Lu, Ganwei

Abstract Prior studies have demonstrated that MM cells with PTEN mutation and high AKT activity are more sensitive to inhibitors of the PI3K/Akt/mTOR pathway. However, the molecular mechanisms that regulate the differential response to these agents are not well characterized. The objective of this study was to determine proteins that are different between MM cell lines with higher AKT activity (OPM2) and lower AKT activity (MM.1S) in response to the AKT inhibitor, perifosine. Methods: MM cell lines (MM.1S and OPM2) were treated with serial concentration of perifosine (KRX-0401, Keryx, NY, NY, provided by the NCI). Proteomic analysis using the nanoscale BD Clontech antibody-based protein microarray technique was performed using cells treated with perifosine (10uM for 16 hrs) or vehicle (sterile water) as control. Apoptosis was determined using Annexin V/PI FACS analysis at 24 and 48 hrs. The treatment time and concentration were chosen so that it did not induce more than 25% apoptosis to ensure adequate analysis of changes in signaling pathways. The antibody microarray is a technique that detects differences in protein abundance between the treated and control sample with each experiment by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 human monoclonal antibodies. Two microarray slides were used for each experiment. 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, CA). The data was normalized to the mean INR of the two cell lines. Proteins whose expression level changed relative to control greater than 1.3 fold were determined. Unsupervised clustering demonstrated a different protein signature between MM.1S and OPM2 in response to perifosine. There were 144 proteins differentially expressed by 1.3 fold between MM.1S and OPM2. Proteins that were downregulated in OPM2 as compared to MM.1S included those in the PI3K pathway and cell cycle regulation such as PTEN, p70S6Kinase, the AKT substrate GSK-3, eEF-2 kinase, eIF-4g, Ku-80, cyclin A, E2F-2, CDK2, CDK7, and c-myc; proteins involved in apoptosis such as p21WAF, caspase 4 and 8, FADD, and PARP; kinases such as PKAc, PKA RI, ERK2, and JNK1; and other proteins regulating apoptosis and proliferation including p53, the NF-kB inhibitor IkB, and the heat shock protein HSP70. The nanoscale protein array is a useful and rapid technique that may be used to identify differences between resistant and sensitive cells to novel therapeutic agents. We identified proteins that are differentially expressed between MM cells sensitive and relatively resistant to the AKT inhibitor perifosine. Further analysis of the role of these proteins in the mechanism of resistance/sensitivity to perifosine is being performed. Future use of inhibitors of NF-kB (bortezomib) or heat shock protein inhibitors in conjunction with perifosine may overcome resistance induced by these proteins in MM cells with low AKT activity. 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 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]

Abstract Purpose: The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway and the heat shock protein family are up-regulated in multiple myeloma and are both regulators of the cyclin D/retinoblastoma pathway, a critical pathway in multiple myeloma. Inhibitors of mTOR and HSP90 protein have showed in vitro and in vivo single-agent activity in multiple myeloma. Our objective was to determine the effects of the mTOR inhibitor rapamycin and the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on multiple myeloma cells. Experimental Design: Multiple myeloma cell lines were incubated with rapamycin (0.1-100 nmol/L) and 17-AAG (100-600 nmol/L) alone and in combination. Results: In this study, we showed that the combination of rapamycin and 17-AAG synergistically inhibited proliferation, induced apoptosis and cell cycle arrest, induced cleavage of poly(ADP-ribose) polymerase and caspase-8/caspase-9, and dysregulated signaling in the phosphatidylinositol 3-kinase/AKT/mTOR and cyclin D1/retinoblastoma pathways. In addition, we showed that both 17-AAG and rapamycin inhibited angiogenesis and osteoclast formation, indicating that these agents target not only multiple myeloma cells but also the bone marrow microenvironment. Conclusions: These studies provide the basis for potential clinical evaluation of this combination for multiple myeloma patients.

Abstract The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1–dependent migration was regulated by the PI3K and ERK/MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.