Poster abstracts
Poster number 46 submitted by Victor Hernandez
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
Victor A. Hernandez (Pharmaceutics and Pharmacology, The Ohio State University ), Jessika Carvajal (Pharmaceutics and Pharmacology, The Ohio State University ), Jonathan L. Papa, Nicholas Shkolnikov (Pharmaceutics and Pharmacology, The Ohio State University ), Hatice Gulcin Ozer (Biomedical Informatics, The Ohio State University ), Jack C. Yalowich, Terry S. Elton (Pharmaceutics and Pharmacology, The Ohio State University ), Junan Li (Pharmaceutics and Pharmacology, The Ohio State University )
Abstract:
The human DNA topoisomerase IIα (170 kDa, TOP2α/170) is an essential enzyme that resolves DNA topological entanglements that form during chromosome condensation and segregation, making it indispensable for the survival of highly proliferating cells including cancer cells. Hence, TOP2α is a prominent target for anticancer therapies. Some of the most widely used TOP2 targeting drugs such as etoposide, stabilize a TOP2α/170-DNA cleavage complex preventing the religation of broken DNA strands and exerting cytotoxic effects by the accumulation of DNA damage.
Acquired chemoresistance to TOP2 targeting drugs continues to be a major obstacle in cancer treatment in the clinic. Our lab 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. TOP2α/90 is the translation product of novel alternatively spliced mRNA via intron 19 (I19) retention and processing. TOP2α/90 is a determinant of acquired resistance to etoposide through a dominant-negative effect related to heterodimerization with TOP2α/170.
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, CRISPR/Cas9 was utilized for gene editing of TOP2α in K/VP.5 cells to enhance I19 removal and circumvent acquired TOP2α-mediated drug resistance. Gene edited clones were identified by qPCR and verified by sequencing. 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. In addition, gene editing to optimize the TOP2α E19/I19 SS in K/VP.5 cells circumvented resistance to TOP2α-targeted agents.
Conversely, experiments were performed whereby the E19/I19 SS in etoposide sensitive K562 cells was gene edited to decrease/eliminate splicing at this boundary. Inhibiting splicing of the E19/I19 SS in K562 cells resulted in a decrease in full-length TOP2α/170 expression and induction of resistance to etoposide. Together, results support the role of alternative splicing at the E19/I19 boundary as a determinant of resistance to TOP2α-targeting agents.
Keywords: CRISPRCas9, Alternative RNA Splicing , Drug Resistance