A novel small molecule NJH-13 induces pyroptosis via the Ca²⁺ driven AKT-FOXO1-GSDME signaling pathway in NSCLC by targeting TRPV5

Introduction: Pyroptosis represents a mode of programmed necrotic cell death (PCD), mediated by members of gasdermin family (GSDMs), such as GSDME. It is emerging as a promising approach for combating cancer. Notably, GSDME is the key modulator for the switch between apoptosis and pyroptosis in cells. However, GSDME is often downregulated in many malignancies, including lung adenocarcinoma.

Objective: To identify novel pyroptosis inducers in non-small cell lung cancer (NSCLC) and dissect the underlying mechanism.

Methods: Pyroptosis was examined by live cell imaging, PI/Hoechst/Annexin V staining, LDH release assay, ELISA, and western blot assays. DARTS, CETSA, molecular docking was used to identify the target of NJH-13. RNA-seq, qPCR, chromatin immunoprecipitation (ChIP), dual luciferase assays were used elucidate the mechanism.

Results: In this study, NJH-13, an N-containing heterocycle, was screened out and identified to possess the ability to activate GSDME, consequently triggering pyroptosis in NSCLC cells. By using the DARTS strategy, transient receptor potential cation channel subfamily V member 5 (TRPV5) was identified as a potential target of NJH-13. NJH-13 increased intracellular calcium level and triggered oxidative stress, both of which are critical events leading to pyroptosis mediated by GSDME. Mechanistically, NJH-13 enhanced the transcription of GSDME via the protein kinase B (AKT)/forkhead box transcription factor O1 (FOXO1) signaling pathway. ChIP revealed that FOXO1 bound directly to the promoter region of GSDME, thus triggering the GSDME-mediated pyroptosis. Pharmacological and genetic activation of AKT or inhibition of FOXO1 partially rescued NJH-13-induced pyroptotic cell death. Moreover, NJH-13 treatment suppressed tumor growth in vivo.

Conclusion: Taken together, our results revealed that TRPV5 is a distinctive target for manipulating pyroptosis and provided evidence that NJH-13 functions as a potential anti-cancer agent capable of triggering pyroptosis in NSCLC cells.