The Chemotherapeutic Agent CX-5461 Affects Cell Viability Through a Topoisomerase II Poison-Like Mechanism

Open Access
- Author:
- Dennis, Kady
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Justin R Pritchard, Thesis Supervisor
Joseph C. Reese, Thesis Honors Advisor - Keywords:
- CX-5461
mechanism of action
RNA Polymerase I inhibitor
Topoisomerase II poison - Abstract:
- Small molecule inhibitors have astounding potential for treating cancer, but it has become increasingly clearer that they can produce many off-target effects within a cell. Having a proper understanding of a drug’s mechanism of cytotoxicity is critical for its informed use in the clinic and for basic science research. The anti-cancer chemotherapeutic, CX-5461, was initially developed as an RNA Polymerase I inhibitor, but recent evidence has surfaced showing its capability to induce G-quadruplex formation and cause DNA damage. To further explore this controversy and properly identify CX-5461’s mechanism of cytotoxicity, we performed a validated short hairpin RNA signature drug classification assay, where we identified CX-5461’s mechanism of action as a topoisomerase II poison. Additional genetic and biochemical assays, both in vitro and in a purified system, were able to confirm our findings by comparing to a known topoisomerase II poison, doxorubicin. These findings highlight the importance of using multiple methodologies to confirm a drug’s true mechanism of action, as the misguided usage of the drug can have far reaching impacts. This misclassification could account for CX-5461’s mediocre efficacy against hematologic malignancies in clinical trials and its usage as an RNA Polymerase I functional probe in research labs. Future work could aim to further confirm its classification via a CRISPR knockout drug screen, identify biomarkers for CX-5461 sensitivity, test for its effect on other cellular mechanisms, and re-evaluate other RNA Polymerase I inhibitors for a similar misconception. The work completed here contributes to the field of biochemistry and molecular biology, as it exemplifies the importance of proper identification of drug interactions within cells and the implications of this in the clinic and lab.