Topoisomerase levels determine chemotherapy response in vitro and in vivo

Abstract

Topoisomerase poisons are chemotherapeutic agents that are used extensively for treating human malignancies. These drugs can be highly effective, yet tumors are frequently refractory to treatment or become resistant upon tumor relapse. Using a pool-based RNAi screening approach and a well characterized mouse model of lymphoma, we explored the genetic basis for heterogeneous responses to topoisomerase poisons in vitro and in vivo. These experiments identified Top2A expression levels as major determinants of response to the topoisomerase 2 poison doxorubicin and showed that suppression of Top2A produces resistance to doxorubicin in vitro and in vivo. Analogously, using a targeted RNAi approach, we demonstrated that suppression of Top1 produces resistance to the topoisomerase 1 poison camptothecin yet hypersensitizes cancer cells to doxorubicin. Importantly, lymphomas relapsing after treatment display spontaneous changes in topoisomerase levels as predicted by in vitro gene knockdown studies. These results highlight the utility of pooled shRNA screens for identifying genetic determinants of chemotherapy response and suggest strategies for improving the effectiveness of topoisomerase poisons in the clinic.

Fig. 1.

A rapid RNAi enrichment screen identifies mediators of doxorubicin resistance. (A) The GFP competition assay. Differential survival of shRNA-transduced cells (green) relative to control cells (colorless) is assayed by changes in the percentage GFP in the surviving cell population. (B) GFP competition assay data from lymphoma cells infected with the indicated shRNA either untreated or 24 h after doxorubicin (DXR) treatment at the indicated doses. (C) Immunoblotting of lysates from lymphoma cells transduced with shRNAs targeting p53, Chk2, and Top2A either untreated or treated for 8 h with 31 ng/ml doxorubicin to stabilize p53. Tubulin serves as a loading control.

Fig. 2.

Suppression of Top2A expression causes resistance to topoisomerase 2 poisons in vitro. (A and B) Flow cytometric analyses of lymphoma cells expressing shTop2A 668 (A) or shp53 1224 (B) after 24 h of the indicated drug treatments. DXR, doxorubicin; ETOP, etoposide; MAF, maphosphamide; CPT, camptothecin. (C) Lymphoma cells, transduced singly with four independent Top2A shRNAs, were puromycin-selected and treated with doxorubicin for 24 h at the indicated doses. Viability was assayed by flow cytometry (FSC versus SSC) and plotted relative to untreated controls. Error bars are ±SEM from three replicates. (D) Immunoblotting of lymphoma cell lysates expressing no short hairpin (Vector) or Top1, Top2A, or p53 shRNAs in the presence or absence of doxorubicin (DXR; 15.6 ng/ml for 8 h).

Fig. 3.

Top2A knockdown causes doxorubicin resistance in vivo. In vivo competition assay is shown. (A) GFP flow cytometry plots. Lymphoma cells were infected in vitro with GFP-tagged shTop2A 668 or 849, shp53, or vector control constructs (A Left). These cells were injected into the tail vein of syngeneic recipient mice (five mice per cohort) and were monitored daily for tumors by palpation. Upon tumor onset (day 0), one mouse from each cohort was killed, and lymphoma cells were assayed for percentage GFP⁺ (A Middle). The remaining mice were treated with doxorubicin (10 mg/kg i.p. injection), and tumors were harvested upon relapse and assayed for percentage GFP (A Right). (B) Kaplan–Meier tumor-free survival curves. Vector, shTop2A, and shp53 tumors were FACS-sorted to 100% GFP⁺ before injection into recipient mice and DXR-treated as for A at day 0.

Fig. 4.

Top1 knockdown causes camptothecin resistance in vitro and in vivo. (A) Top1 knockdown causes resistance to camptothecin but hypersensitizes to the topoisomerase 2 poisons, doxorubicin and etoposide, as shown by a GFP competition assay 24 h after drug treatment. (B) In vitro viability assays of puromycin-selected (shRNA-containing) cells for four independent shRNAs targeting Top1, after 24-h camptothecin treatment. Error bars are ±SEM from three replicates. (C) Immunoblotting of Eμ-Myc;Arf^−/− lymphoma cell lysates with or without camptothecin (31 nM CPT, 8 h). (D) Kaplan–Meier survival curve. Eμ-Myc;Arf^−/− lymphomas were infected in vitro with vector control or shTop1 2215 and were FACS-sorted to 100% GFP⁺ before injection into recipient mice. Upon lymphoma onset (day 0) mice were treated with irinotecan (CPT-11), a clinically relevant camptothecin derivative (50 mg/kg intraperitoneal injection daily for 2 days) and monitored for survival.

Fig. 5.

Top1 knockdown can sensitize to doxorubicin treatment in vivo. (A) Top1 knockdown sensitizes Eμ-Myc;Arf^−/− lymphomas to doxorubicin in vivo, as shown by an increased in vivo tumor-free survival after doxorubicin treatment (10 mg/kg, day 0). shTop1 data are pooled from four shTop1 1600 and four shTop1 2215 mice. (B) Predicted changes in topoisomerase expression levels occur spontaneously during treatment failure in vivo. Immunoblotting analysis of untreated lymphomas and postdoxorubicin-treated relapses from A.