Unbiased phosphoproteomics reveals downregulation of proliferative signaling after daratumumab treatment. (A) RPMI-8226 cells were treated with 20 μM daratumumab (Dara) or IgG1 isotype control for 20 minutes (n = 3 each) and then harvested for unbiased phosphoproteomics with immobilized metal affinity chromatography enrichment for phosphopeptide enrichment. Plot displays results of kinase substrate enrichment analysis, indicating modest decrease in phosphorylation of numerous predicted substrates of MAPK pathway kinases as well as cyclin-dependent kinases (cutoff, P < .05; log₂ fold-change > |0.5|). (B) Western blot in RPMI-8226 of MAPK (ERK1/2) (Thr202/Tyr204) relative to total MAPK demonstrates modest decrease in MAPK phosphorylation after 5, 10, or 15 minutes of Dara treatment; magnitude of change normalized to IgG1 control at each time point (red) appears consistent with phosphoproteomic data. (C) Western blot of MM.1S cells treated with Dara and blotted for p-AKT (Ser473) and total AKT, with quantification of p-AKT relative to total AKT and normalized to IgG1 at each time point. All images representative of 2 independent western blots.