CXCR4-targeted nanotoxins induce GSDME-dependent pyroptosis in head and neck squamous cell carcinoma

Background: Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC. Methods: T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4⁺ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining. Results: First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4⁺ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy. Conclusions: Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.