W. Michael Kuehl

Multiple myeloma: evolving genetic events and host interactions

Multiple myeloma is a neoplasm of terminally differentiated B cells (plasma cells) in which chromosome translocations frequently place oncogenes under the control of immunoglobulin enhancers. Unlike most haematopoietic cancers, multiple myeloma often has complex chromosomal abnormalities that are reminiscent of epithelial tumours. What causes full-blown myeloma? And can our molecular understanding of this common haematological malignancy be used to develop effective preventive and treatment strategies?

Genetics and Cytogenetics of Multiple Myeloma A Workshop Report

Much has been learned regarding the biology and clinical implications of genetic abnormalities in multiple myeloma. Because of recent advances in the field, an International Workshop was held in Paris in February of 2003. This summary describes the consensus recommendations arising from that meeting with special emphasis on novel genetic observations. For instance, it is increasingly clear that translocations involving the immunoglobulin heavy-chain locus are important for the pathogenesis of one-half of patients. As a corollary, it also clear that the remaining patients, lacking IgH translocations, have hyperdiploidy as the hallmark of their disease. Several important genetic markers are associated with a shortened survival such as chromosome 13 monosomy, hypodiploidy, and others. The events leading the transformation of the monoclonal gammopathy of undetermined significance (MGUS) to myeloma are still unclear. One of the few differential genetic lesions between myeloma and MGUS is the presence of ras mutations in the latter. Gene expression platforms are capable of detecting many of the genetic aberrations found in the clonal cells of myeloma. Areas in need of further study were identified. The study of the genetic aberrations will likely form the platform for targeted therapy for the 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.

A monoclonal gammopathy precedes multiple myeloma in most patients

Preexisting plasma cell disorders, monoclonal gammopathy of undetermined significance, or smoldering myeloma are present in at least one-third of multiple myeloma patients. However, the proportion of patients with a preexisting plasma cell disorder has never been determined by laboratory testing on prediagnostic sera. We cross-referenced our autologous stem cell transplantation database with the Department of Defense Serum Repository. Serum protein electrophoresis, immunofixation electrophoresis, and serum free light-chain analysis were performed on all sera collected 2 or more years before diagnosis to detect a monoclonal gammopathy (M-Ig). In 30 of 90 patients, 110 prediagnostic samples were available from 2.2 to 15.3 years before diagnosis. An M-Ig was detected initially in 27 of 30 patients (90%, 95% confidence interval, 74%-97%); by serum protein electrophoresis and/or immunofixation electrophoresis in 21 patients (77.8%), and only by serum free light-chain analysis in 6 patients (22.2%). Four patients had only one positive sample within 4 years before diagnosis, with all preceding sera negative. All 4 patients with light-chain/nonsecretory myeloma evolved from a light-chain M-Ig. A preexisting M-Ig is present in most multiple myeloma patients before diagnosis. Some patients progress rapidly through a premalignant phase. Light-chain detected M-Ig is a new entity that requires further study.

Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma.

The oncogene c-maf is translocated in approximately 5%-10% of multiple myelomas. Unexpectedly, we observed c-maf expression in myeloma cell lines lacking c-maf translocations and in 50% of multiple myeloma bone marrow samples. By gene expression profiling, we identified three c-maf target genes: cyclin D2, integrin beta7, and CCR1. c-maf transactivated the cyclin D2 promoter and enhanced myeloma proliferation, whereas dominant inhibition of c-maf blocked tumor formation in immunodeficient mice. c-maf-driven expression of integrin beta7 enhanced myeloma adhesion to bone marrow stroma and increased production of VEGF. We propose that c-maf transforms plasma cells by stimulating cell cycle progression and by altering bone marrow stromal interactions. The frequent overexpression of c-maf in myeloma makes it an attractive target for therapeutic intervention.

Classical and/or alternative NF-κB pathway activation in multiple myeloma

Mutations involving the nuclear factor-kappaB (NF-kappaB) pathway are present in at least 17% of multiple myeloma (MM) tumors and 40% of MM cell lines (MMCLs). These mutations, which are apparent progression events, enable MM tumors to become less dependent on bone marrow signals that activate NF-kappaB. Studies on a panel of 51 MMCLs provide some clarification of the mechanisms through which these mutations act and the significance of classical versus alternative activation of NF-kappaB. First, only one mutation (NFKB2) selectively activates the alternative pathway, whereas several mutations (CYLD, NFKB1, and TACI) selectively activate the classical pathway. However, most mutations affecting NF-kappaB-inducing kinase (NIK) levels (NIK, TRAF2, TRAF3, cIAP1&2, and CD40) activate the alternative but often both pathways. Second, we confirm the critical role of TRAF2 in regulating NIK degradation, whereas TRAF3 enhances but is not essential for cIAP1/2-mediated proteasomal degradation of NIK in MM. Third, using transfection to selectively activate the classical or alternative NF-kappaB pathways, we show virtually identical changes in gene expression in one MMCL, whereas the changes are similar albeit nonidentical in a second MMCL. Our results suggest that MM tumors can achieve increased autonomy from the bone marrow microenvironment by mutations that activate either NF-kappaB pathway.

Molecular pathogenesis of multiple myeloma and its premalignant precursor

Multiple myeloma is a monoclonal tumor of plasma cells, and its development is preceded by a premalignant tumor with which it shares genetic abnormalities, including universal dysregulation of the cyclin D/retinoblastoma (cyclin D/RB) pathway. A complex interaction with the BM microenvironment, characterized by activation of osteoclasts and suppression of osteoblasts, leads to lytic bone disease. Intratumor genetic heterogeneity, which occurs in addition to intertumor heterogeneity, contributes to the rapid emergence of drug resistance in high-risk disease. Despite recent therapeutic advances, which have doubled the median survival time, myeloma continues to be a mostly incurable disease. Here we review the current understanding of myeloma pathogenesis and insight into new therapeutic strategies provided by animal models and genetic screens.

Somatic cell hybridization of mouse myeloma cells

Somatic cell hybrides between different mouse myeloma cell lines have been readili isolated using modifications of existing techniques. The hybrid nature of these cells was established by HAT or HAT-ouabain selective procedures, their chromosome number, and, in one case, H-2 surface antifen expression. Three hybrid cell lines are described here in detail: an IgG2B, K X LgG2a, k; an IgG1, k X IgG2b, k; and an IgG1, k X IgM, Lambda. In all cases, both parental types of H and L chains are expressed in the hybrid cells and no new chains are observed. However, molecules possessing disulfide-bonded mixtures of parental H and/or L chains are seen. Analysis of subclones of these hybrids indicates considerable stability in the expression of the immunoglobulins for up to 13 months. However, segregant clones no longer synthesizing one or more of the parental H or L chains arise frequently.

Critical roles for immunoglobulin translocations and cyclin D dysregulation in multiple myeloma

Summary:  Multiple myeloma (MM) is a tumor of long‐lived bone marrow plasma cells (PCs). Nearly 40% of MM tumors have immunoglobulin H (IgH) translocations involving four recurrent chromosomal loci (oncogenes): 11q13 (cyclin D1), 6p21 (cyclin D3), 4p16 (MMSET and FGFR3), and 16q23 (c‐maf). Other MM tumors have Ig translocations involving different loci, none of which is involved in more than 1% of tumors. At least 25% of MM tumors have no Ig translocation. Unlike normal PCs, MM tumors usually express one of the three cyclin D genes at a high level. Translocations involving 4p16 and 16q23 do not directly target a cyclin D gene, but they are associated with a high level of cyclin D2 expression. Although cyclin D1 is not expressed in normal hematopoietic cells, one‐third of MM tumors ectopically express cyclin D1 in the absence of t(11;14). Despite a low proliferation index in MM, dysregulation of a cyclin D gene seems to be a unifying oncogenic event. Analysis of 34 MM cell lines indicates that tumors having an IgH translocation are significantly over‐represented, whereas tumors that ectopically express cyclin D1 are not represented. We speculate that ectopic cyclin D1 expression without t(11;14) is dependent on tumor‐specific interaction with bone marrow stromal cells.

Rescue of Hippo coactivator YAP1 triggers DNA damage–induced apoptosis in hematological cancers

Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.