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.

Differential expression of c-myb mRNA in murine B lymphomas by a block to transcription elongation.

Expression of c-myb proto-oncogene messenger RNA (mRNA) and protein has been detected principally in tumors and in normal tissue of hematopoietic origin. In each hematopoietic lineage examined, expression of the c-myb gene is markedly downregulated during hematopoietic maturation. However, the mechanism by which differential expression of the c-myb gene is regulated is not known. In murine B-lymphoid tumor cell lines, the amount of steady-state c-myb mRNA is 10 to more than 100 times greater in pre-B cell lymphomas than in B cell lymphomas and plasmacytomas. The downregulation of c-myb mRNA correlates with events at the pre-B cell-B cell junction. Differential expression of c-myb mRNA levels detected between a pre-B cell lymphoma and a mature B cell lymphoma is now shown to be mediated by a block to transcription elongation in the first intron of the c-myb locus. In addition, this developmentally regulated difference in transcriptional activity is correlated with alterations in higher order chromatin structure as reflected by changes in the patterns of hypersensitivity to deoxyribonuclease I at the 5' end of the c-myb transcription unit. Regulation of transcription elongation may provide a more sensitive mechanism for rapidly increasing and decreasing mRNA levels in response to external stimuli than regulation of the initiation of transcription.

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.

Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.

During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-induced differentiation of these transfectants was inhibited in proportion to the level of exogenous c-myb mRNA expression. By adding or removing ZnCl2 at different times during the induction process, it was possible to show that up-regulation of exogenous c-myb limited to the first 2 days of induction had little or no effect on differentiation. In contrast, continuous expression of exogenous c-myb beginning at any time during the period of induction blocked further differentiation. These results suggest that during HMBA induction of MEL cells, the early down-regulation of c-myb mRNA is not necessary for terminal differentiation, whereas the down-regulation of c-myb at a later time is necessary.

Functional analysis of c-Myb protein in T-lymphocytic cell lines shows that it trans-activates the c-myc promoter.

The function of c-Myb protein was revealed by transfecting an expression vector containing the entire c-Myb protein-coding sequence into the murine CTLL-2 T-cell line. Expressions of high levels of c-Myb protein did not alter the expression of several T-cell markers, c-fos mRNA expression, responses to interleukin-2, and growth characteristics of these cells. Interestingly, expression of the c-myc gene was drastically increased in this clone. Further, the c-myb expression plasmid, but not a frameshift mutant of c-myb, enhanced the expression of a hybrid construct of c-myc promoter linked to a reporter gene by 8- to 14-fold. These results demonstrate a role of c-Myb protein in c-myc gene expression.

Only the DNA binding and transactivation domains of c-Myb are required to block terminal differentiation of murine erythroleukemia cells.

The c-Myb protein is a transcription factor with an apparent but poorly defined role in hematopoietic cell growth and differentiation. The DNA binding and several transcriptional regulatory domains of the c-Myb protein have been defined by transient transfections into nonhematopoietic cell lines. Although the relationship between these domains and transformation has been studied, little is known about the function of these domains during hematopoietic maturation. Up-regulation of stably transfected c-myb in murine erythroleukemia (MEL) cells blocks terminal differentiation when MEL cells are induced to differentiate with N,N'-hexamethylene bisacetamide. To determine which functional domains of c-Myb are necessary and sufficient to block differentiation, mutated c-myb constructs under the control of a murine metallothionein promoter were transfected into C19 MEL cells, and stable clonal cell lines were established. The ability of Myb mutants to block differentiation paralleled their ability to transactivate transcription of a reporter gene containing Myb-responsive elements, by transient transfection into a lymphoid cell line. The smallest c-Myb mutant able to block differentiation consisted of the DNA binding domain juxtaposed to the transactivation domain. Therefore, the DNA binding domain and the transactivation domain are necessary and sufficient for c-Myb to block differentiation in MEL cells.

Mechanism of c-myc regulation by c-Myb in different cell lineages.

Activation of the murine c-myc promoter by murine c-Myb protein was examined in several cell lines by using a transient expression system in which Myb expression vectors activate the c-myc promoter linked to a chloramphenicol acetyltransferase reporter gene or a genomic beta-globin gene. S1 nuclease protection analyses confirmed that the induction of c-myc by c-Myb was transcriptional and affected both P1 and P2 start sites in a murine T-cell line, EL4, and a myelomonocytic line, WEHI-3. Mutational analyses of the c-myc promoter revealed that two distinct regions could confer Myb responsiveness in two T-cell lines, a distal site upstream of P1 and a proximal site within the first noncoding exon. In contrast, only the proximal site was required for other cell lineages examined. Five separate Myb-binding sites were located in this proximal site and found to be important for c-Myb trans activation. DNA binding was necessary for c-myc activation, as shown by the loss of function associated with mutation of Myb's DNA-binding domain and by trans-dominant repressor activity of the DNA binding, trans-activation-defective mutant. The involvement of additional protein factors was addressed by inhibiting protein synthesis with cycloheximide in a conditional expression system in which the activity of presynthesized Myb was under the control of estrogen. These experiments indicate that de novo synthesis of additional proteins was not necessary for c-myc trans activation. Together these data reveal two cell lineage-dependent pathways by which c-Myb regulates c-myc; however, both pathways are mechanistically indistinguishable in that direct DNA binding by Myb is required for activating c-myc whereas neither de novo protein synthesis nor other labile proteins are necessary.

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.

Selective expression of one c-myc allele in two human myeloma cell lines.

Chromosomal translocations or DNA rearrangements affecting c-myc occur in almost all murine plasmacytoma and human Burkitt's lymphoma tumors and are associated with a high incidence of exon 2 missense mutations and selective expression of the affected allele. Screening nine multiple myeloma cell lines, we identified no exon 2 missense mutations but did identify two lines with single, silent mutations in exon 1 and exon 2, respectively. Each of these informative multiple myeloma cell lines selectively expresses only one c-myc allele despite the apparent absence of chromosomal translocations or DNA rearrangements affecting c-myc.

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.

Sequence and expression of a murine cDNA encoding PC326, a novel gene expressed in plasmacytomas but not normal plasma cells.

Using a subtractive cDNA approach we have identified a gene, PC326, expressed in 13 of 14 murine plasmacytoma cell lines, but not in any B- or pre-B-lymphoma cell lines. It expresses 4.6-kb and 5.2-kb mRNAs that encode a 747 amino acid protein containing two highly acidic domains flanking a novel, moderately acidic 20 amino acid sequence that is repeated 7.5 times. Sequence comparison identifies an additional 43 amino acid domain that is homologous to a repeated sequence found in the members of the beta-transducin gene family. The PC326 mRNA is detectable in testis but in no other murine tissues, including plasma cells induced by lipopolysaccharide stimulation of splenocytes. Somatic cell hybrids derived from plasmacytomas and fibroblast or T-cell lines have a fibroblastic or T-cell phenotype respectively. Unlike B-cell-specific genes (e.g. immunoglobulin), the expression of which is extinguished in these hybrids, PC326 mRNA appears to be irreversibly turned on in these hybrids. Since PC326 is not expressed in normal plasma cells, it appears that its expression is a cause or consequence of the tumorigenic process that generates murine plasmacytomas.

Identification of consensus genes expressed in plasmacytomas but not B lymphomas.

We have combined subtractive cDNA and PCR technologies to construct and analyze a plasmacytoma minus a highly differentiated B lymphoma subtractive cDNA library. We detected no plasmacytoma-specific clones by hybridization with differential cDNA probes or the subtractive insert. However, random selection of 115 clones has identified 16 quantitatively subtractive and 39 qualitatively subtractive clones. From these clones we have identified 8 potentially interesting genes. One quantitatively subtractive clone (clone 315) identifies an mRNA that is expressed in most plasmacytoma cell lines, but is expressed at an approximately 10-fold lower level in B and pre-B lymphoma cell lines; preliminary evidence suggests that the expression of this gene is increased by IL-6. From the 31 unrelated qualitatively subtractive clones, we have identified two classes of genes that are expressed in one or none of 8 B lymphomas examined: 1) those expressed in most plasmacytoma and pre-B lymphoma cell lines (clone 70 and clone 260); and 2) those expressed in most plasmacytoma cell lines, but not in any of the ten pre-B lymphomas examined (clones 251, 289A, 289B, 326, 291).

Genes expressed selectively in plasmacytomas: markers of differentiation and transformation.

We have analyzed a murine plasmacytoma minus highly differentiated B lymphoma subtractive cDNA library and identified eight genes that are expressed in most plasmacytomas but at a much lower level, or not at all, in most B lymphomas. Four of the genes are markers of the terminal differentiation of B lymphocytes into plasma cells: placental alkaline phosphatase, also expressed in pre-B lymphomas xlr-3, a new X-linked member of the xlr multi-gene family EGP314, a pan-epithelial glycoprotein with sequence features of an adhesion molecule PC315, a gene that is up-regulated by IL6, but without obvious sequence homologies. Two of the genes are not clearly related to normal B cell differentiation, appearing to be associated with malignant transformation of plasma cells: PC326 is a new member of the beta-transducin mosaic protein gene family. It is an X-linked gene, expressed at a very low level in testis, but in no other normal tissue, including LPS- or IL6-induced plasma cells. It has a high level of expression (apparently dysregulated) in most (> 85%) mineral oil induced plasmacytomas. However the likelihood that PC326 is expressed decreases as the tumor latency decreases when different retroviral agents are used to accelerate mineral oil induced plasmacytomagenesis. This suggests that PC326 expression may be a late event in a multi-step process of tumorigenesis. PC251 a new member of the hematopoietic growth factor receptor family, most homologous to IL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Dysregulation of c-myc in multiple myeloma.

Translocation of c-myc to IgH switch regions, or less frequently to one of the IgL loci, is essentially an invariant event in murine plasmacytomas. This results in dysregulation of c-myc, manifested by selective expression of the translocated allele. Human multiple myeloma (MM) has a similarly high incidence of translocations involving IgH switch regions, but c-myc is infrequently involved as a partner in these translocations. However, in screening a panel of 20 MM cell lines, we identified six lines containing two genetically distinguishable c-myc alleles. For these six informative lines (and the corresponding tumor for one line) there is selective expression of one c-myc allele despite the apparent absence of translocation, DNA rearrangement, or amplification involving c-myc. This result suggests frequent tumor specific cis-dysregulation of c-myc in MM by a presently unknown mechanism.

IgH translocations in multiple myeloma: a nearly universal event that rarely involves c-myc.

Dysregulation of c-myc by translocation to the switch regions of the IgH locus occurs in most murine plasmacytomas. Translocations involving 14q32 have been reported in 20-40% of abnormal karyotypes from human multiple myeloma (MM), and involve a variety of loci. Using cytogenetics, FISH and a Southern blot assay, we analyzed 21 MM cells lines and one plasma cell leukemia and identified evidence of a 14q32 translocation in 20/22 samples. The partner loci involved are 11q13 in 6 (associated with cyclin D1 expression), 4p16 in 6 (associated with FGFR3 expression), unidentified in 3 and 1p13, 6, 8q24, 12q24, 16q23, and 21q22 once each. We conclude that conventional karyotypes underestimate the frequency of 14q32 translocations in MM, where they appear to be a nearly universal event. The translocations most frequently involve IgH switch regions, and include two recurrent partner loci (11q13 and 4p16) and a promiscuous array of other partner loci. Although c-myc appears to be cis-dysregulated frequently in MM, it is only rarely translocated to the IgH locus.

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.

Sequence and expression of murine cDNAs encoding Xlr3a and Xlr3b, defining a new X-linked lymphocyte-regulated Xlr gene subfamily.

Using a subtractive cDNA approach we have identified two nearly identical genes, Xlr3a and Xlr3b (X-linked lymphocyte regulated), expressed at a consistently high level in 14 out of 14 murine plasmacytoma cell lines, at a high level in 1 out of 8 B-lymphoma cell lines, and at a very low level in 2 out of the 8 B-lymphoma cell lines. The messages are not detected in 10 pre-B-lymphoma cell lines. These genes express 2.0-kb mRNAs that encode 226-amino-acid proteins that are extremely basic, with an estimated pI of 8.1 and 9.0, respectively. By sequence comparison they are homologous to Xlr1, an acidic nuclear protein that is produced in lymphoid cell lines corresponding to the late stages of lymphocyte differentiation. Xlr2 is a highly homologous gene that is expressed in differentiating male germ cells. Xlr3a and Xlr3b are members of a new subfamily in the Xlr multigene family. Like Xlr1, they are up-regulated during B-cell terminal differentiation in normal and neoplastic B-cells, and cross-hybridize with a message in testis RNA. Also, like Xlr1, they do not cross-hybridize with human genomic DNA.

Isolation and characterization of myosin from cloned rat glioma and mouse neuroblastoma cells.

Myosin has been isolated from the clonal lines of murine neuroblastoma and rat glioma cells. Partial characterization of the two cellular myosins indicates that both possess the following properties: (1) the same elution position as rabbit skeletal muscle myosin by Sepharose 4B chromatography; (2) the presence of heavy (molecular weight about 200,000) and light subunit polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis; (3) EDTA and Ca2+ activated but Mg2+-inhibited ATPase activity in 0.6 M KCl; and (4) binding to rabbit skeletal muscle F-actin which is inhibited by Mg2+-ATP. For both mouse neuroblastoma and rat glioma cells, approximately 0.5-1.5% of the total cell protein is present as myosin. Cellular myosin appears to be indistinguishable in quantity and biochemical properties regardless of whether it is isolated from monolayer or suspension neuroblastoma cells.