Plasma cells expression from smouldering myeloma to myeloma reveals the importance of the PRC2 complex, cell cycle progression, and the divergent evolutionary pathways within the different molecular subgroups

Abstract Abstract 2811 Poster Board II-787 Introduction The recurrent translocation t(4;14)(p16;q32) occurs in less than 20% of patients with newly diagnosed Multiple Myeloma (MM) and is associated with a poor clinical outcome following either conventional or high-dose chemotherapy. Recently, it has been reported that patients carrying t(4;14) are prognostically heterogeneous and that the novel agents bortezomib and lenalidomide may overcome the poor prognosis related to this cytogenetic abnormality. In the present study, we analyzed the gene expression profile of patients who carried or not t(4;14) and were primarily treated with a bortezomib-based regimen. Patients and methods Two hundred thirty six patients with MM who received a combination of bortezomib-thalidomide-dexamethasone (VTD) as first-line therapy were evaluated for the presence at diagnosis of t(4;14). Of these, 41 patients (17.3%) were t(4;14) positive. On an intention-to-treat basis, the rate of CR and near CR (nCR) to VTD induction therapy among patients carrying t(4;14) was 41%, a value higher than the 29% observed among t(4;14) negative patients. In 218 patients for whom data on t(4;14), del(13q) and del(17p) were available, the differential gene expression of CD138+ enriched plasma cells was evaluated by means of expression microarray using the Affymetrix platform. The analysis was performed in t(4;14) negative patients and patients carrying t(4;14), either alone or combined with other abnormalities; t(4;14) negative patients included those with del(13q) alone and with any of these abnormalities. Results In 27 patients, t(4;14) was associated with either del(13q) (24 patients) or del(17p) (3 patients); the remaining 14 patients carried t(4;14) alone. The expression profiles of patients carrying either t(4;14) alone or t(4;14) combined with del(13q) significantly clustered apart when compared with those of cytogenetic negative patients. Similarly, the expression profiles of patients with del(13) alone clustered with those of cytogenetic negative patients. De-regulated expression of similar molecular pathways was demonstrated in patients carrying t(4;14) alone or combined with del(13q). Thus, the analysis of gene expression profiles according to response or no response to VTD was performed in two subgroups of patients, including those carrying t(4;14) alone or combined with del(13q) and those carrying either del(13q) alone or without cytogenetic abnormalities. By comparing the lists of genes differentially expressed (P '0.05) in patients who responded (e.g. those who achieved CR+nCR) and failed to respond (NR) to VTD according to the presence or absence of t(4;14), we found that the differential expression of 3719 genes characterized CR+nCR vs NR patients in the t(4;14) positive subgroup. At the opposite, the differential expression of 3182 genes characterized CR+nCR vs NR patients in the t(4;14) negative subgroup. 271 genes which were common to the two groups of genes were excluded from the list of genes found to be differentially expressed in t(4;14) positive patients who responded to VTD. Among these patients, we observed the de-regulated expression of genes involved in cell cycle progression (e.g. MDM2, CDK6 and SMAD2), Wnt signalling pathway (e.g. FZD7, WNT10A, MMP7,WNT2B, WNT6, WNT9A and DAAM2), and Hedgehog signalling pathway (GAS1, STK36 and GLI1). Overall, genes involved in cell cycle progression resulted over-expressed, thus suggesting a more aggressive phenotype of t(4;14) positive plasma cells of responder patients; nevertheless, the overall down-regulation of genes involved in Wnt and Hedgehog signalling pathways (known to be involved in the maintenance of a putative tumoral stem cell compartment) might mitigate this phenotype and predispose t(4;14) positive plasma cells to more favourably respond to VTD induction therapy. Supported by: BolognAIL, Fondazione Carisbo, Progetto di Ricerca Finalizzata (M.C). Disclosures: No relevant conflicts of interest to declare.

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Differential Regulation of D-Type Cyclins by Insulin-Like Growth Factor-I and Serum in Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v108.11.3435.3435 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3435-3435

Author(s):

Janet Glassford ◽

Eric W.-F. Lam ◽

Kwee L. Yong

Keyword(s):

Cell Cycle ◽

Cyclin D1 ◽

Cell Cycle Progression ◽

Plasma Cells ◽

Cyclin Dependent Kinase ◽

Cycle Progression ◽

Myeloma Cells ◽

Exogenous Growth ◽

Igf I ◽

Exogenous Growth Factors

Abstract Dysregulation of a D-type cyclin gene is an early and universal event in multiple myeloma (MM), but given the low proliferative activity in this disease, the functional significance of these genetic lesions is unclear. In this study we first examined the expression and regulation of D-type cyclins and other cell-cycle regulatory proteins in a panel of human myeloma cell lines (HMCLs) and in primary normal and malignant CD138+ plasma cells by Western blotting. D-type cyclins, cyclin dependent kinase-4 (CDK4), CDK6, p27, the retinoblastoma protein (pRb) and proliferating cell nuclear antigen (PCNA) were absent in normal bone marow (BM) CD138+ cells (n=5) and heterogeneously expressed in both HMCLs (n=11) and in primary CD138+ MM cells (n=20; 16 from BM aspirates and 4 extra-medullary). Furthermore, expression of these proteins was positively associated with disease progression. Nine of the primary malignant samples were, like normal CD138+ cells, negative for D-type cyclins, their associated kinases or pRB. Of these, one was from a newly diagnosed patient, six were from patients with stable disease and two from patients on treatment. The remaining eleven MM patient samples had heterogeneous expression of D-type cyclins and other cell cycle regulators, four of which had detectable CDK 4/6-phosphorylated pRb. All four of these were from patients with progressive disease, and three were extra-medullary. Furthermore, we demonstrate for the first time in both HMCLs and primary CD138+ MM cells, that cyclins D1 and -D2 are positively regulated by IGF-I and foetal calf serum leading to increased phosphorylation of pRb on CDK4/6 specific sites and an increase in cells in S (DNA synthesis) phase of the cell cycle. In addition, p27 was down-regulated by IGF-I and FCS in some HMCLs suggesting that this cyclin dependent kinase inhibitor also contributes to cell cycle regulation in myeloma cells. However, at low stoichiometric concentrations observed in primary malignant plasma cells, p27 was up-regulated by mitogenic stimuli, consistent with its role in stabilising cyclin D/CDK complexes. Immunoprecipitation analysis revealed that cyclins D1 and -D2 were present in complexes with both CDKs −4 and −6, suggesting that both of these kinases mediate the effect of cyclin D1 or cyclin D2 on pRb phosphorylation and cell cycle entry in these cells. Unlike HMCLs in which cyclin D2 was the primary controller of cell cycle exit/entry (MM1-S and NCI-H929), HMCLs harbouring an 11q13 IgH translocation (KMS12-BM and U266) were refractory to serum withdrawal, suggesting that 11q13 may confer growth factor independent expression of cyclin D1. To determine if cyclin D1 expression via 11q13 was sufficient to promote cell cycle progression, we functionally inactivated it using siRNA in KMS12-BM. Knock-down of cyclin D1 coincided with decreased CDK4/6-specific pRb phosphorylation and an increase in cells arrested in G1 phase of the cell cycle, confirming that cyclin D1 expression via 11q13 leads to cell cycle progression in MM cells, independent of exogenous growth factors. In summary, we show that D-type cyclins are functional in MM, differentially responsive to exogenous growth factors and that their expression is positively associated with aggressive disease.

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