Fig. 7

LSD1 and proteasome inhibition have anti-multiple myeloma activity in vivo. A U266 cell lines were cultured on a layer of GFP⁺ HS-5 stromal cells and treated with the indicated concentrations of SP2509, CFZ, or the combination. Percentage of MM cells was measured overtime by FACS analysis after anti-CD138-APC staining and the ratio with GFP + cells was calculated. Data are the means ± s.d. of four independent experiments B KMS-28 and PBMCs from six healthy donors were treated or not with 2.5 nM of CFZ and increasing concentration of SP2509, as reported. Cell viability was estimated by FACS analysis with Annexin V-PI staining, 72 hpt. Data are the means ± s.d. of 6 independent experiments. C Buffy coats derived from bone marrow aspirates of MM patients were treated with CFZ (2.5 nM) in combination or not with SP2509 (4 µM). Cell viability was estimated by flow cytometry measuring PI and CD138⁺ cells 72 hpt. Histograms represent the percentage of viable cells normalized vs DMSO samples. Data are the means ± s.e.m. of 8 independent MM patients. D Growth rate fold change (normalized on tumor volume measured 8 days post-treatment) of KMS-28-TTA_shLSD1 cells injected subcutaneously into the flanks of NSG mice. When tumor masses became palpable, mice were randomized for treatment with vehicle (n = 22), 4 mg/kg CFZ (n = 19), 0.25 mg/ml DOXY (n = 11), or a combination of both compounds (n = 7) over 3 weeks. The timeline above shows the schedule of treatment followed for in vivo treatments. E Fluorescent microscopy images (×10 magnification) of DsRed⁺KMS-28 TTA xenografts at 24 hpi (left) and 72 hpi (right) into the yolk of zebrafish embryos treated with indicated compounds. F Dot-plot shows the trend in tumor burden at 72 hpi, normalized to tumor area at 24 hpi (DMSO = 24 embryos; CFZ = 24 embryos; SP2509 = 24 embryos; SP2509/CFZ = 30 embryos). (**P < 0.01; ***P < 0.001; ****P < 0.0001). PBMC, Peripheral blood mononuclear cell; s.e.m.: standard error of the mean; hpi: hours post-injection