Project description:The pathogenesis of multiple myeloma (MM) is thought to involve at least two pathways, which generate hyperdiploid (HRD) or nonhyperdiploid (NHRD) tumors, respectively. Apart from chromosome content, the two pathways are distinguished by five primary immunoglobulin heavy chain (IGH) rearrangements (4p16, FGFR3, and MMSET; 6p21, CCND3; 11q13, CCND1; 16q23, MAF; 20q12, MAFB) that are present mainly in NHRD tumors. To determine the prevalence and structures of IGH, immunoglobulin (IG) light chain, and MYC genomic rearrangements in MM, we have done comprehensive metaphase fluorescent in situ hybridization analyses on 48 advanced MM tumors and 47 MM cell lines. As expected, the prevalence of the five primary IGH rearrangements was nearly 70% in NHRD tumors, but only 12% in HRD tumors. However, IGH rearrangements not involving one of the five primary partners, and IG light chain rearrangements, have a similar prevalence in HRD and NHRD tumors. In addition, MYC rearrangements, which are thought to be late progression events that sometimes do not involve an IG heavy or light chain locus, also have a similar prevalence in HRD and NHRD tumors. In contrast to the primary IGH rearrangements, which usually are simple balanced translocations, these other IG rearrangements usually have complex structures, as previously described for MYC rearrangements in MM. We conclude that IG light chain and MYC rearrangements, as well as secondary IGH rearrangements, make similar contributions to the progression of both HRD and NHRD MM tumors.
Project description:To better define the molecular basis of multiple myeloma (MM), we performed unsupervised hierarchic clustering of mRNA expression profiles in CD138-enriched plasma cells from 414 newly diagnosed patients who went on to receive high-dose therapy and tandem stem cell transplants. Seven disease subtypes were validated that were strongly influenced by known genetic lesions, such as c-MAF- and MAFB-, CCND1- and CCND3-, and MMSET-activating translocations and hyperdiploidy. Indicative of the deregulation of common pathways by gene orthologs, common gene signatures were observed in cases with c-MAF and MAFB activation and CCND1 and CCND3 activation, the latter consisting of 2 subgroups, one characterized by expression of the early B-cell markers CD20 and PAX5. A low incidence of focal bone disease distinguished one and increased expression of proliferation-associated genes of another novel subgroup. Comprising varying fractions of each of the other 6 subgroups, the proliferation subgroup dominated at relapse, suggesting that this signature is linked to disease progression. Proliferation and MMSET-spike groups were characterized by significant overexpression of genes mapping to chromosome 1q, and both exhibited a poor prognosis relative to the other groups. A subset of cases with a predominating myeloid gene expression signature, excluded from the profiling analyses, had more favorable baseline characteristics and superior prognosis to those lacking this signature.
Project description:Plasma cell leukemia (PCL) is an aggressive and rare hematological malignancy that originates either as primary disease (pPCL) or as a secondary leukemic transformation (sPCL) of multiple myeloma (MM). We report here the genetic aberrations and survival of 80 patients with pPCL or sPCL and make comparisons with 439 cases of MM. pPCL presents a decade earlier than sPCL (54.7 vs 65.3 years) and is associated with longer median overall survival (11.1 vs 1.3 months; P<0.001). 14q32 (IgH) translocations are highly prevalent in both sPCL and pPCL (82-87%); in pPCL IgH translocations almost exclusively involve 11q13 (CCND1), supporting a central etiological role, while in sPCL multiple partner oncogenes are involved, including 11q13, 4p16 (FGFR3/MMSET) and 16q23 (MAF), recapitulating MM. Both show ubiquitous inactivation of TP53 (pPCL 56%; sPCL 83%) by coding mutation or 17p13 deletion; complemented by p14ARF epigenetic silencing in sPCL (29%). Both show frequent N-RAS or K-RAS mutation. Poor survival in pPCL was predicted by MYC translocation (P=0.006). Survival in sPCL was consistently short. Overall pPCL and sPCL are different disorders with distinct natural histories, genetics and survival.
Project description:In multiple myeloma, karyotopic 14q32 translocations have been identified at a variable frequency (10-60% in different studies). In the majority of cases, the partner chromosome has not been identified (14q+), and in the remaining cases, a diverse array of chromosomal partners has been implicated, with 11q13 being the most common. We developed a comprehensive Southern blot assay to identify and distinguish different kinds of immunoglobulin heavy chain (IgH) switch recombination events. Illegitimate switch recombination fragments (defined as containing sequences from only one switch region) are potential markers of translocation events into IgH switch regions and were identified in 15 of 21 myeloma cell lines, including seven of eight karyotyped lines that have no detectable 14q32 translocation. From all nine lines or tumor samples analyzed further, cloned illegitimate switch recombination fragments were confirmed to be IgH switch translocation breakpoints. In three of these cases, the translocation breakpoint was shown to be present in the primary tumor. These translocation breakpoints involve six chromosomal loci: 4p16.3 (two lines and the one tumor); 6; 8q24.13; 11q13.3 (in three lines); 16q23.1; and 21q22.1. We suggest that translocations into the IgH locus (i) are frequent (karyotypic 14q32 translocations and/or illegitimate switch recombination fragments are present in primary tumor samples and in 19 of 21 lines that we have analyzed); (ii) occur mainly in switch regions; and (iii) involve a diverse but nonrandom array (i.e., frequently 11q13 or 4p16) of chromosomal partners. This appears to be the most frequent genetic abnormality in multiple myeloma.
Project description:Two oncogenic pathways have been hypothesized for multiple myeloma (MM) and premalignant monoclonal gammopathy of undetermined significance (MGUS) tumors: a nonhyperdiploid pathway associated with a high prevalence of IgH translocations and a hyperdiploid pathway associated with multiple trisomies of 8 chromosomes. Cyclin D1, D2, or D3 expression appears to be increased and/or dysregulated in virtually all MM tumors despite their low proliferative capacity. Translocations can directly dysregulate CCND1 (11q13) or CCND3 (6p21), or MAF (16q23) or MAFB (20q11) transcription factors that target CCND2. Biallelic dysregulation of CCND1 occurs in nearly 40% of tumors, most of which are hyperdiploid. Other tumors express increased CCND2, either with or without a t(4;14) translocation. Using gene expression profiling to identify 5 recurrent translocations, specific trisomies, and expression of cyclin D genes, MM tumors can be divided into 8 TC (translocation/cyclin D) groups (11q13, 6p21, 4p16, maf, D1, D1+D2, D2, and none) that appear to be defined by early, and perhaps initiating, oncogenic events. However, despite subsequent progression events, these groups have differing gene expression profiles and also significant differences in the prevalence of bone disease, frequency at relapse, and progression to extramedullary tumor.
Project description:We have sequenced 463 presenting cases of myeloma entered into the UK Myeloma XI study using whole exome sequencing. Here we identify mutations induced as a consequence of misdirected AID in the partner oncogenes of IGH translocations, which are activating and associated with impaired clinical outcome. An APOBEC mutational signature is seen in 3.8% of cases and is linked to the translocation-mediated deregulation of MAF and MAFB, a known poor prognostic factor. Patients with this signature have an increased mutational load and a poor prognosis. Loss of MAF or MAFB expression results in decreased APOBEC3B and APOBEC4 expression, indicating a transcriptional control mechanism. Kataegis, a further mutational pattern associated with APOBEC deregulation, is seen at the sites of the MYC translocation. The APOBEC mutational signature seen in myeloma is, therefore, associated with poor prognosis primary and secondary translocations and the molecular mechanisms involved in generating them.
Project description:BACKGROUND: Multiple myeloma is a plasma-cell tumor with heterogeneity in molecular abnormalities and treatment response. DESIGN AND METHODS: We have assessed whether human myeloma cell lines have kept patients' heterogeneity using Affymetrix gene expression profiling of 40 human myeloma cell lines obtained with or without IL6 addition and could provide a signature for stratification of patient risk. RESULTS: Human myeloma cell lines, especially those derived in the presence of IL6, displayed a heterogeneity that overlaps that of the patients with multiple myeloma. Human myeloma cell lines segregated into 6 groups marked by overexpression of MAF, MMSET, CCND1, FRZB with or without overexpression of cancer testis antigens (CTA). Cell lines of CTA/MAF and MAF groups have a translocation involving C-MAF or MAFB, cell lines of groups CCND1-1 and CCND1-2like have a t(11;14) and cell lines of group MMSET have a t(4;14). The CTA/FRZB group comprises cell lines that had no or no recurrent 14q32 translocation. Expression of 248 genes accounted for human myeloma cell line molecular heterogeneity. Human myeloma cell line heterogeneity genes comprise genes with prognostic value for survival of patients making it possible to build a powerful prognostic score involving a total of 13 genes. CONCLUSIONS: Human myeloma cell lines derived in the presence of IL6 recapitulate the molecular diversity of multiple myeloma that made it possible to design, using human myeloma cell line heterogeneity genes, a high-risk signature for patients at diagnosis. We propose this classification to be used when addressing the physiopathology of multiple myeloma with human myeloma cell lines.
Project description:Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. MMSET, identified by its fusion to the IgH locus in t(4;14) MM, is universally overexpressed in t(4;14) MM. In order to identify cell surface biomarkers associated with t(4;14) MM for small molecule or antibody based therapies, we knocked down MMSET expression with shRNA and generated a cell line pair from KMS11, a t(4;14) MM cell line. We used quantitative mass spectrometry to identify plasma membrane proteins associated with MMSET overexpression. Using this approach, 50 cell surface proteins were identified as differentially expressed between KMS11 and KMS11/shMMSET. Western blot and flow cytometry analysis indicated SLAMF7 was over-expressed in t(4;14) MM cell lines and down-regulated by MMSET shRNAs. SLAMF7 expression was also confirmed in primary t(4;14) MM samples by flow cytometry analysis. Quantitative RT-PCR and ChIP analysis indicated MMSET might regulate the transcription level of SLAMF7 and be an important functional element for SLAMF7 promoter activity. Furthermore, SLAMF7 shRNA could induce G1 arrest or apoptosis and reduce clonogenetic capacity in t(4;14) MM cells. Overall, these results illustrated SLAMF7 might be a novel cell surface protein associated with t(4;14) MM. It is potential to develop t(4;14) MM targeted therapy by SLAMF7 antibody mediated drug delivery.
Project description:Dysregulation of cyclin D2 contributes to the pathogenesis of multiple myeloma, and can occur through translocations that activate MAF/MAFB or MMSET/FGFR3. However, cyclin D2 induction can also be seen in the absence of such translocations, such as in patients with hyperdiploid disease, through unknown mechanisms. In UniGene cluster data-mining and ECgene analysis, we found that zinc-finger with KRAB and SCAN domains 3 (ZKSCAN3), a novel transcription factor, is overrepresented in this malignancy, and three consensus ZKSCAN3 binding sites were found in the cyclin D2 promoter. Analysis of a panel of myeloma cell lines, primary patient samples and datasets from Oncomine and the Multiple Myeloma Genomics Portal (MMGP) revealed expression of ZKSCAN3 messenger RNA (mRNA) in a majority of samples. Studies of cell lines by western blotting, and of primary tissue microarrays by immunohistochemistry, showed ZKSCAN3 protein expression in a majority, and in a manner that paralleled messenger levels in cell lines. ZKSCAN3 overexpression was associated with increased gene copy number or genomic DNA gain/amplification in a subset based on analysis of data from the MMGP, and from fluorescence in situ hybridization studies of cell lines and primary samples. Overexpression of ZKSCAN3 induced cyclin D2 promoter activity in a MAF/MAFB-independent manner, and to an extent that was influenced by the number of consensus ZKSCAN3 binding sites. Moreover, ZKSCAN3 protein expression correlated with cyclin D2 levels in cell lines and primary samples, and its overexpression induced cyclin D2. Conversely, ZKSCAN3 suppression using small hairpin RNAs (shRNAs) reduced cyclin D2 levels, and, importantly, inhibited myeloma cell line proliferation. Finally, ZKSCAN3 was noted to specifically bind to oligonucleotides representing sequences from the cyclin D2 promoter, and to the endogenous promoter itself in myeloma cells. Taken together, the data support the conclusion that ZKSCAN3 induction represents a mechanism by which myeloma cells can induce cyclin D2 dysregulation, and contribute to disease pathogenesis.
Project description:The loci encoding B and T cell Ag receptors are generally distinct in commonly studied mammals, with each receptor's gene segments limited to intralocus, cis chromosomal rearrangements. The nurse shark (Ginglymostoma cirratum) represents the oldest vertebrate class, the cartilaginous fish, with adaptive immunity provided via Ig and TCR lineages, and is one species among a growing number of taxa employing Ig-TCR? rearrangements that blend these distinct lineages. Analysis of the nurse shark Ig-TCR? repertoire found that these rearrangements possess CDR3 characteristics highly similar to canonical TCR? rearrangements. Furthermore, the Ig-TCR? rearrangements are expressed with TCR?, canonically found in the TCR? heterodimer. We also quantified BCR and TCR transcripts in the thymus for BCR (IgHV-IgHC), chimeric (IgHV-TCR?C), and canonical (TCR?V-TCR?C) transcripts, finding equivalent expression levels in both thymus and spleen. We also characterized the nurse shark TCR?? locus with a targeted bacterial artifical chromosome sequencing approach and found that the TCR? locus houses a complex of V segments from multiple lineages. An IgH-like V segment, nestled within the nurse shark TCR? translocus, grouped with IgHV-like rearrangements we found expressed with TCR? (but not IgH) rearrangements in our phylogenetic analysis. This distinct lineage of TCR?-associated IgH-like V segments was termed "TAILVs." Our data illustrate a dynamic TCR? repertoire employing TCR?Vs, NARTCRVs, bona fide trans-rearrangements from shark IgH clusters, and a novel lineage in the TCR?-associated Ig-like V segments.