Donata Verdelli

Molecular Classification of Multiple Myeloma: A Distinct Transcriptional Profile Characterizes Patients Expressing CCND1 and Negative for 14q32 Translocations

PURPOSE The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups. MATERIALS AND METHODS The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. RESULTS Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures. CONCLUSION Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.

Gene expression profiling of plasma cell dyscrasias reveals molecular patterns associated with distinct IGH translocations in multiple myeloma.

Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the α-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

A SNP microarray and FISH‐based procedure to detect allelic imbalances in multiple myeloma: An integrated genomics approach reveals a wide gene dosage effect

Multiple myeloma (MM) is characterized by marked genomic heterogeneity. Beyond structural rearrangements, a relevant role in its biology is represented by allelic imbalances leading to significant variations in ploidy status. To elucidate better the genomic complexity of MM, we analyzed a panel of 45 patients using combined FISH and microarray approaches. We firstly generated genome‐wide profiles of 41 MMs and four plasma cell leukemias, using a self‐developed procedure to infer exact local copy numbers (CNs) for each sample. Our analysis allowed the identification of a significant fraction of patients showing near‐tetraploidy. Furthermore, a conventional hierarchical clustering analysis showed that near‐tetraploidy, 1q gain, hyperdiploidy, and recursive deletions at 1p and chromosomes 13, 14, and 22 were the main aberrations driving samples grouping. Moreover, mapping information was integrated with gene expression profiles of the tumor samples. A multiclass analysis of transcriptional profiles characterizing the different clusters showed marked gene‐dosage effects, particularly concerning 1q transcripts; this finding was also confirmed by a nonparametric analysis between normalized gene expression levels and local CN variations (1027 highly‐significant correlated genes). Finally, we identified several loci in which gene expression correlated with the occurrence of loss of heterozygosity. Our results provide insights into the composite network linking genome structure and transcriptional features in MM.

Molecular characterization of human multiple myeloma cell lines by integrative genomics: insights into the biology of the disease.

To investigate the patterns of genetic lesions in a panel of 23 human multiple myeloma cell lines (HMCLs), we made a genomic integrative analysis involving FISH, and both gene expression and genome‐wide profiling approaches. The expression profiles of the genes targeted by the main IGH translocations showed that the WHSC1/MMSET gene involved in t(4;14)(p16;q32) was expressed at different levels in all of the HMCLs, and that the expression of the MAF gene was not restricted to the HMCLs carrying t(14;16)(q32;q23). Supervised analyses identified a limited number of genes specifically associated with t(4;14) and involved in different biological processes. The signature related to MAF/MAFB expression included the known MAF target genes CCND2 and ITGB7, as well as genes controlling cell shape and cell adhesion. Genome‐wide DNA profiling allowed the identification of a gain on chromosome arm 1q in 88% of the analyzed cell lines, together with recurrent gains on 8q, 18q, 7q, and 20q; the most frequent deletions affected 1p, 13q, 17p, and 14q; and almost all of the cell lines presented LOH on chromosome 13. Two hundred and twenty‐two genes were found to be simultaneously overexpressed and amplified in our panel, including the BCL2 locus at 18q21.33. Our data further support the evidence of the genomic complexity of multiple myeloma and reinforce the role of an integrated genomic approach in improving our understanding of the molecular pathogenesis of the disease. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Upregulation of translational machinery and distinct genetic subgroups characterise hyperdiploidy in multiple myeloma

Karyotypic instability, including numerical and structural chromosomal aberrations, represents a distinct feature of multiple myeloma (MM). About 40–50% of patients display hyperdiploidy, defined by recurrent trisomies of non‐random chromosomes. To molecularly characterise hyperdiploid (H) and nonhyperdiploid (NH) MM, we analysed the gene expression profiles of 66 primary tumours, and used fluorescence in situ hybridisation to investigate the major chromosomal alterations. The differential expression of 225 genes mainly involved in protein biosynthesis, transcriptional machinery and oxidative phosphorylation distinguished the 28 H‐MM from the 38 NH‐MM cases. The 204 upregulated genes in H‐MM mapped mainly to the chromosomes involved in hyperdiploidy, and the 29% upregulated genes in NH‐MM mapped to 16q. The identified transcriptional fingerprint was robustly validated on a publicly available gene expression dataset of 64 MM cases; and the global expression modulation of regions on the chromosomes involved in hyperdiploidy was verified using a self‐developed non‐parametric statistical method. H‐MM could be further divided into two distinct molecular and transcriptional entities, characterised by the presence of trisomy 11 and 1q‐extracopies/chromosome 13 deletion respectively. These data reinforce the importance of combining molecular cytogenetics and gene expression profiling to define a genomic framework for the study of MM pathogenesis and clinical management.

Characterization of oncogene dysregulation in multiple myeloma by combined FISH and DNA microarray analyses

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF, and MAFB genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23), and t(14;20)(q32;q12), respectively, in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) by DNA microarray analysis and compared the results with the presence of translocations as assessed by dual‐color FISH or RT‐PCR. A t(4;14) was found in 6 MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in 1 MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in 1 PCL. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased MMSET expression was found in 1 MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.

The oral protein-kinase C β inhibitor enzastaurin (LY317615) suppresses signalling through the AKT pathway, inhibits proliferation and induces apoptosis in multiple myeloma cell lines

Deregulation of the protein kinase C (PKC) signalling pathway has been implicated in tumor progression. Here we investigated the PKC inhibitor enzastaurin for its activity against multiple myeloma (MM) cells. Enzastaurin suppresses cell proliferation in a large panel of human myeloma cell lines (HMCLs), with IC50 values ranging from 1.3 to 12.5 µM and induces apoptosis, which is prevented by the ZVAD-fmk broad caspase inhibitor. These results are consistent with decreased phosphorylation of AKT and GSK3-β, a downstream target of the AKT pathway and a pharmacodynamic marker for enzastaurin. Furthermore, enzastaurin cytotoxicity is retained when HMCLs were cocultured with multipotent mesenchymal stromal cells. Enzastaurin has additive or synergistic cytotoxic effects with bortezomib or thalidomide. Considering the strong anti-myeloma activity of enzastaurin in vitro and in animal models and its safe toxicity profile, phase II studies in MM patients of enzastaurin alone or in combination with other drugs are warranted.

Molecular targeting of the PKC‐β inhibitor enzastaurin (LY317615) in multiple myeloma involves a coordinated downregulation of MYC and IRF4 expression

The protein kinase C (PKC) pathway has been shown to play a role in the regulation of cell proliferation in several haematological malignancies, including multiple myeloma (MM). Recent data have shown that a PKC inhibitor, enzastaurin, has antiproliferative and proapoptotic activity in a large panel of human myeloma cell lines (HMCLs). In order to further characterise the effect of enzastaurin in MM, we performed gene expression profiling of enzastaurin‐treated KMS‐26 cell line. We identified 62 upregulated and 32 downregulated genes that are mainly involved in cellular adhesion (CXCL12, CXCR4), apoptosis (CTSB, TRAF5, BCL2L1), cell proliferation (IGF1, GADD45A, BCMA (B‐cell maturation antigen), CDC20), transcription regulation (MYC, MX11, IRF4), immune and defence responses. Subsequent validation by Western blotting of selected genes in four enzastaurin‐treated HMCLs was consistent with our microarray analysis. Our data indicate that enzastaurin may affect important processes involved in the proliferation and survival of malignant plasma cells as well as in their interactions with the bone marrow microenvironment and provide a preclinical rationale for the potential role of this drug in the treatment of MM. Copyright

Identification of a novel IGH-MMSET fusion transcript in a human myeloma cell line with the t(4;14)(p16.3;q32) chromosomal translocation.

We described a novel IGH-MMSET fusion transcript in the context of the molecular characterization of the recently established human myeloma cell lines (HMCLs) KMS26, KMS28BM and KMS34, found to express high levels of FGFR3 transcripts (Otsuki et al, 2002). We further confirmed this evidence by Western blot analysis showing expression of fgfr3 in all three cell lines, albeit at different levels. Activating mutations within specific domains of the FGFR3 gene were not found in all three cell lines by using the protocols previously described (Intini et al, 2001). Finally, KMS28BM expressed the fgfr3-IIIb isoform, similar to that reported in KMS18 (Ronchetti et al, 2001) (data not shown). The presence of t(4;14) was investigated by dual-colour fluorescence in situ hybridization using a combination of BAC clones specific for FGRF3, MMSET and IGH loci. Dissociation of FGFR3 and MMSET signals and association of FGFR3 and IGH loci were detected in all cell lines (data not shown). Analysis of IGH-MMSET fusion transcripts was performed by reverse transcription polymerase chain reaction (RT-PCR) as previously described (Malgeri et al, 2000). Fragments specific for the MB4-1 and MB4-3 type breakpoints were detected in KMS34 and KMS26, respectively, whereas a fragment smaller than the MB4-3 type breakpoint was found in KMS28BM. Its direct sequencing using the IGH and ms6r primers (respectively 54 and 108 bp in length) revealed the presence of MMSET exon 6 but the absence of the entire MMSET exon 5 (data not shown), thus strongly suggesting the occurrence of a novel breakpoint within intron 5 of the gene (Fig 1A). To characterize this novel transcript, we attempted the molecular cloning of the 4p16Æ3 breakpoint in KMS28BM as previously described (Richelda et al, 1997). A 10 kb BamHIrearranged JH fragment containing 4p16Æ3 sequences was isolated (Fig 1B) and the sequencing analysis of a HindIII fragment positive for an Sl probe revealed two particular findings. First, the Sl sequence was joined to an Sc1 sequence, thus suggesting that the recombination event occurred after a legitimate class switch recombination, a finding previously observed in an HMCL and two primary cases harbouring t(4;14) (Fenton et al, 2003). Secondly, the 4p16Æ3 sequence juxtaposed to Sc1 was derived from the 4p16Æ3 cosmid clone L75b9c (starting at nucleotide 554) (see scheme in Fig 1B). This breakpoint was therefore telomeric to the untranslated MMSET exon 1a, occurred 295 bp upstream of the KMS11 breakpoint (Richelda et al, 1997) (see schema in Fig 1C), and should have accounted for an MB4-1 type transcript. To clarify this finding, we investigated the arrangement of the genomic region containing MMSET exon 6 by Southern blotting. Rearranged fragments were detected with both EcoRI and BamHI restriction enzymes (Fig 1C). The cloning of the 12 kb EcoRI fragment demonstrated that MMSET intron 5 sequence ended 112 bp upstream of exon 6 (nucleotide 18729 of cosmid L190b4) (Fig 1C) and was juxtaposed to a sequence derived from the more telomeric cosmid L184d6 (starting at nucleotide 16069), thus indicating the occurrence of an internal deletion of 13141 nucleotides. Both breakpoint sites occurred within Alu repeat regions. We suggest that the 4p16Æ3 breakpoint in KMS28BM occurred within the 5¢ regulatory region of the MMSET gene, and was followed by an internal deletion involving translated exons 3–5, leading to the generation of a novel IGH-MMSET fusion transcript. However, putative truncated MMSET proteins generated by this transcript are similar to those predicted in t(4;14) cases harbouring the MB4-3 (Malgeri et al, 2000). Furthermore, this novel fusion transcript may represent a rare event as it was not detected in eight HMCLs and 26 primary MMs with t(4,14) that were investigated in our laboratory (Malgeri et al, 2000 and data not shown).

Molecular events underlying interleukin-6 independence in a subclone of the CMA-03 multiple myeloma cell line.

We explored the molecular mechanisms involved in the establishement of CMA‐03/06, an IL‐6‐independent variant of the multiple myeloma cell line CMA‐03 previously generated in our Institution. CMA‐03/06 cells grow in the absence of IL‐6 with a doubling time comparable with that of CMA‐03 cells; neither the addition of IL6 (IL‐6) to the culture medium nor co‐culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL‐6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL‐6 independence of CMA‐03/06 cells is not apparently due to the development of an autocrine IL‐6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA‐03/06 cells showed an activated pattern of the NF‐κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA‐03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA‐03/06 cells display a higher sensibility to NF‐κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL‐6 independence of CMA‐03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell‐line CMA‐03 and its variant CMA‐03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL‐6‐independent growth of myeloma cells.