Ghobrial, Irene M.

Abstract The bone marrow microenvironment in MM is characterized by the presence of upregulated osteoclast activity (OCL) and increased angiogenesis. We have recently demonstrated that the HSP90 inhibitor 17-AAG (provided by the NCI) and the mTOR inhibitor rapamycin (LC Laboratories, MA) have synergistic inhibitory activity on MM cells. The objective of this study was to determine the effect of rapamycin, 17-AAG and the combination on OCL formation and angiogenesis. Rapamycin (0.01–100nM), 17-AAG (10–1000nM) and the combination was tested using an in vitro human OCL formation assay and a human angiogenesis assay (AngioKit, TCS Cellworks, UK). Nonadherent human marrow mononuclear cells (1 x 105/100 μL) were plated in 96-well plates in the presence or absence of DMSO, rapamycin, 17-AAG or the combination. RANKL (100ng/ml) and MCSF (20ng/ml) were added to all wells except control media and MCSF. After 3 weeks, cells were fixed, and the number of OCL-like multinucleated cells were scored. To test the effect of the agents on early OCL precursors, we added the inhibitory agents on days 1, 7 or 14 of the culture. The AngioKit is comprised of human endothelial cells in a 24 well plate. The endothelial cells proliferate and then migrate through the matrix to form tubular structures and anastomosing tubules by 2 weeks. Two control wells were treated with VEGF (+ve control) and two with suramin (−ve control). The optimized medium and test samples were replaced on days 4, 7, and 9 after initial treatment. On day 11, cultures were fixed and stained with antibodies to CD31 to detect vessel formation. The degree of tubule formation was quantitated using computerized image analysis (Angiosys, TCS Cellworks, UK). Single agent rapamycin (20–100nM) inhibited OCL formation by 35% as compared to control in all tested doses indicating that PI3K/mTOR is an important regulator of OCL formation. The effect was similar on day 7 and day 14 indicating that this pathway is important for early and late OCL formation. 17-AAG 100–600nM significantly inhibited OCL formation with 100nM 17-AAG inducing 12% OCL formation as compared to control, while 300 and 600nM completely abrogated OCLs (0% OCLs). This effect was similar at day 7. However, when 17-AAG was added on day 14, it only induced 50–60% reduction in OCL formation indicating that 17-AAG affects early OCL formation. The combination of the two agents completely inhibited OCL formation on day 1 and 7 and led to a 65% reduction in OCLs when added on day 14 of the culture. Rapamycin showed a marked decrease in angiogenesis (similar to the negative control suramin), even at the lowest level tested of 0.01nM. 17-AAG demonstrated some inhibition of angiogenesis at 10 nM, and completely abrogated angiogenesis at 500–1000nM. In summary, rapamycin and 17-AAG inhibit OCL formation and angiogenesis. The effect of 17-AAG was on early OCL formation while rapamycin was on both early and late OCL. These results are contradictory to previous data indicating that 17-AAG increases OCL activity in MM. Rapamycin had a significant inhibitory effect on angiogenesis even at low doses. These results demonstrate that the use of rapamycin analogues and 17-AAG in clinical trials may have a therapeutic effect, not only on MM cells, but also on the bone marrow microenvironment. Supported in part by an ASH Scholar Award and an MMRF grant.

Abstract Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder. For selected patients high dose chemotherapy with peripheral blood stem cell support is effective, but even in those patients, only 50% derive organ responses. Other patients are too sick to undergo that therapy. Thalidomide has limited utility in this disease, largely because of its toxicity profile in patients with AL. Lenalidomide, Celgene’s lead clinical compound in a new group of drugs called IMiDs®, is highly active in patients with multiple myeloma, especially in conjunction with dexamethasone. We sought to determine the toxicity and efficacy of lenalidomide in patients with AL. Methods: Patients with symptomatic AL who had a measurable plasma cell disorder (defined as serum M-spike ≥ g/dL urine M-spike >200 mg/24 hours; or involved immunoglobulin free light chain (FLC) ≥10 mg/dL and an abnormal FLC ratio) and adequate organ reserve defined as a creatinine ≤3 mg/dL, absolute neutrophil count ≥1000, platelet count ≥75000, and a hemoglobin ≥8 g/dL were eligible. Patients were started on lenalidomide 25 mg/day for 21 days followed by a 7 day rest (1 cycle). Dose modifications were made based on toxicity. If there was evidence of progression before 3 months or no evidence of hematologic response after 3 cycles, dexamethasone 40 mg p.o. days 1–4 and 15–18 was added. Results: Twenty-three patients were enrolled between 10/28/04 and 4/7/05; 14 were previously treated. Patient characteristics are shown in Table. Organ involvement was as follows: cardiac (61%); renal (70%), hepatic (22%); nerve (13%). Nine patients withdrew from study before completing 3 months of therapy. The reasons were: cancel (1); death (4); adverse events (2); and progression (2). At the time of our preliminary analysis in July 2005, an additional 3 patients have withdrawn from study: death (1) and patient refusal (2). Of the 5 patients who died, 4 had severe cardiac involvement and at least 3 organs involved by amyloid. The median follow-up for the eleven patients remaining on study is 6.2 months. Of the 12 patients who have crossed the 3 month treatment landmark, there are 7 hematologic partial responses (4 confirmed and 3 unconfirmed), two renal responses and one liver response. Of these same 12 patients, all but one has had dexamethasone added to their treatment program. The most common grade 3–4 adverse advents at least possibly attributable to lenalidomide were neutropenia (43%), thrombocytopenia (26%), rash (17%), dyspnea (9%), fatigue (9%), and edema (4%). Expansion of the trial is planned to accrue an additional 10 evaluable patients. Conclusions: Early results suggest that lenalidomide ± dexamethasone has activity in patients with primary systemic amyloidosis. Supported in part by the Caliguiri Fund for Amyloidosis Research, Robert A. Kyle Hematologic Malignancies Program, and Celgene. Patient Characteristics Median Range Age 64 44–88 Major Organs 2 0–3 Involved FLC, mg/dl 23 4.1–278 Serum alb, g/dl 2.8 1.2–3.7 Urine protein, g/24 hrs 3.7 0.02–14.3 Creatinine, mg/dl 1.3 0.7–2.6 Alkaline phos IU/l (nml <115) 99 57–1729 NY Heart Class 1 1–3 Troponin T, mcg/l 0.03 0.01–0.55 NT-proBNP, ng/l 1419 109–42844 LV Septum, mm 13 9–24 LV EF, % 63 22–72

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 The objective of this study was to determine the molecular alterations that occur at the protein level in patients with WM in order to identify novel targets of therapy, determine new markers of prognosis, and begin to delineate the pathogenesis of WM. Five bone marrow samples were obtained after informed consent from patients with symptomatic WM. Two bone marrow samples were obtained from age-matched controls and were pooled. All samples were purified with anti-CD19+ beads with over 90% purity. Protein quantification was performed and 1ug of protein was obtained for each sample and control. The nanoscale protein micorarray technique (BD Clontech, CA) was used to measure changes in the patterns of protein expression between WM samples and control lymphocytes. This is a new technique that detects differences in protein abundance between the tumor and control samples by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 monoclonal antibodies against human polypeptides. It requires minimal amount of protein. 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, ratios 1 and 2 correspond to slides 1 and 2) for each spot on the array. The INR values were input into GeneSpring 6.0 software (Silicon Genetics, Redwood City CA). The data was 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 samples were of symptomatic WM. There were 3 females and 2 males. The median age was 61 years (range, 47–83). Four patients were newly diagnosed and one had received prior rituximab, CHOP ad thalidomide therapy. Clustering analysis did not demonstrate a difference between newly diagnosed and treated samples. There were 72 proteins up or downregulated by 1.3 fold in all WM samples as compared to control. These included proteins in the PI3K pathway such as VHR, PTP1B, PI3K (p110alpha) and Rb2. Protein kinases such as PKCi, PKCbeta, PKC gamma, PKC delta, PLCgamma were all upregulated in WM samples. Other proteins included the B-cell specific activator protein PAX-5, the ubiquitin protein UBCH6, the STAT kinase STAT4, the GTPase Rho A-binding kinase ROK alpha, and the apoptosis protein SMAC/DIABLO. We demonstrate that several isoforms of the PKC family of proteins are upregulated in WM. PKC proteins regulate apoptosis, proliferation and migration in many cancer cells. These proteins may be useful targets of therapy in future clinical trials in WM. Other inhibitors that may be useful in WM include ubiqutin/proteosome inhibitors such as bortezomib and PI3K pathway inhibitors such AKT or mTOR inhibitors. Our results also confirm the presence of PAX-5 in WM consistent with previous cytogenetic studies. Supported in part by an ASH scholar award and Research Fund for Waldenstrom.

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 It appears that a number of recent manuscripts using protein microarray technology are using equivalent analysis procedures that the gene-expression microarray community implemented in their infancy. That is, utilizing a classic reference design such that the ratio of the sample of interest to a reference sample is the response of interest and assessing fold change to determine differential expression. For example, recent publications have concluded that proteins with a fold change less than 0.7 or greater than 1.3 demonstrate significant down- or up-regulated differential expression, respectively. However, fold change is an unreliable measure of differential expression and statistical models that distinguish true signal from random noise should be utilized instead of fold changes. Over the last half decade a tremendous amount of research has been devoted to gene-expression microarrays to vastly improve on the areas of experimental design, normalization and statistical analyses to assess differential expression and classification and these methods are directly applicable to protein microarray technology. Thus, the objective is to review the statistical methodology that has been developed for two-color cDNA arrays that is directly applicable to protein arrays. Examples are provided from a mantle-cell lymphoma protein-array experiment.

Abstract Waldenström’s macroglobulinemia (WM) is an incurable low-grade lymphoproliferative disorder characterized by bone marrow (BM) infiltration of a clonal population of small B-lymphocytes, plasmacytoid lymphocytes and plasma cells that secrete monoclonal IgM antibody. Because it is difficult to obtain tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported and the genetic basis of the disease remains poorly defined. Therefore, we performed a comprehensive high-resolution study to identify genomic abnormalities involved in WM pathogenesis. Fifty-seven WM patients were analyzed using a combination of array-based comparative genomic hybridization (aCGH)(N=42); gene expression profiling (GEP)(N=22), DNA sequencing (N=24) and cIgM-FISH (N=57). Agilent 244A and Affymetrix U133 platforms were used in the aCGH and GEP experiments, respectively. An NF-κB index was calculated from GEP data, as a surrogate marker of NF-κB transcription activity. NFKB2 sub cellular localization was determined by immunofluorescence staining. Overall, 83% of samples have aCGH defined chromosomal abnormalities, with a median of 3 abnormalities per patient (range 0 to 27). We identified 16 recurrent regions of copy number change found in >5% (3 or more patients); 10 deleted and 6 amplified regions. The most common abnormality was the entire or partial deletion of 6q, identified in 40% of cases. Four nonoverlapped minimal deleted regions were identified on 6q (MDR1 to MDR4), each of them being present in at least 14 of 17 patients with the abnormality. Gain of 6p was the second most common abnormality (17%) and its presence was always concomitant with 6q loss. Other recurrent deletions were 13q14 (10%) and 7q22, 8p, 11q22-q23, 11q23-q24 and 17p11-p13 (7% each). Copy gains were identified as partial or entire gains of chromosomes 18 (17%), 4 (12%) and 3 (10%), 8q (10%) and Xq27.1-q28 (10%). Three patients had either homozygous deletions or LOH with mutations affecting both alleles of TRAF3 on 14q32. TRAF3 inactivation was correlated with an increased NF-kB transcriptional signature and it was associated with activation of the non-canonical NFkB pathway. Additionally, we identified the inactivation of TNFAIP3, another negative regulator of the NF-kB pathways, in one of 24 patients. Finally, interstitial deletions identified in 4 patients at 13q14 identified a 1.1 Mb MDR, including MIRN15A and MIRN16-1. These findings confirm that focal 13q14 deletion is not restrict to B-CLL and that the MIRN15A/MIRN16-1 abnormalities might be common to several indolent B-cell diseases. Overall, we identified TRAF3 and TNFAIP3 inactivation in 5.3% and 2.4%, respectively. To our knowledge, this is the first study showing the inactivation of bona fide tumor suppressor genes in WM patients. Furthermore, these genes are negative regulators of NF-kB signaling pathway, highlighting its biologic importance in sustaining and limiting growth in WM. Mutational activation of this pathway, which is normally activated by ligand-receptor interactions within the BM microenvironment, suggest a therapeutic role for inhibitors of NF-KB pathway activation in the treatment of WM.

Abstract The objective of this study was to analyze protein changes that occur a the molecular level with novel therapeutic agents used in MM in order to identify potential combinations for clinical trials. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression in Kas 6/1 MM cell line after treatment with bortezomib, dexamethasone, CC-5013, and 17-AAG. The antibody array is a new technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto glass slides spotted with 512 different monoclonal antibodies specific for human proteins. Induction of apoptosis was assessed by Annexin/Propidium iodide FACS analysis at 24 and 48 hours using different concentrations of the inhibitors. Protein arrays were performed at concentrations and treatment times that did not induce more than 25% apoptosis to ensure adequate analysis of early changes in signaling pathways. Bortezomib 5nM at 10 hours, dexamethasone 100mM, CC-5013 250nM, and 17-AAG 1mM at 24 hours were used. Control cells were treated with vehicles ETOH or DMSO at the same concentrations as the inhibitors. To assess differential protein expression, the mean of the Cy5/Cy3 ratios for each sample and its control were analyzed using the Clontech software and an internally normalized ratio (INR) was calculated. INR values >1.3 or <0.77 were considered as valid changes in protein abundance for this study. All inhibitors induced the upregulation of apoptosis proteins including BAK, BIK, SMAC/DIABLO, and caspase 14. Several proteins were inhibited by all 4 inhibitors indicating common pathways affected by these agents. These included members of the PI3K pathway (PI3K-p85a, p70S6kinase, and cyclin dependent kinases), MAPKinases (ERK1 and 2), antiapoptotic protein Bclx, heat shock proteins Hsp70 and HSP60, and ubiqutin/NFkB pathway (Ubch6, IKKalpha, and IKBe). These results indicate that inhibitors of the PI3K pathway such as mTOR inhibitors, IKK inhibitors or MAPkinase inhibitors may be used in the future in combination with bortezomib, dexamethasone, CC-5013 or 17-AAG. Fatty acid synthetase was downregulated by bortezomib and dexamethasone indicating potential use of fatty acid synthase inhibitors in combination with bortezomib or dexamethasone in future clinical trials. These results suggest that antibody protein arrays may be used in the rational design of combinations of targeted therapies in MM.

Abstract INTRODUCTION: Previous studies have demonstrated the clinical activity of the mTOR inhibitor RAD001 in low- grade lymphomas. Our preclinical studies demonstrated activity of mTOR inhibitors in Waldenstrom Macroglobulinemia (WM) cell lines and patient samples. This phase II study aimed to determine safety and activity of the oral mTOR inhibitor RAD001 (Novartis Pharmaceutical, MA) in patients with relapsed or refractory WM. METHODS: Patients who had at least one previous therapy for WM, and who had symptomatic relapsed or refractory disease were eligible. NCI CTCAE v3.0 was used for toxicity assessment. All patients received daily RAD001 at 10 mg. A cycle was considered 28 days. Patients were allowed to stay on therapy until progression of disease or excessive toxicity. This study was conducted in a collaborative effort between Dana Farber Cancer Institute (DFCI) and Mayo Clinic College of Medicine. Here, we report the data on the patients accrued at DFCI. RESULTS: 19 pts (15 men and 4 women) have been treated to date. All patients had symptomatic disease and required therapy. The median number of lines of prior treatment was 3 (range 1 – 5) including included rituximab, nucleoside analogues (fludarabine or 2-CDA), combination chemotherapy (e.g. CHOP, CVP), chloramucil, and bortezomib. The median IgM at baseline was 3330 mg/dL (range 1010– 7410). The median follow on RAD001 was 8 months (range 3 – 22 months). Eighteen pts are currently evaluable for response. Best responses to RAD001 after 2 cycles using IgM monoclonal protein were as follows: partial remission in 8 (44%), minimal response in 5 (28%). Progressive disease occurred in 4 (22%) and stable disease occurred in 1 (6%). The overall response rate (PR+MR) was 72%. The median duration of response has not been reached (3–22+ months). Patients tolerated therapy well without significant toxicities. Grade 3 and 4 toxicities included grade 4 thrombocytopenia in 1 patient, grade 3 pneumonia in 1 patient, grade 3 hyperglycemia in 1 patient and grade 3 mucositis in 1 patient. Other adverse events of grade 2 or lower included nail cracking, mucositis, diarrhea, and fatigue. Attributable toxicities otherwise proved manageable with appropriate supportive care, and RAD001 was generally well tolerated. One patient enrolled on the study withdrew consent and changed to hospice care within 3 weeks of therapy, and passed away due to disease progression. CONCLUSIONS: The use of the oral RAD001 single agent RAD001 in patients with relapsed or refractory WM was welltolerated and demonstrated significant activity achieving an overall response rate in 72% of patients. Future studies of combination of this agent with rituximab and bortezomib are currently being planned.

Abstract PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.