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A Defect of the INK4-Cdk4 Checkpoint and Myc Collaborate in Blastoid Mantle Cell Lymphoma–Like Lymphoma Formation in Mice

Published:February 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.01.004
      Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a monoclonal proliferation of lymphocytes with the co-expression of CD5 and CD43, but not of CD23. Typical MCL is associated with overexpression of cyclin D1, and blastoid MCL variants are associated with Myc (alias c-myc) translocations. In this study, we developed a murine model of MCL-like lymphoma by crossing Cdk4R24C mice with Myc-3′RR transgenic mice. The Cdk4R24C mouse is a knockin strain that expresses a Cdk4 protein that is resistant to inhibition by p16INK4a as well as other INK4 family members. Ablation of INK4 control on Cdk4 does not affect lymphomagenesis, B-cell maturation, and functions in Cdk4R24C mice. Additionally, B cells were normal in numbers, cell cycle activity, mitogen responsiveness, and Ig synthesis in response to activation. By contrast, breeding Cdk4R24C mice with Myc-3′RR transgenic mice prone to develop aggressive Burkitt lymphoma–like lymphoma (CD19+IgM+IgD+ cells) leads to the development of clonal blastoid MCL-like lymphoma (CD19+IgM+CD5+CD43+CD23 cells) in Myc/Cdk4R24C mice. Western blot analysis revealed high amounts of Cdk4/cyclin D1 complexes as the main hallmark of these lymphomas. These results indicate that although silent in nonmalignant B cells, a defect in the INK4-Cdk4 checkpoint can participate in lymphomagenesis in conjunction with additional alterations of cell cycle control, a situation that might be reminiscent of the development of human blastoid MCL.
      Tumorigenesis is a multistep process in which several genetic lesions have to accumulate to produce a fully malignant phenotype. One of the most critical steps is the regulation of cell cycle progression that is managed by a series of cyclins and cyclin-dependent kinases (Cdk). Cyclin D1, D2, and D3 are reported to regulate G1/S progression whereas cyclin B1 and B2 act for G2/M transition. Each cyclin has to associate with a specific Cdk to gain enzymatic activity and to become functionally active. Cdk4 and Cdk6 are chief catalytic subunits of the cyclin D family of proteins that govern G1/S-phase progression.
      • Ruas M.
      • Peters G.
      The p16INK4a/CDKN2A tumor suppressor and its relatives.
      The tumor suppressor protein p16INK4a and all members of the INK4 family bind specifically to Cdk4 or Cdk6 and inhibit the cyclin D/Cdk4 and D/Cdk6 complex formation.
      Deregulation of Cdk4 by overexpression, translocations, and inactivation (by mutations, deletion, or promoter methylation) of their key kinase inhibitors p16INK4a and p15INK4b plays a role in lymphomagenesis especially in mantle cell lymphoma (MCL).
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      • Kasprzycka M.
      • Lai R.
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      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      MCL is a malignant lymphoproliferative disorder derived from naive pregerminal center CD5+ cells.
      • Jares P.
      • Campo E.
      Advances in the understanding of mantle cell lymphoma.
      Several MCL variants have been identified: typical MCL; blastoid variant of MCL; and cyclin D1-negative MCL. Typical MCL are characterized by overexpression of cyclin D1 gene. However the tumorigenetic and transforming property of cyclin D1 is less effective than other oncogenes. Cyclin D1 transgenic animals do not develop spontaneous lymphomas and lymphomagenesis in these animals requires the cooperation of other oncogene such as Myc.
      • Bodrug S.E.
      • Warner B.J.
      • Bath M.L.
      • Lindeman G.J.
      • Harris A.W.
      • Adams J.M.
      Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.
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      • Grzeschiczek A.
      • Kowalski M.B.
      • Möröy T.
      Cyclin D1/bcl-1 cooperates with myc genes in the generation of B-cell lymphoma in transgenic mice.
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      • Amin R.
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      • Cogné M.
      • Denizot Y.
      A myeloma translocation-like model associating CCND1 with the immunoglobulin heavy-chain locus 3′ enhancers does not promote by itself B cell malignancies.
      Blastoid variants of MCL harbor frequent deletion of p16INK4a and overexpression of both Cdk4 and Myc.
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      • Bea S.
      • Ribas M.
      • Hernandez J.M.
      • Bosch F.
      • Pinyol M.
      • Hernandez L.
      • Garcia J.L.
      • Flores T.
      • Conzalez M.
      • Lopez-Guillermo A.
      • Piris M.A.
      • Cardesa A.
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      • Miro R.
      • Campo E.
      Increased number of chromosomal imbalances and high-level DNA amplifications in mantle cell lymphoma are associated with blastoid variants.
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      • Hernandez S.
      • Bea S.
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      • Campo E.
      c-myc mRNA expression and genomic alterations in mantle cell lymphomas and other nodal non-Hodgkin's lymphomas.
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      • Marit G.
      • Dubus P.
      • de Mascarel A.
      • Merlio J.P.
      Blastoid and common variants of mantle cell lymphoma exhibit distinct immunophenotypic and interphase FISH features.
      Cyclin D1-negative MCL do no express high amounts of cyclin D1 but share the same secondary genetic aberrations that typical MCL supporting the concept that they correspond to the same genetic entity.
      • Yatabe Y.
      • Suzuki R.
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      • Seta M.
      • Nakamura S.
      Significance of cyclin D1 overexpression for the diagnosis of mantle cell lymphoma: a clinicopathologic comparison of cyclin D1-positive MCL and cyclin D1-negative MCL-like B-cell lymphoma.
      • Fu K.
      • Weisenburger D.D.
      • Greiner T.C.
      • Dave S.
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      • Gesk S.
      • Siebert R.
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      • Dave B.J.
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      • Smith L.M.
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      • Braziel R.M.
      • Müller-Hermelink H.K.
      • Campo E.
      • Gascoyne R.D.
      • Staudt L.M.
      • Chan W.C.
      Lymphoma/Leukaemia Molecular Profiling Project: Cyclin D1-negative mantle cell lymphoma: a clinicopathologic study based on gene expression profiling.
      • Salaverria I.
      • Zettl A.
      • Bea S.
      • Moreno V.
      • Valls J.
      • Hartmann E.
      • Ott G.
      • Wright G.
      • Lopez-Guillermo A.
      • Chan W.C.
      • Weisenburger D.D.
      • Gascoyne R.D.
      • Grogan T.M.
      • Delabie J.
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      • Montserrat E.
      • Muller-Hermelink H.K.
      • Staud L.M.
      • Rosenwald A.
      • Campo E.
      Specific secondary genetic alterations in mantle cell lymphoma provide prognosis information independent of the gene expression-based proliferation signature.
      • Wlodarska I.
      • Dierickx D.
      • Vanhentenrijk V.
      • van Roosbroeck K.
      • Pospisilova H.
      • Minnei F.
      • Verhoef G.
      • Thomas J.
      • Vandenberghe P.
      • De Wolf-Peeters C.
      Translocations targeting CCND2 CCND3, and MYCN do occur in t(11;14)-negative mantle cell lymphoma.
      Few murine model of MCL are available. Tumor promoter pristane-stimulated old Eμ-Cyclin D1 transgenic mice developed CD19+IgM+CD5+CD23 MCL.
      • Smith M.R.
      • Joshi I.
      • Jin F.
      • Al-Saleem T.
      Murine model for mantle cell lymphoma.
      In this model, the initiating event of deregulated cyclin D1 expression is brought by the Eμ-cyclin D1 transgene. Mice must then undergo changes associated with aging that may sensitize B cells to the effect of pristane that acts as a non-specific mitogenic stimulus or immune stimulant. Interleukin 14 α (IL-14α)/Myc double transgenic mice developed CD19+IgM+CD5+CD23 lymphoma reproducing many features of blastoid variant of MCL (including high expression of Myc and of the B cell growth factor IL-14α).
      • Ford R.J.
      • Shen L.
      • Lin-Lee Y.C.
      • Pham L.V.
      • Multani A.
      • Zhou H.J.
      • Tamayo A.T.
      • Zhang C.
      • Hawthorn L.
      • Cowell J.K.
      • Ambrus Jr, J.L.
      Development of a murine model for blastoid variant mantle-cell lymphoma.
      To improve our knowledge concerning the potential role of Cdk4 in the development of B cell lymphoma, we addressed whether the lack of interaction between Cdk4 and the INK4 family inhibitors impedes B-cell maturation and functions. The Cdk4-Arg24Cys (R24C) mutation abolishes the ability of all four INK4 members to bind Cdk4.
      • Wölfel T.
      • Hauer M.
      • Schneider J.
      • Serrano M.
      • Wölfel C.
      • Klehmann-Hieb E.
      • De Plaen E.
      • Hankeln T.
      • Meyer zum Büschenfelde K.H.
      • Beach D.
      A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma.
      Knock-in mice carrying the Cdk4R24C protein developed a wide spectrum of tumors, mainly sarcomas, carcinomas, and endocrine tumors, but surprisingly, no or rare B-cell lymphoma.
      • Rane S.G.
      • Cosenza S.C.
      • Mettus R.V.
      • Reddy E.P.
      Germ line transmission of the Cdk4R24C mutation facilitates tumorigenesis and escape from cellular senescence.
      • Sotillo R.
      • Dubus P.
      • Martin J.
      • de la Cueva E.
      • Ortega S.
      • Malumbres M.
      • Barbacid M.
      Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.
      In the present study, we report that the germline Cdk4R24C mutation had no effect on normal B-cell development, maturation, proliferation, and function and did not predispose transgenic animals to the development of B-cell malignancies. However, Cdk4 is also a well-established target of Myc.
      • Hermeking H.
      • Rago C.
      • Schuhmacher M.
      • Li Q.
      • Barrett J.F.
      • Obaya A.J.
      • O'Connell B.C.
      • Mateyak M.K.
      • Tam W.
      • Kohlhuber F.
      • Dang C.V.
      • Sedivy J.M.
      • Eick D.
      • Vogelstein B.
      • Kinzler K.W.
      Identification of CDK4 as a target of c-myc.
      Therefore, we investigated whether disruption of Cdk4 regulation by INK4 can constitute a second hit when Myc is overexpressed by crossing Cdk4R24C mice with Myc-3′RR transgenic mice susceptible to develop Burkitt lymphoma (BL)-like lymphomas.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      • Vincent C.
      • Truffinet V.
      • Fiancette R.
      • Petit B.
      • Cogné N.
      • Cogné M.
      • Denizot Y.
      Uncoupling between Ig somatic hypermutation and oncogene mutation in mouse lymphoma.
      • Vincent-Fabert C.
      • Fiancette R.
      • Cogné M.
      • Pinaud E.
      • Denizot Y.
      The IgH 3′ regulatory region and its implication in lymphomagenesis.
      We describe here the development of lymphoid malignancies closely resembling human MCL in Myc/Cdk4R24C mice. The typical hallmark of these lymphomas is the presence of high amounts of Cdk4R24C/cyclin D1 complexes related to their lack of inhibition by p16INK4a and p15INK4b. These data point to Cdk4 as a critical effector of Myc-driven proliferation in B cells and place the INK4 inhibitors as critical modulators of lymphomagenesis. These findings might be of critical interest when considering Cdk4 and/or INK4 inhibitors as potential therapeutic targets.

      Materials and Methods

      Generation of Transgenic Mice

      The study was approved by CNRS and University review committee. The genetically engineered knockin Cdk4R24C mice were generated in a mixed 129/Sv × CD-1 background.
      • Rane S.G.
      • Dubus P.
      • Mettus R.V.
      • Galbreath E.J.
      • Boden G.
      • Reddy E.P.
      • Barbacid M.
      Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in β-islet cell hyperplasia.
      They were crossed during five generations with C57/BL6 mice to obtain the Cdk4R24C mutation in a C57/BL6 background. These Cdk4R24C mice were then crossed with mice carrying a Myc transgene driven by the IgH 3′ regulatory region (Myc-3′RR in a C57/BL6 background)
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      to obtain Myc/Cdk4R24C mice. According to the French law for animal experimentations, mice were sacrificed after the first sign of illness: enlarged lymph nodes or elevated white blood cell (WBC) counts. For WBC counts, a value exhibiting a threefold increase as compared to control was arbitrary chosen as the cutoff to sacrifice mice.

      Southern Blot Analysis

      Genomic DNA was extracted, digested, loaded on agarose gel, transferred onto nylon sheets, hybridized with a 32P-labeled hs4 probe, and autoradiographed to ensure the presence of the Myc-3′RR transgene.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      Genomic DNA digested with EcoRI was analyzed with a 32P-labeled JH4 probe to test lymphoma clonality.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.

      PCR

      PCR experiments for Cdk4R24C mice were performed with specific forward 5′-TAGGTAACACAAAGACTCCC-3′ and reverse 5′-TTTTGGAGAGTGAAGTGCAG-3′ primers. Expected sizes of amplified products were 180 bp and 280 bp for wild-type (wt) and mutated alleles, respectively.

      mRNA Quantification

      RNA was extracted using tri-reagent (Ambion, Austin, TX). Real-time PCR was performed by using TaqMan assay reagents and analyzed on an ABI Prism 7000 system (Applied Biosystems, Foster City, CA). Assay references were Mm00500563-m1 (MSH2); Mm00487769-g1 (MSH6); Mm00449156-m1 (UNG); Mm00453168-m1 (POLη); Mm00450983-m1 (Rev1); Mm01184115-m1 (AID); Mm00487803-m1 (Myc); Mm0043259-m1 (cyclin D1); Mm00477631-m1 (bcl2) Mm00437763-m1 (bcl2l1); Mm00432054-m1 (bcl2l2); Mm00478988-m1 (bcl2l10); Mn00432042-m1 (bad); Mm00437796-m1 (bcl2l11); Mm00432073-m1 (Bid); Mm00433237-m1 (Fas); Mm00438864-m1 (FasL); Mm00432050-m1 (Bax); Mm00432045-m1 (Bak1); Mm00771682-g1 (S100a6); Mm00433970-m1 (HOXB6); Mn00660916-m1 (TBCLD9); and Mm99999915-g1 (GAPDH for normalization of gene expression levels).

      Flow Cytometry Analysis

      Cells were labeled with various antibodies (Southern Biotechnologies, Birmingham, AL) and analyzed on a Coulter XL apparatus (Beckman Coulter, Fullerton, CA). For intracellular labeling, cells were fixed and permeabilized with the Intraprep permeabilization reagent (Beckman Coulter) before incubation with fluorescein isothiocyanate–labeled anti-Ki-67 or irrelevant antibodies (Becton Dickinson).

      Proliferation Assay

      Splenic B cells (2 × 105 cells/well) were cultured (in sixtuplicate) in 96-well plates, either alone or in the presence of lipopolysaccharide (LPS) from Salmonella typhimurium (Sigma, L'Isle d'Abeau Chenes, France) or anti-CD40 (R&D systems, Lille, France) for 72 hours. Cell number was assessed using the CellTiter 96 One Solution Cell Proliferation assay (Promega Corporation, Madison, WI).

      Short Hairpin RNA Transfection

      Splenocytes from Myc/Cdk4R24C mice were transfected with Cdk2 (reference sc-29260-SH), Cdk4 (reference sc-29262-SH), Cdk6 (reference sc-35048-SH), or control short hairpin RNA (shRNA) plasmid (reference sc-108066) according to the manufacturer's recommendations (Santa Cruz Biotechnology, Santa Cruz, CA). Cells (2 × 105 cells/mL) were then cultured in 96-well plates alone or in the presence of 10 μg/mL of LPS for 3 days. The cell number was assessed as described above.

      Cell Cycle Analysis

      Splenocytes from Cdk4R24C mice and Myc/Cdk4R24C mice were grown for 72 hours with 10 μg/mL LPS. Cells were stained with anti-B220-V450–labeled antibodies and propidium iodide. B-cell DNA content was determined by flow cytometry.

      Apoptosis Assay

      After 24 to 72 hours of growth, splenocytes were incubated with 7-amino-actinomycin D and fluorescein isothiocyanate–labeled Annexin V antibodies (Becton Dickinson), and analyzed by flow cytometry.

      Spleen Cell Cultures for Ig Production

      Splenocytes were cultured and stimulated as described.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      Supernatants were stored until Ig evaluations. In vitro–stimulated splenocytes were stained with antibodies (anti–B220-PC5 and anti-Ig subclasses conjugated with fluorescein isothiocyanate) and analyzed by flow cytometry.

      Blood Sampling

      Blood samples were recovered from transgenic mice and wt controls with heparinized needles.

      ELISA

      Culture supernatants and sera from Cdk4R24C mice and wt mice were analyzed for the presence of the various immunoglobulin classes by enzyme-linked immunosorbent assay.
      • Vincent-Fabert C.
      • Truffinet V.
      • Fiancette R.
      • Cogné N.
      • Cogné M.
      • Denizot Y.
      Ig synthesis and class switching do not require the presence of the hs4 enhancer in the 3′ IgH regulatory region.

      Microarray Analysis

      mRNA was extracted from splenic B cells or lymphomas purified with CD19-coupled beads from Miltenyi Biotech (Bergisch Gladbach, Germany). Microarray experiments were done in “Nice–Sophia Antipolis Microarray Facility” (France).
      • Le Brigand K.
      • Barbry P.
      MEDIANTE: a web-based microarray data manager.
      • Le Brigand K.
      • Russell R.
      • Moreilhon C.
      • Rouillard J.M.
      • Jost B.
      • Amiot F.
      • Magnone V.
      • Bole-Feysot C.
      • Rostagno P.
      • Virolle V.
      • Defamie V.
      • Dessen P.
      • Williams G.
      • Lyons P.
      • Rios G.
      • Mari B.
      • Gulari E.
      • Kastner P.
      • Gidrol X.
      • Freeman T.C.
      • Barbry P.
      An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.
      Statistical analysis was made with the Bioconductor open-source software, particularly its Limma package.
      • Moreilhon C.
      • Gras D.
      • Hologne C.
      • Bajolet O.
      • Cottrez F.
      • Magnone V.
      • Merten M.
      • Groux H.
      • Puchelle E.
      • Barbry P.
      Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.

      Amplification for VH Somatic Hypermutation Experiments

      Genomic DNA lymphomas was amplified using the following primers: forward primer VHJ558 5′-GCGAAGCTTARGCCTGGGRCTTCAGTGAAG-3′ complementary to the VHJ558 segment, and backward primer 5′-AGGCTCTGAGATCCCTAGACAG-3′ corresponding to a sequence in the JH4 segment. The PCR products were cloned into the pGEM-T Easy Vector System (Promega Corporation) and sequenced using an automated laser fluorescent ANA ABI-PRISM sequencer (Perkin-Elmer, Branchburg, NJ).

      Western Blot Analysis

      Cell lysates were analyzed by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking, membranes were incubated with rabbit anti-Cdk2, anti-Cdk4, anti-Cdk6, anti-cyclin D1, anti-cyclin D2, anti-cyclin D3, anti-Cyclin E, and anti-GAPDH antibodies (Santa Cruz Biotechnology), and revealed with horseradish peroxidase–labeled goat anti-rabbit Ig (Bio-Rad, Hercules, CA) by chemoluminescence (ECL plus, Amersham, Little Chalfont, UK).

      Cyclin D1 Isoforms

      Cyclin D1a and cyclin D1b transcripts were investigated as previously reported,
      • Wu J.
      • Wu S.H.
      • Bollig A.
      • Thakur A.
      • Liao D.J.
      Identification of the cyclin D1b mRNA variant in mouse.
      using the following primers: cyclin D1 forward, 5′-CACGATTTCATCGAACA CTT-3′; cyclin D1a reverse, 5′-CCACTTCCCCCTCCTCCTCA-3′; and cyclin D1b reverse, 5′-AGATATGGTCCTATGCTGGCTG-3′reverse. Amplification of β-actin was used as control (forward primer 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and reverse primer 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′).

      Immunohistochemistry

      Morphological analysis was realized on 5-mm-thick sections stained with hematin, eosin and safranin. Cyclin D1 protein was detected with a rabbit polyclonal IgG antibody (H-295; Santa Cruz Biotechnology).

      Results

      B-Cell Lymphopoiesis in Cdk4R24C Mice

      Spleen, bone marrow, liver, gastrointestinal tract, and lungs were normal in Cdk4R24C mice as compared with wt mice over a period of 15 months (data not shown). The Cdk4R24C mutation had no significant impact on B-cell maturation and function. Numbers of B cells in bone marrow, spleen, and blood were normal (Table 1). Their proliferation in response to LPS or anti-CD40 and their apoptosis rate were similar to those of B cells of control mice (see Supplemental Figure S1 at http://ajp.amjpathol.org). Finally their in vitro and in vivo ability to secrete all of the various Ig isotypes were normal, and their ability to switch in response to cytokine was not affected (Table 2).
      Table 1B-Cell Phenotyping in Cdk4R24C Mice
      wtCdk4R24C mice
      Bone marrow
       WCC (×106)8.9 ± 1.88.4 ± 3.1
       % B220+ cells15.3 ± 1.516.4 ± 1.0
       % CD19+ cells among B220+cells67.7 ± 8.073.1 ± 4.2
       % CD117+ cells among B220+cells2.2 ± 0.61.3 ± 0.1
       % CD25+ cells among B220+cells16.9 ± 0.118.1 ± 3.7
       % IgM+IgDcells among B220+cells14.0 ± 0.817.7 ± 2.1
       % CD11b+ cells68.7 ± 3.365.1 ± 3.4
       % CD4+ cells2.5 ± 0.32.3 ± 1.0
       % CD8+ cells1.4 ± 0.12.3 ± 0.1
      Spleen
       WCC (x106 per organ)8.9 ± 1.88.4 ± 3.1
       % B220+ cells29.4 ± 4.624.8 ± 8.8
       % CD19+ cells among B220+cells80.7 ± 4.480.3 ± 6.4
       % IgM+ cells among B220+ cells85.2 ± 4.084.5 ± 5.2
       % IgD+ cells among B220+ cells81.4 ± 5.471.1 ± 7.8
       % CD11b+ cells4.4 ± 0.86.4 ± 3.8
       % CD4+ cells25.5 ± 5.521.6 ± 4.6
       % CD8+ cells13.4 ± 3.07.88 ± 1.4
      Blood
       WCC (×106 per mL)11.8 ± 2.010.8 ± 1.7
       % B220+ cells23.9 ± 4.316.3 ± 3.5
       % IgM+ cells among B220+ cells87.0 ± 1.686.2 ± 1.8
       % IgD+ cells among B220+ cells89.4 ± 1.091.2 ± 1.2
      Table 2Ig Production from B Cells of Cdk4R24C Mice and wt Controls
      Ig isotypeCdk4R24C micewt miceSignificance
      Plasma Ig levelsIgM191.6 ± 38.2358.3 ± 71.8P = 0.06
      IgG11700.0 ± 77.41333.3 ± 234.7P = 0.33
      IgG2b1900.0 ± 73.02333.3 ± 197.7P = 0.06
      IgG3262.5 ± 67.5492.0 ± 135.4P = 0.87
      IgE60.6 ± 4.568.8 ± 6.5P = 0.22
      IgA2133.3 ± 348.92716.6 ± 462.1P = 0.19
      Surface Ig expressionIgG15.8 ± 1.3%9.9 ± 3.5%P = 0.52
      IgG2b11.3 ± 6.0%7.0 ± 3.1%P = 0.36
      IgG32.5 ± 1.1%1.2 ± 0.14%P = 0.1
      IgA2.0 ± 0.2%2.8 ± 0.5%P = 0.26
      Ig secretionIgM2713.0 ± 370.01800.0 ± 643.0P = 0.27
      IgG169.0 ± 7.330.6 ± 11.3P = 0.06
      IgG2b18.0 ± 3.512.0 ± 2.3P = 0.27
      IgG312.6 ± 4.36.3 ± 1.8P = 0.18
      IgA5.6 ± 0.67.3 ± 0.3P = 0.09

      B-Cell Proliferation in Myc/Cdk4R24C Mice

      The occurrence of lymphoma was very rare in Cdk4R24C mice.
      • Rane S.G.
      • Cosenza S.C.
      • Mettus R.V.
      • Reddy E.P.
      Germ line transmission of the Cdk4R24C mutation facilitates tumorigenesis and escape from cellular senescence.
      • Sotillo R.
      • Dubus P.
      • Martin J.
      • de la Cueva E.
      • Ortega S.
      • Malumbres M.
      • Barbacid M.
      Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.
      Since Cdk4 is a well-known partner of Myc,
      • Hermeking H.
      • Rago C.
      • Schuhmacher M.
      • Li Q.
      • Barrett J.F.
      • Obaya A.J.
      • O'Connell B.C.
      • Mateyak M.K.
      • Tam W.
      • Kohlhuber F.
      • Dang C.V.
      • Sedivy J.M.
      • Eick D.
      • Vogelstein B.
      • Kinzler K.W.
      Identification of CDK4 as a target of c-myc.
      • Menssen A.
      • Hermeking H.
      Characterization of the c-myc-regulated transcriptome by SAGE: identification and analysis of c-myc target genes.
      we evaluated whether Myc could cooperated in lymphomagenesis in Cdk4R24C mice. We crossed Myc-3′RR transgenic mice (reported to develop BL-like lymphoma by 6 to 12 months of age)
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      with Cdk4R24C mice to obtain Myc/Cdk4R24C mice. The impact of the Cdk4R24C mutation was firstly investigated on Myc transcript expression. As shown in Figure 1A, splenic B cells from young (12-week-old) Cdk4R24C mice had higher (P < 0.05, Mann-Whitney U-test) amounts of Myc transcripts than wt ones. These Myc transcripts were of the order of magnitude of those found in young Myc-3′RR transgenic mice. B splenocytes from Myc/Cdk4R24C exhibited higher Myc transcript levels than Cdk4R24C and Myc-3′RR ones. In Myc-3′RR transgenic mice, overexpression of Myc enforces B splenocyte S-phase entry on activation.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      We further assessed the proliferating fraction of B cells using Ki-67 immunofluorescence staining after addition of LPS in Myc/Cdk4R24C and Cdk4R24C B cells. The percentage of Ki-67+B220+ cells was markedly increased after 24 hours of stimulation in Myc/Cdk4R24C B cells (Figure 1B) showing elevated cell cycle entry capacity. To determine whether Myc/Cdk4R24C B cells exhibited increased proliferation potential following mitogen stimulation, Myc/Cdk4R24C splenic B cells were stimulated for 72 hours with LPS or anti-CD40. Myc/Cdk4R24C B cells did not display a significant increase of growth (assessed by the MTS assay) as compared to Cdk4R24C B cells (Figure 1C). One potential explanation was that the promiscuous S-phase entry triggered by Myc expression was countered by a high rate of apoptosis. Consistent with this notion, we noted increased accumulation of apoptotic cells (<2N) in Myc/Cdk4R24C B cells as compared with Cdk4R24C B cells (Figure 1D). This phenomenon has been previously reported in B cells expressing a constitutively nuclear cyclin D1.
      • Gladden A.B.
      • Woolery R.
      • Wasik M.A.
      • Diehl J.A.
      Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma.
      It might also be linked to the high amounts of Myc transcripts found in Myc/Cdk4R24C B cells, Myc being well known to increase apoptosis.
      Figure thumbnail gr1
      Figure 1Myc transcripts, cell cycle entry and growth in Myc/Cdk4R24C mice. A: Real-time PCR analysis of Myc transcripts in purified splenic B cells from wt, Cdk4R24C, Myc-3′RR, and Myc/Cdk4R24C mice (n = 5, all groups). Gene expression levels were normalized to GAPDH transcripts. Amounts of Myc transcripts were compared to sample with the lowest level of Myc (a wt mouse, which was arbitrary labeled 1). Differences were investigated by using the Mann-Whitney U-test. *P < 0.05 versus wt mice, P < 0.05 versus with Cdk4R24C mice. B: Splenocytes from wt, Cdk4R24C, Myc-3′RR, and Myc/Cdk4R24C mice were grown for 24 hours with (in gray) or without (in white) 10 μg/mL LPS. Left: Percentages of B220+Ki-67+ cells were assessed by flow cytometry. Mean ± SEM of four independent experiments in duplicate. *P < 0.05 versus unstimulated cells (Mann-Whitney U-test), P < 0.05. Right: Flow cytometry analysis of B220+Ki-67+ cells from LPS-stimulated splenocytes of Cdk4R24C mice and Myc/Cdk4R24C mice. Numbers indicated the percentage of Ki-67+ cells among B220+ B cells. One representative experiment of four is shown. C: Splenocytes from wt, Myc-3′RR, Cdk4R24C, and Myc/Cdk4R24C mice were grown for 72 hours with (gray) or without (white) 10 μg/mL LPS or 5 μg/mL anti-CD40 antibody. Proliferation was assessed using the MTT assay. Mean ± SEM of six independent experiments performed six times. *P < 0.001 as compared to unstimulated cells (Mann-Whitney U-test), P < 0.01. D: Splenocytes from Cdk4R24C mice and Myc/Cdk4R24C mice) were grown for 72 hours with 10 μg/mL LPS. Cells were then stained with anti–B220-V450–labeled antibodies and propidium iodide, and B-cell DNA content was determined by flow cytometry.

      Characteristics of the Malignancy in Myc/Cdk4R24C Mice

      Myc-3′RR transgenic mice develop with age BL-like lymphomas with invasion of spleen and bone marrow with leukemic cells; gastrointestinal tract and lungs remain unaffected. In soma cases, invasion of leukemic cells were also documented in liver.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      In Myc/Cdk4R24C mice, invasion of lymphoma cells were documented in spleen and bone marrow; liver, gastrointestinal tract, and lungs remain unaffected (data not shown). A histopathological image of the spleen of a Myc/Cdk4R24C lymphoma mouse and of a wt mouse is reported in Figure 2A. The life span of Myc/Cdk4R24C mice was similar to that of Myc/3′RR mice (Figure 3A), but animals did not develop clonal lymph node BL-like lymphoma as Myc-3′RR mice did.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      WBC counts in Cdk4R24C mice did not vary with time over a period of 1 year (data not shown) and were similar to those found in wt mice and Myc/Cdk4R24C mice at the age of 2 months (around 10 × 103 cells/μL). At the age of 4 months, Myc/Cdk4R24C mice began to exhibit elevated WBC counts (Figure 3B) and enlarged spleen (Figure 3C). WBC counts in Myc/Cdk4R24C lymphoma mice were lower (P < 0.05, Mann-Whitney U-test) than those found in Myc-3′RR/p53+/− lymphoma mice recently reported to develop at the age of 3 months a wide pattern of B-cell lymphomas.
      • Fiancette R.
      • Rouaud P.
      • Vincent-Fabert C.
      • Laffleur B.
      • Magnone V.
      • Cogné M.
      • Denizot Y.
      A p53 defect sensitizes various stages of B cell development to lymphomagenesis in mice carrying an IgH 3′ regulatory region-driven c-myc transgene.
      In Myc/Cdk4R24C lymphoma mice, blood smear stained with May-Grunwald Giemsa showed an increased number of circulating cells with uniform size and rather round shape. Nuclei were round with clumped chromatin. Cytoplasm was scant and basophilic (Figure 3D). Flow cytometry was used to characterize these cells. A CD19+B220+IgM+IgD+ population was found (Figure 4). As reported in Figure 5A, flow cytometry analysis indicated that B-cell lymphoma of Myc/Cdk4R24 mice expressed strong levels of surface IgM as compared with their normal counterpart; human MCL tend to express surface Ig relatively strongly.
      • Molot R.J.
      • Meeker T.C.
      • Wittwer C.T.
      • Perkins S.L.
      • Segal G.H.
      • Masih A.S.
      • Braylan R.C.
      • Kjeldsberg C.R.
      Antigen expression and polymerase chain reaction amplification of mantle cell lymphomas.
      Staining was negative for CD4, CD8, CD21, CD25, CD93, CD117, CD138, and Thy1,2 (data not shown). The expanded B-cell population co-expressed CD43 and CD5, but not CD23 (Figure 4). This cell-surface labeling was classical to that found in human MCL; B-cell chronic lymphoid lymphoma (B-CLL) coexpress CD5, CD43, and CD23.
      • Parrens M.
      • Belaud-Rotureau M.A.
      • Fitoussi O.
      • Carerre N.
      • Bouabdallah K.
      • Marit G.
      • Dubus P.
      • de Mascarel A.
      • Merlio J.P.
      Blastoid and common variants of mantle cell lymphoma exhibit distinct immunophenotypic and interphase FISH features.
      • Garcia-Conde J.
      • Cabanillas F.
      Mantle cell lymphoma: a lymphoproliferative disorder associated with aberrant function of the cell cycle.
      This MCL-like phenotype markedly differed from the BL-like phenotype of Myc-3′RR mice (B220+CD19+IgM+IgD+CD43CD5CD23; Figure 4).
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      Analysis of bone marrow and spleen of Myc/Cdk4R24C animals revealed the presence of the same B220+IgM+IgD+CD19+CD5+CD43+CD23 population (data not shown). Leukemic cells with the same phenotype were also dramatically abundant in the peritoneal cavity of Myc/Cdk4R24C mice (29.5 ± 6.7 × 106 cells, n = 3) as compared with wt animals (0.5 ± 0.2 × 106 cells; n = 3). The use of a JH4 probe on Southern blot analyses of spleen extracts revealed rearranged bands in addition to the germline band, indicating that spleen was invaded by leukemic cells from a clonal origin (Figure 3E). The proliferation rate of these lymphoma cells was estimated by investigating the nuclear proliferation–associated Ag Ki-67, a nuclear protein present during G1, S, G2, and M phases of the cell cycle. About 25% to 35% of lymphoma cells from Myc/Cdk4R24C mice expressed the Ki-67 Ag (Figure 3F). In comparison, more than 70% of BL-like cells in Myc-3′RR were Ki-67+.
      Figure thumbnail gr2
      Figure 2histological tissue section and cyclin D1 immunostaining of spleen of Myc/Cdk4R24C lymphoma mice. A: Representative histological tissue section of spleen of wt (left) and Myc/Cdk4R24C lymphoma mice (right). Effacement of germinal centers replaced with lymphoma cells was evidenced in spleen of Myc/Cdk4R24C lymphoma mice. B: Immunostaining with antibodies against the cyclin D1 protein wt (left) MCL-like lymphoma mice (right). (Original magnification ×200.)
      Figure thumbnail gr3
      Figure 3Analysis of Myc/Cdk4R24C mice. A: Survival curves of 32 Myc/Cdk4R24C mice, 29 Myc-3′RR mice, and 20 Cdk4R24C animals. B: Number of circulating white blood cells (WBC) in Myc/Cdk4R24C mice at 2, 3, 4, and 5 months and Myc/p53+/− lymphoma mice
      • Fiancette R.
      • Rouaud P.
      • Vincent-Fabert C.
      • Laffleur B.
      • Magnone V.
      • Cogné M.
      • Denizot Y.
      A p53 defect sensitizes various stages of B cell development to lymphomagenesis in mice carrying an IgH 3′ regulatory region-driven c-myc transgene.
      at 3 months. The dotted line indicates the arbitrary cutoff chosen to sacrifice mice (a threefold increase as compared to control values). C: Spleen of Myc/Cdk4R24C mice and healthy mice. D: Blood smears stained with May-Grunwald Giemsa from Myc/Cdk4R24C leukemic mouse and healthy mouse. E: Southern blot analysis to examine IgH gene configuration with a JH4 probe. Genomic DNA was prepared and digested with EcoRI from spleen cells of Cdk4R24C mice (lanes C1, C2, and C3) and splenic cells of four Myc/Cdk4R24C leukemic mice (lanes T1, T2, T3, and T4). Dotted line indicates the location of the germline band. Asterisks indicate the location of recombined bands in tumor (T) samples. F: Analysis of B220+Ki-67+ cells in lymphoma from Myc/Cdk4R24C and Myc-3′RR mice. One representative experiment of four is shown.
      Figure thumbnail gr4
      Figure 4Flow cytometry analysis of leukemic cells in Myc/Cdk4R24C mice and Myc-3′RR mice. Myc/Cdk4R24C-derived lymphoma (left) and Myc-3′RR–derived lymphoma (middle) were labeled with various antibodies and analyzed by flow cytometry. Right panels show which markers were assessed: B220+IgM+ cells (A), B220+IgD+ cells (B), B220+CD19+ cells (C), B220+CD43+ cells (D), B220+CD5+ cells (E), B220+CD23+ cells (F). One representative experiment of 10 is shown.
      Figure thumbnail gr5
      Figure 5Expression of IgM, effect of Cdk shRNA depletion on growth and cyclin D1 subtype in MCL-like lymphoma of Myc/Cdk4R24C mice. A: Flow cytometry analysis of splenocytes from Myc/Cdk4R24C mice. Cells were labeled with anti–B220-PC5 and IgM-PE antibodies. Normal B cells and lymphoma B cells are indicated. B: Lymphoma cells from Myc/Cdk4R24C mice were transfected with Cdk2, Cdk4, or Cdk6 shRNA according to the manufacturer's recommendations. Cells (2 × 105 cells/mL) were cultured in replicates of six in 96-well plates with 10 μg/mL LPS for 3 days. Cell number was assessed using the CellTiter 96 One Solution Cell Proliferation assay. Results are reported as variation of growth as compared to control cells. Mean ± SEM of three experiments for Cdk2 and Cdk6 and two experiments for Cdk4. C: PCR analysis of cyclin D1 a and b subtypes in lymphoma of Myc/Cdk4R24C mice. Analysis of β actin transcripts were used as control. Results from eight lymphomas are reported. Lane 5: size marker.

      Analysis of Somatic Hypermutation in MCL-Like Lymphoma of Myc/Cdk4R24C Mice

      The rate of somatic hypermutation (SHM) in VH region in human MCL is low suggesting a weak influence of the germinal center microenvironment.
      • Jares P.
      • Campo E.
      Advances in the understanding of mantle cell lymphoma.
      We analyzed SHM in lymphomas of Myc/Cdk4R24C mice. Results from 5 tumors (63 sequences) are summarized in Supplemental Figure S2 (available at http://ajp.amjpathol.org). All were essentially unmutated, strengthening the hypothesis that mouse MCL-like lymphoma, similarly to human ones, derived from a naive pregerminal center B cell. Several enzymatic activity transcripts (including AID, UNG, MSH2, MSH6, Rev-1, and POLη) are implicated in SHM generation.
      • Neuberger M.S.
      • Rada C.
      Somatic hypermutation: activation-induced deaminase for C/G followed by polymerase η for A/T.
      In agreement with its unmutated VH status, low AID transcripts were detected in Myc/Cdk4R24C-derived MCL-like lymphoma, whereas no differences were found for MSH2, MSH6, UNG, POLη, and Rev1 transcripts (see Supplemental Figure S2, available at http://ajp.amjpathol.org).

      Gene Expression Profile of Myc/Cdk4R24C-Derived Lymphomas

      The gene expression profile of lymphomas in Myc/Cdk4R24C mice was compared with normal B splenocytes using an array of 44,000 genes. The microarray data presented in this article have been submitted to the Gene Expression Omnibus database under the accession number GSE32230. Among these, 533 genes significantly differed (147 up-regulated and 386 down-regulated, as shown in Supplemental Table S1, available at http://ajp.amjpathol.org). Lymphomas showed differential expression of multiple genes involved in cell cycle regulation, cell growth, apoptosis, and differentiation. Noticeably, lymphomas demonstrated a selective up-regulation of several genes previously found to be up-regulated in various leukemias or tumors, such as Itgb1, Il18, Hoxb6, Thbs3, Nrp2, Rab3b, Ass1, Tagln2, Pvrl2, Ltbp4, and Tns1. Some genes reported as tumor inhibitors were down-regulated such as Gpr171, Gadd45g, and Tsc22d1. As previously shown in human MCL,
      • Rummel M.J.
      • de Vos S.
      • Hoelzer D.
      • Koeffler H.P.
      • Hofmann W.K.
      Altered apoptotic pathways in mantle cell lymphoma.
      the apoptotic status of Myc/Cdk4R24C lymphomas was reduced by the down-regulation of the key pro-apoptotic gene Fas. Quantitative real-time PCR analysis confirmed the down-regulation (P < 0.05, Mann-Whitney U-test) of Fas transcripts in MCL-like lymphomas (relative expression of 2.3 ± 0.45; mean ± SEM of eight lymphomas) as compared to normal B splenocytes (relative expression of 5.21 ± 0.65; five mice). As previously shown in human MCL,
      • Mûllauer L.
      • Mosberger I.
      • Chott A.
      Fas ligand expression in nodal non-Hodgkin's lymphoma.
      quantitative real-time PCR analysis documented up-regulation (P < 0.05) of FasL (CD178) transcripts in MCL-like lymphomas of Myc/Cdk4R24C mice (relative expression of 14.3 ± 8.2; eight lymphomas) as compared to normal B splenocytes (relative expression of 1.3 ± 0.3; six mice). The apoptotic status of Myc/Cdk4R24C lymphomas is also reduced by the up-regulation of several anti-apoptotic genes such as Tmeff1, Smyd2, Twsg1, and Itgb1 (see Supplemental Table S1, available at http://ajp.amjpathol.org). By contrast, gene expression profiles did not indicate differences for members of the bcl-2 family (data not shown). Quantitative real-time PCR analysis confirmed no significant change of transcripts for several pro-apoptotic genes (such as bad, bid, bax, and bak1) and anti-apoptotic genes (such as bcl2, bcl-xl, bcl2l2, bcl2l10, and bcl2l11) (data not shown).

      Comparison of Gene Expression Profile of Myc/Cdk4R24C- and Myc-3′RR–Derived Lymphomas

      The gene expression profile of MCL-like lymphoma of Myc/Cdk4R24C mice was compared with the one of BL-like lymphoma of Myc-3′RR mice using the same array of 44,000 genes. The microarray data presented in this article have been submitted to the Gene Expression Omnibus database under the accession numbers GSE32229 and GSE32230. The 23 most discriminated probes between them are reported in Supplemental Table S1 (available at http://ajp.amjpathol.org). A few numbers of genes would be of interest, noticeably S100A6, HOXB6, and TBC1D9. The S100A6 (S100 calcium protein A6) protein involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation plays an important role in MLL-AF4–associated leukemogenesis.
      • Yamaguchi H.
      • Hanawa H.
      • Uchida N.
      • Inamai M.
      • Sawaguchi K.
      • Mitamura Y.
      • Shimada T.
      • Dan K.
      • Inokuchi K.
      Multistep pathogenesis of leukaemia via the MLL-AF4 chimeric gene/Flt3 gene tyrosine kinase domain (TDK) mutation-related enhancement of S100A6 expression.
      HOXB6 encodes a protein with a homeobox DNA-binding domain; its mRNA expression was exclusively detected in leukemic B cells, but not in normal B-cell progenitors, establishing a new HOXB6 gene expression during transformation.
      • Starkova J.
      • Zamostna B.
      • Mejstrikova E.
      • Krejci R.
      • Drabkin H.A.
      • Trka J.
      Hox gene expression in phenotypic and genotypic subgroups and low HOXA gene expression as an adverse prognostic factor in pediatric ALL.
      TBC1D9 (TBC1 domain family member 9) is a gene with an unknown function found to be overexpressed in human MCL as compared to reactive lymphoid tissue.
      • Islam T.C.
      • Asplund A.C.
      • Lindvall J.M.
      • Nygren L.
      • Liden J.
      • Kimby E.
      • Christensson B.
      • Smith C.I.
      • Sande B.
      High level of cannabinoid receptor 1, absence of regulator of G protein signalling 13 and differential expression of cyclin D1 in mantle cell lymphoma.
      Quantitative real-time PCR analysis confirmed the up-regulation (P < 0.05, Mann-Whitney U-test) of S100A6 transcripts in MCL-like lymphomas (relative expression of 42.6 ± 13.0; mean ± SEM of eight lymphomas) as compared to BL-like lymphomas (relative expression of 15.8 ± 6.1; eight lymphomas), the up-regulation of HOXB6 transcripts in MCL-like lymphomas (relative expression of 10.4 ± 2.1) as compared to BL-like lymphomas (relative expression of 4.1 ± 0.9) and the up-regulation of TBC19D transcripts in MCL-like lymphomas (relative expression of 16.1 ± 4.2) as compared to BL-like lymphomas (relative expression of 5.5 ± 0.9).

      Comparison of Gene Expression Profile of MCL-Like Lymphoma of Myc/Cdk4R24C Mice with Human MCL

      As described above, modifications of Fas/FasL, TBC1, and Myc in MCL-like lymphoma of Myc/Cdk4R24C mice were similar to that found in conventional or blastoid human MCL.
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      • Bea S.
      • Ribas M.
      • Hernandez J.M.
      • Bosch F.
      • Pinyol M.
      • Hernandez L.
      • Garcia J.L.
      • Flores T.
      • Conzalez M.
      • Lopez-Guillermo A.
      • Piris M.A.
      • Cardesa A.
      • Montserrat E.
      • Miro R.
      • Campo E.
      Increased number of chromosomal imbalances and high-level DNA amplifications in mantle cell lymphoma are associated with blastoid variants.
      • Hernandez L.
      • Hernandez S.
      • Bea S.
      • Pinyol M.
      • Ferrer A.
      • Bosch F.
      • Nadal A.
      • Fernandez P.L.
      • Palacin A.
      • Montserrat E.
      • Campo E.
      c-myc mRNA expression and genomic alterations in mantle cell lymphomas and other nodal non-Hodgkin's lymphomas.
      • Parrens M.
      • Belaud-Rotureau M.A.
      • Fitoussi O.
      • Carerre N.
      • Bouabdallah K.
      • Marit G.
      • Dubus P.
      • de Mascarel A.
      • Merlio J.P.
      Blastoid and common variants of mantle cell lymphoma exhibit distinct immunophenotypic and interphase FISH features.
      • Rummel M.J.
      • de Vos S.
      • Hoelzer D.
      • Koeffler H.P.
      • Hofmann W.K.
      Altered apoptotic pathways in mantle cell lymphoma.
      • Mûllauer L.
      • Mosberger I.
      • Chott A.
      Fas ligand expression in nodal non-Hodgkin's lymphoma.
      • Islam T.C.
      • Asplund A.C.
      • Lindvall J.M.
      • Nygren L.
      • Liden J.
      • Kimby E.
      • Christensson B.
      • Smith C.I.
      • Sande B.
      High level of cannabinoid receptor 1, absence of regulator of G protein signalling 13 and differential expression of cyclin D1 in mantle cell lymphoma.
      We then compared the gene expression profiling of MCL-like lymphoma of Myc/Cdk4R24C mice to those previously reported for human MCL either using microarray,
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      or gene imbalance.
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      Table 3
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      highlights the 37 genes found to be up-regulated (11 genes) or down-regulated (26 genes) both in MCL-like lymphoma of Myc/Cdk4R24C mice and human MCL.
      Table 3Comparison of Gene Expression Profile of MCL-Like Lymphoma of Myc/Cdk4R24C Mice and Human MCL
      GeneChange
      As compared with normal B cells.
      DescriptionReferences
      AHNAKIncreaseNucleoprotein
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      ,
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      CDC14BIncreaseProtein of cell division
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      ITGB7IncreaseIntegrin
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      TNFRSFIncreaseMember of tumor necrosis factor superfamily
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      OSBPIncreaseOxysterol binding protein
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      ANKRDIncreaseAnkyrin repeat domain
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      ZBTBIncreaseZing finger and BTB domain containing
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      IL18IncreaseInterleukin 18
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      LTBPIncreaseLatent transforming growth factor beta binding protein
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      TRIMIncreaseMember of the tripartite motif family
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      EVIIncreaseEctopic viral integration site
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      ENPPDecreaseEctonucleotide pyrophosphatase/phosphodiesterase
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      SLAMFDecreaseSignalling lymphocytic activation molecule family member
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      RGSDecreaseRegulator og G protein
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      IL21RDecreaseInterleukin 21 receptor
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      CR2DecreaseComplement component receptor 2
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      STAT4DecreaseSignal transducer and activator of transcription 4
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      CPMDecreaseCarboxypeptidase M
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      SPMY1DecreaseSprouty homolog 1
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      ICOSLDecreaseInducible T cell costimulator ligand
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      CCLDecreaseChemokine (C-C motif) ligand
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      LTADecreaseLymphotoxin alpha
      • Rizzatti E.G.
      • Falcao R.P.
      • Panepucci R.A.
      • Proto-Siqueira R.
      • Anselmo-Lima W.T.
      • Okamoto O.K.
      • Zago M.A.
      Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT. WNT and TGFβ signalling pathways.
      IL4RDecreaseInterleukin 4 receptor
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      CCR6DecreaseC-C motif chemokine receptor 6
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      CCNG2DecreaseCyclin G2
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      TGFβDecreaseTransforming growth factor beta
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      CD72DecreaseSignal transduction membrane protein
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      MERTKDecreasec-mer proto-oncogene tyrosine kinase
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      VAV1DecreaseVAV oncogene family
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      EGRDecreaseEarly growth response transcription factor
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      NFκβDecreaseNuclear factor of κ light polypeptide gene enhancer
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      SYNGRDecreaseMember of a family of transmembrane synaptic vesicle proteins
      • Ek S.
      • Högerkorp C.M.
      • Dictor M.
      • Ehinger M.
      • Borrebaeck C.A.K.
      Mantle cell lymphoma express a distinct gene signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells.
      TMEDDecreaseTransmembrane protein transport
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      FBXODecreaseF-box protein 5
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      TMEMDecreaseTransmembrane protein
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      GPRDecreaseG protein coupled receptor
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      GTSDecreaseCathepsin
      • Schraders M.
      • Jares P.
      • Bea S.
      • Schoenmakers E.F.P.M.
      • van Krieken H.J.M.
      • Campo E.
      • Groenen P.J.T.A.
      Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.
      low asterisk As compared with normal B cells.

      Accumulation of Cyclin D1/Cdk4 Complexes in Myc/Cdk4R24C Lymphomas

      D-type cyclins (D1, D2, and D3) are G1-specific cyclins that associate with Cdk4 or Cdk6, and promote restriction point progression during G1 phase.
      • Kim J.K.
      • Diehl J.A.
      Nuclear cyclin D1: an oncogenic driver in human cancer.
      Although cyclin D1 is most frequently implicated in human MCL, overexpression of cyclin D2 and cyclin D3 has also been reported in cyclin D1–negative human MCL.
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      Cyclin D1 overexpression per se is not sufficient to drive neoplastic growth. Mutations that interfere with nuclear exclusion of cyclin D1/Cdk4 complexes during S-phase trigger neoplastic conversion.
      • Kim J.K.
      • Diehl J.A.
      Nuclear cyclin D1: an oncogenic driver in human cancer.
      As already reported in human MCL,
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      Western blot analysis highlighted high amounts of Cdk4 in MCL-like lymphoma from Myc/Cdk4R24C mice (Figure 6). A similar increase was noted in BL-like lymphoma from Myc-3′RR mice. The major differences between MCL-like lymphoma of Myc/Cdk4R24C mice and BL-like lymphoma of Myc-3′RR mice related to high amounts of cyclin D1/Cdk4 complexes in MCL-like lymphoma, but not in BL-like lymphoma (Figure 6); similar levels of free cyclin D1 were found. We observed using immunohistochemistry that the most cyclin D1 protein was present in the cytoplasm of neoplastic cells (Figure 2B), a result that fit well with two recent studies reporting such a phenomenon in MCL cells.
      • Beltran E.
      • Fresquet V.
      • Martinez-Useros J.
      • Richter-Larrea J.A.
      • Sagardoy A.
      • Sesma I.
      • Almada L.L.
      • Montes-Moreno S.
      • Siebert R.
      • Gesk S.
      • Calasanz M.J.
      • Malumbres R.
      • Rieger M.
      • Prosper F.
      • Lossos I.S.
      • Piris M.A.
      • Fernadez-Zapico M.E.
      • Martinez-Climent J.A.
      A cyclin-D1 interaction with BAX underlies its oncogenic role and potential as a therapeutic target in mantle cell lymphoma.
      • Tchakarska G.
      • Roussel M.
      • Troussard X.
      • Sola B.
      Cyclin D1 inhibits mitochondrial activity in B cells.
      Whether similar levels of free cyclin D2 were noted in BL-like lymphoma and MCL-like lymphoma, cyclin D2/Cdk4 complexes were detected in MCL-like lymphoma of Myc/Cdk4R24 mice (Figure 6). In contrast to BL-like lymphoma of Myc-3′RR mice, high levels of cyclin D3 were detected in MCL-like lymphoma of Myc/Cdk4R24 mice (Figure 6). Taken altogether, these results suggest a role for Cdk4 in growth of MCL-like lymphoma cells. Confirming this, shRNA depletion of Cdk4 reduced the in vitro proliferation properties of MCL-like lymphoma cells of Myc/Cdk4R24C mice (Figure 5B). No significant levels of Cdk6 were detected, demonstrating that Cdk4 is the primary catalytic partner for cyclin D1, D2, and D3 in MCL-like lymphoma of Myc/Cdk4R24C mice (Figure 6). Confirming this, shRNA depletion of Cdk6 did not affect the proliferation properties of Myc/Cdk4R24C B cells (Figure 5B). Cyclin E/Cdk2 complexes have important roles in cell cycle progression, although selective Cdk2 inhibition is readily compensated. Cdk2 gain was reported to correlate with unfavorable outcome in human MCL,
      • Jardin F.
      • Picquenot J.M.
      • Parmentier F.
      • Ruminy P.
      • Cornic M.
      • Penther D.
      • Bertrand P.
      • Lanic H.
      • Cassuto O.
      • Humbrecht C.
      • Lemasle E.
      • Wautier A.
      • Bastard C.
      • Tilly H.
      Detection of gene copy number aberrations in mantled cell lymphoma by a single quantitative multiplex PCR assay: clinicopathological relevance and prognosis value.
      and expression of cyclin E was found in cyclin D1-negative MCL.
      • Wlodarska I.
      • Dierickx D.
      • Vanhentenrijk V.
      • van Roosbroeck K.
      • Pospisilova H.
      • Minnei F.
      • Verhoef G.
      • Thomas J.
      • Vandenberghe P.
      • De Wolf-Peeters C.
      Translocations targeting CCND2 CCND3, and MYCN do occur in t(11;14)-negative mantle cell lymphoma.
      As shown in Figure 6, Cdk2 levels were higher in MCL-like lymphoma of Myc/Cdk4R24C than in BL-like lymphoma of Myc-3′RR mice. In agreement, higher levels of cyclin E were found in Myc/Cdk4R24C-derived MCL-like lymphoma. However, no cyclin E/Cdk2 complexes could be documented (Figure 6). shRNA depletion of Cdk2 did not affect the proliferation properties of Myc/Cdk4R24C B cells (Figure 5B), suggesting that if Cdk2 is implicated in B-cell proliferation, its inhibition is readily compensated.
      Figure thumbnail gr6
      Figure 6Cdks and cyclins in Myc/Cdk4R24C and Myc-3′RR lymphomas. Cell lysates were prepared from lymph node MCL-like lymphoma of Myc/Cdk4R24C mice and BL-like lymphoma of Myc-3′RR mice for Western blot analysis. Purified splenic CD19+ cells from wt and Cdk4R24C mice were used as controls. GAPDH serves as an internal loading control. Cdk, cyclins, and their complexes were investigated: Cdk6, Cdk4 (complexed and free), cyclin D1 (complexed and free), cyclin D2 (complexed and free), cyclin D3, Cdk2 (complexed and free), cyclin E. Two representative samples of five are reported.

      Cyclin D1 Isoform and Mutational Status in Myc/Cdk4R24 MCL-Like Lymphomas

      Point mutations in the cyclin D1 gene have been reported to affect its activity.
      • Kim J.K.
      • Diehl J.A.
      Nuclear cyclin D1: an oncogenic driver in human cancer.
      For example, the G/A polymorphism occurring at nucleotide position 870 generates a splice variant of cyclin D1 (cyclin D1b), rendering the Cdk4/cyclin D1b complexes refractory to nuclear export through the cell cycle, thus increasing its oncogenic potency.
      • Kim J.K.
      • Diehl J.A.
      Nuclear cyclin D1: an oncogenic driver in human cancer.
      Cloning and sequencing of cyclin D1 transcripts from six Myc/Cdk4R24C lymphomas highlighted the wt form of the cyclin D1 gene (data not shown). Real-time PCR analysis did not evidence higher cyclin D1 mRNA levels in Myc/Cdk4R24C lymphoma mice as compared to Myc-3′RR ones (data not shown). Taken together, alterations in cyclin D1/Cdk4 complex turnover can lead to aberrant accumulation of nuclear cyclin D1, independent of changes in cyclin D1 transcription and translation. These results demonstrate that stabilization of cyclin D1 (but also D2 and D3) through their altered nuclear trafficking (via the Cdk4R24C mutation) might be the mechanism leading to the generation of MCL-like lymphoma in Myc/Cdk4R24C mice. Finally, two isoforms of cyclin D1, designed a and b, have been described.
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      As shown in Figure 5C, only cyclin D1a transcripts were documented in MCL-like lymphoma of Myc/Cdk4R24C mice. Of interest, cyclin D1b had no cell cycle regulatory properties in B lymphocytes,
      • Lévêque C.
      • Marsaud V.
      • Renoir J.M.
      • Sola B.
      Alternative cyclin D1 forms a and b have different biological functions in the cell cycle of B lymphocytes.
      and human MCL expressed only the cyclin D1a isoform.
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.

      Discussion

      Cdk4 is the chief catalytic subunit of the cyclin D family of proteins that governs G1/S-phase progression.
      • Ruas M.
      • Peters G.
      The p16INK4a/CDKN2A tumor suppressor and its relatives.
      Mice devoid of Cdk4 are viable, but with ageing, develop diabetes mellitus due to a specific defect in pancreatic β-cell proliferation.
      • Rane S.G.
      • Dubus P.
      • Mettus R.V.
      • Galbreath E.J.
      • Boden G.
      • Reddy E.P.
      • Barbacid M.
      Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in β-islet cell hyperplasia.
      • Martin J.
      • Hunt S.L.
      • Dubus P.
      • Sotillo R.
      • Néhmé-Pélluard F.
      • Magnuson M.A.
      • Parlow A.F.
      • Malumbres M.
      • Ortega S.
      • Barbacid M.
      Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number.
      Therefore, Cdk4 is not essential (due to its redundancies with Cdk6 and other Cdks) for G1-phase progression of most cell types although its absence delays entry into S phase.
      • Rane S.G.
      • Dubus P.
      • Mettus R.V.
      • Galbreath E.J.
      • Boden G.
      • Reddy E.P.
      • Barbacid M.
      Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in β-islet cell hyperplasia.
      • Santamaria D.
      • Barrière C.
      • Cerqueira A.
      • Hunt S.
      • Tardy C.
      • Newton K.
      • Caceres J.F.
      • Dubus P.
      • Malumbres M.
      • Barbacid M.
      Cdk1 is sufficient to drive the mammalian cell cycle.
      To understand the role of Cdk4 in neoplastic development, Cdk4R24C knockin mice were engineered.
      • Rane S.G.
      • Dubus P.
      • Mettus R.V.
      • Galbreath E.J.
      • Boden G.
      • Reddy E.P.
      • Barbacid M.
      Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in β-islet cell hyperplasia.
      The mutation only abolishes the ability of Cdk4 to bind to all INK4 family members.
      • Wölfel T.
      • Hauer M.
      • Schneider J.
      • Serrano M.
      • Wölfel C.
      • Klehmann-Hieb E.
      • De Plaen E.
      • Hankeln T.
      • Meyer zum Büschenfelde K.H.
      • Beach D.
      A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma.
      The mutant Cdk4R24C gene may thus function as a dominant oncogene that governs G1/S-phase progression via phosphorylation-mediated inactivation of Rb proteins independently of INK4 negative regulation. The hyperphosphorylated state of the Rb family proteins can be regarded as a physiological inactivation of the Rb family protein functions. In our experiments, we investigated whether impaired cell cycle control attributable to mutant Cdk4R24C would affect B-cell growth and/or maturation and whether the Cdk4 R24C mutation cooperates with Myc in the generation of B-cell lymphoma in transgenic mice. MCL does not occur spontaneously in mice. Thus, the Myc/Cdk4R24C model can provide an interesting opportunity, not only to understand the development of MCL-like lymphoma in the murine lymphoid lineage, but also to understand the impact of the Cdk4R24C mutation in this lineage since the Cdk4R24C mutation, until now, has not been reported in human B-cell lymphomas.
      • Mori N.
      • Yang R.
      • Kawamata N.
      • Miller C.W.
      • Mizoguchi H.
      • Koeffler H.P.
      Absence of R24C mutation of the CDK4 gene in leukemias and solid tumors.
      How Cdk4 and Myc interact to produce this phenotype may provide important clues to the pathogenesis of human MCL.
      Results of the present study clearly show that activated Cdk4R24C had no evident effect by itself on B-cell growth and maturation. Cdk4R24C mice had normal B-cell compartments with normal growth capacities in response to B-cell stimuli such as LPS and anti-CD40. Plasma levels of circulating Ig were similar between Cdk4R24C and wt mice. In vitro class switching and membrane Ig expression in B cells submitted to LPS and/or cytokines were similar in Cdk4R24C mice and wt animals. Finally, the in vitro capacity to secrete Ig was unaffected by the Cdk4R24C mutation. Thus, the dominant mutant oncogene Cdk4R24C did not significantly affect B-cell homeostasis. Therefore, INK4 protein regulations through Cdk4 have no major in vivo function on B-cell proliferation and maturation in young and adult mice. This result is in agreement with previous studies highlighting low incidence of hematopoietic malignancies in Cdk4R24C mice.
      • Rane S.G.
      • Cosenza S.C.
      • Mettus R.V.
      • Reddy E.P.
      Germ line transmission of the Cdk4R24C mutation facilitates tumorigenesis and escape from cellular senescence.
      • Sotillo R.
      • Dubus P.
      • Martin J.
      • de la Cueva E.
      • Ortega S.
      • Malumbres M.
      • Barbacid M.
      Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.
      So far, no B-cell phenotype has been reported (either) in two different Cdk4 knockout mice.
      • Martin J.
      • Hunt S.L.
      • Dubus P.
      • Sotillo R.
      • Néhmé-Pélluard F.
      • Magnuson M.A.
      • Parlow A.F.
      • Malumbres M.
      • Ortega S.
      • Barbacid M.
      Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number.
      • Santamaria D.
      • Barrière C.
      • Cerqueira A.
      • Hunt S.
      • Tardy C.
      • Newton K.
      • Caceres J.F.
      • Dubus P.
      • Malumbres M.
      • Barbacid M.
      Cdk1 is sufficient to drive the mammalian cell cycle.
      • Tsutsui T.
      • Hesabi B.
      • Moons D.S.
      • Pandolfi P.P.
      • Hansel K.S.
      • Koff A.
      • Kiyokawa H.
      Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity.
      We did not perform specific studies on Cdk4-null B cells, because their spontaneous diabetic phenotype impairs in vivo basal-state evaluation of the B-cell compartment and may indirectly alters their immune response. Therefore, our model cannot rule out a possible role of Cdk4 in B cell growth and maturation. Cdk6, but not 4, has been reported to play a key role in cytokine-mediated B-cell proliferation
      • Wagner E.F.
      • Hleb M.
      • Hanna N.
      • Sharma S.
      A pivotal role of cyclin D3 and cyclin-dependent kinase inhibitor p27 in the regulation of IL-2-.IL-4-, or IL-10-mediated human B cell proliferation.
      and B-cell differentiation.
      • Schrantz N.
      • Beney G.E.
      • Auffredou M.T.
      • Bourgeade M.F.
      • Leca G.
      • Vasquez A.
      The expression of p18INK4 and p27kip1 cyclin-dependent kinase inhibitors is regulated differently during human B cell differentiation.
      Cdk2 was predominantly implicated in the proliferation of anti-IgM–stimulated mature B lymphocytes.
      • Solvason N.
      • Wu W.W.
      • Kabra N.
      • Wu W.
      • Lees E.
      • Howard M.C.
      Induction of cell cycle regulatory proteins in anti-immunoglobulin-stimulated mature B lymphoytes.
      Cdk4R24C mice expressed normal levels of Cdk6, the other target of INK4 inhibitors. Surprisingly, the lack of B-cell phenotype on Cdk4R24C mice also suggests that INK4 proteins able to bind and inhibit Cdk6 also have no direct effect on proper B-cell proliferation and differentiation.
      Cdk4R24C mice developed tumors in multiple organs such as liver, spleen, pituitary, pancreas, testis, and lungs only later in life.
      • Rane S.G.
      • Cosenza S.C.
      • Mettus R.V.
      • Reddy E.P.
      Germ line transmission of the Cdk4R24C mutation facilitates tumorigenesis and escape from cellular senescence.
      • Sotillo R.
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      Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.
      The low transforming potential of the Cdk4R24C mutation makes it an ideal tool in crossing experiments with other transgenic mice bearing potential cooperating oncogenes. For example, the mutant Cdk4R24C promotes rapid growth of carcinogen-induced melanoma in hepatocyte growth factor–overexpressing transgenic mice.
      • Tormo D.
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      Rapid growth of invasive metastatic melanoma in carcinogen-treated hepatocyte growth factor/scatter factor-transgenic mice carrying an oncogenic CDK4 mutation.
      The Myc oncogene encodes a transcriptional factor that can drive proliferation by promoting cell cycle entry.
      • Pelengaris S.
      • Khan M.
      • Evan G.
      c-MYC: more than just a matter of life and death.
      Cdk4 has been identified as a target of Myc,
      • Hermeking H.
      • Rago C.
      • Schuhmacher M.
      • Li Q.
      • Barrett J.F.
      • Obaya A.J.
      • O'Connell B.C.
      • Mateyak M.K.
      • Tam W.
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      • Dang C.V.
      • Sedivy J.M.
      • Eick D.
      • Vogelstein B.
      • Kinzler K.W.
      Identification of CDK4 as a target of c-myc.
      and Myc stimulates cyclin E/Cdk2 activity and S-phase entry by abrogating p27kip1 inhibition, in part by redistribution of this inhibitor to cyclin D/Cdk4 complexes.
      • Ausserlechner M.J.
      • Obexer P.
      • Geley S.
      • Kofler R.
      G1 arrest by p16INK4A uncouples growth from cell cycle progression in leukaemia cells with deregulated cyclin E and c-myc expression.
      In turn, lack of Cdk4 inhibited Myc tumorigenic activities as evidenced in various epithelial tumors induced by forced expression of Myc under the K5 promoter.
      • Miliani de Marval P.L.
      • Marcias E.
      • Rounbehler R.
      • Sicinski P.
      • Kiyokawa H.
      • Johnson D.G.
      • Conti C.J.
      • Rodriguez-Puebla M.L.
      Lack of cyclin-dependent kinase 4 inhibits c-myc tumorigenic activities in epithelial tissues.
      Myc/Cdk4R24C mice provide an interesting opportunity to understand the impact of the Cdk4R24C mutation in the lymphoid lineage in conjunction with a Myc overexpression.
      The main characteristics of Myc/Cdk4R24C lymphomas are the following: i) mice develop a systemic B lymphoma/leukemia with a IgM+CD5+CD43+CD23 phenotype; ii) the B-cell malignancy is clonal; iii) cells are not rapidly proliferating; iv) transcriptome analysis of Myc/Cdk4R24C-derived leukemic cells highlighted a lowered apoptotic expression pathway (ie, down-regulation of Fas transcripts) with no evident sign of a high proliferation signature; v) VH genes are essentially unmutated as for naive B cells; and vi) Myc/Cdk4R24C lymphomas overexpressed Myc and cyclin D1/Cdk4 complexes. Of interest, all these features are well-known characteristics of human blastoid MCL. Human blastoid MCL is a leucemic lymphoma with bone marrow infiltration and peripheral blood involvement. Human blastoid MCL derived from a normal CD5+ naive B lymphocyte and is characterized by a blockade of apoptosis and high incidence expression of Myc, Cdk4, cyclin D1, cyclin E, and inactivation of p16INK4a and p18INK4c.
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      • Jares P.
      • Campo E.
      Advances in the understanding of mantle cell lymphoma.
      • Hernandez L.
      • Hernandez S.
      • Bea S.
      • Pinyol M.
      • Ferrer A.
      • Bosch F.
      • Nadal A.
      • Fernandez P.L.
      • Palacin A.
      • Montserrat E.
      • Campo E.
      c-myc mRNA expression and genomic alterations in mantle cell lymphomas and other nodal non-Hodgkin's lymphomas.
      • Wlodarska I.
      • Dierickx D.
      • Vanhentenrijk V.
      • van Roosbroeck K.
      • Pospisilova H.
      • Minnei F.
      • Verhoef G.
      • Thomas J.
      • Vandenberghe P.
      • De Wolf-Peeters C.
      Translocations targeting CCND2 CCND3, and MYCN do occur in t(11;14)-negative mantle cell lymphoma.
      • Quintanilla-Martinez
      • Davies-Hill T.
      • Fend F.
      • Calzada-Wack
      • Sorbara L.
      • Campo E.
      • Jaffe E.S.
      • Raffeld M.
      Sequestration of p27kip1 protein by cyclin D1 in typical and blastic variants of mantle cell lymphoma (MCL): implication for pathogenesis.
      • Pinyol M.
      • Hernandez L.
      • Cazorla M.
      • Balbin M.
      • Jares P.
      • Fernandez P.L.
      • Montserrat E.
      • Cardesa A.
      • Lopez-Otin C.
      • Campo E.
      Deletions and loss of expression of p16INK4a and p21Warf1 genes are associated with aggressive variants of mantle cell lymphomas.
      • Quintanilla -Martinez L.
      • Slotta-Huspenina J.
      • Koch I.
      • Klier M.
      • Hsi E.D.
      • de Leval L.
      • Klapper W.
      • Gesk S.
      • Siebert R.
      • Fend F.
      Differential diagnosis of cyclin D2+ mantle cell lymphoma based on fluorescence in situ hybridization and quantitative real-time-PCR.
      All these features are found in Myc/Cdk4R24C lymphomas (INK4 deficiencies are partially mimicked with the Cdk4R24C mutation). About 25% to 35% of lymphoma cells from Myc/Cdk4R24C mice expressed the Ki-67. It may be low, especially when compared with the one found in BL-like lymphoma cells from Myc-3′RR mice.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      This difference is linked to the high proliferative status of BL in Myc-3′RR mice. Their transcriptome analysis highlighted wide alterations in numerous genes implicated in the proliferation processes.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      These alterations were not found in MCL-like lymphoma from Myc/Cdk4R24C mice. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of human B-cell lymphoma. DLBCL are immunophenotypically heterogeneous; about 10% of DLBCL express the CD5 antigen. Human CD5+ DLBCL are negative for cyclin D1, whereas human MCL are positive.
      • Matolcsy A.
      • Chadburn A.
      • Knowles D.M.
      De novo CD5-positive and Richter's syndrome-associated diffuse large B cell lymphomas are genotypically distinct.
      Human CD5+ DLBCL express Ig VH genes with somatic mutations that are different from MCL.
      • Taniguchi M.
      • Oka K.
      • Hiasa A.
      • Yamaguchi M.
      • Ohno T.
      • Kita K.
      • Shiku H.
      De novo CD5+ diffuse large B-cell lymphomas express VH genes with somatic mutation.
      Lymphomas from Myc/Cdk4R24C are cyclin D1 positive with unmutated VH genes and thus appear different from CD5+ DLBCL and more in accordance with a MCL-like phenotype.
      B cells from Myc-3′RR mice express higher amounts of Myc transcripts than wt mice.
      • Truffinet V.
      • Pinaud E.
      • Cogné N.
      • Petit B.
      • Guglielmi L.
      • Cogné M.
      • Denizot Y.
      The 3′ IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.
      B cells from Cdk4R24C mice express higher levels of Myc as compared with wt mice, and a synergistic Myc transcript expression is found in double Myc/Cdk4R24C B cells. We currently have no satisfactory explanation for this, but there is evidence that these elevated Myc transcripts might have implications for the phenotype of double transgenic mice. Firstly Myc is well known to stimulate Cdk4 expression,
      • Hermeking H.
      • Rago C.
      • Schuhmacher M.
      • Li Q.
      • Barrett J.F.
      • Obaya A.J.
      • O'Connell B.C.
      • Mateyak M.K.
      • Tam W.
      • Kohlhuber F.
      • Dang C.V.
      • Sedivy J.M.
      • Eick D.
      • Vogelstein B.
      • Kinzler K.W.
      Identification of CDK4 as a target of c-myc.
      and cyclin D1/Cdk4 complexes are upstream regulator of Myc.
      • Haas K.
      • Staller P.
      • Geisen C.
      • Bartek J.
      • Eilers M.
      • Möröy T.
      Mutual requirement of CDK4 and Myc in malignant transformation: evidence for cyclin D1/CDK4 and p16INK4A as upstream regulators of Myc.
      High Cdk4 expression and cyclin D1/Cdk4 complexes are found in MCL-like lymphoma in Myc/Cdk4R24C mice. Secondly, Myc is well known to stimulate apoptotic processes.
      • Pelengaris S.
      • Khan M.
      • Evan G.
      c-MYC: more than just a matter of life and death.
      Additional mutations on pathways down-regulating apoptotic events are required to achieve the oncogenic process. Such modifications are found both in MCL-like lymphoma in Myc/Cdk4R24C mice and in human MCL.
      Myc is well known to stimulate growth but also apoptotic processes. We found both elevated cell cycle entry and elevated apoptotic processes in B cells of young Myc/Cdk4R24C mice. Tumorigenesis is a multistep process in which several genetic lesions have to accumulate to produce a fully malignant phenotype. In numerous mature B-cell lymphomas, tumors to emerge need new hits, especially on genes regulating the apoptotic process (such as Fas, members of the Bcl2 family, or p53). Alteration of Fas/FasL pathways is found in MCL-like lymphoma from Myc/Cdk4R24C. Such differences concerning the apoptotic status of pre-malignant B cells versus B-cell lymphomas have been previously reported in Myc transgenic mice.
      • Wang J.
      • Boxer L.M.
      Regulatory elements in the immunoglobulin heavy chain gene 3′-enhancers induce c-myc deregulation and lymphomagenesis in murine B cells.
      Our data revealed a role for Cdk4-INK4 dysregulation in lymphomagenesis but only in the context of a Myc overexpression. Of major interest is that the malignancy that arose in double Myc/Cdk4R24C transgenic mice differed from those initiated by Myc alone, and resembled MCL, whereas those that arose in Myc mice resembled BL. Such a phenomenon has already been reported with Myc transgenic mice expressing constitutive or antigen-stimulated states of the B-cell receptor.
      • Refaeli Y.
      • Young R.M.
      • Turner B.C.
      • Duda J.
      • Field K.A.
      • Bishop J.M.
      The B cell antigen receptor and overexpression of MYC can cooperate in the genesis of B cell lymphomas.
      Thus, another hit (such as the Cdk4R24C mutation, which can partially mimic Cdk4 or cyclin D1 overexpression and/or INK4 deletion) can profoundly alter Myc-induced lymphomagenesis. MCL is strongly associated with the t(11,14) chromosomal translocation leading to overexpression of cyclin D1.
      • Marzec M.
      • Kasprzycka M.
      • Lai R.
      • Gladden A.B.
      • Wlodarski P.
      • Tomczak E.
      • Nowell P.
      • Deprimo S.E.
      • Sadis S.
      • Eck S.
      • Schuster S.J.
      • Diehl J.A.
      • Wasik M.A.
      Mantle cell lymphoma cells express predominantly cyclin D1 isoform and are higly sensitive to selective inhibition of CDK4 kinase activity.
      However, transgenic animals overexpressing cyclin D1 do not develop lymphomas.
      • Bodrug S.E.
      • Warner B.J.
      • Bath M.L.
      • Lindeman G.J.
      • Harris A.W.
      • Adams J.M.
      Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.
      • Lovec H.
      • Grzeschiczek A.
      • Kowalski M.B.
      • Möröy T.
      Cyclin D1/bcl-1 cooperates with myc genes in the generation of B-cell lymphoma in transgenic mice.
      • Fiancette R.
      • Amin R.
      • Truffinet V.
      • Vincent-Fabert C.
      • Cogné N.
      • Cogné M.
      • Denizot Y.
      A myeloma translocation-like model associating CCND1 with the immunoglobulin heavy-chain locus 3′ enhancers does not promote by itself B cell malignancies.
      In turn, expression of a constitutively nuclear cyclin D1 in murine lymphocytes was reported to generate IgM+ B-cell lymphoma showing that nuclear retention of cyclin D1 is oncogenic in vivo.
      • Gladden A.B.
      • Woolery R.
      • Wasik M.A.
      • Diehl J.A.
      Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma.
      Western blot analysis of Myc/Cdk4R24C MCL-like lymphoma reveals high amounts of Cdk4/cyclin D1 complexes, thus elevated nuclear retention of cyclin D1. Therefore, Myc/Cdk4R24C mice confirm that elevated nuclear retention of cyclin D1 is oncogenic in vivo and is the key step to drive lymphomagenesis toward the MCL subtype. The vast majority of human MCL have elevated cyclin D1 synthesis related to cyclin D1 gene translocation to the IgH locus. Such translocation is not mimicked in our experimental model. A few MCL are cyclin D1 negative but have elevated cyclin D2 or D3 levels. MCL-like lymphomas from Myc/Cdk4R24C mice have elevated cyclin D1/Cdk4 complexes. Despite that elevated levels of cyclin D2 and cyclin D3 were also documented, our MCL-like lymphomas support a model of cyclin D1–positive MCL. The cyclin D1 protein is normally not expressed in human B lymphocytes, despite that low levels of cyclin D1 transcript could be documented.
      • Gladkikh A.
      • Potashnikova D.
      • Korneva E.
      • Khudoleeva O.
      • Vorobjev I.
      Cyclin D1 expression in B-cell lymphomas.
      Elevated cyclin D1 transcript levels have been documented, not only in MCL (which exhibit the t(11;14) translocation), but also in numerous other types of B-cell lymphomas (without the t(11;14) translocation) such as follicular lymphoma, marginal zone lymphomas, DLBCL, and B-cell chronic lymphocytic leukemia.
      • Gladkikh A.
      • Potashnikova D.
      • Korneva E.
      • Khudoleeva O.
      • Vorobjev I.
      Cyclin D1 expression in B-cell lymphomas.
      Cyclin D1 elevation in the absence of t(11;14) translocation suggests more than one method of cyclin D1 up-regulation in malignant B cells such as stabilization of cyclin D1/Cdk4 complexes in our model. There is no positive correlation between cyclin D1 expression and unfavorable MCL outcome. Moderate cyclin D1 levels in blastoid MCL variants are often accompanied by elevated cyclin D2 or cyclin D3 levels.
      • Wlodarska I.
      • Dierickx D.
      • Vanhentenrijk V.
      • van Roosbroeck K.
      • Pospisilova H.
      • Minnei F.
      • Verhoef G.
      • Thomas J.
      • Vandenberghe P.
      • De Wolf-Peeters C.
      Translocations targeting CCND2 CCND3, and MYCN do occur in t(11;14)-negative mantle cell lymphoma.
      • Quintanilla -Martinez L.
      • Slotta-Huspenina J.
      • Koch I.
      • Klier M.
      • Hsi E.D.
      • de Leval L.
      • Klapper W.
      • Gesk S.
      • Siebert R.
      • Fend F.
      Differential diagnosis of cyclin D2+ mantle cell lymphoma based on fluorescence in situ hybridization and quantitative real-time-PCR.
      Such elevations were found in MCL-like lymphomas of the Myc/Cdk4R24C mice. As for human MCL, functional substitution within the cyclin D family and their cooperation in MCL-like lymphoma of Myc/Cdk4R24C mice remain to be further elucidated.
      MCL-like lymphoma of Myc/Cdk4R24C mice express both cyclin D1, cyclin D2, and cyclin D3. There is evidence that all these D-type cyclins and Cdk4/cyclin D complexes may be implicated in lymphoma growth. D-type cyclins have been suggested to play a distinct role in cell growth and differentiation. Cellular levels of D-type cyclins do not remain stable during cell growth but markedly change, not only in response to growth conditions (exponentially phase versus stationary phase), but also in response to the cell-differentiated status or to specific cellular mitogenic stimulus.
      • Bryja V.
      • Pachernik J.
      • Vondracek J.
      • Soucek K.
      • Cajanek L.
      • Horvath V.
      • Holubcova Z.
      • Dvorak P.
      • Hampl A.
      Lineage specific composition of D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4. CDK6 and individual D-type cyclins in differentiating cells of embryonic origin.
      • Paternot S.
      • Dumont J.E.
      • Roger P.P.
      Differential utilization of cyclin D1 and cyclin D3 in the distinct mitogenic stimulations by growth factors and TSH of human thymocytes in primary culture.
      During cell growth, Cdk4/cyclin D1 complexes may be reduced in favor of Cdk4/cyclin D2 or C3 ones. For example, cyclin D3 replaces cyclin D1 during differentiation of the leukemia cell line HL-60.
      • Bartkova J.
      • Lukas J.
      • Strauss M.
      • Bartek J.
      Cyclin D3: requirement for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation.
      Some cells exhibit high levels of cyclin D1 and D2 localized in the cytoplasm, bound but not activating Cdk4.
      • Bryja V.
      • Pachernik J.
      • Vondracek J.
      • Soucek K.
      • Cajanek L.
      • Horvath V.
      • Holubcova Z.
      • Dvorak P.
      • Hampl A.
      Lineage specific composition of D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4. CDK6 and individual D-type cyclins in differentiating cells of embryonic origin.
      Cdk4/cyclin D1 and CDK4/cyclin D3 complexes differently impact some phosphorylated Rb functions.
      • Bryja V.
      • Pachernik J.
      • Vondracek J.
      • Soucek K.
      • Cajanek L.
      • Horvath V.
      • Holubcova Z.
      • Dvorak P.
      • Hampl A.
      Lineage specific composition of D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4. CDK6 and individual D-type cyclins in differentiating cells of embryonic origin.
      • Paternot S.
      • Dumont J.E.
      • Roger P.P.
      Differential utilization of cyclin D1 and cyclin D3 in the distinct mitogenic stimulations by growth factors and TSH of human thymocytes in primary culture.
      Finally, cyclin D3 is expressed in various nonproliferating cell types.
      • Bartkova J.
      • Lukas J.
      • Strauss M.
      • Bartek J.
      Cyclin D3: requirement for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation.
      Taken altogether, these results suggest that dramatic switches in the expression of individual D-type cyclins and associated Cdk4 complexes may occur during lymphoma development in Myc/Cdk4R24C mice and that further experimental studies (especially concerning p21cip1 and p27kip1 sequestration) would be of importance to solve the question.
      In conclusion, the data presented here demonstrate that the Cdk4R24C mutant can cooperatively transform B cells in concert with a Myc gene. They establish Cdk4 as a proto-oncogene whose activity appears to depend on a specific cooperating partner and link disturbances in the regulation of cell cycle progression to the development of B-cell malignancies. In Myc-3′RR mice, Myc is specifically expressed from the pre-B to the mature B-cell stages
      • Guglielmi L.
      • Le Bert M.
      • Truffinet V.
      • Cogné M.
      • Denizot Y.
      Insulators to improve expression of a 3′IgH LCR-driven reporter gene in transgenic mouse models.
      ; the 3′RR being active in late B-cell lymphopoiesis.
      • Vincent-Fabert C.
      • Fiancette R.
      • Cogné M.
      • Pinaud E.
      • Denizot Y.
      The IgH 3′ regulatory region and its implication in lymphomagenesis.
      • Pinaud E.
      • Marquet M.
      • Fiancette R.
      • Péron S.
      • Vincent-Fabert C.
      • Denizot Y.
      • Cogné M.
      The IgH locus 3′ regulatory region: pulling the strings from behind.
      Moreover, we demonstrate that disturbed Cdk4/INK4 regulation lead to a phenotype switch of Myc-driven B-cell lymphomas from BL-like lymphoma to MCL-like lymphoma. Although closely reproducing many features of blastoid MCL, Myc/Cdk4R24C mice provide a novel and efficient model to dissect the molecular events leading to MCL and an important tool to test potential therapeutic agents.

      Acknowledgments

      We thank Sylvie Desforges and Bernadette Remerand for help with animal care. We are deeply grateful to Mariano Barbacid [Centro Nacional de Investigaciones Oncolügicas (CNIO), Madrid, Spain] for providing the Cdk4R24C mouse strain. We acknowledge the technological expertise of the Nice Sophia–Antipolis Functional Genomics Platform. We also thank Pascal Barbry for helpful discussions and support during this work.

      Supplementary data

      • Supplemental Figure S1

        Proliferation and apoptosis in B cells of Cdk4R24Cmice. A: PCR analysis of Cdk4R24C mice. The wt and mutated alleles had 180 and 280 bp length, respectively. B-D: Splenocytes from Cdk4R24C mice (in gray) and wt animals (in white) were grown for 3 days alone (control) or with LPS (B), anti-CD40 (C), or both (D). Growth was evaluated with the MTS assay. Mean ± SEM of six independent experiments in sextuplicate. *P < 0.01 versus unstimulated cells (Mann-Whitney U-test). E: Splenocytes from Cdk4R24C mice (•) and wt animals (○) were grown for 3 days. Percentage of B220+ living cells were reported as mean ± SEM of three independent experiments in duplicate.

      • Supplemental Figure S2

        The mutation status of VH rearranged genes was investigated in five Myc/Cdk4R24C lymphomas (63 sequences). All were essentially unmutated as shown in (A) and (B). Mutation types are reported in (C). (D) Real-time PCR analysis of AID, MSH2, MSH6, UNG, POLη, and Rev1 transcripts in purified CD19+ splenocytes from four Cdk4R24C mice and four Myc/Cdk4R24C lymphomas.

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