The varied distribution and impact of RAS codon and other key DNA alterations across the translocation cyclin D subgroups in multiple myeloma

Purpose The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups. Materials and Methods The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. Results Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures. Conclusion Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.

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Bone Marrow and Peripheral Blood Dendritic Cells From Patients With Multiple Myeloma Are Phenotypically and Functionally Normal Despite the Detection of Kaposi’s Sarcoma Herpesvirus Gene Sequences

Blood ◽

10.1182/blood.v93.5.1487.405a35_1487_1495 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1487-1495 ◽

Cited By ~ 2

Author(s):

Noopur Raje ◽

Jianlin Gong ◽

Dharminder Chauhan ◽

Gerrard Teoh ◽

David Avigan ◽

...

Keyword(s):

Multiple Myeloma ◽

Dendritic Cells ◽

Gene Sequence ◽

Patient Specific ◽

Normal Donor ◽

Cyclin D ◽

Gene Sequences ◽

Hla Dr ◽

Viral Cyclin ◽

Kshv Gene

Multiple myeloma (MM) cells express idiotypic proteins and other tumor-associated antigens which make them ideal targets for novel immunotherapeutic approaches. However, recent reports show the presence of Kaposi’s sarcoma herpesvirus (KSHV) gene sequences in bone marrow dendritic cells (BMDCs) in MM, raising concerns regarding their antigen-presenting cell (APC) function. In the present study, we sought to identify the ideal source of DCs from MM patients for use in vaccination approaches. We compared the relative frequency, phenotype, and function of BMDCs or peripheral blood dendritic cells (PBDCs) from MM patients versus normal donors. DCs were derived by culture of mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The yield as well as the pattern and intensity of Ag (HLA-DR, CD40, CD54, CD80, and CD86) expression were equivalent on DCs from BM or PB of MM patients versus normal donors. Comparison of PBDCs versus BMDCs showed higher surface expression of HLA-DR (P = .01), CD86 (P = .0003), and CD14 (P = .04) on PBDCs. APC function, assessed using an allogeneic mixed lymphocyte reaction (MLR), demonstrated equivalent T-cell proliferation triggered by MM versus normal DCs. Moreover, no differences in APC function were noted in BMDCs compared with PBDCs. Polymerase chain reaction (PCR) analysis of genomic DNA from both MM patient and normal donor DCs for the 233-bp KSHV gene sequence (KS330233) was negative, but nested PCR to yield a final product of 186 bp internal to KS330233 was positive in 16 of 18 (88.8%) MM BMDCs, 3 of 8 (37.5%) normal BMDCs, 1 of 5 (20%) MM PBDCs, and 2 of 6 (33.3%) normal donor PBDCs. Sequencing of 4 MM patient PCR products showed 96% to 98% homology to the published KSHV gene sequence, with patient specific mutations ruling out PCR artifacts or contamination. In addition, KHSV-specific viral cyclin D (open reading frame [ORF] 72) was amplified in 2 of 5 MM BMDCs, with sequencing of the ORF 72 amplicon revealing 91% and 92% homology to the KSHV viral cyclin D sequence. These sequences again demonstrated patient specific mutations, ruling out contamination. Therefore, our studies show that PB appears to be the preferred source of DCs for use in vaccination strategies due to the ready accessibility and phenotypic profile of PBDCs, as well as the comparable APC function and lower detection rate of KSHV gene sequences compared with BMDCs. Whether active KSHV infection is present and important in the pathophysiology of MM remains unclear; however, our study shows that MMDCs remain functional despite the detection of KSHV gene sequences.

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Molecular Classification of Multiple Myeloma: A Distinct Transcriptional Profile Characterizes Patients Expressing CCND1 and Negative for 14q32 Translocations.

Blood ◽

10.1182/blood.v106.11.1548.1548 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1548-1548

Author(s):

Luca Agnelli ◽

Silvio Bicciato ◽

Michela Mattioli ◽

Sonia Fabris ◽

Daniela Intini ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Expression Profiles ◽

Gene Expression Pattern ◽

Marker Genes ◽

Molecular Characteristics ◽

Cyclin D ◽

Expression Data ◽

Chromosome 13Q ◽

13Q Deletion

Abstract The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM), being at least one of them deregulated in almost all MM tumors. A recently proposed TC classification1 grouped MM patients into five classes on the basis of their cyclins D expression profiles and the presence of the main translocations involving the immunoglobulin heavy-chain (IGH) locus at 14q32. The aim of our study was to identify the putative transcriptional fingerprints associated with the deregulation of the different D-type cyclins and the presence of IGH translocations. The cyclin D expression levels obtained by high-density oligonucleotide microarray analysis of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes, along with the molecular characteristics. The cyclin D expression data were validated by means of real-time quantitative PCR analysis; fluorescence in-situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. A multi-class classification analysis was performed on the gene expression data and used to identify the transcriptional fingerprints of the 5 TC groups. 112 probe sets were selected as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. In particular, TC1, TC4 and TC5 groups were characterized by the molecular signatures associated with the primary IGH translocations target genes. The TC2 group, showing significantly extra copies of the CCND1 locus (P=5.9×10−3) and neither IGH translocations nor the chromosome 13q deletion (P=1.7×10−3), was characterized by the overexpression of 30 genes, mainly involved in protein biosynthesis at translational level. Among the most specifically modulated transcripts within the group we identified a novel gene containing a BTB/POZ domain, typical of many zinc finger transcription factors and associated with transcriptional repression activity. A meta-analysis performed on two publicly available MM datasets, containing almost 250 cases, validated the identified gene expression signatures with a global classification rate (indicating the correct prediction of the TC class for the independent set) of 86% and 90%, respectively. Our data contribute to the understanding of the molecular and biological features of distinct MM subtypes; the identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.

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Identification of a Potent Natural Triterpenoid Inhibitor of Proteosome Chymotrypsin-Like Activity and NF-κB with Specific Anti-Myeloma Activity in Vitro and in Vivo

Blood ◽

10.1182/blood.v112.11.3679.3679 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3679-3679

Author(s):

Rodger E. Tiedemann ◽

Jonathan J Keats ◽

Jessica Schmidt ◽

Chang-Xin Shi ◽

Yuan X Zhu ◽

...

Keyword(s):

Multiple Myeloma ◽

Significant Proportion ◽

Dual Function ◽

Cyclin D ◽

Cyclin D2 ◽

Chemical Library ◽

Myeloma Cells ◽

Specific Suppression ◽

Synthetic Promoters

Abstract As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that inhibit signaling pathways driving cyclin D2 promoter transactivation, assaying more than 4,000 compounds. The top-ranked compound from these studies was a natural triterpenoid, pristimerin. Pristimerin markedly suppressed cyclin D2 promoter activity (>90%) in 3T3 fibroblast cells and inhibited cyclin D1, D2 and D3 protein expression in myeloma tumor cells. Strikingly, the early (4 hour) transcriptional response of myeloma cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors (P<10−9) (Connectivity Map Build 2, www.broad.mit.edu/cmap), with rapid induction of heat shock proteins (HSP70 >90-fold), activating transcription factor (ATF) 3 and CHOP. Enzymatic assays performed with purified 20S proteosome, or with total cellular extract, confirm that pristimerin rapidly and specifically inhibits chymotrypsin-like 20S proteosome activity at low concentration (<100nM), causing sustained inhibition lasting >6 hours. Consistent with inhibition of proteosome function, pristimerin causes rapid and sustained accumulation of high molecular weight poly-ubiquitinated protein in myeloma cell lines. Notably, related cytotoxic triterpenoid drugs, such as the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me, RTA 402) or betulinic acid or the ginsenosides - all of which show promising anti-cancer activities and are currently in clinical trials for advanced lymphoma, leukemia or solid malignancies - commonly inhibit NF-kB activation via direct inhibition of IKKα or IKKβ. In contrast drugs that function as proteosome inhibitors also commonly suppress NF-kB function instead by impairing degradation of ubiquitinated IkB. Immunoblotting for phosphorylated IkB confirms that pristimerin, like other triterpenoids, acts upstream of IkB to inhibit its phosphorylation, although pristimerin simultaneously inhibits proteosome activity with marked potency to diminish the clearance of ubiquitinated IkB. As a result of this two-fold stabilization of IkB, pristimerin causes overt and specific suppression of NF-kB mediated transcription, measured by a panel of transcriptional reporters with synthetic promoters containing 5x repeats of generic binding sites for NF-kB, AP-1, CREB or TCF4. Importantly, specific suppression of constitutive NF-kB transcriptional activity was pronounced in myeloma cells with inherent NF-kB pathway activation resulting from bi-allelic deletion of the TRAF3 tumor suppressor. Constitutive activation of the NF-kB pathway occurs in a significant proportion of primary myeloma tumors, most commonly via inactivation of TRAF3. Selective silencing of NF-kB driven transcription in myeloma cells may mediate the potent suppression of cyclin D proteins induced by this compound. Significantly, multiple myeloma cells are exquisitely sensitive to both proteosome inhibition or NFkB pathway inhibition. Consistent with these twin vulnerabilities, pristimerin is potently and selectively lethal to primary myeloma cells from patients (IC50<100nM) grown in mixed lineage culture and inhibits the growth of xenografted human plasmacytoma tumors grown in mice, providing a strong rationale for pharmaceutical development of triterpenoid dual-function proteosome-plus-NF-kB pathway inhibitors as therapeutics for multiple myeloma and related human malignancies.

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

Immunological Reviews ◽

10.1034/j.1600-065x.2003.00052.x ◽

2003 ◽

Vol 194 (1) ◽

pp. 96-104 ◽

Cited By ~ 83

Author(s):

P. Leif Bergsagel ◽

W. Michael Kuehl

Keyword(s):

Multiple Myeloma ◽

Cyclin D

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Kinetin Riboside Is a Novel Targeted Inhibitor of CCND1 and CCND2 Transactivation with Substantial Preclinical Anti-Myeloma Activity.

Blood ◽

10.1182/blood.v108.11.367.367 ◽

2006 ◽

Vol 108 (11) ◽

pp. 367-367

Author(s):

Rodger E. Tiedemann ◽

Xinliang Mao ◽

Ronald J. Marler ◽

Craig B. Reeder ◽

Aaron Schimmer ◽

...

Keyword(s):

Multiple Myeloma ◽

Cyclin D1 ◽

Myeloma Cell ◽

S Phase ◽

Luciferase Reporter ◽

Clinical Activity ◽

G1 Arrest ◽

Cyclin D ◽

Cyclin D2 ◽

Myeloma Cells

Abstract Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood;106:296). To identify novel pharmaceutical inhibitors of cyclin D2 (CCND2) transactivation we therefore screened the Lopac, Prestwick and Spectrum libraries of drugs and natural compounds (n>4000) using NIH 3T3 cells stably expressing the CCND2 promoter driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Kinetin riboside, a nucleoside analogue and plant cytokinin hormone, as a novel inhibitor of CCND1+CCND2 transactivation. By immunoblotting, Kinetin riboside induced rapid suppression of cyclin D1 and D2 proteins (<6 hours) in H929, JJN3, Kms11 and U266 human myeloma cell lines (HMCL) that all over-express either cyclin D1 or D2 due to a range of transforming events relevant to myeloma (including deregulation of FGFR3, MMSET or c-Maf oncogenes, or translocation of CCND1 to the IgH locus). Similar results were obtained in primary CD138+ purified myeloma cells from 5/6 patients. To verify that cyclin D1 and D2 suppression induced by Kinetin riboside is a direct effect and does not occur secondary to cellular arrest in S-phase (when cyclin D protein levels decline) we examined the effects of Kinetin riboside on the cell cycle profile of HMCL. Within 20 hours Kinetin riboside caused the proportion of cells entering S-phase to fall by 50–70% in all HMCL tested, consistent with primary cyclin D suppression and secondary G0/G1 arrest. By MTT assay, Kinetin riboside is cytotoxic to HMCL with an IC50 of <1.7 mg/L (5uM) in 8/12 lines and <5mg/L (15uM) in 11/12 HMCL. By comparison, toxicity studies in Balb/c mice confirm that Kinetin riboside is tolerated in vivo at a dose of 80–100mg/kg i.p. or 25mg/kg i.v. Importantly, Kinetin riboside shows potent synergy with dexamethasone in HMCL that are poorly responsive to one or other agent and Kinetin riboside activity persists during co-culture with myeloma growth factors IL-6, IGF-1 and Baff. Moreover, when tested against unsorted patient bone marrow samples, Kinetin riboside preferentially killed CD138+ myeloma cells at up to 5–8 fold greater rate than normal marrow progenitors. Kinetin riboside induced myeloma cell death is mediated by apoptosis and is associated with caspase 9 cleavage and annexin V binding. Mechanistically, we show that kinetin riboside blocks CCND2 promoter transactivation induced by cAMP or by regulatory phosphoproteins (activated by Forskolin or the PP2A inhibitor, Cantharadin, respectively) and also blocks cis-activation of CCND1 induced by translocation to the IgH enhancer and trans-activation of CCND2 induced by cMaf or FGFR3 over-expression, indicating that kinetin riboside acts at a distal level to block transactivation of CCND1+CCND2 induced by multiple factors. Gene expression profiling reveals that KinR causes rapid induction of the transcriptional repressor, cAMP Response Element Modifier (CREM), which has been reported to bind the cyclin D2 promoter to regulate cyclin D2 expression (Muniz, 2006, Biol Reprod.), providing a putative mechanism for targeted suppression of CCND1+CCND2. Together these studies demonstrate a novel targeted mechanism and substantial pre-clinical activity for kinetin riboside and provide a rationale for clinical evaluation of this drug to improve the outcome of multiple myeloma.

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A High Throughput Screen Identified Cyproheptadine That Decreases D-Type Cyclins, Arrests Cells in the G1 Phase, and Induces Apoptosis in Multiple Myeloma and Leukemia Cells.

Blood ◽

10.1182/blood.v110.11.1507.1507 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1507-1507

Author(s):

Xinliang Mao ◽

A. Keith Stewart ◽

Rose Hurren ◽

Marcela Gronda ◽

Kyle Lee ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Leukemia Cell ◽

Mitochondrial Pathway ◽

G1 Phase ◽

G1 Arrest ◽

Cyclin D ◽

Caspase Activation ◽

Leukemia Cell Lines ◽

Receptor Inhibitor

Abstract Cyclin Ds are key regulators of the cell cycle that are frequently over-expressed in multiple myeloma and leukemia and act to promote the phosphorylation of Rb, thereby facilitating the transition from G1 to S phase. Over-expression of cyclin Ds increases cell proliferation and chemoresistance. In contrast, reducing cyclin Ds levels directly or indirectly through G1 arrest can decrease cellular proliferation and induce apoptosis. To identify novel pharmaceutical inhibitors of cyclin D transactivation, we screened the LOPAC and Prestwick libraries of drugs and natural compounds (n = 2400) using NIH 3T3 cells stably expressing the Cyclin D2 promoter-driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Cyproheptadine as a novel inhibitor of Cyclin D2 transactivation. Cyproheptadine has been used previously for the treatment of atopic dermatitis and anorexia, but its ability to inhibit cyclin D expression has not previously been reported. By immunoblotting, Cyproheptadine decreased expression of cyclin D1, D2 and D3 proteins in human myeloma and leukemia cell lines at low micromolar concentrations. Consistent with effects on the cyclin Ds, Cyproheptadine arrested cells in the G1 phase at concentrations associated with reduction in cyclin D expression. Decreased cyclin D expression and G1 arrest can induce apoptosis, so we tested the effects of Cyproheptadine on cell viability. Myeloma and leukemia cell lines were treated with increasing concentrations of Cyproheptadine and viability was measured by the MTS assay. Cyproheptadine reduced the viability of 7/10 myeloma and 7/8 AML cells lines with an IC50 ranging from 10–25μM. In contrast, it was less toxic to HeLa or NIH3T3 cells with IC20 > 50 μM. Cyproheptadine also reduced the viability of primary myeloma (8/8) and AML patient samples (7/9) with an IC50 <25 μM, but was less toxic to normal hematopoietic cells (IC20 > 50 μM). In a MDAY-D2 mouse model of leukemia, treatment with Cyproheptadine (50mg/kg/d) abolished formation of malignant leukemic ascites without untoward toxicity. Cyproheptadine-induced cell death was associated with reductions in mitochondrial membrane potential. Furthermore, reductions of pro-caspases -3 and -9 were observed prior to the reduction in pro-caspase-8, indicating that Cyproheptadine activates the mitochondrial pathway of caspase activation. Cyproheptadine is a known H1 histamine and serotonin receptor inhibitor, but pre-incubation with histamine, serotonin, or a combination of histamine and serotonin did not abrogate Cyproheptadine-induced cell death. Moreover, the structurally related H1 receptor inhibitor loratadine did not decrease cyclin D expression or reduce cell viability. Therefore, the pro-apoptotic activity of Cyproheptadine is not due to a competitive inhibition of the H1 and/or serotonin receptors, suggesting that Cyproheptadine has additional targets. In summary, Cyproheptadine arrests cells in G1, reduces cyclin D expression, and induces apoptosis via the mitochondrial pathway of caspase activation. Given the prior safety and toxicity record of Cyproheptadine, this drug could be rapidly advanced into clinical trial for the treatment of hematologic malignancies.

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Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-κB with antimyeloma activity in vitro and in vivo

Blood ◽

10.1182/blood-2008-09-179796 ◽

2009 ◽

Vol 113 (17) ◽

pp. 4027-4037 ◽

Cited By ~ 62

Author(s):

Rodger E. Tiedemann ◽

Jessica Schmidt ◽

Jonathan J. Keats ◽

Chang-Xin Shi ◽

Yuan Xiao Zhu ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcriptional Response ◽

Dual Function ◽

Cyclin D ◽

Activating Transcription Factor 3 ◽

Chemical Library ◽

Myeloma Cells ◽

Human Multiple Myeloma

Abstract As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-κB activation via inhibition of IKKα or IKKβ, whereas proteosome inhibitors instead suppress NF-κB function by impairing degradation of ubiquitinated IκB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-κB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-κB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50 < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib—providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-κB inhibitors as therapeutics for human multiple myeloma and related malignancies.

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