Strategies to Circumvent Topoisomerase IIα Intron 19 Intronic Polyadenylation (IPA) in Acquired Etoposide Resistance Human Leukemia K562 Cells

Abstract

<b>Abstract ID 20741</b> <b>Poster Board 251</b> DNA topoisomerase IIα (TOP2α, 170kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction at mitosis by producing transient DNA double-stranded breaks and is a significant target for DNA damage stabilizing anti-cancer agents like etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposide-resistant human leukemia K562 clonal subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90 is translated from TOP2α mRNA due to Intron19 (I19) intronic polyadenylation (IPA). TOP2α/90 heterodimerizes with TOP2α/170 and is a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Previously we gene-edited/optimized the weak TOP2α I19 5′ splice site in K/VP.5 cells using CRISPR/Cas9 with homology-directed repair (HDR). In gene-edited cells, TOP2α/90 levels were reduced, TOP2α/170 levels were restored, and drug resistance was reversed. Given that no TOP2α sequence differences were identified in K562 vs K/VP.5 cells, we hypothesize that decreased TOP2α/170 and increased TOP2α/90 in K/VP.5 cells is related to IPA and aberrant expression of RNA cleavage/polyadenylation factor(s). Based on 3′-Rapid Amplification of cDNA Ends (3′-RACE), polyadenylation site (PAS) #3 (out of 7 PASs found in I19) is the major site utilized in K/VP.5 which leads to IPA. Therefore, CRISPR/Cas9/HDR and anti-sense morpholinos were utilized to mutate/block the PAS #3 in TOP2α I19 to eliminate/reduce IPA. We first mutated PAS #3 from ‘ATTTAA’ to ‘ACCCAA’ in K/VP.5 cells using CRISPR/Cas9/HDR editing, and successfully obtained one, two and three-allele edited clones assessed by qPCR and Sanger sequencing [Note: three TOP2α alleles are present in K/VP.5 cells]. In the PAS #3 one-allele edited clone, a ∼50% decrease of TOP2α/90 and a 4-fold increase of TOP2α/170 mRNA/protein was detected compared to K/VP.5 cells. Growth inhibitory assays demonstrated a 5-fold reversal of etoposide resistance. CRISPR/Cas9/HDR re-transfection of the one-allele edited clone resulted in generation/isolation of PAS #3 two-allele and three-allele edited clones, validated by qPCR and Sanger sequencing. Experiments are planned to assess TOP2α/170 and TOP2α/90 mRNA/protein levels in the new gene-edited clones as well as to evaluate circumvention of drug resistance by etoposide-induced growth inhibition and DNA damage (Comet assay) studies. In separate experiments, a customized anti-sense morpholino was used to hybridize and block TOP2α I19 PAS #3 in K/VP.5 cells. Results showed a 50% decrease in TOP2α/90 level 48 hr after transfection with the antisense morpholino compared to a control morpholino. Taken together, results suggest that by mutating/blocking PAS #3, aberrant trans-acting factors regulating I19 IPA can no longer recognize the PAS thereby preventing TOP2α I19 IPA resulting in restoration of full-length TOP2α/170 levels and circumvention of chemoresistance in K/VP.5 cells. Future strategies will be directed to identify putative aberrant splicing/RNA cleavage/polyadenylation factor(s) expressed in K/VP.5 cells utilizing an unbiased MS2 (bacteriophage coat protein) tag-trap pull-down strategy and proteomics analysis.