Early B-Cell-Specific Inactivation of ATM Synergizes with Ectopic Cyclind1 Expression to Promote Pre-Germinal Center B-Cell Lymp

ORIGINAL ARTICLE Early B-cell-speciﬁc inactivation of ATM synergizes with ectopic CyclinD1 expression to promote pre-germinal center B-cell lymphomas in mice

K Yamamoto1,2, BJ Lee1,CLi1, RL Dubois1, E Hobeika3,4, G Bhagat5 and S Zha1,5,6

Ataxia telangiectasia-mutated (ATM) kinase is a master regulator of the DNA damage response. ATM is frequently inactivated in human B-cell non-Hodgkin lymphomas, including ~ 50% of mantle cell lymphomas (MCLs) characterized by ectopic expression of CyclinD1. Here we report that early and robust deletion of ATM in precursor/progenitor B cells causes cell autonomous, clonal mature B-cell lymphomas of both pre- and post-germinal center (GC) origins. Unexpectedly, naive B-cell-specific deletion of ATM is not sufficient to induce lymphomas in mice, highlighting the important tumor suppressor function of ATM in immature B cells. Although EμCyclinD1 is not sufficient to induce lymphomas, EμCyclinD1 accelerates the kinetics and increases the incidence of clonal lymphomas in ATM-deficient B-cells and skews the lymphomas toward pre-GC-derived small lymphocytic neoplasms, sharing morphological features of human MCL. This is in part due to CyclinD1-driven expansion of ATM-deficient naive B cells with genomic instability, which promotes the deletions of additional tumor suppressor genes (i.e. Trp53, Mll2, Rb1 and Cdkn2a). Together these findings define a synergistic function of ATM and CyclinD1 in pre-GC B-cell proliferation and lymphomagenesis and provide a prototypic animal model to study the pathogenesis of human MCL.

Leukemia (2015) 29, 1414–1424; doi:10.1038/leu.2015.41

INTRODUCTION during both V(D)J recombination and CSR.4–7 In the absence of ATM, Mature B-cell lymphomas represent 85% of non-Hodgkin lym- physiological DSBs at Ig and T-cell receptor (TCR) loci accumulate, phomas (NHL) and many harbor characteristic chromosomal leading to greatly increased risk of leukemia and lymphoma in 8 translocations involving the immunoglobulin (Ig) loci. These Ataxia-telangiectasia patients with germline ATM inactivation. ATM translocations result from mis-repair of DNA double-stranded is also somatically inactivated in human lymphoid malignancies, breaks (DSBs) generated during V(D)J recombination or class most notably in ~ 50% of mantle cell lymphomas (MCLs), 45% of switch recombination (CSR). V(D)J recombination assembles the T-cell pro-lymphocytic leukemia, 10–20% of chronic lymphocytic – productive Ig genes from germline V, D and J gene segments in leukemia, and 5% of diffuse large B-cell lymphomas (DLBCLs).9 12 immature bone marrow B cells. CSR occurs in mature B-cells in ATM-null mice recapitulate many phenotypes of Ataxia- specialized structures-germinal centers (GCs) and allows expres- telangiectasia patients and routinely succumb to aggressive T-cell sion of different antibody isotypes (for example, IgG1 or IgE) with lymphomas at 3–4 months of age with translocations involving the different effector functions.1 Naive B cells also undergo somatic TCR loci, suggesting that ATM suppresses T-cell lymphomas by hypermutation (SHM) of the Ig variable region in GCs to achieve reducing genomic instability during V(D)J recombination.13 How- higher affinities. While V(D)J recombination and CSR are initiated ever, the developmental stage and the role of ATM inactivation in by lymphocyte-specific enzymes, both reactions generate DNA B-cell lymphomagenesis is not yet well understood. DSB intermediates that are repaired by ubiquitously expressed Among all B-cell non-Hodgkin lymphomas, MCL has the highest DNA repair mechanisms. Thus, defects in DNA repair or DNA frequency of bi-allelic inactivation of ATM.9 MCL is a mature B-cell damage response lead to accumulation of DSB intermediates lymphoma composed of small- to medium-sized lymphocytes which, if not repaired appropriately, lead to oncogenic chromo- with frequent (~30%) involvement of the gastrointestinal tract. somal translocations in human mature B-cell lymphomas by The variable regions of the Ig are unmutated in the majority of transposing the strong Ig promoters/enhancers adjacent to MCL cases, consistent with a pre-GC origin. MCL is characterized cellular oncogenes (for example, c-MYC, BCL2).2 by deregulated expression of D-type cyclins, especially CyclinD1, Ataxia telangiectasia-mutated (ATM) kinase encodes a serine/ via the characteristic t(11;14) chromosomal translocation that joins threonine protein kinase, which is activated by DSBs to establish CCND1 with the active Ig-heavy chain gene (IGH) promoter and cell cycle checkpoints and promote DNA repair.3 Through this enhancer. Yet, ectopic expression of wild-type (WT) CyclinD1 is mechanism, ATM promotes efficient and precise DNA repair insufficient to induce MCL in mice,14,15 implying additional genetic

1Institute for Cancer Genetics, College for Physicians and Surgeons, Columbia University, New York, NY, USA; 2Graduate Program for Pathobiology and Molecular Medicine, College for Physicians and Surgeons, Columbia University, New York, NY, USA; 3Centre for Biological Signaling Studies BIOSS, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany; 4Department of Molecular Immunology, Albert-Ludwigs-Universität Freiburg and Max Planck Institute for Immunobiology, Freiburg, Germany; 5Department of Pathology and Cell Biology, College for Physicians and Surgeons, Columbia University, New York, NY, USA and 6Department of Pediatrics, College for Physicians and Surgeons, Columbia University, New York, NY, USA. Correspondence: Dr S Zha, Institute for Cancer Genetics, College for Physicians and Surgeons, Columbia University, 1130 St Nicholas Avenue RM503B, New York, NY 10032, USA. E-mail: [email protected] Received 31 October 2014; revised 22 January 2015; accepted 6 February 2015; accepted article preview online 13 February 2015; advance online publication, 20 March 2015 Pre-germinal center B-cell Lymphomas in mice K Yamamoto et al 1415 alterations are necessary. Effective treatment for MCL is not RESULTS available, and animal models recapitulating molecular features of Mouse models with B-cell-specific deletion of ATM human MCL are yet to be developed. fi To circumvent early lethality due to thymic lymphomas in Here we report that progenitor B-cell-speci c inactivation of germline Atm knockout mice (Atm− / −), we generated B-cell- ATM results in indolent and monoclonal mature B-cell lympho- specific deletion of Atm using CD21Cre, CD19Cre or Mb1+/Cre in proliferations that recapitulate the morphological spectrum of combination with the ATM conditional allele (ATMC).23 CD21Cre fi ATM-de cient human B-cell non-Hodgkin lymphoma, including allele17 mediates specific and robust ATM deletion in IgM+-naive B both GC and non-GC-derived lymphomas. EμCyclinD1 transgene cells and CD19Cre+ATMC/C (ref. 18) results in ATM deletion ranging markedly accelerates the proliferation of genomic instable ATM- from 60% in bone marrow pre-B cells to ~ 100% in naive splenic B deficient naive B cells, leading to early clonal expansion of pre-GC cells (Supplementary Figure 1A). Despite efficient deletion of ATM naive B cells and pre-GC lymphomas, and identifies a cooperative in naive splenic B cells in both CD21Cre+ATMC/C and CD19 effect of these two lesions in immature and naive B cells for Cre+ATMC/C mice as evidenced by Southern blot analyses, CSR lymphomagenesis. defects and genomic instability (Supplementary Figures 1A–1C), none of the CD21Cre+ATMC/C (n = 23) or CD19Cre+ATMC/C (n = 36) mice developed definitive B-cell lymphoproliferations in MATERIALS AND METHODS 428 month follow-up period (Supplementary Figure 1D), by Mice which time the bone marrow samples were virtually devoid of ATMC/C,16 EμCyclinD1 transgenic,14 CD21Cre,17 CD19Cre18 and Mb1Cre (ref. 19) B cells. mice were previously described. All experimental cohorts carry transgene On the basis of this observation and the postulated ‘early’ (CD21Cre, EμCyclinD1) or Cre knock-in (Mb1Cre or CD19Cre) in hetero- deletion of ATM in human MCL,25 we focused on Mb1Cre,19 which zygosity. All animal experiments were performed in accordance with is the earliest B-cell-specific Cre allele available, which leads to Columbia University Institutional Animal Care and Use Committee. specific and robust cre activation in early pro-B/pre-B-cells.26 We generated four cohorts, Mb1+/creATM+/+(C) (hereafter referred to − − Flow cytometry and immunohistochemistry analyses as M) Mb1+/CreATMC/C( )EμCyclinD1 (MA), Mb1+/cre(+)ATM+/+(C) + +/cre C/C( − ) + Flow cytometry analyses were performed on single cell suspensions from EμCyclinD1 (MD/D) and Mb1 ATM EμCyclinD1 (MAD). the spleen, bone marrow, lymph nodes as previously described.20 First, we confirmed the efficient and specific deletion of the ATM Antibodies include CD43(S7), CD5(53–7.3), CD138(281.2), CD21/35(7G6), gene and protein in splenic B cells from MA mice by Southern CD23(B3B4), CD11b/Mac-1(M1/70) and IgK from BD Pharmingen (San (Figure 1a) and western blotting (Figure 1b), respectively. In B cells Diego, CA, USA); B220(RA3–6B2), CD19(eBio1D3), CD8a(53–6.7), CD4(L3T4), purified from MA mice, irradiation induced phosphorylation of CD3ε(145-2C11) and TCRβ(H57-597) from eBioscience (San Diego, CA, Kap1, an ATM-specific substrate,27 was largely abolished confirming USA); Ter119 and Gr1(RB6-8C3) from BioLegend (San Diego, CA, USA); IgM the loss of ATM kinase activity (Figure 1c). Meanwhile, T cells from and IgD from Southern Biotech (Birmingham, AL, USA) and peptide nucleic MA or MAD mice were devoid of the development defects acid from Vector Labs (Burlingame, CA, USA). Immunohistochemistry fi 28— fi fi associated with ATM de ciency namely reduced surface CD3/ staining was performed on formalin xed, paraf n-embedded tissue TCRβ expression and reduced CD4 or CD8 single-positive T cells in sections as described previously using the following antibodies: B220, CD3, the thymus-consistent with normal ATM function in T cells from MA PAX5, BCL6, IRF4 and CD138.21 Immunofluorescence on paraffin- or MAD mice (Figure 1d). Similarly, myeloid (Gr1+ or CD11b+)and embedded tissues was performed using antibodies against Ki67 (SP6, + Abcam, Cambridge, MA, USA), and B220 (RA-3B62, BD Pharmingen). erythroid (Ter119 ) lineages were also unaffected in the bone Images were acquired with a Nikon Eclipse 80i microscope (Nikon, Melville, marrow and spleen of MA and MAD mice (Supplementary Figure 2A). NY) and processed on NIS-Elements AR 3.10 software (Nikon). Together, these data support the specificandefficient deletion of ATM in developing B-cells. In the Mb1+/Cre mice, the Cre knock-in disrupts the endogenous Mb1/CD79a gene in the targeted allele.19 As Southern blot and western blot analyses − − Mb1/CD79a is essential for B-cell development and Mb1/CD79a / For Southern blot analyses, genomic DNA from the spleen, tumor and B-cells arrest at the pro/pre- B-cell stage,29,30 we also confirmed corresponding kidneys were digested with EcoRI (for JH4, c-Myc, constant normal B-cell development and spleen cellularity in control MD/D, region of TCRδ probe) KpnI (for 3′ ATM CKO probe) or HindIII (for the IgK +/Cre 13,22,23 μ MA and MAD mice (all carrying heterozygous Mb1 alleles) and probe). To avoid cross-reactivity with the E CyclinD1 transgene that +/Cre fi includes a fragment from JH4 to μ enhancer, we generated a new JH4 only used Mb1 for all breeding and nal tumor cohorts probe by PCR amplification with the following primers: 5′-TGGTGACAATTT (Figure 1d, Supplementary Figure 2B). Finally, ectopic expression of + CAGGGTCA-3′ and 5′-TTGAGACCGAGGCTAGATGC-3′. For western blots, CyclinD1 in both B and T cells was also verified in EμCyclinD1 MD stimulated splenic B or T cells were probed with antibodies against ATM and MAD mice by western blotting (Figure 1b). (1:2000, MAT3, Sigma, St Louis, MO, USA), pKap1 (1:2000, Bethyl, Montgomery, TX, USA), Kap1/TIF1β (1:1000, Cell Signaling, Danvers, MA, B-cell-specific deletion of ATM leads to B-cell autonomous β USA), -Actin (1:10 000, A5316, Sigma) and CyclinD1 (1:1000, Abcam). lymphomas of both GC and non-GC phenotype To determine the role of ATM deficiency in B-cell lymphomagen- CSR and FISH analyses esis, we followed a cohort of MA mice with weekly palpation to Splenic B-cells from 8–12-week-old mice were purified with anti-CD43 identify any abnormal growths. During the 20-month follow-up, magnetic-activated cell sorting (MACS) beads and stimulated with LPS/IL4 12.5% (3/24) of MA mice developed overt splenomegaly and 20 for 2–4 days as described previously. Metaphases were obtained at day lymphadenopathy, with frequent intestinal involvement (67%, 2/3) 4.5 after stimulation and stained with Cy3-conjugated peptide nucleic acid – 24 (Figures 2a c, Table 1). Flow cytometry analyses revealed probe against 3 × telomere sequence as previously described. Tumor monoclonal expansion of CD19+ (often B220+) B cells, which metaphases were harvested after 6 h incubation in RPMI1640 with15% was further confirmed by Southern blot analyses that detected fetal bovine serum in the presence of colcemid (100 ng/ml, Invitrogen, CA, fi USA). Chromosome 12 paint was obtained from Applied Spectral Imaging clonal IgH rearrangements in all con rmed cases (Figure 2d, ′ ′ Supplementary Figures 3A and B). In the same period, none of the (Carlsbad, CA, USA) and the 5 c-myc (C10) and 5 IgH (BAC207) locus- Cre/+ +/+(C/+) specific FISH (fluorescence in situ hybridization) probes were described Mb1 ATM mice developed any signs of tumors. At previously.22 Images were acquired with Zeiss Axio Imager Z2 equipped 420 month of age, all tumor cohorts were euthanized for with a CoolCube1 camera (Carl Zeiss, Thornwood, NY, USA) and an endpoint analyses. One additional MA mouse (2023) was found to automatic stage system, and processed with Isis and Metafer4 software have a B-cell lymphoma based on clonal IgH rearrangement and packages (MetaSystems, Newton, MA, USA). histology (Figure 2c, Supplementary Figures 3A and B), bringing

Mb1+/CreATM+/C Mb1+/CreATMC/- Mb1+/CreATMC/C

Kid BM Thy Spl B Kid BM Thy Spl B T Kid BM Thy Spl B

WT -

- C -

MD MA MAD

IR - + - + - + MD MA MAD

T B T B T B ATM

ATM P-Kap1 (S824)

CyclinD1 Kap1

β-Actin β-Actin

BM Spleen Thymocytes

Ctrl

ATM-/-

+/cre +/C Mb1 ATM EμCyclinD1+ (MD)

Mb1+/creATMC/C (MA)

Mb1+/creATMC/C EμCyclinD1+ (MAD) B220 B220 CD4 B220 Cell No.

CD43 IgM IgM CD8 Surface TCRβ Figure 1. B-cell-speciﬁc deletion of ATM in Mb1+/CreATMC/ − and Mb1+/CreATMC/ − EμCylinD1+ mouse models. (a) Southern blot analyses of the Atm locus with genomic DNA harvested from the kidney (Kid), thymus (Thy), bone marrow (BM), total spleen cells (Spl), LPS/IL-4-stimulated splenic B-cells (B) and Con A stimulated splenic T cells (T). (b) Western blot analyses for CyclinD1 and ATM in stimulated splenic B and T cells harvested from MD, MA and MAD mice. (c) Phosphorylation of Kap1 in LPS/IL4 stimulated B cells (day 3) with or without irradiation (IR,10 Gy). Protein lysate is collected 2 h after IR. (d) Representative ﬂow cytometry analyses of bone marrow (BM), spleen and thymocytes from Atm− / − as well as MA, MAD and MD mice.

Leukemia (2015) 1414 – 1424 © 2015 Macmillan Publishers Limited Pre-germinal center B-cell Lymphomas in mice K Yamamoto et al 1417 3468 MAD Ctrl 2023 MA 70 1cm 100 B-Lymphoma ≥20M 60 B-Lymphoma ≤20M 7/15 80 50 60 40 30 40 B-cell Lymphoma % survival 1/21 3468 MAD Ctrl MA (24) 20 ns 20 Analyzed % Mice 2/12 3/18 MD/D (12) 10 3/24 MAD (18) * 0 0 0 5 10 15 20 MA MD/D MAD Time (M)

Ctrl MA MAD 2583 2594 3468 4271 2569 4161

IgM B220

CD19

3557 3560 3710 4159 4161 4271 3468 3613 3713

KSS K S K S K S K S K S K S K B* K

JH4

- GL

Jĸ4

- GL

5’-MycA

TCR

Figure 2. MA and MAD mice develop clonal B-cell lymphoproliferations. (a) Tumor-free survival of MA, MAD and MD/D cohorts up to 20 month of age. Number of mice followed in each cohort is listed in the parentheses. *Po0.05 per Mantel–Cox/log-rank test for statistical significance. (b) Lifetime risk for B-cell lymphoproliferations in MA, MAD and MD/D mice. The gray boxes represent tumors discovered within 20-month follow-up period and the white boxes represent tumors discovered at endpoint analyses (420-month). (c) Representative images of enlarged spleen and lymph node (from MAD mouse 3468) and small intestine (right, from MA mouse 2023). The black arrow heads point to the lymph nodes. (d) Representative flow cytometry analyses of spleen from tumor-bearing MA and MAD mice. (e) Southern blot analyses of DNA harvested from Kidney (K) and Spleen (S) from MAD mice. Asterisk denotes mouse 3613, in which the tumor primarily resided in bone marrow (B). Mouse 3468 has leukemia with significant infiltration in the kidney, thus the presence of clonal rearrangements. Blue arrow heads point to clonal rearrangements. Probe for the constant region of TCRδ is used as loading control. GL: germ line. the lifetime risk for monoclonal lymphoproliferative disease to IgH for SHM and found evidence of SHM in 2/4 MA DLBCL, 4/24 (16.7%) in MA mice. Histopathologic and immunophenotypic indicating a post-GC cell of origin. On the basis of these data, we analyses revealed diffuse large B-cell lymphomas (DLBCL) of conclude that deletion of ATM in early pre-/pro- B-cells leads to splenic or nodal origin in the MA mice, with one manifesting a GC infrequent mature B-cell lymphomas of heterogeneous cell origins phenotype (BCL6+, IRF4 − ) and three exhibiting a non-GC with long latency. These results support a role of ATM as a tumor phenotype (BCL6 ± , IRF4+) (Supplementary Figure 3C, Table 1). suppressor gene during early B-cell development and also Given the GC and non-GC immunophenotypes, we analyzed the highlight the need for additional genetic hits.

Table 1. Pathological characteristics of clonal B-cell lymphoproliferations of MD/D, MA and MAD mice

No. Genotype Age (M) Diagnosis Organs SHM Surface marker (FACS) IHC

B220 IgM Others PAX5 BCL6 IRF4

2594 MA 18 DLBCL S, Ln − +++ + PNA+ ND + − 2583 MA 20 DLBCL S,Ln − ++ +− + 2023 MA 25 DLBCL S, Ln + (1/710) −− ND + + 3465 MA 19 DLBCL S, K + (3/710) + + IgD − + − + 3468 MAD 13 DLBCL S,Ln, PB, Liv, K − +++ +++ ND − + 3710 MAD 23 LPD S − +++ +++ IgD − ND − ± 3557 MAD 24 LPDa S + (2/710) − +++ + − ± 3613 MAD 16 LPDa,b S, BM − +++ +++ + − ± 4271 MAD 21 LPDa,b S − + +++ + − ± 4159 MAD 21 LPDa,b S − + +++ + − ± 4161 MAD 21 LPDa,b S, Ln + (1/710) − +++ CD5+ + − ± 3560 MAD 24 LPDa,b S − ++ +− ± 3713 MAD 17 LPDb S − + + CD5+ + − ± 2569 MAD 23 LPDb S, Ln − +++ +++ + − ± 2012 MD/D 26 DLBCL S, Ln, PB, K + (3/710) +++ +++ IgD − ND − + 2603 MD/D 24 LPDa S − + +++ CD5lo ND − + Abbreviations: DLBCL, diffuse large B-cell lymphomas; FACS, ﬂuorescence-activated cell sorting; IgD, immunoglobulin D; IgM, immunoglobulin M; IHC, immunohistochemistry; LPD, lymphoproliferative disorder; PNA, peanut agglutinin; MA, Mb1+/CreATMC/C(− )EµCyclinD1 −; MAD, Mb1+/creATMC/C( − ) EµCyclinD1+; MD/D, Mb1+/cre(+)ATM+/+(C)EµCyclinD1+; ND, not determined; SHM, somatic hypermutation. Organs involved: BM, bone marrow; K, kidney; Liv, liver; LN, lymph node; PB, peripheral blood; S, spleen. IRF4 ± indicates mixed IRF4 expression. aRed pulp inﬁltration. bVariable mantle and marginal zone expansion.

CyclinD1 overexpression accelerates B-cell lymphoproliferations in GCs. Of note, 5/10(50%) MAD tumors showed significant infiltration MA mice and skews lymphomas toward pre-GC origin of the red pulp by small- to medium-sized lymphocytes, resembling AmongallhumanB-cellnon-Hodgkin lymphomas, MCL, character- the pattern of splenic involvement by human chronic lymphocytic ized by ectopic expression of CyclinD1, has the highest frequency of leukemia and MCL (Table 1, Figure 3). The immunophenotype of bi-allelic inactivation of ATM.9 Given the early deletion of ATM during the DLBCL indicated a non-GC B-cell origin (BCL6 − , IRF4+), as did MCL development, it was proposed that ATM deficiency might that of the lymphoproliferative disorders (PAX5+, BCL6 − ,IRF4± increase IgH-CyclinD1 translocation to promote MCL.9 Alternatively, and CD138 − ). Consistent with these observations, whereas 50% ectopic CyclinD1 expression might synergize with ATM deficiency to (2/4) of MA lymphomas showed IgH variable region gene promote cellular proliferation at the price of genomic instability to mutations, only 2/10 (20%) of the MAD lymphomas showed drive B-cell lymphomagenesis. To test this, we bred MA mice with evidence of SHM. These findings suggest that CyclinD1 over- EμCyclinD1 transgenic mice to generate the MAD and control MD/D expression synergizes with ATM deficiency to promote predomi- mice. At 2 months of age, B-cell number and B-cell development in nantly pre-GC B-cell lymphoproliferations recapitulating some MAD mice is comparable with MA and MD controls (Figure 1d). By morphological and immune phenotypes of human MCL. 20-months of age, 3/18 (16.7%) MAD mice had developed overt splenomegaly and lymphadenopathy with occasional intestinal MAD tumors share a subset of molecular features with involvement and leukemic presentation (Figures 2a–c, Table 1). The human MCL tumor-free survival of MAD mice was significantly shorter than either To further characterize the genetic lesions that underlie lympho- MA or MD/D control groups (Figure 2a). Moreover, at the endpoint magenesis in the MAD mice, we performed comparative genomic analyses (420 month), 46.6% (7/15) of MAD mice showed clonal hybridization (CGH) analyses on three MAD, one MA and one MD B-cell expansions by flow cytometry analyses and clonal IgH and IgK tumors with high tumor content (450% by fluorescence-activated rearrangements by Southern blotting (Figures 2d and e), bringing the cell sorting). CGH identified a large number of gross chromosomal lifetime risk for B-cell lymphoproliferations in MAD mice to 55.6% gains and losses in all tumors (Figure 4a and Supplementary (10/18) (Table 1). This is in sharp contrast to the control MD/D mice, Figure 4A). Specifically, two MAD tumors (3468 and 4161) display which did not develop overt splenomegaly within the 20-month focal deletion involving the Trp53 tumor suppressor gene, follow-up and only 2/12 MD/D mice showed evidence of B-cell including clear homozygous deletion in tumor 3468 (Figure 4b lymphoproliferation upon histopathologic analyses at necropsy and Supplementary Figure 4A). Cdkd2a (ink4a/p16) and Rb1 are (Figure 2b, Table 1). By flow cytometry, MA and MAD lymphopro- also deleted in a subset of the MAD and MA lymphomas liferations had similar immunophenotypes, namely CD19+B220hi/ (Figure 4b). MLL2, a haplo-insufficient tumor suppressor gene in loIgMhi/lo (Figure 2c). The B-cell identity of B220-negative lympho- MCL as well as DLBCL, is also partially deleted in all tumors tested proliferations was further validated by PAX5 immunohistochemistry (Figure 4b). Meanwhile, Mdm2, Cdk4, and Notch1 are moderately staining (Figure 3, Supplementary Figure 3C). All tumors were amplified (mostly trisome) in several MAD and MA lymphomas − − CD21/35 CD23 (Supplementary Figure 3D), excluding GC B-cell (Figure 4b). These changes are consistent with recurrent genetic origin. Two out of 10 (20%) MAD tumors expressed CD5, a marker alterations that have been characterized for human MCL.25 that is frequently associated with activated B cells and also Meanwhile, the CGH analyses also identified focal deletions in expressed by human MCL (Supplementary Figure 3D, Table 1). In Igκ (chromosome 4) and Igh (chromosome 12) loci indicative of addition to DLBCL observed in 2/10 (20%) MAD mice, including B-cell origin. Fine mapping of the Igh locus showed that the one exhibiting prominent red pulp involvement, a spectrum of deletions in all three MAD tumors are restricted to the V-D-J indolent lymphoproliferations was noted in the remainder. All portion of the Igh locus and does not involve the switch region eight lymphoproliferative disorders were characterized by variable located downstream (centrometric of chromosome 12) (Figure 4d), degrees of white pulp enlargement, predominantly owing to consistent with the pre-GC origin of the MAD tumors. In contrast, MA follicular mantle and marginal zone expansion without discernable tumor 2023 shows heterozygous deletionwithinswitchregion

3468 DLBCL

H&E B220/CD3 PAX5/BCL6 IRF4

4271 LPD

4161 LPD

3713 LPD

Scale Bar=200μm Figure 3. Histopathologic and immunophenotypic analyses of B-cell lymphoproliferations of MAD mice. Representative photomicrographs showing the spectrum of B-cell lymphoproliferations observed. (3468, H&E) DLBCL. The white pulp is markedly expanded and the architecture effaced by an infiltrate of large pleomorphic lymphocytes. The neoplastic cells express B220 (blue). Scattered small disrupted BCL6+ germinal centers are seen and the neoplastic cells are BCL6 negative but they show IRF4 expression, findings compatible with a DLBCL of non-GC phenotype. (4271, H&E) LPD with red pulp lymphocytic infiltrate. The white pulp is disrupted by expansions of the follicular mantle and marginal zones and a diffuse infiltrate of small-sized lymphocytes is present in the red pulp. The neoplastic lymphocytes show B220 (blue), PAX5 and IRF4(variable) expression and they lack BCL6 expression. (4161, H&E) LPD. Clusters of large lymphocytes are seen surrounding the white pulp and also infiltrating the red pulp. The neoplastic lymphocytes are B220 (blue) and BCL6 negative, but they express PAX5 and show IRF4 (variable) expression. (3713, H&E) LPD. The white pulp is variably disrupted by expansions of the follicular mantle and marginal zones with limited red pulp infiltration. The neoplastic cells express B220 (variable), PAX5 and IRF4 (variable) but they lack BCL6 expression.

(Figure 4d). Together with the positive staining for Bcl6 and the tumors. Finally, targeted Sanger sequencing of 10 MAD and 4 MA presence of SHM, this finding suggests that at least a subset of the tumors did not find mutations in the C-terminal Pro, Glu, Ser, Thr-rich MA tumors derived from GC or post-GC cells. CGH is not able to (PEST) domain of Notch1 or the Su(var)3-9 Enhancer-of-Zeste (SET) detect reciprocal translocations. To test whether reciprocal transloca- domain of Multiple Myeloma SET domain (MMSET), two genes that tion involving IgH locus occurred in any of the tumors, we performed are mutated in ~10% human MCL.9 Together, these findings suggest chromosome 12 paint together with FISH using a probe that that MAD tumors share molecular features with human MCL. hybridizes to the telomeric end of Igh locus (upstream toward the Vh region, 5′ IgH, 207) in two MAD (3468 and 4161) and one MA (2594) fi tumors, for which high-quality metaphases were available. The ATM de ciency enhances genomic instability and increases analyses revealed heterozygous deletion, but not translocation chromosomal fusions in CyclinD1+ B cells involving the 5′IgH in MAD tumor 4161 and MA tumor 2594 To better understand the mechanism by which ectopic CyclinD1 (Figure 4c and Supplementary Figure 4C). FISH with c-Myc probes expression synergizes with ATM deficiency to promote B-cell exclude IgH-Myc translocation in the tumors and CGH analyses lymphoproliferations, we analyzed non-neoplastic B cells from suggested that c-Myc is not grossly amplified in tested MAD or MA 6–12-week-old MAD and MA mice. MCL is thought to originate

3468 MAD Ch.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X Igk p53 IgH

4161 MAD Ch.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X

2012 MD2023 MA4159 MAD4161 MAD3468 MAD Cdkn2a Cdkn2a 3468 MAD 4161 MAD Cdkn2a Cdkn2a Cdkn2a MLL2 MLL2 DAPI Ch.12 5’lgH MLL2 MLL2 Kmt2d/Mll2 MLL2 MLL2 MLL2 Normal MLL2 Rb1 Rb1 5’ IgH Rb1 Rb1 Deleted Rb1 Rb1 Rb1 Rb1 Rb1 Rb1 Rb1 Rb1 Rb1 Trp53 Trp53 DAPI Ch.12 5’c-Myc Trp53 Trp53 Cdk4 Cdk4 Mdm2 Mdm2 Mdm2 Mdm2 Notch1 Notch1 Notch1 Notch1 Notch1 Notch1 Notch1 C-myc Myc

Average Signal Intensity ≤-1 -0.6 0 0.6 ≥1 Scale: 10μm

3 3468 MAD 4159 MAD 2 4161 MAD 2023 MA 3’ Centromere 5’ Telomere 1 2012 MD 0 -0.6 -1 -2 -3 Average Signal Intensity Average

1-152 1-20 1-4 Eμ Cμ Cα

VH DH JH Figure 4. Comparative genomic hybridization (CGH) and FISH analyses of MA and MAD tumors. (a) Landscape representation of CGH on MAD tumors-3468 and 4161, with green arrows indicating particular regions of interest. Chromosome (Ch.) numbers indicated at the top. The arrows indicate loci of interest (e.g. Igκ, Igh and Trp53). The y axis (average signal intensity) represents the log ratio of copy number between tumor vs normal at given probe. (b) Heatmap showing focal deletion of Trp53, Cdkn2a, MLL2 and Rb and potential trisomy of Mdm2 and Cdk4. It also shows normal copy number of c-Myc. Different rows represent unique probes used for CGH analyses. (c) Chromosome (Ch.) 12 paint analyses combined with either Telomeric Igh FISH (BAC207 at the Vh region) or c-Myc on MAD tumors-3468 and 4161. The diagram on the right represents the mono-allelic deletion of the Igh in tumor 4161. (d) CGH analysis of the Igh locus of MAD, MA, MD tumors. Demarcations of each region within the Igh locus are shown below. Average signal intensity of −0.6 indicates the value corresponding to heterozygous loss of the allele.

Leukemia (2015) 1414 – 1424 © 2015 Macmillan Publishers Limited Pre-germinal center B-cell Lymphomas in mice K Yamamoto et al 1421 from pre-GC, mantle zone B cells, which have yet to encounter Two kinds of breaks can be quantified by the telomere-FISH assay: CSR and SHM.31 As ATM deficiency reduces CSR efficiency,32,33 we chromosomal breaks, which refer to breaks at both sister- hypothesized that ectopic expression of CyclinD1 and ATM chromatids, and chromatid breaks, which refer to breaks in only deficiency together might be deleterious to cells undergoing one of the two sister-chromatids (Figure 5b). The frequencies of CSR, thus effectively trapping naive B cells at the pre-GC stage of chromosomal breaks as well as chromatid breaks were compar- development/maturation. To test this hypothesis, we measured able in MA and MAD mice and significantly higher than those CSR in purified B cells derived from MA, MD, and MAD mice as well observed in MD mice (Figure 5b, Supplementary Table 1). as WT or Atm− / − control mice in vitro. Ectopic expression of Moreover, the frequency of dicentric chromosomes generated CyclinD1 by itself did not measurably affect CSR. In vitro CSR to by end–end fusion of chromosomal breaks was significantly IgG1, measured by flow cytometry analyses, was reduced to higher in activated B cells derived from MAD mice than in those ~ 50% of WT levels in MAD as well as MA and Atm− / − B-cells from the MA or MD mice (Figures 5b and c, Supplementary Table 1), (Figure 5a), suggesting ATM deficiency compromised CSR in WT suggesting possible increased proclivity of chromosomal translo- and CyclinD1+ B cells at similar levels. Furthermore, irradiation cations. We conclude that ATM deficiency induces genomic induced G1/S and G2/M checkpoints in LPS/IL4-activated B cells instability in CyclinD1+ B cells. are normal in WT and CyclinD1+ B cells and comparable in Atm− / −, MA and MAD B cells (Supplementary Figures 5A and B), Ectopic CyclinD1 expression increases naive B-cell proliferation suggesting that ATM deficiency compromised DNA damage and rescues the B-cell lymphocytopenia in older MA mice induced checkpoints in both WT and CyclinD1+ B cells. Next, we Given the majority of the MAD and MA lymphomas are IgM+ and assessed the degree of genomic instability in LPS/IL4-activated B thus derived from cells that have not yet undergone CSR, we cells from Atm− / −, MA and MAD mice by telomere-FISH analyses. compared the naive B-cell population in young (1–3-month-old) vs

Chromosomal Chromatid Normal Breaks Breaks Dicentric +LPS/IL-4 Day 4 Ctrl ATM-/- MD MA MAD B220

IgG1

120

100

80 ns

60 Chromatid Breaks

40 Chromosomal Breaks + Relative to Ctrl (Day 4) 1 20 P=0.0011

% IgG 0 P=0.20 Ctrl MD/D MA MAD ATM-/- 60

*P=0.02 50 10 9 40 8 7 30 6 20 p=0.14 5 4 % Abnormal Metaphase 10 % Dicentric 3 2 0

Chromosomes/Metaphase 1 Ctrl MD/D MA MAD 0 Ctrl MD/D MA MAD Figure 5. Increased chromosomal fusions in stimulated MAD B cells. (a) Representative flow cytometry plots for surface IgG1 expression in LPS/IL4 stimulated B cells and quantification. Efficiency is calculated as a percentage of IgG1+ cells relative to the control. (b) Representative images of cytogenetic abnormalities observed in T-FISH assay in stimulated splenic B cells and the quantification. (c) Quantification of dicentric chromosomes observed in stimulated B cells derived from MD/D, MA, MAD and Atm− / − mice. P-values were calculated using a two- tailed Student’s t-test assuming unequal variances.

© 2015 Macmillan Publishers Limited Leukemia (2015) 1414 – 1424 Pre-germinal center B-cell Lymphomas in mice K Yamamoto et al 1422 mid-age (4–6- and 7–12-month-old) MAD and MA mice. Although on splenic B-cell frequency in WT mice (MD vs control) but all mature B-cell population are present in 6- and markedly rescued the splenic B-cell lymphocytopenia in MA mice 12-month-old MAD, MA and MD/D controls, the frequency of naive (MAD vs MA, Figure 6b). This is intriguing, as the naive B-cell B cells (CD19+ cells) in spleen declined significantly faster in MA population is thought to be the progenitor of MCL lymphomas. We mice, recapitulating the ATM-deficient B-cell lymphocytopenia that then co-stained splenic sections from 6-month-old tumor-free mice worsens over time (Figures 6a and b, Supplementary Figure 6). with B-cell marker-B220 and proliferation marker-Ki67. Quantifica- Ectopic expression of CylinD1 alone has, at most, moderate effects tion of Ki67+B220+ cells revealed a significant reduction of Ki67+

Ctrl 6 Months Old 80 Ctrl MD MA MAD MD/D MA 60 MAD

40 IgM * 20 B220

%CD19+ Cells in Spleen P=0.007 0 CD19 1-3 4-6 7-12 Time (M)

Scale Bar=100μm Ki67 Ctrl MD MA MAD B220 DAPI

Ki67 B220 *Non-B-cell

P=0.89

**P=0.0048

+ 15 B220

+ 10

5 cells/field 0 No. of Ki67

-5 MD MA MAD Figure 6. CyclinD1 expression rescues progressive B-cell loss in MA mice. (a) Representative flow cytometry plots of splenic B220+CD19+/IgM+ B-cell populations in 6-month-old mice. (b) Quantification of CD19+ B-cell populations from non-tumor-bearing mice analyzed at the indicated time points. (c) Representative immunofluorescence (IF) images, and (d) Quantification of Ki67+ (green, nuclear staining) and B220+ (red, membrane staining) cells in spleens harvested from 7-month-old non-tumor mice. Fields (MD (n = 7), MA (n = 15) and MAD (n = 14)) selected for quantification were taken at × 400 total magnification with equivalent B220+ cellularity. P-values were calculated based on individual two-tailed Student’s t-tests assuming unequal variances.

Leukemia (2015) 1414 – 1424 © 2015 Macmillan Publishers Limited Pre-germinal center B-cell Lymphomas in mice K Yamamoto et al 1423 frequency in B cells from MA mice, which is reverted by ectopic deficiency for B-cell transformation. In addition to its checkpoint expression of CyclinD1 in the MAD mice (Figures 6c and d). We function, ATM has important roles in DNA repair, as ATM conclude that ectopic expression of CyclinD1 promotes prolifera- deficiency is detrimental to primary cells owing to ongoing tion of ATM-deficient naive B cells with genomic instability, rescue genomic instability. Here we show that B-cell-specific deletion of the B-cell lymphocytopenia, and promote lymphomagenesis in pre- ATM in MA mice leads to progressive B-cell lymphocytopenia at 6 GC B cells that would otherwise diminish owing to ATM deficiency. and 12 months of age (Figure 6), a phenotype that is overlooked in germline ATM-null mice owing to lethal thymic lymphomas. We μ DISCUSSION further showed that E CyclinD1 promotes the proliferation of naive B-cells and successfully restores B-cell cellularity in older MA Deletion of ATM in early B-cell development is necessary mice (Figure 6). Based on this finding, we propose that CyclinD1 for mature B-cell malignancies promotes B-cell lymphomas by promoting proliferation of Given the mature B-cell phenotypes of most ATM-deficient human ATM-deficient naive B cells with genomic instability, which in B-cell lymphomas and the well-characterized role of ATM during turn maintain the cellularity of tumor-prone ATM-deficient B cells. CSR, it was speculated that ATM maintains genomic stability in GC There are three D-type cyclins (D1, D2 and D3) with overlapping B cells to suppress B-cell lymphomas. However, despite complete functions during embryonic development,41,42 but GC B cells ATM deletion in naive B cells, our attempt to establish an ATM- exclusively depend on CyclinD3 for proliferation and survival,43,44 deficient mature B-cell lymphoma model with naive B-cell-specific which might explain why the pre-GC cells are more susceptible to CD21Cre or less robust pre-B-cell-specific CD19Cre was fruitless. the mitogenic cues unleashed by ectopic CyclinD1. Mb1Cre allele is an early and robust B-cell-specific Cre that leads In summary, we report the first animal model for ATM-deficient to Cre activation in almost all pre-B-cells (in contrast to ~ 60% of B-cell lymphomas and reveal a synergistic role of CyclinD1 pre-B-cells via CD19Cre). In this regard, 16.7% of MA and ~ 55% of expression and ATM deficiency in naive B cells to promote pre- MAD mice developed indolent yet clonal ‘mature’ B-cell lympho- GC B-cell lymphomas. The MA and MAD mature B-cell lymphomas mas (Figure 2b), recapitulating the disease spectrum of human are largely indolent, similar to the Bcl6-driven mouse lymphomas45 lymphomas, especially MCL. Even so, most of the MA and MAD and distinct from Myc-driven aggressive lymphomas. In this regard, tumors are IgM+ and thus have not undergone CSR. Fine mapping MA and MAD lymphomas recapitulate the disease spectrum of of the Igh loci with CGH probe and FISH analyses further revealed human lymphomas, especially MCL,46 and would be an invaluable V(D)J recombination-related, but not CSR-related chromosomal resource to better understand MCL and the clinical progression of fi alterations in MAD tumors. Together these ndings highlight the indolent MCL to an aggressive form in the future. critical tumor suppressor function of ATM in early progenitor or naive B cells, possibly during V(D)J recombination, to prevent mature B-cell lymphomas down the line. In this context, ATM is CONFLICT OF INTEREST known to prevent the persistence and propagation of chromo- The authors declare no conflict of interest. some breaks originating from V(D)J recombination in naive B cells.34 Recent studies reveal that early genetic alterations in hematopoietic stem cells could prime mature B-cell malignancies, ACKNOWLEDGEMENTS 35 including chronic lymphocytic leukemia. We wish to thank Dr Frederick W Alt for providing the ATM conditional mouse model, Dr Klaus Rajewsky for providing the CD19Cre and CD21Cre mice and Dr Michael Reth How ATM promotes lymphomagenesis driven by CyclinD1 for providing the Mb1Cre mice. We also wish to thank Ms Hongyan Tang and Mr Denis Loredan for their technical assistance. We thank Jennifer L Crowe for critical Despite strong evidence for a role of CyclinD1 in lymphomagen- reading of the manuscript. Research reported in this publication was supported by esis, mice with ectopic expression of WT CyclinD1 (MD/D) rarely the NIH/NCI 1RO1CA158073, American Cancer Society (124300-RSG-13-038 DMC) for developed lymphomas. Genomic analyses of human MCL suggest SZ and NIH/NCI PO1 CA174653 for SZ and GB. SZ was a St Baldrick’s Scholar for that ATM deletion coexists with CyclinD1 expression in ~ 50% of Pediatric Cancer and is a Leukemia Lymphomas Society Scholar. MCLs.9 Here we show that ATM deletion promotes B-cell lymphomagenesis in EμCyclinD1 mice.14,36 Although ATM deficiency and the resulting genomic instability might simply promote REFERENCES IgH-CyclinD1 translocation in human MCL, our study suggests that 1 Klein U, Dalla-Favera R. Germinal centres: role in B-cell physiology and loss of ATM also promotes lymphomagenesis after ectopic malignancy. Nat Rev Immunol 2008; 8:22–33. expression of CyclinD1. In this context, we showed that ATM 2 Franco S, Alt FW, Manis JP. 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