Laurent Volpon

MALT1 Small Molecule Inhibitors Specifically Suppress ABC-DLBCL In Vitro and In Vivo

MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.

Cap‐free structure of eIF4E suggests a basis for conformational regulation by its ligands

The activity of the eukaryotic translation initiation factor eIF4E is modulated through conformational response to its ligands. For example, eIF4G and eIF4E‐binding proteins (4E‐BPs) modulate cap affinity, and thus physiological activity of eIF4E, by binding a site distal to the 7‐methylguanosine cap‐binding site. Further, cap binding substantially modulates eIF4Es affinity for eIF4G and the 4E‐BPs. To date, only cap‐bound eIF4E structures were reported. In the absence of structural information on the apo form, the molecular underpinnings of this conformational response mechanism cannot be established. We report here the first cap‐free eIF4E structure. Apo‐eIF4E exhibits structural differences in the cap‐binding site and dorsal surface relative to cap‐eIF4E. Analysis of structure and dynamics of apo‐eIF4E, and changes observed upon ligand binding, reveal a molecular basis for eIF4Es conformational response to these ligands. In particular, alterations in the S4‐H4 loop, distal to either the cap or eIF4G binding sites, appear key to modulating these effects. Mutation in this loop mimics these effects. Overall, our studies have important implications for the regulation of eIF4E.

The Oncogene eIF4E Reprograms the Nuclear Pore Complex to Promote mRNA Export and Oncogenic Transformation

The eukaryotic translation initiation factor eIF4E is a potent oncogene that promotes the nuclear export and translation of specific transcripts. Here, we have discovered that eIF4E alters the cytoplasmic face of the nuclear pore complex (NPC), which leads to enhanced mRNA export of eIF4E target mRNAs. Specifically, eIF4E substantially reduces the major component of the cytoplasmic fibrils of the NPC, RanBP2, relocalizes an associated nucleoporin, Nup214, and elevates RanBP1 and the RNA export factors, Gle1 and DDX19. Genetic or pharmacological inhibition of eIF4E impedes these effects. RanBP2 overexpression specifically inhibits the eIF4E mRNA export pathway and impairs oncogenic transformation by eIF4E. The RanBP2 cytoplasmic fibrils most likely slow the release and/or recycling of critical export factors to the nucleus. eIF4E overcomes this inhibitory mechanism by indirectly reducing levels of RanBP2. More generally, these results suggest that reprogramming the NPC is a means by which oncogenes can harness the proliferative capacity of the cell.

Structural characterization of the Z RING-eIF4E complex reveals a distinct mode of control for eIF4E

The eukaryotic translation initiation factor eIF4E, a potent oncogene, is highly regulated. One class of eIF4E regulators, including eIF4G and the 4E-binding proteins (4E-BPs), interact with eIF4E using a conserved YXXXXLΦ-binding site. The structural basis of this interaction and its regulation are well established. Really Interesting New Gene (RING) domain containing proteins, such as the promyelocytic leukemia protein PML and the arenaviral protein Z, represent a second class of eIF4E regulators that inhibit eIF4E function by decreasing eIF4E’s affinity for its m7G cap ligand. To elucidate the structural basis of this inhibition, we determined the structure of Z and studied the Z-eIF4E complex using NMR methods. We show that Z interacts with eIF4E via a novel binding site, which has no homology with that of eIF4G or the 4E-BPs, and is different from the RING recognition site used in the ubiquitin system. Z and eIF4G interact with distinct parts of eIF4E and differentially alter the conformation of the m7G cap-binding site. Our results provide a molecular basis for how PML and Z RINGs reduce the affinity of eIF4E for the m7G cap and thereby act as key inhibitors of eIF4E function. Furthermore, our findings provide unique insights into RING protein interactions.

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Recognition of the methyl-7-guanosine (m7G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E–cap recognition is mediated via complementary charge interactions of the positively charged m7G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m7G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3–cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growth-promoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.

MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL.

The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in acute myeloid leukemia

The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). Although eIF4E RNA levels are elevated 3- to 10-fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses this activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the coding region. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens.

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate.

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m(7)G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5 μM eIF4E) but much weaker affinity at more typical NMR concentrations (50- ). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central β-sheet and dorsal surface of eIF4E, similar to m(7)G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in drug design efforts and reveal new insights into ligand eIF4E recognition.

NMR assignment of the arenaviral protein Z from Lassa fever virus

The arenavirus protein Z from Lassa fever virus was recently found to inhibit mRNA translation through direct interaction with eIF4E. Here, we report the NMR assignment of this RING-containing protein that was determined by triple resonance NMR techniques.

Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E

Significance Dysregulated nuclear trafficking of oncoproteins contributes to cancer. Here, we study the trafficking of eukaryotic translation initiation factor 4E (eIF4E) in acute myeloid leukemia (AML), where eIF4E is highly elevated and accumulates in the nucleus. Nuclear eIF4E promotes export of networks of transcripts encoding oncoproteins. During clinical responses to eIF4E-targeted therapy in patients who have AML, the distribution of eIF4E becomes almost entirely cytoplasmic. At relapse, eIF4E is again mainly nuclear. Our studies provide a molecular understanding for eIF4E trafficking, through association with importin 8. We provide a molecular basis for importin 8 selectivity for certain forms of eIF4E and demonstrate the relevance of its nuclear localization to its oncogenic potential, thereby positioning the importin 8–eIF4E interaction as a novel therapeutic target. Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m7G)–capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients’ responses. During clinical responses to the m7G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m7G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8–eIF4E complex as a novel therapeutic target.