Upamostat potentiates bortezomib cytotoxicity and delays proteasome recovery in bortezomib-resistant malignant lymphoma cells through modulation of the DDI2/NRF1 axis.

Proteasome inhibitors such as bortezomib (BTZ) are potent against hematological malignancies, but acquired resistance remains a major obstacle to durable clinical responses. DNA damage-inducible 1 homolog 2 (DDI2)-mediated processing of nuclear factor erythroid 2-related factor 1 (NRF1) contributes to adaptive proteasome restoration after inhibition, and nelfinavir (NFV) is the best-characterized small-molecule inhibitor of DDI2. Since previous studies have focused primarily on aspartic protease inhibitors, and several serine protease inhibitors share structural similarities with NFV, we screened selected serine protease inhibitors to modulate the DDI2/NRF1 axis. Among them, Upamostat (UMS), a clinically tested urokinase-type plasminogen activator inhibitor, altered BTZ-induced NRF1 processing, as reflected by a reduced CF/UF ratio, in U937 cells, whereas another urokinase inhibitor, UK-371804, had no such effect, suggesting that this activity was not a general consequence of urokinase inhibition. TSA showed that UMS increased the thermal stability of DDI2, supporting a direct interaction, and docking analysis suggested its accommodation within the retroviral protease-like domain of DDI2. In BTZ-resistant U937 (BTZ-R) cells, basal NRF1 mRNA expression and proteasome activity were elevated. UMS enhanced BTZ-induced reactive oxygen species generation, caspase activation, Annexin V positivity, and loss of cell viability, with stronger synergistic effects in BTZ-R cells than in parental cells. Although UMS did not suppress the early induction of NRF1 or proteasome-subunit transcripts, it significantly delayed recovery of chymotrypsin-like and caspase-like proteasome activities after BTZ washout. These findings identify UMS as a candidate DDI2/NRF1-axis modulator that potentiates BTZ-induced cytotoxicity and delays functional proteasome recovery in BTZ-resistant malignant lymphoma cells.