Cell Line Panel Reveals Repair Pathways Important to DNA Damaging Drugs
Differential effects of topoisomerase inhibitors across the DT40 DNA repair panel. Camptothecin is representative of Top1 inhibitors (its clinical derivatives are irinotecan and topotecan). Etoposide is representative of Top2 inhibitors (other clinical Top2 inhibitors include doxorubicin and mitoxantrone). Note the differential involvement of the DNA end-joining genes Ku70, ligase IV and DNA-dependent protein-kinase (black bars). Each horizontal bar represents a genetically altered cell line. Knockout genes are listed on both sides of the plots. Color correspond to different DNA repair pathways: black, non-homologous end joining (NHEJ); salmon, damage checkpoint; blue, homologous recombination (HR); brown, translesion synthesis (TLS); aqua, PARP1; green, removal of Top1 or Top2 covalent complexes; pink, Fanconi anemia (FA) pathway; orange, DNA polymerases and translesion synthesis; purple, FEN1 (flap endonuclease); red, nucleotide excision repair (NER); light green, DNA helicases; yellow, MSH3 (mismatch repair); white, ATG5 (autophagy). Wild-type cells are shown at the top with a reference value of 0.
DNA topoisomerases I and II (Top1 and Top2) facilitate DNA replication by relaxing supercoiled DNA but use distinct mechanisms. Inhibitors of Top1 and Top2 are used clinically as anticancer drugs to lock these proteins onto DNA, eventually generating DNA double strand breaks and killing cells. Cancer cells can develop resistance to Top1 and Top2 inhibitors by increasing the activities of DNA repair pathways, such as homologous recombination (HR) and nonhomologous end joining (NHEJ). Knowing which pathways are critical for a particular drug could provide insight into the lesions it produces as well as indicate complementary treatment targets to enhance its efficacy. However, numerous genes play important roles in various repair pathways, and comprehensively analyzing all of these genes has proven difficult.
To address this problem, Yves Pommier, M.D., Ph.D., Chief of CCR’s Developmental Therapeutics Branch and collaborators from Kyoto University in Japan assembled a panel of 49 isogenic cell lines derived from the DT40 chicken B cell line. Forty-eight of the lines are deficient in one or two DNA repair genes (Figure), and the final line, which lacks ATG5, has impaired autophagy. Using a high-throughput cell viability assay, the researchers assessed the sensitivities of the mutant lines to three Top1 inhibitors and three Top2 inhibitors as compared to wild type DT40 cells.
The Top1 inhibitors used in the study, camptothecin and the indenoisoquinolines LMP400 and LMP776, which are being developed by NCI in collaboration with Purdue University, all trap Top1-DNA cleavage complexes. The Top2 inhibitors, on the other hand, are of two types, the catalytic inhibitor ICRF-193 and the Top2 poisons etoposide and doxorubicin, which are non-intercalating and intercalating drugs, respectively.
The investigators found that the response patterns from the mutant cell lines to the three Top1 inhibitors were very similar. Likewise, they saw significant correlation among the response patterns to the Top2 inhibitors. In contrast, there was a profound difference in the patterns between the Top1 and Top2 inhibitors, suggesting that the assay could distinguish between the two classes of inhibitors based on the different types of DNA lesions they generated.
The most prominent difference between the Top1 and Top2 inhibitors occurred in cells deficient in the NHEJ genes KU70, LIGASE IV, and DNA-PKcs (black bars at the top of the figure). In these cells there was a significant increase in sensitivity to etoposide but a significant increase in resistance to camptothecin. A similar increase in sensitivity to doxorubicin and ICRF-193 in the cells demonstrated that NHEJ components play a critical role in repairing Top2-induced lesions and may serve as useful drug targets to enhance the activity of Top2 inhibitors.
Interestingly, the NHEJ mutants that were strongly resistant to camptothecin were only weakly resistant to LMP776 and showed neither increased resistance nor sensitivity to LMP400. These results suggest that the three inhibitors, which are thought to have a similar mechanism, may each damage DNA in a slightly different manner, affecting the ability of NHEJ components to access the damaged DNA. Genes that more uniformly increased sensitivity to the Top1 inhibitors, if inhibited, might increase the toxicity of these drugs, included HR genes, such as BRCA1 and XRCC2; RAD18; PARP1; TDP1; CtIP; and Fanconi Anemia pathway genes.
When the researchers looked more closely at the Top2 inhibitor response patterns, they found that cells deficient in HR genes had increased sensitivity to etoposide but more limited responses to doxorubicin or ICRF-193. They also saw an unexpected increase in sensitivity to ICRF-193 in TDP2-deficient cells, suggesting that ICRF-193, a catalytic inhibitor, may actually act like the Top2 poison etoposide, which requires TDP2-mediated repair, or that TDP2 may have a role in NHEJ to remove Top2-DNA complexes. The investigators also noted that the doxorubicin response pattern showed relatively low correlation with those of etoposide and ICRF-193, which may be due to its additional known toxic activities.
Finally, in agreement with previous studies, the research team showed that reduced autophagy in the ATG5-deficient cells protects them from Top2 inhibitors and, to a lesser extent, Top1 inhibitors.
Together, these results identified previously unknown differences between drugs of the same class and point to potential targets for enhancing the effectiveness of Top1 and Top2 inhibitors against cancer cells. In addition, the novel panel of cell lines the researchers have collected should be useful for rationally exploring the mechanisms of additional drugs that damage cellular DNA.Summary Posted: Sat, 02/01/2014
Maede Y, Shimizu H, Fukushima T, Kogame T, Nakamura T, Miki T, Takeda S, Pommier Y, Murai J. Differential and Common DNA Repair Pathways for Topoisomerase I- and II-Targeted Drugs in a Genetic DT40 Repair Cell Screen Panel. Mol Cancer Ther. 2014 Jan;13(1):214-20 PubMed Link