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.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure–property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

In recent years, novel bacterial topoisomerase inhibitors (NBTIs) have been developed as future antibacterials for treating multidrug-resistant bacterial infections. A series of dioxane-linked NBTIs with an amide moiety has been synthesized and evaluated. Compound 3 inhibits DNA gyrase, induces the formation of single strand breaks to bacterial DNA, and achieves potent antibacterial activity against a variety of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Optimization of this series of analogues led to the discovery of a subseries of compounds (22−25) with more potent anti-MRSA activity, dual inhibition of DNA gyrase and topoisomerase IV, and the ability to induce double strand breaks through inhibition of S. aureus DNA gyrase.

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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.

Antibacterial resistance continues its devastation of available therapies. Novel bacterial topoisomerase inhibitors (NBTIs) offer one solution to this critical issue. Two series of amine NBTIs bearing tricyclic DNA-binding moieties as well as amide NBTIs with a bicyclic DNA-binding moiety were synthesized and evaluated against methicillin-resistant Staphylococcus aureus (MRSA). Additionally, these compounds and a series of bicyclic amine analogues displayed high activity against susceptible and drug-resistant Neisseria gonorrhoeae, expanding the spectrum of these dioxane-linked NBTIs.

An essential function of DNA topoisomerase IIα (170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome dysjunction by introducing transient DNA double‐strand breaks (DSBs). 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 utilized to make changes (GAG//GTAAACàGAG//GTAAGT) in the TOP2α gene's suboptimal exon 19/intron 19 5′ splice site (E19/I19 5′ SS). Gene edited clones were identified by qPCR and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 protein, and increased TOP2α/170 protein. Sensitivity to etoposide‐induced DNA damage (γH2AX, comet assays) and growth inhibition was restored to the levels comparable to those in parental K562 cells. Together 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 TOP2‐targeted drugs.