CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIαIntron 19 5′ Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells

Abstract

An essential function of DNA topoisomerase IIa (TOP2a; 170 kDa, TOP2a/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2a/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2a/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2a/170, expresses high levels of a novel C-terminal truncated TOP2a isoform (90 kDa, TOP2a/90). TOP2a/ 90, the translation product of a TOP2a mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2a/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 TOP2a-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAAAC→GAG//GTAAGT) in the TOP2a gene's suboptimal exon 19/intron 19 59 splice site (E19/I19 59 SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2a alleles edited revealed improved I19 removal, decreased TOP2a/90 mRNA/protein, and increased TOP2a/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (gH2AX, 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 TOP2a E19/I19 59 SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2a-targeted drugs. SIGNIFICANCE STATEMENTResults presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 59 splice site in the DNA topoisomerase IIa (TOP2a) gene results in circumvention of acquired drug resistance to etoposide and other TOP2a-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2a 90 kDa isoform and increasing expression of full-length TOP2a 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2a-targeted drugs.

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

“…Etoposide-resistant K/VP.5 cells were selected and cloned subsequent to intermittent and eventually continuous exposure of K562 cells to 0.5 μM etoposide as previously described [ 35 ]. Human leukemia K562, K/VP.5 and gene-edited clonal cells were maintained in DMEM/10% FBS as previously reported [ 11 , 15 , 26 ]. All experiments described below were performed utilizing cells growing in mid-log phase.…”

“…Briefly, sgRNA #1 (0.5 μg) was incubated with 2 μg TrueCut Cas9 Protein v2 and 5 μM TOP2α E19/I19 repair template “Silenced E19/I19 5′SS” for 15 min according to the manufacturer’s instructions. This mixture was then transfected into K562 cells (2.25 × 10 6 cells in 100 μl) by electroporation as reported previously [ 26 ]. Forty-eight hours later, K562 cells (1 x 10 6 ) were lysed for Cas9 targeting and repair efficiency using the GCD assay described above.…”

“…Several studies have established that the strengthening of a weak or suboptimal 5′ SS through mutagenesis could modulate alternative mRNA splicing and IPA [ 19 – 25 ]. Specifically, our laboratory previously improved I19 removal in etoposide resistant K/VP.5 cells [ 26 ] by utilizing CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9) with homology-directed repair (HDR) [ 27 – 32 ] to introduce two specific nucleotide changes ( ) in the human TOP2α gene’s suboptimal exon 19/intron 19 5′ SS boundary (E19/I19 5′ SS). A gene-edited clone with enhanced I19 removal verified by qPCR and RNA seq, exhibited decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein expression [ 26 ].…”

“…Specifically, our laboratory previously improved I19 removal in etoposide resistant K/VP.5 cells [ 26 ] by utilizing CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9) with homology-directed repair (HDR) [ 27 – 32 ] to introduce two specific nucleotide changes ( ) in the human TOP2α gene’s suboptimal exon 19/intron 19 5′ SS boundary (E19/I19 5′ SS). A gene-edited clone with enhanced I19 removal verified by qPCR and RNA seq, exhibited decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein expression [ 26 ]. This gene edited cell line exhibited increased DNA damage in response to etoposide and other TOP2-targeted drugs as well as increased sensitivity to drug induced-growth inhibition [ 26 ].…”

“…A gene-edited clone with enhanced I19 removal verified by qPCR and RNA seq, exhibited decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein expression [ 26 ]. This gene edited cell line exhibited increased DNA damage in response to etoposide and other TOP2-targeted drugs as well as increased sensitivity to drug induced-growth inhibition [ 26 ]. Together, these results indicated that the optimization of the TOP2α E19/I19 5′ SS in K/VP.5 cells by gene-editing circumvented etoposide resistance and confirmed the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs [ 26 ].…”