INVESTIGATING PROTEASOME INHIBITOR RESISTANCE IN MULTIPLE MYELOMA

Multiple Myeloma (MM) is an incurable plasma cell malignancy arising in the bone marrow. The development of drug resistance remains a major cause of treatment failure. Since proteasome inhibitors (PIs) represent a cornerstone of MM therapy, we conducted a gain-of-function CRISPR activation screen to identify functional mediators of resistance to the PI carfilzomib (CFZ). This approach identified the E3 ligase MDM2 and the deubiquitinase ZUP1 among the genes enriched in cells treated with CFZ. MDM2 is known to be upregulated in advanced and relapsed MM. Our results show that MDM2 transactivation conferred CFZ resistance in AMO-1 cells. Pharmacological inhibition of MDM2 with NVP-CGM097 synergized with PIs across multiple MM cell lines independently of TP53 status. Mechanistically, MDM2 inhibition induced p21 upregulation, cellcycle arrest, and reduction of c-MYC expression, accompanied by impaired activation of DNA damage response mediators. Importantly, the combination retained efficacy in MM–stromal coculture models and in primary patient samples, including high risk cases harboring del(17p), while sparing normal peripheral blood mononuclear cells. Analysis of patient datasets further revealed that ZUP1 expression is elevated in MM patients compared to healthy donors, with higher levels correlating with poorer survival. Functionally, sgRNA-mediated transactivation of ZUP1 enhanced MM cell recovery after CFZ-induced cytotoxicity and conferred a competitive growth advantage under PI treatment. Consistent with these findings, ZUP1 cDNA overexpression and gene silencing further confirmed its role in regulating MM cell response to PI. Mechanistically, ZUP1 transactivation altered total ubiquitination, reduced CFZinduced cell cycle arrest, and attenuated DNA damage signaling. Collectively, these findings highlight the central role of the ubiquitin-proteasome system (UPS) and DNA damage signaling in PI resistance and identify MDM2 and ZUP1 as functional drivers in MM