MM patient bone marrow CD138+CD38+ plasma cells are sensitive to melflufen. (A) Drug sensitivity was measured after 72 h incubation with melflufen or with DMSO alone (control). Cell viability was assessed by multicolor high-throughput flow cytometry using annexin V and 7AAD viability markers. The viability (%) of CD138+CD38+ cells was calculated, and dose-response curves drawn. The MM patient samples can be divided into three groups based on the sensitivity of their CD138+CD38+ cells to melflufen: highly sensitive (red: EC50 < 0.1 nM), intermediately sensitive (gray: EC50 0.1–5 nM), not sensitive (black: EC50 > 5 nM). (B) Correlation of CD138+CD38+ cell sensitivity to melflufen (y-axis) with melphalan sensitivity (x-axis). Drug sensitivity was measured using EC50 values. (C) Comparison of CD138+CD38+ cell sensitivity (EC50) between NDMM (dots) and RRMM (triangles) in patient samples treated with melflufen (black; p = 0.0004), melphalan (blue; p = 0.025), selinexor (red; p = 0.44), bortezomib (brown; p = 0.34), and 4-HC (purple; p = 1). DMSO: dimethyl sulfoxide; 4-HC: 4-hydroperoxycyclophosphamide; NDMM: newly diagnosed multiple myeloma; RRMM: relapsed/refractory multiple myeloma.