7MeERT Engages PSMD14-Associated Proteasome Lid Components and Suppresses Glycolysis via K63-Linked Ubiquitination and PKM2 Nuclear Exclusion

INTRODUCTION: Altered energy metabolism is a central feature of cancer. Among ubiquitin-dependent regulatory mechanisms, K63-linked ubiquitination has attracted attention as a non-proteolytic means of modulating metabolic enzymes. PSMD14 (Rpn11), a JAMM-family deubiquitinase located in the 19S proteasome lid, has been linked to malignant progression, but the role of pharmacological perturbation of PSMD14-associated proteasome lid biology in cancer metabolism has not been fully defined. METHODS: To examine the metabolic effects of 7MeERT, an Ertredin derivative, we performed extracellular flux assays in multiple cancer cell types. Drug-protein engagement was assessed using lysate-based DARTS and CETSA. Changes in ubiquitination and PKM2 localization were analyzed by immunoblotting, TUBE2 pulldown, and confocal immunofluorescence analysis, and PSMD14 knockdown was used to examine the mechanism. RESULTS: 7MeERT reduced basal and/or compensatory glycolysis in glioblastoma, triple-negative breast cancer, and pancreatic cancer cells. Lysate-based DARTS and CETSA supported engagement of PSMD14 and other proteasome lid subunits by 7MeERT, whereas the base subunit PSMC2 was unaffected. In contrast to MG132, 7MeERT did not cause broad accumulation of ubiquitinated proteins. Instead, it preferentially increased K63-linked ubiquitination, enhanced PKM2 ubiquitination, and decreased nuclear PKM2 localization. PSMD14 knockdown reproduced the K63-linked ubiquitination, PKM2 localization, and glycolytic phenotypes associated with 7MeERT treatment. In addition, 7MeERT more strongly suppressed the proliferation of TNBC cells with relatively high PSMD14 expression than that of normal mammary epithelial cells. DISCUSSION: These data support a model in which engagement of PSMD14-associated proteasome lid components perturbs non-proteolytic ubiquitin signaling and disrupts PKM2-linked glycolytic regulation in cancer cells. CONCLUSION: 7MeERT suppresses cancer cell glycolysis in association with engagement of PSMD14- associated proteasome lid components and alterations in K63-linked ubiquitination and PKM2 nuclear localization. This mechanism suggests that modulation of PSMD14-associated deubiquitination may provide an approach distinct from conventional proteasome inhibition.