Proteomic Analysis of Multiple Myeloma Identifies Potential Targets for Drug Therapy.

The objective of this study was to investigate the underlying molecular alteration in multiple myeloma at the protein level in order to identify regulators of pathogenesis, discover novel targets of therapy, and compare genetic and proteomic alterations. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between MM and normal plasma cells. The antibody array is a new technique enabling protein differences to be assayed directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto glass slides spotted with 512 different monoclonal antibodies. CD138+purified plasma cells were obtained from cryopreserved bone marrow samples of 12 newly diagnosed patients with MM. The labeling index was high (1% cutoff) in 7 samples and low in the other 5. Control plasma cells were obtained from 9 pooled CD138+ purified normal donor bone marrow plasma cells. Interphase FISH analysis for 17p deletion, 13q deletion, and t(11:14) were performed. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software. Unsupervised clustering identified 4 groups of MM. Changes of protein expression ≥2 fold in 70% of the samples as compared to control were identified. There were 6 proteins differentially expressed between all MM samples and control cells including proteins in the ras signaling pathway (KSR-1), the ubiquitin pathway (Ubc-H6), cyclin-dependent kinases (CDK4), cytokines (IL-6), DNA toposisomerase II, and the rho-interacting serine-threonine kinase CRIK. Proteins differentially expressed in MM groups 1 and 2 compared to normal control included cell cycle regulators (cul-2, MCM6, PCNA, TGFb1), kinases (p70S6K, PKC), and chromatin regulators (Ran, AKAP450, Rad50). Protiens differentially expressed in MM group 3 included cell cycle regulators (CDK2, CLK1, MENA), apoptosis regulators (XIAP, caspase 4, perforin) kinases (IKKa and RAC1 in the Wnt signaling pathway) and P53 regulators, while proteins identified in MM group 4 included NFkB/ubiquitin proteins (IKKa and Ubch6), cell cycle regulators (c-myc, CDK4), p53 pathway proteins (53bp2), ras-signaling proteins (KSR1), and the kinase CRIK. There were no differences in protein expression between the high and low labeling index groups. 13q was identified in 5 (42%), 17 p in 1(8%) and t(11:14) in 1(8%) patients. 80% of the 13 q deletion cases clustered in MM group 1 and 2 patients. Cyclin D-1 was upregulated in 5 (42%) patients including the patient with (11:14) translocation. This is the first proteomic study of patients with MM. The results are consistent with previously identified genetic alterations in MM indicating that this novel technique could be used in identifying molecular changes in MM. It identifies novel proteins dysregulated in MM that differ between the 4 MM groups. These results may be used in the future to individualize therapy based on the proteins dysregulated in each group. For example, IKK inhibitors may be useful in group 3 MM patients, while mTOR inhibitors (upstream of p70S6K) could be used in groups 1 and 2 patients. Future correlations with gene expression arrays and prognosis in a larger cohort of patients is warranted.