G-quadruplex targeting by CX-5461 as a novel therapeutic strategy in testicular germ cell tumors

Testicular germ cell tumors (TGCTs) are highly curable malignancies; however, resistance to cisplatin-based therapy remains a critical clinical challenge. G-quadruplexes (G4s) are specific guanine rich, four-stranded DNA structures that are enriched at distinct genomic regions. Due to their stability, G4s can influence genome stability, gene expression, DNA replication as well as telomere maintenance. G4 stabilization by small molecules has been shown to have anticancer effects in several tumors but remains underexplored in TGCTs. Here, we systematically evaluated the effect of six different G4- ligands (CX-5461, CX-3543, BRACO19, PDS, CM03 and RHPS4) on TGCT cell lines and non-malignant fibroblasts. Among those G4 ligands, CX-5461 showed strongest cellular changes in TGCTs. We observed dose- and time-dependent G4 stabilization upon CX-5461 treatment in TGCT cells, accompanied by impaired RNA polymerase I-dependent transcription and induction of DNA double-strand breaks, whereas only minimal effects were observed in control fibroblasts. These effects led to the activation of p53 signaling, and consequently an upregulation of canonical p53 target genes, including CDKN1A, MDM2, PIDD1, GADD45A, ZMAT3, SESN1 and PPM1D, resulting in G2/M cell-cycle arrest and apoptosis. The presence of potential quadruplex forming sequences (PQSs) in the promoters or gene bodies of these genes suggests that G4 structures may contribute to the p53 transcriptional response. Collectively, these data demonstrate that CX-5461 appears as highly potent G4-ligand inducing p53-dependent molecular cascades leading to cell death in germ cell tumors. Unlike in many other tumors, p53 remains functionally intact in TGCTs, suggesting that CX-5461-mediated activation of the p53 pathway could be selectively targeted as a therapeutic strategy in TGCTs.