activity by proximity ligation assay (PLA), o-propargyl-puromycin (OPP), and polysome profiling. Results The antibody microarray analysis revealed that WT AHI-1 cells have the greatest number of changes in the phospho-proteome and proteome compared to BCR-ABL1+ cells and AHI-1 SH3Δ cells with and without IM. Pathway enrichment analysis identified that the targets with significantly increased differential antibody signal after IM treatment in WT AHI-1 cells were related to the regulation of translation initiation complex (p < 0.0001). Interestingly, our RNA-seq dataset analysis further identified several eukaryotic initiation factor 4F (eIF4F) complex members to be significantly increased in CD34+ CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex (2-fold, p = 0.001). WT AHI-1 cells also showed increased expression and phosphorylation of eIF4G1 (>2-fold) and eIF4B (>2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, and cyclin D3 (a downstream protein of eIF4F translational activity) as compared to BCR-ABL1+ cells, by immunoblotting. These results were similarly demonstrated in IM-resistant cells as compared to IM-sensitive cells (2-5-fold, p < 0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p < 0.0001) and increased TKI sensitivity in IM-resistant cells (p < 0.0021). These cells showed reduced levels of eIF4F complex formation by PLA assay (p = 0.013) and reduced protein expression of cyclin D3 (40%, p = 0.0036) and BCR-ABL1 (70%). Similarly, IM-resistant cells were more sensitive to SBI-756 treatment, an eIF4G1 inhibitor (50% reduction, p = 0.0001) than IM-sensitive cells (30% reduction); these effects were enhanced by a combination of SBI-756 and IM (80%, p < 0.02). Furthermore, SBI-756 treatment reduced PLA foci formation (p = 0.014) and global protein synthesis rates (p < 0.0001). Polysome profiling demonstrated that IM-resistant cells had higher levels of translation activity, which was inhibited by SBI-756 treatment. Most interestingly, SBI-756 treatment reduced protein expression of BCR-ABL1 (70%) and cyclin D3 (40%) in these cells. Conclusion We have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells. eIF4G1 inhibitor treatment sensitises IM-resistant cells to TKI and reduces BCR-ABL1 protein expression, providing potential treatment strategies to overcome TKI resistance.

Abstract 400: Functional, structural and binding studies of the atypical ER-resident protein FKBP7, a potential target in chemoresistant prostate cancer

Abstract Background: Metastatic Castration Resistant Prostate Cancer (mCRPC) is the latest stage of Prostate Cancer (PCa). Despite the use of taxane-based chemotherapies Docetaxel and Cabazitaxel, mCRPC remains lethal due to chemoresistance. We recently identified FKBP7 as a potential therapeutic target of interest in PCa. FKBP7 is overexpressed in taxane-resistant PCa cells and impacts both cell proliferation and Docetaxel efficacy in chemoresistant models. We demonstrated FKBP7 affects translation and binds to the translation initiation complex eIF4F. FKBP7 is an ER-resident FKBP, but its properties remain poorly described. Methods: FKBP7 expression upon chemotherapy treatment was assessed by Western Blot and correlation between FKBP7 expression and clinical prognosis was examined on public cancer databases. Subcellular localization of FKBP7 was addressed by digitonin-based fractionation. Finally, FKBP7-predicted structure was obtained with structural modeling servers and sequence alignments were performed on AliView. We then produced and purified the recombinant catalytic domain of FKBP7 and collected high quality 15N HSQC NMR spectra. Afterwards, we examined its interaction with well-known FKBP ligands. Results: Increased FKBP7 expression was observed upon treatment with several cytotoxic chemotherapies in PCa-cell lines, and TCGA data underlined FKBP7 impacts survival in other cancers than PCa. Together, this suggests an enlarged potential involvement of FKBP7 in adaptive resistance. Mechanistically, in taxane-resistant models, FKBP7 overexpression did not result from higher protein stability but rather from a transcriptional regulation. Preliminary results of subcellular fractionation showed FKBP7 is present in the Endoplasmic Reticulum (ER) and seems to localize in the cytosol, independently of proteasomal degradation. We also confirmed FKBP7 is N45-glycosylated and observed that cytosolic-FKBP7 appears mainly glycosylated. This suggests that the ER-resident FKBP7 could be refluxed in the cytosol depending on its glycosylation state to regulate the activity of the cytosolic eIF4F complex. We are currently validating this hypothesis. At last, structural modeling predicted a FKBP-type fold with a potential FKBP7 substrate specificity as its catalytic pocket presents distinct composition for charge and bulkiness compared to other FKBPs. Accordingly, FKBP7 strongly binds Rapamycin and Everolimus, but surprisingly not FK506, confirming a selective binding profile. Conclusion: Our work depicts the recently identified FKBP7/eIF4F pathway in PCa-resistance and points atypical properties for the ER-resident FKBP7. It also indicates FKBP7 could be a druggable target in adaptive resistance of other solid cancers. Besides, we gathered original data on its structure and specificity for future drug-targeting strategies. Citation Format: Luce Dreno, Carolina Tiraboschi, Sofian Lacoste, Sofia Calpe Gomez, Marine F. Garrido, Matthieu Bertrand, Mariana Tannoury, Eric Jacquet, Naima Nhiri, Yohann Loriot, Karim Fizazi, Daniel Compagno, Nadine Assrir, Ewen Lescop, Anne Chauchereau. Functional, structural and binding studies of the atypical ER-resident protein FKBP7, a potential target in chemoresistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 400.

Targeting Translational Control in Cancer Using Small Molecule Activators of PP2A

BackgroundCancer cells rely on hyperactive protein synthesis to reprogram the proteome to sustain the transformed phenotype. Thus, aberrant protein translation has emerged as a promising therapeutic target. Cap‐dependent translation is an exquisitely regulated process that requires the assembly of the eIF4F translation initiation complex. The rate limiting component of the eIF4F complex is eIF4E, which binds to the 5’ cap structure of mRNAs to initiate translation. Cap‐dependent translation is kept in check by the translation repressor 4E‐BP1; in its active hypophosphorylated form, 4E‐BP1 sequesters eIF4E to prevent translation initiation. Cancer cells use several mechanisms to bypass 4E‐BP1 translational suppres­sion, including hyperactivation of mTORC1 for hyperphos­phorylation and inactivation of 4E‐BP1; inactivation of the 4E‐BP1 phosphatase, PP2A; and profound suppression of 4E‐BP1 expression. We have found that the expression and activity of 4E‐BP1 can be restored in tumor cells using novel small molecule acti­vators of PP2A (SMAPs), a class of compounds that activate selected PP2A tumor suppressors and are being ac­tively developed as anticancer agents (Leonard et al. Cell, 2020). The objective of this study was to investigate the mechanism(s) by which SMAPs lead to restoration of the expression and activity of 4E‐BP1 in cancer cells for inhibition of hyperactive protein translation and tumor suppression.HypothesisBased on our finding that SMAPs promote PP2A‐dependent transcriptional activation of the 4E‐BP1 gene, we hypothesize that PP2A activation modulates the levels and/or activity of key transcription factors that regulate 4E‐BP1 gene expression.Results/DataWe have found that SMAPs (e.g., lead compound DT‐061) upregulate 4E‐BP1 mRNA and protein in tumor cells by inducing transcription of the 4E‐BP1 gene. Importantly, SMAPs promote the accumulation of active 4E‐BP1, capable of suppressing cap‐dependent translation in cancer cells. SMAPs promote PP2A‐dependent loss of Snail/Slug and upregulation of ATF4, transcription factors that have been implicated in negative and posi­tive control of 4E‐BP1 gene ex­pression, respectively. Snail downregulation was prevented by proteasome inhibition, indicating that Snail loss is due to proteasomal degradation. Use of GSK‐3β and PKD1 inhibitors excluded canonical mechanisms of Snail degradation in the effects of SMAPs. siRNA knockdown experiments are underway to explore the role of changes in Snail and ATF4 in SMAP‐induced upregulation of 4E‐BP1. Ongoing studies are also examining the mechanisms of SMAP/PP2A regulation of Snail/Slug and ATF4 in tumor cells.ConclusionsSMAPs promote PP2A‐dependent proteasomal degradation of Snail/Slug protein via a non‐canonical mechanism, as well as upregulation of ATF4. We propose that these effects relieve transcriptional repression of 4E‐BP1 by Snail/Slug while promoting 4E‐BP1 transcription by ATF4, leading to a robust increase in 4E‐BP1 levels. 4E‐BP1 translation repressive activity is ensured by PP2A‐induced 4E‐BP1 dephosphorylation. Thus, SMAPs offer a powerful tool for therapeutic targeting of aberrant protein translation in cancer.

Differential Effects on the Translation of Immune-Related Alternatively Polyadenylated mRNAs in Melanoma and T Cells by eIF4A Inhibition.

Simple SummaryImmune checkpoints blockade has emerged as an effective approach to prevent immune escape of tumor cells, and constitutes a powerful anti-cancer therapeutic strategy. Regulation of the expression of genes encoding immune checkpoint inhibitors has thus become an increasingly important field of study. Beyond transcription, gene expression is regulated at several post-transcriptional levels including pre-mRNA 3′-end processing and mRNA translation. More specifically, the eIF4F translation initiation complex represents an important hub for oncogenic signaling in the etiology of different cancers. The eIF4A RNA helicase component of the eIF4F can be inhibited by the widely characterized small molecule inhibitor silvestrol. Here, we evaluated the effect of eIF4A inhibition with silvestrol on the translation of alternatively polyadenylated mRNAs in melanoma cell lines and activated T cells. We show that silvestrol can selectively inhibit the translation of alternatively polyadenylated isoforms of genes encoding key immune-related proteins.Targeting the translation initiation complex eIF4F, which binds the 5′ cap of mRNAs, is a promising anti-cancer approach. Silvestrol, a small molecule inhibitor of eIF4A, the RNA helicase component of eIF4F, inhibits the translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor, which, in turn, reduces the transcription of the gene encoding one of the major immune checkpoint proteins, i.e., programmed death ligand-1 (PD-L1) in melanoma cells. A large proportion of human genes produce multiple mRNAs differing in their 3′-ends through the use of alternative polyadenylation (APA) sites, which, when located in alternative last exons, can generate protein isoforms, as in the STAT1 gene. Here, we provide evidence that the STAT1α, but not STAT1β protein isoform generated by APA, is required for silvestrol-dependent inhibition of PD-L1 expression in interferon-γ-treated melanoma cells. Using polysome profiling in activated T cells we find that, beyond STAT1, eIF4A inhibition downregulates the translation of some important immune-related mRNAs, such as the ones encoding TIM-3, LAG-3, IDO1, CD27 or CD137, but with little effect on the ones for BTLA and ADAR-1 and no effect on the ones encoding CTLA-4, PD-1 and CD40-L. We next apply RT-qPCR and 3′-seq (RNA-seq focused on mRNA 3′ ends) on polysomal RNAs to analyze in a high throughput manner the effect of eIF4A inhibition on the translation of APA isoforms. We identify about 150 genes, including TIM-3, LAG-3, AHNAK and SEMA4D, for which silvestrol differentially inhibits the translation of APA isoforms in T cells. It is therefore crucial to consider 3′-end mRNA heterogeneity in the understanding of the anti-tumor activities of eIF4A inhibitors.

IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap.

Inositol hexakisphosphate kinase 1 (IP6K1) is a small molecule kinase that catalyzes the conversion of the inositol phosphate IP6 to 5-IP7. We show that IP6K1 acts independently of its catalytic activity to upregulate the formation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein granules that store translationally repressed mRNA. IP6K1 does not localise to P-bodies, but instead binds to ribosomes, where it interacts with the mRNA decapping complex - the scaffold protein EDC4, activator proteins DCP1A/B, decapping enzyme DCP2 and RNA helicase DDX6. Along with its partner 4E-T, DDX6 is known to nucleate protein-protein interactions on the 5' mRNA cap to facilitate P-body formation. IP6K1 binds the translation initiation complex eIF4F on the mRNA cap, augmenting the interaction of DDX6 with 4E-T (also known as EIF4ENIF1) and the cap-binding protein eIF4E. Cells with reduced IP6K1 show downregulated microRNA-mediated translational suppression and increased stability of DCP2-regulated transcripts. Our findings unveil IP6K1 as a novel facilitator of proteome remodelling on the mRNA cap, tipping the balance in favour of translational repression over initiation, thus leading to P-body assembly. This article has an associated First Person interview with the first author of the paper.

Identification of a Highly Deregulated eIF4F Translation Initiation Complex in Drug-Resistant BCR-ABL + Cells By a Phospho-Proteomic Antibody Microarray

Protein-tyrosine kinase inhibitors (TKIs) have been effective for treatment of early stages of chronic myeloid leukemia (CML). However, BCR-ABL-dependent resistance mechanisms and TKI-unresponsive quiescent leukemic stem cells (LSCs) can result in drug resistance and disease relapse. We have demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs. It interacts with BCR-ABL through the AHI-1 WD40 domain and with other proteins, like dynamin-2 (DNM2), through its SH3 domain, to enhance leukemic-initiating activities and TKI resistance. To uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance in CML stem/progenitor cells, an advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL + cells co-transduced with wild-type Ahi-1 or Ahi-1 SH3 Δ mutant in the presence or absence of imatinib (IM). The microarray simultaneously quantified the differences in expression and phosphorylation sites of proteins in multiple signaling pathways using 1325 antibodies in duplicate measurements, and each microarray analysis was performed in duplicate. Significant changes in antibody signals for protein expression or phosphorylation were determined using limma. This analysis revealed that the overexpression of wild-type Ahi-1 (WT Ahi-1) has a profound differential effect, compared to the SH3 domain deleted Ahi-1 (Ahi-1 SH3 Δ), on the expression and phosphorylation status of proteins in BCR-ABL + cells with and without IM treatment. BCR-ABL + cells co-expressing WT Ahi-1 had a greater number of significantly differential antibody signals (7 increases, 42 decreases) when compared to BCR-ABL + cells, while Ahi-1 SH3 Δ expressing cells resulted in fewer significantly differential antibody signals (12 increases, 2 decreases) compared to BCR-ABL + cells. IM treatment resulted in WT Ahi-1 expressing cells having the greatest number of significantly differential antibody signals (7 increases, 56 decreases) compared to BCR-ABL + cells (5 decreases) and those co-expressing Ahi-1 SH3 Δ (2 increases, 9 decreases). Pathway enrichment analysis, using gProfiler, identified that the targets with significantly increased differential antibody signal after IM treatment in WT Ahi-1 cells were related to the regulation of translation initiation complex (p>0.0001). Interestingly, our RNA-seq dataset analysis further identified several members of the eukaryotic initiation factor 4F (eIF4F) complex to be significantly upregulated in CD34 + CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex involved in translation initiation (2-fold, p=0.001), as well as mTOR, a key regulator that controls the assembly of the eIF4F complex. This finding prompted us to further explore the regulation of translation initiation and the members of the eIF4F complex in BCR-ABL + cells. Immunoblotting demonstrated that BCR-ABL + cells co-transduced with WT Ahi-1 showed increased expression of eIF4G1 (3-fold) and eIF4B (2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, compared to BCR-ABL + cells. Additionally, Cyclin D3, a gene reported to be sensitive to eIF4F translational activity, was found to have slightly increased expression (1.4-fold) in WT Ahi-1 cells compared to BCR-ABL + cells. Most interestingly, these results were also demonstrated in IM-resistant CML cells as compared to IM-sensitive cells, with an increase in eIF4G1 expression (2-fold), phosphorylation of eIF4B (5-fold, p=0.003), and Cyclin D3 expression (4-fold, p<0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p<0.0001) and increases TKI sensitivity in IM-resistant cells (3-fold, p<0.05). A new protein interaction between eIF4G1 and the mRNA cap-binding protein eIF4E was further identified in these cells using a proximity ligation assay. Thus, we have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells, which may contribute to the regulation of TKI resistance in CML. DisclosuresNo relevant conflicts of interest to declare.

Deep computational analysis details dysregulation of eukaryotic translation initiation complex eIF4F in human cancers.

eIF4F plays diverse roles in human cancers, which complicate the development of an overarching understanding of its functional and regulatory impacts across tumor types. Typically, eIF4F drives initiation from the mRNA 5' end (cap) and is composed of eIF4G1, eIF4A1, and cap-binding eIF4E. Cap-independent initiation is possible without eIF4E, from internal ribosomal entry sites (IRESs). By analyzing large public datasets, we found that cancers selectively overexpress EIF4G1 more than EIF4E. That expression imbalance supports EIF4G1 as a prognostic indicator in patients with cancer. It also attenuates "housekeeping" pathways that are usually regulated in a tissue-specific manner via cap-dependent initiation in healthy tissues and reinforce regulation of cancer-preferred pathways in cap-independent contexts. Cap-independent initiation is mechanistically attributable to eIF4G1 hyperphosphorylation that promotes binding to eIF4A1 and reduced eIF4E availability. Collectively, these findings reveal a novel model of dysregulated eIF4F function and highlight the clinical relevance of cap-(in)dependent initiation in cancer.

Targeting the DEAD-Box RNA Helicase eIF4A with Rocaglates-A Pan-Antiviral Strategy for Minimizing the Impact of Future RNA Virus Pandemics.

The increase in pandemics caused by RNA viruses of zoonotic origin highlights the urgent need for broad-spectrum antivirals against novel and re-emerging RNA viruses. Broad-spectrum antivirals could be deployed as first-line interventions during an outbreak while virus-specific drugs and vaccines are developed and rolled out. Viruses depend on the host’s protein synthesis machinery for replication. Several natural compounds that target the cellular DEAD-box RNA helicase eIF4A, a key component of the eukaryotic translation initiation complex eIF4F, have emerged as potential broad-spectrum antivirals. Rocaglates, a group of flavaglines of plant origin that clamp mRNAs with highly structured 5′ untranslated regions (5′UTRs) onto the surface of eIF4A through specific stacking interactions, exhibit the largest selectivity and potential therapeutic indices among all known eIF4A inhibitors. Their unique mechanism of action limits the inhibitory effect of rocaglates to the translation of eIF4A-dependent viral mRNAs and a minor fraction of host mRNAs exhibiting stable RNA secondary structures and/or polypurine sequence stretches in their 5′UTRs, resulting in minimal potential toxic side effects. Maintaining a favorable safety profile while inducing efficient inhibition of a broad spectrum of RNA viruses makes rocaglates into primary candidates for further development as pan-antiviral therapeutics.

Targeting eIF4F translation initiation complex with SBI-756 sensitises B lymphoma cells to venetoclax

BackgroundThe BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL).MethodsWe tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex.ResultsTreatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis.ConclusionsOur data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.

Effective Inhibition of MYC-Amplified Group 3 Medulloblastoma Through Targeting EIF4A1.

PurposeIn medulloblastoma (MB), group 3 (G3) patients with MYC amplification tend to exhibit worse prognosis, thus creating a need for novel effective therapies. As the driver and crucial dependency for MYC-amplified G3-MB, MYC has been proven to be a prospective therapeutic target. Here, we aimed to identify novel effective therapeutic strategies against MYC-amplified G3-MB via targeting MYC translation.Materials and MethodsMajor components of translation initiation complex eIF4F were subjected to MB tumor dataset analysis, and EIF4A1 was identified to be a potential therapeutic target of MYC-amplified G3-MB. Validation was performed through genetic or pharmacological approaches with multiple patient-derived tumor models of MYC-amplified G3-MB in vitro and in vivo. Underlying mechanisms were further explored by Western blot, quantitative real-time PCR and mass spectrometry (MS) analyses.ResultsMB tumor datasets analyses showed that EIF4A1 was significantly up-regulated in G3-MB patients relative to normal cerebella, positively correlated with MYC in G3-MB at transcriptional level and a crucial cancer dependency in MYC-amplified G3-MB cells. Targeting EIF4A1 with a CRISPR/Cas9 approach or small-molecule inhibitor silvestrol effectively attenuated growth in multiple preclinical models of MYC-amplified G3-MB via blocking proliferation and inducing apoptosis. Mechanistically, EIF4A1 inhibition effectively impeded MYC expression at translational level, and its potency was positively associated with MYC level. Whole-proteome MS analysis of silvestrol-treated cells further unveiled other biological functions and pathways influenced by EIF4A1 inhibition.ConclusionOur investigation shows that interrupting MYC translation by EIF4A1 inhibition could be a potential effective therapeutic approach when treating patients with MYC-amplified G3-MB.

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.

An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells

Cancer persister cells tolerate anticancer drugs and serve as the founders of acquired resistance and cancer relapse. Here we show that a subpopulation of BRAFV600 mutant melanoma cells that tolerates exposure to BRAF and MEK inhibitors undergoes a reversible remodelling of mRNA translation that evolves in parallel with drug sensitivity. Although this process is associated with a global reduction in protein synthesis, a subset of mRNAs undergoes an increased efficiency in translation. Inhibiting the eIF4A RNA helicase, a component of the eIF4F translation initiation complex, abrogates this selectively increased translation and is lethal to persister cells. Translation remodelling in persister cells coincides with an increased N6-methyladenosine modification in the 5′-untranslated region of some highly translated mRNAs. Combination of eIF4A inhibitor with BRAF and MEK inhibitors effectively inhibits the emergence of persister cells and may represent a new therapeutic strategy to prevent acquired drug resistance.

Abstract 324: Translation Determines the Acute Cardiac Response to Ischemia/Reperfusion

Introduction: Since transcription is a relatively slow process, translation of preexisting mRNA networks might affect cardiac gene expression thereby rapidly adapting the myocardium to stress before changes in transcription take place. The mTOR kinase links nutrient and energy levels to protein synthesis by regulation of mRNA translation. Inhibition of mTOR complex 1, the responsible pathway for translational regulation, protects against reperfusion injury, however the underlying mechanisms are unknown. Through rapid restoration of myocardial energy levels upon reperfusion an mTOR-dependent translatome could adapt early changes in gene expression inducing a maladaptive gene network, independent from transcription, which mediates reperfusion injury. Hypothesis: Reperfusion induces an mTOR-mediated overshooting protein synthesis response that contributes to reperfusion injury by rapidly and selectively translating preexisting mRNAs. Methods and Results: To induce reperfusion injury mice were subjected to 60 minutes of LAD ligation and increasing times of reperfusion. Puromycin incorporation showed an overshooting protein synthesis response of cardiac myocytes upon reperfusion that was especially prevalent in the peri-infarct region. Protein synthesis was induced by increased formation of the eIF4F translation initiation complex through the mTORC1-4EBP1 signaling axis. Cardiac myocyte specific ribosome profiling combined with RNA-seq of hearts 2 days after reperfusion showed that the acute cardiac response to reperfusion was mediated by translation of specific mRNAs encoding proteins which determine early events of reperfusion such as leukocyte infiltration and inflammation. Inhibition of the mTORC1-4EBP1-eIF4F axis attenuated this specific protein synthesis response. Importantly, 4EGI-1, a specific inhibitor of eIF4F complex formation, bypassed off-target effects of previously used mTOR inhibitors and protected cardiac myocytes from cell death upon reperfusion. Conclusions: During reperfusion protein synthesis is strongly induced in the peri-infarct region by mTORC1 and contributes to reperfusion injury by rapidly translating specific mRNAs that mediate the early gene expression response to reperfusion.

Abstract 2698: eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma

Abstract Dysregulated messenger RNA (mRNA) translation drives the pathogenesis of multiple hematological malignancies. In lymphoma this includes the upregulation of key driver oncogenes and anti-apoptotic proteins (e.g., MYC, CCND1/3, BCL2 and MCL1) that contain a highly structured 5’-untranslated region (UTR) in their mRNA requiring enhanced eIF4A helicase activity for translation. eIF4A is a component of the eIF4F translation initiation complex and catalyzes the ATP-dependent unwinding of RNA duplexes and facilitates 43S ribosome scanning within the 5’-UTR. The activation of oncogenic signaling pathways, including RAS and PI3K, enhance eIF4A activity through phosphorylation of eIF4B, eIF4G and PDCD4 which facilitates formation of eIF4F and full activation of eIF4A. The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, promoting the translation of oncogenes with complex 5’-UTRs that are required for tumor cell proliferation, survival and metastasis. eFT226 is a potent and sequence selective eIF4A1 inhibitor that promotes eIF4A1 binding to specific 5’-UTR polypurine and/or G-quadraplex recognition motifs leading to a selective block in ribosome mRNA scanning. The sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating &amp;gt;100-fold greater sensitivity for reporter constructs containing a polypurine motif in the 5’-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex with increased drug residence time between eFT226, eIF4A1 and RNA oligonucleotides containing polypurine motifs. The ability of eFT226 to inhibit MYC or MCL1 expression was found to be dependent on the presence of their respective 5’-UTR supporting a translational regulation mechanism dependent on recognition elements within the 5’-UTR. eFT226 shows potent anti-proliferative activity (GI50 &amp;lt; 15 nM) against a panel of B-cell lymphoma cell lines. Treatment with eFT226 leads to coordinated inhibition of MYC, CCND1/3, BCL2 or MCL1 protein expression resulting in significant anti-tumor activity. eFT226 has good pharmacokinetic properties and exhibits significant in vivo activity across a panel of diffuse large B cell lymphoma (DLBCL), and Burkitt lymphoma tumor models with ≤1 mg/kg/week IV administration. Further evaluation of predictive markers of sensitivity or resistance has shown that tumors with mTOR mediated activation of eIF4A are most sensitive to eFT226. In addition, tumors with PTEN mutations do not exhibit activated eIF4A and are generally resistant to induction of apoptosis by eFT226, resulting in reduced in vivo efficacy. The association of eFT226 activity with PI3K/mTOR pathway activation and mutational status provides a means to identify patient subsets during clinical development. Clinical trials in patients with lymphoma and other malignancies are planned. Citation Format: Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Eric Sung, Ana Parra, Jocelyn Staunton, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Justin T. Ernst, Siegfried H. Reich, Kevin R. Webster. eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma [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 2698.

XPO1 inhibitor KPT-330 synergizes with Bcl-xL inhibitor to induce cancer cell apoptosis by perturbing rRNA processing and Mcl-1 protein synthesis

XPO1 (exportin1) mediates nuclear export of proteins and RNAs and is frequently overexpressed in cancers. In this study, we show that the orally bioavailable XPO1 inhibitor KPT-330 reduced Mcl-1 protein level, by which it synergized with Bcl-xL inhibitor A-1331852 to induce apoptosis in cancer cells. KPT-330/A-1331852 combination disrupted bindings of Mcl-1 and Bcl-xL to Bax, Bak, and/or Bim, elicited mitochondrial outer membrane permeabilization, and triggered apoptosis. KPT-330 generally mitigated mRNA expression and protein synthesis rather than mRNA nuclear export or protein stability of Mcl-1. KPT-330 inhibited mTORC1/4E-BP1 and Mnk1/eIF4E axes, which disrupted the eIF4F translation initiation complex but was dispensable for Mcl-1 reduction and KPT-330/A-1331852 combination-induced apoptosis. Mature rRNAs are integral components of the ribosome that determines protein synthesis ability. KPT-330 impeded nucleolar rRNA processing and reduced total levels of multiple mature rRNAs. Reconstitution of XPO1 by expressing degradation-resistant C528S mutant retained rRNA amount, Mcl-1 expression, and Bcl-xL inhibitor resistance upon KPT-330 treatment. KPT-330/A-1331852 combination suppressed growth and enhanced apoptosis of non-small cell lung cancer xenografts. Therefore, we clarify the reason of apoptosis resistance of cancer cells to XPO1 inhibition and develop a potential strategy for treating solid tumors.