Molecular Pathogenesis Of Multiple Myeloma Research Articles

Characterization of Hematopoietic Progenitor Kinase 1 (HPK1) in Multiple Myeloma: a Player in Pathogenesis?,

Abstract 3956Hematopoietic progenitor kinase 1 (HPK1) is a differentially regulated MAP4 kinase (MAP4K) in B lymphocytes (Geisberger et al., 2002; Königsberger et al., 2010). In germinal centres (GCs), B-cell differentiation, selection and Ig-class-switch are modulated by TNF-receptor family members, subsequently activating GC kinases like HPK1 by corresponding adaptor molecules (TRADD and TRAF), or by activated RTKs linked to SH2/SH3 adaptors. Initiating signaling events in the molecular pathogenesis of multiple myeloma (MM) are currently under investigation. A first proteome wide analysis implicated a potential contribution of HPK1 to the pathogenesis of MM demonstrating that selective knock down of HPK1 (MAP4K1) caused a lethal phenotype in MM cells (Tiedemann et al. 2010). Here, we studied expression and regulation of HPK1 in MM and effects on MM phenotype by modulation of HPK1 activity in vitro and in vivo.HPK1 mRNA and protein expression was examined in a series of human MM cell lines and murine MPC-11 MM cells compared to human B-lymphocytes, B-CLL cells and other non-hematological tissues. Subsequently, HPK1 was overexpressed in MPC-11 cells in a kinase-active (HPK1high) and kinase-deficient (K46E) variant. In addition, HPK1 was knocked down by RNAi in MPC-11 cells (HPK1low). Subsequently, we performed analyses of the transcriptome and proteome, in particular with attention to the kinome, in order to further characterize HPK1-modulated MM cells. In addition, transformation of MM phenotype was assayed by thymidine uptake and induced apoptosis in vitro as well as by variances of subcutaneous tumor growth in murine model with regard to the different HPK1 activities in the respective MM cells (HPK1wt, HPK1high, HPK1K46E, HPK1low and corresponding controls).HPK1 expression varied in MM cell lines tested (high: MPC-11, RPMI-8226; mid: U-266, NCI-H929, OPM-2, JJN-3, KMS-11, ANBL-6, MM1.S, OCI-MY5, MM1.R; low: L363, KMS-18, UTMC-2). HPK-1 mRNA and protein expression was found to be significantly (2-5fold) lower in purified CD138+/CD38high cells from bone marrow aspirates of MM patients as in CD19+ peripheral blood B-lymphocytes or CD19+/CD20+/CD5+ B-CLL cells. In cells derived from non-hematological tissues HPK1 was found below the detection limit. For further analyses, MPC-11 cells were generated overexpressing HPK1. Of note, kinase-active HPK1high cells showed decreased sensitivity to induced apoptosis by glucocorticoid receptor inhibition compared to HPK1wt and HPK1K46E cells. In addition, we found a consecutive increased phosphorylation of downstream signaling proteins in HPK1high cells (MEKK1, ASK1 or p38, JNK, TAK1, MEK1/2, p70S6K alpha, MAPKAPK2). Pro-apoptotic effects by hyperactivation of JNK and p38 were blocked by HPK1-induced consecutive overexpression of cell-cycle-associated genes (GADD45 gamma, Cip1, MCL-1). This observation may contribute to inhibition of caspase-dependent pathways and protect myeloma cells from dexamethasone-induced apoptosis. These in vitro findings were correlated with an accelerated tumor growth and higher tumor burden in vivo (194% vs. 100% HPK1high vs. HPK1wt and respective K46E control, day 7). In contrast, HPK1 knock down by siRNA resulted in a decline of phosphorylation of cAMP response element binding protein-kinase (CREB) and TGF beta-activated kinase-1 (TAK1), whereas pJNK1/2, pERK1, p70S6K alpha und pMAPKAPK2 were not modulated compared to HPK1wt cells. Also no significant alteration was observed in HPK1low cells regarding proliferation and apoptosis in vitro.In conclusion, HPK1 is constitutively expressed in MM cells and shows regulatory activity concerning intracellular survival signaling in MM cells. HPK1 may contribute to the malignant phenotype so that a HPK1-targeted approach will be promising. To confirm this hypothesis, a HPK1 knock down approach in vivo is recommended. Disclosures:No relevant conflicts of interest to declare.

MEK and MAF in myeloma therapy

Despite increased understanding of molecular pathogenesis of multiple myeloma and implementation of therapies such as bortezomib and thalidomide, only 10% of patients survive more than 10 years after diagnosis. Until recently, new therapies for myeloma have not been developed based on a detailed understanding of the molecular pathology of the disease. In this issue of Blood, Annunziata et al report a rationale for the use of mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitors in the subset of myeloma patients expressing high levels of the MAF oncogene.

Novel therapeutic strategies in multiple myeloma: role of the heat shock protein inhibitors

Despite advances in understanding the molecular pathogenesis of multiple myeloma and promising new therapies, almost all patients eventually relapse with resistant disease. There is therefore a strong rationale for combining novel therapies that target intrinsic molecular pathways mediating multiple myeloma cell resistance. One such protein family is the heat shock proteins (HSP), especially the HSP90 family. Heat shock protein inhibitors have been identified as promising cancer treatments as, while they only inhibit a single biologic function, the chaperone-protein association, their effect is widespread as it results in the destruction of numerous client proteins. This article reviews the preclinical and clinical data, which support the testing of HSP90 inhibitors as cancer drugs and update the reader on the current status of the ongoing clinical trials of HSP90 inhibitors in multiple myeloma.

Integrative Genomic Approach Identifies Deregulated MicroRNAs in Human Myeloma Cell Lines.

The recent discovery of microRNAs (miRNAs), small noncoding RNAs involved in the regulation of cell cycle, survival, and differentiation programmes, has added a further level of complexity to normal and cancer cell biology. Loss or amplification of miRNA genes by broad cytogenetic abnormalities or minute molecular aberrations has been observed in a variety of human malignancies, with the consequent altered expression of these regulatory genes. Additionally, approximately one third of miRNAs are located within the intronic regions of coding transcription units, and recent evidence indicates that the expression of these miRNAs largely coincides with the transcription of the corresponding host genes. To date, no evidence of deregulated miRNA expression has been reported in multiple myeloma (MM). To provide insights into miRNA biology in MM, we performed an integrative analysis of genome-wide, gene expression and miRNA expression profilings in a panel of 16 human myeloma cell lines (HMCLs). Global miRNA and mRNA expression data were generated on Agilent miRNA microarrays (representing 470 human mature miRNAs) and GeneChip® HG-U133A arrays, respectively, and both quantile-normalized. Genome-wide profiling data were generated on GeneChip® Human Mapping 250K NspI arrays and copy number (CN) values were inferred using the circulary binary segmentation (DNAcopy R Bioconductor package). To measure the correlation between the expression levels of each miRNA and the corresponding CN value or host gene expression, conventional non-parametric analyses were performed (Kendall's tau and Wilcoxon rank-sum tests). As regards miRNA gene CN, the most frequent alteration identified was represented by gain/amplification (for all miRNA genes investigated, an increased CN was present in at least 3 HMCLs, with an average frequency of 58%), followed by loss (5%) and biallelic deletion (0.3%). Our analysis revealed that 14 different miRNA transcripts (miR-15a, miR-19a, miR-21, miR-22, miR-30d, miR-99b, miR-130b, miR-132, miR-140, miR-185, miR-339, miR-491, miR-503, miR-768-3p) had concordant levels with the inferred CN value of the corresponding miRNA gene. Notably, the identified miRNAs mapped to different genomic regions, some of which are involved in recurrent CN alterations in MM, such as 8q24, 19q13.33, or chromosome arms 13q, 16q, 17p, 17q, 22q, and for some of the miRNAs a role in other types of cancer has already been suggested. As regards intragenic miRNAs, 187 miRNA/host gene pairs were obtained after localizing miRNAs within the absolute 5′ and 3′ regions of genes represented on the HG-U133A arrays; 25 of these showed a significant correlation between miRNA and mRNA levels. Among the most correlated miRNA/hostgene pairs we identified miR-152/COPZ2, miR-342-3p/EVL, miR-335/MEST, miR-25 and miR-106b/MCM7. For some of the identified pairs, miRNA expression levels were validated by means of Q-RT-PCR. In conclusion, we showed that miRNA expression in HMCLs could be affected by the presence of genomic lesions or may correlate with host-gene modulation, suggesting a possible role in the molecular pathogenesis of MM. Our integrative approach represents the basis for further investigations, also in primary tumors, aimed at functionally characterizing specific miRNAs in MM.

The road to cure in multiple myeloma

For the past several decades, multiple myeloma (MM), the second most common hematologic malignancy, has been considered a fatal disease with a median survival of 5 years. However, recent advances in the understanding of the molecular pathogenesis of MM have led to new treatment strategies.

Chromosome 1p21 Deletion Defines a Subgroup of Multiple Myeloma with Poor Clinical Outcomes Independent of del (13q), del(p53), t(4;14) or 1q21 Amplification.

Amplifications involving chromosome 1q and deletions involving 1p are frequent events in multiple myeloma (MM). The pathogenesis and clinical significance of these anomalies is largely unknown but CKS1B amplification at 1q21 detected in 30–40% of MM patients is associated with disease progression. As karyotyping and SNP based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we used FISH combined with cytoplasmic light chain detection (cIg-FISH) to investigate the prevalence and prognostic significance of del(1p21) in a cohort of 186 MM patients undergoing autologous stem cell transplant. CIg-FISH detected hemizygous 1p21 deletions in 18% of the cases. The median percentage of clonal plasma cells harboring del(1p21) was 55% (range 20–95%). The presence of 1p21 deletions was strongly correlated with CKS1B amplification (p=0.004), t(4;14) (p= 0.027), and del(p53) (p=0.04), but not with del(13q) or t(11;14). There was no association between del (1p21) and other biological factors including age, gender, Hb, albumin, C-reactive protein, beta-2 microglobulin level, isotype or bone marrow plasmacytosis. Patients with 1p21 deletions had significantly shorter progression-free (median 10.5 vs. 25.4 months, p=0.0001) and overall survivals (median 33.9 months vs. not reached, p=0.001) than those without such deletions. On multivariate analysis, del(1p21) was an independent risk factor for progression free (p< 0.0001) and overall survivals (p=0.0005) after adjusting for other genetic risk factors including del(13q), del(p53), t(4;14) and CKS1B amplification. Our results indicate that del(1p21) is a novel genetic risk factor and warrant inclusion of this genetic aberration in the risk-stratification of MM. Further studies are required to identify candidate tumor suppressor gene(s) at the 1p21 locus and explore their role in the molecular pathogenesis of MM.

Evaluation of XL999, a Potent Inhibitor of FGFR3, for the Potential Treatment of t(4;14) Positive Multiple Myeloma.

Recent advances in our understanding of the molecular pathogenesis of Multiple Myeloma (MM) have provided novel therapeutic targets for treatment of this incurable malignancy. The association of Fibroblast Growth Factor Receptor 3 (FGFR3) with the t(4;14) translocation that occurs uniquely in approximately 15% of MM patients, coupled with the demonstration of the transforming potential of this receptor tyrosine kinase (RTK), make this a particularly attractive target for the development of therapeutic agents for this poor prognosis group. Indeed, inhibition of activated FGFR3 induces apoptosis and inhibits growth in mice of FGFR3-expressing MM tumors, providing further validation of this RTK as a therapeutic target in t(4;14) MM. XL999 is a novel small molecule inhibitor of kinases involved in tumor cell growth and angiogenesis, and is currently in a Phase 1 clinical trial in patients with non-small cell lung cancer. XL999 inhibits members of the class III-V RTKs, including FGFR1 and 3, VEGFR2, PDGFR? and ?, KIT, and FLT3, and exhibits pharmacodynamic target modulation, anti-angiogenesis, and efficacy in multiple tumor models in mice (Eur. J. Cancer Suppl. 2: 141). We report here the FGFR3-targeting characteristics and anti-myeloma activity of XL999. XL999 inhibits FGFR3 with low nanomolar potency in biochemical enzyme assays. Therefore, we examined the activity of XL999 in several cell-based mechanistic assays of FGFR3 activity. The IL-6 dependent cell line, B9, was engineered to express wild-type or activated mutants (Y373C, K650E, G384D and J807C) of FGFR3 found in MM. XL999 differentially inhibited FGF-mediated growth of B9 cells expressing wild-type and mutant receptors, with IC50 values in the low nM range. Growth of these cells could be rescued by IL-6, indicating selectivity of XL999 for FGFR3. The activity of XL999 against FGFR3 was also examined in t(4;14) positive MM cell lines expressing FGFR3. XL999 inhibited the proliferation and viability of KMS-11 cells (FGFR3-Y373C) and OPM-2 cells (expressing FGFR3-K650E) with low nM potency. Importantly, inhibition by XL999 was still observed in the presence of IL-6 or IGF1, potent growth factors for MM cells. In addition, XL999 potently inhibited the phosphorylation of FGFR3 in OPM-2 and KMS-11 tumors after a single oral dose in pharmacodynamic studies in nude mice. U266 and 8226 cells, which lack FGFR3 expression, displayed minimal growth inhibition by XL999, demonstrating that at effective concentrations, XL999 is selective and exhibits minimal nonspecific cytotoxicity to FGFR3 negative MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest, dose-dependent inhibition of downstream ERK phosphorylation, and induction of apoptosis in FGFR3-positive cells. In vitro analysis of XL999 combined with melphalan, bortezomib, or dexamethasone applied simultaneously to KMS11 cells indicated a strong synergistic interaction with dexamethasone, and additivity with either melphalan or bortezomib. Finally, XL999 produced cytotoxic responses in 5/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. Collectively, these results suggest that XL999 may hold potential for patients with MM, particularly in combination with other agents.

Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets

Multiple myeloma is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. The bone marrow microenvironment promotes multiple myeloma cell growth and resistance to conventional therapies. Although multiple myeloma remains incurable, novel targeted agents, used alone or in combination, have shown great promise to overcome conventional drug resistance and improve patient outcome. Recent oncogenomic studies have further advanced our understanding of the molecular pathogenesis of multiple myeloma, providing the framework for new prognostic classification and identifying new therapeutic targets.

Towards a new age in the treatment of multiple myeloma

Multiple myeloma (MM) is an incurable disease characterized by the proliferation of end-stage B lymphocytes (plasma cells, PCs). As a consequence of myeloma growth in the bone marrow, a number of signaling pathways are activated that trigger malignant PC proliferation, escape from apoptosis, migration, and invasion. Thanks to new insights into the molecular pathogenesis of MM, novel approaches aimed at targeting these abnormally activated cascades have recently been developed and others are under study. These strategies include the inhibition of membrane receptor tyrosine kinases, inhibition of the proteasome/aggresome machinery, inhibition of histone deacetylases, inhibition of farnesyltransferases, targeting of molecular chaperones, and others. We will herein review and discuss these novel biological approaches with particular emphasis on those based on biochemical pathways which drive cell signaling. By providing the rationale for innovative therapeutic strategies, the above mechanisms represent targets for new compounds being tested in the management of this disease.

Molecular pathogenesis of multiple myeloma

Molecular pathogenesis of multiple myeloma

Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis

AbstractTo define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4,RB1, anddisabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-κB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM. (Blood. 2003;102:4504-4511)

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.

Multiple Myeloma: Monoallelic Deletions of the Tumor Suppressor Genes TP53 and RB1 in Long-Term Follow-Up

Recently, a working-model of a stepwise malignant transformation in the molecular pathogenesis of multiple myeloma (MM) was proposed, involving the tumor suppressor gene TP53 and retinoblastoma gene (RB1) as prominent components of cell cycle control. To further define the role of TP53 and RB1 in disease progression, we retrospectively analyzed by fluorescence in situ hybridization (FISH) cytological material from 16 patients who underwent sequential bone marrow biopsies during the course of their disease. For TP53, no deletions were detected at presentation or during follow-up. It is possible that the patients reported here represent a subset with relatively long survival, and therefore did not demonstrate the TP53 deletions that had been reported in patients with a very poor prognosis. For RB1, monoallelic deletion was demonstrated in nine patients. In each case, the deletion appeared already in the first biopsy analyzed. The presence of a deletion did not affect the rate of tumor progression or the length of follow-up, and thus prognosis. Monoallelic deletions of RB1 appear to be a frequent and early event in the pathogenesis of MM, without obvious relevance for disease progression.