ALBERT

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: The identification of novel tumor-associated antigens, especially those shared among patients, is urgently needed to improve the efficacy of immunotherapy for multiple myeloma (MM). In this study, we examined whether Dickkopf-1 (DKK1), a protein that is not expressed in most normal tissues but is expressed by tumor cells from almost all patients with myeloma, could be a good candidate. We identified and synthesized DKK1 peptides for human leukocyte antigen (HLA)–A*0201 and confirmed their immunogenicity by in vivo immunization in HLA-A*0201 transgenic mice. We detected, using peptidetetramers, low frequencies of DKK1 peptide-specific CD8-positive (CD8+) T cells in patients with myeloma and generated peptide-specific T-cell lines and clones from HLA-A*0201-positive (HLA-A*0201+) blood donors and patients with myeloma. These T cells efficiently lysed peptide-pulsed but not unpulsed T2 or autologous dendritic cells, DKK1-positive (DKK1+)/HLA-A*0201+ myeloma cell lines U266 and IM-9, and, more importantly, HLA-A*0201+ primary myeloma cells from patients. No killing was observed on DKK1+/HLA-A*0201-negative (HLA-A*0201−) myeloma cell lines and primary myeloma cells or HLA-A*0201+ normal lymphocytes, including B cells. These results indicate that these T cells were potent cytotoxic T cells and recognized DKK1 peptides naturally presented by myeloma cells in the context of HLA-A*0201 molecules. Hence, our study identifies DKK1 as a potentially important antigen for immunotherapy in MM.

Description: Introduction: Natural killer (NK) cell activity is regulated by a dynamic balance between inhibitory and activating receptors that recognize ligands on target cells. Human leukocyte antigen (HLA)-class I, particularly HLA-C and -Bw4 molecules, are key ligands transmitting inhibitory signals to NK cells. NK cells avidly lyse tumor cells that do not display such inhibitory KIR-ligands. The proteasome is responsible for the generation of peptides that bind to and stabilize class I molecules at the cell surface. We hypothesized that bortezomib, a partial proteasome inhibitor that is clinically approved for the treatment of refractory/relapsed myeloma (MM), could reduce HLA expression on MM cells and thus enhance NK cell-mediated cytotoxicity. Methods: HLA-class I or HLA-C expression was assessed using flow cytometry, after gating on AnnexinV/PI double negative cells, and/or confocal microscopy. Expression of other proteins was measured by flow cytometry using specific mAb. NK cell-mediated lysis of myeloma cells was measured by 51Cr-release. Results: Bortezomib at clinically attainable concentrations down-regulated HLA-class I expression on MM cells in a time- and dose-dependent fashion. Reduction of class I post-10 nM bortezomib treatment was observed in all myeloma cell lines tested (n=10), by a median of 49% (range: 19–66%). A similar decrease of HLA-class I was obtained in 10–50 nM bortezomib treated primary MM cells (n=6). Bortezomib significantly enhanced the sensitivity of MM cells to allogeneic and autologous NK cell-mediated lysis. Further, the level of reduction in HLA-class I expression correlated well with increased susceptibility to lysis by NK cells. The level of down-regulation of HLA-class I induced by bortezomib was reproduced by incubating MM cells with HLA-blocking antibody and resulted in equipotent enhancement of NK cell-mediated lysis (Figure 1). The extent of HLA-class I down-regulation by bortezomib was therefore biologically relevant. Down-regulation of HLA-class I was also observed in vivo on purified MM cells 48 hours after a single dose of bortezomib, by a median of 47% (range: 16–63%, n=6, P= .002). HLA-C expression (the principal NK cell inhibitory ligand) was rescued by exogenous provision of HLA-C binding peptides providing a mechanistic explanation for the effect of bortezomib on HLA-class I expression. Finally, we did not observe bortezomib-mediated enhancement of NK cell-mediated lysis of myeloma through receptors other than the KIR receptor family, including tumor necrosis factor related apoptosis-inducing ligand, NKG2D and natural cytotoxicity receptors. HLA-class I down-regulation was not observed in renal cell and breast carcinoma cell lines, which is in keeping with the remarkable activity of bortezomib in myeloma. Our findings have clear therapeutic implications for MM and other NK cell-sensitive malignancies in the context of both allogeneic and autologous adoptively transferred NK cells. Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis

Description: Gene expression profiles that reflect unique aspects of biologic phenotypes and characterize the heterogeneity of monoclonal gammopathies may facilitate the development of a ‘signature’ that predicts the evolution of MGUS to multiple myeloma (MM). Furthermore, while most patients with MM are initially sensitive to therapy, resistance invariably develops. Thus, a need exits for novel therapeutic strategies targeting resistance-associated deregulated molecular pathways. Using previously-described methodologies that employ DNA microarray data in a cohort of 877 annotated samples from patients with plasma cell dyscrasias (MGUS, untreated MM and refractory MM), a complex multi-gene expression profile (‘metagene’) of 120 genes was identified that predicts risk of progression, from MGUS to MM. This analysis used an initial ‘test’ cohort of patients with MGUS that represented patients followed for a median of 36.4 months. At follow up, 11% of these patients were identified as having disease progression. Leave-one-out cross validation analysis within the test cohort revealed 95% accuracy. The ‘metagene model’ was also evaluated in a large independent validation cohort. Overexpressed genes in the predictive model represented those involved in the proteasome, RAS, and MAP-kinase pathways. In addition, we used gene expression signatures that reflect the deregulation of major oncogenic signaling pathways (Ras, PI3kinase, Akt, Src, ß-catenin, E2F, and Myc) and pathways representative of the tumor microenvironment (Hypoxia, Angiogenesis, Chromosomal Instability, and TNF-alpha) to identify patterns of pathway activation unique to individual patients with plasma cell dyscrasias. This analysis revealed that patterns of pathway activation clearly defined the stages of myeloma progression. As an example, Myc deregulation was unique to refractory MM (p 〈 0.001) as compared to Ras and PI3K activation in MGUS patients (p 〈 0.01). Also, unique to refractory MM, a majority of the tumors (〉 96%) had deregulation of multiple signaling pathways, suggesting a complex oncogenic process. In particular, these samples demonstrated deregulation in Ras (78%), Myc (70%), Src (63%), E2F (52%) and ß-catenin (52%) pathways. In contrast, normal plasma cells showed none to minimal deregulation of the oncogenic pathways. As a proof of concept, we then used cell proliferation assays to show that the predicted deregulation of Ras, Src and PI3 kinase pathways was directly proportional (p 〈 0.01, log rank test) to the sensitivity of cell lines (n = 17) to agents (FTS, SU6656, and LY4002 respectively) that specifically target these pathways. This suggests that in addition to identifying a high-risk cohort of patients with MGUS, such a strategy provides a novel approach to targeted therapeutics in refractory MM - by guiding the appropriate use of pathway specific inhibitors. Finally, results of Kaplan Meier analyses using gene expression-based classifiers and risk stratification models as well as further in vitro data involving manipulation of other oncogenic pathways and signatures relevant to myeloma (e.g. hypoxia, chromosomal instability and angiogenesis) will be also be presented.

Description: The heparan sulfate proteoglycan syndecan-1 is expressed by myeloma cells and shed into the myeloma microenvironment. High levels of shed syndecan-1 in myeloma patient sera correlate with poor prognosis and studies in animal models indicate that shed syndecan-1 is a potent stimulator of myeloma tumor growth and metastasis. Overexpression of extracellular endosulfatases, enzymes which remove 6-O sulfate groups from heparan sulfate chains, diminishes myeloma tumor growth in vivo. Together, these findings identify syndecan-1 as a potential target for myeloma therapy. Here, 3 different strategies were tested in animal models of myeloma with the following results: (1) treatment with bacterial heparinase III, an enzyme that degrades heparan sulfate chains, dramatically inhibited the growth of primary tumors in the human severe combined immunodeficient (SCID-hu) model of myeloma; (2) treatment with an inhibitor of human heparanase, an enzyme that synergizes with syndecan-1 in promoting myeloma progression, blocked the growth of myeloma in vivo; and (3) knockdown of syndecan-1 expression by RNAi diminished and delayed myeloma tumor development in vivo. These results confirm the importance of syndecan-1 in myeloma pathobiology and provide strong evidence that disruption of the normal function or amount of syndecan-1 or its heparan sulfate chains is a valid therapeutic approach for this cancer.

Description: We have demonstrated that canonical and non-canonical Wnt signaling occurs in myeloma cells (Qiang et al., 2005) and overexpression of Wnt3a in myeloma cells inhibits the osteolytic phenotype and also tumor growth in vivo (Qiang et al Blood, Abstract #3420, 2006). To further investigate the mechanisms that contribute to this process we have expanded our in vivo data by showing that while H929 cells stably expressing Wnt3a (H929/W3a) leads to reduced tumor growth in the in-vivo SCID-hu bone graft model compared with H929 vector alone transfected control cells (H929/EV), there was no significant difference in the subcutaneous growth of the two cell lines in SCID mice. Taken together these data suggests that alteration of the human bone marrow microenvironment is central to Wnt-mediated reduction in tumor growth in bone. We next employed an in-vitro co-culture model in which the mouse osteoprogenitor cell line, C2C12, and human osteoblast cell line, Saos-2 were co-cultured with either H929/Wnt3 or H929/EV cells. QPCR analysis demonstrated that osteoprotegerin (OPG) mRNA expression (relative OPG mRNA to GAPDH) in C2C12 cells co-cultured with H929/W3a was significantly elevated compared with H929/EV (mean±SD: 14.34±0.97 vs 8.43±0.16; P

Description: The genetic lesions important in the pathogenesis and prognosis of multiple myeloma (MM) continue to be elucidated. TP53 is deleted in approximately 10% of cases and is associated with a poor prognosis in MM. As a transcription factor, TP53 regulates the expression of genes involved in a variety of cellular functions, including cell-cycle arrest, DNA repair, and apoptosis. In the current study, we identified novel TP53 target genes and demonstrated the clinical relevance of alterations in this signaling pathway in disease progression. First, we examined the expression of 122 genes that were identified as direct TP53 target genes by paired-end diTags (PET) analysis and whose p53-dependent expression had been validated. The target genes were profiled in a collection of 351 newly diagnosed MM samples using the Affymetrix U133Plus2.0 microarrays. In this set of tumors, TP53 expression in the lowest 10th percentile was associated with a significantly shorter event-free (P = 0.0004) and overall survival (P = 0.0001). However, expression of only a few of the previously identified TP53 target genes correlated with TP53 expression in MM cells. This suggested that TP53 may regulate a distinct set of genes in MM. To elucidate the TP53 regulatory networks in MM, we used lentiviral transduction to over express TP53 in four MM cell lines (OCI-MY5, JJN3, ARP-1 and Delta 47). An increase in steady-state TP53 level was confirmed by Western blot analysis 24 hours post lentiviral transduction and massive cell death occurred within 36 hours. Gene expression profiling revealed that a total of 85 genes were affected by TP53 expression: 50 were up-regulated and 35 were down-regulated 1.5-fold or greater in at least 3 of 4 MM cell lines. Expression analysis of primary MM revealed that the 85 genes also exhibited differential expression in comparison of those with the lowest relative to the highest TP53 expression, suggesting that TP53 may directly or indirectly regulate the expression of these genes. Of the 85 genes, 4 genes (ZMAT3, TNFRSF10B, TRIM22, and NOTCH1) were in the group of 122 P53 target genes identified by PET analysis. Consistent with known TP53 cellular functions, we found 69 of the 85 TP53 regulated genes in MM to be involved in apoptosis, cell cycle regulation, cell growth and differentiation, DNA repair and chromatin modification, and transcription regulation. Unsupervised hierarchical clustering of 351 newly diagnosed MM and 90 relapsed MM with the 85 TP53 associated genes identified 2 subgroups in each analysis. The subtype associated with lower TP53 expression had a significantly shorter EFS (P = 0.0006) and OS (P = 0.0010) in newly diagnosed MM and shorter post-relapse survival in relapsed disease (P 〈 0.0001). Thus we have identified MM-specific TP53 target genes by expression profiling in both cultured myeloma cells and primary tumors that correlate with clinical outcome. The identification and characterization of these pathways may lead to a better understanding of the critical role of TP53 loss in cancer.

Description: Serum-free light chain (SFLC) levels are useful for diagnosing nonsecretory myeloma and monitoring response in light-chain–only disease, especially in the presence of renal failure. As part of a tandem autotransplantation trial for newly diagnosed multiple myeloma, SFLC levels were measured at baseline, within 7 days of starting the first cycle, and before both the second induction cycle and the first transplantation. SFLC baseline levels higher than 75 mg/dL (top tertile) identified 33% of 301 patients with higher near-complete response rate (n-CR) to induction therapy (37% vs 20%, P = .002) yet inferior 24-month overall survival (OS: 76% vs 91%, P 〈 .001) and event-free survival (EFS: 73% vs 90%, P 〈 .001), retaining independent prognostic significance for both EFS (HR = 2.40, P = .008) and OS (HR = 2.43, P = .016). Baseline SFLC higher than 75 mg/dL was associated with light-chain–only secretion (P 〈 .001), creatinine level 176.8 μM (2 mg/dL) or higher (P 〈 .001), beta-2-microglobulin 297.5 nM/L (3.5 mg/L) or higher (P 〈 .001), lactate dehydrogenase 190 U/L or higher (P 〈 .001), and bone marrow plasmacytosis higher than 30% (P = .003). Additional independent adverse implications were conferred by top-tertile SFLC reductions before cycle 2 (OS: HR = 2.97, P = .003; EFS: HR = 2.56, P = .003) and before transplantation (OS: HR = 3.31, P = .001; EFS: HR = 2.65, P = .003). Unlike baseline and follow-up analyses of serum and urine M-proteins, high SFLC levels at baseline—reflecting more aggressive disease—and steeper reductions after therapy identified patients with inferior survival.

Description: Multiple myeloma (MM) plasma cells, but not those from healthy donors and patients with monoclonal gammopathy of undetermined significance or other plasma cell dyscrasias involving the bone marrow, express the Wnt-signaling antagonist DKK1. We previously reported that secretion of DKK1 by MM cells likely contributes to osteolytic lesions in this disease by inhibiting Wnt signaling, which is essential for osteoblast differentiation and survival. The mechanisms responsible for activation and regulation of DKK1 expression in MM are not known. Herein, we could trace DKK1 expression changes in MM cells to perturbations in the JNK signaling cascade, which is differentially modulated through oxidative stress and interactions between MM cells with osteoclasts in vitro. Despite its role as a tumor suppressor and mediator of apoptosis in other cell types including osteoblasts, our data suggest that DKK1, a stress-responsive gene in MM, does not mediate apoptotic signaling, is not activated by TP53, and its forced overexpression could not inhibit cell growth or sensitize MM cells to apoptosis following treatment with thalidomide or lenalidomide. We conclude that specific strategies to modulate persistent activation of the JNK pathway may be beneficial in preventing disease progression and treating myeloma-associated bone disease by inhibiting DKK1 expression.