The notion that plasma cells (PCs) are terminally differentiated has prevented intensive research in multiple myeloma (MM) about their phenotypic plasticity and differentiation. Here, we demonstrated in healthy individuals (n = 20) that the CD19 − CD81 expression axis identifies three bone marrow (BM)PC subsets with distinct age-prevalence, proliferation, replication-history, immunoglobulin-production, and phenotype, consistent with progressively increased differentiation from CD19+CD81+ into CD19 − CD81+ and CD19 − CD81 − BMPCs. Afterwards, we demonstrated in 225 newly diagnosed MM patients that, comparing to normal BMPC counterparts, 59% had fully differentiated (CD19 − CD81 −) clones, 38% intermediate- Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts differentiated (CD19 − CD81+) and 3% less-differentiated (CD19+CD81+) clones. The latter patients had dismal outcome, and PC differentiation emerged as an independent prognostic marker for progression-free (HR: 1.7; P = 0.005) and overall survival (HR: 2.1; P = 0.006). Longitudinal comparison of diagnostic vs minimal-residual-disease samples (n = 40) unraveled that in 20% of patients, less-differentiated PCs subclones become enriched after therapy-induced pressure. We also revealed that CD81 expression is epigenetically regulated, that less-differentiated clonal PCs retain high expression of genes related to preceding B-cell stages (for example: PAX5), and show distinct mutation profile vs fully differentiated PC clones within individual patients. Together, we shed new light into PC plasticity and demonstrated that MM patients harbouring less-differentiated PCs have dismal survival, which might be related to higher chemoresistant potential plus different molecular and genomic profiles.

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respectively BCL2 and MCL1. We also intended to decipher the mechanism of action of this combination and to identify resistance factors. Methods: For this study, we treated a panel of 25 Human Myeloma cell lines (HMCLs) and a cohort of more than 40 myeloma patient samples with Venetoclax and S63845 to identify those resistant to both agents. This group of resistant HMCLs and patient samples was treated with the combination S63845/Venetoclax at low doses. siRNA assays were performed to identify the role of effectors BAX and BAK and BH3-only proteins BIM and NOXA in combination-induced apoptosis. To understand the mechanism of action of this combination, we also studied the activation of BAX and BAK using specific antibodies. Finally, the role playing by BCLXL in the resistance to the S63845/Venetoclax combination was studied by both silencing and overexpression experiments. Results: One third of our cell line collection as well as our patient cohort was resistant to each BH3 mimetic. The combination was highly effective and synergic at low doses in three fourth of these cells. We demonstrated that the efficiency of the combination was equally dependent on BAX and BAK and that the simultaneous silencing of both effectors completely abrogated the combination effect. Conversely, the response to S63845 was mainly dependent on BAK. Interestingly, we also showed that the combination-induced apoptosis was associated with a hetero-complex formation between BAX and BAK. The BH3-only protein BIM, but not NOXA, was also shown to play a role in the combination response. Finally, BCLXL remained a resistance factor to the S63845/Venetoclax combination in agreement with its role in the resistance to both MCL1 and BCL2 BH3 mimetics alone. Summary/Conclusion: Our work demonstrates the interest of using the combination of two BH3 mimetics targeting both MCL1 and BCL2 in MM cells otherwise resistant to each inhibitor alone. Using low doses, this therapeutic strategy could potentially minimize side effects. We also demonstrated the crucial role of BAX and BAK in this BH3 mimetic combination-induced cell death. This is in agreement with the hetero-complex formation of BAX and BAK observed during myeloma cell apoptosis resulting from the combination treatment.