Doxorubicin-mediated retardation of aggresome formation enhances Carfilzomib-induced cell death synergistically by augmenting ER stress and proapoptotic signaling.

Multiple myeloma (MM), the second most common hematological malignancy, is associated with anemia, renal dysfunction, bone pain, and hypercalcemia. Proteasome inhibitors are a mainstay in MM therapy, but resistance, often mediated by aggresome formation, remains a challenge. Aggresomes sequester misfolded proteins and facilitate their clearance via autophagy. This study focused on identifying anticancer agents that inhibit aggresome formation in response to carfilzomib and exploring their mechanisms of action. We employed MM cell lines U266B1, LP-1, RPMI-8266, and NCI-H929, assessing cell viability using Trypan blue and aggresome structures through immunofluorescence. We quantified protein levels of aggresome regulators, ER and Golgi stress markers, and pro-apoptotic factors using western blotting. Results indicated that doxorubicin significantly enhances carfilzomib-induced cell death and disrupts aggresome formation in U266B1 cells. Mechanistic analyses revealed that doxorubicin downregulated structural components and promotors of aggresome formation, including Vimentin, HDAC6, p62, HSP70, BAG3, HOOK2, and Dynein. Furthermore, doxorubicin potentiated carfilzomib-induced MM cell death by enhancing ER and Golgi stress responses and promoting apoptosis. Notably, doxorubicin did not potentiate proteasome inhibitor-mediated MM cell death in the absence of aggresome-inducing activity. Combined treatment with carfilzomib and doxorubicin failed to induce synergistic cell death in three MM cell lines (LP-1, RPMI-8266, and NCI-H929) lacking aggresome-forming capability, which exhibited higher sensitivity to carfilzomib than U266B1 cells. In conclusion, our findings suggest that doxorubicin enhances carfilzomib-induced cell death by inhibiting aggresome formation and that the combined treatment with carfilzomib and doxorubicin may be an effective strategy to target MM cells with aggresome-forming activity.