Cap-dependent mRNA Translation Research Articles

Dual targeting of glutamine and serine metabolism in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.

The human eIF4E:4E-BP2 complex structure for studying hyperphosphorylation.

The cap-dependent mRNA translation is dysregulated in many kinds of cancers. The interaction between eIF4E and eIF4G through a canonical eIF4E-binding motif (CEBM) determines the efficacy of the cap-dependent mRNA translation. eIF4E-binding proteins (4E-BPs) share the CEBM and compete with eIF4G for the same binding surface of eIF4E and then inhibit the mRNA translation. 4E-BPs function as tumor repressors in nature. Hyperphosphorylation of 4E-BPs regulates the structure folding and causes the dissociation of 4E-BPs from eIF4E. However, until now, there has been no structure of the full-length 4E-BPs in complex with eIF4E. The regulation mechanism of phosphorylation is still unclear. In this work, we first investigate the interactions of human eIF4E with the CEBM and an auxiliary eIF4E-binding motif (AEBM) in eIF4G and 4E-BPs. The results unravel that the structure and interactions of the CEBM are highly conserved between eIF4G and 4E-BPs. However, the extended CEBM (ECEBM) in 4E-BPs forms a longer helix than that in eIF4G. The residue R62 in the ECEBM of 4E-BP2 forms salt bridges with E32 and E70 of eIF4E. The residue R63 of 4E-BP2 forms two special hydrogen bonds with N77 of eIF4E. Both of these interactions are missing in eIF4G. The AEBM of 4E-BPs folds into a β-sheet conformation, which protects V81 inside a hydrophobic core in 4E-BP2. In eIF4G, the AEBM exists in a random coil state. The hydrophilic residues S637 and D638 of eIF4G open the hydrophobic core for solvents. The results show that the ECEBM and AEBM may be responsible for the competing advantage of 4E-BP2. Finally, based on our previous work (J. Zeng, F. Jiang and Y. D. Wu, J. Chem. Theory Comput., 2017, 13, 320), the human eIF4E:4E-BP2 complex (eIF4E:BP2P18-I88) including all reported phosphorylation sites is predicted. The eIF4E:BP2P18-I88 complex is different from the existing experimental eIF4E:eIF4G complex and provides an important structure for further studying the regulation mechanism of phosphorylation in 4E-BPs.

Long-Acting E. coli-Derived Asparaginase Potentiates the Anti-Leukemic Effect of BCL2 Inhibition, but Not MCL1 Inhibition, in Preclinical Models of Acute Myeloid Leukemia

Background: Asparaginases are enzymes that deplete circulating asparagine and glutamine from plasma and can therefore be used therapeutically for cancers that are sensitive to amino acid depletion. Commercially available asparaginases are isolated fromeither E. coli or Erwinia chrysanthemi (also called crisantaspase), the latter having a higher glutaminase activity. Our previous studies have shown that pegcrisantaspase (PegC), a long-acting pegylated crisantaspase, synergizes with the BCL2 inhibitor, Venetoclax (Ven), to kill several AML cell lines and patient-derived primary AML cells with complex karyotype in vitro and in vivo in a xenograft mouse model. Whether long-acting E. coli-derived asparaginases will have similar anti-AML activity as a single agent or in combination with BCL-2 inhibition, and the potential of asparaginases to synergize with inhibitors of MCL1, a key resistance factor of BCL2 inhibition, has yet to be determined. Methods: Using a panel of 4 human AML cell lines (MOLM14, MV411, MonoMac6, HL60) as well as primary AML patient samples, weestablished the anti-leukemic activity of the BCL2 inhibitor S55746 and the MCL1 inhibitor S63845 alone and in combination with the long-acting E. coli asparaginase, calaspargase pegol-mknl (CalPegA). To determine the ability of S55746 or S63845 with or without CalPegA to inhibit mRNA translation, an established mechanism of Ven-PegC anti-leukemic activity, we measured the dissociation of the translation initiation factor eIF4E from 4EBP1, a negative regulator of cap-dependent mRNA translation, which then allows the formation of translation initiation complexes. The in vivo anti-leukemic efficacy of S55746 and S63845 alone and in combination with CalPegA was tested using an orthotopic AML xenograft mouse model with luciferase-expressing MV411 cells. Results: We found that CalPegA treatment resulted in a dose-dependent inhibition of AML cell proliferation, with IC 50 values ranging from 0.78 to 2.8 IU/mL. Single agent S55746 and S63845 treatment inhibited AML cell proliferation with IC 50 values ranging from 0.01 to 6.7 µM and 0.0001 to 0.08 µM, respectively. In 3 of the human AML cell lines (MOLM-14, MV4-11, and MonoMac6), the addition of a small dose (IC 20) of CalPegA potentiated the anti-leukemic effect of S55746, reducing the IC 50 by approximately 1- 4-fold, and by 10-fold in HL60 cells (Figure 1). This potentiation was also observed in 3 out of 4 primary AML patient samples tested, with the IC 50 of S55746 reduced by 4.5, 18, and 156-fold by the addition of CalPegA. The activity of S63845 was not substantially impacted by the addition of CalPegA in either AML cell lines (Figure 1) or primary samples. Immunoprecipitation of 4EBP1 revealed that treatment with a combination of either S63845 or S55746 with CalPegA increased the binding of eIF4E to 4EBP1 compared to single agent treatments, suggesting an inhibition of translational complex formation. In a MV411 tumor-bearing model, treatment withS55746-CalPegA combination reduced tumor burden over time significantly more than either single agent. S63845 treatment was highly effective at inhibiting leukemia growth over time and was not improved by the addition of CalPegA, as tumor burden in the S63845 and S63845 + CalPegA-treated groups was not significantly different over the course of the study (Figure 2). Conclusions: We report that the E. coli asparaginase, CalPegA, enhances the anti-leukemic effect of the BCL2 inhibitor, S55746, but does not impact the activity of the MCL1 inhibitor, S63845, in AML cell lines, patient derived primary AML samples, and in an AML xenograft mouse model. Ongoing studies are further investigating the anti-leukemic mechanism of S55476/S63845 + CalPegA and examining the efficacy and pharmacodynamic/pharmacokinetic effects of these agents in a patient derived xenograft model of AML.

Spatially distinct molecular patterns of gene expression in idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a heterogeneous disease that is pathologically characterized by areas of normal-appearing lung parenchyma, active fibrosis (transition zones including fibroblastic foci) and dense fibrosis. Defining transcriptional differences between these pathologically heterogeneous regions of the IPF lung is critical to understanding the distribution and extent of fibrotic lung disease and identifying potential therapeutic targets. Application of a spatial transcriptomics platform would provide more detailed spatial resolution of transcriptional signals compared to previous single cell or bulk RNA-Seq studies.MethodsWe performed spatial transcriptomics using GeoMx Nanostring Digital Spatial Profiling on formalin-fixed paraffin-embedded (FFPE) tissue from 32 IPF and 12 control subjects and identified 231 regions of interest (ROIs). We compared normal-appearing lung parenchyma and airways between IPF and controls with histologically normal lung tissue, as well as histologically distinct regions within IPF (normal-appearing lung parenchyma, transition zones containing fibroblastic foci, areas of dense fibrosis, and honeycomb epithelium metaplasia).ResultsWe identified 254 differentially expressed genes (DEGs) between IPF and controls in histologically normal-appearing regions of lung parenchyma; pathway analysis identified disease processes such as EIF2 signaling (important for cap-dependent mRNA translation), epithelial adherens junction signaling, HIF1α signaling, and integrin signaling. Within IPF, we identified 173 DEGs between transition and normal-appearing lung parenchyma and 198 DEGs between dense fibrosis and normal lung parenchyma; pathways dysregulated in both transition and dense fibrotic areas include EIF2 signaling pathway activation (upstream of endoplasmic reticulum (ER) stress proteins ATF4 and CHOP) and wound healing signaling pathway deactivation. Through cell deconvolution of transcriptome data and immunofluorescence staining, we confirmed loss of alveolar parenchymal signals (AGER, SFTPB, SFTPC), gain of secretory cell markers (SCGB3A2, MUC5B) as well as dysregulation of the upstream regulator ATF4, in histologically normal-appearing tissue in IPF.ConclusionsOur findings demonstrate that histologically normal-appearing regions from the IPF lung are transcriptionally distinct when compared to similar lung tissue from controls with histologically normal lung tissue, and that transition zones and areas of dense fibrosis within the IPF lung demonstrate activation of ER stress and deactivation of wound healing pathways.

Overcoming Venetoclax (Ven) Resistance through Glutamine (Gln) Depletion: Final Analysis of the Phase 1 Trial of Ven and Pegcrisantaspase (PegC) Combination in Relapsed and Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Ven-based therapies have been an advancement in the front-line treatment of AML; however development of resistance is common and the efficacy of Ven-containing regimens in R/R AML has been far less promising. We reported in vitro and in vivo depletion of Gln induced by long-acting Erwinia asparaginase, PegC, inhibited proliferation of complex karyotype (CK) AML and synergistically enhanced the antiapoptotic activity of Ven-mediated antagonism of Bcl-2 by decreasing the expression of proteins such as Mcl-1, whose translation is cap-dependent. Here we report the final results of our phase 1 clinical trial (NCT04666649) of VenPegC for treatment of R/R AML including patients (pts) previously treated with Ven. Methods: Pts received Ven 400 mg on D1-28 and PegC (500, 750, 1000 IU/m 2 in cohorts 1-3, IV) D1 & D15 every 4 weeks (wk). Primary study endpoints were incidences of regimen limiting toxicities (RLT) and RP2D. Secondary endpoints included rate of composite complete remissions. Correlative studies included monitoring plasma amino acid levels and asparaginase activity, and expression of proteins regulating cap-dependent mRNA translation in bone marrow (BM) mononuclear cells pre- and post-VenPegC treatment. Results: As of July 31, 2023, 26 pts were enrolled of whom 24 received at least 1 dose of PegC and 17 completed at least 1 cycle of PegC (2 doses). The median age was 61 (range 24-79), 16 (62%) were female; 9 (35%) had CK-AML and 5 (19%) had TP53 mutations; 18 (69%) were previously treated with Ven. Median prior lines of treatment were 2 (range 1-5). No RLT was observed in cohort 1 (n=3). Cohort 2 was expanded to 6 pts due to transient asymptomatic hypertriglyceridemia. Two pts in cohort 3 (n=3) developed grade 3 hyperbilirubinemia determined as RLT. Expansion cohort included 11 pts who were treated at MTD/RP2D (PegC 750 IU/m 2 plus Ven 400 mg) given as out-pt without a need for hospitalization. No pancreatitis, thrombohemorrhagic events, or silent inactivation were observed in the entire study population. Overall response rate (ORR) for pts who received ≥1 cycle of PegC was 47% (8/17), Fig 1. CR+ CRh+CRi rate was 29% (5/17); 1 pt had a partial remission with full count recovery and is currently receiving cycle 6; 2 pts had stable disease. Four pts showed hematologic improvement. Twelve (71%) pts who received ≥1 cycle of PegC were previously treated with Ven and were Ven-refractory. ORR in Ven-refractory pts was 42% (5/12); of the 5 pts who harbored a RAS or PTPN11 mutation and previously treated with Ven, 60% responded. Remarkably, all pts (3/3) who had a RUNX1 mutation achieved a CR; Pt 6 (relapsed AML with NRAS/cKIT/RUNX1 mutations) achieved an MRD-negative CRh and underwent an allogeneic stem cell transplant (alloHSCT); Pt 12 (primary refractory AML with TP53/NRAS/RUNX1 mutations) achieved a clearance of BM blasts after 1 dose of PegC and achieved MRD-negative CR at the end of cycle 1, and Pt 15 (post-alloHSCT relapsed AML with NRAS/PTPN11/CSF3R/RUNX1 mutations) achieved a CRi and received 10 cycles of VenPegC. Pt 31 (post-alloHSCT relapsed AML with FLT3-ITD) who had persistent disease after gilteritinib achieved an MRD-negative CRi, pending 2 nd alloHSCT. Nadir plasma asparaginase activity of >0.1 IU/mL was achieved in cycle 1 for all pts who received ≥1 cycle of VenPegC. All pts with available results achieved undetectable asparagine (Asn) level by day 7 and Asn remained undetectable throughout the study. Plasma Gln reduction from baseline occurred in all pts (except 1) within cycle 1. Pts in cohort 1 reached nadir Gln after initial PegC exposure in an average of 22 days compared to 16 days in cohort 2. In general, pts who achieved CR had the lowest absolute plasma Gln levels. Available Glu and Gln are shown in Fig.1 for pts who achieved a CR/PR. Out of the 8 assessed pts (4 responders & 4 non-responders), VenPegC inhibited cap-dependent translation after 1 cycle in responders, as evidenced by decreased phosphorylated eIF4E (active, pheIF4E) in 4/4 pts and decreased phosphorylated 4EBP1 (inactive, ph4EBP1), a negative regulator of eIF4E, in 2/4 pts. Conversely, pheIF4E and ph4EBP1 expression was increased in 3/4 and 4/4 of the non-responders, respectively (Fig.2). Conclusion: VenPegC is a novel regimen that can induce complete remission in some heavily pretreated R/R AML patients, even with previous exposure to Ven. Pts with RUNX1 mutation, whose translation depends on pheIF4E, may benefit further from this regimen.

Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

Cancer cell plasticity enables cell survival in harsh physiological environments and fate transitions such as the epithelial-to-mesenchymal transition (EMT) that underlies invasion and metastasis. Using genome-wide transcriptomic and translatomic studies, an alternate mechanism of cap-dependent mRNA translation by the DAP5/eIF3d complex is shown to be essential for metastasis, EMT, and tumor directed angiogenesis. DAP5/eIF3d carries out selective translation of mRNAs encoding EMT transcription factors and regulators, cell migration integrins, metalloproteinases, and cell survival and angiogenesis factors. DAP5 is overexpressed in metastatic human breast cancers associated with poor metastasis-free survival. In human and murine breast cancer animal models, DAP5 is not required for primary tumor growth but is essential for EMT, cell migration, invasion, metastasis, angiogenesis, and resistance to anoikis. Thus, cancer cell mRNA translation involves two cap-dependent mRNA translation mechanisms, eIF4E/mTORC1 and DAP5/eIF3d. These findings highlight a surprising level of plasticity in mRNA translation during cancer progression and metastasis.

Reversal of memory and autism-related phenotypes in Tsc2+/- mice via inhibition of Nlgn1.

Tuberous sclerosis complex (TSC) is a rare monogenic disorder co-diagnosed with high rates of autism and is caused by loss of function mutations in the TSC1 or TSC2 genes. A key pathway hyperactivated in TSC is the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which regulates cap-dependent mRNA translation. We previously demonstrated that exaggerated cap-dependent translation leads to autism-related phenotypes and increased mRNA translation and protein expression of Neuroligin 1 (Nlgn1) in mice. Inhibition of Nlgn1 expression reversed social behavior deficits in mice with increased cap-dependent translation. Herein, we report elevated translation of Nlgn1 mRNA and an increase in its protein expression. Genetic or pharmacological inhibition of Nlgn1 expression in Tsc2 +/- mice rescued impaired hippocampal mGluR-LTD, contextual discrimination and social behavior deficits in Tsc2 +/- mice, without correcting mTORC1 hyperactivation. Thus, we demonstrate that reduction of Nlgn1 expression in Tsc2 +/- mice is a new therapeutic strategy for TSC and potentially other neurodevelopmental disorders.

Host 5'-3' Exoribonuclease XRN1 Acts as a Proviral Factor for Measles Virus Replication by Downregulating the dsRNA-Activated Kinase PKR.

Many negative-sense RNA viruses, including measles virus (MeV), are thought to carry out much of their viral replication in cytoplasmic membraneless foci known as inclusion bodies (IBs). The mechanisms by which IBs facilitate efficient viral replication remain largely unknown but may involve an intricate network of regulation at the host-virus interface. Viruses are able to modulate such interactions by a variety of strategies including adaptation of their genomes and "hijacking" of host proteins. The latter possibility broadens the molecular reservoir available for a virus to enhance its replication and/or antagonize host antiviral responses. Here, we show that the cellular 5'-3' exoribonuclease, XRN1, is a host protein hijacked by MeV. We found that upon MeV infection, XRN1 is translocated to cytoplasmic IBs where it acts in a proviral manner by preventing the accumulation of double-stranded RNA (dsRNA) within the IBs. This leads to the suppression of the dsRNA-induced innate immune responses mediated via the protein kinase R (PKR)-integrated stress response (ISR) pathway. IMPORTANCE Measles virus remains a major global health threat due to its high transmissibility and significant morbidity in children and immunocompromised individuals. Although there is an effective vaccine against MeV, a large population in the world remains without access to the vaccine, contributing to more than 7,000,000 measles cases and 60,000 measles deaths in 2020 (CDC). For negative-sense RNA viruses including MeV, one active research area is the exploration of virus-host interactions occurring at cytoplasmic IBs where viral replication takes place. In this study we present evidence suggesting a model in which MeV IBs antagonize host innate immunity by recruiting XRN1 to reduce dsRNA accumulation and subsequent PKR kinase activation/ISR induction. In the absence of XRN1, the increased dsRNA level acts as a potent activator of the antiviral PKR/ISR pathway leading to suppression of global cap-dependent mRNA translation and inhibition of viral replication.

4E‐BP1/2 Deletion Enhances mRNA Cap‐binding Complex Assembly and Protein Synthesis in Immobilized Skeletal Muscle But is Not Sufficient to Prevent Muscle Atrophy

Skeletal muscle atrophy in long‐term care patients is positively correlated with worse health outcomes and patient mortality. Immobilization‐induced muscle atrophy is due to an imbalance between protein synthesis and protein degradation. The imbalance favors degradation due to anabolic resistance within the muscle. Targeting regulators of protein synthesis to increase mRNA translation under immobilized conditions may improve the protein synthesis/degradation imbalance and ameliorate immobilization‐induced skeletal muscle atrophy. 4E‐BP1 and 4E‐BP2 (eukaryotic initiation factor 4E (eIF4E) binding proteins 1 and 2) are important regulators of mRNA translation initiation that bind to eIF4E and prevents its binding to the scaffold protein eIF4G, an essential and rate‐limiting step of cap‐dependent mRNA translation. Previous studies have implicated eIF4E and its binding proteins in disuse‐induced repression of muscle protein synthesis. Consequently, we hypothesize that eliminating 4E‐BP1/2 may enhance mRNA translation and protect against disuse‐induced muscle atrophy.The hindlimb muscles of 8–9‐week‐old C57Bl/6 mixed‐sex wild‐type (WT) (20.25±2.39 g; n=19) and global 4E‐BP1/4E‐BP2 double knockout (DKO) (20.55±2.65 g; n=19) mice were compared after 3 days of unilateral hindlimb immobilization. Each mouse had one hindlimb immobilized in the plantarflexed position while the contralateral nonimmobilized hindlimb served as a control. Food and water were provided to the mice ad libitum. The hindlimb muscles were removed after a 4‐6 h fast, and the weights were compared between both genotypes for both the immobilized and free limbs. The gastrocnemius muscles were homogenized, eIF4E was immunoprecipitated, and eIF4E, eIF4G, and 4E‐BP1 in the immunoprecipitated sample was assessed by western blot analysis. In the immobilized gastrocnemius of the DKO mice the amount of eIF4G associated with eIF4E was higher than in control mice (p=0.001). In addition, mRNA translation, as assessed in a separate study by the incorporation of puromycin (0.04 μmol puromycin/g of body mass) into protein in the gastrocnemius, was also higher in muscle of the immobilized leg of DKO (n=24) compared to control mice (n=26) (17.1% increase, p=0.012). Surprisingly, eIF4E binding protein deficiency did not prevent muscle atrophy, as the mass of the gastrocnemius, plantaris, and soleus muscles was reduced to a similar extent in the immobilized compared to the control leg of both DKO and wildtype mice. To assess a possible role for upregulation of protein degradation in disuse‐induced muscle atrophy, expression of the E3 ubiquitin ligase, MAFbx‐1 (muscle atrophy f‐box 1) was also assessed. MAFbx‐1 expression was elevated in the gastrocnemius of the immobilized compared to the contralateral non‐immobilized limb, though no significant difference was found between genotypes. More research into other components of the mRNA cap binding complex, e.g., eIF4A, and additional biomarkers of protein degradative pathways may reveal more details on the mechanism of immobilization‐induced muscle atrophy.

Translatome changes in acute myeloid leukemia cells post exposure to pegcrisantaspase and venetoclax

The clinical outcomes of patients with acute myeloid leukemia (AML) treated with available therapy remain unsatisfactory. We recently reported that the BCL-2 inhibitor venetoclax synergizes with pegcrisantaspase (Ven-PegC) and exhibits remarkable in vivo efficacy in a preclinical model of AML with complex karyotype. The Ven-PegC combination blocks synthesis of proteins in AML cells by inhibiting cap-dependent translation of mRNA. To further explore the impact of Ven-PegC on protein translation, we used polysome profiling and high-throughput RNA sequencing to characterize Ven-PegC-dependent changes to the translatome. Here we report that the translation of five mRNAs, including two microRNAs, one rRNA, and two mitochondrial genes, was altered after exposure to all three treatments (Ven, PegC, and Ven-PegC). We focused our translatome validation studies on six additional genes related to translational efficiency that were modified by Ven-PegC. Notably, Ven-PegC treatment increased the RNA translation and protein levels of Tribbles homologue 3 (TRIB3), eukaryotic translation initiation factor 3 subunit C (eIF3C), doublesex and mab-3-related transcription factor 1 (DMRT1), and salt-inducible kinase 1 (SIK1). We validated the observed changes in gene/protein expression in vitro and confirmed our cell line-based studies in the bone marrow of an AML patient-derived xenograft model after Ven-PegC treatment. These results support examining alterations in the translatome post chemotherapy to offer insight into the drug's mechanism of action and to inform future therapeutic decisions.

Perspective: Modulating the integrated stress response to slow aging and ameliorate age-related pathology.

Healthy aging requires the coordination of numerous stress signaling pathways that converge on the protein homeostasis network. The Integrated Stress Response (ISR) is activated by diverse stimuli, leading to phosphorylation of the eukaryotic translation initiation factor elF2 in its α-subunit. Under replete conditions, elF2 orchestrates 5' cap-dependent mRNA translation and is thus responsible for general protein synthesis. elF2α phosphorylation, the key event of the ISR, reduces global mRNA translation while enhancing the expression of a signature set of stress response genes. Despite the critical role of protein quality control in healthy aging and in numerous longevity pathways, the role of the ISR in longevity remains largely unexplored. ISR activity increases with age, suggesting a potential link with the aging process. Although decreased protein biosynthesis, which occurs during ISR activation, have been linked to lifespan extension, recent data show that lifespan is limited by the ISR as its inhibition extends survival in nematodes and enhances cognitive function in aged mice. Here we survey how aging affects the ISR, the role of the ISR in modulating aging, and pharmacological interventions to tune the ISR. Finally, we will explore the ISR as a plausible target for clinical interventions in aging and age-related disease.

Cap-independent translation: A shared mechanism for lifespan extension by rapamycin, acarbose, and 17α-estradiol.

We hypothesized that rapamycin (Rapa), acarbose (ACA), which both increase mouse lifespan, and 17α‐estradiol, which increases lifespan in males (17aE2) all share common intracellular signaling pathways with long‐lived Snell dwarf, PAPPA‐KO, and Ghr−/− mice. The long‐lived mutant mice exhibit reduction in mTORC1 activity, declines in cap‐dependent mRNA translation, and increases in cap‐independent translation (CIT). Here, we report that Rapa and ACA prevent age‐related declines in CIT target proteins in both sexes, while 17aE2 has the same effect only in males, suggesting increases in CIT. mTORC1 activity showed the reciprocal pattern, with age‐related increases blocked by Rapa, ACA, and 17aE2 (in males only). METTL3, required for addition of 6‐methyl‐adenosine to mRNA and thus a trigger for CIT, also showed an age‐dependent increase blunted by Rapa, ACA, and 17aE2 (in males). Diminution of mTORC1 activity and increases in CIT‐dependent proteins may represent a shared pathway for both long‐lived‐mutant mice and drug‐induced lifespan extension in mice.

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.

Eukaryotic initiation factor 4 gamma 2 contributes to neuropathic pain through down-regulation of Kv1.2 and the mu opioid receptor in mouse primary sensory neurones

BackgroundNerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to neuropathic pain genesis. Eukaryotic initiation factor 4 gamma 2 (eIF4G2) is a general repressor of cap-dependent mRNA translation. Whether DRG eIF4G2 participates in nerve injury-induced alternations in gene expression and nociceptive hypersensitivity is unknown. MethodsThe expression and distribution of eIF4G2 mRNA and protein in mouse DRG after spinal nerve ligation (SNL) were assessed. Effects of eIF4G2 siRNA microinjected through a glass micropipette into the injured DRG on the SNL-induced DRG mu opioid receptor (MOR) and Kv1.2 downregulation and nociceptive hypersensitivity were examined. In addition, effects of DRG microinjection of adeno-associated virus 5-expressing eIF4G2 (AAV5-eIF4G2) on basal DRG MOR and Kv1.2 expression and nociceptive thresholds were analysed. ResultseIF4G2 protein co-expressed with Kv1.2 and MOR in DRG neurones. Levels of eIF4G2 mRNA (1.7 [0.24] to 2.3 [0.14]-fold of sham, P<0.01) and protein (1.6 [0.14] to 2.5 [0.22]-fold of sham, P<0.01) in injured DRG were time-dependently increased on days 3–14 after SNL. Blocking increased eIF4G2 through microinjection of eIF4G2 siRNA into the injured DRG attenuated SNL-induced downregulation of DRG MOR and Kv1.2 and development and maintenance of nociceptive hypersensitivities. DRG microinjection of AAV5-eIF4G2 reduced DRG MOR and Kv1.2 expression and elicited hypersensitivities to mechanical, heat and cold stimuli in naïve mice. ConclusionseIF4G2 contributes to neuropathic pain through participation in downregulation of Kv1.2 and MOR in injured DRG and is a potential target for treatment of this disorder.

CDK1, the Other ‘Master Regulator’ of Autophagy

Autophagy and cap-dependent mRNA translation are tightly regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signalling complex in response to nutrient availability. However, the regulation of these processes, and mTORC1 itself, is different during mitosis, and this has remained an area of significant controversy; for example, studies have argued that autophagy is either repressed or highly active during mitosis. Recent studies have shown that autophagy initiation is repressed, and cap-dependent mRNA translation is maintained during mitosis despite mTORC1 activity being repressed. This is achieved in large part by a switch from mTORC1- to cyclin-dependent kinase 1 (CDK1)-mediated regulation. Here, we review the history and recent advances and seek to present a unifying model to inform the future study of autophagy and mTORC1 during mitosis.

Abstract IA13: An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis

Abstract While translation initiation of most mammalian mRNAs is mediated by a 5' cap structure that binds eIF4E, inactivation/silencing of eIF4E, or quantitative sequestration of eIF4E by 4E-BPs through mTORC1 inhibition, does not impair translation of many capped mRNAs. This suggests an alternate mechanism may exist for cap-dependent but eIF4E/mTORC1-independent translation. DAP5 (NAT1, eIF4G2, p97) is an eIF4G family member that lacks the N-terminal binding domains for eIF4E and PABP. DAP5 was shown to mediate translation initiation through internal ribosome entry sites (IRES elements). We recently showed that DAP5 utilizes the cap binding activity of eIF3d to facilitate cap-dependent translation of a large number of capped mRNAs, making it a previous unknown major mechanism of cap-dependent but eIF4E/mTORC1-independent mRNA translation. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factor and cell receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair, oncogenesis, translation initiation, and cancer cell metastasis. Up to 20% of cellular mRNAs utilize DAP5-eIF3d for cap-dependent mRNA translation. Mass spectrometry and in vivo cross-linking studies found that eIF3d is a direct binding partner of DAP5 but does not bind strongly to eIF4GI or II family members that bind eIF4E. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E and mTORC1-independent capped-mRNA translation. Given the prevalence of oncogenic and survival mRNAs directed in translation by DAP5-eIF3d, we analyzed the NCI human tissue genome cancer atlas (TCGA) and found that high expression of DAP5 mRNA is strongly associated with estrogen receptor-negative (ER-) breast cancer metastasis and poor survival. We therefore investigated the role of DAP5 in preclinical mouse models of highly metastatic ER- breast cancer. Reduction of DAP5 expression by ~70% almost eliminated metastasis to the lung and significantly increased survival with no impact on primary tumor growth. These and other studies indicate that the DAP5-eIF3d complex is a significant alternate mechanism for cap-dependent, eIF4E/mTORC1-independent mRNA translation despite lacking eIF4E and PABP binding, that DAP5 directs translation of many capped mRNAs that are involved in cell stress and survival pathways, is essential for promoting breast cancer metastasis, and that most mRNAs translated by DAP5 do not utilize an IRES. Citation Format: Columba de la Parra, Amanda Ernlund, Amandine Alard, Kelly Ruggles, Beatrix Ueberheide, Olga Katsara, Robert J. Schneider. An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis [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 IA13.

5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5' UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

MTORC1 inhibition promotes human Treg differentiation via privileged mRNA translation

Abstract Maturation of regulatory T cells (Tregs) in peripheral sites is known to require TGFβ exposure and inhibition of protein kinase mTORC1. It is well known that mTOR inhibition is associated with repression of cap-dependent mRNA translation, which represents the major mechanism for protein synthesis, leaving unanswered how Tregs carry-out essential translation for development and immune suppression activity. To answer this question, we performed genome-wide transcription and translation profiling in CD4+CD127dim/− CD25+Tregs derived from anti-CD3/CD28-activated human naïve CD4+ T cells, treated with the mTORC1 inhibitor RAD001 and/or TGFβ. We found that TGFβ activated both Treg differentiation and immune suppression genes, while mTORC1 inhibition selectively blocked translation of most T cell mRNAs except those induced by TGFβ, including FOXP3, CTLA-4, CD101 or CD103, locking in Treg lineage commitment and immune suppression function. These canonical Treg fate-determining mRNAs were resistant to mTORC1 inhibition, an effect mediated in part by their 5′-untranslated regions through an alternate form of appears to be cap-dependent, eIF4E-independent mRNA translation. In conclusion, TGFβ transcriptional reprogramming together with mTORC1-independent translational reprogramming enable a privileged translation mechanism by which activated CD4+ T cells become Tregs.

The expression of lacrimal androgen-binding proteins in mice Pseudomonas aeruginosa keratitis.

To investigate the expression of lacrimal androgen-binding proteins (ABPs) in mice Pseudomonas aeruginosa (P. aeruginosa) keratitis. P. aeruginosa mice model from different gender was developed by intra-stromal injection. The expression of lacrimal ABPs in lacrimal gland specimens from P. aeruginosa keratitis mice was detected by the quantitative polymerase chain reaction (qRT-PCR). Corneal virulence was evaluated based on clinical scores. To study the mechanism of lacrimal ABPs' expression, experimental subjects were pre-treated with 4E-BP1 inhibitor, and were used to evaluate the expression levels by qRT-PCR. Compared with control groups, the expression of ABPα, ABPη and ABPζ in lacrimal gland from P. aeruginosa keratitis mice had no meaningful changes, while ABPε and ABPδ were significantly higher at 1d after infection. The expression of ABPδ in lacrimal gland of male mice was higher than female mice, regardless of whether or not P. aeruginosa keratitis occurred. After 4E-BP1 inhibitor subconjunctival injection or lacrimal injection, the expression of ABPδ and ABPε has no significant change compared with the control group. ABPδ and ABPε secreted by mice lacrimal gland may involve in the progress of alleviating the severity of corneal damage in P. aeruginosa keratitis. The expression of ABPδ and ABPε upon P. aeruginosa infection is independent of cap-dependent mRNA translation activated by 4E-BP1.

G-quadruplex located in the 5'UTR of the BAG-1 mRNA affects both its cap-dependent and cap-independent translation through global secondary structure maintenance.

The anti-apoptotic BAG-1 protein isoforms are known to be overexpressed in colorectal tumors and are considered to be potential therapeutic targets. The isoforms are derived from alternative translation initiations occuring at four in-frame start codons of a single mRNA transcript. Its 5′UTR also contains an internal ribosome entry site (IRES) regulating the cap-independent translation of the transcript. An RNA G-quadruplex (rG4) is located at the 5′end of the BAG-1 5′UTR, upstream of the known cis-regulatory elements. Herein, we observed that the expression of BAG-1 isoforms is post-transcriptionally regulated in colorectal cancer cells and tumors, and that stabilisation of the rG4 by small molecules ligands reduces the expression of endogenous BAG-1 isoforms. We demonstrated a critical role for the rG4 in the control of both cap-dependent and independent translation of the BAG-1 mRNA in colorectal cancer cells. Additionally, we found an upstream ORF that also represses BAG-1 mRNA translation. The structural probing of the complete 5′UTR showed that the rG4 acts as a steric block which controls the initiation of translation at each start codon of the transcript and also maintains the global 5′UTR secondary structure required for IRES-dependent translation.