The Novel Nucleoside Analogue ProTide NUC-7738 Overcomes Cancer Resistance Mechanisms In Vitro and in a First-In-Human Phase I Clinical Trial.

PURPOSE: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3'-Deoxyadenosine (3'-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. EXPERIMENTAL DESIGN: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. RESULTS: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. CONCLUSIONS: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

LARP1 isoform expression in human cancer cell lines.

LARP1 is an oncogenic RNA-binding protein required for ribosome biogenesis and cancer cell survival. From published in vitro studies, there is disparity over which of two different LARP1 protein isoforms (termed the long LI-LARP1 and short SI-LARP1) is the canonical. Here, after conducting a series of biochemical and cellular assays, we conclude that LI-LARP1 (NM_033551.3 > NP_056130.2) is the dominantly expressed form. We observe that SI-LARP1 (NM_015315.5> NP_056130.2) is epigenetically repressed and that this repression is evolutionarily conserved in all but a small subclade of mammalian species. As with other LARP family members, there are multiple potential LARP1 mRNA isoforms that appear to be censored within the nucleus. The capacity of the cell to modulate splicing and expression of these apparently 'redundant' mRNAs hints at contextually specific mechanisms of LARP1 expression.

Subcellular mRNA Localization Regulates Ribosome Biogenesis in Migrating Cells.

Translation of ribosomal protein-coding mRNAs (RP-mRNAs) constitutes a key step in ribosome biogenesis, but the mechanisms that modulate RP-mRNA translation in coordination with other cellular processes are poorly defined. Here, we show that subcellular localization of RP-mRNAs acts as a key regulator of their translation during cell migration. As cells migrate into their surroundings, RP-mRNAs localize to the actin-rich cell protrusions. This localization is mediated by La-related protein 6 (LARP6), an RNA-binding protein that is enriched in protrusions. Protrusions act as hotspots of translation for RP-mRNAs, enhancing RP synthesis, ribosome biogenesis, and the overall protein synthesis in migratory cells. In human breast carcinomas, epithelial-to-mesenchymal transition (EMT) upregulates LARP6 expression to enhance protein synthesis and support invasive growth. Our findings reveal LARP6-mediated mRNA localization as a key regulator of ribosome biogenesis during cell migration and demonstrate a role for this process in cancer progression downstream of EMT.

Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response.

In eukaryotes, tRNAs are transcribed in the nucleus and exported to the cytosol, where they deliver amino acids to ribosomes for protein translation. This nuclear-cytoplasmic movement was believed to be unidirectional. However, active shuttling of tRNAs, named tRNA retrograde transport, between the cytosol and nucleus has been discovered. This pathway is conserved in eukaryotes, suggesting a fundamental function; however, little is known about its role in human cells. Here we report that, in human cells, oxidative stress triggers tRNA retrograde transport, which is rapid, reversible, and selective for certain tRNA species, mostly with shorter 3' ends. Retrograde transport of tRNASeC, which promotes translation of selenoproteins required to maintain homeostatic redox levels in cells, is highly efficient. tRNA retrograde transport is regulated by the integrated stress response pathway via the PERK-REDD1-mTOR axis. Thus, we propose that tRNA retrograde transport is part of the cellular response to oxidative stress.

The Deadly Bite of STAT3.

The Tasmanian devils' facial tumor disease (DFTD) is a transmissible cancer that spreads by biting and threatens extinction of this marsupial. In this issue of Cancer Cell, Kosack et al. describe how overexpression of ERBB and uncontrolled activation of STAT3 drive DFTD growth and immune evasion.

Three human aminoacyl-tRNA synthetases have distinct sub-mitochondrial localizations that are unaffected by disease-associated mutations.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key enzymes in the mitochondrial protein translation system and catalyze the charging of amino acids on their cognate tRNAs. Mutations in their nuclear genes are associated with pathologies having a broad spectrum of clinical phenotypes, but with no clear molecular mechanism(s). For example, mutations in the nuclear genes encoding mt-AspRS and mt-ArgRS are correlated with the moderate neurodegenerative disorder leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) and with the severe neurodevelopmental disorder pontocerebellar hypoplasia type 6 (PCH6), respectively. Previous studies have shown no or only minor impacts of these mutations on the canonical properties of these enzymes, indicating that the role of the mt-aaRSs in protein synthesis is mostly not affected by these mutations, but their effects on the mitochondrial localizations of aaRSs remain unclear. Here, we demonstrate that three human aaRSs, mt-AspRS, mt-ArgRS, and LysRS, each have a specific sub-mitochondrial distribution, with mt-ArgRS being exclusively localized in the membrane, LysRS exclusively in the soluble fraction, and mt-AspRS being present in both. Chemical treatments revealed that mt-AspRs is anchored in the mitochondrial membrane through electrostatic interactions, whereas mt-ArgRS uses hydrophobic interactions. We also report that novel mutations in mt-AspRS and mt-ArgRS genes from individuals with LBSL and PCH6, respectively, had no significant impact on the mitochondrial localizations of mt-AspRS and mt-ArgRS. The variable sub-mitochondrial locations for these three mt-aaRSs strongly suggest the existence of additional enzyme properties, requiring further investigation to unravel the mechanisms underlying the two neurodegenerative disorders.

Molecular Signatures of Regression of the Canine Transmissible Venereal Tumor.

The canine transmissible venereal tumor (CTVT) is a clonally transmissible cancer that regresses spontaneously or after treatment with vincristine, but we know little about the regression mechanisms. We performed global transcriptional, methylation, and functional pathway analyses on serial biopsies of vincristine-treated CTVTs and found that regression occurs in sequential steps; activation of the innate immune system and host epithelial tissue remodeling followed by immune infiltration of the tumor, arrest in the cell cycle, and repair of tissue damage. We identified CCL5 as a possible driver of CTVT regression. Changes in gene expression are associated with methylation changes at specific intragenic sites. Our results underscore the critical role of host innate immunity in triggering cancer regression.

Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.

Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures.

Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Released selective pressure on a structural domain gives new insights on the functional relaxation of mitochondrial aspartyl-tRNA synthetase.

Mammalian mitochondrial aminoacyl-tRNA synthetases are nuclear-encoded enzymes that are essential for mitochondrial protein synthesis. Due to an endosymbiotic origin of the mitochondria, many of them share structural domains with homologous bacterial enzymes of same specificity. This is also the case for human mitochondrial aspartyl-tRNA synthetase (AspRS) that shares the so-called bacterial insertion domain with bacterial homologs. The function of this domain in the mitochondrial proteins is unclear. Here, we show by bioinformatic analyses that the sequences coding for the bacterial insertion domain are less conserved in opisthokont and protist than in bacteria and viridiplantae. The divergence suggests a loss of evolutionary pressure on this domain for non-plant mitochondrial AspRSs. This discovery is further connected with the herein described occurrence of alternatively spliced transcripts of the mRNAs coding for some mammalian mitochondrial AspRSs. Interestingly, the spliced transcripts alternately lack one of the four exons that code for the bacterial insertion domain. Although we showed that the human alternative transcript is present in all tested tissues; co-exists with the full-length form, possesses 5'- and 3'-UTRs, a poly-A tail and is bound to polysomes, we were unable to detect the corresponding protein. The relaxed selective pressure combined with the occurrence of alternative splicing, involving a single structural sub-domain, favors the hypothesis of the loss of function of this domain for AspRSs of mitochondrial location. This evolutionary divergence is in line with other characteristics, established for the human mt-AspRS, that indicate a functional relaxation of non-viridiplantae mt-AspRSs when compared to bacterial and plant ones, despite their common ancestry.

Pathogenic implications of human mitochondrial aminoacyl-tRNA synthetases.

Mitochondria are considered as the powerhouse of eukaryotic cells. They host several central metabolic processes fueling the oxidative phosphorylation pathway (OXPHOS) that produces ATP from its precursors ADP and inorganic phosphate Pi (PPi). The respiratory chain complexes responsible for the OXPHOS pathway are formed from complementary sets of protein subunits encoded by the nuclear genome and the mitochondrial genome, respectively. The expression of the mitochondrial genome requires a specific and fully active translation machinery from which aminoacyl-tRNA synthetases (aaRSs) are key actors. Whilst the macromolecules involved in mammalian mitochondrial translation have been under investigation for many years, there has been an explosion of interest in human mitochondrial aaRSs (mt-aaRSs) since the discovery of a large (and growing) number of mutations in these genes that are linked to a variety of neurodegenerative disorders. Herein we will review the present knowledge on mt-aaRSs in terms of their biogenesis, their connection to mitochondrial respiration, i.e., the respiratory chain (RC) complexes, and to the mitochondrial translation machinery. The pathology-related mutations detected so far are described, with special attention given to their impact on mt-aaRSs biogenesis, functioning, and/or subsequent activities. The collected data to date shed light on the diverse routes that are linking primary molecular possible impact of a mutation to its phenotypic expression. It is envisioned that a variety of mechanisms, inside and outside the translation machinery, would play a role on the heterogeneous manifestations of mitochondrial disorders.

A human pathology-related mutation prevents import of an aminoacyl-tRNA synthetase into mitochondria.

Mutations in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase, a key enzyme for mitochondrial translation, are correlated with leukoencephalopathy. A Ser45 to Gly45 mutation is located in the predicted targeting signal of the protein. We demonstrate in the present study, by in vivo and in vitro approaches, that this pathology-related mutation impairs the import process across mitochondrial membranes.