A gene expression signature distinguishes innate response and resistance to proteasome inhibitors in multiple myeloma.

Extensive interindividual variation in response to chemotherapy is a major stumbling block in achieving desirable efficacy in the treatment of cancers, including multiple myeloma (MM). In this study, our goal was to develop a gene expression signature that predicts response specific to proteasome inhibitor (PI) treatment in MM. Using a well-characterized panel of human myeloma cell lines (HMCLs) representing the biological and genetic heterogeneity of MM, we created an in vitro chemosensitivity profile in response to treatment with the four PIs bortezomib, carfilzomib, ixazomib and oprozomib as single agents. Gene expression profiling was performed using next-generation high-throughput RNA-sequencing. Applying machine learning-based computational approaches including the supervised ensemble learning methods Random forest and Random survival forest, we identified a 42-gene expression signature that could not only distinguish good and poor PI response in the HMCL panel, but could also be successfully applied to four different clinical data sets on MM patients undergoing PI-based chemotherapy to distinguish between extraordinary (good and poor) outcomes. Our results demonstrate the use of in vitro modeling and machine learning-based approaches to establish predictive biomarkers of response and resistance to drugs that may serve to better direct myeloma patient treatment options.

Ectopic expression of wild-type FGFR3 cooperates with MYC to accelerate development of B-cell lineage neoplasms.

The t(4;14) translocation in multiple myeloma (MM) simultaneously dysregulates two apparent oncogenes: fibroblast growth factor receptor 3 (FGFR3) controlled by the 3' immunoglobulin heavy chain enhancer on der(14) and MMSET controlled by the intronic Emu enhancer on der(4). Although all MM tumors and cell lines with a t(4;14) translocation have dysregulated MMSET, about 25% do not express FGFR3. Therefore, the function of dysregulated wild-type (WT) FGFR3 in the pathogenesis of MM remains unclear. We developed a murine transgenic (TG) model in which WT FGFR3 is overexpressed in B lymphoid cells. Although high levels of FGFR3 resulted in lymphoid hyperplasia in about one-third of older mice, no increase in tumorigenesis was observed. However, double TG FGFR3/Myc mice develop mature B lymphoma tumors that occur with a higher penetrance and shorter latency than in single TG Myc mice (P=0.006). We conclude that expression of high levels of WT FGFR3 can be oncogenic and cooperate with MYC to generate B lymphoid tumors. This suggests that dysregulated FGFR3 expression is likely to be essential at least for the early stages of pathogenesis of MM tumors that have a t(4;14) translocation.

International Myeloma Working Group molecular classification of multiple myeloma: spotlight review.

Myeloma is a malignant proliferation of monoclonal plasma cells. Although morphologically similar, several subtypes of the disease have been identified at the genetic and molecular level. These genetic subtypes are associated with unique clinicopathological features and dissimilar outcome. At the top hierarchical level, myeloma can be divided into hyperdiploid and non-hyperdiploid subtypes. The latter is mainly composed of cases harboring IgH translocations, generally associated with more aggressive clinical features and shorter survival. The three main IgH translocations in myeloma are the t(11;14)(q13;q32), t(4;14)(p16;q32) and t(14;16)(q32;q23). Trisomies and a more indolent form of the disease characterize hyperdiploid myeloma. A number of genetic progression factors have been identified including deletions of chromosomes 13 and 17 and abnormalities of chromosome 1 (1p deletion and 1q amplification). Other key drivers of cell survival and proliferation have also been identified such as nuclear factor- B-activating mutations and other deregulation factors for the cyclin-dependent pathways regulators. Further understanding of the biological subtypes of the disease has come from the application of novel techniques such as gene expression profiling and array-based comparative genomic hybridization. The combination of data arising from these studies and that previously elucidated through other mechanisms allows for most myeloma cases to be classified under one of several genetic subtypes. This paper proposes a framework for the classification of myeloma subtypes and provides recommendations for genetic testing. This group proposes that genetic testing needs to be incorporated into daily clinical practice and also as an essential component of all ongoing and future clinical trials.

Genetic events in the pathogenesis of multiple myeloma.

The genetics of myeloma has been increasingly elucidated in recent years. Recurrent genetic events, and also biologically distinct and clinically relevant genetic subtypes of myeloma have been defined. This has facilitated our understanding of the molecular pathogenesis of the disease. In addition, some genetic abnormalities have proved to be highly reproducible prognostic factors. With the expanding therapeutic armamentarium, it is time to include genetic assessment as part of clinical evaluation of myeloma patients to guide management. In this review we examine the role of various genetic abnormalities in the molecular pathogenesis of myeloma, and the use of such abnormalities in disease classification, prognosis and clinical management.

Frequent inactivation of the cyclin-dependent kinase inhibitor p18 by homozygous deletion in multiple myeloma cell lines: ectopic p18 expression inhibits growth and induces apoptosis.

Multiple myeloma (MM) is a clonal neoplasm of plasma cells which offers an excellent model to study multistep molecular oncogenesis. In 20-25% of primary tumors and cell lines examined, cyclin D1 is overexpressed due to the translocation t(11;14)(q13;q32). We have characterized cyclin-dependent kinase inhibitor p15 (CDKN2B), p16 (CDKN2A) and p18 (CDKN2C) deletions in cyclin D1-expressing and non-expressing MM cell lines. p18 was found to be frequently deleted (38%); in some cases p18 deletions coexisted with hemizygous p16 deletion. To examine the function of p18 as a putative tumor suppressor in myeloma cells, a zinc-inducible p18 construct was stably transfected into KMS12, a MM cell line with biallelic p18 and monoallelic p16 deletions as well as cyclin D1 overexpression. Ectopic expression of p18 caused 40-45% growth suppression as determined by trypan blue exclusion and MTS assays. p18 induction also resulted in apoptosis, suggesting that inhibition of the cyclin D1/CDK/pRb pathway in these tumor cells could be a crucial step toward the induction of tumor regression via apoptotic cell death. This cell cycle pathway is thus frequently mutated and provides a potentially novel target for gene therapeutic or pharmacologic approaches to human myeloma.

Multiple myeloma.

Multiple myeloma (MM) is a malignancy of the plasma cell characterized by migration and localization to the bone marrow where cells then disseminate and facilitate the formation of bone lesions. Unfortunately, while treatment of this disease is effective in palliating the disease, and even prolonging survival, this disease is generally regarded as incurable. Understanding the basic biology of myeloma cells will ultimately lead to more effective treatments by developing target based therapy. In Section I, Dr. Bergsagel discusses the molecular pathogenesis of MM and shares insights regarding specific chromosomal translocations and their role in the genesis and progression of MM. New information regarding FGFR3 as an oncogene as well as how activating mutations may contribute to disease evolution and may be an important target for novel therapeutics of MM is presented. In Section II, Dr. Anderson elaborates on novel therapeutic approaches to MM also targeting fundamental genetic abnormalities in MM cells. Both preclinical and clinical studies of novel agents including PS-341 and IMiDs are highlighted. In Section III, Dr. Harousseau discusses the role of autologous stem cell transplant in MM. He highlights clinical trials addressing the question of conditioning regimens and the impact of tandem transplants. He also addresses the role of allogeneic BMT and the use of attenuated dose conditioning regimens (so called mini-allogeneic transplants) in the treatment of MM. In Section IV, Dr. Dalton provides an overview of the current state of myeloma therapy and summarizes the different and exciting approaches being undertaken to cure this disease.

Chromosome translocations in multiple myeloma.

Multiple myeloma (MM), a malignant tumor of somatically mutated, isotype-switched plasma cells (PC), usually arises from a common benign PC tumor called Monoclonal Gammopathy of Undetermined Significance (MGUS). MM progresses within the bone marrow, and then to an extramedullary stage from which MM cell lines are generated. The incidence of IgH translocations increases with the stage of disease: 50% in MGUS, 60-65% in intramedullarly MM, 70-80% in extramedullary MM, and >90% in MM cell lines. Primary, simple reciprocal IgH translocations, which are present in both MGUS and MM, involve many partners and provide an early immortalizing event. Four chromosomal partners appear to account for the majority of primary IgH translocations: 11q13 (cyclin D1), 6p21 (cyclin D3), 4p16 (FGFR3 and MMSET), and 16q23 (c-maf). They are mediated primarily by errors in IgH switch recombination and less often by errors in somatic hypermutation, with the former dissociating the intronic and 3' enhancer(s), so that potential oncogenes can be dysregulated on each derivative chromosome (e.g., FGFR3 on der14 and MMSET on der4). Secondary translocations, which sometimes do not involve Ig loci, are more complex, and are not mediated by errors in B cell specific DNA modification mechanisms. They involve other chromosomal partners, notably 8q24 (c-myc), and are associated with tumor progression. Consistent with MM being the malignant counterpart of a long-lived PC, oncogenes dysregulated by primary IgH translocations in MM do not appear to confer an anti-apoptotic effect, but instead increase proliferation and/or inhibit differentiation. The fact that so many different primary transforming events give rise to tumors with the same phenotype suggests that there is only a single fate available for the transformed cell.

TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.

Activation of the TCL1 oncogene has been implicated in T cell leukemias/lymphomas and recently was associated with AIDS diffuse large B cell lymphomas (AIDS-DLBCL). Also, in nonmalignant lymphoid tissues, antibody staining has shown that mantle zone B cells expressed abundant Tcl1 protein, whereas germinal center (GC; centrocytes and centroblasts) B cells showed markedly reduced expression. Here, we analyze isolated B cell subsets from hyperplastic tonsil to determine a more precise pattern of Tcl1 expression with development. We also examine multiple B cell lines and B lymphoma patient samples to determine whether different tumor classes retain or alter the developmental pattern of expression. We show that TCL1 expression is not affected by Epstein-Barr virus (EBV) infection and is high in naïve B cells, reduced in GC B cells, and absent in memory B cells and plasma cells. Human herpesvirus-8 infected primary effusion lymphomas (PEL) and multiple myelomas are uniformly TCL1 negative, whereas all other transformed B cell lines tested express moderate to abundant TCL1. This observation supports the hypothesis that PEL, like myeloma, usually arise from post-GC stages of B cell development. Tcl1 protein is also detected in most naïve/GC-derived B lymphoma patient samples (23 of 27 [85%] positive), whereas most post-GC-derived B lymphomas lack expression (10 of 41 [24%] positive). These data indicate that the pattern of Tcl1 expression is distinct between naïve/GC and post-GC-derived B lymphomas (P < 0.001) and that the developmental pattern of expression is largely retained. However, post-GC-derived AIDS-DLBCL express TCL1 at a frequency equivalent to naïve/GC-derived B lymphomas in immune-competent individuals (7 of 9 [78%] positive), suggesting that TCL1 down-regulation is adversely affected by severe immune system dysfunction. These findings demonstrate that TCL1 expression in B cell lymphoma usually reflects the stage of B cell development from which they derive, except in AIDS-related lymphomas.

Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma.

The t(4;14) translocation occurs frequently in multiple myeloma (MM) and results in the simultaneous dysregulated expression of 2 potential oncogenes, FGFR3 (fibroblast growth factor receptor 3) from der(14) and multiple myeloma SET domain protein/Wolf-Hirschhorn syndrome candidate gene 1 from der(4). It is now shown that myeloma cells carrying a t(4;14) translocation express a functional FGFR3 that in some cases is constitutively activated by the same mutations that cause thanatophoric dysplasia. As with activating mutations of K-ras and N-ras, which are reported in approximately 40% of patients with MM, activating mutations of FGFR3 occur during tumor progression. However, the constitutive activation of ras and FGFR3 does not occur in the same myeloma cells. Thus the activated forms of these proteins appear to share an overlapping role in tumor progression, suggesting that they also share the signaling cascade. Consistent with this prediction, it is shown that activated FGFR3-when expressed at levels similar to those seen in t(4;14) myeloma-is an oncogene that acts through the MAP kinase pathway to transform NIH 3T3 cells, which can then generate tumors in nude mice. Thus, FGFR3, when overexpressed in MM, may be not only oncogenic when stimulated by FGF ligands in the bone marrow microenvironment, but is also a target for activating mutations that enable FGFR3 to play a ras-like role in tumor progression.

Recurrent immunoglobulin gene translocations identify distinct molecular subtypes of myeloma.

BACKGROUND: Chromosome translocations involving the immunoglobulin heavy chain gene (IgH) on 14q32 are a seminal event in the pathogenesis of many B-cell malignancies. Since myeloma is a post-germinal center tumor of mature, isotype switched plasma cells, we hypothesized that 14q32 translocations would usually involve IgH switch regions. MATERIALS AND METHODS: We analyzed a panel of 21 human myeloma cell lines using a Southern blot assay to detect illegitimate rearrangements involving the switch regions. We then cloned the breakpoints, developed probes for FISH analysis, and characterized the oncogenes dysregulated by the translocations. RESULTS: Only half of the cell lines demonstrated a 14q32 abnormality by conventional karyotypic analysis, but we were able to identify translocations involving IgH switch regions in 15 of 21 lines, including all of the lines in which a 14q32 translocations was not identified by conventional karyotypic analysis. Six cell lines have an Ig translocation involving 11q13 with overexpression of cyclin D1. Six cell lines have an Ig translocation involving 16q23 with overexpression of c-maf. Five lines have an Ig translocations involving 4p16 with overexpression of FGFR3 and a novel gene, MMSET. The 4p16 breakpoints occur within the 5' introns of MMSET, and are associated with IgH-MMSET hybrid mRNA transcripts. The remaining five cell lines have translocations involving other loci, including: 6p25 (MUM1), 8q24 (c-myc), and 21q22 (?AML1). CONCLUSIONS: Recurrent Ig translocations identify at least three distinct molecular subtypes of myeloma. Our long-term goal is to determine if there are phenotypic, prognostic and therapeutic differences associated with these molecular subtypes.

Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations.

Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Published 2000 Wiley-Liss, Inc.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma.

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

Concurrent activation of a novel putative transforming gene, myeov, and cyclin D1 in a subset of multiple myeloma cell lines with t(11;14)(q13;q32).

Through the application of the NIH/3T3 tumorigenicity assay to DNA from a gastric carcinoma, we have identified a novel transforming gene, designated myeov (myeloma overexpressed gene in a subset of t[11;14]-positive multiple myelomas). Sequence analyses did not reveal any homology with sequences present in the GenBank, except the deduced protein structure predicts a transmembrane localization. Myeov was mapped to chromosome 11q13 and localized by DNA fiber fluorescence in situ hybridization (FISH) 360-kilobase (kb) centromeric of cyclin D1. In 3 of 7 multiple myeloma (MM) cell lines with a t(11;14)(q13;q32) and cyclin-D1 overexpression, Northern blot analysis revealed overexpression of myeov as well. In all 7 cell lines, the translocation breakpoint was mapped within the 360-kb region between myeov and cyclin D1. DNA fiber FISH with a contig of probes covering the constant region of the immunoglobulin heavy chain (IgH) revealed that exclusively in the 3 myeov-overexpressing cell lines (KMS-12, KMS-21, and XG-5), either the 5' E(mu) enhancer or the most telomeric 3' Ealpha enhancer was juxtaposed to myeov. Although cyclin D1 overexpression represents a characteristic feature of all MM cell lines with t(11;14), our results demonstrate aberrant expression of a second putative oncogene in a subset of these cases, due to juxtaposition to IgH enhancers. The clinical relevance of this dual activation remains to be elucidated. (Blood. 2000;95:2691-2698)

Insertion of excised IgH switch sequences causes overexpression of cyclin D1 in a myeloma tumor cell.

Oncogenes are often dysregulated in B cell tumors as a result of a reciprocal translocation involving an immunoglobulin locus. The translocations are caused by errors in two developmentally regulated DNA recombination processes: V(D)J and IgH switch recombination. Both processes share the property of joining discontinuous sequences from one chromosome and releasing intervening sequences as circles that are lost from progeny cells. Here we show that these intervening sequences may instead insert in the genome and that during productive IgH mu-epsilon switch recombination in U266 myeloma tumor cells, a portion of the excised IgH switch intervening sequences containing the 3' alpha-1 enhancer has inserted on chromosome 11q13, resulting in overexpression of the adjacent cyclin D1 oncogene.

The t(4;14) translocation in myeloma dysregulates both FGFR3 and a novel gene, MMSET, resulting in IgH/MMSET hybrid transcripts.

Previously we reported that a karyotypically silent t(4;14)(p16. 3;q32.3) translocation is present in about 25% of multiple myeloma (MM) tumors, and causes overexpression of FGFR3, which is 50 to 100 kb telomeric to the 4p16 breakpoints. Frequent FGFR3 kinase activating mutations in MM with t(4;14) translocations substantiate an oncogenic role for FGFR3. We now report that the 4p16 breakpoints occur telomeric to and within the 5' introns of a novel gene, MMSET (Multiple Myeloma SET domain). In normal tissues, MMSET has a complex pattern of expression with a short form (647 amino acids [aa]) containing an HMG box and hath region, and an alternatively spliced long form (1365 aa) containing the HMG box and hath region plus 4 PHD fingers and a SET domain. Although t(4;14) translocation results in IgH/MMSET hybrid transcripts, overexpression of MMSET also occurs from endogenous promoters on 4p16. Given the homology to HRX/MLL1/ALL1 at 11q23 that is dysregulated by translocations in acute leukemia, we hypothesize that dysregulation of MMSET contributes to neoplastic transformation in MM with t(4;14) translocation. This is the first example of an IgH translocation that simultaneously dysregulates two genes with oncogenic potential: FGFR3 on der(14) and MMSET on der(4).

Frequent dysregulation of the c-maf proto-oncogene at 16q23 by translocation to an Ig locus in multiple myeloma.

Dysregulation of oncogenes by translocation to an IgH (14q32) or IgL (kappa, 2p11 or lambda, 22q11) locus is a frequent event in the pathogenesis of B-cell tumors. Translocations involving an IgH locus and a diverse but nonrandom array of chromosomal loci occur in most multiple myeloma (MM) tumors even though the translocations often are not detected by conventional cytogenetic analysis. In a continuing analysis of translocations in 21 MM lines, we show that the novel, karyotypically silent t(14;16)(q32.3;q23) translocation is present in 5 MM lines, with cloned breakpoints from 4 lines dispersed over an approximately 500-kb region centromeric to the c-maf proto-oncogene at 16q23. Another line has a t(16;22)(q23;q11), with the breakpoint telomeric to c-maf, so that the translocation breakpoints in these 6 lines bracket c-maf. Only these 6 lines overexpress c-maf mRNA. As predicted for dysregulation of c-maf by translocation, there is selective expression of one c-maf allele in 2 informative lines with translocations. This is the first human tumor in which the basic zipper c-maf transcription factor is shown to function as an oncogene.

Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3.

Dysregulation of oncogenes by translocation to the IgH locus (14q32) is a seminal event in the pathogenesis of B-cell tumours. In multiple myeloma (MM), translocations to the IgH locus have been reported at an incidence of 20-60%. For most translocations, the partner chromosome is unknown (14q+); for the others, a diverse array of chromosomal partners have been identified, with 11q13 (cyclin D1) the only chromosome that is frequently involved. Recently, we developed a Southern-blot assay that detects translocation breakpoint fragments in most MM tumours, including those with no translocation detected by conventional karyotyping. In a continuing analysis of translocation in 21 myeloma cell lines and primary tumours, we show that the novel, karyotypically silent translocation t(4;14)(p16.3;q32.3) is present in five lines and at least three of ten primary tumours. The chromosome-4 breakpoints are clustered in a 70-kb region centromeric to the fibroblast growth factor receptor 3 gene (FGFR3), the apparent dysregulated oncogene. Two lines and one primary tumour with this translocation selectively express an FGFR3 allele containing activating mutations identified previously in thanatophoric dwarfism. We propose that after the t(4;14) translocation, somatic mutation during tumour progression frequently generates in FGFR3 protein that is active in the absence of ligand.