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. 2013 Aug 8;12(17):2867–2875. doi: 10.4161/cc.25922

Histone H2AX suppresses translocations in lymphomas of Eμ-c-Myctransgenic mice that contain a germline amplicon of tumor-promoting genes

Angela Fusello 1,2, Julie Horowitz 1,2,3, Katherine Yang-Iott 1,2, Brenna L Brady 1,2,3, Bu Yin 1,2,4, Marta AW Rowh 1,2,3, Eric Rappaport 5, Craig H Bassing 1,2,3,4,*
PMCID: PMC3899199  PMID: 23966158

Abstract

The DNA damage response (DDR) can restrain the ability of oncogenes to cause genomic instability and drive malignant transformation. The gene encoding the histone H2AX DDR factor maps to 11q23, a region frequently altered in human cancers. Since H2ax functions as a haploinsufficient suppressor of B lineage lymphomas with c-Myc amplification and/or translocation, we determined the impact of H2ax expression on the ability of deregulated c-Myc expression to cause genomic instability and drive transformation of B cells. Neither H2ax deficiency nor haploinsufficiency affected the rate of mortality of Eμ-c-Myc mice from B lineage lymphomas with genomic deletions and amplifications. Yet H2ax functioned in a dosage-dependent manner to prevent unbalanced translocations in Eμ-c-Myc tumors, demonstrating that H2ax functions in a haploinsufficient manner to suppress allelic imbalances and limit molecular heterogeneity within and among Eμ-c-Myc lymphomas. Regardless of H2ax copy number, all Eμ-c-Myc tumors contained identical amplification of chromosome 19 sequences spanning 20 genes. Many of these genes encode proteins with tumor-promoting activities, including Cd274, which encodes the PD-L1 programmed death ligand that induces T cell apoptosis and enables cancer cells to escape immune surveillance. This amplicon was in non-malignant B and T cells and non-lymphoid cells, linked to the Eμ-c-Myc transgene, and associated with overexpression of PD-L1 on non-malignant B cells. Our data demonstrate that, in addition to deregulated c-Myc expression, non-malignant B lineage lymphocytes of Eμ-c-Myc transgenic mice may have constitutive amplification and increased expression of other tumor-promoting genes.

Keywords: DNA damage response, Eμ-c-Myc transgenic mice, genomic instability, histone H2AX, lymphoma

Introduction

DNA double-strand breaks (DSBs) arise when the sugar backbones of both DNA strands are fractured in sufficient proximity to interrupt base pairing and liberate DNA ends. DSBs are common and hazardous genomic lesions. They are caused by DNA replication errors in every S phase and by reactive oxygen species generated during cellular metabolism in all cell cycle phases. Liberated DNA ends can be degraded by nucleases and/or separate from one another and aberrantly join with ends released from other DSBs, causing genomic deletions and/or chromosome translocations. Formation of dicentric chromosome translocations can lead to genomic amplifications. Since genomic instability can drive malignant cellular transformation, eukaryotic cells have evolved DDR mechanisms to sense and repair DSBs to facilitate proper repair or induce apoptosis when damage is too severe. A conserved feature of the DDR in response to DSBs is activation of the Ataxia Telangiectasia mutated (ATM) kinase and ATM-mediated phosphorylation of histone H2AX in chromatin around DSBs to form γ-H2AX and create binding sites for other DDR proteins.1 Deregulated expression of proto-oncogenes can induce DSBs through increasing cellular metabolism and promoting DNA replication stress.2-4 Malignant transformation of such cells is associated with disruption of ATM-mediated signaling and accumulation of genomic instability, suggesting that ATM-dependent DDR mechanisms may delay or prevent the ability of oncogenes to cause genomic instability and drive malignant transformation.2-4 Support of this notion was provided by subsequent data that genomic instability and transformation caused by aberrant expression of the c-Myc proto-oncogene is accelerated in Atm-deficient mice.5-7

The C-MYC proto-oncogene encodes a transcription factor that regulates the expression of many genes, including several involved in the control of proliferation and metabolism.8 C-MYC also associates with DNA pre-replication complexes to control DNA replication initiation and origin activity.9 Aberrant C-MYC expression in pre-malignant cells can lead to increased levels of DSB, defective DSB repair, and genomic instability.2,10-12 The first evidence of deregulated C-MYC expression in human cancer was found in Burkitt Lymphoma (BL) cells containing IGH/C-MYC translocations.13 Since its creation almost 30 y ago,14 the Eμ-c-Myc transgenic mouse strain with deregulated c-Myc expression in lymphocytes has been used to model BL and investigate mechanisms that cooperate with or suppress C-MYC driven malignant transformation. Eμ-c-Myc mice succumb to disseminated clonal or oligoclonal B lineage lymphomas of the pre-B or mature B cell stage that generally lack translocations but contain genetic mutations that disrupt cell cycle checkpoints and apoptotic pathways.7,14,15 Increased activation of ATM and phosphorylation of H2AX are detectable in normal and malignant B lineage cells of Eμ-c-Myc transgenic mice.6,7 As compared with Eμ-c-Myc mice, Eμ-c-Myc:Atm−/− mice succumb more rapidly to B lymphoid tumors with translocations,7 demonstrating that ATM-dependent DDR mechanisms restrain the ability of deregulated c-Myc to cause genomic instability and drive malignant transformation of B lineage lymphocytes.

Histone H2ax is a dosage-dependent suppressor of translocations and tumors in mice.16,17 H2AFX, the gene encoding H2AX, maps to the 11q23 cytogenetic region that is frequently altered in human cancers,16 including sporadic breast cancers with mono-allelic H2AFX deletion,18 suggesting that H2AX likely exhibits similar haploinsufficient tumor suppressor functions in humans. H2ax−/− cells exhibit defects in DSB repair and an impaired G2/M checkpoint.19-21 Although H2ax−/− and H2ax+/− cells accumulate chromosome translocations, neither H2ax−/− nor H2ax+/− mice are tumor-prone.16,17 In contrast, H2ax−/−p53−/− and H2ax+/−p53−/− mice rapidly succumb to lymphomas with genomic instability.16,17 Most H2ax−/−p53−/− and H2ax+/−p53−/− B lineage lymphomas contain translocations that fuse the immunoglobulin heavy chain (Igh) locus to the c-myc proto-oncogene, leading to amplification and/or de-regulated expression of c-myc.16,17 These translocations arise through aberrant repair of Igh DSBs induced during V(D)J recombination or class switch recombination with c-myc DSBs.16,17 Formation of γ-H2AX holds together and protects DNA ends from nuclease degradation to facilitate DSB repair and prevent genomic instability.22-25 Thus, H2AX may suppress lymphomas with Igh:c-myc translocations only by inhibiting the formation of these oncogenic lesions. However, considering that deregulated expression of c-Myc and other oncogenes leads to γ-H2ax formation,2-5,7,9,12 histone H2ax also might function downstream of Igh/c-Myc translocations to inhibit oncogene-driven genomic instability and tumor progression. Therefore, to determine whether H2ax can restrain the ability of deregulated c-Myc expression to cause genomic instability and drive malignant transformation, we evaluated the impact of H2ax deficiency and haploinsufficiency upon the tumor phenotype of Eμ-c-Myc transgenic mice.

Results

H2ax deficiency and haploinsuficiency had no impact on the rate at which Eμ-c-Myc transgenic mice succumb to clonal B lineage lymphomas

To determine the impact of H2ax deficiency and haploinsufficiency upon tumor predisposition of Eμ-c-Myc transgenic mice, we generated and aged cohorts of 16 Eμ-c-Myc+/−, 12 Eμ-c-Myc+/−H2ax+/−, and 11 Eμ-c-Myc+/−H2ax−/− mice. Eμ-c-Myc+/− mice succumbed to lymphomas between 53 and 130 d of age, with 50% survival at 75 d (Fig. 1A). Eμ-c-Myc+/−H2ax+/− mice developed fatal lymphomas between 53 and 122 d of age, with 50% survival at 80 d, while Eμ-c-Myc+/−H2ax−/− mice succumbed to lymphomas between 42 and 167 d of age, with 50% survival at 85 d (Fig. 1A). There were no significant differences in rates of mortality from lymphoma among Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− mice. Flow cytometry analyses indicated that the lymphomas in these mice were B lineage lymphomas of the pre-B or mature B cell stage, with no differences in stage distribution among Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− mice (data not shown). Most lymphoid tumors were widely disseminated among several lymph nodes, the thymus, the spleen, and other organs, with no obvious differences among mice of each genotype (data not shown). These data demonstrate that neither H2ax deficiency nor haploinsufficiency affects the rate of mortality of Eμ-c-Myc transgenic mice from disseminated B lineage lymphomas.

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Figure 1. Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− mice exhibit similar predisposition to clonal and oligoclonal B lineage lymphomas. (A) Kaplan–Meier curve showing the tumor-free survival of the 17 Eμ-c-Myc+/−, 12 Eμ-c-Myc+/−H2ax+/−, and 12 Eμ-c-Myc+/−H2ax−/− cohort mice over time. There were no significant differences in survival between mice of any genotype. (B) Southern blot analysis of Igh rearrangements in representative Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− tumors. The schematic of the EcoRI fragment of the JH locus indicates the relative positions of the DQ52 segment, the 4 JH segments, and the 3′JH probe. The blot shows a Southern analysis of EcoR1-digested genomic DNA isolated from the indicated tumors, or kidney from wild-type mice as a control. The positions of the Igh germline (GL) and Eμ-c-Myc (Eμ) bands are indicated.

Southern blot analysis of Igh gene rearrangements can be used to assess whether B lineage lymphomas arise from clonal expansion and transformation of one or more cells. Therefore, we next conducted Southern blot analysis of Igh gene rearrangements in Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− lymphomas. We found that most tumors contained 2 Igh gene rearrangements (Fig. 1B), indicating that they arose from clonal expansion and transformation of one B lineage lymphocyte. However, some harbored 3 or 4 Igh gene rearrangements (Fig. 1B), revealing that they most likely developed from the clonal expansion and transformation and expansion of 2 distinct cells. We detected no significant difference in the percentage of clonal and oligo-clonal B lineage lymphomas among Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− tumors. These data show that neither H2ax deficiency nor haploinsufficiency affects predisposition of Eμ-c-Myc transgenic mice to spontaneous development of clonal or oligoclonal B lineage lymphomas

H2ax deficiency and haploinsuficiency lead to increased frequencies of translocations in Eμ-c-Myc B lineage lymphomas

To determine the impact of H2ax deficiency and haploinsufficiency on the ability of c-Myc to cause translocations in B lineage lymphomas, we conducted spectral karyotyping (SKY) on Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− tumors. No translocations were detected in any of the 6 Eμ-c-Myc tumors assayed (Fig. 2A and Table 1). In contrast, translocations were present in 4 of the 8 Eμ-c-Myc+/−H2ax+/− tumors and 7 of the 8 Eμ-c-Myc+/−H2ax−/− tumors assayed (Fig. 2A and Table 1). One Eμ-c-Myc+/−H2ax+/− tumor harbored a clonal non-reciprocal translocation, while 3 others contained non-clonal non-reciprocal translocations (Table 1). Three Eμ-c-Myc+/−H2ax−/− tumors contained one or more clonal non-reciprocal translocation, and another harbored a clonal reciprocal translocation (Table 1). In addition, 6 Eμ-c-Myc+/−H2ax−/− lymphomas harbored one or more non-clonal non-reciprocal translocation, and another one contained a non-clonal reciprocal translocation (Table 1). Notably, the frequency of tumors with non-reciprocal translocations inversely correlated with H2ax copy number (Fig. 2B). In addition, detached centromeres that arise from un-repaired or mis-repaired DSBs were only detected in Eμ-c-Myc+/−H2ax−/− B lineage lymphomas (Fig. 2C). These data indicate that histone H2AX functions in a haploinsufficient manner to prevent translocations that create allelic imbalances and molecular heterogeneity in and among Eμ-c-Myc malignancies.

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Figure 2. H2ax suppresses unbalanced translocations in B lineage lymphomas of Eμ-c-Myc+/− mice. (A and B) SKY images of metaphases from (A) Eμ-c-Myc+/− tumor #650 and (B) Eμ-c-Myc+/−H2ax−/− tumor #179 with the non-clonal t(15;3) and clonal t(17;16) translocations circled. (C) Graph depicting the frequencies of translocations in metaphases from 6 Eμ-c-Myc+/−, 8 Eμ-c-Myc+/−H2ax+/−, and 8 Eμ-c-Myc+/−H2ax−/− lymphomas. (D) Graph depicting the frequencies of detached centromeres in metaphases from 6 Eμ-c-Myc+/−, 8 Eμ-c-Myc+/−H2ax+/−, and 8 Eμ-c-Myc+/−H2ax−/− lymphomas.

Table 1. SKY analysis of Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax+/− tumors.

Tumor number Tumor genotype Clonal translocations Non-clonal translocations
60 Eμ-c-Myc+/− none none
588 Eμ-c-Myc+/− none none
650 Eμ-c-Myc+/− none none
842 Eμ-c-Myc+/− none none
879 Eμ-c-Myc+/− none none
896 Eμ-c-Myc+/− none none
182 Eμ-c-Myc+/−H2ax+/− none none
183 Eμ-c-Myc+/−H2ax+/− none none
546 Eμ-c-Myc+/−H2ax+/− none t(4;5)
547 Eμ-c-Myc+/−H2ax+/− none none
552 Eμ-c-Myc+/−H2ax+/− none t(14;1)
723 Eμ-c-Myc+/−H2ax+/− none none
857 Eμ-c-Myc+/−H2ax+/− none t(3;19
863 Eμ-c-Myc+/−H2ax+/− t(17;5) none
179 Eμ-c-Myc+/−H2ax−/− t(17;16) t(17;5), t(15;3), t(5;16),
t(15;5), t(7;5)
186 Eμ-c-Myc+/−H2ax−/− t(3;9), t(9;3)* t(12;6), t(15;16), t(15;5),
t(15;3), t(15;14), t(9;16),
t(3;16), t(6;16), t(9;6)
606 Eμ-c-Myc+/−H2ax−/− none none
674 Eμ-c-Myc+/−H2ax−/− none t(17;13), t(17;19), t(16;5)
675 Eμ-c-Myc+/−H2ax−/− none t(5;16), t(17;16)
861 Eμ-c-Myc+/−H2ax−/− t(11;5), t(13;16) t(6;7), t(7;6)*
920 Eμ-c-Myc+/−H2ax−/− t(17;5) t(1;5)
927 Eμ-c-Myc+/−H2ax−/− none t(10;16)
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*Reciprocal translocations.

H2ax deficiency and haploinsuficiency had no impact on the frequencies of genomic deletions and amplifications in Eμ-c-Myc B lineage lymphomas

To determine the impact of H2ax deficiency and haploinsufficiency on the ability of c-Myc to cause genomic deletions and amplifications in B lineage lymphomas, we conducted comparative genomic hybridization (CGH) on DNA isolated from 4 Eμ-c-Myc+/−, 4 Eμ-c-Myc+/−H2ax+/−, and 8 Eμ-c-Myc+/−H2ax−/− tumors, including those already analyzed by SKY. For this CGH analysis, we used control reference DNA from kidneys of wild-type littermate mice to avoid potential complications from kidney infiltration of malignant B lineage cells in Eμ-c-Myc mice. We observed similar numbers of deletions and amplifications in each tumor assayed (Table 2). The frequencies, sizes, and distribution of these lesions were similar among all tumors (data not shown). Some deletions and amplifications were present in more than one tumor, suggesting that regions could be prone to DSBs and/or contain genes that, upon their deletion or amplification, contribute to c-Myc driven malignant transformation. This GCH analysis also confirmed that non-reciprocal translocations identified by SKY caused loss of genomic sequences (Fig. 3A). Collectively, these data demonstrate that H2AX expression has no discernable influence on the ability of the Eμ-c-Myc transgene to cause genomic deletions or amplifications that create allelic imbalances in transformed B lineage cells.

Table 2. CGH analysis of Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax+/− tumors.

Tumor number Tumor
genotype		 Number of deletions Number of amplifications
60 Eμ-c-Myc+/− 30 45
588 Eμ-c-Myc+/− 2 18
842 Eμ-c-Myc+/− 20 28
896 Eμ-c-Myc+/− 60 22
182 Eμ-c-Myc+/−H2ax+/− 38 27
547 Eμ-c-Myc+/−H2ax+/− 3 12
552 Eμ-c-Myc+/−H2ax+/− 60 27
723 Eμ-c-Myc+/−H2ax+/− 24 20
179 Eμ-c-Myc+/−H2ax−/− 9 22
186 Eμ-c-Myc+/−H2ax−/− 42 24
606 Eμ-c-Myc+/−H2ax−/− 34 8
674 Eμ-c-Myc+/−H2ax−/− 4 8
675 Eμ-c-Myc+/−H2ax−/− 20 26
861 Eμ-c-Myc+/−H2ax−/− 37 11
920 Eμ-c-Myc+/−H2ax−/− 41 28
927 Eμ-c-Myc+/−H2ax−/− 79 30
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Figure 3. The Eμ-c-Myc transgene is linked to germline amplification of growth-promoting genes on chromosome 19. (A) CGH images of chromosome 19 signals from Eμ-c-Myc+/−H2ax−/− tumors #674, #179, and #606. The green and red signals indicate copy number increases or decreases, respectively. Deletion of chromosome 19 sequences in tumor #674 was associated with a non-clonal, non-reciprocal t(17;19) translocation. (B) FISH images of Chr19Amp hybridization on chromosome 19 in a metaphase from Eμ-c-Myc+/− tumor #723. (C and D) FISH images of Chr19Amp on one copy of chromosome 19 in metaphases from Eμ-c-Myc+/− or wild-type (C) B lymphocytes or (D) T lymphocytes. (E) FISH images of Chr19Amp on one copy of chromosome 19 in metaphases from Eμ-c-Myc+/− or wild-type MEFs from littermates. (E) FISH images of Chr19Amp and c-Myc on one copy of chromosome 19 in metaphases from Eμ-c-Myc+/− MEFs. (F) Flow cytometric analysis of cell surface PD-L1 expression on non-malignant bone marrow and splenic B lymphocytes in Eμ-c-Myc+/− and wild-type mouse littermates. This experiment was conducted on 3 mice of each genotype. Asterisks and lines indicate significant differences where P < 0.0025.

Eμ-c-Myc transgenic mice contain a germline amplicon of tumor-promoting genes

Strikingly, every Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− tumor that we analyzed by CGH contained an identical 3 megabase (Mb) amplification on chromosome 19 that spans 20 known genes (Fig. 3A). This region of chromosome 19 exhibits synteny to a region of human chromosome 9 (9p24) that is often amplified in human primary mediastinal B-cell lymphoma (PMBL) and Hodgkins lymphoma (HL) cells.26-33 The common region of amplification among these human and Eμ-c-Myc malignancies contains 15 genes, many of which are known to promote cell growth (Table 3).34 Since this amplicon was identical in all 20 tumors containing the Eμ-c-Myc transgene, and we used reference DNA from littermate wild-type mice, we suspected that these sequences were amplified in the germline of our Eμ-c-Myc+/− mice. Consistent with this notion, we detected this amplicon in kidney DNA of Eμ-c-Myc+/− mice, with no evidence of lymphoma in the bone marrow or spleen using reference DNA from a wild-type mouse kidney (data not shown). To identify the amplicon in single cells, we conducted fluorescence in situ hybridization (FISH) with a probe that hybridizes to genomic sequences in this amplicon, referred to as the Chr19Amp probe. We first performed this assay on metaphase spreads prepared from Eμ-c-Myc+/− tumors to show that we could detect amplification of Chr19Amp-hybridizing sequences on one copy of chromosome 19 (Fig. 3B). We then analyzed metaphase spreads prepared from bone marrow B lineage cells and thymus T lineage cells of young Eμ-c-Myc+/− mice with no evidence of lymphoma in the bone marrow or spleen. We observed Chr19Amp signals on one copy of chromosome 19 in every metaphase of non-malignant B and T cells from Eμ-c-Myc+/− mice, but not in any metaphases from littermate wild-type control mice (Fig. 3C and D), indicating that the amplicon is present in all non-malignant lymphocytes of Eμ-c-Myc transgenic mice.

Table 3. Genes in the amplicon on chromosome 19 of Eμ-c-Myc+/− mice.

Gene Encoded protein
Glis3 GLIS family zing finger 3
Slc7a7 Solute carrier family 7, member 7
Ppapdc2 Phosphatidic acid phosphatase type 2 domain containing 2
Cdc37l1 Cell division cycle 37-like
Ak3 Adenylate kinase 3
Rcl1 RNA terminal phosphate cyclase-like 1
Jak2 Janus kinase 2
Insl6 Insulin-like 6
Cd274 Programmed cell death ligand 1
Pdcd7lg2 Programmed cell death ligand 2
Mlana Melanoma antigen recognized by T cells
Ranbp6 RAN binding protein 6
Il33 Interleukin 33
Trpd52l3 Tumor protein D52-like 3
Uhrf2 Ubiquitin-like, containing PHD and RING finger domains 2
Gldc Glycine decarboxylase
Mbl2 Mannose-binding lectin protein C
Dkk1 Dickkopf homolog 1
Prkg1 Protein kinase, cGMP-dependent, type 1
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To assess whether the chromosome 19 amplicon and Eμ-c-Myc transgene are linked and present in the germline, we generated and analyzed metaphase spreads of mouse embryonic fibroblasts (MEFs) from Eμ-c-Myc+/− mice and wild-type mouse littermates. We conducted FISH with a chromosome 19 paint and a probe that spans c-Myc, which resides on chromosome 15. In addition to 2 normal c-Myc probe signals, we detected a c-Myc probe signal on chromosome 19 in Eμ-c-Myc+/− but not in wild-type MEFs (Fig. 3E), revealing that the Eμ-c-Myc transgene is integrated into chromosome 19 in our Eμ-c-Myc+/− mice. To determine whether the amplicon is present in the germline of Eμ-c-Myc mice, and, if so, whether the Eμ-c-Myc transgene is integrated near this lesion, we conducted FISH with c-Myc and Chr19Amp probes. In addition to normal c-Myc probe signals (on chromosome 15) and a Chr19Amp probe signal on one chromosome, we saw co-localization Chr19Amp and c-Myc probe signals on another chromosome in each metaphase in Eμ-c-Myc+/−, but not wild-type, MEFs (Fig. 3F). These data demonstrate that the chromosome 19 amplicon is present in the germline of Eμ-c-Myc+/− mice, and that the Eμ-c-Myc transgene is integrated near these amplified sequences.

Amplification of the syntenic region in human PMBL and HL cells leads to increased expression of the amplified genes, including Cd274, which encodes the PD-L1 programmed death ligand that induces T cell apoptosis and enables cancer cells to escape immune surveillance.35 Thus, to determine whether the chromosome 19 amplicon in our Eμ-c-Myc transgenic mice leads to increased expression of an amplified gene, we used flow cytometry to quantity PD-L1 expression on the surface of bone marrow and splenic B lineage cells of young Eμ-c-Myc+/− mice without evidence of lymphoma. We found a 4–5-fold greater level of PD-L1 expression on non-malignant B lymphocytes in our Eμ-c-Myc+/− mice as compared with wild-type mouse littermates (Fig. 3G), showing that the germline amplification of Cd274 in our Eμ-c-Myc+/− mice leads to enhanced PD-L1 expression. This finding demonstrates that, in addition to deregulated c-Myc expression, the non-malignant B lineage cells of our Eμ-c-Myc transgenic mice have increased expression of at least one tumor-promoting gene in the chromosome 19 amplicon.

Discussion

We have shown that, although neither H2ax deficiency nor haploinsufficiency alters the rate of mortality of Eμ-c-Myc+/− mice from B lineage lymphomas with genomic deletions and amplifications, H2ax functions in a haploinsufficient manner to suppress unbalanced clonal and non-clonal translocations in Eμ-c-Myc+/− tumors. This result is different from previous findings that Atm inactivation in Eμ-c-Myc transgenic mice accelerates the rate of onset of B cell lymphomas and causes unbalanced clonal and non-clonal translocations.6,7 The distinct cancer phenotypes of Eμ-c-Myc, Eμ-c-Myc:Atm−/−, and Eμ-c-Myc:H2ax−/− mice indicates that ATM-dependent, H2AX-independent mechanisms suppress initiation and/or progression of Eμ-c-Myc B cell lymphomas, whereas ATM-dependent, H2AX-dependent mechanisms prevent generation and/or outgrowth of unbalanced translocations in Eμ-c-Myc tumors. In addition to H2AX, ATM phosphorylates and activates many proteins in response to DSBs, including the p53 tumor suppressor protein that controls the G1/S cell cycle checkpoint and induces apoptosis or senescence when DNA damage is too severe.36 Accordingly, Atm-deficient, but neither H2ax-deficient nor H2ax-haploinsufficient, cells exhibit impaired p53-mediated DNA damage responses.20 Considering that non-malignant B cells of Eμ-c-Myc mice exhibit increased p53 activation, Eμ-c-Myc tumors frequently harbor inactivating p53 mutations, and p53 deficiency accelerates the onset of lymphomas in Eμ-c-Myc mice,15,37 our data suggests that activation of p53 is the critical ATM-dependent, H2AX-independent DDR mechanism that delays oncogene-driven B cell transformation in Eμ-c-Myc mice. However, due to amplification of chromosome 19 oncogenes in Eμ-c-Myc transgenic mice, additional studies are required to determine whether H2AX-dependent DDR mechanisms suppress malignant transformation of B cells, in which deregulated c-Myc expression is the only oncogenic lesion.

We have shown that decreases in H2ax copy number lead to unbalanced clonal and non-clonal translocations in B cell lymphomas of Eμ-c-Myc+/− mice without altering the frequencies of genomic deletions and amplifications in these tumors. Genomic instability in Eμ-c-Myc+/−, Eμ-c-Myc+/−H2ax+/−, and Eμ-c-Myc+/−H2ax−/− lymphomas likely arises from increased frequencies of DSBs caused by aberrant DNA replication and elevated reactive oxygen species in cells with deregulated c-Myc expression. Translocations are only generated through aberrant joining of DNA ends liberated from DSBs induced on distinct chromosomes,38 while deletions and amplifications can form through aberrant joining of DNA ends from replication-associated DSBs induced on the same chromosome.39,40 Proliferating H2ax+/− and H2ax−/− cells accumulate translocations at higher frequencies than wild-type cells.16,17 Inactivation of p53 has no effect upon the frequencies of these translocations, yet leads to development of H2ax-deficient and haploinsufficient lymphomas with oncogenic translocations.16,17,22,23 In response to DSBs, ATM phosphorylates H2AX to promote chromatin changes that stabilize broken DNA strands to facilitate normal DSB repair and prevent liberated DNA ends from separating and aberrantly joining to generate translocations during cell cycle progression.22-24 We conclude that these ATM-dependent, H2AX-dependent chromatin changes suppress formation of unbalanced translocations in Eμ-c-Myc B lineage lymphomas, regardless of whether p53 DNA damage responses remain active. Although the unbalanced translocations that arise from decreased H2ax copy number do not accelerate the mortality rate of Eμ-c-Myc mice from lymphoma, these lesions create allelic imbalances that increase molecular heterogeneity in and among Eμ-c-Myc tumors. Therefore, regardless of whether H2ax deficiency or haploinsufficiency causes genomic instability that drives oncogene-mediated transformation in other contexts, our data indicates that mono-allelic H2AFX deletion in human cancers may cause genomic changes that contribute to relapse and development of drug resistance cancer cells.

Our discovery that the Eμ-c-Myc transgene integration in our mice is linked with amplification of chromosome 19 sequences spanning Cd274 and other tumor-promoting genes has important and broad implications. Several independent Eμ-c-Myc transgenic mouse lines were created in 1985 and found to exhibit similar predisposition to B lineage lymphomas.14 Eμ-c-Myc mice have been widely distributed and used over the past 28 y to model BL and elucidate molecular mechanisms that cooperate with or suppress C-MYC-driven lymphomagenesis. The chromosome 19 amplification could have arisen during breeding of descendant Eμ-c-Myc transgenic mice in our colony, or in a colony from which our mice derived. Alternatively, the original integration of the Eμ-c-Myc transgene in the founder animal from which our Eμ-c-Myc mice descended could have occurred in association with duplication of these chromosome 19 sequences. This latter possibility might explain why gene expression signatures of Eμ-c-Myc tumors have been found to resemble diffuse large B cell lymphomas like PMBLs rather than BL.41 We showed that non-malignant B lineage cells of our Eμ-c-Myc transgenic mice have increased expression of at least one tumor-promoting gene in the chromosome 19 amplicon. In this context, our data demonstrate that, in addition to deregulated c-Myc expression, non-malignant B lineage cells of some Eμ-c-Myc transgenic mice may have constitutive amplification and increased expression of other tumor-promoting genes. Consequently, some conclusions attributed to deregulated c-Myc expression through studies using Eμ-c-Myc mice may need re-evaluation to determine potential contributions from germline amplification of chromosome 19 genes.

Materials and Methods

Mice

Eμ-c-Myc mice14 on a C57BL/6 background were obtained from Dr John Cleveland. We generated and have maintained H2ax+/− and H2ax−/− mice on a 129S6/SvEv background.16 We purchased and maintained wild-type 129S6/SvEv mice (Taconic). We bred Eμ-c-Myc+/− males with H2ax−/− females to generate Eμ-c-Myc+/−H2ax+/− males that were crossed with wild-type or H2ax−/− females to generate Eμ-c-Myc+/− or Eμ-c-Myc+/−H2ax+/− and Eμ-c-Myc+/−H2ax−/− mice. All mouse experiments were performed in accordance with national guidelines and regulations and approved by the Institutional Animal Care and Use Committee of the Children's Hospital of Philadelphia.

Southern blotting

Genomic DNA (20–30 μg) of B lineage lymphomas or kidneys from wild-type animals was digested with 100 units of EcoRI (New England Biolabs), separated on a 1.0% TAE agarose gel, transferred onto Zeta-probe membrane (BioRad), and hybridized with a 32P-labeled JH probe as described.16

Cytogenetics

Metaphase spreads were prepared as described.24 Spectral karyotyping and chromosome painting were performed by manufacturer’s instructions (Applied Spectral Imaging [ASI]). FISH Bacterial Artificial Chromosome probes were labeled using a Biotin-Nick Translation Mix kit (Roche). The Chr19Amp BAC RP24–118N10 was purchased from the Children’s Hospital of Oakland Research Institute. The c-myc 307D14 BAC has been described.42 Slides were examined under an Olympus BX61 microscope (magnification: 600×), controlled by a LAMBDA 10-B Smart Shutter from Sutter Instrument. Images were captured using a Sutter Instrument LAMBDA LS light source and an ASI COOL-1300QS camera, and analyzed through ASI Case Data Manager Version 5.5.

Comparative genomic hybridization

Comparative genomic hybridization was conducted on DNA (0.5–1.5 μg) of lymphomas or kidneys from wild-type or Eμ-c-Myc+/− mice according to the manufacturer's instructions using the following Agilent kits: Genomic DNA Enzymatic Labeling, Genomic DNA Purification, Oligo aCGH/ChIP-on-chip Hybridization, SurePrint G3 Mouse Genome CGH Microarray 4× 180 K, and Stabilization and Drying Solution Kit. Hybridized arrays were scanned with the Agilent High-Resolution C Scanner, which had been upgraded to scan down to 2 micron resolution. TIFF files from the scanner were processed using Agilent Feature Extraction software (version 10.7.1.1), including manual correction of spot finding and generation of quality control metrics. Extracted intensities were analyzed, visualized, and annotated using Agilent’s Genomic Workbench (version 5.0.14).

PD-L1 Flow cytometry

Single-cell suspensions from 4–6-wk-old mice were stained in 2% PBS with the following antibodies from BD PharMingen: PE/Cy7-conjugated anti-B220, APC-conjugated anti-IgM, PE-conjugated anti-PD-L1, and/or PE-conjugated IgG2a, Igλ isotype control. Data were collected using a FACSCalibur and CellQuest software from BD Biosciences and analyzed using FlowJo software (Tree Star).

Acknowledgments

We thank Amy DeMicco for technical help with figure construction. This research was supported by Training Grant TG GM-07229 of the University of Pennsylvania (BLB.); the Cancer Research Institute Pre-doctoral Emphasis Pathway in Tumor Immunology Training Grant awarded to the University of Pennsylvania (BY); the Training Program Rheumatic Disease of the University of Pennsylvania 5T32-AR007442-23 (MAWR); and a Leukemia and Lymphoma Society Scholar Award, and the National Institutes of Health R01 Grants CA125195 and CA136470 (CHB).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

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