Background:Irrespective of the remarkable progress throughout the last decade, multiple myeloma (MM) remains incurable in the vast majority of patients. This might be due to the fact that several key drivers of MM cannot be effectively targeted. The polycomb group protein BMI‐1 represents a prominent example: initially linked to the pathogenesis of MM more than a decade ago, with close associations to high‐risk genes such as MYC and FOXM1, targeting of BMI‐1 is still hindered by the lack of clinically effective compounds.Aims:We provide first insights into the pre‐clinical efficacy of BMI‐1 modulators in MM using a comprehensive set of in vitro and in vivo models.Methods:PTC‐028 and PTC596 were studied in human MM cell lines (HMCLs) using cytotoxicity, colony formation, co‐culture, qPCR, Western Blot, flow cytometry, ELISA, and lentiviral transduction experiments. In vivo, PTC596 was explored in the 5TGM1 murine model of MM.Results:BMI‐1 modulators decreased the level of BMI‐1 protein within 24 h of treatment and demonstrated potent activity in parental and PI‐resistant HMCLs (n = 16) (median IC50 40/57 nM for PTC‐028/PTC596, respectively). IC50s are >10‐fold reduced compared to the previously reported BMI‐1 modulator PTC‐209 (median IC50 680 nM, P < 0.05). Similar potency was observed in co‐culture and colony formation assays. Importantly, a decrease of BMI‐1 protein levels significantly correlated with IC50s (R>0.8, P < 0.01).Surprisingly, BMI‐1 overexpression did not rescue MM cells from BMI‐1 modulators suggesting that alternate mechanisms mediate anti‐survival effects. We therefore performed time course experiments demonstrating potent mitotic arrest 6–24 h post treatment associated with elevated expression of Cyclin B1, AURKA and BIRC5 as well as downregulation of MCL1. Prolonged mitosis was followed by the induction of apoptosis verified by the presence of Annexin V positive cells, cleaved caspases 8 and 9, cleaved PARP, loss of MCL1 protein and depolarization of the mitochondrial membrane potential. Having established mitotic arrest as a key mechanism of BMI‐1 modulators, we next studied central MM signalling cascades. We noted a significant reduction of MYC and AKT activity (but not ERK and GSK3b). Downregulation of MYC and FOXM1 was also observed at protein level demonstrating that BMI‐1 modulators affect key MM genes.Drug combination studies with established agents (IMiDs/Dex/PIs/MEL) showed cell line specific effects. Intriguingly, we observed synergism with BH3 mimetics (targeting BCL2/BCLxL/MCL1) and epigenetic modulators (targeting EZH2/CBP/EP300/BRD4/HDACs). A1331852 and GSK343 were the most promising combination partners, suggesting that mitotic arrest as well as impaired polycomb repressive complex 1 activity due to loss of BMI‐1 might sensitize MM cells to BCLxL and EZH2 inhibition, respectively.Finally, we examined the in vivo activity of PTC596 in the 5TGM1 model demonstrating a dose dependent reduction of BM infiltration and complete eradication of MM cells at 30 mg/kg/biweekly. This remarkable activity was confirmed in an independent experiment demonstrating similar potency (i.e. no detectable MM cells post PTC596 treatment).Summary/Conclusion:We demonstrate promising pre‐clinical activity of BMI‐1 modulators in MM. Moreover, our data suggest that BMI‐1 levels could serve as predictive marker, reveal BMI‐1 modulators as potent anti‐mitotic agents that target key MM genes (e.g. MYC) and show strong synergism with experimental anti‐MM drugs. These results strongly support the clinical evaluation of this novel drug class in myeloma.