Compound C inhibits nonsense-mediated RNA decay independently of AMPK

The nonsense mediated RNA decay (NMD) pathway safeguards the integrity of the transcriptome by targeting mRNAs with premature translation termination codons (PTCs) for degradation. It also regulates gene expression by degrading a large number of non-mutant RNAs (including mRNAs and noncoding RNAs) that bear NMD-inducing features. Consequently, NMD has been shown to influence development, cellular response to stress, and clinical outcome of many genetic diseases. Small molecules that can modulate NMD activity provide critical tools for understanding the mechanism and physiological functions of NMD, and they also offer potential means for treating certain genetic diseases and cancer. Therefore, there is an intense interest in identifying small-molecule NMD inhibitors or enhancers. It was previously reported that both inhibition of NMD and treatment with the AMPK-selective inhibitor Compound C (CC) induce autophagy in human cells, raising the possibility that CC may be capable of inhibiting NMD. Here we show that CC indeed has a NMD-inhibitory activity. Inhibition of NMD by CC is, however, independent of AMPK activity. As a competitive ATP analog, CC does not affect the kinase activity of SMG1, an essential NMD factor and the only known kinase in the NMD pathway. However, CC treatment down-regulates the protein levels of several NMD factors. The induction of autophagy by CC treatment is independent of ATF4, a NMD target that has been shown to promote autophagy in response to NMD inhibition. Our results reveal a new activity of CC as a NMD inhibitor, which has implications for its use in basic research and drug development

Pan-cancer analysis reveals recurrent BCAR4 gene fusions across solid tumors

UNLABELLED: Chromosomal rearrangements often result in active regulatory regions juxtaposed upstream of an oncogene to generate an expressed gene fusion. Repeated activation of a common downstream partner-with differing upstream regions across a patient cohort-suggests a conserved oncogenic role. Analysis of 9,638 patients across 32 solid tumor types revealed an annotated long noncoding RNA (lncRNA), Breast Cancer Anti-Estrogen Resistance 4 (BCAR4), was the most prevalent, uncharacterized, downstream gene fusion partner occurring in 11 cancers. Its oncogenic role was confirmed using multiple cell lines with endogenous BCAR4 gene fusions. Furthermore, overexpressing clinically prevalent BCAR4 gene fusions in untransformed cell lines was sufficient to induce an oncogenic phenotype. We show that the minimum common region to all gene fusions harbors an open reading frame that is necessary to drive proliferation. IMPLICATIONS: BCAR4 gene fusions represent an underappreciated class of gene fusions that may have biological and clinical implications across solid tumors

RAG-induced DNA double-strand breaks signal through Pim2 to promote pre-B cell survival and limit proliferation

Interleukin 7 (IL-7) promotes pre–B cell survival and proliferation by activating the Pim1 and Akt kinases. These signals must be attenuated to induce G1 cell cycle arrest and expression of the RAG endonuclease, which are both required for IgL chain gene rearrangement. As lost IL-7 signals would limit pre–B cell survival, how cells survive during IgL chain gene rearrangement remains unclear. We show that RAG-induced DNA double-strand breaks (DSBs) generated during IgL chain gene assembly paradoxically promote pre–B cell survival. This occurs through the ATM-dependent induction of Pim2 kinase expression. Similar to Pim1, Pim2 phosphorylates BAD, which antagonizes the pro-apoptotic function of BAX. However, unlike IL-7 induction of Pim1, RAG DSB-mediated induction of Pim2 does not drive proliferation. Rather, Pim2 has antiproliferative functions that prevent the transit of pre–B cells harboring RAG DSBs from G1 into S phase, where these DNA breaks could be aberrantly repaired. Thus, signals from IL-7 and RAG DSBs activate distinct Pim kinase family members that have context-dependent activities in regulating pre–B cell proliferation and survival

Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics

Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense mediated decay (NMD) is the cell's natural surveillance mechanism, that detects and destroys PTC containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using RT-qPCR, we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation (c.715 C > T; p.[R239*]). These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their iPSC-derived RPE. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wildtype levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre–B cell receptor (pre–BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre–BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre–B cells. Here, we show that RAG DSBs inhibit pre–BCR signals through the ATM- and NF-κB2–dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre–BCR signaling. This regulatory circuit prevents the pre–BCR from inducing additional Igl chain gene rearrangements and driving pre–B cells with RAG DSBs into cycle. We propose that pre–B cells toggle between pre–BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes

Modeling colorectal cancer: A bio-resource of 50 patient-derived organoid lines

Background and Aim Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. To improve outcomes for these patients, we need to develop new treatment strategies. Personalized cancer medicine, where patients are treated based on the characteristics of their own tumor, has gained significant interest for its promise to improve outcomes and reduce unnecessary side effects. The purpose of this study was to examine the potential utility of patient-derived colorectal cancer organoids (PDCOs) in a personalized cancer medicine setting. Methods Patient-derived colorectal cancer organoids were derived from tissue obtained from treatment-naïve patients undergoing surgical resection for the treatment of CRC. We examined the recapitulation of key histopathological, molecular, and phenotypic characteristics of the primary tumor. Results We created a bio-resource of PDCOs from primary and metastatic CRCs. Key histopathological features were retained in PDCOs when compared with the primary tumor. Additionally, a cohort of 12 PDCOs, and their corresponding primary tumors and normal sample, were characterized through whole exome sequencing and somatic variant calling. These PDCOs exhibited a high level of concordance in key driver mutations when compared with the primary tumor. Conclusions Patient-derived colorectal cancer organoids recapitulate characteristics of the tissue from which they are derived and are a powerful tool for cancer research. Further research will determine their utility for predicting patient outcomes in a personalized cancer medicine setting

Novel regulation of the nonsense-mediated RNA decay pathway

The nonsense mediated RNA decay (NMD) pathway maintains the integrity of cellular RNAs and controls gene expression. NMD is essential for vertebrate development and defects in NMD are associated with a variety of neurodevelopmental disorders and cancers. NMD activity is tightly regulated and is altered in response to environmental and developmental signals. To better study this dynamic pathway and to identify clinically relevant regulators of its activity, we developed a dual-color bioluminescent NMD reporter that rapidly and accurately quantifies NMD activity in mammalian cells. Using this reporter, we performed a chemical screen for small-molecule modulators of NMD activity and identified the cardiac glycosides (CGs) as potent repressors of NMD activity. Further studies on the mechanism of action of these drugs led to the finding that intracellular calcium, a key cellular signaling molecule, potently regulates NMD, with increases in intracellular calcium repressing NMD. The regulation of NMD by calcium may be exploited to treat certain genetic diseases and cancers. Regulation of NMD is particularly important to the cellular stress response. Stresses such as hypoxia, amino acid deprivation, and ER stress induce a reduction in NMD activity that promotes the expression of genes that help the cell to cope with these environmental insults. We investigated the regulation of NMD and its role in the cellular response to DNA damage and osmotic shock. We found that NMD is suppressed by persistent, but not transient, DNA damage. Conditions that constantly induce damage, such as excessive mitogenic signaling or the presence of genotoxic agents, or that prevent its swift repair, such as mutations in repair factors, generate persistent DNA lesions. Telomeres are another prominent source of persistent DNA damage because telomeric damage is difficult to repair, and telomere erosion from repeated cell divisions also elicits a protracted DNA damage response (DDR). The inhibition of NMD by persistent DNA damage is mediated in part by p38 MAP kinase signaling and augments the expression of ATF3, a stress-inducible transcription factor, by stabilizing its mRNAs. We found that osmotic shock also causes NMD inhibition but in a p38-independent manner. These results reveal a novel p38-dependent pathway that regulates NMD activity in response to persistent DNA damage which contributes to gene expression changes in damaged cells

Control of gene expression through the nonsense-mediated RNA decay pathway

Abstract Nonsense-mediated RNA decay (NMD) was originally discovered as a cellular surveillance pathway that safeguards the quality of mRNA transcripts in eukaryotic cells. In its canonical function, NMD prevents translation of mutant mRNAs harboring premature termination codons (PTCs) by targeting them for degradation. However, recent studies have shown that NMD has a much broader role in gene expression by regulating the stability of many normal transcripts. In this review, we discuss the function of NMD in normal physiological processes, its dynamic regulation by developmental and environmental cues, and its association with human disease

Viral bronchiolitis management in hospitals in the UK

Background Viral bronchiolitis is the leading cause of hospitalisation in infants less than a year old. The United Kingdom (UK) National Institute for Health and Care Excellence (NICE) published a guideline for the management of viral bronchiolitis in June 2015. Objectives This study aimed to prospectively survey the management of viral bronchiolitis in hospital Trusts in the UK to provide a baseline of practice prior to the publication of the 2015 NICE bronchiolitis guideline against which future practice can be assessed. Study design An electronic, structured questionnaire was sent to hospital paediatricians in the UK prior to the publication of the NICE bronchiolitis guideline via the Royal College of Paediatrics and Child Health e-portfolio system to assess the quality of Trust’s viral bronchiolitis management guidelines. Results Paediatricians from 111 (65% of all) UK Trusts completed an electronic questionnaire. 91% of Trusts had a bronchiolitis guideline. Overall only 18% of Trusts would be fully compliant with the NICE guideline. Between 43–100% of Trusts would be compliant with different sections of the guideline. There was variation in hospital admission criteria with respect to the need for supplemental oxygen (oxygen saturations <88% to <95%). ‘Unnecessary’ medications (especially bronchodilators, nebulised hypertonic saline and antibiotics) and investigations (chest x-ray and blood gas) were regularly advised. 72% of Trusts advised respiratory virus testing in all hospitalised infants and 64% created bronchiolitis bays to cohort infants. Conclusions There was wide variation in the management of infants with bronchiolitis in Trusts. Most bronchiolitic infants are not managed optimally in hospitals. Future guidelines should include advice on virus testing and isolation/cohorting