George, Diane

Abstract The objective of this study was to determine the molecular alterations that occur at the protein level in patients with PTLD in order to identify novel targets of therapy, determine new markers of prognosis, and begin to delineate the pathogenesis of PTLD. Six tumor samples were obtained after informed consent from adult patients with PTLD post solid organ transplant. Four benign lymph nodes were obtained from age-matched controls. Three of the samples and their two controls were obtained from frozen tissue and the other three with two matching controls were obtained form paraffin embedded tissue. Immunohistochemistry using anti-CD19+ antibodies was performed and laser microdissection or slide scraping was used to obtain the tissue. Deparaffinization was performed using xylene and ethanol serial dilutions. Protein quantification was performed and 1ug of protein was obtained for each sample and its control. The nanoscale protein micorarray technique (BD Clontech, CA) was used to measure changes in the patterns of protein expression between PTLD samples and control lymphocytes. This is a new technique that detects differences in protein abundance between the disease and control samples by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 human monoclonal antibodies. It requires minute amounts of proteins. Two microarray slides were used for each experiment and a control experiment of control versus control was performed for normalization of the data. The slides were scanned using the Axon GenePix 4000B scanner. Two ratios were generated from the spot images for each protein target. The mean of the ratios of Cy5/Cy3 of both slides were analyzed using Clontech software and used to calculate an Internally Normalized Ratio (INR = Ratio1/Ratio2) for each spot on the array. The INR values were input into GeneSpring 6.0 software (Silicon Genetics, Redwood City CA). The data were normalized to the mean INR of the control samples. Proteins whose expression fold change relative to control was greater than 1.3 fold were determined. All cases represented monomorphic PTLD. Five were confirmed EBV positive (EBNA positive). The tissues were obtained from multiple organs including liver, gastric, cerebral, renal and lymph nodes. There were 22 proteins upregulated in the frozen sections as compared to the control and 157 proteins upregulated by 1.3 fold in the paraffin embedded sections as compared to control. Most of these proteins were common between the paraffin embedded and frozen sections. Proteins that were dysregulated included: proteins in the PI3K pathway and cell cycle regulation such as p70S6K, VHR, the AKT substrate GSK-3, cyclin C, MCM6, eIF-4E, CDK1, CDK7, eE-F-2 kinase; kinases such as JNKK1, PKC-lambda, RhoA kinase ROKalpha, ERK1, PKAc, PKArIIa and PKCbeta; apoptosis related proteins such as caspase 8, NF-kB, and MDM2; TGF signaling protein p300, and the heat shock protein HSP90. These results provide insight into pathways that are dysregulated in PTLD and can be targeted in future clinical trials with specific inhibitors such as PI3K, PKC, NF-kB or HSP90 inhibitors.

Abstract We have previously demonstrated that the chemokine receptor CXCR4 and its ligand SDF-1 are important regulators of migration in MM. The objective of this study was to investigate the role of the CXCR4 inhibitor AMD3100 (Sigma, MO) on proliferation, survival, migration, adhesion and invasion in MM. MM cell lines (MM.1S, OPM2 and Kas6/1) were exposed to serial dilutions of SDF-1 (1–100nM) in the presence or absence of the CXCR4 inhibitor AMD3100 (20–100uM) incubated for 16 hrs or the CXCR4 inhibitory antibody MAB171 (RDSystems, MN) 10–800uM or its IgG2 isotype control. Inhibition of proliferation was measured using the MTT proliferation assay. Apoptosis was determined using Annexin V/PI FACS analysis. Migration was determined using a transwell migration assay (Costar, Corning, NY). Cells treated with serial concentrations of AMD3100 or vehicle (sterile water) were washed, placed in serum free medium for two hours, then placed in the migration chambers with 1% FCS medium in the presence of SDF–1 20nM (a concentration previously determined to induce maximum migration in MM cells). After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, CA) with 96 well plated coated with fibronectin and using MM cells treated with serial dilutions of AMD3100 or vehicle in the presence or absence of serial concentrations of SDF-1 (10–30nM). Invasion was determined using the invasion assay (EMD Biosciences, CA). Serial concentrations of SDF-1 induced a bell-shaped migration curve in MM cells with 10–30nM inducing maximum migration (324% compared to untreated control) while higher doses of SDF-1 (100nM) did not induce migration in all MM cells tested. AMD3100 induced a dose dependent inhibition of migration in MM cells treated with 20nM SDF-1. The maximum effect was demonstrated at 25uM with 48% migration as compared to control. Higher doses of AMD3100 (50–100uM) did not further inhibit migration. The anti-CXCR4 antibody demonstrated a dose dependent inhibition of migration. Anti-CXCR4 10uM inhibited migration to 53% and 200uM to 35%. The effects of higher doses of anti-CXCR4 were not significantly different as compared to 200uM. Serial concentrations of SDF-1 (10–100) induced a dose-response increase in adhesion to fribronectin with 10nM increasing adhesion by 202% as compared to untreated cells and 100nM 462% indicating activation of alpha4 beta1 integrin on MM cells. AMD3100 100uM inhibited adhesion at all doses by 55% as compared to control. Using the invasion assay, SDF-1 100nM induced maximal invasion of MM cells. The effect of SDF-1 on invasion was not abrogated by AMD3100. AMD3100 did not induce significant apoptosis or inhibition of proliferation at all tested doses, in the presence or absence of 20–100nM SDF-1. In summary, AMD3100 inhibited migration and adhesion of MM cells, but not invasion indicating that invasion occurs through non-CXCR4 dependent mechanisms. AMD3100 did not induce apoptosis or inhibition of proliferation indicating that its effect is specific on migration and adhesion. Further experiments to test its role on homing of MM cells in vivo are underway. The future use of this novel agent as an inhibitor of homing of MM cells in clinical trials may be warranted. Supported in part by an ASH Scholar Award and an MMRF grant.

Abstract Waldenstrom Macroglobulinemia (WM) is characterized by the presence of lymphoplasmacytic cells in the bone marrow, and often in the lymph nodes. The mechanisms by which cells migrate/home to the bone marrow are poorly understood. The chemokine receptor CXCR4 and its ligand SDF-1 have been implicated in the migration and homing of lymphocytes. The objective of this study was to begin to identify the role of CXCR4 in WM. Paraffin embedded tissues were obtained after informed consent from 10 bone marrow samples of asymptomatic WM/monoclonal gammopathy of undetermined significance (MGUS) (n=5) and symptomatic WM (n=5). Four normal bone marrow samples were obtained for control. CXCR4 expression was determined using anti-CXCR4 antibody (Affinity Bioreagents, Golden, CO) 1:200 dilution for 16hrs, and a scale of 0–3+ was used to determine relative expression in the lymphocytes. Anti-CD20 antibody was used to determine the presence of lymphocytes in the samples. To determine whether the receptor is functional, migration was performed with the WM cell line (WSU-WM, kind gift from Dr. Al-Khatib, Wayne State University) and using a transwell migration assay (Costar, Corning, NY) with serial dilutions of SDF-1 (10-100nM). Comparison between the asymptomatic and symptomatic WM groups was performed by c2 test, and Mann-Whitney test, as appropriate. The baseline patient characteristics are displayed in table 1. CXCR4 expression was present on the surface of scattered lymphocytes in the normal bone marrow (median expression 1+). The expression of CXCR4 was increased in the asymptomatic/MGUS cases of WM (median expression 2+). In contrast, CXCR4 expression was downregulated in 4/5 cases of symptomatic WM specifically all the ones with extensive involvement of the bone marrow with lymphoplasmacytic cells (median expression 0). SDF-1 induced a bell- shaped response of migration in the WM cells with the maximal migration occurring at 1-10nM SDF-1. SDF-1 1-10nM induced 340% increase in migration as compared to untreated cells. However, high doses of SDF-1 100nM did not induce any migration as compared to untreated cells indicating inhibition of migration in response to high doses of SDF-1. In summary, we demonstrate that the CXCR4 receptor is functional and induces migration indicating that it plays a role in the migration of WM cells to the bone marrow. In addition, we demonstrate that CXCR4 expression is downregulated in WM with high infiltration of cells in the bone marrow as compared to those with minimal infiltration. These results may be due to the high level of SDF-1 in the bone marrow that induce downregulation of CXCR4. High levels of SDF-1 may downregulate the receptor and inhibit migration in order to confine the cells within the bone marrow. Supported in part by an ASH scholar award and the RFW. Patient characteristics Asymptomatic WM/MGUS, (range), (N=5) Symptomatic WM, (range) (N=5) Median age at diagnosis 67.6 (55.8–78) 71.5 (61–77) Median IgM level (mg/dL) 997 (904–1490) 5410 (2000–7820) Median Hb (gm/dL) 12.6 (11.1–15.1) 10.8 (9.3–11.6) Organomegaly (liver or spleen) N 20% Lymphadenopathy N 40% Median involvement of tumor cells in the BM in % 5% 30(10–60)

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]