ALBERT

Description: Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell–derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.

Description: Abstract 1804 Poster Board I-830 Introduction MicroRNAs (miRNAs) are non-coding RNAs that regulate mRNA expression. miRNAs often act synergistically to repress target genes and their deregulation can contribute to the initiation and progression of a variety of cancers. The clinical relationship between global expression miRNA and mRNA in cancer has not been studied in detail. Methods We used whole genome microarray analyses of CD138-enriched plasma cells from 52 newly diagnosed cases of multiple myeloma (MM) to correlate miRNA expression profiles with a validated mRNA-based risk stratification, proliferation index, and pre-defined gene sets. Results In stark contrast to mRNAs, we discovered that all tested and expressed miRNAs were significantly up-regulated in high-risk disease as defined by a validated 70-gene risk score (P

Description: Abstract 1786 Poster Board I-812 Background Multiple Myeloma (MM) is characterized by a clonal proliferation of antibody producing malignant plasma cells. Complete or partial monoallelic deletion of chromosome 13, is commonly observed in tumor cells of patients with monoclonal gammopathy of unknown significance and in over 50% of MM patients, as well as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Recurrent loss of a minimal common region (MCR) of 10 megabases at 13q14, in MM and CLL suggests the MCR harbors a tumor suppressor gene(s) (TSG) with biological and clinical relevance. Within this MCR resides the Ret Finger Protein 2 (RFP2) encoding gene, which produce an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). Because of its copy number-dependent expression, its strong and unique promoter, and its associated inferior survival with reduced expression in MM, RFP2 represents a candidate TSG. Nevertheless, its role and targets have not yet been established. Here we describe a functional analysis of RFP2 in MM cells. Methods: The MMS1 MM cell line lacks chromosome 13 deletion. To study the effects of loss of RFP2 in this line we used the PLKO- GFP lentiviral vector to stably transduce a RFP2 shRNA. Flow cytometer selected cell lines exhibit significantly reduced expression of RFP2 relative transduced shRNA controls or to the parental line. Cell growth rate was measured using trypan blue counting, soft agar colony formation and thymidine incorporation. Cell cycle analysis and apoptosis were measured by flow cytometry after staining with PI or Annexin-V PE and 7AAD, respectively. Intracellular signal modulation was demonstrated by Western blotting. Results At day six post transduction, 75-95% of MMS1 cells were GFP positive. RFP2 downregulation induced an impairment of cell growth with a G2 phase arrest and a profound apoptosis (over 50% at day six as compared with less than 15% of controls). This effect was mediated through ER stress evidenced by upregulation of p-eIF2a and Bip, and the induction of Caspase-8, 9 and 3 cleavage. RFP2 complementation did not produce by itself a significant growth promoting effect, but was able to rescue the knockdown-induced growth retardation. The above described presence of ER stress, combined with the previous reports that RFP2 has E3 ubiquitin ligase activity prompted us to assess total protein ubiquitination. Concordant with its effects on ER stress, RFP2 downregulation was associated with significantly higher levels of poly-ubiquitinated proteins. Subsequently, we were able to document a significant reduction (60% inhibition) in 20S proteasome activity in RFP2 down regulated cells. Proteasome inhibition by RFP2 down regulation was confirmed in other MM cell lines and was partially abrogated by restoring RFP2 levels by overexpression. Importantly, RFP2 down regulated cells were more sensitive to bortezomib; indeed proteasome inhibition was synergistic with RFP2 downregulation in MM cells. The above results prompted us to study the mechanism whereby RFP2 impacts survival and proliferation of MM cells. Inhibition of the NF-kappa-B (NFκB) pathway is a hallmark of proteasome-related growth retardation and apoptosis and is a key pathway in MM. We show that NFkB luciferase reporter assay was associated with significant activity reduction with RFP2 downregulation. To define the mechanism of this process, we examined the level of NFkB related proteins in nuclear and cytoplasmic fractions. Interestingly, the most prominent effect observed in RFP2 down regulated cells was increased levels of IkBá in the nucleus. Altogether, these results support our supposition that the effects of RFP2 downregulation are mediated through an inhibition of the NFkB pathway that is associated with increased nuclear IkBa as well as a decrease in 20S proteasome activity. Conclusions RFP2 is a gene mapping to a deletion hotspot at 13q14 and reduced RNA expression is associated with poor survival in MM. Functional studies revealed that shRNA mediated knockdown of RFP2 in MM causes growth retardation and apoptosis, mediated by ER stress and a G2 arrest, mediated by proteasome inhibition and reduced NFkB activity. Although RFP2 did not prove itself to be a tumor suppressor gene in our studies, targeting RFP2 may represent a novel therapeutic approach in MM and other lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Using data derived from the U133Plus2.0 microarray (U2), we recently constructed a 17-gene model predictive of high-risk multiple myeloma (MM). In the model, 13% of newly diagnosed cases were considered to have high-risk MM with 24 month overall survival estimates of 50% and 90% in the high- and low-risk groups, respectively (p

Description: In multiple myeloma (MM), the proteasome inhibitor bortezomib induces mesenchymal stem cells (MSC) toward osteoblast differentiation. However, it is full unclear about the mechanism(s) underlying bortezomib in this process. Wnt/beta-catenin pathway plays a pivotal role in osteoblast differenciation and bone development. We have demonstrated that inhibition of Wnt/beta-catenin signaling by MM-derived Dkk1 suppresses osteoblast progenitor cell differentiation into osteoblasts (Qiang et al, Bone 2008) and deregulate RANKL and OPG expression in osteoblast cells (Qiang et al Blood 2008a). Increase Wnt signaling by overexpression of Wnt3a in myeloma cells diminished MM-trigged bone lesion in mouse model (Qiang et al Blood 2008b). In the present study we revealed that bortezomib promotes MSC differentiation into osteoblast cells via Wnt-independent activation of beta-catenin/TCF signaling. E-cadherin pull-down assay and subsequently immunoblotting analysis demonstrated that bortezomib induced increases in both free and active forms of beta-catenin protein in cytoplasm and nuclear in bell-shaped dose- and time-dependent manner in mouse and human osteoblast progenitor cell lines including C2C12, C3H10T1/2, Saos-2 and MG63. Similar results were illustrated in primary human 2 cases of normal MSC and MSC from 8 cases of MM pateints. Bortezomib induced increase in ubiquitinated beta-catenin was evidenced by obvious seen slow migration bands of beta-catenin protein in SDS-PAGE gel analysis indicating that bortezomib increased beta-catenin protein by modification of proteasome-mediated degradation of beta-catenin. Increase in cytoplasm and nuclear beta-catenin protein response to bortezomib treatment in the osteoblast cell lines and 4 cases MM derived MSC was further confirmed by immunofluorescent analysis. RT-PCT analysis of TCF family revealed that abundant TCF1 and TCF4 mRNA were expressed in all tested cell lines and in a primary normal MSC, and MM-derived MSC. Bortezomib treatment also resulted in TCF transcriptional activity in bell-shaped, dose-dependent pattern as determined by luciferase activity in these cells transfected with TOPflash plasmid DNAs. Maximal responses to bortezomib were seen at 12.5 nM for both C2C12 (p

Description: Introduction: Expression NCAM1, a cell adhesion molecule involved in neuron-neuron adhesion, is also expressed by multiple myeloma (MM PC). Osteolytic bone lesions are a hallmark of MM and elevated expression of NCAM in MM PC has been correlated with this process (Ely and Knowles, 2003). We have previously reported that MM PC express DKK1 and MM blocks osteoblast differentiation in a DKK1-specific manner, suggesting that secretion of the Wnt signaling inhibitor plays a role in MM bone disease. Herein we used gene expression microarrays and tissue microarrays (TMAs) to investigate the simultaneous expression of DKK1 and NCAM in MM and MGUS. Methods: The study population consisted of 198 newly diagnosed MM and 44 MGUS. RNA from CD138-selected plasma cells was hybridized to Affymetrix U133Plus microarrays and data processed with Affymetrix Microarray Suite GCOS1.1 software. TMAs were constructed from formalin-fixed, paraffin embedded bone marrow biopsies. Serial sections of TMA were immunostained for CD138, NCAM and DKK1. TMAs were scanned using ScanScope using 20x lens, assessed using TMA lab software (Aperio Technologies) and scored as an average number of cells in the context of CD138 staining. Results: When put in context of a recently described molecular classification (Zhan et al., 2006), NCAM and DKK1 were found significantly co-over-expressed (DKK1+/NCAM+) in HYPERDIPLOID MM (P 〈 0.01); NCAM+/DKK1− was typical for MMSET-spike MM (P

Description: Background: Many prognostic models have been proposed for staging patients with MM, most recently the ISS system utilizing B2M and albumin. Patients and Methods: In the context of Total Therapy 2 (TT2) for newly diagnosed MM, we identified 220 patients in whom 4 standard prognostic factors (SPF: B2M, albumin, LDH, hemoglobin), imaging data (MRI-defined focal lesions [MRI-FL]), metaphase-derived cytogenetic abnormalities (CA), FISH-derived amp1q21 and del13q14, and gene expression profiling (GEP)-derived data are available. The baseline characteristics and clinical outcome of the 220 patients are similar to those of the entire population of 668 patients receiving TT2. The median follow-up of the 220 patients is 42mo compared to 52mo for all 668 patients. Five multivariate analysis-based prognostic models were derived, utilizing SPF only (model I), with progressive addition of CA (model II), MRI (model III), FISH (model IV) and GEP (model V). Results: In model I, B2M, LDH and hemoglobin, but not albumin were independently significantly associated with overall survival (OS) (R2=22.5%); upon introduction of CA (model II) (p=.008), only LDH remained significant (p=.033) (R2=28.3%); addition of MRI (model III) was not significant (R2=31.1%); addition of FISH (model IV) was significant for amp1q21 (p=.003) but not for del13q13, with CA remaining significant (p=.023) (R2=38.1%); when GEP was added (model V), the 70 gene-derived model (p

Description: Deletion of chromosome 17p13 is a poor risk feature in MM, presumably resulting from a loss-of-hetrozygosity of the TP53 locus. However, details on the role of TP53 in the pathogenesis of MM and its relevance as a prognostic variable remain uncertain. We determined TP53 mRNA expression, DNA sequence integrity and copy number in newly diagnosed and relapsed disease and correlated these features with disease progression and outcome. Among 351 newly diagnosed cases treated on TT2, a randomized trial of high-dose chemotherapy and autotransplants +/− thalidomide. TP53 gene expression, as determined by Affymetrix microarray of CD138-selected cells, varied from a high of 5,241 to a low of 10 (mean 1,460). TP53 expression lower than 727, present in 10% of tumors, was associated with shorter event-free survival (EFS: P=0.0012) and overall survival (OS: P=0.0006) with a median follow-up of 40 months. Short EFS (P221 umol/L), and LDH (〉191 IU/L), Amp1q21, del13, MRI lesions (〉3), and high-risk molecular groups (PR, MS, and MF) (P 〈 .05) in the TT2 cohort. There was no significant difference in cytogenetic abnormalities, B2M (〉4 mg/L), albumin (33%). On multivariate regression analysis, low TP53 expression was an independent variable predicting shorter survival (EFS: P=0.044; OS: P=0.019), even in the context of high-risk molecular entities and ISS. We next used interphase FISH to correlate TP53 gene copy number with expression in randomly selected cases from the TT2 cohort. 17p13 deletion was observed in 88% (27% with bi-allelic deletion) of 17 cases from the group of 35 (10% of total) with the lowest TP53 expression, while deletion was present in 36% in 22 cases with low-, 12% in 17 cases with mid-level, and 6% in 17 samples with high-expression of TP53 (P

Description: Abstract 743 Background There has been considerable recent focus upon the molecular classification of myeloma. However, the prognostic impact of molecular changes has mostly been assessed from small and/or incomplete studies from single institutions or groups. There has been no large scale analysis of molecular features linked to ISS stage. Methods In order to clarify the overall impact of molecular changes we undertook a collective analysis of 9,897 patients through the International Myeloma Working Group (IMWG). Within this population 2,295 patients had presence of cytogenetic abnormalities (Any CA); 1,713 hypodiploidy; 1,673 hyperdiploidy; 2,309 cytogenetic deletion 13; 3,226 deletion 13 by FISH; 1,573 FISH t(4;14); 1,486 FISH del p17; 1, 683 FISH t(11:14); and 366 FISH t(4;16). Enrolled patients had complete clinical and treatment details available including baseline standard prognostic factors, ISS stage, as well as both progression free survival (PFS) and overall survival (OS) information. Data came from 14 sites: 3 from the US and the remainder from Europe, Asia, and Latin America as for the ISS staging system analyses. Univariate and multivariate analyses were performed. Results Each of the known adverse molecular features had a negative impact upon both PFS and OS (p=002 -

Description: High-risk multiple myeloma (HRMM) is routinely defined by laboratory parameters alone or in combination in the Durie-Salmon and, more recently, the ISS staging systems. The Bartl grade, a cell morphology-based staging system, has seen limited use. The presence of abnormal cytogenetics, high BrdU labeling index, interphase FISH abnormalities, and flow cytometric measures have also been used. A molecular-based classification and risk stratification of MM may improve the definition of HRMM. Global gene expression profiling (GEP) with of CD138-selected plasma cells followed by unsupervised hierarchical cluster analysis revealed that MM comprises a spectrum of seven distinct reproducible subtypes. A validated molecular classification schema has been defined as follows: (MS = t(4;14); MF = t(14;16) or t(14;20); CD-1 = t(11;14) or t(6;14) and CD-2 = t(11;14) or t(6;14) with high CD20 and/or VPREB3), hyperdiploidy (HY = high DKK1, FRZB, NCAM1, TNFSF10), low bone disease (LB = NF-kB signature, high CCND2, CST6, and IL6R) and proliferation (PR = high MIK67, CCNB1, CCNB2, TOP2A, and TYMS). Correlating GEP with outcome in two independent cohorts permitted the identification of a high-risk signature (UAMS 17-gene model), present in approximately 13% of newly diagnosed disease. GEP and high-resolution comparative genomic hybridization in 92 cases confirmed that the altered expression of the 17 genes in the model is driven by 1q gains and 1p losses. This high-risk signature is evident in a subset of all 7 molecular subtypes and negatively influences outcome. For example, low-risk MS disease fares much better than high-risk MS disease. We recently reported that the addition of bortezomib to TT3 has significantly improved outcome in low-risk MS disease, thereby demonstrating the value of GEP in evaluating benefits of new treatments that might be otherwise masked. When subjected to multivariate analysis including the International Staging System (ISS) and a gene expression-based proliferation index (GEP PI), the UAMS 17-gene model remained a significant predictor of outcome. Mulligan and colleagues developed outcome classifiers for relapsed disease treated with single agent bortezomib or high dose dexamethasone improved upon the risk stratification provided by the ISS. These predictive models showed some specificity for bortezomib. Using U133A data from newly diagnosed disease treated with ASCT, the Mayo clinic group validated the UAMS 17-gene model, but also showed that the t(4;14) translocation remained a significant adverse variable. The IFM recently reported on a 15-gene model of high-risk (IFM 15-gene model) related to cell proliferation. Multivariate showed that the UAMS 17-gene model was significant in all datasets, while the IFM 15-model was significant in a limited number. This difference might be attributed to the dependence of the IFM model to cell proliferation. GEP on 71 paired diagnostic and relapse samples indicate that the UAMS 17-gene model score increases in 80% of the cases and a low-risk to high-risk conversion in 14 of 24 (58%) severely impacted post-relapse survival. Expression of TP53 is a surrogate for 17p13 deletion, and TP53 expression below a specific threshold (seen in approximately 10% of newly diagnosed disease) imparts a poor prognosis in low-risk – but not high-risk – MM, defined by the UAMS 17-gene model. In conclusion, while the majority of patients with MM can anticipate long-term disease control, approximately 25% of patients with molecularly defined HRMM do not benefit from current approaches.