Background and objective
G-quadruplex (G4) structures, formed by guanine-rich nucleic acids, are implicated in tumor progression and represent promising therapeutic targets. In laryngeal cancer, the profile and clinical relevance of G4s, as well as the efficacy of G4 ligands, remain unexplored. This study aims to examine the clinical significance of G4 accumulation and investigate the antitumor and immunomodulatory mechanisms of the G4 ligand in laryngeal cancer.
Methods
G4 formation was analyzed in laryngeal cancer and adjacent normal tissues by immunohistochemistry (IHC), and its correlation with patient prognosis was evaluated by Kaplan-Meier survival curves. In vitro, the effects of G4 ligand CX-5461 and Pyridostatin (PDS) on cell proliferation, apoptosis, and cell cycle were assessed. Tumor cells were co-cultured with dendritic cells (DC), and the immunogenic cell death (ICD) of tumor cells was detected by ELISA and immunofluorescence. The DC maturation was also examined by flow cytometry analysis and ELISA. In vivo, the antitumor efficacy of G4 ligand alone and in combination with anti-PD-1 was validated in immunocompetent mouse xenograft models. The tumor immune microenvironment was analyzed by flow cytometry.
Results
Clinical data revealed significantly higher G4 levels in laryngeal tumor tissues compared to adjacent normal tissues, with elevated G4 levels closely associated with poor prognosis. In vitro, CX-5461 and PDS induced apoptosis and G2/M cell cycle arrest. CX-5461 significantly inhibited tumor growth in the subcutaneous tumor model of mice, but PDS was poor in vivo. Furthermore, CX-5461 can promote DNA double-strand breaks in SNU1076 and SNU899 cells, increase the expression level of γ-H2AX, and cause the cells' calreticulin (CRT) to protrude outward while increasing the secretion of high mobility group box 1 (HMGB1). When tumor cells treated with CX-5461 are co-cultured with DC cells, it can promote the increase in the expression of CD80 and CD86 on DC cells, and also increase the secretion of cytokines IL-6, IL-12, and TNF-α. In vivo, CX-5461 significantly suppressed tumor growth and enhanced infiltration of CD8+ T cells, CD45+ immune cells, and DCs in the tumor microenvironment. Combined therapy with CX-5461 and anti-PD-1 demonstrated a striking synergistic antitumor effect in vivo.
Conclusions
G4 is a novel prognostic biomarker for laryngeal cancer. CX-5461 exerts anti-laryngocarcinoma effects by inducing ICD and reshaping the tumor immune microenvironment, providing a strong preclinical rationale for combining CX-5461 with immune checkpoint blockade in laryngeal cancer treatment.
G-quadruplexes (G4s) are non-canonical secondary nucleic acid structures formed by the folding of guanine-rich (G-rich) sequences, which are widely distributed in the promoters of oncogenes (e.g., MYC, KRAS), the initiation regions of DNA replication, and telomeres, and participate in tumorigenesis and progression by regulating transcription, replication, and genomic stability [1], [2], [3]. Recent studies have shown that aberrant G4 formation can also modulate cellular functions through multiple pathways, including impairing mitochondrial function [4], promoting cellular reactive oxygen species (ROS) accumulation [5], and altering epigenetic regulation [6], [7]. Accumulating evidence indicates that G4s are abnormally enriched in various solid tumors (e.g., breast cancer, liver cancer) and hematological malignancies, and their dynamic formation is tightly linked to tumor cell proliferation and metastasis [8], [9], [10], [11], [12], [13], [14].
G-quadruplex ligands are a class of molecules that specifically recognize and stabilize G4 structures, playing critical roles in regulating gene expression and maintaining genomic stability. Most of these ligands engage in π-π stacking interactions with the G-tetrad core of G4s via their planar aromatic ring systems, and further enhance binding selectivity through hydrogen bonds, electrostatic interactions, and van der Waals forces with the G4 grooves [3]. To date, diverse types of G4 ligands have been developed, including synthetic small molecules (e.g., Pyridostatin (PDS) [15], [16], CX-5461 [17], [18], BRACO-19 [19], [20], natural products (e.g., quercetin [21], [22], berberine [23], [24]), and metal complexes [25], [26]. Functionally, G4 ligands can induce telomere shortening, repress oncogene transcription, or disrupt RNA metabolism, thereby inhibiting tumor cell proliferation. This has positioned G4 targeting as a promising avenue for anticancer drug discovery.
CX-5461, also known as Pidnarulex, is derived from the fluoroquinolone compound QQ58 [27], designed to bind to G4s by Hurley's group in 2001. CX-5461 was first reported to exhibit potent anticancer activity in 2011 [28]. Currently, CX-5461 is the sole G4 ligand undergoing active Phase I/II evaluation [28]; monotherapy studies are testing its tolerable dosing in homologous recombination-deficient solid tumours (NCT04890613), assessing safety in MYC-aberrant lymphomas (NCT07069699), and evaluating systemic response in metastatic solid cancers (NCT06606990); concurrently, two pivotal combination trials are accruing: one investigating synergy with cemiplimab in refractory microsatellite stable colorectal cancer (NCT07147231), and another probing safety alongside trastuzumab deruxtecan in HER2-positive solid tumors and breast cancer (NCT07137416). Preclinical studies have confirmed that CX-5461 can also inhibit RNA polymerase I-driven ribosomal DNA (rDNA) transcription [17], [29], [30], [31] and induce topoisomerase poisoning [32]; furthermore, subsequent studies demonstrated that those effects are ultimately driven by the direct G4 stabilization by CX-5461 at G-rich transcribed genomic regions [33], [34]. Notably, CX-5461 and other G4 ligands can also induce DNA damage in tumor cells and enhance antitumor immunity via the cGAS-STING pathway [35], [36], [37], [38], suggesting that G4 ligands may exert immunomodulatory effects by triggering immunogenic cell death (ICD) in cancer cells.
The 2024 statistics from the Global Cancer Observatory show that there were 1,020,472 new cases and 475,065 deaths of head and neck tumors each year, with the incidence and mortality rates continuing to rise [39]. Laryngeal cancer is the second most common head and neck malignancy [40]; its anatomical location means treatment profoundly impacts respiratory, phonatory, and swallowing functions. Despite therapeutic advances, ∼60% of patients present with advanced-stage (III/IV) disease, and recurrence/metastasis rates remain high. Alarmingly, laryngeal cancer is one of the few malignancies with declining 5-year overall survival (66% to 63% over 40 years) [41]. Critically, the existing profile, clinical significance of G4s in laryngeal cancer, and the antitumor potential of G4 ligands in this disease remain completely uncharacterized.
To address this critical knowledge gap, we systematically evaluated the prognostic value of G4s in a clinical laryngeal cancer cohort, then investigated the cytotoxic and immunomodulatory effects of the G4 ligand in vitro and in vivo, and further explored its synergistic potential with anti-PD-1 immunotherapy. This study establishes G4s as a novel prognostic biomarker for laryngeal cancer and reveals that CX-5461 exerts dual antitumor effects via direct cytotoxicity and ICD-mediated immune activation, providing a strong preclinical rationale for translating G4-targeted combination immunotherapy into clinical practice.