Nonsense-mediated Decay Targets Research Articles

Abstract 3986: Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in small cell lung cancer

Abstract Cancer immunotherapy has revolutionized the field of oncology and is one of the most promising treatments for lung cancer. Small cell lung cancer (SCLC) is highly aggressive and patients often relapse due to chemotherapy resistance. In the recent years, chemotherapy has been combined with immune checkpoint inhibition (ICI) for SCLC treatment, leading to improved responses in some patients, but also leaving a high number of non-responders to address. Paradoxically, SCLC has one of the highest tumor mutational burden, including remarkably abundant frameshifts (FS) which could create highly immunogenic neoantigens (NeoAg). However, FS-harbouring transcripts are often degraded via Nonsense-Mediated Decay (NMD). We therefore aimed to inhibit the NMD pathway to increase the immunogenicity of SCLC. Our lab has so far profiled the mutational landscape of more than 400 SCLC patients and generated over 100 patient-derived xenotransplant models. We analyzed genome and transcriptome sequencing data of multiple tumors to interrogate the NMD target repertoire in SCLC. We determined tumor-specific mutations and predicted NeoAg based on the HLA-I-binding ability of mutant peptides. Upon NMD inhibiton, we validated the expression and presentation of NeoAg via transcriptomics, proteomics and HLA-I-immunopeptidomics. NMD-sensitive mutated transcripts included a wide range of somatic alterations beyond FS-mutations, suggesting an overall increase of potential tumor-specific NeoAg. We furthermore evaluated immunogenicity after NMD perturbation employing in vitro co-cultures as well as immunocompetent in vivo mouse models. Inhibition of NMD in tumor cells resulted in an HLA-I-dependent enhancement of T cell-mediated tumor killing in vitro, and in vivo experiments showed reduced tumor growth accompanied by increased immune infiltration upon controlled induction of NMD inhibition. Altogether, NMD inhibition in SCLC led to an upregulation of NeoAg expression in tumor cells accompanied by enhanced T cell responses in vitro and in vivo. Our strategy provides a novel approach for the treatment of low immunogenic tumors which could be combined with ICI to enhance the efficacy of immunotherapy. Citation Format: Lucia A. Torres Fernández, Volker Böhm, Lukas Maas, Anna Schöllhorn, Joel Kaufmann, Joshua D'Rozario, Laura Kaiser, Niels Gehring, Martin Peifer, Cécile Gouttefangeas, Roman K. Thomas, Julie George. Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in small cell lung cancer [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 3986.

Turnip crinkle virus-encoded suppressor of RNA silencing suppresses mRNA decay by interacting with Arabidopsis XRN4.

Plant cells employ intricate defense mechanisms, including mRNA decay pathways, to counter viral infections. Among the RNA quality control (RQC) mechanisms, nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop decay (NSD) pathways play critical roles in recognizing and cleaving aberrant mRNA molecules. Turnip crinkle virus (TCV) is a plant virus that triggers mRNA decay pathways, but it has also evolved strategies to evade this antiviral defense. In this study, we investigated the activation of mRNA decay during TCV infection and its impact on TCV RNA accumulation. We found that TCV infection induced the upregulation of essential mRNA decay factors, indicating their involvement in antiviral defense and the capsid protein (CP) of TCV, a well-characterized viral suppressor of RNA silencing (VSR), also compromised the mRNA decay-based antiviral defense by targeting AtXRN4. This interference with mRNA decay was supported by the observation that TCV CP stabilized a reporter transcript with a long 3' untranslated region (UTR). Moreover, TCV CP suppressed the decay of known NMD target transcripts, further emphasizing its ability to modulate host RNA control mechanisms. Importantly, TCV CP physically interacted with AtXRN4, providing insight into the mechanism of viral interference with mRNA decay. Overall, our findings reveal an alternative strategy employed by TCV, wherein the viral coat protein suppresses the mRNA decay pathway to facilitate viral infection.

SUZ domain–containing proteins have multiple effects on nonsense-mediated decay target transcripts

Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain-containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1. When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1: R3H domain-containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in an SUZ domain-dependent manner. This indicated that the SUZ domain-containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ domain-containing proteins play a complex role in regulating protein output from transcripts targeted by NMD.

Nonsense-mediated decay machinery in Plasmodium falciparum is inefficient and non-essential.

Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite Plasmodium falciparum shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag the P. falciparum orthologs of two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). We localize both PfUPF1 and PfUPF2 to puncta within the parasite cytoplasm and show that these proteins interact with each other and other mRNA-binding proteins. Using RNA-seq, we find that although these core NMD orthologs are expressed and interact in P. falciparum, they are not required for degradation of nonsense transcripts. Furthermore, our work suggests that the majority of intron retention in P. falciparum has no functional role and that NMD is not required for parasite growth ex vivo. IMPORTANCE In many organisms, the process of destroying nonsense transcripts is dependent on a small set of highly conserved proteins. We show that in the malaria parasite, these proteins do not impact the abundance of nonsense transcripts. Furthermore, we demonstrate efficient CRISPR-Cas9 editing of the malaria parasite using commercial Cas9 nuclease and synthetic guide RNA, streamlining genomic modifications in this genetically intractable organism.

The G3BP1-UPF1-Associated Long Non-Coding RNA CALA Regulates RNA Turnover in the Cytoplasm.

Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.

A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation.

The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using cross-linking immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon–intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial myelodysplasia (MDS)/acute myeloid leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease.

Inhibition of Nonsense-mediated Decay Causes Hyperalgesic Priming in Mice

RNA stability permeates neurobiology. Yet, the contribution of a dominant pathway of RNA metabolism termed nonsense-mediated decay (NMD) to pain is unclear. NMD safeguards against translation of mRNAs that harbor premature termination codons. It also controls the stability of roughly 10% of typical protein-coding mRNAs. NMD hinges on the activity of a conserved kinase designated SMG-1. We sought to determine the role of SMG-1 and NMD in pain. We found that SMG-1 is expressed in sensory neurons throughout the DRG. We identified SMG-1 targets in DRG neurons using high-throughput sequencing. We analyzed multiple features present in mRNA to establish the characteristics of NMD substrates in DRG neurons. The most dominant feature was structural content in the 3’UTR. This implies that factors that bind to structured motifs likely recruit NMD machinery to specific mRNAs. We identify numerous NMD targets linked to pain including components of the integrated stress response such as ATF-4. We found that inhibition of SMG-1 with a small molecule results in hyperalgesic priming in mice. Given that ATF-4 is targeted by NMD, we next asked if priming was due to activation of the integrated stress response. Indeed, a small molecule inhibitor of the integrated stress response called ISRIB prevented priming triggered by inhibition of NMD. Collectively, our results indicate that (i) inhibition of NMD is pro-nociceptive, (ii) priming induced by NMD blockade requires the integrated stress response, (iii) and structure is a defining feature of NMD targets. This work establishes a clear link between NMD and pain-associated behaviors in mice. RNA stability permeates neurobiology. Yet, the contribution of a dominant pathway of RNA metabolism termed nonsense-mediated decay (NMD) to pain is unclear. NMD safeguards against translation of mRNAs that harbor premature termination codons. It also controls the stability of roughly 10% of typical protein-coding mRNAs. NMD hinges on the activity of a conserved kinase designated SMG-1. We sought to determine the role of SMG-1 and NMD in pain. We found that SMG-1 is expressed in sensory neurons throughout the DRG. We identified SMG-1 targets in DRG neurons using high-throughput sequencing. We analyzed multiple features present in mRNA to establish the characteristics of NMD substrates in DRG neurons. The most dominant feature was structural content in the 3’UTR. This implies that factors that bind to structured motifs likely recruit NMD machinery to specific mRNAs. We identify numerous NMD targets linked to pain including components of the integrated stress response such as ATF-4. We found that inhibition of SMG-1 with a small molecule results in hyperalgesic priming in mice. Given that ATF-4 is targeted by NMD, we next asked if priming was due to activation of the integrated stress response. Indeed, a small molecule inhibitor of the integrated stress response called ISRIB prevented priming triggered by inhibition of NMD. Collectively, our results indicate that (i) inhibition of NMD is pro-nociceptive, (ii) priming induced by NMD blockade requires the integrated stress response, (iii) and structure is a defining feature of NMD targets. This work establishes a clear link between NMD and pain-associated behaviors in mice.

Global SLAM-seq for accurate mRNA decay determination and identification of NMD targets.

Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5% of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives, instead of steady-state RNA level changes. With SLAM-seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2% of our candidates being identified in previous studies. This indicates that SLAM-seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.

The total mRNA concentration buffering system in yeast is global rather than gene-specific.

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances, this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae. We also assessed mRNA concentrations and their synthesis rates in nonsense-mediated decay (NMD) targets in euploid strains. We found that the altered synthesis rates in the genes from the aneuploid chromosome and the changes in their mRNA stabilities were not counterbalanced. In addition, the stability of NMD targets was not specifically compensated by the changes in synthesis rate. We conclude that there is no genetic compensation of NMD mRNA targets in yeast, and total mRNA buffering uses mostly a global system rather than a gene-specific one.

Nonsense-mediated decay factor SMG7 sensitizes cells to TNFα-induced apoptosis via CYLD tumor suppressor and the noncoding oncogene Pvt1.

Nonsense-mediated decay (NMD) proteins are responsible for the surveillance and degradation of aberrant RNAs. Suppressor with morphogenetic effect on genitalia 7 (SMG7) is an NMD complex protein and a regulator of tumor necrosis factor (TNF)-induced extrinsic apoptosis; however, this unique function has not been explored in detail. In this study, we show that loss of Smg7 leads to unrestricted expression of long noncoding RNAs (lncRNAs) in addition to NMD targets. Functional analysis of Smg7-/- cells showed downregulation of the tumor suppressor cylindromatosis (CYLD) and diminished caspase activity, thereby switching cells to nuclear factor-κB (NF-κB)-mediated protection. This positive relationship between SMG7 and CYLD was found to be widely conserved in human cancer cell lines and renal carcinoma samples from The Cancer Genome Atlas. In addition to CYLD suppression, upregulation of lncRNAs Pvt1 and Adapt33 rendered cells resistant to TNF, while pharmacologic inhibition of NF-κB in Pvt1-overexpressing TNF-resistant cells and Smg7-deficient spheroids re-established TNF-induced lethality. Thus, loss of SMG7 decouples regulation of two separate oncogenic factors with cumulative downstream effects on the NF-κB pathway. The data highlight a novel and specific regulation of oncogenic factors by SMG7 and pinpoint a composite tumor suppressor role in response to TNF.

The Multifunctional Long-Distance Movement Protein of Pea Enation Mosaic Virus 2 Protects Viral and Host Transcripts from Nonsense-Mediated Decay.

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant's vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly.

SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript. The first half acts as dominant-negative isoform suppressing poison cassette exon inclusion and instead promoting the retention of flanking introns containing repeated SRSF7 binding sites. Massive SRSF7 binding to these sites and its oligomerization promote the assembly of large nuclear bodies, which sequester SRSF7 transcripts at their transcription site, preventing their export and restoring normal SRSF7 protein levels. We further show that hundreds of human and mouse NMD targets, especially RNA-binding proteins, encode potential Split-ORFs, some of which are expressed under specific cellular conditions.

RNA degradomes reveal substrates and importance for dark and nitrogen stress responses of Arabidopsis XRN4.

XRN4, the plant cytoplasmic homolog of yeast and metazoan XRN1, catalyzes exoribonucleolytic degradation of uncapped mRNAs from the 5′ end. Most studies of cytoplasmic XRN substrates have focused on polyadenylated transcripts, although many substrates are likely first deadenylated. Here, we report the global investigation of XRN4 substrates in both polyadenylated and nonpolyadenylated RNA to better understand the impact of the enzyme in Arabidopsis. RNA degradome analysis demonstrated that xrn4 mutants overaccumulate many more decapped deadenylated intermediates than those that are polyadenylated. Among these XRN4 substrates that have 5′ ends precisely at cap sites, those associated with photosynthesis, nitrogen responses and auxin responses were enriched. Moreover, xrn4 was found to be defective in the dark stress response and lateral root growth during N resupply, demonstrating that XRN4 is required during both processes. XRN4 also contributes to nonsense-mediated decay (NMD) and xrn4 accumulates 3′ fragments of select NMD targets, despite the lack of the metazoan endoribonuclease SMG6 in plants. Beyond demonstrating that XRN4 is a major player in multiple decay pathways, this study identified intriguing molecular impacts of the enzyme, including those that led to new insights about mRNA decay and discovery of functional contributions at the whole-plant level.

RNA virus evasion of nonsense-mediated decay.

Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3’ untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3’ UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3’ UTR. Several additional carmovirus 3’ UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5’ end of their 3’ UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3’ UTRs.

Intron-retained transcripts of the spinal muscular atrophy genes, SMN1 and SMN2

BackgroundThe SMN genes, SMN1 and SMN2, are highly homologous genes which are related to the development or clinical severity of spinal muscular atrophy. Some alternative splicing patterns of the SMN genes have been well documented. In 2007, an SMN1 transcript with a full sequence of intron 3 was reported as the first intron-retained SMN transcript. MethodsIntron-retained SMN transcripts in various cells and tissues were studied using reverse transcription (RT)-PCR. HeLa cells were used for subcellular localization of the transcripts and protein expression analysis with Western blotting. ResultsTwo intron-retained SMN transcripts were detected, which contain full sequences of intron 2b or intron 3. These transcripts were produced from SMN1 and SMN2, and ubiquitously expressed in human cells and tissues. Western blotting analysis showed no proteins derived from the intron-retained transcripts. Fractionation analysis showed that these intron-retained transcripts were localized mainly in the nucleus. Contrary to our expectation, the intron-retained transcript levels decreased during the treatment of cycloheximide, an inhibitor of nonsense-mediated decay (NMD), suggesting that they were not targets of NMD. ConclusionIntron 2b-retained SMN transcript and intron3-retained SMN transcript were ubiquitously expressed in human cells and tissues. The intron-retained transcripts were mainly localized in the nucleus and decreased through non-NMD pathway.

Abstract B060: Small molecule inhibitor of pre-mRNA splicing evokes antitumor activity via MDM4-p53 pathway

Abstract Proper control of gene expression at the level of transcription and RNA processing is a fundamental process of tumor cell growth. Here we showed that a novel pre-mRNA splicing modulator, Cdc2-like kinase (CLK) inhibitor T-025, suppressed tumor cell growth in vitro and in vivo. To reveal the mechanisms linking aberrant splicing and tumor growth suppression by T-025, we combined transcriptomic RNA-seq and proteomic p-SILAC data. We identified MDM4 as a mediator for triggering p53-dependent apoptosis in response to T-025. CLK inhibition led to exon 6 skipping of MDM4 mRNA, a potential target for nonsense-mediated decay, decreasing MDM4 protein. Consistent with these results, overexpression of MDM4 or knockdown of p53 attenuated growth-suppressive effects of T-025. We then investigated the relationship between genomic features and sensitivity to T-025 by evaluating the antiproliferative effect of T-025 against 240 cancer cell lines. Strikingly, expression of MDM4 was correlated with that of CLK2 and cell sensitivity to T-025. In addition, we found that copy number variation and mRNA expression of MYC were correlated with cell sensitivity to T-025. T-025 exhibited greater sensitivity in MYC-expressing cells and MYC-driven spontaneous breast cancer models. Together, we demonstrated that MDM4-p53 signaling and MYC-activation have key roles in antitumor activity of CLK inhibitor. Citation Format: Kazuho Nishimura, Masahiro Yaguchi, Yukiko Yamamoto, Shunsuke Ebara, Kawakita Yoichi, Ryo Mizojiri, Yusuke Nakayama, Kozo Hayashi, Shuichi Miyakawa, Kenichi Iwai, Toshiyuki Nomura. Small molecule inhibitor of pre-mRNA splicing evokes antitumor activity via MDM4-p53 pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B060.

Abstract B125: Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7

Abstract Refractory Anemia with Ringed Sideroblasts (RARS), a subtype of Myelodysplatic Syndrome (MDS), occurs with a high frequency of hotspot mutations in HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, Targets of rapamycin 1 domains) domains of SF3B1. This protein component of the U2 snRNP complex of the spliceosome is essential in the proper selection and usage of 3' splice sites. RNAseq analysis of MDS and other tumor types in which SF3B1 hotspot mutations have been found show that alternative 3' splice site usage is the predominant cause of RNA transcript aberration. These modifications can result in mRNAs encoding novel peptides, or they can introduce premature termination codons into the pre-mRNA, most likely directing it to the Nonsense Mediated Decay (NMD) pathway for degradation. Using a predictive tool to determine the likelihood of a given aberrant transcript to be targeted for NMD, we determined that nearly 50% of the SF3B1-mutant-associated aberrant transcripts were candidates for degradation. We confirmed this experimentally by treating isogenic Nalm-6 cells (engineered by AAV homology to express SF3B1 K700E or K700K) with or without cycloheximide, an agent known to inhibit translation and RNA degradation by NMD. Investigation of the resulting RNAseq data showed significant rescue of gene expression only for the transcripts predicted to be NMD targets. Ingenuity Pathway Analysis indicated that many of the downregulated genes in SF3B1 mutant samples were involved in differentiation, which has been shown to be dysregulated in MDS. We tested the idea that such modifications in the transcriptome confer selective advantage or impair differentiation in SF3B1 mutant cells. We began by manipulating the expression of ABCB7, one of the genes identified in our RNAseq analysis to be downregulated by aberrant splicing and subsequent NMD. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and, indirectly, in heme production. Additionally, loss of function of ABCB7 is causal in X-linked sideroblastic anemia and has been implicated in RARS MDS. We discovered in our SILAC proteomic analysis that ABCB7 protein was dramatically decreased in K700E SF3B1 Nalm-6 cells relative to K700K Nalm-6, in agreement with our RNAseq analysis. Using doxycycline-inducible shRNA expression, we knocked down ABCB7 mRNA and protein expression in TF-1 erythroblasts. These cells show significant decreases in erythropoeitin (EPO)-induced differentiation when expressing exogenous K700E SF3B1, but not K700R (a very conservative mutation) or WT SF3B1. With direct knock down of ABCB7, we observed a similar phenotype - impairment of EPO-induced differentiation in ABCB7 shRNA-induced cells by Day 7, with no overall decline in cell viability. Interestingly, knock down of SF3B1 expression with shRNA also reduces ABCB7 mRNA. However, it also promotes cell death. This is consistent with the heterozygous nature of SF3B1 hotspot mutations; severe loss of SF3B1 function is deleterious. We propose that hotspot SF3B1 mutants promote aberrant splicing of multiple genes, inducing a general “spliceosomal sickness” in addition to downregulating key genes (e.g. ABCB7) responsible for erythroid differentiation impairment, such as that observed in RARS. Citation Format: Rachel B. Darman, Samantha A. Perino, Michael Seiler, Shouyong Peng, Jacob Feala, Peter Fekkes, Gregg F. Keaney, Kaiko Kunii, Linda Lee, Kian Huat Lim, Yoshiya Oda, Khin Myint, Esther A. Obeng, Ermira Pazolli, Eun Sun Park, John Yuan Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Yoshiharu Mizui, Benjamin L. Ebert, Peter G. Smith, Silvia Buonamici. Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B125.

Abstract 2118: Nonsense-mediated decay regulates the expression of tumor suppressor transcripts and cancer pathways in non-small cell lung cancer cell lines

Abstract Background: Nonsense-mediated decay (NMD) is an RNA surveillance pathway that selectively degrades mRNAs bearing premature translation termination codons (PTCs). In addition, NMD also regulates the expression of a subset of physiological mRNAs containing upstream open reading frames (uORFs), spliceable introns in the 3′ untranslated region (3′-UTR) and those that undergo aberrant alternative splicing. Cancer cells produce many mRNAs harboring PTCs because of high frequency of mutations either in the coding region or in the splice sites that result in aberrant splicing. Thus, cancer cells may depend on NMD for survival. Inhibiting NMD provides an attractive cancer therapeutic strategy as it rescues mRNAs bearing NMD features, some of which may produce functional proteins. Therefore, it is important to identify NMD targets and their NMD-inducing features in cancer cells. In the current study, by using NMD inhibition approach, we identified mRNA transcripts and pathways that are regulated by NMD in non-small cell lung cancer (NSCLC) cell lines. Methods: To identify NMD targets, UPF1, a key component of NMD pathway was depleted by RNAi in NSCLC cell lines. mRNAs regulated by NMD were identified by micro array and RNAseq approaches and selected NMD targets were confirmed by real-time PCR. Reverse phase protein array (RPPA) was performed to identify cancer pathways affected by NMD inhibition. Migration, proliferation and clonogenic assays were performed to assess the effect of NMD inhibition on cancer phenotype. Results: Our microarray and RNAseq analysis results indicated that in NSCLC cell lines, NMD regulates the expression of a number of physiological transcripts derived from non-mutant genes. These include transcripts involved in integrated stress response, apoptotic pathways and tumor suppressor activity. We showed that tumor suppressors DMTF1, and p53 undergo NMD-susceptible alternative splicing in NSCLC cell lines and that NMD-regulated p53 isoforms are partially functional. Our results also demonstrated that mRNA transcripts derived from TSC1 and GADD45A, tumor suppressors that negatively regulate mTOR pathway, are also upregulated upon NMD inhibition in NSCLC cell lines. Concomitantly, RPPA analysis revealed that NMD inhibition resulted in downregulation of components involved in mTOR pathway. NMD inhibition also resulted in reduced cell viability and migration. Conclusions: NMD regulates the expression of physiological transcripts involved in integrated stress response, apoptosis and tumor suppressor activity and impacts cancer phenotype in NSCLC cell line models. Our findings suggest that NMD could serve as an attractive therapeutic target. Citation Format: Jayanthi P. Gudikote, Hao Zhao, Jing Wang, Youhong Fan, Lixia Diao, Lauren A. Byers, Uma Giri, Monique Nilsson, John V. Heymach. Nonsense-mediated decay regulates the expression of tumor suppressor transcripts and cancer pathways in non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2118. doi:10.1158/1538-7445.AM2015-2118

Target Discrimination in Nonsense-Mediated mRNA Decay Requires Upf1 ATPase Activity

RNA quality-control pathways get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the adenosine triphosphatase (ATPase) cycle of the SF1 helicase Upf1 is required for mRNA discrimination during nonsense-mediated decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD-translation and termination codon-proximal poly(A) binding protein-depend on the ATPase activity of Upf1 to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be a widely used activity in target RNA discrimination.

Differential recruitment of splice variants from SR pre-mRNAs to polysomes during development and in response to stresses.

We have previously shown that precursor mRNAs (pre-mRNAs) of serine/arginine-rich (SR) proteins are extensively alternatively spliced to generate approximately 100 distinct splice variants from 14 SR genes and that the splicing pattern of SR pre-mRNAs changes in different organs and in response to abiotic stresses. About half of the splice variants are potential targets of nonsense-mediated decay (NMD) and 25 splice forms were confirmed to be real NMD targets. However, it is not known whether (i) all splice variants are recruited to polysomes for translation; (ii) there is a preferential recruitment of specific splice isoforms to polysomes; and (iii) there is a differential recruitment of splice variants during development and in response to stresses. To address these questions, we analyzed the association of SR splice variants with polysomes from seedlings, different organs and seedlings exposed to heat and cold stress. In seedlings, about one-third of the splice variants (22 out of 72) are not recruited to polysomes. Among those associated with polysomes, the functional isoforms that code for full-length proteins and some candidate putative and confirmed NMD targets were identified. There was preferential recruitment of some splice forms over others. Predominant recruitment of functional isoforms along with a few NMD candidates was found in different organs. Furthermore, we observed differential recruitment of isoforms in different organs. Heat and cold stress enhanced or reduced recruitment of specific splice variants. Our studies reveal differential recruitment of SR splice variants to polysomes under normal conditions, during development and in response to stresses.