Circumvention of acquired resistance to topoisomerase II-targeted anticancer agents in HL-60 leukemia cells by prevention of intronic polyadenylation.

DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is a nuclear enzyme that plays a key role in chromosomal segregation at mitosis by catalyzing transient DNA double-stranded breaks, allowing replicated DNA duplexes to separate. Given its importance during mitosis and high expression level in proliferating cells, TOP2α/170 is a prominent target for anticancer therapy. However, the effectiveness of TOP2-targeted agents is often compromised by acquired drug resistance, which in patients is most associated with decreased TOP2α/170 protein expression, thereby diminishing drug-induced TOP2α-DNA complexes and cytotoxic DNA damage. Mimicking this clinical chemoresistance phenotype, reduced TOP2α/170 mRNA/protein was previously reported in an acquired HL-60 cell line associated with production of a C-terminal truncated 160 kDa protein (TOP2α/160). In this report, intronic polyadenylation (IPA) within intron 33 (I33) of the TOP2α/170 gene, responsible for production of TOP2α/160, was recapitulated by quantitative real-time polymerase chain reaction, 3'-rapid amplification of cDNA ends, and Sanger sequencing. HL-60/MX2 cells are crossresistant to multiple TOP2-targeted agents. To circumvent drug resistance, CRISPR/Cas9 gene editing with homology-directed repair was used to mutate/enhance the weak 5' splice site at the exon 33/I33 junction of the TOP2α gene to improve splicing out of I33 and to suppress IPA. The resulting splice site gene-edited clone, designated MX2/SS-Edit, expressed reduced TOP2α/160 mRNA/protein, increased TOP2α/170 mRNA/protein, and exhibited partial restoration of sensitivity to mitoxantrone, etoposide, and amsacrine. Together, results indicated that strengthening the weak TOP2α exon 33/I33 splice site by knock-in gene editing attenuated IPA offering a strategy to circumvent resistance to TOP2-targeted therapies. SIGNIFICANCE STATEMENT: Results presented here validated drug resistance in the HL-60/MX2 leukemia cell line driven by intronic polyadenylation (IPA) within intron 33 of the DNA topoisomerase IIα (TOP2α) gene, which produced a truncated and predominantly cytoplasmic TOP2α protein isoform (TOP2α/160). Using CRISPR/Cas9/homology-directed repair gene editing, the weak exon 33/intron 33 5' splice site was enhanced to suppress IPA, which restored expression of full-length protein (TOP2α/170) and led to a gain-of-function in drug sensitivity, offering a potential strategy to overcome drug resistance.