Inhibition Of Nonsense-mediated mRNA Decay Research Articles

Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributionsof most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

Abstract 5625: Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts

Abstract Introduction: Frameshift insertions/deletions (fs-indels) can lead to the generation of immunogenic non-self-peptides which contribute to anti-tumor immune responses. However, fs-indels often generate premature termination codons (PTCs), leading to mRNA degradation by the nonsense mediated mRNA decay (NMD) pathway. Importantly, some PTC-containing transcripts naturally escape NMD degradation and produce highly immunogenic neoantigens. Therefore, we hypothesize that NMD inhibition could be used to increase the cellular pool of fs-indels derived neoantigens in cancer cells, and enhance anti-tumor immunogenicity. NMD also regulates other cellular processes by degrading a subset of non-PTC-containing self-transcripts. Therefore, by focusing NMD inhibition towards the degradation of PTC-transcripts only, toxicity may be limited in a clinical setting. We investigate how targeting different NMD proteins affects the degradation of fs-transcripts across several cancers types. To assess the clinical relevance of NMD inhibition, we study how it specifically affects p53 fs-transcripts, since p53 is the most mutated gene in cancer and fs-mutant p53 has been shown to be highly immunogenic. Methods: Core NMD proteins were depleted across a panel of cell lines from different cancer types and transcriptomics, flow cytometry, qPCR and western blot were used to evaluate changes in the expression of NMD-targeted transcripts and NMD members. Proteomics and immunopeptidomics were used to study protein expression and HLA-peptide presentation arising from fs-transcripts. A Translating RNA Imaging by Coat protein Knock-off (TRICK) assay was developed to study the translation of NMD-targeted p53 mutants upon NMD inhibition. This assay can precisely quantify the translation of p53 mutant RNA upon different treatments and cellular stresses at single-molecule resolution. Results: We found an increase in expression of PTC-transcripts upon silencing of multiple NMD members. Our data suggests that some NMD members are better targets for inhibition of NMD PTC-surveillance, whilst others are more appropriate for inhibition of NMD-mediated gene expression regulation. These data support the recently proposed hypothesis that NMD is a branched pathway, where each branch targets specific subsets of transcripts depending on the cell type. These results are clinically relevant as the method by which NMD is inhibited could help optimize the generation of immunogenic neoantigens whilst minimizing toxicity. The TRICK assay has shown that inhibiting NMD surveillance reduces the degradation of p53 fs-transcripts and increases their protein expression. Conclusion: We have identified NMD pathway members that can be targeted to efficiently inhibit the surveillance role of NMD and prevent the degradation of fs-transcripts that could generate highly immunogenic neoantigens. Citation Format: Shanila Fernandez Patel, Roberto Vendramin, Danwen Qian, Yue Zhao, Lorena Ligammari, Krupa Thakkar, Jun Murai, Eva Grönroos, Jeremy Carlton, Kevin Litchfield. Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5625.

Reclassification of variants of tumor suppressor genes based on Sanger RNA sequencing without NMD inhibition.

Introduction: RNA sequence analysis can be effectively used to identify aberrant splicing, and tumor suppressor genes are adequate targets considering their loss-of-function mechanisms. Sanger sequencing is the simplest method for RNA sequence analysis; however, because of its insufficient sensitivity in cases with nonsense-mediated mRNA decay (NMD), the use of cultured specimens with NMD inhibition has been recommended, hindering its wide adoption. Method: The results of Sanger sequencing of peripheral blood RNA without NMD inhibition performed on potential splicing variants of tumor suppressor genes were retrospectively reviewed. For negative cases, in which no change was identified in the transcript, the possibility of false negativity caused by NMD was assessed through a review of the up-to-date literature. Results: Eleven potential splice variants of various tumor suppressor genes were reviewed. Six variants were classified as pathogenic or likely pathogenic based on the nullifying effect identified by Sanger RNA sequencing. Four variants remained as variants of uncertain significance because of identified in-frame changes or normal expression of both alleles. The result of one variant was suspected to be a false negative caused by NMD after reviewing a recent study that reported the same variant as causing a nullifying effect on the affected transcript. Conclusion: Although RNA changes found in the majority of cases were expected to undergo NMD by canonical rules, most cases (10/11) were interpretable by Sanger RNA sequencing without NMD inhibition due to incomplete NMD efficiency or allele-specific expression despite highly efficient NMD.

253 Functional and RNA-level recovery of CFTR nonsense variants with nonsense-mediated mRNA decay inhibition, readthrough, and elexacaftor-tezacaftor-ivacaftor

253 Functional and RNA-level recovery of CFTR nonsense variants with nonsense-mediated mRNA decay inhibition, readthrough, and elexacaftor-tezacaftor-ivacaftor

Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot-Marie-Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene.

Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot-Marie-Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy.

Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with roles in cellular stress responses, differentiation, and viral defense. It functions in both quality control and post-transcriptional regulation of gene expression. NMD has also emerged as a modulator of cancer progression, although available evidence supports both a tumor suppressor and a pro-tumorigenic role, depending on the model. To further investigate the role of NMD in cancer, we knocked out the NMD factor SMG7 in the HT1080 human fibrosarcoma cell line, resulting in suppression of NMD function. We then compared the oncogenic properties of the parental cell line, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. We also tested the effect of a drug inhibiting the NMD factor SMG1 to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays and a mouse xenograft tumor model, we showed that suppression of NMD function severely compromises the oncogenic phenotype. Molecular pathway analysis revealed that NMD suppression strongly reduces matrix metalloprotease 9 (MMP9) expression and that MMP9 re-expression partially rescues the oncogenic phenotype. Since MMP9 promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation may contribute to the reduced tumorigenicity of NMD-suppressed cells. Collectively, our results highlight the potential value of NMD inhibition as a therapeutic approach.

Inhibition of nonsense-mediated mRNA decay may improve stop codon read-through therapy for Duchenne muscular dystrophy.

Duchene muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are genetic neuromuscular disorders that affect skeletal and cardiac muscle resulting from mutations in the dystrophin gene (DMD), coding for dystrophin protein. Read-through therapies hold great promise for the treatment of genetic diseases harboring nonsense mutations, such as DMD/BMD, as they enable a complete translation of the affected mRNA. However, to date, most read-through drugs have not achieved a cure for patients. One possible explanation for the limitation of these therapies for DMD/BMD is that they rely on the presence of mutant dystrophin mRNAs. However, the mutant mRNAs containing premature termination codons are identified by the cellular surveillance mechanism, the nonsense-mediated mRNA decay (NMD) process, and are degraded. Here, we show that the combination of read-through drugs together with known NMD inhibitors have a synergistic effect on the levels of nonsense-containing mRNAs, among them the mutant dystrophin mRNA. This synergistic effect may enhance read-through therapies' efficacy and improve the current treatment for patients.

IL-6/STAT3 signaling in tumor cells restricts the expression of frameshift-derived neoantigens by SMG1 induction

BackgroundThe quality and quantity of tumor neoantigens derived from tumor mutations determines the fate of the immune response in cancer. Frameshift mutations elicit better tumor neoantigens, especially when they are not targeted by nonsense-mediated mRNA decay (NMD). For tumor progression, malignant cells need to counteract the immune response including the silencing of immunodominant neoantigens (antigen immunoediting) and promoting an immunosuppressive tumor microenvironment. Although NMD inhibition has been reported to induce tumor immunity and increase the expression of cryptic neoantigens, the possibility that NMD activity could be modulated by immune forces operating in the tumor microenvironment as a new immunoediting mechanism has not been addressed.MethodsWe study the effect of SMG1 expression (main kinase that initiates NMD) in the survival and the nature of the tumor immune infiltration using TCGA RNAseq and scRNAseq datasets of breast, lung and pancreatic cancer. Different murine tumor models were used to corroborate the antitumor immune dependencies of NMD. We evaluate whether changes of SMG1 expression in malignant cells impact the immune response elicited by cancer immunotherapy. To determine how NMD fluctuates in malignant cells we generated a luciferase reporter system to track NMD activity in vivo under different immune conditions. Cytokine screening, in silico studies and functional assays were conducted to determine the regulation of SMG1 via IL-6/STAT3 signaling.ResultsIL-6/STAT3 signaling induces SMG1, which limits the expression of potent frameshift neoantigens that are under NMD control compromising the outcome of the immune response.ConclusionWe revealed a new neoantigen immunoediting mechanism regulated by immune forces (IL-6/STAT3 signaling) responsible for silencing otherwise potent frameshift mutation-derived neoantigens.

Nonsense-mediated mRNA decay inhibition synergizes with MDM2 inhibition to suppress TP53 wild-type cancer cells in p53 isoform-dependent manner

The restoration of the normal function of the tumour suppressors, such as p53, is an important strategy in tumour therapeutics. Nonsense-mediated mRNA decay (NMD) inhibition by NMD inhibitor (NMDi) upregulates functional p53 isoforms, p53β and p53γ, and activates the p53 pathway. XR-2, a novel mouse double minute 2 homolog (MDM2) inhibitor, can disrupt the interaction between p53 and MDM2, thus decreasing the MDM2-mediated degradation of p53 and increasing the p53 protein levels. However, the combined effects of these two agents have not been thoroughly explored. This study combined XR-2 and NMDi in four TP53 wild-types and four TP53-mutated cancer cell lines. The combination of these two agents achieved significant synergistic effects on TP53 wild-type cancer cell lines by transactivating p53 target genes, inducing apoptosis, cell-cycle arrest and DNA damage repair. The p53β isoform induced by NMDi enhances the transactivation ability of p53α induced by XR-2, which partially explains the mechanism of the synergistic effects of XR-2 and NMDi. This study identified a combination treatment of NMDi and XR-2 which could serve as a novel cancer therapeutic approach for MDM2-overexpressed TP53 wild-type cancers and delineated a future therapy based on the further reactivation of p53.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Gene-specific nonsense-mediated mRNA decay targeting for cystic fibrosis therapy

Low CFTR mRNA expression due to nonsense-mediated mRNA decay (NMD) is a major hurdle in developing a therapy for cystic fibrosis (CF) caused by the W1282X mutation in the CFTR gene. CFTR-W1282X truncated protein retains partial function, so increasing its levels by inhibiting NMD of its mRNA will likely be beneficial. Because NMD regulates the normal expression of many genes, gene-specific stabilization of CFTR-W1282X mRNA expression is more desirable than general NMD inhibition. Synthetic antisense oligonucleotides (ASOs) designed to prevent binding of exon junction complexes (EJC) downstream of premature termination codons (PTCs) attenuate NMD in a gene-specific manner. We describe cocktails of three ASOs that specifically increase the expression of CFTR-W1282X mRNA and CFTR protein upon delivery into human bronchial epithelial cells. This treatment increases the CFTR-mediated chloride current. These results set the stage for clinical development of an allele-specific therapy for CF caused by the W1282X mutation.

Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay.

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense‐mediated mRNA decay (NMD). UPF3B has been demonstrated to support NMD, presumably by bridging an exon junction complex (EJC) to the NMD factor UPF2. The role of UPF3A has been described either as a weak NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not substantially change global NMD activity. In contrast, the co‐depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome‐wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. In rescue experiments, UPF2 or EJC binding‐deficient UPF3B largely retained NMD activity. However, combinations of different mutants, including deletion of the middle domain, showed additive or synergistic effects and therefore failed to maintain NMD. Collectively, UPF3A and UPF3B emerge as fault‐tolerant, functionally redundant NMD activators in human cells.

AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice.

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV)is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

Elucidation of the Mechanism of Host NMD Suppression by HTLV-1 Rex: Dissection of Rex to Identify the NMD Inhibitory Domain.

The human retrovirus human T-cell leukemia virus type I (HTLV-1) infects human T cells by vertical transmission from mother to child through breast milk or horizontal transmission through blood transfusion or sexual contact. Approximately 5% of infected individuals develop adult T-cell leukemia/lymphoma (ATL) with a poor prognosis, while 95% of infected individuals remain asymptomatic for the rest of their lives, during which time the infected cells maintain a stable immortalized latent state in the body. It is not known why such a long latent state is maintained. We hypothesize that the role of functional proteins of HTLV-1 during early infection influences the phenotype of infected cells in latency. In eukaryotic cells, a mRNA quality control mechanism called nonsense-mediated mRNA decay (NMD) functions not only to eliminate abnormal mRNAs with nonsense codons but also to target virus-derived RNAs. We have reported that HTLV-1 genomic RNA is a potential target of NMD, and that Rex suppresses NMD and stabilizes viral RNA against it. In this study, we aimed to elucidate the molecular mechanism of NMD suppression by Rex using various Rex mutant proteins. We found that region X (aa20–57) of Rex, the function of which has not been clarified, is required for NMD repression. We showed that Rex binds to Upf1, which is the host key regulator to detect abnormal mRNA and initiate NMD, through this region. Rex also interacts with SMG5 and SMG7, which play essential roles for the completion of the NMD pathway. Moreover, Rex selectively binds to Upf3B, which is involved in the normal NMD complex, and replaces it with a less active form, Upf3A, to reduce NMD activity. These results revealed that Rex invades the NMD cascade from its initiation to completion and suppresses host NMD activity to protect the viral genomic mRNA.

Nonsense-mediated mRNA decay modulates Arabidopsis flowering time via the SET DOMAIN GROUP 40-FLOWERING LOCUS C module.

The nonsense-mediated mRNA decay (NMD) surveillance system clears aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. Although loss of the core NMD proteins UP-FRAMESHIFT1 (UPF1) and UPF3 leads to late flowering in Arabidopsis, the underlying mechanism remains elusive. Here, we showed that mutations in UPF1 and UPF3 cause temperature- and photoperiod-independent late flowering. Expression analyses revealed high FLOWERING LOCUS C (FLC) mRNA levels in upf mutants; in agreement with this, the flc mutation strongly suppressed the late flowering of upf mutants. Vernalization accelerated flowering of upf mutants in a temperature-independent manner. FLC transcript levels rose in wild-type plants upon NMD inhibition. In upf mutants, we observed increased enrichment of H3K4me3 and reduced enrichment of H3K27me3 in FLC chromatin. Transcriptome analyses showed that SET DOMAIN GROUP 40 (SDG40) mRNA levels increased in upf mutants, and the SDG40 transcript underwent NMD-coupled alternative splicing, suggesting that SDG40 affects flowering time in upf mutants. Furthermore, NMD directly regulated SDG40 transcript stability. The sdg40 mutants showed decreased H3K4me3 and increased H3K27me3 levels in FLC chromatin, flowered early, and rescued the late flowering of upf mutants. Taken together, these results suggest that NMD epigenetically regulates FLC through SDG40 to modulate flowering time in Arabidopsis.

SMG5-SMG7 authorize nonsense-mediated mRNA decay by enabling SMG6 endonucleolytic activity

Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.

Characterisation of the Semliki Forest Virus-host cell interactome reveals the viral capsid protein as an inhibitor of nonsense-mediated mRNA decay.

The positive-sense, single-stranded RNA alphaviruses pose a potential epidemic threat. Understanding the complex interactions between the viral and the host cell proteins is crucial for elucidating the mechanisms underlying successful virus replication strategies and for developing specific antiviral interventions. Here we present the first comprehensive protein-protein interaction map between the proteins of Semliki Forest Virus (SFV), a mosquito-borne member of the alphaviruses, and host cell proteins. Among the many identified cellular interactors of SFV proteins, the enrichment of factors involved in translation and nonsense-mediated mRNA decay (NMD) was striking, reflecting the virus' hijacking of the translation machinery and indicating viral countermeasures for escaping NMD by inhibiting NMD at later time points during the infectious cycle. In addition to observing a general inhibition of NMD about 4 hours post infection, we also demonstrate that transient expression of the SFV capsid protein is sufficient to inhibit NMD in cells, suggesting that the massive production of capsid protein during the SFV reproduction cycle is responsible for NMD inhibition.

Effect of small molecule eRF3 degraders on premature termination codon readthrough.

Premature termination codon (PTC) readthrough is considered a potential treatment for genetic diseases caused by nonsense mutations. High concentrations of aminoglycosides induce low levels of PTC readthrough but also elicit severe toxicity. Identifying compounds that enhance PTC readthrough by aminoglycosides or reduce their toxicity is a continuing challenge. In humans, a binary complex of eukaryotic release factors 1 (eRF1) and 3 (eRF3a or eRF3b) mediates translation termination. They also participate in the SURF (SMG1-UPF1-eRF1-eRF3) complex assembly involved in nonsense-mediated mRNA decay (NMD). We show that PTC readthrough by aminoglycoside G418 is considerably enhanced by eRF3a and eRF3b siRNAs and cereblon E3 ligase modulators CC-885 and CC-90009, which induce proteasomal degradation of eRF3a and eRF3b. eRF3 degradation also reduces eRF1 levels and upregulates UPF1 and selectively stabilizes TP53 transcripts bearing a nonsense mutation over WT, indicating NMD suppression. CC-90009 is considerably less toxic than CC-885 and it enhances PTC readthrough in combination with aminoglycosides in mucopolysaccharidosis type I-Hurler, late infantile neuronal ceroid lipofuscinosis, Duchenne muscular dystrophy and junctional epidermolysis bullosa patient-derived cells with nonsense mutations in the IDUA, TPP1, DMD and COL17A1 genes, respectively. Combination of CC-90009 with aminoglycosides such as gentamicin or ELX-02 may have potential for PTC readthrough therapy.

The Effect of SF3B1 Mutation on the DNA Damage Response and Nonsense-Mediated mRNA Decay in Cancer.

Recurrent mutations in splicing factor 3B subunit 1 (SF3B1) have been identified in several malignancies and are associated with an increased expression of 3’ cryptic transcripts as a result of alternative branchpoint recognition. A large fraction of cryptic transcripts associated with SF3B1 mutations is expected to be sensitive for RNA degradation via nonsense-mediated mRNA decay (NMD). Several studies indicated alterations in various signaling pathways in SF3B1-mutated cells, including an impaired DNA damage response (DDR) in chronic lymphocytic leukemia (CLL). In this study, we investigated isogenic cell lines and treatment naïve primary CLL samples without any TP53 and/or ATM defect, and found no significant effects of SF3B1 mutations on the ATM/p53 response, phosphorylation of H2AX and sensitivity to fludarabine. Cryptic transcripts associated with SF3B1 mutation status were observed at relatively low levels compared to the canonical transcripts and were validated as target for mRNA degradation via NMD. Expression of cryptic transcripts increased after NMD inhibition. In conclusion, our results confirm involvement of NMD in the biological effects of SF3B1 mutations. Further studies may elucidate whether SF3B1-mutant patients could benefit from NMD modulatory agents.

Loss of the fragile X syndrome protein FMRP results in misregulation of nonsense-mediated mRNA decay.

Loss of the fragile X protein FMRP is a leading cause of intellectual disability and autism1,2, but the underlying mechanism remains poorly understood. We report that FMRP deficiency results in hyperactivated nonsense-mediated mRNA decay (NMD)3,4 in human SH-SY5Y neuroblastoma cells and fragile X syndrome (FXS) fibroblast-derived induced pluripotent stem cells (iPSCs). We examined the underlying mechanism and found that the key NMD factor UPF1 binds directly to FMRP, promoting FMRP binding to NMD targets. Our data indicate that FMRP acts as an NMD repressor. In the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD. Transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. Consistent with this, small-molecule-mediated inhibition of hyperactivated NMD, which typifies iPSCs derived from patients with FXS, restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable-either directly or indirectly-to misregulated NMD.