New Topoisomerase I mutations are associated with resistance to camptothecin

Abstract

BackgroundTopoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance.MethodsWe previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing.ResultsIn SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells.ConclusionsThese results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.

Rottlerin potentiates camptothecin-induced cytotoxicity in human hormone refractory prostate cancers through increased formation and stabilization of topoisomerase I-DNA cleavage complexes in a PKCδ-independent pathway

“…The correlation between TOP1cc formation and DNA damage response has been reported in a wide variety of cancer cells when exposed to topoisomerase I inhibitors [27,28]. Moreover, it has been reported that in SN38 (an active metabolite of irinotecan) resistant cancer cell clones, the presence of topoisomerase I mutations did not change the expression level and activity of topoisomerase I, but decrease the TOP1cc associated with a reduction in DSBs when challenged with SN38 [29]. These studies suggest that the formation of TOP1cc is critical to DNA damage and apoptosis caused by topoisomerase I inhibitors.…”

Enhanced Delivery of SN-38 to Human Tumor Xenografts with an Anti-Trop-2–SN-38 Antibody Conjugate (Sacituzumab Govitecan)

“…Levels of SN-38 were found in the tumor of animals given IMMU-132, and as the topoisomerase I activity of SN-38 is enhanced in Sphase cells, maintaining an SN-38 presence for 3 days compared with 8 hours would be another advantage. Because resistance to irinotecan therapy can occur for a variety of reasons (24)(25)(26)(27), it is uncertain whether the ability to enhance SN-38 delivery with this ADC will affect resistance in a clinically meaningful manner. We Figure 3.…”

Sorafenib Overcomes Irinotecan Resistance in Colorectal Cancer by Inhibiting the ABCG2 Drug-Efflux Pump

“…These cell lines have been obtained in 2000, were amplified and frozen, and one aliquot of each was thawed for this project, although no authentication was done by the authors. The SN-38-resistant HCT116 and SW48 cell clones were obtained as previously described (7,9,12). Briefly, the reference SN-38-sensitive HCT116 cell clone (HCT116-s) was exposed to 10 nmol/L SN-38 and cloned to obtain the HCT116-SN6 and HCT116-A2 clones.…”

“…Like other camptothecin derivatives, SN-38 is an inhibitor of topoisomerase I, a nuclear enzyme needed for replication and transcription through relaxation of supercoiled DNA (4,5). Cellular mechanisms causing irinotecan/SN-38 resistance have been reported for each step of the CPT-11 pathway (6)(7)(8)(9). Among them, it has been shown that cultured cells that are resistant to camptothecin derivatives have reduced intracellular drug accumulation, mediated by the ATPbinding cassette (ABC) transporter ABCG2 (10), especially in colon cancer cells (11,12).…”