Fig. 5

SUMOylation inhibition decreases miR-551b and miR-25 level. A miR-551b and miR-25 levels were decreased by TAK-981 treatment in MM1S and MM1R cells. MM1S and MM1R cells were treated with Vehicle (Veh), 0.1 μM TAK-981 (TAK), 1 μM Dex (Dex), or 0.1 μM TAK-981 with 1 μM Dex (combo) for 48 h. miR level was measured by Taqman q-PCR. B Knockdown of SAE2 or UBC9 decreases the expression of miR-551b and miR-25. MM1R cells were transfected with siRNA targeting SAE2 (SiSAE2) or UBC9 (SiUBC9), or non-targeting control (SiCtrl) for 72 h, miR levels were measured by qPCR. C TAK-981 treatment decreases miR-551b and miR-25 levels in vivo. Tumor tissue from MM1R xenograft mice (in Fig. 2C) was used for RNA extraction and miR measurement. D TAK-981 treatment decreased miR-551b and miR-25 level in primary MM cells in dose-dependent manner in primary MM cells treated with TAK- 981. Primary MM cells from 5 relapsing patients were treated with TAK-981 at indicated concentrations for 48 h. miR-551b and miR-25 level was measured by q-PCR. Data presented as mean ± SD, n = 5. E TAK-981 treatment affects miR-551b and miR-25 downstream genes protein level. Western blot presents protein level in MM1R cells treated with vehicle (Veh) and 0.1 μM TAK-981 (TAK-981) for 48 h. Relative protein level was quantified by Image J and labeled below each blot. F GSEA shows cell cycle regulators are suppressed but apoptosis and autophagy related pathways are upregulated upon TAK-981 treatment. GSEA false discovery rate (FDR)-q values, p-values and normalized enrichment scores (NES) are labeled. G UBA2 level correlates with ZFP36 and ULK1 expression in patient specimens. Analysis of cohort (GSE2658) of 559 MM patients (Supplemental Fig. S1B). Patients with high SAE2 (UBA2; UBA2high group) showed lower ZFP36 levels (left) and ULK1 level (right) than patients with low SAE2 (UBA2; UBA2low group). Data were analyzed using unpaired Student t tests: Data presented as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001