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: Total Therapy 2 examined the clinical benefit of adding thalidomide up-front to a tandem transplant regimen for newly diagnosed patients with multiple myeloma. When initially reported with a median follow-up of 42 months, complete response rate and event-free survival were superior among the 323 patients randomized to thalidomide, whereas overall survival was indistinguishable from that of the 345 patients treated on the control arm. With further follow-up currently at a median of 72 months, survival plots segregated 5 years after initiation of therapy in favor of thalidomide (P = .09), reaching statistical significance for the one third of patients exhibiting cytogenetic abnormalities (CAs; P = .02), a well-recognized adverse prognostic feature. The duration of complete remission was also superior in the cohort presenting with CAs such that, at 7 years from onset of complete remission, 45% remained relapse-free as opposed to 20% on the control arm (P = .05). These observations were confirmed when examined by multivariate analysis demonstrating that thalidomide reduced the hazard of death by 41% among patients with CA-positive disease (P = .008). Because two thirds of patients without CAs have remained alive at 7 years, the presently emerging separation in favor of thalidomide may eventually reach statistical significance as well.

Description: Induction of osteolytic bone disease in multiple myeloma (MM) is caused by activation of osteoclastogenesis and suppression of osteoblastogenesis. Bone formation is reduced mainly through production of inhibitors of osteoblast differentiation by MM cells and by impaired osteogenic differentiation of endogenous mesenchymal stem cells (MSCs). Recently, human placenta has emerged as a potentially valuable source of progenitor cells for multiple therapeutic purposes, including bone repair and cancer (Parolini et al., Stem Cells26:300–311, 2008). The aim of the study was to investigate the effects of human placenta-derived adherent cells (PDAC™) on MM bone disease and tumor growth in the SCID-rab mouse model for MM. PDAC™ are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Bone disease was evaluated by measurements of bone mineral density (BMD) and visualized by X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and live animal imaging. For in vivo tracking PDAC™ or our stroma-dependent BN MM cell line was transduced with a luciferase/GFP reporter in a lentiviral vector. In the first in vivo experiment, 10 SCID-rab mice were engrafted with a patient’s MM cells. Following establishment of MM and detection of bone disease, luciferase-expressing PDAC™ (1×106 cells/bone) or phosphate-buffered saline (PBS) were injected directly into implanted myelomatous bones in SCID-rab mice (5 mice/group). At experiment’s end (5 wk after cytotherapy) PDAC™ could be detected in mice by live animal imaging. Whereas in control mice, BMD of the implanted bone was reduced from pretreatment levels by 8±4%, administration of PDAC™ resulted in increased BMD of the implanted bone in all mice by 132±20% from pretreatment levels (p

Description: The Dickkopf-1 (DKK1) gene product inhibits osteoblast activity by blocking Wnt signaling. Elevated levels of DKK1 in bone marrow plasma and peripheral blood are associated with the presence of osteolytic bone lesions in patients with multiple myeloma (Tian E, et al. N Engl J Med.2003, 349:2483), evidently by inhibiting osteoblast differentiation and, as a result, stimulating osteoclastogenesis driven by osteoblast progenitors’ enhanced production of RANKL and reduced expression of OPG. DKK1-neutralizing antibody suppressed bone resorption induced by inflammatory joint disease and myeloma in murine models (Diarra, Nature Medicine2007, 13:156, Yaccoby, Blood2007, 109:2106), and ectopic Wnt3a expression prevented myeloma induced bone resoption (Qiang, Blood2008;112:374). To further investigate whether DKK1 is essential for producing myeloma bone disease, we used 5 myeloma cell lines with various expression levels of DKK1 in our SCID-hu model. U266 and CAG cells, which express minimal amounts of DKK1 mRNA by qRT-PCR corresponding to 5ng/ml in spent media by ELISA, caused severe bone resorption, whereas ARP1, H929 and OPM2 which express 55, 61, and 320 fold higher levels of DKK1 mRNA (and 80 ng/ml protein for ARP1 cells) had no effects on the bones. OPM2 cells overexpressing DKK1 following transduction with lentiviral vector (127 fold higher than U266 cells), also did not cause bone resorption. To understand if the lack of relationship between DKK1 expression by myeloma cell lines and bone resorption reflected the level of expression, we used the prostate cancer cell line PC3 that expresses 100 times higher DKK1 mRNA than DKK1-transduced OPM2 cells. These cells caused severe bone destruction in the SCID-hu model. DKK1 expression by PC3 was inhibited by 80% by constitutive or conditional (tet-on) shRNA-lentivirus, and resulted in corresponding reduction (75%, p=0.02) in loss of bone mineral density compared with control cells transduced with conditional or constitutive control shRNA lentivirus. PC3 cells recovered from the tumors had 50% reduced levels of DKK1 mRNA. Myeloma associated osteolytic bone disease results from inhibition of osteoblast differentiation, resulting in reduced bone formation and overcharged osteoclastogenesis by osteoblast progenitors. It thus appears that a quantitative threshold exists below which DKK1 inhibition of Wnt is not sufficient to cause osteolytic bone disease. Alternatively, it is possible that the DKK1 produced by the MM cell lines is not funtional, or that factors other than inhibition of Wnt signaling by DKK1 can also be responsible for producing osteolytic bone disease. We are currently investigating whether direct stimulation of osteoclastic activity by the myeloma cell lines that cause osteolysis but express low DKK1 can explain this observed discrepancy, or if they inhibit osteoblast differentiation by other mechanisms. This work was supported in part by NIH grant CA55816.

Description: Introduction: The mechanism of action and resistance to chemotherapy is poorly understood and measures of efficacy typically rely on clinical outcome data. Recent advances suggest that prospective gene expression profiling (GEP) can be used to more accurately define not only short-term but lasting treatment benefits. We recently reported that baseline tumor cell gene signatures, encompassing 70 and as few as 17 genes, can discriminate risk groups of myeloma patients both in the untreated and previously treated settings. However, a subset of predicted low-risk cases followed an aggressive clinical course accompanied by a shift from 70-gene-defined low-to high-risk over time, either reflecting clonal evolution or outgrowth of aggressive clones present, but undetectable, at diagnosis. Accurately identifying this patient population is a first step in preempting such transformation. We hypothesized that changes in gene expression patterns of tumor cells following a short term in-vivo challenge with a single agent chemotherapeutic might expose these latently aggressive cells. Unlike in-vitro testing, clinical drug administration also allows for assessing tumor cell perturbation in the context of host interactions. Building on recent observations that clinical outcome in myeloma patients could be correlated with 48hr GEP changes induced in vivo following single agent administration of thalidomide, lenalidomide, and dexamethasone, we now examined whether such short-term tumor-cell GEP and proteomic alterations could fine-tune clinical outcome prediction beyond the well established 70-gene-based baseline prediction model. Methods: Affymetrix U133Plus2.0 microarray analysis and mass spectrometry were performed on CD138-enriched tumor cells prior to and 48hr following a single test-dose application of bortezomib at 1.0mg/m2 in 142 newly diagnosed patients with MM. A total of 1051 genes (P 〈 .005) were differentially expressed at 48 hours. Both change in expression, adjusting for baseline expression, and post-drug expression levels of each gene were examined for correlations with event-free survival in a Cox proportional hazards model. Post-drug expression was chosen and 113 genes were retained (p =1 (unfavorable) was used to create a score which, in the context of running log rank statistics, was used to classify patients into high- and low-risk groups. The independent prognostic power of the score for event-free and overall survival was investigated, together with baseline prognostic variables, by multivariate analysis. This method was tested in a 10-fold cross-validation procedure using the same data set. The model is currently being validated in an independent set of 100 cases and results will be reported. Results: Changes in the expression of proteasome genes, and their related proteins, predominated a list of 113 outcome-related genes. A high-risk score, associated with upregulation of proteasome genes, seen in 24% of cases, was associated with median survival of less than 24 months, dramatically contrasting with a 3-year survival estimate of greater than 80% in the 76% in whom proteasome genes were not activated (p

Description: Myeloma is intimately associated with osteolytic bone disease, in part by inhibiting mesenchymal stem cell (MSC) differentiation into osteoblasts via blocking Wnt signaling (Tian, N Engl J Med2003;349:2483). Stimulation of osteoblast activity by bortezomib is associated with anti myeloma effects in patients (Zangari, Br J Haematol2005;131:71) and by MSC infusion in a model system (Yaccoby, Haematologica2006;91:192). To further evaluate the potential therapeutic utility of MSCs, we investigated the effects of MSCs on myeloma cell survival and the genomic consequences of MSC-myeloma cell interactions. CD138-purified myeloma (MM) cells from 16 consented patients were cocultured with MSCs derived from the bone marrow of 3 healthy donors (MSC 1, 2, &3, provided by Dr. D. Prockop, Tulane University). In 7 co-culture experiments, MSCs supported MM cell survival for 6–8 days compared to MM cells cultured alone, with a median viable cell count increasing by 2.3 fold v controls (1.2–4.1, p=0.003); in 4, coculture suppressed MM survival (median=0.5, range 0.3–0.7, p=0.016); and in 5 there was no effect (median=0.9, range 0.9–1.0). In 3 experiments, the effects of the different MSCs on MM cell survival varied inconsistently among the 3 MSCs, from 0.5 to 2.5 fold surviving cells. In order to understand this heterogeneity, we investigated the genomic consequences of MM-MSC interactions. RNA was extracted immediately after mixing (t=0) and following 18 hours co-culture (t=18), and changes in gene expression analyzed using the Affymetrix microarray system. Since myeloma cells adhere tightly to MSCs, we analyzed expression changes in 1,708 genes expressed only in MM cells and 4862 expressed only in MSCs. At t=18, 250 MM cell genes were changed by 〉2 fold compared with t=0 (222 up and 28 down regulated), and 1,036 MSC genes were changed 〉2 fold (1018 up and 18 downregulated). Each of the 3 MSCs also had unique genes expressed (40, 85 and 162 for MSC 1, 2, &3, respectively), of which expression of 12, 30, and 39, respectively, was changed by 〉2 fold. Analysis of 776 MM- and 2398 MSC-related genes after a signal cutoff of 500 with Ingenuity Pathways Analysis software showed changes in the expression of several groups of interrelated genes following co-culture. For MM, these included ERKs, AKT, NFKB, E2F1, and FOS. The transcription regulators BTG2, ACTN2, CALR, E2F1, E2F5, and ATF7 were up regulated, while FOS and FOXM1 were down regulated. Groups changed in MSCs included IL1B, CCND1, MYC, CTBP1, EGFR, RUNX1, HRAS, and SMAD3. There were minor differences in changes of the expression patterns of some of the probesets between the three MSCs, likely related to alternative splicing or to variations in the 3′-UTR. These studies continue in order to discern possible differences in the interactions between MM cells and MSCs, and to identify whether MM cells or MSC determine the outcome of these differences.

Description: Smoldering multiple myeloma (SMM) is usually followed expectantly without therapy. We conducted a phase 2 trial in 76 eligible patients with SMM, combining thalidomide (THAL, 200 mg/d) with monthly pamidronate. In the first 2 years, THAL dose reduction was required in 86% and drug was discontinued in 50%. Within 4 years, 63% improved, including 25% qualifying for partial response (PR); by then, 34 patients had progressed and 17 required salvage therapy. Unexpectedly, attaining PR status was associated with a shorter time to salvage therapy for disease progression (P 〈 .001), perhaps reflecting greater drug sensitivity of more aggressive disease. Low beta-2-microglobulin levels less than 2 mg/L were independently associated with superior overall and event-free survival. Four-year survival and event-free survival estimates of 91% and 60%, respectively, together with a median postsalvage therapy survival of more than 5 years justify the conduct of a prospective randomized clinical trial to determine the clinical value of preemptive therapy in SMM. Trial registered at http://www.clinicaltrials.gov under identifier NCT00083382.