Patient-derived tumoroids and proteomic signatures: tools for early drug discovery.

Onco-virotherapy is an emergent treatment for cancer based on viral vectors. The therapeutic activity is based on two different mechanisms including tumor-specific oncolysis and immunostimulatory properties. In this study, we evaluated onco-virotherapy in vitro responses on immunocompetent non-small cell lung cancer (NSCLC) patient-derived tumoroids (PDTs) and healthy organoids. PDTs are accurate tools to predict patient's clinical responses at the in vitro stage. We showed that onco-virotherapy could exert specific antitumoral effects by producing a higher number of viral particles in PDTs than in healthy organoids. In the present work, we used multiplex protein screening, based on proximity extension assay to highlight different response profiles. Our results pointed to the increase of proteins implied in T cell activation, such as IFN-γ following onco-virotherapy treatment. Based on our observation, oncolytic viruses-based therapy responders are dependent on several factors: a high PD-L1 expression, which is a biomarker of greater immune response under immunotherapies, and the number of viral particles present in tumor tissue, which is dependent to the metabolic state of tumoral cells. Herein, we highlight the use of PDTs as an alternative in vitro model to assess patient-specific responses to onco-virotherapy at the early stage of the preclinical phases.

In vitro vascularized immunocompetent patient-derived model to test cancer therapies.

This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived peripheral blood mononuclear cells (PBMCs) permits to achieve a physiologically relevant tumor microenvironment. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA), and inhibitory receptors of T cells functions.

Vectorized Treg-depleting αCTLA-4 elicits antigen cross-presentation and CD8+ T cell immunity to reject 'cold' tumors.

BACKGROUND: Immune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of patients with cancer including those with poorly immune infiltrated 'cold' tumors are resistant to currently available ICB therapies. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved αCTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for αCTLA-4; ICB and regulatory T cell (Treg) depletion are both thought to contribute efficacy and toxicity in available, systemic, αCTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe αCTLA-4 therapies have been lacking. Here we assess and identify spatially restricted exposure to a novel strongly Treg-depleting, checkpoint-blocking, vectorized αCTLA-4, as a highly efficacious and potentially safe strategy to target CTLA-4. METHODS: A novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for monoclonal antibodies (mAbs) and targets associated with superior Treg-depleting activity. A tumor-selective oncolytic vaccinia vector was then engineered to encode this novel, strongly Treg-depleting, checkpoint-blocking, αCTLA-4 antibody or a matching surrogate antibody, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (VVGM-αCTLA-4). RESULTS: The identified 4-E03 antibody showed significantly stronger Treg depletion, but equipotent checkpoint blockade, compared with clinically validated αCTLA-4 ipilimumab against CTLA-4-expressing Treg cells in a humanized mouse model in vivo. Intratumoral administration of VVGM-αCTLA-4 achieved tumor-restricted CTLA-4 receptor saturation and Treg depletion, which elicited antigen cross-presentation and stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic αCTLA-4 antibody therapy. Efficacy correlated with FcγR-mediated intratumoral Treg depletion. Remarkably, in a clinically relevant mouse model resistant to systemic ICB, intratumoral VVGM-αCTLA-4 synergized with αPD-1 to reject cold tumors. CONCLUSION: Our findings demonstrate in vivo proof of concept for spatial restriction of Treg depletion-optimized immune checkpoint blocking, vectorized αCTLA-4 as a highly effective and safe strategy to target CTLA-4. A clinical trial evaluating intratumoral VVGM-αhCTLA-4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has commenced.

An antisense noncoding RNA enhances translation via localized structural rearrangements of its cognate mRNA.

A large portion of eukaryotic genes are associated with noncoding, natural antisense transcripts (NATs). Despite sharing extensive sequence complementarity with their sense mRNAs, mRNA-NAT pairs elusively often evade dsRNA-cleavage and siRNA-triggered silencing. More surprisingly, some NATs enhance translation of their sense mRNAs by yet unknown mechanism(s). Here, we show that translation enhancement of the rice (Oryza sativa) PHOSPHATE1.2 (PHO1.2) mRNA is enabled by specific structural rearrangements guided by its noncoding antisense RNA (cis-NATpho1.2). Their interaction in vitro revealed no evidence of widespread intermolecular dsRNA formation, but rather specific local changes in nucleotide base pairing, leading to higher flexibility of PHO1.2 mRNA at a key high guanine-cytosine�(GC) regulatory region inhibiting translation, ∼350-nt downstream of the start codon. Sense-antisense RNA interaction increased formation of the 80S complex in PHO1.2, possibly by inducing structural rearrangement within this inhibitory region, thus making this mRNA more accessible to 60S. This work presents a framework for nucleotide resolution studies of functional mRNA-antisense pairs.

Modulation of Shoot Phosphate Level and Growth by PHOSPHATE1 Upstream Open Reading Frame.

Inorganic orthophosphate (Pi) is an essential nutrient for plant growth, and its availability strongly impacts crop yield. PHOSPHATE1 (PHO1) transfers Pi from root to shoot via Pi export into root xylem vessels. In this work, we demonstrate that an upstream open reading frame (uORF) present in the 5' untranslated region of the Arabidopsis (Arabidopsis thaliana) PHO1 inhibits its translation and influences Pi homeostasis. The presence of the uORF strongly inhibited the translation of a PHO1 5'UTR-luciferase construct in protoplasts. A point mutation removing the PHO1 uORF (ΔuORF) in transgenic Arabidopsis resulted in increased association of its mRNA with polysomes and led to higher PHO1 protein levels, independent of Pi availability. Interestingly, deletion of the uORF led to higher shoot Pi content and was associated with improved shoot growth under low external Pi supply and no deleterious effects under Pi-sufficient conditions. We further show that natural accessions lacking the PHO1 uORF exhibit higher PHO1 protein levels and shoot Pi content. Increased shoot Pi content was linked to the absence of the PHO1 uORF in a population of F2 segregants. We identified the PHO1 uORF in genomes of crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aesativum), and we verified the inhibitory effect of the rice PHO1 uORF on translation in protoplasts. Our work suggests that regulation of PHO1 expression via its uORF might be a genetic resource useful-both in natural populations and in the context of genome editing-toward improving plant growth under Pi-deficient conditions.

The transcription and export complex THO/TREX contributes to transcription termination in plants.

Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3'UTR extensions in several endogenous genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.

Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional Elongation.

Transcript elongation factors associate with elongating RNA polymerase II (RNAPII) to control the efficiency of mRNA synthesis and consequently modulate plant growth and development. Encountering obstacles during transcription such as nucleosomes or particular DNA sequences may cause backtracking and transcriptional arrest of RNAPII. The elongation factor TFIIS stimulates the intrinsic transcript cleavage activity of the polymerase, which is required for efficient rescue of backtracked/arrested RNAPII. A TFIIS mutant variant (TFIISmut) lacks the stimulatory activity to promote RNA cleavage, but instead efficiently inhibits unstimulated transcript cleavage by RNAPII. We could not recover viable Arabidopsis (Arabidopsis thaliana) tfIIs plants constitutively expressing TFIISmut. Induced, transient expression of TFIISmut in tfIIs plants provoked severe growth defects, transcriptomic changes and massive, transcription-related redistribution of elongating RNAPII within transcribed regions toward the transcriptional start site. The predominant site of RNAPII accumulation overlapped with the +1 nucleosome, suggesting that upon inhibition of RNA cleavage activity, RNAPII arrest prevalently occurs at this position. In the presence of TFIISmut, the amount of RNAPII was reduced, which could be reverted by inhibiting the proteasome, indicating proteasomal degradation of arrested RNAPII. Our findings suggest that polymerase backtracking/arrest frequently occurs in plant cells, and RNAPII-reactivation is essential for correct transcriptional output and proper growth/development.

Prediction of regulatory long intergenic non-coding RNAs acting in trans through base-pairing interactions.

BACKGROUND: Long intergenic non-coding RNAs (lincRNAs) can act as regulators of expression of protein-coding genes. Trans-natural antisense transcripts (trans-NATs) are a type of lincRNAs that contain sequence complementary to mRNA from other loci. The regulatory potential of trans-NATs has been poorly studied in eukaryotes and no example of trans-NATs regulating gene expression in plants are reported. The goal of this study was to identify lincRNAs, and particularly trans-NATs, in Arabidopsis thaliana that have a potential to regulate expression of target genes in trans at the transcriptional or translational level. RESULTS: We identified 1001 lincRNAs using an RNAseq dataset from total polyA+ and polysome-associated RNA of seedlings grown under high and low phosphate, or shoots and roots treated with different phytohormones, of which 550 were differentially regulated. Approximately 30% of lincRNAs showed conservation amongst Brassicaceae and 25% harbored transposon element (TE) sequences. Gene co-expression network analysis highlighted a group of lincRNAs associated with the response of roots to low phosphate. A total of 129 trans-NATs were predicted, of which 88 were significantly differentially expressed under at least one pairwise comparison. Five trans-NATs showed a positive correlation between their expression and target mRNA steady-state levels, and three showed a negative correlation. Expression of four trans-NATs positively correlated with a change in target mRNA polysome association. The regulatory potential of these trans-NATs did not implicate miRNA mimics nor siRNAs. We also looked for lincRNAs that could regulate gene expression in trans by Watson-Crick DNA:RNA base pairing with target protein-encoding loci. We identified 100 and 81 with a positive or negative correlation, respectively, with steady-state level of their predicted target. The regulatory potential of one such candidate lincRNA harboring a SINE TE sequence was validated in a protoplast assay on three distinct genes containing homologous TE sequence in their promoters. Construction of networks highlighted other putative lincRNAs with multiple predicted target loci for which expression was positively correlated with target gene expression. CONCLUSIONS: This study identified lincRNAs in Arabidopsis with potential in regulating target gene expression in trans by both RNA:RNA and RNA:DNA base pairing and highlights lincRNAs harboring TE sequences in such activity.

Control of Cognate Sense mRNA Translation by cis-Natural Antisense RNAs.

Cis-Natural Antisense Transcripts (cis-NATs), which overlap protein coding genes and are transcribed from the opposite DNA strand, constitute an important group of noncoding RNAs. Whereas several examples of cis-NATs regulating the expression of their cognate sense gene are known, most cis-NATs function by altering the steady-state level or structure of mRNA via changes in transcription, mRNA stability, or splicing, and very few cases involve the regulation of sense mRNA translation. This study was designed to systematically search for cis-NATs influencing cognate sense mRNA translation in Arabidopsis (Arabidopsis thaliana). Establishment of a pipeline relying on sequencing of total polyA+ and polysomal RNA from Arabidopsis grown under various conditions (i.e. nutrient deprivation and phytohormone treatments) allowed the identification of 14 cis-NATs whose expression correlated either positively or negatively with cognate sense mRNA translation. With use of a combination of cis-NAT stable over-expression in transgenic plants and transient expression in protoplasts, the impact of cis-NAT expression on mRNA translation was confirmed for 4 out of 5 tested cis-NAT:sense mRNA pairs. These results expand the number of cis-NATs known to regulate cognate sense mRNA translation and provide a foundation for future studies of their mode of action. Moreover, this study highlights the role of this class of noncoding RNAs in translation regulation.

Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants.

Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.

Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame.

In the late phase of the HIV virus cycle, the unspliced genomic RNA is exported to the cytoplasm for the necessary translation of the Gag and Gag-pol polyproteins. Three distinct translation initiation mechanisms ensuring Gag production have been described with little rationale for their multiplicity. The Gag-IRES has the singularity to be located within Gag ORF and to directly interact with ribosomal 40S. Aiming at elucidating the specificity and the relevance of this interaction, we probed HIV-1 Gag-IRES structure and developed an innovative integrative modelling strategy to take into account all the gathered information. We propose a novel Gag-IRES secondary structure strongly supported by all experimental data. We further demonstrate the presence of two regions within Gag-IRES that independently and directly interact with the ribosome. Importantly, these binding sites are functionally relevant to Gag translation both in vitro and ex vivo. This work provides insight into the Gag-IRES molecular mechanism and gives compelling evidence for its physiological importance. It allows us to propose original hypotheses about the IRES physiological role and conservation among primate lentiviruses.

LOOP IIId of the HCV IRES is essential for the structural rearrangement of the 40S-HCV IRES complex.

As obligatory intracellular parasites, viruses rely on cellular machines to complete their life cycle, and most importantly they recruit the host ribosomes to translate their mRNA. The Hepatitis C viral mRNA initiates translation by directly binding the 40S ribosomal subunit in such a way that the initiation codon is correctly positioned in the P site of the ribosome. Such a property is likely to be central for many viruses, therefore the description of host-pathogen interaction at the molecular level is instrumental to provide new therapeutic targets. In this study, we monitored the 40S ribosomal subunit and the viral RNA structural rearrangement induced upon the formation of the binary complex. We further took advantage of an IRES viral mutant mRNA deficient for translation to identify the interactions necessary to promote translation. Using a combination of structure probing in solution and molecular modeling we establish a whole atom model which appears to be very similar to the one obtained recently by cryoEM. Our model brings new information on the complex, and most importantly reveals some structural rearrangement within the ribosome. This study suggests that the formation of a 'kissing complex' between the viral RNA and the 18S ribosomal RNA locks the 40S ribosomal subunit in a conformation proficient for translation.

mRNAs that specifically interact with eukaryotic ribosomal subunits.

The accuracy of start codon selection is determined by the translation initiation process. In prokaryotes the initiation step on most mRNAs relies on recruitment of the small ribosomal subunit onto the initiation codon by base pairing between the mRNA and the 16S rRNA. Eukaryotes have evolved a complex molecular machinery involving at least 11 initiation factors, and mRNAs do not directly recruit the small ribosomal subunit. Instead the initiation complex is recruited to the 5' end of the mRNA through a complex protein network including eIF4E that interacts with the 5' cap structure and poly-A binding protein that interacts with the 3'end. However, some viral and cellular mRNAs are able to escape this pathway by internal recruitment of one or several components of the translation machinery. Here we review those eukaryotic mRNAs that have been reported to directly recruit the 40S ribosomal subunit internally. In the well characterized cases of viral IRESes, a specific RNA structure is involved in this process, and in addition to recruitment of the ribosome, the mRNA also manipulates the ribosome structure to stimulate the first translocation step. We also review recently described IRES/ribosome interactions in cases where the molecular mechanism leading to translation initiation has yet to be described. Finally we evaluate the possibility that mRNA may recruit the 40S ribosomal subunit through base pairing with the 18S rRNA.

40S recruitment in the absence of eIF4G/4A by EMCV IRES refines the model for translation initiation on the archetype of Type II IRESs.

Initiation of translation on Type II IRESs, such as those of EMCV and FMDV viruses, has been well documented in the recent years. For EMCV, the current model argues for a mechanism in which the key interaction necessary for the pre-initiation complex recruitment is eIF4G binding to the central J-K domains of EMCV-IRES. Here we demonstrate that, in contrast with the current model, the molecular mechanism of EMCV-IRES involves direct recruitment of the 40S subunit. Importantly, we identified a specific structural element that prevents the correct positioning of the initiation codon in the close vicinity of the ribosomal P site. This work clarifies how this interaction could not be anticipated by earlier studies and allows us to propose a new model for initiation complex assembly on EMCV-IRES. The role attributed to eIF4G/4A can thus be refined as stabilizing/promoting the conformational changes that are necessary for IRES function, thus resembling the role conventionally assigned to ITAFs. This raises the interesting possibility that IRESs are primarily ribosome binders, some of which having partly lost the ability to fold into the active structure without the help of proteins.

Structural analyses of Avocado sunblotch viroid reveal differences in the folding of plus and minus RNA strands.

Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.

Structural investigation of HIV-1 genomic RNA dimerization process reveals a role for the Major Splice-site Donor stem loop.

The 5'UnTranslated Region (5'UTR) of HIV-1 genomic RNA, which precedes the Gag coding sequence, fulfills several roles during the lentivirus life cycle. This 335 nucleotides leader contains many stable structures that are crucial for the regulation of genetic expression at the level of transcription, splicing, and translation. In the late steps of the virus cycle, i.e. virions formation, the genomic RNA serves as propagated genome and its encapsidation in new particles relies on its ability to form non-covalent dimers. Dimerization is proposed to be initiated by the intermolecular pairing of a self-complementary sequence located in the apical loop of the DIS hairpin (Dimer Initiation Sequence). The regulation of this phenomenon and the extraordinary stability of the dimers imply that structural elements other than this kissing complex remain to be identified. Here, we show that swapping the Gag open reading frame (ORF) by reporter genes interferes with dimers formation efficiency. Importantly, the nature of the ORF alters specific structures of the 5'UTR. By using a systematic "SHAPE" approach, we pointed out that sequences within the Major Splice Site are involved in the dimerization process. Furthermore, by the use of an antisense oligonucleotide specific for the MSD associated to a SHAPE analysis of the 5'UTR structure, we demonstrated that interfering with the MSD results both in an impaired dimerization and in modifications of the 5'UTR structure. All together these data support a recently proposed model in which intramolecular base pairings are important determinants for the dimerization process. We further conclude that much care should be taken when comparing translation activity of reporter constructs with the viral situation.

Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors.

The 5' leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA harbors an internal ribosome entry site (IRES) that is functional during the G2/M phase of the cell cycle. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. We used 'standard' chemical and enzymatic probes and an 'RNA SHAPE' analysis to model the structure of the HIV-1 5' leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. We also assessed the impact of mutations on IRES function. Strikingly, mutations did not significantly affect IRES activity suggesting that the requirement for pre-formed stable secondary or tertiary structure within the HIV-1 IRES may not be as strict as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5' leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins.

Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides.

Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein. In this study, we explored the possibility of using aminoglycoside antibiotics to induce the production of a full-length functional p53 protein from a gene carrying a PTC. We identified a human cancer cell line containing a PTC, for which high levels of readthrough were obtained in the presence of aminoglycosides. Using these cells, we demonstrated that aminoglycoside treatment stabilized the mutant mRNA, which would otherwise have been degraded by non-sense-mediated decay, resulting in the production of a functional full-length p53 protein. Finally, we showed that aminoglycoside treatment decreased the viability of cancer cells specifically in the presence of nonsense-mutated p53 gene. These results open possibilities of developing promising treatments of cancers linked with non-sense mutations in tumor suppressor genes. They show that molecules designed to induce stop-codon readthrough can be used to inhibit tumor growth and offer a rational basis for developing new personalized strategies that could diversify the existing arsenal of cancer therapies.