DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2/90 expression decreases drug-induced TOP2-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2/170. Alternative processing of TOP2 pre-mRNA, and subsequent synthesis of TOP2/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2/170-DNA covalent complexes in response to TOP2-targeting agents.
DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells since it resolves DNA topologic entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated TOP2α/90 isoform, detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide (J Pharmacol Exp Ther 2017;360:152-63). TOP2α/90 (786 amino acids) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 (Tyr805) required to generate double-strand DNA breaks as well as the nuclear localization signals present in the TOP2α/170 isoform (1531 amino acids). The function of TOP2α/90 is unknown. Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Importantly, co-immunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, while siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated K562 or K/VP.5 cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by soft agar colony formation assays. qPCR and immunoassays demonstrated expression of TOP2α/90 mRNA and protein in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents. Citation Format: Ragu Kanagasabai, Soumendra Karmahapatra, Yang Yu, Victor A. Hernandez, Corey A. Kientz, Evan E. Kania, Terry S. Elton, Jack C. Yalowich. The novel C-terminal truncated 90-kDa isoform of topoisomerase IIα, TOP2α/90, is a determinant of etoposide resistance in K562 leukemia cells via heterodimerization with the TOP2α/170 isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 904.
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.
Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localizedin situbiotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNAXistas models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization ofXistand other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.
DNA topoisomerase IIα (170 kDa, TOP2α/170) is a key homodimeric enzyme resolving DNA topologic entanglements during chromosome dysjunction by introducing transient DNA double‐strand breaks (DSB). TOP2α/170 is also a prominent clinical target for anticancer drugs, such as etoposide (VP‐16), whose clinical efficacy is often compromised due to chemoresistance. We recently demonstrated that parental K562 cells and cloned K562 cells (K/VP.5) with acquired resistance to VP‐16 express a novel truncated TOP2α/90 (90 kDa) isoform dramatically overexpressed in drug‐resistant K/VP.5 cells (JPET 360(1): 152–163, 2017). Recent results also demonstrated overexpression of TOP2α/90 in isogenic HL‐60 AML cells resistant to the TOP2α‐targeting agents, mAMSA and mitoxantrone.TOP2α/90 is the C‐terminal truncated translation product of TOP2α mRNA lacking the active‐site Tyr805 required for the generation of DSBs. TOP2α/90 mRNA retains a portion of intron 19 indicating altered RNA processing and an exon‐intron read‐through. TOP2α/90 was detectable in the nucleus and heterodimerizes with TOP2α/170. Forced expression of TOP2α/90 in K562 cells decreased VP‐16–induced DNA damage and cytotoxicity while siRNA knockdown of TOP2α/90 in K/VP.5 cells increased VP‐16 activity (Mol. Pharmacol. 93: 515–525, 2018). Together results suggest that TOP2α/90 is a resistance determinant/biomarker through a dominant negative effect. qPCR analyses of paired/matched AML patient samples (pre‐treatment & relapse) revealed that, in 3 of 4 patients, there was a statistically significant increase in the ratio of expression of TOP2α/90 mRNA compared to TOP2α/170, after relapse.We hypothesize that an intrinsically weak exon 19/intron 19 (E19/I19) splice site (ss) in TOP2α pre‐mRNA results in partial I19 retention and formation of truncated TOP2α/90. We further posit that therapeutic genome to modify I19 will provide a tractable strategy to circumvent TOP2α‐mediated drug resistance.To investigate the influence of a weak TOP2α E19/I19 ss (GAG/gtaaac)in I19 retention, a minigene (MG1+) expression construct with a consensus I19 5′ ss (CAG/gtaagt) was generated. Transfection of K/VP.5 cells with MG1+ resulted in a reduction of I19 retention (assessed by qPCR), accompanied by an increase in the levels of a properly spliced amplicon indicative of TOP2α/170 mRNA. These findings set the stage for CRISPR/Cas9 experiments to determine whether editing the I19 5′ ss of the TOP2α gene will result in decreased I19 retention, diminished TOP2α/90 generation, and increased VP‐16 sensitivity in K/VP.5 cells. To date, CRISPR/Cas9 targeting has resulted in successful targeting/mutation of E19/I19 to a consensus ss sequence. Selection of an edited cell clone is in process. Our results contribute to a better understanding of alternative TOP2α mRNA processing leading to the development of resistance to TOP2α poisons. Characterization of alternative RNA processing of the TOP2α mRNA will lead to strategies to circumvent acquired drug resistance. These and future results may also allow for evaluation of TOP2α/90 as a biomarker for drug resistance, prognosis, and/or guide TOP2α‐targeted therapies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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