Cinobufagin overcomes bortezomib resistance in multiple myeloma strains by targeting SEC62/TRPM4-mediated NECSO.

Background: Necrosis by sodium overload (NECSO) is a poorly understood, novel form of cell death implicated in cancer. TRPM4 is the only protein currently linked to NECSO and is downregulated in bortezomib-resistant multiple myeloma (MM) patients and cell lines.

Purpose: To investigate the potential of cinobufagin to overcome bortezomib resistance in MM and elucidate its underlying mechanism of action, particularly regarding TRPM4 and NECSO induction.

Study Design: This study combined in vitro investigations using bortezomib-resistant MM cell lines (8226-BTZR, KMS-11-BTZR) and in vivo xenograft mouse models with comprehensive molecular interaction studies.

Methods: Cell proliferation assays, xenograft tumor growth monitoring, and immunohistochemistry (CD138/Ki67) were used to assess cinobufagin's effects. NECSO induction was evaluated mechanistically. TRPM4 expression was analyzed. The interaction between cinobufagin and its target was identified using LiP-MS, molecular docking, and molecular dynamics simulations, and validated by MST and CETSA assays. Protein-protein interactions (SEC62/TRPM4), ubiquitination status, and proteasomal degradation of TRPM4 were assessed by SPR, molecular dynamics simulations and immunoprecipitation assay. The role of SEC62 was confirmed using knockdown experiments.

Results: Cinobufagin effectively inhibited proliferation of bortezomib-resistant MM cells in vitro and suppressed tumor growth while reducing CD138/Ki67 expression in vivo. Mechanistically, cinobufagin induced NECSO. It upregulated TRPM4 expression and was found to directly bind SEC62. SEC62 interacts with TRPM4, promoting its ubiquitination and proteasomal degradation. Cinobufagin disrupts the SEC62/TRPM4 interaction, thereby stabilizing TRPM4 by inhibiting its ubiquitin-proteasome-mediated degradation. SEC62 knockdown attenuated cinobufagin's effects, confirming SEC62's role in mediating the reversal of bortezomib resistance.

Conclusion: These findings identify TRPM4 as a promising therapeutic target in bortezomib-resistant MM. Cinobufagin overcomes bortezomib resistance by modulating the SEC62-TRPM4 axis, stabilizing TRPM4, and uniquely inducing the previously unexploited NECSO cell death pathway. This highlights cinobufagin's significant therapeutic potential.