Post-transcriptional gene regulation: From mechanisms to RNA chemistry and therapeutics.

A better understanding of the RNA biology and chemistry is necessary to then develop new RNA therapeutic strategies. This review is the synthesis of a series of conferences that took place during the 6th international course on post-transcriptional gene regulation at Institut Curie. This year, the course made a special focus on RNA chemistry.

Metabolism-dependent secondary effect of anti-MAPK cancer therapy on DNA repair.

Amino acid bioavailability impacts mRNA translation in a codon-dependent manner. Here, we report that the anti-cancer MAPK inhibitors (MAPKi) decrease the intracellular concentration of aspartate and glutamate in melanoma cells. This coincides with the accumulation of ribosomes on codons corresponding to these amino acids and triggers the translation-dependent degradation of mRNAs encoding aspartate- and glutamate-rich proteins, involved in DNA metabolism such as DNA replication and repair. Consequently, cells that survive MAPKi degrade aspartate and glutamate likely to generate energy, which simultaneously decreases their requirement for amino acids due to the downregulation of aspartate- and glutamate-rich proteins involved in cell proliferation. Concomitantly, the downregulation of aspartate- and glutamate-rich proteins involved in DNA repair increases DNA damage loads. Thus, DNA repair defects, and therefore mutations, are at least in part a secondary effect of the metabolic adaptation of cells exposed to MAPKi.

Post-transcriptional polyadenylation site cleavage maintains 3'-end processing upon DNA damage.

The recognition of polyadenylation signals (PAS) in eukaryotic pre-mRNAs is usually coupled to transcription termination, occurring while pre-mRNA is chromatin-bound. However, for some pre-mRNAs, this 3'-end processing occurs post-transcriptionally, i.e., through a co-transcriptional cleavage (CoTC) event downstream of the PAS, leading to chromatin release and subsequent PAS cleavage in the nucleoplasm. While DNA-damaging agents trigger the shutdown of co-transcriptional chromatin-associated 3'-end processing, specific compensatory mechanisms exist to ensure efficient 3'-end processing for certain pre-mRNAs, including those that encode proteins involved in the DNA damage response, such as the tumor suppressor p53. We show that cleavage at the p53 polyadenylation site occurs in part post-transcriptionally following a co-transcriptional cleavage event. Cells with an engineered deletion of the p53 CoTC site exhibit impaired p53 3'-end processing, decreased mRNA and protein levels of p53 and its transcriptional target p21, and altered cell cycle progression upon UV-induced DNA damage. Using a transcriptome-wide analysis of PAS cleavage, we identify additional pre-mRNAs whose PAS cleavage is maintained in response to UV irradiation and occurring post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows certain pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.

The multiple roles of LDH in cancer.

High serum lactate dehydrogenase (LDH) levels are typically associated with a poor prognosis in many cancer types. Even the most effective drugs, which have radically improved outcomes in patients with melanoma over the past decade, provide only marginal benefit to those with high serum LDH levels. When viewed separately from the oncological, biochemical, biological and immunological perspectives, serum LDH is often interpreted in very different ways. Oncologists usually see high serum LDH only as a robust biomarker of a poor prognosis, and biochemists are aware of the complexity of the various LDH isoforms and of their key roles in cancer metabolism, whereas LDH is typically considered to be oncogenic and/or immunosuppressive by cancer biologists and immunologists. Integrating these various viewpoints shows that the regulation of the five LDH isoforms, and their enzymatic and non-enzymatic functions is closely related to key oncological processes. In this Review, we highlight that serum LDH is far more than a simple indicator of tumour burden; it is a complex biomarker associated with the activation of several oncogenic signalling pathways as well as with the metabolic activity, invasiveness and immunogenicity of many tumours, and constitutes an extremely attractive target for cancer therapy.

Compartment-specific and ELAVL1-coordinated regulation of intronic polyadenylation isoforms by doxorubicin.

Intronic polyadenylation (IPA) isoforms, which contain alternative last exons, are widely regulated in various biological processes and by many factors. However, little is known about their cytoplasmic regulation and translational status. In this study, we provide the first evidence that the genome-wide patterns of IPA isoform regulation during a biological process can be very distinct between the transcriptome and translatome, and between the nucleus and cytosol. Indeed, by 3'-seq analyses on breast cancer cells, we show that the genotoxic anticancer drug, doxorubicin, preferentially down-regulates the IPA to the last-exon (IPA:LE) isoform ratio in whole cells (as previously reported) but preferentially up-regulates it in polysomes. We further show that in nuclei, doxorubicin almost exclusively down-regulates the IPA:LE ratio, whereas in the cytosol, it preferentially up-regulates the isoform ratio, as in polysomes. Then, focusing on IPA isoforms that are up-regulated by doxorubicin in the cytosol and highly translated (up-regulated and/or abundant in polysomes), we identify several IPA isoforms that promote cell survival to doxorubicin. Mechanistically, by using an original approach of condition- and compartment-specific CLIP-seq (CCS-iCLIP) to analyze ELAVL1-RNA interactions in the nucleus and cytosol in the presence and absence of doxorubicin, as well as 3'-seq analyses upon ELAVL1 depletion, we show that the RNA-binding protein ELAVL1 mediates both nuclear down-regulation and cytosolic up-regulation of the IPA:LE isoform ratio in distinct sets of genes in response to doxorubicin. Altogether, these findings reveal differential regulation of the IPA:LE isoform ratio across subcellular compartments during drug response and its coordination by an RNA-binding protein.

Glycolysis Dependency as a Hallmark of SF3B1-Mutated Cells.

SF3B1 mutations are recurrent in cancer and result in aberrant splicing of a previously defined set of genes. Here, we investigated the fate of aberrant transcripts induced by mutant SF3B1 and the related functional consequences. We first demonstrate that mutant SF3B1 does not alter global nascent protein synthesis, suggesting target-dependent consequences. Polysome profiling revealed that 35% of aberrantly spliced transcripts are more translated than their corresponding canonically spliced transcripts. This mostly occurs in genes with enriched metabolic functions. Furthermore, LC-MS/MS analysis showed that mutant SF3B1 impacts the abundance of proteins involved in metabolism. Functional metabolic characterization revealed that mutant SF3B1 decreases mitochondrial respiration and promotes glycolysis to compensate for defective mitochondrial metabolism. Hence, mutant SF3B1 induces glycolysis dependency, which sensitizes cells to glycolysis inhibition. Overall, we provide evidence of the oncogenic involvement of mutant SF3B1 in uveal melanoma through a metabolic switch to glycolysis, revealing vulnerability to glycolysis inhibitors as a promising therapeutic strategy.

At the crossroads of RNA biology, genome integrity and cancer.

This article is the synthesis of the scientific presentations that took place during two international courses at Institute Curie, one on post-transcriptional gene regulation and the other on genome instability and human disease, that were joined together in their 2021 edition. This joined course brought together the knowledge on RNA metabolism and the maintenance of genome stability.

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

Targeting the translation initiation complex eIF4F, which binds the 5' cap of mRNAs, is a promising anti-cancer approach. Silvestrol, a small molecule inhibitor of eIF4A, the RNA helicase component of eIF4F, inhibits the translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor, which, in turn, reduces the transcription of the gene encoding one of the major immune checkpoint proteins, i.e., programmed death ligand-1 (PD-L1) in melanoma cells. A large proportion of human genes produce multiple mRNAs differing in their 3'-ends through the use of alternative polyadenylation (APA) sites, which, when located in alternative last exons, can generate protein isoforms, as in the STAT1 gene. Here, we provide evidence that the STAT1α, but not STAT1ß protein isoform generated by APA, is required for silvestrol-dependent inhibition of PD-L1 expression in interferon-γ-treated melanoma cells. Using polysome profiling in activated T cells we find that, beyond STAT1, eIF4A inhibition downregulates the translation of some important immune-related mRNAs, such as the ones encoding TIM-3, LAG-3, IDO1, CD27 or CD137, but with little effect on the ones for BTLA and ADAR-1 and no effect on the ones encoding CTLA-4, PD-1 and CD40-L. We next apply RT-qPCR and 3'-seq (RNA-seq focused on mRNA 3' ends) on polysomal RNAs to analyze in a high throughput manner the effect of eIF4A inhibition on the translation of APA isoforms. We identify about 150 genes, including TIM-3, LAG-3, AHNAK and SEMA4D, for which silvestrol differentially inhibits the translation of APA isoforms in T cells. It is therefore crucial to consider 3'-end mRNA heterogeneity in the understanding of the anti-tumor activities of eIF4A inhibitors.

A PD-1/PD-L1 Proximity Assay as a Theranostic Marker for PD-1 Blockade in Patients with Metastatic Melanoma.

PURPOSE: Less than 50% of patients with melanoma respond to anti-programmed cell death protein 1 (anti-PD-1), and this treatment can induce severe toxicity. Predictive markers are thus needed to improve the benefit/risk ratio of immune checkpoint inhibitors (ICI). Baseline tumor parameters such as programmed death ligand 1 (PD-L1) expression, CD8+ T-cell infiltration, mutational burden, and various transcriptomic signatures are associated with response to ICI, but their predictive values are not sufficient. Interaction between PD-1 and its main ligand, PD-L1, appears as a valuable target of anti-PD-1 therapy. Thus, instead of looking at PD-L1 expression only, we evaluated the predictive value of the proximity between PD-1 and its neighboring PD-L1 molecules in terms of response to anti-PD-1 therapy. EXPERIMENTAL DESIGN: PD-1/PD-L1 proximity was assessed by proximity ligation assay (PLA) on 137 samples from two cohorts (exploratory n = 66 and validation n = 71) of samples from patients with melanoma treated with anti-PD-1±anti-CTLA-4. Additional predictive biomarkers, such as PD-L1 expression (MELscore), CD8+ cells density, and NanoString RNA signature, were also evaluated. RESULTS: A PD-1/PD-L1 PLA model was developed to predict tumor response in an exploratory cohort and further evaluated in an independent validation cohort. This score showed higher predictive ability (AUC = 0.85 and 0.79 in the two cohorts, respectively) for PD-1/PD-L1 PLA as compared with other parameters (AUC = 0.71-0.77). Progression-free and overall survival were significantly longer in patients with high PLA values (P = 0.00019 and P < 0.0001, respectively). CONCLUSIONS: The proximity between PD-1 and PD-L1, easily assessed by this PLA on one formalin-fixed paraffin-embedded section, appears as a new biomarker of anti-PD-1 efficacy.

The plasticity of mRNA translation during cancer progression and therapy resistance.

Translational control of mRNAs during gene expression allows cells to promptly and dynamically adapt to a variety of stimuli, including in neoplasia in response to aberrant oncogenic signalling (for example, PI3K-AKT-mTOR, RAS-MAPK and MYC) and microenvironmental stress such as low oxygen and nutrient supply. Such translational rewiring allows rapid, specific changes in the cell proteome that shape specific cancer phenotypes to promote cancer onset, progression and resistance to anticancer therapies. In this Review, we illustrate the plasticity of mRNA translation. We first highlight the diverse mechanisms by which it is regulated, including by translation factors (for example, eukaryotic initiation factor 4F (eIF4F) and eIF2), RNA-binding proteins, tRNAs and ribosomal RNAs that are modulated in response to aberrant intracellular pathways or microenvironmental stress. We then describe how translational control can influence tumour behaviour by impacting on the phenotypic plasticity of cancer cells as well as on components of the tumour microenvironment. Finally, we highlight the role of mRNA translation in the cellular response to anticancer therapies and its promise as a key therapeutic target.

In situ detection of the eIF4F translation initiation complex in mammalian cells and tissues.

The eukaryotic translation initiation complex eIF4F plays an important role in gene expression. The methods that are used to monitor the formation of the eIF4F complex are usually indirect and provide no information on its subcellular localization. This protocol describes a proximity ligation assay-based procedure allowing the direct in situ visualization of the eIF4F complex, as well as its absolute quantification per cell using adapted image analysis software. For complete details on the use and execution of this protocol, please refer to Boussemart et al. (2014).

Non-homologous end-joining at challenged replication forks: an RNA connection?

Defective DNA replication, known as 'replication stress', is a source of DNA damage, a hallmark of numerous human diseases, including cancer, developmental defect, neurological disorders, and premature aging. Recent work indicates that non-homologous end-joining (NHEJ) is unexpectedly active during DNA replication to repair replication-born DNA lesions and to safeguard replication fork integrity. However, erroneous NHEJ events are deleterious to genome stability. RNAs are novel regulators of NHEJ activity through their ability to modulate the assembly of repair complexes in trans. At DNA damage sites, RNAs and DNA-embedded ribonucleotides modulate repair efficiency and fidelity. We discuss here how RNAs and associated proteins, including RNA binding proteins, may regulate NHEJ to sustain genome stability during DNA replication.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Persistent Cancer Cells: The Deadly Survivors.

Persistent cancer cells are the discrete and usually undetected cells that survive cancer drug treatment and constitute a major cause of treatment failure. These cells are characterized by their slow proliferation, highly flexible energy consumption, adaptation to their microenvironment, and phenotypic plasticity. Mechanisms that underlie their persistence offer highly coveted and sought-after therapeutic targets, and include diverse epigenetic, transcriptional, and translational regulatory processes, as well as complex cell-cell interactions. Although the successful clinical targeting of persistent cancer cells remains to be realized, immense progress has been made in understanding their persistence, yielding promising preclinical results.

ZRANB2 and SYF2-mediated splicing programs converging on ECT2 are involved in breast cancer cell resistance to doxorubicin.

Besides analyses of specific alternative splicing (AS) variants, little is known about AS regulatory pathways and programs involved in anticancer drug resistance. Doxorubicin is widely used in breast cancer chemotherapy. Here, we identified 1723 AS events and 41 splicing factors regulated in a breast cancer cell model of acquired resistance to doxorubicin. An RNAi screen on splicing factors identified the little studied ZRANB2 and SYF2, whose depletion partially reversed doxorubicin resistance. By RNAi and RNA-seq in resistant cells, we found that the AS programs controlled by ZRANB2 and SYF2 were enriched in resistance-associated AS events, and converged on the ECT2 splice variant including exon 5 (ECT2-Ex5+). Both ZRANB2 and SYF2 were found associated with ECT2 pre-messenger RNA, and ECT2-Ex5+ isoform depletion reduced doxorubicin resistance. Following doxorubicin treatment, resistant cells accumulated in S phase, which partially depended on ZRANB2, SYF2 and the ECT2-Ex5+ isoform. Finally, doxorubicin combination with an oligonucleotide inhibiting ECT2-Ex5 inclusion reduced doxorubicin-resistant tumor growth in mouse xenografts, and high ECT2-Ex5 inclusion levels were associated with bad prognosis in breast cancer treated with chemotherapy. Altogether, our data identify AS programs controlled by ZRANB2 and SYF2 and converging on ECT2, that participate to breast cancer cell resistance to doxorubicin.

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

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

Redox modifications of cysteine-containing proteins, cell cycle arrest and translation inhibition: Involvement in vitamin C-induced breast cancer cell death.

Vitamin C (VitC) possesses pro-oxidant properties at high pharmacologic concentrations which favor repurposing VitC as an anti-cancer therapeutic agent. However, redox-based anticancer properties of VitC are yet partially understood. We examined the difference between the reduced and oxidized forms of VitC, ascorbic acid (AA) and dehydroascorbic acid (DHA), in terms of cytotoxicity and redox mechanisms toward breast cancer cells. Our data showed that AA displayed higher cytotoxicity towards triple-negative breast cancer (TNBC) cell lines in vitro than DHA. AA exhibited a similar cytotoxicity on non-TNBC cells, while only a minor detrimental effect on noncancerous cells. Using MDA-MB-231, a representative TNBC cell line, we observed that AA- and DHA-induced cytotoxicity were linked to cellular redox-state alterations. Hydrogen peroxide (H2O2) accumulation in the extracellular medium and in different intracellular compartments, and to a lesser degree, intracellular glutathione oxidation, played a key role in AA-induced cytotoxicity. In contrast, DHA affected glutathione oxidation and had less cytotoxicity. A "redoxome" approach revealed that AA treatment altered the redox state of key antioxidants and a number of cysteine-containing proteins including many nucleic acid binding proteins and proteins involved in RNA and DNA metabolisms and in energetic processes. We showed that cell cycle arrest and translation inhibition were associated with AA-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlated with AA differential cytotoxicity in breast cancer cells, suggesting a potential predictive value of PRDX1. This study provides insight into the redox-based mechanisms of VitC anticancer activity, indicating that pharmacologic doses of VitC and VitC-based rational drug combinations could be novel therapeutic opportunities for triple-negative breast cancer.