Abstract 904: 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

Kanagasabai, Ragu; Karmahapatra, Soumendra Krishna; Yu, Yang; Hernández, Víctor Agmo; Kientz, Corey A.; Kania, Evan E; Elton, Terry S.; Yalowich, Jack C.

doi:10.1158/1538-7445.am2018-904

Cited by 2 publications

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“…Cytotoxicity of etoposide and other TOP2-targeted agents was attenuated in K/VP.5 cells secondary to reduction of TOP2α/170and TOP2ß-DNA covalent cleavage complexes (Ritke et al, 1994;Burgess et al, 2008;Pilati et al, 2012;Ganapathi and Ganapathi, 2013;Capeloa et al, 2020, Austin et al, 2018Austin et al, 2021) as well as heterodimerization of TOP2α/170 with the truncated isoform TOP2α/90 (Kanagasabai et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…For genome editing of the TOP2β/ (Kanagasabai et al, 2017(Kanagasabai et al, , 2018Hernandez et al, 2021). To reduce NHEJ, immediately after electroporation, cells were plated in media with 1 μM HDR Enhancer V2 (cat.…”

Section: Generation Of Amentioning

confidence: 99%

“…1081061, IDT) for 20 min 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(Kanagasabai et al, , 2018Hernandez et al, 2021).…”

Section: Generation Of Amentioning

confidence: 99%

“…To ThermoFisher Scientific) as previously described (Kanagasabai et al, 2017(Kanagasabai et al, , 2018Hernandez et al, 2021). qPCR experiments (total reaction volume 10 µL) were performed using Taqman Gene Expression hydrolysis probes (ThermoFisher Scientific) 13 (Kanagasabi et al, 2017(Kanagasabi et al, , 2018Hernandez et al, 2021) and as shown in Supplemental Table 1.…”

Section: Generation Of Amentioning

confidence: 99%

“…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;Hernandez et al, 2021;Elton et al, 2022), which functions as an additional determinant of resistance via heterodimerization with TOP2α/170 (Kanagasabai et al 2018). TOP2ß/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.…”

Section: Introductionmentioning

confidence: 99%

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

Carvajal-Moreno,

Wang,

Hernandez

et al. 2024

J Pharmacol Exp Ther

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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 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 TOP2ß/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 (HDR) to disrupt production of full length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain (qPCR) and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2ß/180 alleles mutated, revealed partial or complete loss of TOP2ß mRNA/protein, respectively. The loss of TOP2ß/180 protein correlated with decreased XK469-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 TOP2ß/180 alleles modified. No crossresistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

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Section: Discussionmentioning

confidence: 99%

Section: Generation Of Amentioning

confidence: 99%

Section: Generation Of Amentioning

confidence: 99%

Section: Generation Of Amentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Carvajal-Moreno,

Wang,

Hernandez

et al. 2024

J Pharmacol Exp Ther

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

Wang,

Carvajal-Moreno,

Zhao

et al. 2024

Mol Pharmacol

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DNA topoisomerase IIα (TOP2α, 170kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage stabilizing anti-cancer agents such as etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposideresistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90) which heterodimerized with TOP2α/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3′-Rapid Amplification of cDNA Ends (3′-RACE), we confirmed TOP2α/90 as the translation product of a TOP2α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was utilized. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide (AMO) was used to hybridize/block the PAS in TOP2α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2α/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/Cas9 homology-directed repair (HDR) was used to mutate the cryptic I19 PAS (AATAAAACCCAA) to prevent IPA. Gene-edited clones exhibited increased TOP2α/170 and decreased TOP2α/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2α-targeted drugs.Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2α-targeting drugs.

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Abstract 904: 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

Cited by 2 publications

References 0 publications

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

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

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

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