ALBERT

Description: Dogs given 1200 r of total body irradiation were cross-circulated with dogs having normal marrow function. Irradiated controls died in from 4 to 11 days with marrow aplasia. Dogs cross-circulated daily for 6 to 9 days showed histologic evidence of bone marrow repopulation after 1 week. Male dogs cross-circulated with female partners showed typical female drumsticks on mature granulocytes after repopulation had occurred. Cytogenetic studies of an irradiated male dog cross-circulated with a female partner showed all mitotable cells from the bone marrow and peripheral blood to be of female donor type. Allogeneic bone marrow engraftment was associated with an early and severe secondary syndrome which resulted in the death of the animals in the second week. When methotrexate was given, survival was increased to 3 weeks. It was concluded that (1) cross circulation provided leukocytes and platelets adequate for support during the period of radiation-induced marrow aplasia, (2) allogeneic marrow engraftment was produced consistently by cells transferred in the peripheral blood of the normal cross circulation partner, (3) the grafts were associated with an early and severe form of secondary disease, and (4) methotrexate given during the early period of engraftment reduced the severity of the secondary disease.

Description: Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

Description: Key Points CYR61/CCN1 is a bone marrow microenvironmental biomarker for myeloma progression and for transformation of MGUS and asymptomatic disease to overt myeloma. CCN1 reduces myeloma bone disease and tumor growth and is a potential therapeutic target for myeloma.

Description: Abstract 5014 Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev's direct effect on MM cell survival. To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation. These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug's cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Jumping translocations of 1q12 (JT1q12) provide a mechanism for the deletion of 17p in cytogenetically defined high-risk myeloma. Sequential JT1q12s introduce unexpected copy number gains and losses in receptor chromosomes during subclonal evolution.

Description: Abstract 3717 Human placenta has emerged as a valuable, uncontroversial source of transplantable cells for many cytotherapeutic purposes, including modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDAC) are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Multiple myeloma (MM) is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. The aim of the study was to evaluate the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDAC in the SCID-rab model of MM-associated bone disease. SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which primary human myeloma cells were directly injected (Yatta et al., Leukemia 2004; Yaccoby et al., Blood 2007). Bone disease was evaluated by measurements of bone mineral density (BMD) and X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and histologically. For in vivo tracking PDAC were transduced with a luciferase/GFP reporter in a lentiviral vector. SCID-rab mice engrafted with primary myeloma cells from 2 patients. Upon establishment of MM growth, PDAC (1×106 cells/bone) or vehicle were injected into the implanted myelomatous bone (Patient's 1, 5 mice/group; Patient's 2, 7 mice/group). While BMD of the implanted bones was significantly reduced in control hosts, intralesional PDAC cytotherapy significantly increased BMD of the implanted bones from pretreatment levels by 〉37% (p

Description: Sickle cell disease (SCD) results in chronic hypoxia and secondarily increased erythropoietin concentrations. Leukocytosis and activated monocytes are also observed in SCD in absence of infection or vaso-occlusion (steady state), the reasons for which are unknown. We found that erythroid cells produced placenta growth factor (PlGF), an angiogenic growth factor belonging to the vascular endothelial growth factor (VEGF) family, and its expression was induced in bone marrow CD34+ progenitor cells in the presence of erythropoietin. Furthermore, the steady state circulating PlGF levels in subjects with severe SCD (at least 3 vaso-occlusive crises [VOCs] per year) were 18.5 ± 1.2 pg/mL (n = 9) compared with 15.5 ± 1.2 pg/mL (n = 13) in those with mild SCD (fewer than 3 VOCs per year) and 11.3 ± 0.7 pg/mL (n = 9) in healthy controls (P 〈 .05), suggesting a correlation between PlGF levels and SCD severity. In addition, PlGF significantly increased mRNA levels of the proinflammatory cytochemokines interleukin-1β, interleukin-8, monocyte chemoattractant protein-1, and VEGF in peripheral blood mononuclear cells (MNCs) of healthy subjects (n = 4; P 〈 .05). Expression of these same cytochemokines was significantly increased in MNCs from subjects with SCD at steady state (n = 14), compared with healthy controls. Of the leukocyte subfractions, PlGF stimulated monocyte chemotaxis (P 〈 .05, n = 3). Taken together, these data show for the first time that erythroid cells intrinsically release a factor that can directly activate monocytes to increase inflammation. The baseline inflammation seen in SCD has always been attributed to sequelae secondary to the sickling phenomenon. We show that PlGF contributes to the inflammation observed in SCD and increases the incidence of vaso-occlusive events.

Description: Inhibition of integrins αvβ3 and αvβ5 in human brain microvascular endothelial cells (HBMECs) by the function-blocking peptide RGDfV induces loss of spreading on vitronectin, cell detachment, and apoptosis. We demonstrate that cell detachment is not required for apoptosis because plating on bovine serum albumin–blocked poly-L-lysine (allows attachment, but not integrin ligation and cell spreading) also induced apoptosis. Latrunculin B (LatB), which inhibits F-actin polymerization, induced transient loss of HBMEC spreading on vitronectin, but not their detachment, and induced apoptosis despite recovery of cell spreading. However, LatB did not cause apoptosis in 5 tumor cell lines. In HBMECs, both LatB and RGDfV induced transient Y412 and Y245 phosphorylation of endogenous c-Abl, a nonreceptor tyrosine kinase that reciprocally regulates F-actin. LatB also induced nuclear translocation of c-Abl in HBMECs. STI-571 (imatinib), a targeted therapy for BCR-ABL1+ leukemias and inhibitor of c-Abl, platelet-derived growth factor receptor, and c-Kit, decreased endothelial apoptosis. LatB-induced HBMEC apoptosis, and its inhibition by STI-571 also occurred in a 3-dimensional collagen model, supporting physiologic relevance. Last, siRNA to c-Abl (but not nonspecific siRNA) also inhibited RGDfV- and LatB-induced apoptosis. Thus, endogenous c-Abl mediates endothelial apoptosis induced by inhibition of integrins αvβ3/αvβ5 or by LatB-induced disruption of F-actin.

Description: Abstract 808 Background: Bone disease is one of the most debilitating complications in patients with multiple myeloma (MM). The molecular mechanisms by which MM triggers bone disease are not fully understood. We have previously demonstrated that Dkk1 is highly expressed in primary MM plasma cells, and associated with bone disease in MM patients by inhibiting Wnt signaling-promoted mesenchymal stem cell differentiation and osteoprotegerin production in osteoblast cells. We have also reported that increase in Wnt signaling in the bone marrow microenvironment by overexpression of Wnt3a in myeloma cells or administration of rWnt3a, or indirectly increasing Wnt signaling by administration of anti-Dkk1 neutralizing antibody also decreased in osteoclast numbers. However, Dkk1 is less frequently expressed in MM cell lines that are derived mostly from late stage of MM; and injection of these MM cell lines into human fetal bone also is able to induce bone lesion in MM animal model. These results indicate that additional factors may be involved in induction of the bone disease at the stage of the disease. The members of the sFRPs family of secreted proteins (including sFRP-1, -2, -3 and -4) directly bind to Wnts, thereby preventing Wnts from binding to the cellular Wnt receptor complex. It has also been reported that sFRP-1 and -2 augment canonical Wnt3a activated signaling in fibroblast. MM cells from pateints with advanced bone lesions express sFRP2 mRNA. Like sFRP2, sFRP3 mRNA is highly expressed in MM plasma cells, but it's function in MM bone disease remains unknown. We sought to investigate the role of sFRP3 in MM-triggered bone lesions using the osteoblast (OB) cell lines CH3T1/2 and C2C12, and serum from MM pateints those MM cells expressed high level of sFRP3. Methods/Results: RT-PCR analysis showed that sFRP3 is expressed in primary MM plasma cells and certain MM cell lines. Recombinant sFRP3 protein did not inhibit, but synergized with Wnt3a to increase beta-catenin protein, while Dkk1 significantly inhibited this process. Similarly, sFRP3 treatment of OB cells increase Wnt-3a-induced TCF transcript activity in OB cells transfected with TOPflash luciferase report constructs. sFRP3 also increased MSC differentiation, as evidenced by increase in alkaline phosphatase activity (ALP) and increased in mineralization by Alizarin red staining. sFRP3 treatment also increases OPG mRNA and protein production in these cells. Similar to sFRP3, sFRP1 and sFRP2 synergistically acted with Wnt3a to induce MSC differentiation and OPG expression in osteoblasts, while Dkk1 significantly inhibited these processes. To confirm the synergistic effects of sFRPs with canonical Wnt signaling on MSC differentiation, we employed R-podin1, a well-known agonist of canonical Wnt signaling. Treatments of MSC cells with R-podin1 led to increase in beta-catenin protein and TCF transcriptional activity and in ALP activity, and increase in OPG mRNA and protein. Pretreatment of the cells with sFRP2 and sFPP3 proteins further enhanced the function of R-podin1. In contrast, Dkk1 protein showed negative effect on R-Spodin1 functions, indicating that sFRP2 and sFRP3 synergized with R-Spodin1 to induce activation of canonical Wnt signaling and subsequent MSC differentiation and OPG production. Conclusion: Taken together, these data suggest that sFRP2 and sFRP3 augment canonical Wnt signaling to induce MSC differentiation and indirectly inhibit osteoclastogenesis by regulating OPG in MSC cells. These results also indicate that Dkk1 may be most important in MM-induced bone disease. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Description: Syndecan-1 (CD138) is a transmembrane heparan sulfate–bearing proteoglycan expressed by most myeloma plasma cells that regulates adhesion, migration, and growth factor activity. In patients with myeloma, shed syndecan-1 accumulates in the bone marrow, and high levels of syndecan-1 in the serum are an indicator of poor prognosis. To test the effect of soluble syndecan-1 on tumor cell growth and dissemination, ARH-77 B-lymphoid cells were engineered to produce a soluble form of syndecan-1. Controls included vector only (neo)–transfected cells and cells transfected with full-length syndecan-1 complementary DNA that codes for the cell surface form of syndecan-1. Assays reveal that all 3 transfectants have similar growth rates in vitro, but cells expressing soluble syndecan-1 are hyperinvasive in collagen gels relative to controls. When injected into the marrow of human bones that were implanted in severe combined immunodeficient mice, tumors formed by cells expressing soluble syndecan-1 grow faster than tumors formed by neo-transfected cells or by cells expressing cell surface syndecan-1. In addition, cells bearing cell surface syndecan-1 exhibit a diminished capacity to establish tumors within the mice as compared with both neo- and soluble syndecan-1–transfected cells. Tumor cell dissemination to a contralateral human bone is detected significantly more often in the tumors producing soluble syndecan-1 than in controls. Thus, high levels of soluble syndecan-1 present in patients with myeloma may contribute directly to the growth and dissemination of the malignant cells and thus to poor prognosis.