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.

Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intrinsically weak or potentially mutated. The specific case of TOP2α exon 19/intron 19 splice site editing is discussed in etoposide-resistant human leukemia K562 cells as a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. This example supports the importance of aberrant IPA in acquired drug resistance to TOP2α-targeted drugs. In addition, these results demonstrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant splicing/polyadenylation.

microRNAs (miRNAs) are short, noncoding RNAs that inhibit translation by binding primarily to the 3′‐untranslated region (3′‐UTR) of mRNA. The enzyme TOP2α induces covalent complexes with DNA and produces transient double‐strand DNA breaks crucial for processes such as replication and normal chromosomal dysjunction at mitosis. TOP2α is an important target for clinically effective anticancer agents, such as etoposide (VP‐16) since these drugs stabilize the otherwise short‐lived enzyme‐DNA covalent complexes, thereby inducing cytotoxic DNA damage. However, the efficacy of these agents is limited by chemoresistance. Our lab has characterized acquired resistance to VP‐16 in human leukemia K562 cells. This cloned resistant cell line, K/VP.5, contains reduced levels of TOP2α compared to parental K562 cells. The goal of this project is to test the hypothesis that TOP2α levels are decreased in K/VP.5 cells, in part, through miRNA‐mediated mechanisms. Pooled miRNA qPCR profiling experiments were performed to investigate the expression levels of ~500 miRNAs in K562 and K/VP.5 cells. hsa‐miR‐9‐3p and ‐5p (miR‐9‐3p and ‐5p) were overexpressed in K/VP.5 cells compared to K562 cells. The TOP2α 3′‐UTR harbors putative miRNA recognition elements (MRE) for these miRNAs. Therefore, these miRNAs were chosen for further study. To assess post‐transcriptional regulation of TOP2α by miRNAs, a dual luciferase reporter plasmid harboring the entire 3′‐UTR of TOP2α mRNA (998 bp) was constructed (psiTOP2α/UTR). Transfection with psiTOP2α/UTR demonstrated decreased luciferase expression in K/VP.5 compared with K562 cells (p<0.001), suggesting altered post‐transcriptional regulation in resistant cells. K562 cells that were co‐transfected with psiTOP2α/UTR and miR‐9‐5p or ‐3p mimic resulted in a decrease in luciferase expression only for miR‐9‐5p (p<0.001). Mutating the putative miR‐9‐5p seed sequence prevented the decrease in luciferase activity, demonstrating a direct interaction of this miRNA with the MRE of TOP2α. Immunoblotting for TOP2α in K562 cells transfected with miR‐9‐3p or ‐5p mimic resulted in decreased TOP2α protein compared to mock transfected K562 cells (miR‐9‐3p; p<0.05, miR‐9‐5p; p=0.01). In contrast, immunoblotting for TOP2α in K/VP.5 cells transfected with miR‐9‐3p or ‐5p inhibitor resulted in an increase of TOP2α protein (p<0.05), strongly suggesting a role for both miRNAs in acquired resistance to VP‐16. Our findings indicate that miR‐9‐3p and ‐5p reduce TOP2α expression levels. In addition, results presented here contribute to the elucidation of chemoresistance mechanisms and have the translational potential for circumvention of drug resistance by modulation of miRNA concentrations. Support or Funding Information Patrick and Jane O'Neill Endowed Scholarship, Honors and Scholars Enrichment Grant, Research Scholars Award This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA topoisomerase IIα (TOP2α/170; 170kDa) and Topoisomerase IIβ (TOP2β/180; 180kDa) are targets for a number of anticancer drugs, whose clinical efficacy is attenuated by chemoresistance. Our laboratory selected for an etoposide resistant K562 clonal subline, designated K/VP.5. These cells exhibited decreased TOP2α/170 and TOP2β/180 expression. We previously demonstrated that a microRNA-9 (miR-9)mediated post transcriptional mechanism plays a role in drug resistance via reduced TOP2α/170 protein in K/VP.5 cells. Here, it is hypothesized that a similar miR-9 mechanism is responsible for decreased TOP2β/180 levels in K/VP.5 cells. Both miR-9-3p and miR-9-5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by microRNA sequencing and quantitative PCR (qPCR). The 3′-untranslated region (3′-UTR) of TOP2β/180 contains miRNA recognition elements (MRE) for both miRNAs. Cotransfection of K562 cells with a luciferase reporter plasmid harboring TOP2β/180 3′-UTR plus miR-9-3p or miR-9-5p mimics resulted in statistically significant decreased luciferase expression. miR-9-3p and miR-9-5p MRE mutations prevented this decrease validating direct interaction between these miRNAs and TOP2β/180 mRNA.Transfection of K562 cells with miR-9-3p/5p mimics led to decreased TOP2β protein levels without a change in TOP2β/180 mRNA and resulted in reduced TOP2β-specific XK469-induced DNA damage. Conversely, K/VP.5 cells transfected with miR-9-3p/5p inhibitors led to increased TOP2β/180 protein without a change in TOP2β/180 mRNA and resulted in enhancement of XK469-induced DNA damage. Taken together, these results strongly suggest that TOP2β/180 mRNA is translationally repressed by miR-9-This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward.

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.