A Novel Transgenic Mouse Model of the Human Multiple ... · as an oncogene in a murine model of the t(14;16)(q32;q23) translocation. Materials and Methods Mice We made 2 constructs

Molecular and Cellular Pathobiology

A Novel Transgenic Mouse Model of the Human MultipleMyeloma Chromosomal Translocation t(14;16)(q32;q23)

Naoki Morito1, Keigyou Yoh1, Atsuko Maeda2, Takako Nakano2, Akiko Fujita1, Manabu Kusakabe2,Michito Hamada2, Takashi Kudo2, Kunihiro Yamagata1, and Satoru Takahashi2

AbstractMultiple myeloma (MM) is a currently incurable neoplasm of terminally differentiated B cells. The

translocation and/or overexpression of c-MAF have been observed in human MM. Although c-MAF mightfunction as an oncogene in human MM, there has been no report thus far describing the direct induction of MMby c-MAF overexpression in vivo. In this study, we have generated transgenic (TG) mice that express c-Mafspecifically in the B-cell compartment. Aged c-Maf TG mice developed B-cell lymphomas with some clinicalfeatures that resembled those of MM, namely, plasma cell expansion and hyperglobulinemia. Quantitative RT-PCR analysis demonstrated that Ccnd2 and Itgb7, which are known target genes of c-Maf, were highly expressedin the lymphoma cells. This novel TG mouse model of the human MM t(14;16)(q32;q23) chromosomaltranslocation should serve to provide new insight into the role of c-MAF in tumorigenesis. Cancer Res; 71(2);339–48. �2011 AACR.

Introduction

Multiple myeloma (MM) is an incurable neoplasm of term-inally differentiated B cells that is characterized by mono-clonal expansion of malignant plasma cells. The commonpremalignant stage of MM is a monoclonal gammopathy ofundetermined significance (MGUS), which progresses to MMat the rate of 1% of patients per year (1). Extensive genomicanalysis has revealed that approximately 50% of patients withMGUS and MM have primary translocations in the clonalplasma cells that involve the immunoglobulin heavy chain(IGH) locus on chromosome 14q32 (2). Chromosomal trans-locations that involve the juxtaposition of the IGH locus withthe loci for CCND1 (3), FGFR3 (4), or MMSET (5) result in theoverexpression of these genes. It was first reported a decadeago that c-MAF is expressed at high levels in MM cells thatcarry the translocation t(14;16)(q32;q23), in which the IGHlocus is fused with the c-MAF gene locus (6). In addition, c-MAF is overexpressed in 50% of MM cell lines and patients (7).Themaf proto-oncogene was identified originally within the

genome of the avian musculoaponeurotic fibrosarcoma virusAS42 (8). The product of the Maf gene and other members ofthe Maf family share a conserved basic-region leucine zipper

(bZIP) motif that mediates dimer formation and DNA bindingto the Maf recognition element (MARE; ref. 9). Large Mafproteins, such as c-Maf, Mafb, Mafa/L-Maf/Smaf, and Nrl, alsocontain an acidic domain that mediates transcriptional acti-vation and plays a key role in cellular differentiation (8–12). c-Maf encodes a T-helper cell type 2 (Th2)-specific transcriptionfactor that activates the expression of interleukin (IL)-4 in Tcells (13). It is also involved in the regulation of lens fiber celldifferentiation (14–16). Recently, we have found that trans-genic (TG) mice that express c-Maf specifically in the T-cellcompartment develop T-cell lymphoma (17). Furthermore,60% of human T-cell lymphomas, classified as angioimmuno-blastic T-cell lymphomas, were found to overexpress c-MAF(17, 18).

On the basis of these facts, we hypothesized that over-expression of c-Maf in the B-cell lineage would enhance thedevelopment of an MM-like disease. In this study, we havegenerated c-Maf TG mice that overexpress c-Maf in the B-cellcompartment, using the Igh promoter and Em enhancer. Wefound that these mice developed B-cell lymphoma, whichprovided the first direct evidence that c-Maf can functionas an oncogene in a murine model of the t(14;16)(q32;q23)translocation.

Materials and Methods

MiceWe made 2 constructs for the Em c-Maf transgene (Fig. 1A).

For construct-1, we used the Em enhancer and the VH pro-moter, as described previously (19). A 1.5-kb full-length cDNAthat encoded the murine c-Maf protein was inserted into avector that contained the VH promoter and Em enhancer.Construct-1 was injected into fertilized eggs from BDF1 miceto generate TG mice and then backcrossed 4 times into

Authors' Affiliations: Departments of 1Nephrology and 2Anatomy andEmbryology, Life System Medical Sciences, Graduate School of Compre-hensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

Corresponding Author: Satoru Takahashi, Department of Anatomy andEmbryology, Life System Medical Sciences, Graduate School of Compre-hensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsu-kuba 305 8577, Japan. Phone: 81-29-853-7516; Fax: 81-29-853-6965;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-1057

�2011 American Association for Cancer Research.

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C57BL/6J. For construct-2, the VH promoter, Em enhancer, and30Ek enhancer were used. Construct-2 was injected intofertilized eggs from C57BL/6J mice to generate TG mice. Micewere maintained with a C57BL/6J genetic background in

specific pathogen-free conditions in a Laboratory AnimalResource Center. All experiments were carried out accordingto the Guide for the Care and Use of Laboratory Animals at theUniversity of Tsukuba.

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Figure 1. Generation of mice that overexpress c-Maf. A, constructs of the Em c-Maf transgene. The c-Maf cDNA was inserted into a vector that containedthe VH gene promoter (Vp), and an Em enhancer (Em), or one that contained Vp, Em, and a 30Ek enhancer (Ek). The probe for Southern blot analysis, therestriction enzyme sites (EcoRI; E, and HindIII; H), and the predicted sizes of the endogenous gene and the transgene are indicated. Transgene construct-1was used for TG line 524, whereas TG lines 99 and 68 were generated with construct-2. B, a, Southern blot analysis of the endogenous and c-Maf TG genesin Em c-Maf TG mice. The 4.0-kb endogenous and 1.3-kb TG gene fragments are shown for TG mice. B, b, analysis of c-Maf mRNA in B cells. The amountofc-MafmRNA in thesamples fromTGmicewashigher than that in a sample fromWTmice.B, c,Westernblot analysisof splenicB220þBcells fromEmc-MafTGmice (524, 99, and 68) and WT mice. C, immunofluorescence analysis of splenocyte subpopulations from Em c-Maf TG mice at 20 weeks of age. The totalnumbers of T cells (CD3þ) and B cells (B220þ) did not differ between the spleens of WT and Em c-Maf TG mice. There were no apparent differences in thesubpopulations of B cells, namely, follicular cells (B220þCD23highCD21�) and marginal zone (MZ) B cells (B220þCD23�CD21high). D, proliferative potentialof B cells after treatment with LPS. Enriched splenic B220þ cell populations obtained fromWT and Em c-Maf TG mice at 20 weeks of age were cultured in thepresence of LPS, and proliferation was assayed by using a CellTiter 96 AQueous One Solution Cell Proliferation Assay. The mean absorbance at 490 nm ispresented.

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Cancer Res; 71(2) January 15, 2011 Cancer Research340




Southern hybridization analysisHigh-molecular-weight DNA was prepared from the tail of

each mouse, and 10 mg of this DNA was digested with EcoRIand HindIII and then Southern hybridization was performed,as described previously (17). To investigate Igh gene rearran-gement, DNA from the tail and other tissues was examined bySouthern hybridization. DNA that had been digested withEcoRI was hybridized with the JH4 fragment, as describedpreviously (20).

Isolation of mouse B220þ cells and reversetranscriptase PCR to analyze transgene expressionB220þ splenic B cells were isolated usingmagnetic microbe-

ads from Miltenyi Biotec. Total RNA was prepared from theB220þ splenic B cells of 10 weeks old TG mice or their wild-type (WT) littermates using TRIzol reagent according to themanufacturer's instructions (Invitrogen). Total RNA (1 mg)was reverse transcribed into cDNA using SuperScript IIIReverse Transcriptase (Invitrogen), and 1 mL of this 20 mLreaction mixture was used for PCR. The c-Maf sequence wasamplified using the primers 50-CCTGCCGCTTCAA-GAGGGTGC-30 and 50-TCGCGTGTCACACTCACATG-30,which yielded a 225-bp product. The sequence of Hprt (hypox-anthine-guanine phosphoribosyl transferase), which was usedas a control, was amplified using the primers 50-CAAACTTTGCTTTCCCTGGT-30 and 50-CAAGGGCATATC-CAACAACA-30, which yielded a 250-bp product.

Western blot analysisNuclearextractswerepreparedfromtheB220þsplenicBcells

of 10 weeks old TG orWTmice. The extracts were fractionatedby size on a 10% SDS-polyacrylamide gel, transferred to apolyvinylidene difluoride membrane (Fluoro Trans; Pall Bio-Support Division), and incubated with primary and secondaryantibodies. To detect the c-Maf protein, a rabbit antibodyagainst mouse c-Maf was used as the primary antibody andperoxidase-conjugated goat anti-rabbit IgG (Zymed Labora-tories) was used as the secondary antibody. The anti-c-Mafantibodywas kindly providedbyDrMasaharuSakai (Universityof Hokkaido, Sapporo, Japan). To normalize the results withrespect to theamountofprotein ineachsample, agoatantibodyagainst mouse Lamin B (Santa Cruz Biochemicals) was used.

Flow cytometric analysisSingle-cell suspensions were prepared from the spleen and

the bone marrow of each mouse. Multicolor flow cytometricanalysis was performed using an LSR Flow Cytometer andCellQuest software (Becton Dickinson). The phycoerythrin(PE)-labeled rat antibodies against mouse CD3, CD138,CD23, and IgD, the FITC-labeled rat antibodies against mouseB220, CD21, and IgM, and APC-labeled rat antibody againstmouse B220 were obtained from BD Pharmingen.

Cell culture of B cellsB220þ cells (2 � 105) were plated in triplicate on 96-well

plates before stimulation with lipopolysaccharide (LPS; 10mg/mL; Sigma) and anti-mouse CD40 (1 mg/mL; BD Pharmigen).After 16 hours of stimulation, the viable cell number was

determined using a CellTiter 96 AQueous One Solution CellProliferation Assay (Promega). Aliquots of 40 mL of CellTiter96 AQueous One Solution were added to each well andincubated for an additional 3 hours before the final absor-bance at 490 nm was determined.

Measurement of serum immunoglobulins, paraproteindetermination, and X-ray analysis

The amounts of IgG, IgM, and IgA were determined byELISA, as described previously (21). Paraproteins (M spikes,extra gradients) were determined using cellulose acetateelectrophoresis methods by SRL, Inc. X-ray analysis of boneswas carried out using a Faxitron X-ray Specimen Radio-graphic System and Kodak X-OMAT-TL film.

Histopathologic analysisEach mouse was bled under ether anesthesia. At autopsy,

organs were fixed with 10% formalin in 0.01 mol/L phosphatebuffer (pH 7.2) and embedded in paraffin. Sections werestained with hematoxylin and eosin (H&E) and periodicacid-Schiff (PAS) stain for histopathologic examination bylight microscopy. For dual immunofluorescent staining, therabbit antibody against mouse c-Maf, FITC-labeled goat anti-rabbit IgG (green), and PE-labeled rat anti-mouse CD138antibody (red) were used. The anti-CD138 and anti-B220antibodies were purchased from BD Pharmingen. The immu-nohistochemical analysis of CD138 and anti-B220 were per-formed by the avidin-biotin-peroxidase complex stainingtechnique. For immunofluorescent analysis, frozen sectionswere stained with FITC-labeled goat antibodies against mouseimmunoglobulins IgG, IgM, IgA, k light chain, and l light chain(ICN Pharmaceuticals).

Quantitative reverse transcriptase PCRTotal RNA (1 mg) was reverse transcribed into cDNA.

Each reaction was done in duplicate. The quantity of cDNAin each sample was normalized to the amount of hprt cDNA.For the PCR, we used SYBR Premix Ex Taq II (TAKARA Bio)according to the manufacturer's instructions. The amplifica-tion was carried out in a Thermal Cycler Dice Real TimeSystem (TAKARA Bio). The following primer pairs were used:c-Maf forward: 50-CTGCCGCTTCAAGAGGGTGCAGC-30

and c-Maf reverse: 50-GATCTCCTGCTTGAGGTGGTC-30;Ccnd2 forward: 50-CCTCTTCTTCACACTTTTGTTGGTC-30

and Ccnd2 reverse: 50-CTACCTCCCGCAGTGTTCCT-30; Itgb7forward: 50-CAACCTGGACTCACCCGAAG-30 and Itgb7reverse: 50-CTACCTCCCGCAGTGTTCCT-30; Prdm1 forward:50-AAGCCGAGGCATCCTTACC-30 and Prdm1 reverse: 50-GTCCAAAGCGTGTTCCCTTC-30; Pax5 forward: 50-ACAAACGCCAAAACCCTACC-30 and Pax5 reverse: 50-CTGCT-GTACTTTTGTCCGAATGA-30; Xbp1 forward: 50-GGAGCAG-CAAGTGGTGGATT-30 and Xbp1 reverse: 50-CAGCGTGTCC-ATTCCCAAG-30: Myc forward: 50-GATTTCCTTTGGGCGTT-GG-30 and Myc reverse: 50-GAGGTCATAGTTCCTGTTGGT-GAAG-30; Irf4 forward: 50-GGTGTACAGGATTGTTCCAGA-GG-30 and Irf4 reverse: 50-GCAGAGAGCCATAAGGTGCTG-30; Spp1 forward: 50-GCTTGGCTTATGGACTGAGGT-30 andSpp1 reverse: 50-TCAGAAGCTGGGCAACAGG-30; Hprt

Mouse Model of the t(14;16)(q32;q23) Translocation

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forward: 50-TTGTTGTTGGATATGCCCTTGACTA-30 and Hprtreverse: 50-AGGCAGATGGCCACAGGACTA-30.

Statistical analysisResults were expressed as the mean � SE. Data were

compared by 1-way ANOVA and the Bonferroni correctionwas applied routinely. Significant differences between thegroups of mice were analyzed using the Wilcoxon test forpaired samples, and values of P < 0.05 were consideredstatistically significant. Survival rates were compared usingthe Kaplan–Meier method.

Results

Generation of Em c-Maf TG mice that express c-Mafspecifically in B cells

To generate TG mice that express high levels of c-Mafspecifically in B cells, the murine c-Maf coding sequencewas inserted into a vector that contained the VH genepromoter, an Em enhancer sequence, and a 30Ek enhancersequence (Fig. 1A). We generated 2 constructs (the first didnot contain the 30Ek enhancer) and obtained 17 F0 TG micewith a BDF1 or C57BL/6J genetic background. Subse-quently, 3 TG mouse lines with a C57BL/6J genetic back-ground were generated and screened by PCR, using tailDNA as the template. Genomic DNA was analyzed bySouthern blotting to confirm the integrity and copy numberof the transgene in each TG line. The EcoRI/HindIII frag-ment that contained the c-Maf transgene was 1.3 kb inlength, whereas the corresponding fragment for the endo-genous c-Maf gene was 4.0 kb. The results of densitometricanalyses revealed that TG line 524, which was generatedusing construct-1, contained approximately 4 copies of thetransgene, whereas TG lines 99 and 68, which were gener-ated with construct-2, both contained approximately 2copies (Fig. 1B, a). These transgenes were stably trans-mitted to the progeny.

Overexpression of c-Maf in TG miceTo confirm that the transgene was expressed, the levels of c-

Maf mRNA and protein in splenic B cells from the TG lineswere monitored by reverse transcriptase PCR (RT-PCR) andWestern blotting (Fig. 1B, b and c). The c-Maf mRNA wasoverexpressed in all the TG mice that were tested (Fig. 1B, b).Correspondingly, Western blot analysis of B220þ splenic Bcells confirmed that the levels of c-Maf protein were elevated(Fig. 1B, c). c-Maf protein was not detected in WT B cells inthis analysis.

B-cell development and proliferative potential in Emc-Maf TG mice

To investigate the effect of c-Maf overexpression on B-celldifferentiation, we carried out flow cytometric analysis ofsplenocytes isolated from TG mice at 20 weeks of age. Flowcytometry showed that the total numbers of T cells (CD3þ)and B cells (B220þ) in the spleens of Em c-Maf TG mice wereunaltered relative to WT mice (Fig. 1C). In addition, we couldnot find any apparent differences in the subpopulations of B

cells, namely, follicular cells (B220þCD23highCD21�) and mar-ginal zone (MZ) B cells (B220þCD23� CD21high; Fig. 1C). Next,we analyzed the proliferative potential of WT and Em c-Maf TGB cells in vitro. The proliferative potential of Em c-Maf TG Bcells was relatively high, but there was no substantial differ-ence in the response to LPS (Fig. 1D) or CD40 (data not shown)between the two groups.

Aged Em c-Maf TG mice developed lymphomaThe Em c-Maf TG mice appeared healthy up to 50 weeks

of age. However, they subsequently developed tumors. Themean age at diagnosis as lymphoma by histologic analysiswas 80.1 weeks. We confirmed that 28% (18/64) of the Emc-Maf TG mice developed lymphoma (Fig. 2A). We couldnot detect any apparent difference in survival betweenthe mice that were generated with the two different con-structs, construct-1 for TG524 and construct-2 for TG 99and 68.

Lymphomas observed in Em c-Maf TG mice were of B-cell origin

Em c-Maf TG mice appeared healthy up to 50 weeks of age,but they developed tumors subsequently. We performed his-tologic analysis to identify the origin of the lymphoma cells.The spleens of the Em c-Maf TG mice were enlarged approxi-mately 2- to 5-fold relative to those of the WT mice. Thespleens of the mice with lymphoma were infiltrated bynumerous cells, which partially destroyed the normal tissuearchitecture (Fig. 2B). Cells that had infiltrated showed plasmacell-like morphology (Fig. 2B). Moreover, immunohistochem-ical analysis revealed that the infiltrating cells were B220þ

(Fig. 2C) and also expressed CD138 (Fig. 2C). We confirmedthat the infiltrating cells were positive for both CD138 and c-Maf by immunofluorescence staining (Fig. 2C). To investigatethe characteristics of the lymphoma cells, we performed flowcytometry (Fig. 2C and D). We found the number ofB220þCD138þCD21þCD23þIgMþIgD� cells was increased inthe TG mice relative to the WT mice. We also found that thenumber of mature plasma cells (B220lowCD138þ) wasincreased in the TG mice (Fig. 2C).

Hypergammmaglobulinemia in aged Em c-Maf TG miceApproximately 50% (7/14) of the Em c-Maf TG mice of line

524 showed a clonal M spike in the serum between 20 and50 weeks of age (Fig. 3A). At 120 weeks old, 35% (5/14) of theseTG mice had developed lymphoma. The remaining mice thatshowed a clonal M spike (2/14) showed no evidence oflymphoma at 120 weeks. The plasma levels of both IgG andIgM, as measured by ELISA, were increased significantly inaged Em c-Maf TG mice (Fig. 3B). The mean amount of totalIgG in the TG mice was 1,760 � 302 (mg/dL), whereas in thecontrol mice this value was 934 � 142 (mg/dL; P < 0.05). Inaddition, the mean amount of total IgM in the TG mice was1,083 � 223 (mg/dL), whereas in the control mice this valuewas 564 � 92 (mg/dL). However, there was no obviouselevation of total IgA in the TG mice. It must be noted thatonly 1 of the 14 mice showed high levels of both IgG and IgM(Fig. 3B). Notably, these changes in the serum were associated

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with increased numbers of mature plasma cells (B220low

CD138þ) in the bone marrow; the proportion of plasma cellsas a percentage of the total number of bone marrow cells wasabout 5% to 20% in the TGmice compared with less than 5% inthe non-TG controls (Fig. 3C). We also found the number ofB220þCD138þ cells in the bone marrow was increased(Fig. 3C). However, we could not identify any obvious osteo-lytic lesions by X-ray analysis (Fig. 3C).

Em c-Maf TG mice showed clinical features thatresembled those of patients with plasma cell disordersIn Em c-Maf TG mice with hyperglobulinemia that were

older than 60 weeks, renal tubular casts and tubular obstruc-tion were observed (Fig. 4A), which resembled human mye-

loma kidney (so-called cast nephropathy; ref. 22). Moreover,glomerular changes that were characterized by mesangialwidening due to the deposition of PAS-positive material wereobserved in aged Em c-Maf TG mice (Fig. 4A); this resembledmonoclonal immunoglobulin deposition disease (22). Theserenal lesions were similar to the pathologic manifestationsthat are present in humanMMand other plasma cell disordersin which the systemic chronic overproduction of immunoglo-bulins and the accumulation of light chains, paraproteins, andother immunoglobulin fragments are observed (22). Thedepositions contained immunoglobulin light and heavychains, which consisted of either polyclonal heavy and lightchains or clonal IgG or IgM heavy chains and k light chains(Fig. 4B).

Figure 2. Em c-Maf TG micedeveloped B-cell lymphoma. A,cumulative lymphoma-freesurvival rate curves of Em c-MafTG and WT mice. Statisticallysignificant differences (P 0.05)were detected between the twogroups. B, the number of plasmacell-like lymphoma cells in thespleens of Em c-Maf TG mice wasincreased compared with that inthe controls (WT). Disruption of thenormal follicles and accumulationof lymphoma cells were observedin spleens from the TGmice (H&E).C, the B-cell lymphoma cells(B220þ) also expressed CD138, asshown by immunohistochemicalstaining of B220 and CD138.Immunofluorescence showed thatplasma cells (CD138þ, red) thathad infiltrated into the spleens ofEm c-Maf TG mice also expressedc-Maf (green), but this was notobserved in the spleens of WTmice. Flow cytometry revealedthat the number of B220þCD138þ

cells was increased in the spleensof Em c-Maf TG mice that carriedlymphoma. The number of matureplasma cells (B220lowCD138þ) inthe spleen was also increased. D,B220þCD138þ cells in the spleensof Em c-Maf TG mice werecharacterized as B220þCD138þ-CD21þCD23þIgMþIgD� by flowcytometry.

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Autonomous proliferation in nude mice of lymphomacells from Em c-Maf TG mice

To ascertain whether the infiltrating tumor cells in Emc-Maf TG mice could proliferate autonomously, we isolatedmononuclear cells from the spleens of these mice and injectedthe isolated cells into nude mice via the tail vein (106 cells permouse). Recipient mice displayed prominent hepatomegaly,splenomegaly, and/or enlarged lymph nodes throughout theirbodies within 12 weeks of transplantation. A typical example isshown in Figure 5A. The tumor cells could also be trans-planted into syngeneic C57BL/6J mice. To assess clonality, weexamined the rearrangement of the Igh locus in the c-Maf TG

lymphoma cells. Southern blot analysis was carried out using amurine JH4 probe (Fig. 5B). We extracted DNA from the tailsand spleens of the mice. Monoclonal rearrangement of the Ighlocus was found in DNA that had been isolated from thespleens of the Em c-Maf TGmice (Fig. 5B, lanes 3–6). Histologicanalysis of the spleen from these recipient mice revealed thatthe tumor cells showed a plasma cell-like morphology, that is,the same as the original lymphoma cells (Fig. 5C).

Expression of c-Maf target genes in Em c-Maf TG miceTo gain insight into the molecular mechanism by which

plasma cell dyscrasia develops, we measured the level of

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Figure 3. Hypergamma-globulinemia, bone marrowplasmacytic infiltration, and X-rayanalysis of the bones in Em c-MafTG mice. A, representative resultsof electrophoresis of the serumproteins. Note the presence of anM spike (arrow) in Em c-Maf TGmice at 50 weeks old. B, markedelevation of serumimmunoglobulin levels in Em c-MafTG mice. Sera from WT (*; n ¼ 7)and Em c-Maf TG (TG; *; n ¼ 14)mice at 50 weeks old wereanalyzed by ELISA (top). The plotshows both IgG and IgM levels inEm c-Maf TG mice at 50 weeksold. Only 1 of the 14 mice showedhigh levels of both IgG and IgM(bottom). C, representative bonemarrow biopsies from WT and Emc-Maf TG mice were analyzed bylight microscopy (H&E) to revealincreased plasma cell infiltrates inthe marrow of Em c-Maf TG mice.FACS analysis also revealed thatthe numbers of mature plasmacells (B220lowCD138þ) andB220þCD138þ cells wereincreased in the bone marrow inthe TG mice. Obvious osteolyticlesions could not be found in50 weeks old Em c-Maf TG miceby X-ray analysis.

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mRNA expression from c-Maf and its target genes in Em c-MafTGmice, using a quantitative RT-PCR assay (Fig. 6). In humanMM, CCND2 and ITGB7 are known to be targets of c-MAF (7,23). As we have described previously, deregulated expressionof Ccnd2 and Itgb7 is also observed in the T-cell lymphomas ofTG mice that overexpress c-Maf in the T-lymphoid compart-ment (17). Therefore, we analyzed the expression of Ccnd2 andItgb7 in Em c-Maf TGmice. The level of c-Maf expression in thelymphoma-infiltrated lymph nodes of Em c-Maf TG mice was3- to 5-fold greater than the level in WT B cells (B220þ; Fig. 6).We confirmed the percentage of B220þ cells in the lymphnodes was more than 70%. We also found that the expressionof both Ccnd2 and Itgb7 was elevated in Em c-Maf TG mice(Fig. 6), which was consistent with the results of previousreports in human MM (7).Next, we examined the levels of the B-cell–associated

transcription factors, Prdm1 (Blimp-1), Xbp1, and Pax5.Blimp-1 is a transcriptional repressor that plays a critical role

in the terminal differentiation of B cells into antibody-secret-ing plasma cells (24). Overexpression of Blimp-1 has beenshown to drive plasma cell differentiation (24–26). Xbp1 is abZIP transcription factor and a major regulator of plasma celldifferentiation (27). It is known that Em Xbp1s mice developMM/MGUS (28). In addition, Pax5 is known to inhibit plas-macytic development and also to repress the expression ofXbp1, which is needed for plasma cell formation and immu-noglobulin secretion (29). By RT-PCR analysis (Fig. 6), wefound that Prdm1 and Xbp1 expression were increased butPax5 expression was decreased in the TG mice as comparedwith the control mice, which might correlate with the plasmacell differentiation.

It is known that the dysregulation of MYC (c-MYC) expres-sion can induce the progression of MGUS to MM (1). Inaddition, it has been reported that myeloma cells are comple-tely dependent on the transcription factor IRF4 (interferonregulatory factor 4), despite that fact that most myeloma cells

Figure 4. Renal involvement in Emc-Maf TG mice. A, renal tissuesections from 60 weeks old Em c-Maf TG mice with lymphoma andWT mice were stained with PAS.Left, renal tubules; right, glomeruli.B, glomerular immunoglobulindeposition. Serial frozen sectionsof renal tissue from WT and Em c-Maf TG mice were analyzed byimmunofluorescent staining usingspecific antibodies against mouseimmunoglobulin k and l lightchains, as well as antibodiesagainst IgG, IgM, and IgA heavychains.

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do not harbor mutations, translocations, or amplifications ofthe IRF4 locus (30). Moreover, IRF4 itself is a direct target ofMYC that generates an autoregulatory circuit in myeloma cells(30). However, neitherMyc nor Irf4 expression was elevated inthe Em c-Maf TG mice. We also analyzed Spp1 (osteopontin)expression, because we could not find any obvious osteolyticlesions in theEm c-MafTGmice.SPP1, which is highly expressedin MM, plays a critical role in bone disease by protecting thebone fromdestruction (31).We observed that the expression ofSpp1 was upregulated in Em c-Maf TG mice (Fig. 6).

Discussion

It is important to emphasize that v-maf is a classicaloncogene that was identified in an avian transforming virus,AS42 (8). Large Maf proteins, such as c-Maf, Mafb and Mafa,can efficiently transform primary fibroblasts in vitro (9, 32–34).Previously, we generated c-Maf TG mice that overexpress c-Maf in the T-cell compartment, using the VA vector thatcontains the human CD2 promoter and locus control region.The VA c-Maf TG mice developed T-cell lymphoma, thusproviding evidence that c-Maf can function as an oncogenein T cells in vivo (17). Work over the last decade has providedevidence that c-MAF might play crucial roles in the pathogen-

esis of MM. It has been reported that the translocation and/oroverexpression of c-MAF are identified frequently in humanMM (6, 7, 23). Translocations that involve MAFA (23, 35) orMAFB [t(14;20)(q32;q12); refs. 35, 36], which encode other largeMAF proteins, have also been identified. Altogether, translo-cations that involve a large MAF transcription factor are foundin 8% to 10% of MMs: c-MAF is translocated in 5%,MAFB in 2%,and MAFA in less than 1% of cases (23, 35). Although c-MAFtranslocations are observed in only 5% to 10% of MMs, c-MAFis overexpressed in 50% of cases (7, 23).

In this study, we demonstrated that aged c-Maf TG micedeveloped B-cell lymphomas with some clinical features thatresembled those of human MM, namely, expansion of plasmacells, hyperglobulinemia, and renal involvement. Although wefound that plasma cells (B220lowCD138þ) were increased inthe spleen and bone marrow of the TG mice, the lymphomacells in the spleens were B220þCD138þCD21þCD23þIgMþ-IgD�. The phenotype of some Em c-Maf TG mice partiallyresembled that of types of B-cell lymphoma that containplasmablasts and/or plasma cells rather than plasma celldyscrasias, such as Waldenstr€om's macroglobulinemia (WM;also known as lymphoplasmacytic lymphoma) or MALT(mucosa-associated lymphoid tissue) lymphoma. WM is clas-sified as an indolent form of B-cell lymphoma. Infiltration with

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Figure 5. Autonomousproliferation of c-Maf–inducedlymphoma cells. A, mononuclearcells that had been isolated fromthe spleens of Em c-Maf TG miceand transplanted into nude miceinfiltrated the spleens of therecipient mice. Enlarged liver andspleen were observed in therecipient mouse. B, Southern blotanalyses of lymphoma cells fromthe spleens of Em c-Maf TG miceusing a JH4 probe after digestionwith EcoRI. Lane 1 (DNA from tailof a WT mouse), lane 2 (DNA fromthe tail of an Em c-Maf TG mouse),and lanes 3 to 6 (DNA from theenlarged spleens from Em c-MafTG mice) show the presence of aDNA fragment corresponding tothe germ line JH4 gene (solidarrow). Lanes 3 to 6 show DNAfragments that corresponded torearrangements of the Igh gene(asterisk). Lanes 2 to 6 show aDNA fragment that correspondedto the Igh transgene (hollowarrow). C, transplanted tumor cellsshowed a plasma cell–likeappearance in the spleens of therecipients, as shown by H&Estaining.

Morito et al.





clonal lymphoplasmacytic cells, predominantly in the bonemarrow, and an IgM monoclonal gammopathy are diagnosticfindings of WM (37). MALT lymphoma is a distinct subtype ofMZB-cell lymphomas. The largenumber of plasma cells amongMALT lymphoma cells is a well-known phenomenon knownas plasmacytic differentiation (38). With the demonstrationthat the tumors express both mature B-cell (B220þ) andplasma cell (CD138þ) markers, it should be noted that theyfall within a spectrum of mouse plasma cell–related tumors(anaplastic, plasmablastic, and plasmacytic plasmacytoma)

described previously (39, 40, 41). The Em c-Maf TG mouse isa murine model of the human MM t(14;16)(q32;q23) chromo-somal translocation. Primary early-onset reciprocal chromo-somal translocations in MM occur most frequently at IGH on14q32. Meanwhile, secondary late-onset translocations andgene mutations have been implicated in MM progression(42). The fact that Em c-Maf TG mice displayed immaturephenotypes rather than classical MM might suggest thatc-MAF translocation alone is not sufficient for the developmentof classical MM. Experimental efforts to generate mousemodels of MM have typically involved the targeted expressionof an oncogene in the B-cell compartment by TG approaches(28). These strategies have generally yielded B-cell malignan-cies that display immature phenotypes or plasmacytomasrather than classical MM (28). Given that c-MAF is known tobe translocated and/or overexpressed in human MM, the Emc-Maf TG mouse may offer an ideal system to study thepathogenesis of human MM. Aged Em c-Maf TG mice devel-oped B-cell lymphomas rather than plasma cell dyscrasias.In the majority of cases, TG mice that express an oncogene orproto-oncogene specifically in the B-cell compartmentdevelop plasmacytoma and not classical MM. This is the casefor v-abl (43), Il-6 (44), and Bcl-2/c-Myc (45) mice; however,Em Xbp1s (28) TG mice are the exception. Recently, it wasreported that TG mice in which the c-Myc gene was subject toconditional Aid (activation-induced deaminase)-dependentactivation developed the MM/MGUS phenotype (46). We donot understand the underlying differences in the promotion oftumor formation between mice and humans.

Further studies will be needed to define the role of c-Maf inthe development of both human and mouse MM. Clarificationof the mechanism by which c-Maf contributes to oncogenesismay ultimately facilitate the discovery of more specific thera-pies to prevent the progression of MM.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

A grant-in-aid for Scientific Research on Priority Areas and Genome NetworkProject from the Ministry of Education, Culture, Sports, Science, and Technol-ogy, Japan, a grant-in-aid for Exploratory Research (22650090), a grant from TheNaito Foundation, and a grant from the Astellas Foundation for Research onMetabolic Disorders.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 26, 2010; revised September 17, 2010; accepted November2, 2010; published OnlineFirst January 11, 2011.

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2011;71:339-348. Published OnlineFirst January 11, 2011.Cancer Res Naoki Morito, Keigyou Yoh, Atsuko Maeda, et al. Myeloma Chromosomal Translocation t(14;16)(q32;q23)A Novel Transgenic Mouse Model of the Human Multiple

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