High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines

Ketley, Ami; Chen, Catherine Z.; Li, Xin; Arya, Sukrat; Robinson, Thelma; Granados-Riveron, Javier T; Udosen, I.; Morris, Glenn E.; Holt, Ian; Furling, Denis; Chaouch, Soraya; Haworth, Ben; Southall, Noel; Shinn, Paul; Zheng, Wei; Austin, Christopher P.; Hayes, Christopher J.; Brook, David

doi:10.1093/hmg/ddt542

Cited by 61 publications

(50 citation statements)

References 49 publications

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“…Such molecules may, in addition to CUGexp RNA, target other yet-unidentified cellular components critical for DM1 pathogenesis. Interestingly, the most recent report from the Brook laboratory indicates that targeting protein kinases with small molecules results in alleviation of molecular hallmarks of DM1 34 . This was correlated with the disappearance of nuclear CUGexp RNA foci without degradation of the mutant transcripts or their translocation to the cytoplasm.…”

Section: Introductionmentioning

confidence: 97%

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

et al. 2014

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Expandable (CTG)n repeats in the 3′ UTR of the DMPK gene are a cause of myotonic dystrophy type 1 (DM1), which leads to a toxic RNA gain-of-function disease. Mutant RNAs with expanded CUG repeats are retained in the nucleus and aggregate in discrete inclusions. These foci sequester splicing factors of the MBNL family and trigger upregulation of the CUGBP family of proteins resulting in the mis-splicing of their target transcripts. To date, many efforts to develop novel therapeutic strategies have been focused on disrupting the toxic nuclear foci and correcting aberrant alternative splicing via targeting mutant CUG repeats RNA; however, no effective treatment for DM1 is currently available. Herein, we present results of culturing of human DM1 myoblasts and fibroblasts with two small-molecule ATP-binding site-specific kinase inhibitors, C16 and C51, which resulted in the alleviation of the dominant-negative effects of CUG repeat expansion. Reversal of the DM1 molecular phenotype includes a reduction of the size and number of foci containing expanded CUG repeat transcripts, decreased steady-state levels of CUGBP1 protein, and consequent improvement of the aberrant alternative splicing of several pre-mRNAs misregulated in DM1.

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Section: Introductionmentioning

confidence: 97%

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

et al. 2014

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“…The timing of appearance of certain mis-spliced events correlates well with disease severity measured as a loss of muscle strength 4 which makes them very useful biomarkers. This hallmark of DM pathogenesis is most informative when the efficacy of investigated compounds is measured to demonstrate their therapeutic value [25][26][27] . Thus far, profiling of aberrantly spliced transcripts has been performed with high-throughput methods such as specific oligonucleotide-based microarrays and next-generation sequencing, or with traditional RT-PCR and agarose gel quantification of PCR products.…”

Section: Discussionmentioning

confidence: 99%

Quantitative methods to monitor RNA biomarkers in myotonic dystrophy

Wojciechowska¹,

Sobczak²,

Sedehizadeh³

et al. 2018

Neuromuscular Disorders

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Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3′-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeatcontaining RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPK exp RNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPK exp RNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPK exp RNA.Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are adult onset muscular dystrophies leading to disability and shortened lifespan. Both belong to a larger group of human disorders associated with mutational expansions of simple repetitive sequences within specific genes. The mutation that causes DM1 results from the expansion of CTG repeats in the 3′-UTR of the DMPK gene; whereas DM2 is associated with intronic elongation of CCTG repeats in ZNF9/CNBP gene 1,2 . Pathogenesis of both diseases is mediated by a toxic RNA gain-of-function mechanism manifested by nuclear retention of expanded CUG-and CCUG-harboring RNAs which aggregate within nucleoprotein foci 3 . Their adverse effects are mediated through sequestration of various proteins including the muscleblind-like (MBNL) family of splicing factors. These proteins are functionally depleted by the expanded repeats in DM1 and DM2 which leads to abnormalities in many pathways of RNA metabolism including alternative splicing, a molecular hallmark of DM [4][5][6][7][8] . In DM adult skeletal muscle abnormal expression of embryonic splicing isoforms has been reported for a few hundred genes 4 . Given the importance of alternative splicing as one of the biomarkers of disease severity and therapeutic response, accurate quantification of splicing variants could facilitate their refinement for clinical applications. Specific oligonucleotide-based microarrays and next-generation sequencing have been used for profiling and identification of many aberrantly spliced transcripts. However, their routine application for precise quantification remains limited due to low quantitative parameters of these methods, restriction to highly expressed genes, high costs a...

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“…These candidate anticancer drugs are currently under clinical investigations for various types of cancer. Due to the growing demand for the existing drugs in anticancer drug discovery, a number of collections of existing drugs are now available commercially or noncommercially, which are amenable for high throughput screening (Chong et al 2006;Diamandis et al 2007;Doudican et al 2008;Huang et al 2011;Hothi et al 2012;Robinson et al 2013;Czyz et al 2014;Ketley et al 2014;Mei et al 2014) (Table 3).…”

Section: Experimental Models Of Cscs and Drug Repositioningmentioning

confidence: 99%

Existing drugs and their application in drug discovery targeting cancer stem cells

Lv¹,

Shim²

2015

Arch. Pharm. Res.

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Despite standard cancer therapies such as chemotherapy and targeted therapy have shown some efficacies, the cancer in many cases eventually relapses and metastasizes upon stopping the treatment. There is a small subpopulation of cancer cells within tumor, with specific characters similar to those found in stem cells. This group of cancer cells is known as tumor-initiating or cancer stem cells (CSCs), which have an ability to self-renew and give rise to cancer cell progeny. CSCs are related with drug resistance, metastasis and relapse of cancer, hence emerging as a crucial drug target for eliminating cancer. Rapid advancement of CSC biology has enabled researchers to isolate and culture CSCs in vitro, making the cells amenable to high-throughput drug screening. Recently, drug repositioning, which utilizes existing drugs to develop potential new indications, has been gaining popularity as an alternative approach for the drug discovery. As existing drugs have favorable bioavailability and safety profiles, drug repositioning is now actively exploited for prompt development of therapeutics for many serious diseases, such as cancer. In this review, we will introduce latest examples of attempted drug repositioning targeting CSCs and discuss potential use of the repositioned drugs for cancer therapy.

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High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines

Cited by 61 publications

References 49 publications

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

Quantitative methods to monitor RNA biomarkers in myotonic dystrophy

Existing drugs and their application in drug discovery targeting cancer stem cells

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