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

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.

Circumvention of Topoisomerase IIα Intron 19 Intronic Polyadenylation in Acquired Etoposide-Resistant Human Leukemia K562 Cells

“…The relative mRNA expression levels of TOP2α/170 and TOP2α/90 in each cell line were normalized to TATA-binding protein (TaqMan assay Hs99999910_m1; ThermoFisher Scientific) expression using the 2 -∆∆Ct method (Schmittgen and Livak, 2008). Finally, qPCR experiments were conducted using custom Taqman Gene Expression hydrolysis probes (Supplemental Table 1) spanning all human TOP2α gene exon/intron boundaries with weak splice sites to investigate whether IPA occurred in multiple locations in the TOP2α gene (Kanagasabai et al, 2017(Kanagasabai et al, , 2018Hernandez et al, 2021Hernandez et al, , 2022.…”

“…Briefly, sgRNA (0.5 µg) and Cas9 protein (2 µg) were incubated for 20 min at room temperature to form ribonucleoprotein complexes, and 5 µM TOP2α of an I19 PAS #3 repair template was then added. This mixture was then transfected into K/VP.5 cells (1.5 x 10 6 cells in 100 µl) by electroporation with the Amaxa Nucleofactor II system (Nucleofector Kit V; Lonza, Basel, Switzerland) as reported previously (Hernandez et al 2021(Hernandez et al , 2022. Forty-eight hours later, a small fraction of the transfected K/VP.5 cells were lysed for determination of Cas9 targeting/NHEJ repair efficiency using the genomic cleavage detection (GCD) assay described below in the next section.…”

“…Our prior studies demonstrated the importance of both TOP2α/170 and TOP2α/90 protein levels as determinants of sensitivity/resistance to TOP2α-targeting agents (Hernandez et al, 2021(Hernandez et al, , 2022. Therefore, we next used CRISPR/Cas9/HDR (Jinek et al, 2012;Mali et al, 2013;Liang et al, 2017) to introduce specific gene edits in the TOP2α I19 PAS #3 hexamer to attenuate IPA (i.e., decrease TOP2α/90 protein levels)…”

“…Although many chemoresistant mechanisms have been described (Zahreddine et al, 2013;Cree et al, 2017), acquired resistance to TOP2α-targeted drugs is often associated with decreased expression levels of TOP2α170 and/or altered sub-cellular localization, which greatly reduces the cytotoxicity of these drugs due to the diminished formation and accumulation of TOP2α170-DNA cleavage complexes in the nucleus (Burgess et al, 2008;Pilati et al, 2012;Ganapathi and Ganapathi, 2013;Capelôa et al, 2020. Our laboratory (Kanagasabai et al, 2017(Kanagasabai et al, , 2018Hernandez et al, 2021Hernandez et al, , 2022Elton et al, 2022) and others (Harker et al, 1995;Mo and Beck, 1997) demonstrated that intronic polyadenylation (IPA) plays an important role in mediating TOP2 poison/inhibitor acquired resistance in several human leukemia cell lines. IPA generates composite terminal exons through the inhibition/modulation of the 5′ splice site (5′ SS) at the exon/intron boundary and subsequent utilization of a cryptic poly(A) site (PAS) harbored within the intron which can be translated into truncated proteins (Tian et al, 2007;Lee et al, 2018;Singh et al, 2018).…”

Genome editing in cotton: challenges and opportunities

“…Cas13a's RNA-cleaving characteristics may be used for posttranscriptional suppression with similar efficacy to RNA interference (RNAi) techniques of RNA silencing, but with greater specificity and the capacity to cleave nuclear transcripts, which is limited with RNAi (Sun et al 2022). Due to alternative splicing, the transcription of single DNA sequence generates several splicing isoforms, hence targeting DNA with CRISPR systems affects all mRNA isoforms (Hernandez et al 2022). Cas13a enables the investigation of a single isoform's function or interference with its impact without affecting the activity of the other isoforms.…”