SIRT6 expression and prognostic relevance in MM. (A) Immunohistochemical analysis of 3 representative BM specimens derived from normal and MM patients (ND #1, 2, and 5 and MM #1, 4, and 8) show SIRT6 expression (positive cells are brown). Original magnification ×20 (×100 in insets). (B) Immunofluorescence showing subcellular distribution of SIRT6 in MM cell lines and patient MM cells (n = 5). (C) MM.1S and U266 cells were transfected with SIRT6 GFP-tagged plasmid. SIRT6 protein was visualized with an antibody directed against GFP, and cells were counterstained with DAPI to visualize nuclei. (D) Western blot analysis (1 of 3 representative blots) confirms the nuclear localization of SIRT6 in MM cell lines, but not in peripheral blood mononuclear cells (PBMCs) collected from healthy donors and MM patients. Cell lysates from cytoplasm and nuclear fractions were analyzed for SIRT6 expression. GAPDH and nucleolin were used as loading controls for the cytoplasmic and nuclear fractions, respectively. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (E) SIRT6 locus (human chromosome 19p13.3) copy-number heatmap for 45 (left side) MM cell lines (Mayo Clinic database) and 254 (right side) MM patients (Multiple Myeloma Research Consortium [MMRC]) (http://www.broad.mit.edu/mmgp). Color indicates degree of copy-number loss (blue) or gain (red). (F) SIRT6 expression in plasma cells from patients with MM (data set GSE2658). Increased SIRT6 expression is observed in high-risk compared with low-risk MM (unpaired t test; ****P < .0001). (G) Kaplan-Meier plots showing prognostic relevance of SIRT6 expression on overall survival of MM patients (based on GSE4581; deposited by Dr J. D. Shaughnessy Jr, University of Arkansas for Medical Sciences). The patient group with higher SIRT6 expression (blue line) had shorter overall survival than the patient cohort with lower SIRT6 expression (red line) (log-rank test).