SummaryMultiple myeloma (MM) evolves from a highly prevalent premalignant condition termed MGUS. The factors underlying the malignant transformation of MGUS are unknown. We report a MGUS/MM phenotype in transgenic mice with Eμ-directed expression of the XBP-1 spliced isoform (XBP-1s), a factor governing unfolded protein/ER stress response and plasma-cell development. Eμ-XBP-1s elicited elevated serum Ig and skin alterations. With age, Eμ-xbp-1s transgenics develop features diagnostic of human MM, including bone lytic lesions and subendothelial Ig deposition. Furthermore, transcriptional profiles of Eμ-xbp-1s lymphoid and MM cells show aberrant expression of known human MM dysregulated genes. The similarities of this model with the human disease, coupled with documented frequent XBP-1s overexpression in human MM, serve to implicate XBP-1s dysregulation in MM pathogenesis.

Deregulated Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, yet the development of targeted therapies to disrupt the aberrant transcription has proven difficult because the pathway incorporates large protein interaction surfaces and regulates many homeostatic functions. Therefore, we have directed our efforts toward blocking the interaction with BCL9, a co-activator for β-catenin-mediated transcription that is highly expressed in tumors but not in the cells of origin. BCL9 drives β-catenin signaling through direct binding mediated by its α-helical homology domain-2. We developed a Stabilized Alpha-Helix of BCL9 (SAH-BCL9), which we show targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and exhibits mechanism-based anti-tumor effects. SAH-BCL9 also suppresses tumor growth, angiogenesis, invasion, and metastasis in mouse xenograft models of Colo-320 colorectal carcinoma and INA-6 multiple myeloma. By inhibiting the BCL9/β-catenin interaction and selectively suppressing oncogenic Wnt transcription, SAH-BCL9 may serve as a novel prototype therapy for cancers driven by deregulated Wnt signaling.

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.

Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances β-catenin–mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.

No abstract

Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/ additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P ‫؍‬ .007) and overall survival was significantly increased (P < . IntroductionMultiple myeloma (MM) is a B-cell disease characterized by accumulation of malignant plasma cells in the bone marrow (BM), bone lesions, and immunodeficiency. Genetic analysis shows that approximately 55% to 60% of MM patients have a hyperdiploid karyotype, which confers a better prognosis than nonhyperdiploid disease. 1 The most frequent chromosomal abnormalities observed in nonhyperdiploid MM are translocations between immunoglobulin heavy chain gene located on chromosome 14q32 and an oncogene chromosome 11q13 (CYCLIN D1), 4p16.3 (FGFR3 and MMSET), 6p21 (CYCLIN D3), 16q23 (MAF), or 20q11 (MAFB); or less frequently, the immunoglobulin light chain locus (2p12, or 22q11). 2 During cell proliferation, activation of each cell-cycle phase is dependent on the progress and completion of the previous one. Regulation of the cell cycle involves detecting and repairing genetic damage, as well as controlling various checkpoints to prevent uncontrolled cell division. MM cells are further influenced by the BM microenvironment because adhesion of MM cells to extracellular-matrix proteins supports cell adhesion-mediated drug resistance. In addition, binding of MM cells to BM accessory cells induces secretion of cytokines, which further promote growth, survival, and migration of tumor cells, as well as resistance to conventional chemotherapy. 2,3 Aberrant or overexpression of D-type cyclins is ubiquitous in MM, 4,5 and Aurora kinases regulate cell-cycle checkpoints 6 and cell cycle-regulatory molecules, including cyclins and cyclindependent kinases. [7][8][9] Aurora serine/threonine kinases localize in the centrosome and play a crucial role in cell division by regulating chromatid segregation in mitotic cells 10 ; moreover, defective chromatid segregation causes genetic instability, leading to tumorigenesis. 11 They were first identified in Xenopus Eg2, yeast Ipl1, and Drosophila aurora. The human genome expresses 3 members of the mitotic Aurora kinase family: Aurora-A serine/threonine kinases, Aurora-B serine/threonine kinases, and Aurora-C s...

Multiple myeloma (MM) is a cancer of plasma cells with complex molecular characteristics that evolves from monoclonal gammopathy of undetermined significance, a highly prevalent premalignant condition. MM is the second most frequent hematologic cancer in the United States, and it remains incurable, thereby highlighting the need for new therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, shared across different MM subtypes. Here we report that Wnt/␤-catenin is one such pathway. We document the involvement of ␤-catenin in cell-cycle regulation, proliferation, and invasion contributing to enhanced proliferative and metastatic properties of MM. The pleiotropic effects of ␤-catenin in MM correlate with its transcriptional function, and we demonstrate regulation of a novel target gene, Aurora kinase A, implicating ␤-catenin in G2/M regulation. ␤-catenin and Aurora kinase A are present in most MM but not in normal plasma cells and are expressed in a pattern that parallels progression from monoclonal gammopathy of undetermined significance to MM. Our data provide evidence for a novel functional link between ␤-catenin and Aurora kinase A, underscoring a critical role of these pathways in MM disease progression. (Blood. 2009;114:2699-2708) IntroductionMultiple myeloma (MM) is a neoplasm of plasma cells that infiltrates the bone marrow (BM). Despite recent advances in its treatment, it remains incurable, with a median survival of 6 years. 1 MM accounts for more than 10% of all hematologic malignancies and is the second most frequent hematologic cancer in the United States. It is typically preceded by an age-progressive condition termed monoclonal gammopathy of undetermined significance (MGUS), which is present in 1% to 10% of adults older than 25 years of age and progresses to malignant MM at a rate of 0.5% to 3% annually. 1,2 This disease is characterized by frequent chromosomal aberrations and mutations in several oncogenes and tumor-suppressor genes. 3,4 The Wnt/␤-catenin pathway is significant in cancer development because numerous human malignancies such as colorectal, hepatocellular, and breast cancer harbor activating mutations in critical components of this pathway. 5,6 Normally, the Wnt signaling pathway is active during embryogenesis, hematopoietic stem cell growth, cell differentiation, and tissue development. ␤-catenin, a central effector of the Wnt pathway, is involved in both nuclear and cytoplasmic functions. 7,8 In the absence of Wnt ligands, ␤-catenin is targeted by a complex consisting of adenomatous polyposis coli, axin, glycogen synthase kinase-3␤, and casein kinase 1␣ that phosphorylate and mark it for degradation by the ubiquitinproteasome pathway. 9,10 Upon Wnt stimulation, however, the kinase complex is dissociated, and ␤-catenin is not targeted for destruction. The active form of ␤-catenin translocates to the nucleus and, in association with lymphoid enhancer factor (LEF)/Tcell factor (TCF) proteins, activates transcription...

Recent studies have identified vimentin, a type III intermediate filament, among genes differentially expressed in tumours with more invasive features, suggesting an association between vimentin and tumour progression. The aim of this study, was to investigate whether vimentin expression in colon cancer tissue is of clinical relevance. We performed immunostaining in 142 colorectal cancer (CRC) samples and quantified the amount of vimentin expression using computer-assisted image analysis. Vimentin expression in the tumour stroma of CRC was associated with shorter survival. Overall survival in the high vimentin expression group was 71.2% compared with 90.4% in the low-expression group (P ¼ 0.002), whereas disease-free survival for the high-expression group was 62.7% compared with 86.7% for the low-expression group (P ¼ 0.001). Furthermore, the prognostic power of vimentin for disease recurrence was maintained in both stage II and III CRC. Multivariate analysis suggested that vimentin was a better prognostic indicator for disease recurrence (risk ratio ¼ 3.5) than the widely used lymph node status (risk ratio ¼ 2.2). Vimentin expression in the tumour stroma may reflect a higher malignant potential of the tumour and may be a useful predictive marker for disease recurrence in CRC patients.