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Use of CRISPR/Cas9 with homology-directed repair to silence the human Topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells

ABSTRACT: Use of CRISPR/Cas9 with homology-directed repair to silence the human Topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells

PROVIDER: PRJNA827579 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18779860_1.fastq.gz	Fastqsanger.gz
	SRR18779860_2.fastq.gz	Fastqsanger.gz
	SRR18779861_1.fastq.gz	Fastqsanger.gz
	SRR18779861_2.fastq.gz	Fastqsanger.gz
	SRR18779862_1.fastq.gz	Fastqsanger.gz

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Project description:DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5' splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5' splice site in parental K562 cells by CRISPR/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. Forced expression of TOP2α/90 in the gene-edited K562 cells further decreased etoposide-induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the important role of both TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents.

Project description:An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT ) in the TOP2α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.

Project description:Chemotherapy resistance is one of the reasons for the loss of the eye in retinoblastoma (RB) pa-tients. RB chemotherapy resistance has been investigated in different cell culture models like WERI RB1. Furthermore, chemotherapy resistant RB subclones like the etoposide resistant WERI ETOR cell line have been established to improve understanding of chemotherapy resistance in RB. Ob-jective of this study was to characterize the cell line models of an etoposide sensitive WERI RB1 and its etoposide resistant subclone WERI ETOR by proteomics analysis. Subsequently, quantitative proteomic data served for correlation analysis with known drug perturbation profiles. . WERI RB1 and WERI ETOR were cultured and prepared for quantitative mass spectrometry. Comparative proteomic profiling was performed with electrospray ionization tandem mass spectrometry in data-independent acquisition mode (SWATH). The raw SWATH files were processed using neural networks in library free mode along with machine learning algorithms. Pathway enrichment was performed using the REACTOME pathway resource and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. Also, a drug connec-tivity analysis using the L1000 database was used to correlate the mechanism-of-action (MOA) for different anticancer reagents to WERI RB1 / WERI ETOR signatures. A total 4,756 proteins were identified across all samples which revealed a distinct clustering between groups. Of these pro-teins, 64 proteins were significantly altered (q < 0.05 & log2FC |>2|, 22% higher in WERI ETOR). Pathway analysis showed an enriched metabolic pathway for retinoid metabolism and transport pathway in WERI ETOR and for sphingolipid de novo biosynthesis in WERI RB1. In addition, this study showed similar protein signatures of topoisomerase inhibitors and WERI ETOR as well as of ATPase inhibitors, acetylcholine receptor antagonists and VEGFR inhibitors and WERI RB1. In this study, WERI RB1 and WERI ETOR were characterized as a cell line model for chemotherapy re-sistance in RB by using data-independent mass spectrometry. The global proteome revealed the activation of sphingolipid de novo biosynthesis in WERI RB1 and potential treatment options for etoposide resistant RB.

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Use of CRISPR/Cas9 with homology-directed repair to silence the human Topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells

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