The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning

Abstract

Small molecules can affect many cellular processes. The disambiguation of these effects to identify the causative mechanisms of cell death is extremely challenging. This challenge impacts both clinical development and the interpretation of chemical genetic experiments. CX-5461 was developed as a selective RNA polymerase I inhibitor, but recent evidence suggests that it may cause DNA damage and induce G-quadraplex formation. Here we use three complimentary data mining modalities alongside biochemical and cell biological assays to show that CX-5461 exerts its primary cytotoxic activity through topoisomerase II poisoning. We then show that acquired resistance to CX-5461 in previously sensitive lymphoma cells confers collateral resistance to the topoisomerase II poison doxorubicin. Doxorubicin is already a frontline chemotherapy in a variety of hematopoietic malignancies, and CX-5461 is being tested in relapse/refractory hematopoietic tumors. Our data suggest that the mechanism of cell death induced by CX-5461 is critical for rational clinical development in these patients. Moreover, CX-5461 usage as a specific chemical genetic probe of RNA polymerase I function is challenging to interpret. Our multimodal data-driven approach is a useful way to detangle the intended and unintended mechanisms of drug action across diverse essential cellular processes.

Keywords: CX-5461; chemotherapy; mechanism of action; systems biology; topoisomerase.

Fig. 1.

CX-5461 PRISM data correlates with TOP2A poisons and elicits a DNA damage response. (A) Correlation plots between RNA polymerase I sgRNA CERES phenotypes versus CX-5461 PRISM (log2). Clockwise from Top Left for POLR1A, POLR1B, POLR1C, and POLR1E. (B) Correlation plots between PRISM data of CX-5461 versus voreloxin and etoposide, the no. 2 and no. 6 top hits, respectively. (C) Structures of CX-5461 and other quinolone topoisomerase inhibitors, from Left to Right, CX-5461, Ciprofloxacin, and voreloxin, also known as Vosaroxin. The quinolone group of Ciprofloxacin is outlined by a red box. (D) Quantification of γ-H2AX staining as assessed by flow cytometry 12 h posttreatment with the indicated drug at their respective IC50 values. Each dot represents a different replicate, with three biological replicates, each with three technical replicates, per cell line, except A549 which had only two biological replicates. Data are represented as mean ± SEM of the fold change of % γ-H2AX⁺ relative to DMSO and P values were calculated via ANOVA with Holm–Sidak’s multiple comparison’s test (*P < 0.05, **P < 0.01, or ***P < 0.001).

Fig. 2.

CX-5461 classifies as a Top2A poison via Eµ-Myc RNAi drug classification signatures. (A) Dose–response of Eµ-Myc p19^arf−/− to CX-5461 as determined by flow cytometry with propidium iodide exclusion as a live/dead marker. Data are represented as mean ± SEM relative to vehicle treatment with three technical replicates. (B) The RNAi signatures of representative Top2A poisons (Top), representative transcription/translation inhibitors (Bottom), and CX-5461 (Middle). K-nearest neighbors-based algorithmic results are shown on the Right side. (C) PCA of the average eight shRNA signatures of all drugs in our reference dataset. Different colors represent the distinct classification categories. Percent variance explained by each component is represented on the axes. (D) PCA of all DNA damage drug eight shRNA signature replicates in our reference set. Black represents all other DNA damage reference set drugs, blue represents replicates from Top2 poisons, and red represents the replicates for CX-5461.

Fig. 3.

Chemical genetic and biochemical characterization of CX-5461 cytotoxicity. (A) GFP competition assays with shTOP1 and shTOP2A treated with canonical topoisomerase inhibitors doxorubicin and camptothecin, canonical RNA polymerase I inhibitors actinomycin D, mithramycin A, and BMH-21 and CX-5461. Vincristine and 5-fluorouracil are negative controls. Heatmap values represent the mean log2(RI) of at least three biological replicates. (B) Quantification of γ-H2AX staining as assessed by flow cytometry 12 h posttreatment with the indicated drug at their respective IC50 values. Each dot represents a different replicate, with three biological replicates and three technical replicates. Data are represented as mean ± SEM of the fold change of % γ-H2AX⁺ relative to DMSO and P values were calculated via Kruskal–Wallis with Dunn’s multiple comparison’s test (*P < 0.05, **P < 0.01). (C) GFP competition assays with two validated RPL11 hairpins treated with canonical topoisomerase inhibitors doxorubicin and camptothecin, canonical RNA polymerase I inhibitors actinomycin D, mithramycin A, and BMH-21 and CX-5461. Rapamycin and 5-fluorouracil serve as additional positive controls as they are known ribosome biogenesis stress inducers. Vincristine and cisplatin are negative controls. Heatmap values represent the mean log2(RI) of at least three biological replicates. (D) Quantification of foci/nucleus of γ-H2AX immunofluorescence staining of A375 cells treated at an IC50 dose of the indicated drugs for 12 h. Data are represented as mean ± SEM with >30 nuclei for each condition and P values were calculated via Kruskal–Wallis with Dunn’s multiple comparison’s test (*P < 0.05, **P < 0.01). (E) GFP competition assays with multiple shTop2A hairpins and shFirefly (shFF) hairpin as a control in A375 cells with flow cytometry conducted 72 h after treatment at IC50 with the indicated drugs. Data are represented as mean ± SEM with three biological replicates for each condition and P values were calculated with Kruskal–Wallis with Dunn’s multiple comparison’s test (*P < 0.05, **P < 0.01). (F) Quantification of in vitro kinetoplast DNA decatenation in the presence of the indicated inhibitors. Data are represented as mean ± SEM for each condition and P values were calculated via two-tailed Welch’s t test (*P < 0.05).

Fig. 4.

Evolution of CX-5461–doxorubicin collateral resistance in a model of lymphoma. (A) Schematic for resistant clone creation. First, Eµ-Myc p19^arf−/− cells were dosed with either CX-5461 or doxorubicin to kill 99% (LD99) of cells at 48 h. At that time, single surviving cells were sorted into 96-well plates containing media with a concentration of drug that would normally eliminate 20% (LD20) of naïve cells. As wells become confluent, about 12 d after sorting, clones were expanded and redosed with the initial drug to determine if a resistant clone was isolated. Resistant clones were then dosed with all four drugs: doxorubicin, CX-5461, actinomycin D, and vincristine. (B) Full dose–responses for the top six CX-5461 resistant clones and the parental line (in red). Each dot represents mean ± SEM. (C) Full dose–responses for the top doxorubicin-resistant clones and the parental line (in red). Each dot represents mean ± SEM.