Formation Of Initiation Complex Research Articles

Abstract 1522: Cxcr3 chemokine receptor crosstalk with EGFR recurs independently of the Cxcl10 chemokine to signal for cell migration in the absence of the tumor suppressor ING4

Abstract Cxcr3 is a G-protein coupled receptor expressed on various cell types including cancer cells. Cxcr3 is activated via binding of the chemokine Cxcl10, which mediates immune cell migration during normal immune response. Pharmacological inhibition of Cxcr3 was shown to mitigate metastasis in a murine breast cancer model, suggesting it is critical for cancer metastasis, but the mechanism is not well understood. We have previously shown that exogenous Cxcl10 induced migration of ING4-deleted breast cancer cells, but not of ING4-intact cells. Cxcl10 signaling required the growth factor receptor Egfr, suggesting a crosstalk between Cxcr3 and Egfr. Confocal immunofluorescent (IF) colocalization assays showed that Cxcr3 and Egfr rapidly and transiently colocalized upon Cxcl10 treatment, which recurred only in ING4-deleted cells, suggesting that the recurrent receptor association may drive Cxcl10-induced cell migration. Here we characterized the temporal dynamics of the Cxcl10-induced Cxcr3/EGFR receptor associations and migration using IF and transwell migration assays, respectively. We also used inhibitors of Cxcr3 (AMG-487), Egfr (erlotinib and SAH-EJ1), and Gβγ (gallein) in order to delineate the signaling mechanism. The IF results demonstrated that, the first Cxcr3/EGFR association occurred rapidly within 10 minutes of Cxcl10 treatment, which disappeared by 1 hour post Cxcl10 treatment. The Cxcr3/Egfr complex formation recurred again transiently at 8 hours post Cxcl10 treatment. This transient receptor complex formation repeatedly occurred at 8-hour intervals up to 24 hours, suggesting a cyclic complex formation. 1 hour treatment of cells with Cxcl10 induced the cyclic formation of the Cxcr3/Egfr complexes, indicating that continuous presence of Cxcl10 was not required for recurrent Cxcr3/EGFR association. Likewise, 1 hour Cxcl10 treatment of cells induced cell migration, indicating that the initial binding of Cxcl10 to Cxcr3 was the critical event in the subsequent recurrent receptor colocalization and cell migration. Inhibition of the early Cxcr3/Egfr complex formation by AMG-487 or SAH-EJ1 blocked subsequent receptor association and cell migration, corroborating the idea that the initial receptor complex formation is the critical event. Erlotinib did not block the receptor colocalization, but inhibited cell migration when added for the initial 1 hour time duration of Cxcl10 treatment, but not when added after 4 hours. These results indicated that the early Cxcr3/Egfr complex signaled via the Egfr kinase. In contrast, gallein inhibited cell migration irrespective of when it was added, indicating that Gβγ was a required signaling component downstream of Cxcr3/EGFR. These findings present the Cxcl10/Cxcr3/Egfr/Gβγ signaling axis as the potential therapeutic targets for ING4-deficient aggressive breast cancer. Citation Format: Emily Tsutsumi, Suwon Kim. Cxcr3 chemokine receptor crosstalk with EGFR recurs independently of the Cxcl10 chemokine to signal for cell migration in the absence of the tumor suppressor ING4 [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 1522.

The context of the ribosome binding site in mRNAs defines specificity of action of kasugamycin, an inhibitor of translation initiation

Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some Escherichia coli proteins. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug. In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action.

N-Heterocyclic Carbene-Phosphinidenide Complexes as Hydroboration Catalysts.

The reactions of the N-heterocyclic carbene-phosphinidene adducts (NHC)PSiMe3 and (NHC)PH with the dinuclear ruthenium and osmium complexes [(η6-p-cymene)MCl2]2 (M = Ru, Os) afforded the half-sandwich complexes [(η6-p-cymene){(NHC)P}MCl] and [(η6-p-cymene){(NHC)PH}MCl2] with two- and three-legged piano-stool geometries, respectively (NHC = IDipp, IMes; IDipp = 1,3-bis(2,6-diisopropylphenyl)imidazolin-2-ylidene; IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazolin-2-ylidene). The complexes were initially tested as precatalysts for the hydroboration of benzonitrile, and the most active species, the ruthenium complex [(η6-p-cymene){(IMes)P}RuCl], was further used for the efficient hydroboration of a wide range (ca. 50 substrates) of nitriles, carboxylic esters, and carboxamides in neat pinacolborane (HBpin) under comparatively mild reaction conditions (60-80 °C, 3-5 mol % catalyst loading). Preliminary mechanistic and kinetic studies are reported, and stoichiometric reactions with HBpin indicate the initial formation of the monohydride complex [(η6-p-cymene){(IMes)P}RuH] as the putative catalytically active species.

The gatekeeper of Yersinia type III secretion is under RNA thermometer control.

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37°C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5’-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37°C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs.

Cyclin-dependent kinase 7 is essential for spermatogenesis by regulating retinoic acid signaling pathways and the STAT3 molecular pathway.

Spermatogenesis is a complex process that requires precise regulation. Phosphorylation plays a role in spermatogenesis by regulating protein structure and activity. This study focused on cyclin-dependent kinase 7 (CDK7), and explored its function and molecular mechanisms in spermatogenesis in vitro in a cell line and in vivo in a mouse model. Inhibition of CDK7 activity affected spermatogonia proliferation and differentiation, and we found that CDK7 regulates retinoic acid (RA)-mediated c-KIT expression to play a role in spermatogonia. Then, we demonstrated that inhibition of CDK7 affected meiosis initiation, DNA repair, and synaptonemal complex formation in meiosis progression, and CDK7 played this role by regulating RA-mediated STRA8 and REC8 signaling pathways. Moreover, inhibition of CDK7 impacted spermatid differentiation and resulted in decreased counts, decreased motility, and increased head deformity of sperm. We demonstrated that CDK7 affects germ cell apoptosis and sperm motility by activating STAT3 and that STAT3 further regulates Cortactin expression to influence the nuclear elongation, chromatin condensation, and acrosome formation of sperm. Additionally, EP300 was identified as another potential target phosphorylated by CDK7 that participates in chromatin condensation. Our results demonstrated the important role of CDK7 in all key aspects of spermatogenesis, potentially providing an effective target for clinical diagnosis and pathogenesis.

Phosphorylation of eukaryotic initiation factor eIFiso4E enhances the binding rates to VPg of turnip mosaic virus.

Binding of phosphorylated eIFiso4E with viral genome-linked protein (VPg) of turnip mosaic virus was examined by stopped-flow, fluorescence, circular dichroism (CD) spectroscopy, and molecular docking analysis. Phosphorylation of eIFiso4E increased (4-fold) the binding rates as compared to unphosphorylated eIFiso4E with VPg. Stopped-flow kinetic studies of phosphorylated eIFiso4E with VPg showed a concentration-independent conformational change. The dissociation rate was about 3-fold slower for eIFiso4E∙VPg complex upon phosphorylation. Phosphorylation enhanced the association rates and lowered the dissociation rates for the eIFiso4E∙VPg binding, with having higher preferential binding to eIFiso4Ep. Binding rates for the interaction of eIFiso4Ep with VPg increased (6-fold) with an increase in temperature, 278 K to 298 K. The activation energies for binding of eIFiso4Ep and eIFiso4E with VPg were 37.2 ± 2.8 and 52.6 ± 3.6 kJ/mol, respectively. Phosphorylation decreased the activation energy for the binding of eIFiso4E to VPg. The reduced energy barrier suggests more stable platform for eIFiso4Ep∙VPg initiation complex formation, which was further supported by molecular docking analysis. Moreover, far-UV CD studies revealed that VPg formed complex with eIFiso4Ep with substantial change in the secondary structure. These results suggested that phosphorylation, not only reduced the energy barrier and dissociation rate but also enhanced binding rate, and an overall conformational change, which provides a more stable platform for efficient viral translation.

Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence-An Experimental Study of the Human Voltage-Dependent Anion Channel.

Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage-dependent anion channel (VDAC), which is a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function that quantifies the feasibility of the formation of the translation initiation complex through the ribosome–mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome is proposed. The scoring function enables one to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at University of Vienna, Austria.

DNA Intercalators Inhibit Eukaryotic Ribosomal RNA Synthesis by Impairing the Initiation of Transcription.

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system.

Isolation and Identification of Pseudo Seven-Coordinate Ru(III) Intermediate Completing the Catalytic Cycle of Ru-bda Type of Water Oxidation Catalysts

Isolation and Identification of Pseudo Seven-Coordinate Ru(III) Intermediate Completing the Catalytic Cycle of Ru-bda Type of Water Oxidation Catalysts

Hydrosilylation of Carbonyl Compounds Catalyzed by a Nickel Complex Bearing a PBP Ligand

AbstractThe efficient catalytic hydrosilylation of ketones and aldehydes has been investigated using a nickel pincer hydride complex supported by a diphosphino‐boryl ligand (PBP). It was found that the presence of the boryl group within the skeleton of the ligand has a beneficial effect on the catalytic activities observed for ketones compared to related pincer systems. The analysis of the reaction mechanism allows for the synthesis and characterization of a nickel alkoxide derivative by insertion of the carbonyl moiety into the Ni−H bond. Combined experimental and theoretical analysis (DFT) support a reaction mechanism that involves the initial formation of an alkoxide complex followed by reaction with the silane to release the corresponding silyl ether and regenerate the catalyst.

The dynamic cycle of bacterial translation initiation factor IF3.

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.

Schizosaccharomyces pombe Ppr10 and Mpa1 together mediate mitochondrial translational initiation

Pentatricopeptide repeat (PPR) proteins are a large family of proteins that act primarily at different posttranscriptional steps of organellar gene expression. We have previously found that the Schizosaccharomyces pombe PPR protein mpal10 interacts with mitochondrial translational activator Mpa1, and both are essential for mitochondrial protein synthesis. However, it is unclear how these two proteins function in mitochondrial protein synthesis in S. pombe. In this study, we further investigated the role of Ppr10 and Mpa1 in mitochondrial protein synthesis. Mitochondrial translational initiation requires two initiation factors, Mti2 and Mti3, which bind to the small subunit of the mitochondrial ribosome (mt-SSU) during the formation of the mitochondrial translational initiation complex. Using sucrose gradient sedimentation analysis, we found that disruption of ppr10, mpa1, or the PPR motifs in Ppr10 impairs the association of Mti2 and Mti3 with the mt-SSU, suggesting that both Ppr10 and Mpa1 may be required for the interaction of Mti2 and Mti3 with the mt-SSU during the assembly of mitochondrial translational initiation complex. Loss of Ppr10 perturbs the association of mitochondrially encoded cytochrome b (cob1) and cytochrome c oxidase subunit 1 (cox1) mRNAs with assembled mitochondrial ribosomes. Proteomic analysis revealed that a fraction of Ppr10 and Mpa1 copurified with a subset of mitoribosomal proteins. The PPR motifs of Ppr10 are necessary for its interaction with Mpa1 and that disruption of these PPR motifs impairs mitochondrial protein synthesis. Our results suggest that Ppr10 and Mpa1 function together to mediate mitochondrial translational initiation.

MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty.

The family of Poly(A)-binding proteins (PABPs) regulates the stability and translation of messenger RNAs (mRNAs). Here we reported that the three members of PABPs, including PABPC1, PABPC3 and PABPC4, were identified as novel substrates for MKRN3, whose deletion or loss-of-function mutations were genetically associated with human central precocious puberty (CPP). MKRN3-mediated ubiquitination was found to attenuate the binding of PABPs to the poly(A) tails of mRNA, which led to shortened poly(A) tail-length of GNRH1 mRNA and compromised the formation of translation initiation complex (TIC). Recently, we have shown that MKRN3 epigenetically regulates the transcription of GNRH1 through conjugating poly-Ub chains onto methyl-DNA bind protein 3 (MBD3). Therefore, MKRN3-mediated ubiquitin signalling could control both transcriptional and post-transcriptional switches of mammalian puberty initiation. While identifying MKRN3 as a novel tissue-specific translational regulator, our work also provided new mechanistic insights into the etiology of MKRN3 dysfunction-associated human CPP.

Structural studies of RNase M5 reveal two-metal-ion supported two-step dsRNA cleavage for 5S rRNA maturation

ABSTRACT All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3ʹ- and 5ʹ-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3ʹ-before-5ʹ order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.

EIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning.

eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning.

Iron-promoted dealkylative carbene aminocyclization of δ-arylamino-α-diazoesters.

Herein, we report a novel methodology to access N-aryl proline derivatives using amino-tethered α-diazoesters and cheap, readily available iron salts. Mechanistically, the aminocyclization reaction involves the initial formation of an iron-carbene complex followed by a nucleophilic attack of the aniline nitrogen atom to give an ammonium ylide intermediate, which finally undergoes the iron-promoted dealkylation.

An RNA pseudoknot is essential for standby-mediated translation of the tisB toxin mRNA in Escherichia coli.

In response to DNA damage, Escherichia coli cells activate the expression of the toxin gene tisB of the toxin–antitoxin system tisB-istR1. Of three isoforms, only the processed, highly structured +42 tisB mRNA is active. Translation requires a standby site, composed of two essential elements: a single-stranded region located 100 nucleotides upstream of the sequestered RBS, and a structure near the 5′-end of the active mRNA. Here, we propose that this 5′-structure is an RNA pseudoknot which is required for 30S and protein S1-alone binding to the mRNA. Point mutations that prevent formation of this pseudoknot inhibit formation of translation initiation complexes, impair S1 and 30S binding to the mRNA, and render the tisB mRNA non-toxic in vivo. A set of mutations created in either the left or right arm of stem 2 of the pseudoknot entailed loss of toxicity upon overexpression of the corresponding mRNA variants. Combining the matching right-left arm mutations entirely restored toxicity levels to that of the wild-type, active mRNA. Finally, since many pseudoknots have high affinity for S1, we predicted similar pseudoknots in non-homologous type I toxin–antitoxin systems that exhibit features similar to that of tisB-IstR1, suggesting a shared requirement for standby acting at great distances.

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.

Selective translation by alternative bacterial ribosomes

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.