Intronic Polyadenylation in Acquired Cancer Drug Resistance Circumvented by Utilizing CRISPR/Cas9 with Homology-Directed Repair: The Tale of Human DNA Topoisomerase IIα

Abstract

Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intrinsically weak or potentially mutated. The specific case of TOP2α exon 19/intron 19 splice site editing is discussed in etoposide-resistant human leukemia K562 cells as a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. This example supports the importance of aberrant IPA in acquired drug resistance to TOP2α-targeted drugs. In addition, these results demonstrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant splicing/polyadenylation.

Maximizing the Efficacy of CRISPR/Cas Homology-Directed Repair Gene Targeting

“…First, after developing an experimental CRISPR/Cas/HDR hypothesis, serious consideration must be given to the appropriate cellular system to utilize and the number of gene alleles of interest that may need be edited to obtain a resulting altered phenotype. If the cell line of choice is not well characterized, sequence analysis of CRISPR-edited cells may help determine the number of edited and non-edited alleles [91,92,111]. Second, the location of the targeted gene knock-in must be analyzed to determine if a Cas9/12 PAM site(s) is/are harbored within 20 nt of the DNA sequence to be edited (both DNA strands should be analyzed) [93][94][95][96][97][98][99][100].…”

“…Regarding the polarity of the ssODN donor templates with respect to the gRNA (see Figure 1), an NT strand ssODN donor template is preferred for PAM-distal mutations and a T strand ssODN donor template is ideal for PAM-proximal mutations [100]. Finally, blocking PAM mutations (i.e., NGG → NCC) should always be introduced in ssODN repair templates to further increase HDR efficiency [94,99,100] and to allow for potential repeated rounds of CRISPR/Cas/HDR transfections when editing multiple alleles in cell lines, which exhibits aneuploidy (Figure 3) [91,92,111].…”

“…Compared to parental K562 cells, etoposide-resistant K/VP.5 cells contain reduced TOP2α/170 levels and express high levels of a novel C-terminal truncated TOP2a isoform (90 kDa, TOP2α/90) [88,89]. TOP2α/90 is the translation product of a short TOP2α mRNA that is generated from a cryptic poly(A) site harbored in intron 19 (i.e., I19 intronic polyadenylation; I19 IPA) [90,91]. TOP2α/90 lacks the active site tyrosine 805 harbored in exon 20 of full-length TOP2α/170 necessary for TOP2α-mediated DNA strand breaks [88][89][90][91].…”

“…TOP2α/90 is the translation product of a short TOP2α mRNA that is generated from a cryptic poly(A) site harbored in intron 19 (i.e., I19 intronic polyadenylation; I19 IPA) [90,91]. TOP2α/90 lacks the active site tyrosine 805 harbored in exon 20 of full-length TOP2α/170 necessary for TOP2α-mediated DNA strand breaks [88][89][90][91]. We hypothesized that, by utilizing CRISPR/Cas/HDR to enhance the TOP2α gene's suboptimal exon 19/intron 19 5´ SS (E19/I19 5´ SS), removal of intron 19 would be enhanced, which in turn would result in decreased TOP2α/90 mRNA/protein, increased TOP2α/170 mRNA/protein, and circumvention of etoposide resistance [92].…”

“…Donor templates containing two blocking PAM mutations (i.e., NGG → NCC) resulted in the highest HDR efficiency (Figure 3). Finally, another important indication for blocking PAM mutations in HDR repair templates is to ensure that when multiple rounds CRISPR/Cas/HDR transfections are required to edit all gene-specific alleles in cell lines that exhibit aneuploidy; the previously edited alleles will not be re-cut in the subsequent rounds of transfection [91,92].…”