Fig. 3. Defining a myeloma surface signature of proteasome inhibitor resistance.

A Cell surface proteomics was performed on evolved bortezomib-resistant (BtzR) and carfilzomib resistant (CfzR) myeloma cell lines (AMO1 BtzR (n = 3); AMO1 CfzR (n = 3), L363 BtzR (n = 2), L363 CfzR (n = 3), RPMI-8226 BtzR (n = 1)) and aggregated in comparison to wild-type cell lines (AMO1 (n = 3), L363 (n = 2), RPMI-8226 (n = 1)), n denotes number of biological replicates. Significantly changed proteins in PI-resistant lines shown in blue (log₂-fold change > |1|; p < 0.05). Source data in Supplementary Data 3. B Validation by flow cytometry of most-changed surface proteins in AMO1 cells. Representative data of n = 2 independent experiments. C mRNA data in the MMRF CoMMpass database (Release IA14) from paired diagnosis and first-relapse tumor cells (n = 50), where all patients had received a PI as part of their induction regimen. p-value by two-sided t-test. Upper and lower hinges correspond to 25 and 75 percentiles, upper and lower whiskers extend to highest values within 1.5*IQR of the hinge, and center line indicates the median. D Immunohistochemistry for CD53 on myeloma plasma cells in bone marrow core biopsies from UCSF patients before and after Btz treatment (n = 13 patients). H-scoring (see “Methods”) averaged from two independent hematopathologists (E.R. and S.P.). Magnification = ×60, scale bar length = 100 µm. Error bars represent +/− SD and center line represents the mean. p-value by two-sided t-test. E–G Flow cytometry illustrating knockout of CD53 (E), CD50 (F), and EVI2B (G) in MM.1S cells. Representative of n = 2 technical replicates. H MM.1S engineered with knockouts and scramble guide RNA control were treated with Bortezomib for 48 h (n = 3 technical replicates). 95% confidence interval of IC50s from Graphpad (see “Methods”). Error bars represent +/− SD. p-values by Extra sum-of-squares F test. For D and H, source data are provided as a Source data file.