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Modulating RNA Splicing of DNA Topoisomerase IIα in Human Leukemia K562 Cells: Use of CRISPR/Cas9 Gene Editing to Impact Sensitivity/Resistance to the Anticancer Agent Etoposide

Hernandez, Victor A
http://orcid.org/0000-0001-5017-9422
http://rave.ohiolink.edu/etdc/view?acc_num=osu1630504949546357

Abstract Details

2021, Doctor of Philosophy, Ohio State University, Pharmaceutical Sciences.
The human DNA topoisomerase IIα (170 kDa, TOP2α/170) enzyme is essential in proliferating cells by functioning as a homodimer resolving DNA topological entanglements that form during chromosome condensation, replication, and segregation. The TOP2α/170 homodimer disentangles DNA by introducing transient double strand breaks in a DNA helix via a transesterification reaction between the active site Tyr805 from each TOP2α/170 subunit and the phosphodiester DNA backbone, creating the intermediate state known as the TOP2α/170-DNA cleavage complex. An intact DNA duplex passes through the cleaved DNA. Subsequently, the DNA strands are religated restoring the integrity of the DNA and preparing the TOP2α/170 dimer for another catalytic cycle. TOP2α/170 enzymatic activity is indispensable for the survival of highly proliferating cells including cancer cells. This has made TOP2α a prominent target for anticancer therapies. Some of the most widely used topoisomerase II targeting drugs such as etoposide, mitoxantrone, amsacrine and doxorubicin, stabilize the TOP2α/170-DNA cleavage complex preventing the religation of the DNA strands. As a result, these agents exert their cytotoxic effects by the accumulation of double strand DNA breaks which ultimately lead to the initiation of apoptotic pathways. Acquired chemoresistance to topoisomerase II targeting drugs continues to be a major obstacle in cancer treatment in the clinic. In order to characterize the mechanisms of resistance to etoposide, our laboratory developed etoposide resistant human leukemia K562 cells, designated K/VP.5 in which levels of TOP2α/170 were decreased along with identification of a novel C-terminal truncated isoform of TOP2α, TOP2α/90. This 90 kDa protein is present in both in K562 and etoposide resistant K/VP.5 cells with expression levels increased ~3-fold in K/VP.5 cells. TOP2α/90 is the translation product of novel alternatively spliced mRNA via intron 19 (I19) retention and processing, confirmed by 3'-RACE, PCR, and sequencing. TOP2α/90 is missing 770 amino acids of the C-terminal portion of the enzyme. Therefore, TOP2α/90 lacks the active site Tyr805, dimerization domains and nuclear localization signals found in TOP2α/170. TOP2α/90, like TOP2α/170, was found primarily in the nucleus and forms heterodimers with TOP2α/170. Forced TOP2α/90 overexpression in K562 cells resulted in decreased etoposide-induced DNA damage whereas TOP2α/90 knockdown in K/VP.5 cells increased etoposide-induced DNA damage. These results indicated that TOP2α/90 was a determinant of acquired resistance to etoposide through a dominant-negative effect related to heterodimerization with TOP2α/170. Given that TOP2α/90 is a result of I19 retention, it was hypothesized that the E19/I19 splice site (SS) is inefficiently recognized by the spliceosome and results in decreased levels of TOP2α/170 and drug resistance. Therefore, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) was utilized for gene editing of the E19/I19 boundary in the TOP2α gene from K/VP.5 cells to enhance I19 removal, restore levels of TOP2α/170 and circumvent acquired TOP2α-mediated drug resistance. Gene edited clones were identified by qPCR and verified by sequencing and restriction enzyme digestion. Characterization of a K/VP.5 clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 protein, and increased TOP2α/170 protein expression levels. Sensitivity to etoposide-induced DNA damage and growth inhibition was restored to levels comparable to those in parental K562 cells. Sensitivity to growth inhibition of a number of additional TOP2α-targeted drugs was also restored in the gene-edited K/VP.5 clone. These results indicated that gene editing to improve the TOP2α E19/I19 SS in K/VP.5 cells circumvented resistance to TOP2α-targeted agents. To further solidify the role of I19 retention as a determinant of acquired drug resistance to etoposide, converse experiments were performed whereby the E19/I19 SS in drug sensitive parental K562 cells was gene edited to eliminate splicing at this boundary. Inhibiting splicing of the E19/I19 SS in K562 cells resulted in a decrease in TOP2α/170 expression and induction of resistance to etoposide. Together, results support the role of alternative splicing as a determinant of resistance to TOP2α-targeting agents. This work will lead logically to future studies to investigate the role of splicing factors and other effectors involved in regulating TOP2α splicing/intron retention that are responsible for acquired drug resistance and potentially lead to tractable strategies to circumvent resistance in a clinical setting.
Christopher Coss (Committee Member)
Jack Yalowich (Advisor)
Dawn Chandler (Committee Member)
207 p.
Pharmaceuticals; Pharmacology; Pharmacy Sciences
CRISPR/Cas9; Topoisomerase II; Etoposide; Drug resistance; Alternative Pre-mRNA splicing; Intron Retention and Processing; Modulating mRNA Splicing; Circumvention of Drug Resistance by Gene Editing; AML; CML

Recommended Citations

Citations

  • Hernandez, V. A. (2021). Modulating RNA Splicing of DNA Topoisomerase IIα in Human Leukemia K562 Cells: Use of CRISPR/Cas9 Gene Editing to Impact Sensitivity/Resistance to the Anticancer Agent Etoposide [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1630504949546357

    APA Style (7th edition)

  • Hernandez, Victor. Modulating RNA Splicing of DNA Topoisomerase IIα in Human Leukemia K562 Cells: Use of CRISPR/Cas9 Gene Editing to Impact Sensitivity/Resistance to the Anticancer Agent Etoposide. 2021. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1630504949546357.

    MLA Style (8th edition)

  • Hernandez, Victor. "Modulating RNA Splicing of DNA Topoisomerase IIα in Human Leukemia K562 Cells: Use of CRISPR/Cas9 Gene Editing to Impact Sensitivity/Resistance to the Anticancer Agent Etoposide." Doctoral dissertation, Ohio State University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=osu1630504949546357

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.