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The Journal of Pharmacology and Experimental Therapeutics logoLink to The Journal of Pharmacology and Experimental Therapeutics
. 2024 May;389(2):186–196. doi: 10.1124/jpet.123.002038

Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβ in Human Leukemia K562 Cells: Generation of a Resistance Phenotype

Jessika Carvajal-Moreno 1, Xinyi Wang 1, Victor A Hernandez 1, Milon Mondal 1, Xinyu Zhao 1, Jack C Yalowich 1,✉, Terry S Elton 1,✉
PMCID: PMC11026151  PMID: 38508753

Abstract

DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

SIGNIFICANCE STATEMENT

Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

Introduction

The genome in humans encodes two type II topoisomerase enzymes, DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) and DNA topoisomerase IIβ (TOP2β; 180 kDa, TOP2β/180) with 68% sequence identity (Austin et al., 1993). Both enzymes function as homodimers and introduce short-lived DNA double-strand breaks (DSBs) to regulate biologic events, such as DNA replication, transcription, chromosomal segregation, and recombination (Deweese and Osheroff, 2009; Nitiss, 2009). TOP2α/170, located on chromosome 17 (Tsai-Pflugfelder et al., 1988), is cell cycle dependent (Woessner et al., 1991) and is required for normal chromosomal dysjunction during mitosis (Pommier et al., 2016), whereas TOP2β/180 (Drake et al., 1987), located on chromosome 3 (Tan et al., 1992), is continuously expressed throughout the cell cycle (Woessner et al., 1991) and functions prominently in transcriptional control of developmental genes (Cowell et al., 2012; Bollimpelli et al., 2017; Austin et al., 2018, 2021; Madabhushi, 2018).

Both TOP2α/170 and TOP2β/180 are targets for clinically effective anticancer agents (Errington et al., 1999; Edwardson et al., 2015; Austin et al., 2018; Heinicke et al., 2018; Shanbhag and Ambinder, 2018; Economides et al., 2019), albeit with differential drug activity for one isoform over another (Austin et al., 2018). These agents, known as TOP2 poisons/interfacial inhibitors (Pommier and Marchand, 2011) (e.g., etoposide, mitoxantrone, amsacrine, doxorubicin, etc.) stabilize the enzyme-DNA covalent complexes formed by a phospho-tyrosyl bond between the TOP2 active site (tyrosine 805 in TOP2α/170; tyrosine 821 in TOP2β/180) and the 5′-phosphate of cleaved DNA, and prevent the religation of these scissile DNA strand breaks, thus causing the accumulation of DNA damage and subsequent cytotoxicity (Pommier et al., 2010). The clinical efficacy of TOP2 inhibitors is often limited by intrinsic and acquired chemoresistance, which predominantly include reduction of TOP2α/170 and/or TOP2β/180 expression levels or altered sub-cellular distribution (Harker et al., 1991, 1995; Ritke and Yalowich, 1993; Ritke et al., 1994; Herzog et al., 1998; Mirski et al., 2000; Burgess et al., 2008; Pilati et al., 2012; Ganapathi and Ganapathi, 2013; Hermanson et al., 2013; Capelôa et al., 2020). Our laboratory previously demonstrated that acquired resistance to etoposide in a human K562 leukemia cell line, K/VP.5, is associated with decreased TOP2α/170 and TOP2β/180 mRNA/protein (Ritke and Yalowich, 1993; Ritke et al., 1994; Kanagasabai et al., 2017; Carvajal-Moreno et al., 2023).

For TOP2α/170, reduced expression in K/VP.5 cells is due, in part, to a weak exon 19/intron 19 splice site and subsequent intronic polyadenylation (IPA), based on use of a cryptic polyadenylation sequence, resulting in translation of a C-terminal truncated isoform, TOP2α/90 (Kanagasabai et al., 2017; Elton et al., 2020, 2022; Hernandez et al., 2021), which functions as an additional determinant of resistance via heterodimerization with TOP2α/170 (Kanagasabai et al., 2018). TOP2β/180 also contains a weak splice site at the exon 19/intron 19 border but does not contain consensus polyadenylation signal sequences within intron 19. For TOP2β/180, reduced mRNA/protein in K/VP.5 cells is due to a microRNA-9 (miR-9)-mediated posttranscriptional mechanism and plays a role in drug resistance (Carvajal-Moreno et al., 2023).

To further establish the role of the TOP2β/180 in drug resistance to TOP2-targeted agents, CRISPR/Cas9 with homology directed repair (HDR) was used in K562 cells to introduce a premature stop codon at the end of exon 19 while at the same time enhancing the exon 19/intron 19 splice site in parental K562 cells. The aim was to produce a truncated TOP2β/180 isoform or a gene knockout for evaluation of gene-edited acquisition of drug resistance to TOP2-targed agents. Results demonstrated partial reduction of TOP2β/180 mRNA/protein in a gene-edited clone with one allele edited and complete elimination of TOP2β/180 mRNA/protein in a clone when all four TOP2β/180 alleles in K562 cells (Cioe et al., 1981; Zhou et al., 2019) were mutated. Evidence was consistent with nonsense-mediated decay (NMD) of TOP2β/180 mRNA transcribed in gene-edited clones accounting for disruption of TOP2β/180 protein expression. No effects on etoposide-induced DNA strand breaks and growth inhibitory effects in gene-edited clones were observed, while partial resistance was observed to both mitoxantrone and (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) (XK469) (Gao et al., 1999; Mensah-Osman et al., 2003; Alousi et al., 2007), a TOP2β/180 selective agent. Together, results indicated the importance of TOP2β/180 in drug sensitivity/resistance and revealed further the differential isoform activity of TOP2-targeted agents in K562 cells.

Material and Methods

Cell Culture.

Human leukemia K562, and gene-edited clonal cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. 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 (Ritke and Yalowich, 1993). All experiments described below were performed utilizing cells growing in log phase.

Generation of a TOP2β/180 Truncation/Knockout by CRISPR/Cas9/HDR Genome Editing in K562 Cells.

In an attempt to generate C-terminal truncated TOP2β isoform (i.e., a 90 kD TOP2β missing the active site tyrosine, Tyr 821 needed to produce TOP2β/180-DNA covalent cleavage complexes) or a TOP2β/180 knockdown/knockout, a stop codon (TAC) was created in the TOP2β/180 gene at the E19/I19 boundary (GGAGAA//GTAAGC→GGATAC//GTAAGC) by utilizing a sgRNA localized near the E19/I19 boundary, synthesized by Integrated DNA Technologies (IDT), Coralville, Iowa (Supplemental Table 1), ALT-R-S.p. HiFi Cas9, and a 180-nucleotide symmetric single-stranded oligonucleotide repair template (Alt-R HDR Donor Oligo to improve HDR efficiency; IDT; Supplemental Table 1) designated TOP2β/180 E19/I19 Stop Codon Mut/No PAM/+RsaI. This repair template equally spans TOP2β/180 E19/I19 and harbors the desired two nucleotide changes to generate a stop codon. Moreover, this repair template harbored three additional nucleotide changes. One nucleotide change (TGG→TCG) eliminated the protospacer-adjacent motif (PAM; see Fig. 1) to avoid the recutting of edited alleles upon the repeated transfections necessary to edit, at the E19/I19 boundary, all four TOP2β/180 alleles present in the K562 cell line (Cioe et al., 1981; Zhou et al., 2019). Two additional mutations were added to the repair template to introduce a restriction site for RsaI for screening purposes (see Fig. 1).

Fig. 1.

Fig. 1.

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Strategy to use CRISPR/Cas9/HDR to gene edit the TOP2β/180 E19/I19 boundary in K562 cells. (A) The TOP2β/180 E19I19 gene boundary sequence is shown. The wild-type E19/I19 sequences to be edited by CRISPR/Cas9 are bolded and underlined. The PAM site is indicated in purple. (B) The proposed sequence changes to generate a stop codon at the end of TOP2β/180 E19 are bolded, underlined, and denoted in blue (GAA→TAG). The proposed sequence change to eliminate the PAM site (TGG→TCG) is bolded, underlined and also denoted in blue. The proposed sequence change to introduce a RsaI restriction enzyme site PAM site (GCAT→GTAC) is bolded, underlined and denoted in green. Effective editing of the TOP2β/180 gene would allow RsaI digestion at the E19/I19 boundary. (C) To introduce the proposed changes described above (bolded, underlined and denoted in color) in the TOP2β/180 E19/I19 boundary, a ssODN HDR template (denoted TOP2β180 E19/I19 Stop Codon Mut/No PAM/+RsaI) was synthesized. The splice 5′ splice site (5′ SS) score is denoted for wild-type and mutated TOP2β based on a maximum entropy model (Yeo and Burge, 2004) indicating enhancement in the mutated TOP2β.

For genome editing of the TOP2β/180 E19/I19 boundary, TOP2β/180 E19/I19 sgRNA (0.5 µg) was incubated with ALT-R-S.p. HiFi Cas9 (20 µg) and TOP2β/180 E19/I19 Stop Codon Mut/No PAM (5 µM) HDR repair template for 20 minutes. This mixture was then transfected into K562 cells (2.25 × 106) in 100 µl) by electroporation as reported previously (Kanagasabai et al., 2017, 2018; Hernandez et al., 2021). To reduce non-homologous end joining (NHEJ), immediately after electroporation, cells were plated in media with 1 μM HDR Enhancer V2 (cat. no. 10007921, IDT). Forty-eight hours later, K562 cells were lysed for Cas9 targeting and repair efficiency using the genomic cleavage detection (GCD) assay described below. After verification of successful on-target genome editing, the remaining transfected K562 cells were plated using limiting dilution cloning in ten 96-well plates (0.8 cells per well). Aliquots (∼25–50,000 cells from single-cell clones were subsequently lysed with GCD buffer (see below) ∼2 weeks after plating. Supernatants were assayed for HDR by genomic quantitative polymerase chain reaction (qPCR) utilizing a validated custom wild-type TOP2β/180 E19/I19 boundary qPCR hybridization probe (5′-TCATCATGGAGAAGTAAGCATTAAG-3′) (ThermoFisher Scientific; Assay ID: APDJZJP) and the mutant custom TOP2β/180 E19/I19 boundary probe (5′-TCATCATGGATAGGTAAGTACTAAC-3′; Assay ID APH6GYF) specific for the edited TOP2β/180 E19I19 boundary sequence, to identify colonies with TOP2β/180 edited alleles. After the first round of transfection, multiple colonies with one TOP2β/180 E19/I19 Stop Codon Mut/No PAM edited allele were identified by qPCR and confirmed by Sanger sequencing. A single TOP2β/180 allele edited clone, designated K562/Stop Codon-Edit-1 (K562/SC-Edit-1), was subjected to an additional round of transfection with TOP2β/180 sgRNA, ALT-R-S.p. HiFi Cas9, the TOP2β/180 repair template, and HDR Enhancer V2 followed by limiting dilution cloning. Genomic qPCR utilizing both the wild-type and mutant TOP2β/180 E19/I19 boundary probes was used to identify a K562 clone with four TOP2β/180 alleles gene-edited, now designated K562/SC-Edit-4.

Genomic Cleavage Detection.

The TOP2β/180 gene Exon 19/Intron 19 (E19/I19) boundary sequence was targeted with a sgRNA oligonucleotide as described above. The TOP2β/180 E19/I19 sgRNA was chemically modified to increase editing efficiency and to prevent nuclease degradation.

The E19/I19 sgRNA (0.5 µg) was incubated with ALT-R-S.p. HiFi Cas9 (20 µg, cat. no. 1081061, IDT) for 20 minutes at room temperature to form Cas9 protein/sgRNA ribonucleoprotein complexes. These complexes were subsequently transfected into K562 cells by electroporation technology (Nucleofector Kit V; Lonza, Basel, Switzerland) according to the manufacturer’s instructions and as reported previously (Kanagasabai et al., 2017, 2018; Hernandez et al., 2021).

To determine if the Cas9 protein/sgRNA complexes efficiently created on-target DSBs within the TOP2β/180 E19/I19 boundary sequence, transfected K562 cells (1 × 106) were lysed forty-eight hours after transfection using cell lysis GCD buffer/Proteinase K (GeneArt Genomic Cleavage Detection Kit; cat. no. A24372; ThermoFisher, Waltham, MA). Genomic DNA (1 µl of lysate) at the TOP2β/180 locus from E19 to exon 20 (E20) was then PCR amplified (25 µl reaction volume) using the following primers: TOP2β/180 E19 For (5′-TTTAAACCTGGCCAGCGGAAG-3′) and TOP2β/180 E20 Rev (5′-GTGAAAATATAACGAGGGCTTGCAGC-3′). The PCR amplicons were subsequently denatured, reannealed, and incubated with T7 endonuclease I (i.e., structure-selective enzyme that recognizes and cleaves mismatched DNA) to detect insertions/deletions created by NHEJ. The digested and non-digested PCR products were fractionated by electrophoresis on a 2% agarose gel, and images were captured under ultraviolet light after staining with 0.5 mg/ml ethidium bromide using the ChemiDoc XRS1 imaging system and analyzed by ImageLab software (Bio-Rad Laboratories). To calculate the cleavage efficiency of TOP2β/180 gRNA/Cas9, the following equation was used: cleavage efficiency = {1 – [(1 – fraction cleaved)1/2]} x 100, where fraction cleaved = (sum of cleaved band intensities)/(sum of the cleaved and parental band intensities).

qPCR Assay.

Total RNA was isolated from K562, CRISPR/Cas9-edited K562/SC-Edit-1, and K562/SC-Edit-4 cells using the RNA Easy Plus Mini Kit (cat. no. 74134; Qiagen, Germantown, MD). To ensure complete removal of contaminating DNA, an on-column digestion of DNA with RNAse-free DNase (cat. no. 79254; Qiagen) was integrated during RNA purification. RNA (1 µg) was reverse transcribed using random hexamers and MultiScribe Reverse Transcriptase (High-Capacity cDNA Reverse Transcription Kit, cat. no. 4368814; ThermoFisher Scientific) as previously described (Kanagasabai et al., 2017, 2018; Hernandez et al., 2021). qPCR experiments (total reaction volume 10 µl) were performed using Taqman Gene Expression hydrolysis probes (ThermoFisher Scientific) (Kanagasabai et al., 2017, 2018; Hernandez et al., 2021) and as shown in Supplemental Table 1. Relative mRNA expression levels of TOP2β/180 and TOP2α/170 in each clonal cell line were normalized to TATA-binding protein (TaqMan assay Hs99999910_m1; ThermoFisher Scientific) expression using the 2-ΔΔCt method (Schmittgen and Livak, 2008).

In separate experiments, total RNA was isolated from CRISPR/Cas9 edited K562/SC-Edit-1 and K562/SC-Edit-4 treated with either DMSO or 25 µM non-sense mediated decay inhibitor NMDI-14 (cat. no. 307519-88-6; Sigma-Aldrich) for 6 hours, followed by reverse transcription qPCR utilizing a TaqMan hydrolysis probe specific for the TOP2β/180 mRNA E19/I19 boundary (Supplemental Table 1).

RsaI Restriction Enzyme Analysis of CRISPR/Cas9-Edited K562 Cells.

K562, K/SC-Edit-1, and K/SC-Edit-4 cells were lysed using cell GCD lysis buffer/Proteinase K as described above. Genomic DNA (1 µl of lysate) was PCR amplified (50 µl reaction volume) at the TOP2β/180 E19/I19/E20 locus using the following primers: TOP2β/180 E19 For (5′-TTTAAACCTGGCCAGCGGAAG-3′) and TOP2β/180 E20 Rev (5′-GTGAAAATATAACGAGGGCTTGCAGC-3′). Ten microliters of the PCR reaction was then digested by RsaI (cat. no. R0525S, New England Biolabs) according to the manufacturer’s instructions. The digested and non-digested PCR products were fractionated by electrophoresis on a 2% agarose gel, and images were captured as described above.

Immunoassays.

Cellular extracts from K562, K/VP.5, and CRISPR/Cas9 edited K562/SC-Edit-1 and K562/SC-Edit-4 cells were subjected to western blot analysis as previously described (Kanagasabai et al., 2017, 2018; Hernandez et al., 2021). Protein (16 µg) from cell extracts were loaded into each well, and an equal volume of Precision Plus Protein Color Standards was also run for molecular mass reference (cat. no. 1610374; Bio-Rad Laboratories, Hercules, CA). Membranes were incubated overnight at 4°C with one of the following primary antibodies: a mouse monoclonal antibody raised against amino acids 1341–1626 of TOP2β/180 of human origin (H-8) (cat. no. sc-25330 Santa Cruz Biotechnology, Santa Cruz, CA; used at 1:350 dilution), a rabbit polyclonal antibody raised against the human TOP2α/170 amino acids 14–27 (cat. no. C10345 Assay Biotechnology, Sunnyvale, CA, used at 1:1000 dilution), a mouse monoclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (cat. no. sc-47724; Santa Cruz Biotechnology; used at 1:5000 dilution), a mouse γH2AX (phosphorylated Ser-139 residue of the H2A histone family member X) monoclonal antibody (cat. no. sc-25330; Santa Cruz Biotechnology; used at 1:500 dilution). The membranes were subsequently incubated at room temperature for ∼3 hours with a donkey anti-rabbit or an anti-mouse secondary antibody (Jackson Immuno Research, West Grove, PA; used at 1:5000 dilution). Finally, antibody-labeled TOP2α/170, TOP2β/180, γH2AX and GAPDH were detected using defined dilutions of the Clarity Max chemiluminescence kit (Bio-Rad Laboratories Hercules, CA). All immunoassay images were acquired with the ChemiDoc XRS+ imaging system and analyzed with ImageLab software (Bio-Rad Laboratories).

Growth Inhibitory Assays.

Growth inhibitory assays were performed as previously described (Kanagasabai et al., 2017, 2018; Hernandez et al., 2021). Log-phase K562 cells, and gene-edited K562 clonal cells were adjusted to 1–1.5 × 105 cells per mL and incubated for 48 hours with DMSO as control solvent (final concentration 0.5%), with 0.01–15 µM etoposide with 1–250 µM of XK469, with 0.01-25 µM mAMSA, and with 0.02–200 nM mitoxantrone. Cells numbers were counted on a model Z1 Coulter counter (Beckman Coulter, Danvers, MA). Percentage of growth inhibition for each concentration of drug was determined based on comparison with DMSO control growth. The concentration–response (inhibition) curves to a four-parameter logistic equation were generated using Sigmaplot 14.5 (Systat Software, Inc., San Jose, CA) with the 50% inhibition concentration calculated and reported (Table 1).

TABLE 1.

Growth inhibitory effects of anticancer drugs in K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells

Anticancer agent K562 Cells (IC50)a SC-Edit-1 Cells (IC50) SC-Edit-4 Cells (IC50) Relative Resistanceb (SC-Edit-1/K562) Relative Resistance (SC-Edit-4/K562)
μM μM μM
XK469 7.47 ± 1.45 (10)c 8.10 ± 2.58 (3) 17.40 ± 5.43 (6) 1.08 2.33
Mitoxantrone 0.0030 ± 0.0005 (6) 0.0028 ± 0.0099 (3) 0.0066 ± 0.0021 (3) 0.93 2.20
Etoposide 0.072 ± 0.022 (12) 0.071 ± 0.011 (3) 0.071 ± 0.018 (7) 0.99 0.99
mAMSA 0.047 ± 0.021 (5) N.D. 0.049 ± 0.012 (5) N.D. 1.04
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aFifty percent inhibitory concentration (IC50) in a 48-hour growth inhibition assay.

bIC50 of K562/SC-Edit-1 or K562/SC-Edit-4 cells divided by that of the parental K562 cell line.

cMean ± S.D.; numbers in parentheses, number of independent experiments performed on different days.

DNA Damage (Comet) Assays.

Alkaline (pH 13, detecting primarily single-strand DNA breaks) and neutral (pH 8, detecting double-strand DNA breaks) single-cell gel electrophoresis (Comet) assays were performed according to the manufacturer’s protocol (CometAssay Kit, cat. no. 4250-050-K; Trevigen, Gaithersburg, MD) and as previously described by our laboratory (Kanagasabai et al., 2017, 2018; Hernandez et al., 2021). Briefly, K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells were washed and resuspended in buffer (25-50 nM HEPES, 10 mM glucose, 1 mM MgCl2, 5 mM KCl, 130 mM NaCl, 5 mM monosodium phosphate, pH 7.4). Cells were subsequently incubated with 250 µM XK469, etoposide or DMSO (solvent control) for 1 hour at 37°C. The treated cells were washed with ice-cold buffer and resuspended to 0.28 × 106 cells/ml and then further diluted in low melt agarose. After alkaline (pH 13) or neutral (pH 9) electrophoresis (of ∼2000 cells) and subsequent staining with a fluorescence DNA intercalating dye, SYBR Gold, the migrating fragments (comet tail) from the nucleoid (comet head) were visualized and the images captured by fluorescence microscopy. The Olive tail moment (Olive, 2002) was quantified by the ImageJ processing program with the open-source software tool Open Comet (Gyori et al., 2014). Olive tail moments from more than 100 cells per sample condition were determined.

Data Analysis.

Statistical analysis was performed using Sigma-Plot 14.5. All data are expressed as the mean ± standard deviation (S.D.). Unless noted otherwise, groupwise differences were analyzed using a two-tailed paired Student’s t test with no adjustment for multiple comparisons. A P value less than 0.05 was considered statistically significant.

Results

CRISPR/Cas9/HDR: Strategy to Gene-edit TOP2β/180 in Human Leukemia K562 Cells.

Our laboratory previously demonstrated reduced levels of TOP2β/180 in an acquired etoposide-resistant K562 clonal cell line, K/VP.5 (Kanagasabai et al., 2017), due to microRNA-9-3p/5p dysregulation (Carvajal-Moreno et al., 2023). In the present study, we undertook a TOP2β gene-editing project by CRISPR/Cas9/HDR transfections (Jinek et al., 2012; Mali et al., 2013; Liang et al., 2017) in drug sensitive parental K562 cells to further establish the role of this topoisomerase paralog in drug resistance. The strategy is outlined in Fig. 1 to introduce a stop codon at the E19/I19 boundary (AGAA//GTAA→ ATAG//GTAA) along with an enhanced 5′ splice site for generation of a truncated protein or to completely ablate TOP2β production (Figs. 1B and 2A). To more efficiently screen gene edited clones, substitutions (Fig. 1B, denoted in green) were designed in the I19 sequence (GCAT→GTAC) to create a RsaI restriction enzyme site and to better allow for custom qPCR probe discrimination between wild-type K562 cells and CRISPR/Cas9/HDR edited K562 cells. In addition, an I19 nucleotide substitution (TGG→TCG) was designed to eliminate a PAM site (Fig. 1, A and B) preventing Cas9 recutting of gene-edited alleles upon re-transfection required to gene-edit all four TOP2β/180 alleles in K562 cells (Cioe et al., 1981; Zhou et al., 2019). A symmetric 180-nucleotide ssODN HDR template was synthesized to contain the indicated nucleotide changes (Fig. 1C; denoted as TOP2β/180 E19/I19 Stop Codon Mut/No PAM) with 90 nucleotide homology arms to both TOP2β/180 E19 and I19. The HDR template was used in all CRISPR/Cas9/HDR transfection experiments described below.

Fig. 2.

Fig. 2.

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Effects of TOP2β/180 sgRNA on Cas9 cleavage at the TOP2β/180 E19/I19 boundary sequence. (A) Sequence of the TOP2β/180 E19I19 boundary is shown. The PAM site and the corresponding sgRNA are shown in purple. The purple arrow denotes where Cas9 will generate a DSB. (B) Schematic representation of the E19 through I19 region of the TOP2β/180 gene. The blue arrow denotes the site where TOP2β/180 gRNA directed Cas9 cleavage and NHEJ generation of insertions/deletions can occur. Gray arrows denote the forward (For) and reverse (Rev) primers used for the GCD assay. (C) Ethidium bromide-stained agarose gel fractionated GCD PCR amplicons before and after treatment with T7 endonuclease I. The parental and T7 endonuclease I cleaved daughter PCR amplicons are indicated, and their respective sizes are denoted.

Initially, to determine the Cas9 cleavage efficiency at the indicated PAM/sgRNA site, the sgRNA was complexed with Cas9 and transfected into K562 cells (Fig. 2A). Forty-eight hours after transfection, cells were assayed for insertions/deletions generated by Cas9 cleavage and NHEJ (Qi et al., 2013) by PCR using an E19 forward primer and a reverse primer on E20 to amplify the CRISPR/Cas9 target region (Fig. 2B). PCR amplicons were denatured and reannealed with mismatches cleaved by T7 endonuclease I. Results demonstrate that sgRNA efficiently guided Cas9 to the target site with a calculated Cas9 cleavage efficiency of 15.2% (Fig. 2C).

CRISPR/Cas9/HDR: qPCR Selection and Sequence Analysis of Edited TOP2β/180 E19/I19 Clonal Cell Lines.

K562 cells were next transfected with TOP2β/180 sgRNA (Fig. 2A), Cas9 protein, and the HDR template (Fig. 1C), followed by limiting dilution cloning in 96-well plates (0.8 cells per well). After two weeks, cells from single colony wells were lysed and screened by genomic DNA qPCR (Hernandez et al., 2021). To distinguish between the wild-type TOP2β/180 E19/I19 boundary and the CRISPR/Cas9/HDR edited TOP2β/180 E19/I19 boundary, a custom wild-type E19/I19 qPCR hybridization probe (5′-CATCATGGAGAAGTAAGCATTAAGATTGGATT-3′) and a custom mutant TOP2β/180 E19/I19 qPCR hybridization probe containing the edited sequences (Fig. 1C) (5′-CATCATGGATAGGTAAGTACTAAGATTCGATT-3′) were used. Non-transfected K562 cells demonstrated a positive signal using the wild-type TOP2β/180 E19/I19 probe (5′-TCATCATGGAGAAGTAAGCATTAAG-3′, Fig. 3AI, black line) with no signal detected from the mutant (i.e., edited) TOP2β/180 E19/I19 probe (Fig. 3AI, red line). Sanger sequencing verified the wild type TOP2β/180 genomic sequence (Fig. 3BI).

Fig. 3.

Fig. 3.

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qPCR selection and sequence analysis of CRISPR/Cas9/HDR edited TOP2β/180 E19/I19 Stop Codon Mut/No PAM/+RsaI clonal cell lines. (A) Amplification plots of qPCR reactions from K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells (labeled I-III) using wild-type TOP2β/180 E19/I19 and edited-specific mut/E19/I19 boundary qPCR probes. (B) Electropherograms of the genomic sequence of the TOP2β/180 E19/I19 gene boundary in K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells (labeled I–III).

In contrast, for the CRISPR/Cas9/HDR transfected K562 cells lysates (∼100 clonal cell colonies screened), several clones were identified where the qPCR signal for the mutant TOP2β/180 E19/I19 probe was evident (Fig. 3AII; red line for one clone). Sanger sequence analysis indicated that both wild-type and edited genomic sequences were present in the TOP2β/180 E19/I19 boundary at all five of the edited sites (i.e., Stop Codon Mut/No PAM/+RsaI) (Fig. 3BII). Since K562 cells contain four TOP2β/180 alleles (Cioe et al., 1981; Zhou et al., 2019), and the wild-type sequence predominates in the electropherogram of Fig. 3BII, results suggested that only one of the four TOP2β/180 E19/I19 alleles was edited, whereas three alleles remained unchanged. This clone was denoted as K562/SC-Edit-1.

An additional transfection was performed in the K562/SC-Edit-1 clonal cell line with TOP2β/180 sgRNA, Cas9 protein, and the TOP2β/180 HDR template. Forty-eight hours post-transfection, K562/SC-Edit-1 cells were seeded at 0.8 cells/well in 96-well plates. Two weeks later, cells harvested from single clonal colonies were lysed and screened by genomic DNA qPCR and Sanger sequencing. From this second transfection (∼30 colonies screened), a clonal cell line was identified where only the edited TOP2β/180 E19/I19 boundary probe showed a positive signal (Fig. 3AIII, red line) strongly suggesting that all four TOP2β/180 E19/I19 boundary alleles were edited. Sanger sequence analysis confirmed the qPCR results (Fig. 3BIII) that the five desired base pair changes (Fig. 1C) were present in all four TOP2β/180 alleles. This clone was designated K562/SC-Edit-4.

RsaI Analysis Validation of CRISPR/Cas9/HDR Editing of K562/SC-Edit-1 and K562/SC-Edit-4 Cells.

Gene editing two nucleotides within the I19 sequence (GCAT→GTAC) (Fig. 1) introduced a restriction site for RsaI endonuclease (GT↓AC). Therefore, RsaI-mediated cleavage was used as an independent assay to confirm TOP2β/180 gene-editing. Genomic DNA from K562, gene-edited K562/SC-Edit-1, and K562/SC-Edit-4 cell lysates were used as templates for PCR reactions with a forward primer annealing to TOP2β/180 E19 and a reverse primer annealing to TOP2β/180 E20 (Fig. 4A). According to the location of the forward and reverse primers, the expected size of the parental PCR amplicon was 496 bp. After RsaI digestion, the expected sizes of amplicons would be 370 bp and 126 bp (Figs. 4, A and B) in gene-edited lines. As anticipated, RsaI did not result in cleavage in the non-edited K562 cell amplicon (Fig. 4B). Partial RsaI digestion of the 496 bp band was evident in the K562/SC-Edit-1 amplicon with complete digestion of the 496-bp amplicon observed and a concomitant increase in the intensity of cleaved bands in the K562/SC-Edit-4 cell amplicon (Fig. 4B). These results, together with qPCR and sequencing data (Fig. 3), validated that K562/SC-Edit-1 cells contained one edited TOP2β/180 allele, while all four TOP2β/180 alleles were edited in K562/SC-Edit-4 cells.

Fig. 4.

Fig. 4.

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RsaI validation of CRISPR/Cas9 edited TOP2β/180 E19/I19 Stop Codon Mut/No PAM/+RsaI clonal cell lines. (A) Schematic representation of the E19 through E20 portion of the TOP2β/180 gene. Blue arrow denotes site where RsaI generates double strand breaks of the CRISPR-edited PCR amplicons. Gray arrows denote the For and Rev primers used for the identification of CRISPR editing of the TOP2β/180 E19/I19 Stop Codon Mut/No PAM/+RsaI utilizing RsaI endonuclease. (B) Ethidium bromide-stained agarose gel of fractionated RsaI-treated PCR amplicons from K562, K562/SC-Edit-1 and K562/SC-Edit-4 cells DNA. The black denotes the 500 bp marker. The expected sizes of parental and daughter PCR amplicons are shown by red arrows.

TOP2β/180 Expression in Gene-Edited Clones.

Additional qPCR experiments were used to evaluate the impact of introducing a premature stop codon in TOP2β/180 exon 19. Using a wild-type E19/I19 qPCR probe, TOP2β/180 mRNA expression levels were statistically significantly decreased in K562/SC-Edit-1 cells compared with parental K562 cells (P = 0.008) (Fig. 5A). Consistent with gene-editing of all TOP2β/180 alleles, no TOP2β/180 mRNA was detected in K562/SC-Edit-4 cells (Fig. 5A). Separate experiments were conducted to evaluate TOP2β/180 and TOP2α/170 mRNA levels in K562 cells at exponential phase growth indicating that TOP2β/180 mRNA expression was only 20% that of TOP2α/170 (Fig. 5B). In addition, previous results indicated that TOP2β/180 protein was barely detectable in K562 cells compared with TOP2α/170 (Ritke et al., 1994).

Fig. 5.

Fig. 5.

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TOP2β/180 mRNA and protein levels in K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells. (A) qPCR analyses were performed from RNA samples isolated from K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells using a TaqMan hydrolysis probe specific for the TOP2β/180 E19/E20 boundary. Results/data points shown are the mean ± S.D. from five RNA/cDNA isolations/determinations on separate days. All data points represent biologic replicates from the separate paired experiments performed. Averaging results from the paired experiments, there was a statistically significant reduction in TOP2β/180 mRNA expression in K562/SC-Edit-1 compared with K562 cells; P = 0.008. No qPCR signal was detected for TOP2β/180 mRNA for K562/SC-Edit-4. (B) qPCR analyses were performed from RNA samples isolated from K562 cells using TaqMan hydrolysis probes specific for the TOP2β/180 and TOP2α/170 E19/E20 boundary. Results/data points shown are the mean ± S.D. from nine RNA/cDNA isolations/determinations on separate days. All data points represent biologic replicates from the separate paired experiments performed. Averaging results from the paired experiments, there was a statistically significant reduction in TOP2β/180 mRNA expression compared with TOP2α/170 expression in K562 cells (P = <0.001). (C) Representative immunoassay using K562, K/VP.5, K562/SC-Edit-1, and K562/SC-Edit-4 cellular lysates. Blots were probed with antibodies specific for TOP2β/180 and for GAPDH. (D) Expression of TOP2β/180 protein levels in K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells. Results/data points shown are the mean ± S.D. from six experiments for K562 versus K562/SC-Edit-1 performed on separate days and four experiments for K562/SC-Edit-4 cells where no TOP2β/180 protein was detected. Averaging results from the six paired experiments, there was a statistically significant reduction of TOP2β/180 in K562/SC-Edit-1 compared with parental K562 cells; P = 0.046, considering the GAPDH loading control. (E) qPCR analyses were performed from RNA samples isolated from K562/SC-Edit-1 and K562/SC-Edit-4 cells treated with DMSO solvent or nonsense-mediate decay inhibitor NMDI-14 (25 µM) for 6 hours using a TaqMan hydrolysis probe specific for the TOP2β/180 E19/I19 boundary. Results shown are the mean ± S.D. from two to three separate experiments each with duplicate production of cDNAs for analysis. There was a statistically significant increase in TOP2β/180 mRNA expression in K562/SC-Edit-1 (P = 0.039) and K562/SC-Edit-4 cells (P = 0.033) treated with NMDI-14 compared with cells treated with DMSO. *P < 0.05; **P < 0.01, ***P < 0.001.

To further confirm that the CRISPR/Cas9/HDR strategy effectively disrupted production of full length TOP2β/180 protein, total cell lysates were taken from K562, K/VP.5, K562/SC-Edit-1, and K562/SC-Edit-4 cells for immunoassays utilizing a specific TOP2β/180 antibody. Fig. 5C indicated reduced TOP2β/180 protein in drug resistant K/VP.5 cells as demonstrated previously (Kanagasabai et al., 2017; Carvajal-Moreno et al., 2023). In addition, TOP2β/180 protein was reduced in K562/SC-Edit-1 cells and completely absent in K562/SC-Edit-4 cells (Fig. 5C). Averaging results from six independent biologic immunoblotting experiments run on separate days, there was a statistically significant reduction of TOP2β/180 protein in K562/SC-Edit-1 cells to 55% the level expressed in parental K562 cells (P = 0.046) with no expression of TOP2β/180 in K562/SC-Edit-4 cells (Fig. 5D). Together, these results indicated successful gene-editing and disrupted expression of TOP2β/180 mRNA/protein.

K562/SC-Edit-1 and K562/SC-Edit-4 cells were incubated with DMSO solvent control or an inhibitor of nonsense-mediated decay (NMDI-14; 25 µM) followed by qPCR evaluation of TOP2β mRNA using a TaqMan hydrolysis probe specific for the TOP2β/180 mRNA E19/I19 boundary (Supplemental Table 1). Averaging results from two to three separate experiments each with duplicate production of cDNAs, there was a statistically significant increase of TOP2β/180 mRNA in K562/SC-Edit-1 cells (P = 0.039) and in K562/SC-Edit-4 cells (P = 0.033) treated with NMDI-14 (Fig. 5E). These results strongly suggested that the premature stop codon introduced in gene-edited cells resulted in a nonsense-mediated decay process responsible for allele specific loss of TOP2β/180 mRNA/protein.

TOP2β-Targeted Drug Activity in Gene-Edited Cell Lines.

We previously demonstrated that XK469 activity in parental K562 cells correlated with TOP2β/180 protein levels (Carvajal-Moreno et al., 2023) in accord with previous work indicating XK469 specificity at this target (Gao et al., 1999). K562 and K562/SC-Edit-1 cells were incubated with DMSO solvent control or XK469 (100, 250 µM) for 1 hour followed by immunoblot/band depletion assays (Kaufmann and Svingen, 1999) (Fig. 6, A and B). XK469 induced concentration dependent “band depletion” of TOP2β/180 in both cell lines (Fig. 6A, top) consistent with formation of high molecular weight TOP2β/180-DNA covalent complexes that prevent these complexes from entering gels (Kaufmann and Svingen, 1999). In contrast, XK469 incubation did not “band deplete” TOP2α/170 in K562 cells or in K562/SC-Edit-1 cells (Fig. 6a, bottom). Averaging results from four experiments run on separate days, there was a statistically significant depletion of TOP2β/180 protein in K562 cells to 43.2% (P < 0.001) and 42.4% (P = 0.016) of DMSO control at 100 µM and 250 µM XK469, respectively (Fig. 6B). In K562/SC-Edit-1 cells, whose TOP2β/180 protein/mRNA levels were reduced (Fig. 5), there was a statistically significant depletion of TOP2β/180 to 29.1% (P = 0.003) and 12.4% (P = 0.004) of DMS0 control at 100µM and 250µM XK469, respectively (Fig. 6B). In contrast, there was no statistically significant decrease in TOP2α/170 at 100 µM or 250 µM XK469 in either K562 or K562/SC-Edit-1 cells (Fig. 6B). Likewise, XK469 did not “band deplete” TOP2α/170 in K562/SC-Edit-4 cells (Supplemental Fig. 1).

Fig. 6.

Fig. 6.

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Drug Activity in TOP2β/180 Gene-Edited Cell lines. (A) Representative immunoassay (From four experiments performed on separate days) using cellular lysates from K562 and K562/SC-Edit-1 cells treated with DMSO or XK469 (100 and 250µM) for one hour. Blots were probed with antibodies specific for TOP2α/170 and the TOP2β/180 or for GAPDH. (B) TOP2β/180 and TOP2α/170 protein levels in K562 and K562/SC-Edit-1 cells treated with DMSO or XK469 (100 and 250µM) for 1 hour. For TOP2β/180 protein, results shown are the mean ± S.D. from four experiments performed on separate days, in K562 cells comparing DMSO control versus 100 µM XK469 (P < 0.001) and 250 µM XK469 (P = 0.016), respectively; in K562/SC-Edit-1 cells comparing DMSO control versus 100 µM XK469 (P = 0.003) and 250 µM XK469 (P = 0.004), respectively; at 250 µM XK469 comparing K562/SC-Edit-1 versus K562 cells (#, P = 0.033 using a two-tailed Student’s t test). For TOP2α/170 protein, results shown are the mean ± S.D. from four experiments performed on separate days, in K562 cells comparing DMSO control versus 100 µM XK469 (P = 0.398) and 250 µM XK469 (P = 0.201), respectively; in K562/SC-Edit-1 cells comparing DMSO control versus 100 µM XK469 (P = 0.726) and 250µM XK469 (P = 0.949), respectively. All data points represent biologic replicates from the separate experiments. (C) Representative DNA damage experiment (alkaline Comet assay using K562, K562/SC-Edit-1 and K562/SC-Edit-4 cells treated with DMSO, XK469 (250µM), or etoposide (5µM) for one hour; P < 0.001; comparing DMSO versus XK469 (250 µM) for each cell line. (D) Compiled DNA damage experiments (alkaline Comet assays). Results shown are the mean ± S.D. for three experiments performed on separate days, comparing effects of XK469 in parental K562 versus K562/SC-Edit-1 (P = 0.005) and K562/SC-edit-4 cells (P = 0.016), respectively, and comparing effects of etoposide in parental K562 versus K562/SC-edit-1 (P = 0.101) and K562/SC-Edit-4 cells (P = 0.253), respectively. (E) Neutral (pH 9) Comet assays (assessing DNA double-strand breaks) using K562 and K562/SC-Edit-4 cells treated with DMSO, XK469 (250 µM), or etoposide (5 µM) for one hour. Results shown are the mean ± S.D. for five experiments performed on separate days, comparing effects of XK469 in parental K562 versus K562/SC-edit-4 cells (P = 0.046), and comparing effects of etoposide in parental K562 versus K562/SC-Edit-4 cells (P = 0.405). For all experimental conditions in each Comet assay experiment, more than 100 cells were evaluated by OpenComet software (Gyori et al., 2014). (F) Representative immunoblots from whole cell lysates of K562 and K562/SC-Edit-4 cells treated with DMSO, etoposide (50 µM), mitoxantrone (10µM), NK314 (25 µM), mAMSA (50 µM), or XK469 (250 µM) for one hour. Blots were probed with an antibodies specific for γH2AX and GAPDH antibodies. The Clarity max chemiluminescence reagent was use at 1:5 dilution for the blot on the top and at 1:2 dilution for the blot on the bottom. Imaging was for two seconds in both blots. (G) Percent Control γH2AX (γH2AX/GAPDH) from three to nine experiments, each performed on separate days (mean ± S.D.), comparing effects of etoposide (P = 0.076), mitoxantrone (P < 0.001), NK314 (P = 0.802), mAMSA (P = 0.376) in parental K562 versus K562/SC-Edit-4 cells. There was no evidence of XK469-induced γH2AX in any of the replicate experiments. Statistical analysis was performed using a two-tailed paired Student’s t test, as documented in Materials and Methods. *#P < 0.05; **P < 0.01, ***P < 0.001.

Next, alkaline (pH 13) single cell gel electrophoresis (Comet) assays (Olive, 2002), which induce primarily DNA single strand breaks, were performed in K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells assessing the effects of XK469 and etoposide (Figs. 6, C and D). In the representative experiment shown in Fig. 6A, XK469 (250 µM)-induced DNA strand breaks were reduced in both K562/SC-Edit-1 and K562/SC-Edit-4 cells compared with K562 cells. In replicate experiments, compared with parental K562 cells, there was a statistically significant reduction in XK469-induced DNA damage to 31.6% (P = 0.005) and 21.4% (P = 0.016) in K562/SC-Edit-1 and K562/SC-Edit-4 cells, respectively (Fig. 6D). In parallel, etoposide-induced DNA damage was evaluated in K562 cells and the gene-edited clones. No statistically significant reduction in etoposide-induced strand breaks were demonstrated associated with loss of TOP2β/180 protein in the gene-edited cells (Fig. 6D).

In addition, neutral (pH 9) Comet assays for DNA DSBs were performed in K562 and K562/SC-Edit-4 cells to evaluate the effects of XK469 and etoposide (Fig. 6E). Compared with parental K562 cells, there was a statistically significant reduction in XK469-induced DNA damage to 47.2% (P = 0.046) in K562/SC-Edit-4 cells (Fig. 6E). In contrast, there was no statistically significant reduction in etoposide-induced strand breaks in K562/SC-Edit-4 compared with K562 cells. (Fig. 6D).

Drug-induced DSBs were also evaluated by γH2AX formation (Ismail et al., 2007). K562 and K562/SC-Edit-4 cells were incubated for 1 hour with etoposide (50 µM), mitoxantrone (10 µM), NK314 (25 µM), XK469 (250 µM), mAMSA (50 µM), or DMSO solvent control followed by lysis and immunoblotting with anti-γH2AX antibody. In the representative experiments shown in Fig. 6F (top and bottom), etoposide and mAMSA induced γH2AX formation to the same extent in parental K562 and K562/SC-Edit-4 cells. Similarly, NK314, a selective TOP2α-targeted agent (Toyoda et al., 2008), induced similar levels of γH2AX in both cell lines. In contrast, mitoxantrone-induced γH2AX expression was attenuated in K562/SC-Edit-4 compared with parental K562 cells. There was no induction of γH2AX in either cell line treated with XK469 (Fig. 6F, top). In replicate experiments, there was a statistically significant decrease only in mitoxantrone-induced γH2AX expression in K562/SC-Edit-4 compared with K562 cells (P < 0.001) (Fig. 6G).

Forty-eight hour growth inhibition assays were then undertaken in K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells treated continuously with XK469, mitoxantrone, etoposide, or mAMSA. In K562/SC-Edit-1 cells, with 50% reduction of TOP2β/180 protein (Fig. 5D), no resistance was noted to any of the agents tested compared with K562 cells (Table 1). However, compared with K562 cells, K562/SC-Edit-4 cells were 2.3-fold resistant to XK469 and 2.2-fold resistant to mitoxantrone (Table 1). No resistance to etoposide or mAMSA was observed in these K562/SC-Edit-4 cells, which lack TOP2β/180. Doubling times were similar for K562, K562/SC-Edit-1, and K562/SC-Edit-4 cells (Table 2), indicating that disruption of TOP2β/180 expression in these edited-clonal cell lines did not alter their growth characteristics. In Fig. 7, there was a shift to the right of the XK469 concentration-response curve for K562/SC-Edit-4 cells compared with K562 cells (2.3-fold resistant) generated from scattergram analysis of replicate experiments. The growth inhibitory effects of XK469 in K562/SC-Edit-4 cells indicated off-target effects of this presumed TOP2β/180-specific agent since the absence of TOP2β/180 in K562/SC-Edit-4 cells would be expected to yield complete resistance to XK469. Of note, acquired etoposide-resistant K/VP.5 cells, which contain reduced levels of both TOP2α/170 and TOP2β/180 (Ritke and Yalowich, 1993; Ritke et al., 1994; Kanagasabai et al., 2017; Carvajal-Moreno et al., 2023) were 7.4-fold resistant to XK469.

TABLE 2.

Cell line growth characteristics

Cell Line Doubling Time (hours)a
K562 16.7 ± 0.4b
K562/SC-Edit-1 17.2 ± 1.4
K562/SC-Edit-4 16.9 ± 2.3
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aCalculated from log-linear regression plots over 3–4 days of growth.

bMean ± S.D.; average of three independent experiments performed on different days.

Fig. 7.

Fig. 7.

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Growth inhibitory effects of XK469 in K562, K/VP.5, and K/SC-Edit-4 cells. Log-phase cells were incubated with increasing concentrations of XK469 for 48 hours; following which, cells were counted. The extent of growth beyond the starting concentration in drug-treated versus controls was expressed as % Inhibition growth. Results are shown as a scattergram from independent experiments (N = 10, K562 cells; N = 6 K562/SC-Edit-4 cells; N = 6; K/VP.5 cells), performed on separate days.

Discussion

Our laboratory established that acquired resistance to etoposide in a clonal K562 subline, K/VP.5, was associated with a reduction of both TOP2α/170 and TOP2β/180 mRNA/protein (Kanagasabai et al., 2017, 2018; Elton et al., 2020, 2022; Hernandez et al., 2021). Reduced TOP2α/170 expression in K/VP.5 cells results, in part, from a weak splice site at the TOP2α gene exon 19/intron 19 boundary, the presence of polyadenylation sites harbored within intron 19 and subsequent IPA. Intron 19 IPA led to both a reduction in full-length TOP2α/170 levels and the increased production of a C-terminal truncated isoform, TOP2α/90, which lacks the active site Tyr805 required to form TOP2α–DNA covalent complexes (Kanagasabai et al., 2017; Elton et al., 2020, 2022; Hernandez et al., 2021). Cytotoxicity of etoposide and other TOP2-targeted agents was attenuated in K/VP.5 cells secondary to reduction of TOP2α/170- and TOP2β-DNA covalent cleavage complexes (Ritke et al., 1994; Burgess et al., 2008; Pilati et al., 2012; Ganapathi and Ganapathi, 2013; Austin et al., 2018, 2021; Capelôa et al., 2020) as well as heterodimerization of TOP2α/170 with the truncated isoform TOP2α/90 (Kanagasabai et al., 2018).

Like TOP2α, the human TOP2β gene also contains a weak splice site at the exon 19/intron 19 boundary (5′ SS score of 6.95 out of a maximum consensus scored of 13.1; Yeo and Burge, 2004). However, unlike TOP2α, the TOP2β gene does not contain consensus polyadenylation sites within intron 19 and cannot undergo IPA to generate truncated isoforms. Rather, TOP2β/180 mRNA/protein is reduced in K/VP.5 cells mainly due to a microRNA-9 (miR-9)-mediated posttranscriptional mechanism which plays a role in TOP2β/180-mediated drug resistance (Carvajal-Moreno et al., 2023).

To further examine TOP2β/180-mediated drug resistance to TOP2-targeted agents, we introduced a premature stop codon at the end of exon 19 in parental K562 cells by utilizing CRISPR/Cas9/HDR to either: 1) knockdown/knockout this gene through the NMD pathway that recognizes and degrades mRNAs harboring premature stop codons (Embree et al., 2022; Karousis and Muhlemann, 2022) or: 2) produce a C-terminal truncated TOP2β isoform (lacking the exon 20 encoded active site Tyr821 needed to generate TOP2β/180-DNA covalent cleavage complexes) that could mediate drug resistance by a similar mechanism as that found for TOP2α/90; i.e., heterodimerizing with TOP2β/180.

qPCR experiments utilizing a custom specific probe for the gene-edited TOP2β Exon 19/Intron 19 boundary demonstrated extremely low levels of this mRNA in K562/SC-Edit-1 and K562/Edit-4 cells which were increased by use of an inhibitor of nonsense-mediated decay (Fig. 5E). These results strongly suggested that mRNA encoding the putative truncated TOP2β isoform was rapidly degraded. Hence, a truncated TOP2β isoform was not likely produced by our introduced premature stop codon in the TOP2β gene.

Importantly, our results demonstrated reduction of full-length TOP2β/180 mRNA/protein in the gene-edited clone with one allele edited and complete elimination/knockout of TOP2β/180 mRNA/protein in the clone when all four TOP2β/180 alleles present in K562 cells (Cioe et al., 1981; Zhou et al., 2019) were mutated (Fig. 5, A, C, and D). Compared with K562 cells, no change in TOP2α/170 was noted in K562/SC-Edit-1 cells (Fig. 6A, zero drug controls) or in K562-Edit-4 cells (Supplemental Fig. 1).

Using immunoblot “band-depletion” studies, XK469, an identified TOP2β-specific agent (Gao et al., 1999), demonstrated concentration dependent enhancement of TOP2β-DNA covalent complexes in K562 and K562/SC-Edit-1 cells. In contrast, XK469 did not cause band depletion in TOP2α/170 in these (Fig. 6, A and B) or in K562/SC-Edit-4 cells (Supplemental Fig. 1), consistent with the lack of formation of TOP2α/170-DNA complexes. Notably, in K562/SC-Edit-1 cells, which contain reduced TOP2β/180 levels, XK469 (250 µM) induced greater TOP2β/180 band depletion compared with K562 cells (P = 0.033), indicative of the greater ratio of drug-to-TOP2β/180 protein in these gene-edited cells (Fig. 6B).

DNA damage assays (alkaline and neutral Comet assays) demonstrated reduced XK469 strand breakage activity in K562/SC-Edit-1 and K562/SC-Edit-4 cells compared with K562 cells (Fig. 6, C–E) consistent with XK469-mediated TOP2β/180 targeting. However, results suggest XK469 off-target activity since DNA strand breaks, though reduced, were still evident even when TOP2β/180 was completely absent in K562/Edit-4 cells. XK469 specificity for TOP2β/180 has been experimentally questioned previously, suggesting that this agent may also impact TOP2α/170 (Toyoda et al., 2008; Skalicka et al., 2022).

Although low levels of XK469 (250 µM)-induced DSB were observed and were reduced in K562/SC-Edit-4 compared with K562 cells (Fig. 6E), no detectable XK469-induced DSB were detected by analysis of γH2AX formation (Fig. 6F, top). We speculate that the low levels of XK469-induced DSB by Comet assay reflect low TOP2β/180 mRNA/protein expression in K562 cells (especially compared with TOP2α/170; Fig. 5B, Ritke et al., 1994) and were therefore below the level of detection for the γH2AX immunoblot detection assay. In contrast, etoposide (50 µM)-induction of DSB by Comet assay and γH2AX formation were robust and unchanged in K562 compared with gene-edited K562/SC-Edit-4 cells (Figs 6, E–G). Although previous literature indicated etoposide targeting both TOP2 type II topoisomerase paralogs (Hasinoff et al., 2016), with some preference for TOP2α/170 (Errington et al., 1999; Toyoda et al., 2008), the absence of TOP2β/180 in K562/SC-Edit-4 cells likely results in only a small decrease in overall TOP2 levels and explains undiminished DNA damage in response to etoposide. Likewise, NK314 (25 µM), a specific TOP2α/170-targeted agent (Toyoda et al., 2008) was unaffected in its ability to induce DSB assessed by γH2AX formation in K562/SC-Edit-4 cells (Figs. 6, F and G). In contrast, mitoxantrone (10 µM)-induced γH2AX formation was reduced in the TOP2β/180 depleted K562/SC-Edit-4 cells compared with parental K562 cells consistent with literature indicating preferential targeting of TOP2β/180 (Errington et al., 1999; Hasinoff et al., 2016).

Only partial resistance to XK469-induced growth inhibition (2.3-fold) was noted when TOP2β/180 was completely knocked out in K562/SC-Edit 4 cells (Fig. 7). Given the low levels of TOP2β/180 mRNA/protein in K562 cells (Fig. 5B, Ritke et al., 1994), these results are consistent with the partial resistance phenotype to XK469 and a strong indication of off-target effects of this agent. In K/VP.5 cells with reduction of TOP2α/170 protein to ∼10% and reduction of TOP2β/180 to ∼25% of parental K562 cells (Ritke et al., 1994; Kanagasabai et al., 2017; Fig. 5C), XK469-induced even greater resistance (7.4-fold) than in the knockout K562/SC-Edit-4 cells (Fig. 7). This greater fold resistance to XK469 is consistent with targeting at both TOP2α/170 and TOP2β/180, which has been suggested and reported previously (Toyoda et al., 2008; Skalicka et al., 2022).

Weak XK469 activity in forming TOP2α/170- and TOP2β/180-DNA covalent complexes has recently been indicated (Skalicka et al., 2022), which may explain the lack of XK469-induced TOP2α/170 band depletion (Fig. 6, A and B) and weak DNA damage (Fig. 6, D and E) in K562 cells where the preponderance of TOP2α/170 relative to TOP2β/180 may prevent the manifestation of these effects even at the high concentrations (100, 250 µM) of XK469 used. The much higher concentration ratio of XK469-to-TOP2β/180 accounts for observed activities in K562 cells and in the gene-edited K562/SC-Edit-1 cells at the level of TOP2β/180 by band depletion and drug-induced DNA damage (Fig. 6), and growth inhibition (Fig. 7, Table 1). Although the biochemical or structural basis for the differential TOP2 paralog targeting and responses to TOP2β/180 ablation are unclear for XK469 and mitoxantrone on the one-hand (sensitivity to TOP2β/180 levels) and etoposide, NK314, and mAMSA on the other hand (little to no effect of TOP2β/180 gene-editing), our results suggest that the relative levels of the two TOP2 paralogs may be an important determinant of drug sensitivity to TOP2-targeted drugs as was proposed in early studies (Drake et al., 1987).

In summary, by introducing a premature stop codon, we were successful in generating a TOP2β/180 one-allele gene knockdown cell line (i.e., K562/SC-Edit-1) and a TOP2β/180 four-allele gene knockout (i.e., K562/SC-Edit-4). Importantly, these gene edited cells allowed us to demonstrate the importance of TOP2β/180 in drug sensitivity/resistance and further established the differential TOP2α/170 and TOP2β/180 paralog activity of selected TOP2-targeted agents in K562 cells.

Data Availability

The authors declare that all the data supporting the findings of this study are contained within the paper.

Abbreviations

DSB

DNA double-stranded break

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GCD

genomic cleavage detection

HDR

homology directed repair

IDT

Integrated DNA Technologies

IPA

intronic polyadenylation

K/VP.5

etoposide (VP-16)-resistant human K562 leukemia cell line

K562

human leukemia cell line

NHEJ

non-homologous end joining

NMD

non-sense mediated decay

PCR

polymerase chain reaction

PAM

protospacer-adjacent motif

qPCR

quantitative PCR

TOP

topoisomerase IIα protein

XK469

(2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)

Authorship Contributions

Participated in research design: Carvajal-Moreno, Wang, Hernandez, Mondal, Zhao, Yalowich, Elton.

Conducted experiments: Carvajal-Moreno, Wang, Mondal, Zhao.

Performed data analysis: Carvajal-Moreno, Wang, Hernandez, Mondal, Zhao, Yalowich, Elton.

Wrote or contributed to the writing of the manuscript: Carvajal-Moreno, Wang, Yalowich, Elton.

Footnotes

This work was supported by the National Institutes of Health Office of Extramural Research [Grant R01-CA226906-01A1].

The authors declare that they have no conflicts of interest with the contents of this article.

Inline graphicThis article has supplemental material available at jpet.aspetjournals.org.

References

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