Nehme, Alex

Abstract 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.

Abstract The objective of this study was to analyze molecular changes that occur at the protein level in WM and IgM-MGUS in order to identify proteins dysregulated at disease progression and design new therapies. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between WM, IgM-MGUS and normal lymphoplasmacytic cells as control. The antibody array is a novel technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto a glass slide spotted with 512 different monoclonal antibodies specific for human proteins. CD 19+ and CD138+ purified lymphoplasmacytic cells were obtained from cryopreserved bone marrow samples of 5 newly diagnosed patients with WM and 5 patients with IgM-MGUS. Control plasma cells were obtained from 3 pooled CD19+ and CD138+ purified normal donor bone marrow cells. 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 and changes of protein expression ≥2 fold in 60% of the samples as compared to normal control were identified in both WM and MGUS groups. There were 20 proteins upregulated in WM and 21 proteins in MGUS as compared to normal control cells These included proteins from the small GTPases such as the Ras, Rho and Ran families (Rab 4, MONA, RCC1, and CDC42GAP), Kinases/signaling proteins (ROK alpha, p62 dok, JAK, 14-3-3e), cyclin dependent kinase (CDK2), fatty acid synthase (HDAC3), NFkB pathway (IKKalpha, IKBe), and histone deacetylase. The WM and IgM MGUS differed in only 3 proteins using the 2-fold threshold. The WM samples showed overexpression of the heat shock protein Hsp90, the Ras family protein CDC25C, and the chemotaxis protein p43/EMAPII as compared to the MGUS samples. This is the first proteomic study of WM and MGUS patients. It identifies novel proteins dysregulated in WM that may serve in future clinical trials as targets for therapy such as Hsp90, NFkB, HDAC and fatty acid synthase. In addition, it identifies 3 proteins that identify disease progression from MGUS to WM, which could be useful for monitoring MGUS patients and designing targeted therapies to prevent progression to WM.

Abstract To better understand the molecular changes that occur in Waldenstrom macroglobulinemia (WM), we employed antibody-based protein microarrays to compare patterns of protein expression between untreated WM and normal bone marrow controls. Protein expression was defined as a >2-fold or 1.3-fold change in at least 67% of the tumor samples. Proteins up-regulated by >2-fold included Ras family proteins, such as Rab-4 and p62DOK, and Rho family proteins, such as CDC42GAP and ROKα. Other proteins up-regulated by >1.3-fold included cyclin-dependent kinases, apoptosis regulators, and histone deacetylases (HDAC). We then compared the samples of patients with symptomatic and asymptomatic WM and showed similar protein expression signatures, indicating that the dysregulation of signaling pathways occurs early in the disease course. Three proteins were different by >2-fold in symptomatic versus asymptomatic, including the heat shock protein HSP90. Elevated protein expression was confirmed by immunohistochemistry and immunoblotting. Functional significance was validated by the induction of apoptosis and inhibition of proliferation using specific HDAC and HSP90 inhibitors. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated in WM, which both enhance our understanding of disease pathogenesis and represent targets of novel therapeutics. [Cancer Res 2007;67(8):3777–8]