Testicular cancer is a rare but socially significant tumor: it affects men of reproductive age, and its incidence is rising. Existing cisplatin-based chemotherapy is effective, but in some patients the tumor becomes resistant, and then the prognosis deteriorates sharply. Alongside this work, the archive contains studies on reducing treatment toxicity and analyzing the tumor microenvironment in prostate cancer, which seek gentler approaches and biomarkers. This paper goes in the same direction but from a different angle: instead of optimizing doses, it proposes a fundamentally different target, non-canonical DNA structures that are more common in tumor cells than in healthy ones.
The authors took seven germ cell tumor cell lines (seminoma, embryonal carcinoma, choriocarcinoma, and yolk sac tumor) and two healthy fibroblast lines. Cells were treated with six different G-quadruplex ligands at concentrations from 0.1 to 5 µM for 24, 48, or 72 hours, and viability was measured by XTT assay. For the lead compound, CX-5461, they additionally assessed G4 stabilization by flow cytometry with the BG4 antibody, the level of double-strand DNA breaks via phosphorylated histone H2AX, cell cycle phase distribution, and the proportion of apoptotic cells. The transcriptomic response was studied by 3'-RNA sequencing, and potential G4 structures in p53 target genes were predicted with the pqsfinder program.
CX-5461 was the most selective: its IC50 for tumor cells at 72 hours was 0.1-1.6 µM (except for the GCT72 line at 3.8 µM), whereas for healthy fibroblasts it exceeded 16 µM. Treatment with CX-5461 dose-dependently increased the BG4 signal in tumor cells by 1.5-3.8 times but did not change it in fibroblasts. The 47S pre-rRNA, a product of RNA polymerase I, decreased in all tumor lines, most strongly in NT2/D1. The level of γH2AX increased 2.7-4.9 times in embryonal carcinoma cells and did not change in fibroblasts. The proportion of cells in G2/M phase increased from 20-33% to 45-85% at 2 µM CX-5461. Apoptosis reached 45-55% in embryonal carcinoma cells, whereas in fibroblasts it remained below 10%. RNA sequencing revealed activation of the p53 pathway in all lines, with seven common target genes (CDKN1A, MDM2, PIDD1, GADD45A, ZMAT3, SESN1, PPM1D) containing from 12 to 292 potential G4 structures in promoters and from 332 to 1936 in gene bodies.
The work shows that G-quadruplexes are a real vulnerability of germ cell testicular tumors that can be exploited for therapy. Unlike neighboring studies focused on doses and the microenvironment, this one for the first time systematically tested six G4 ligands on a panel of lines representing all histological subtypes of testicular cancer and showed that CX-5461 acts through activation of the intact p53 pathway. This is important because TP53 mutations are extremely rare in testicular cancer, unlike in most other solid tumors. The selectivity for tumor cells with minimal impact on healthy fibroblasts makes CX-5461 a promising candidate for clinical trials, especially in cases of cisplatin resistance.
The next logical step is to test whether CX-5461 can restore sensitivity to cisplatin in resistant lines and in animal models. It also remains to be determined which specific G4 structures in the promoters and gene bodies of the p53 pathway are functionally important for the response. If these structures can be mapped, it will become possible to predict a tumor's sensitivity to G4 ligands from its genome and to develop combination regimens with minimal toxicity.
The results could influence the development of targeted therapy for testicular cancer and, more broadly, the understanding of the role of G-quadruplexes in carcinogenesis and the DNA damage response.
Test the synergy of CX-5461 with cisplatin on resistant lines; validate the role of predicted G4s in regulating p53 genes using genome editing methods.
The work is directly related to the unresolved problem of overcoming cisplatin resistance in germ cell testicular tumors, as well as to the search for selective targets that could reduce the long-term toxicity of therapy in young patients.
G-quadruplexes are four-stranded DNA structures that form in guanine-rich regions. They resemble stacked square plates and can block the work of enzymes that copy DNA.
Not published on arXiv. Here are its page, the PDF and the formalization: you can check it yourself, not only read our account.