Abstract 2972: Trabectedin exerts its potent and selective antitumor effects by irreversibly covalently bonding to duplex stem loops in G-quadruplexes

Abstract Trabectedin, a marine-derived anti-tumor agent from the ecteinascidin family, shows effectiveness when treating cancers such as Ewing Sarcoma, small cell lung cancer (SCLC) and ovarian cancer. It features a unique carbinolamine group that forms reversible bonds with the N2 position of guanine, distinguishing it from typical DNA alkylating agents. Structurally, trabectedin also includes a C-subunit that widens the DNA minor groove, inducing transcriptional replication stress and genome instability. Given the sensitivity of Ewing Sarcoma to trabectedin and the role of G-quadruplexes (G4s) in binding EWSR1—especially those with longer stem loops—this study aims to redefine trabectedin’s molecular target by exploring its potential interaction with G4 structures. Building on these insights, we designed experiments using trabectedin in combination with wild-type (WT) and mutant MYCN G4s to assess whether trabectedin could irreversibly bond covalently to a consensus sequence within a duplex stem loop associated with a G4. Circular dichroism (CD) analysis confirmed that trabectedin stabilized a G4 containing a consensus covalent bonding site within the associated hairpin loop. Mass spectrometry (MS) demonstrated that a guanine in the trabectedin consensus binding site is essential for this irreversible covalent binding. Additionally, CD and MS showed that when trabectedin was bound covalently to the hairpin stem loop, it displaced GSA0932, a molecule that binds non-covalently to both the core structure and stem loop. Finally, we observed that trabectedin treatment significantly increased G4 frequency within cells. On a final note, trabectedin irreversibly binds to the N2 of guanine within a duplex stem loop of a G4, fully accounting for its downstream effects on transcription-dependent replication stress and genomic instability. While this data specifically pertains to trabectedin, other members of the ecteinascidins, which possess structurally distinct C-subunits, appear to exhibit varied downstream effects, such as on transcription. These differences may arise from variations in sequence selectivity at the stem-loop level, leading to the targeting of distinct G-quadruplex structures. Citation Format: Alena Safarova, Marta Martínez Diez, Raquel M. Altares, Pablo M. Aviles, Marcelo Lima Ribeiro, Carmen Cuevas, Laurence H. Hurley. Trabectedin exerts its potent and selective antitumor effects by irreversibly covalently bonding to duplex stem loops in G-quadruplexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2972.