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Blood First Edition Paper, prepublished online September 18, 2015; DOI 10.1182/blood-2015-02-628164
AID-associated DNA repair pathways regulate malignant transformation in a
murine model of BCL6-driven diffuse large B cell lymphoma
Xiwen Gu,1 Carmen J. Booth,2 Zongzhi Liu,3 and Matthew P. Strout1
1
Yale Cancer Center, Section of Hematology, 2Section of Comparative Medicine, and
3
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510
USA.
Running title: AID-associated DNA repair and lymphomagenesis
Correspondence:
Matthew P. Strout
Yale Cancer Center, Section of Hematology
Yale University School of Medicine
300 George Street
New Haven, CT 06511
Phone: 203-737-6062
Fax: 203-785-7232
E-mail: [email protected]
Word count, abstract: 259
Word count, text: 4,068
Figures/tables: 6 figures/1 table
References: 59
1
Copyright © 2015 American Society of Hematology
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Key Points
- The combined effects of AID-associated base excision and mismatch repair delay the
development of BCL6-driven DLBCL.
- Uracil DNA glycosylase (UNG) single deficiency prevents the development of BCL6driven DLBCL.
2
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Abstract
Somatic hypermutation and class switch recombination of the immunoglobulin (Ig)
genes occur in germinal center (GC) B cells and are initiated through deamination of
cytidine to uracil by activation-induced cytidine deaminase (AID). Resulting uracilguanine mismatches are subsequently processed by UNG-mediated base-excision
repair (BER) and MSH2-mediated mismatch repair (MMR) to yield mutations and DNA
strand lesions. Although off-target AID activity also contributes to oncogenic point
mutations and chromosome translocations associated with GC and post-GC B cell
lymphomas, the role of downstream AID-associated DNA repair pathways in the
pathogenesis of lymphoma is unknown. Here, we show that simultaneous deficiency of
UNG and MSH2 or MSH2 alone causes genomic instability and a shorter latency to the
development of BCL6-driven diffuse large B cell lymphoma (DLBCL) in a murine model.
Despite its mutagenic role during immune diversification, the additional development of
several other types of BCL6-independent malignancies underscores the critical role of
MMR in maintaining general genomic stability. In contrast, absence of UNG alone is
highly protective and prevents the development of BCL6-driven DLBCL. We further
demonstrate in the DLBCLs that clonal and non-clonal mutations arise within non-Ig AID
target genes in the combined absence of UNG and MSH2 and that DNA strand lesions
can arise in an UNG-dependent manner but are offset by MSH2. These findings lend
insight into a complex interplay whereby potentially lymphomagenic UNG activity and
general genomic instability are opposed by the protective influence of MSH2, producing
a net protective effect that promotes SHM and CSR of the Ig genes while
simultaneously attenuating malignant transformation of GC B cells.
3
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Introduction
Acquired somatic mutations and chromosome translocations are a hallmark of cancer
that arise as a pathologic result of a DNA repair response to a genotoxic event.1 In
contrast, the introduction of non-templated nucleotides and DNA double-strand breaks
(DSBs) is part of the normal developmental program in germinal center (GC) B cells.
Somatic hypermutation (SHM) and class switch recombination (CSR) of the
immunoglobulin (Ig) genes promotes antibody diversification in GC B cells but the
nature of these genetic remodeling events makes these cells uniquely vulnerable to
malignant transformation.2 Numerous types of B cell malignancies arise from GC and
post-GC B cells including diffuse large B cell lymphoma (DLBCL), Burkitt lymphoma,
follicular lymphoma (FL), lymphoplasmacytic lymphoma, multiple myeloma, and
Hodgkin lymphoma.3 SHM and CSR are both initiated by activation-induced cytidine
deaminase (AID),4 a GC B cell enzyme that lacks strict target specificity and is also able
to introduce mutations and DSBs into non-Ig genes throughout the genome.5-12 A role
for AID in lymphomagenesis is supported by the presence of characteristic somatic
mutations within numerous oncogenes associated with human GC and post-GC B cell
malignancies.13-20 In addition, a prominent feature of these cancers are chromosome
translocations that arise as a consequence of AID-mediated DSBs within the Ig heavy
chain (IgH) class switch (S) region and a partner oncogene such as BCL6 and
cMYC.2,21 Further evidence implicating a direct role for AID in lymphomagenesis stems
from several mouse models where the development and phenotype of B cell lymphoma
is dependent on AID.10,22-24
Despite its DNA modification properties, it is well established that AID does not act
alone.25 Base-excision repair (BER) and mismatch repair (MMR) pathways are required
to process AID-generated uracil-guanine (U-G) mismatches into mutations and DNA
strand lesions.9,26-29 During SHM, removal of uracil by uracil DNA glycosylase (UNG)
creates an abasic site that serves as a template for DNA replication which could result
in any nucleotide substitution.30 Alternatively, the abasic site can be excised by
apurinic/apyridimic endonucleases (APE1 and APE2) and filled in by low-fidelity trans4
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lesion DNA polymerases.31,32 The MMR heterodimer MutSα (MSH2 and MSH6) can
also recognize and facilitate removal of the U-G mismatch with exonuclease 1 (EXO1)
activity creating a gap that is filled in by low-fidelity DNA polymerases.33 This permits
spreading of mutations to surrounding A/T base pairs. During CSR, DNA single-strand
breaks (SSBs) on opposite strands within IgH S regions are created through uracil
removal by UNG and APE activity resulting in staggered DSBs if located in close
proximity.31 If distantly located, these SSBs provide entry points for MutSα recruitment
of EXO1 with consequent strand resection.34 Resulting DSBs are subsequently ligated
by canonical non-homologous and alternative end-joining.35 These events are also
thought to be responsible for strand lesions that lead to chromosome translocations.2
There are no other known repair pathways involved in the resolution of AID-generated
U-G mismatches and it is unknown how these pathways contribute to malignant
transformation of GC B cells. To explore this question, we utilized a murine model to
examine BCL6-driven AID-dependent GC B cell lymphomagenesis in the absence of
UNG (BER) and MSH2 (MMR).
Materials and Methods
Mice
All mice were bred onto a C57BL/6 background. IµHABcl6, Ung+/-, and Msh2+/- mice
were used to generate IµHABcl6 Ung+/- Msh2+/- and Ung-/- Msh2+/- mice.36-39
Intercrossing of the offspring generated IµHABcl6, IµHABcl6 Ung-/-, IµHABcl6 Msh2-/-,
IµHABcl6 Ung-/- Msh2-/- mice as well as control Ung-/-, Msh2-/-, and Ung-/- Msh2-/- mice.
Genotyping was performed by PCR as previously described.26,36 Mice were housed in
the Yale Animal Resource Center and all procedures involving mice were approved by
the Yale Institutional Animal Care and Use Committee (Yale IACUC protocol #11403).
Statistical analysis of survival was performed by Kaplan-Meier survival and log-rank
(Mantel-Cox) tests to assess whether survival differences were significant. The MannWhitney test was used to compare median DLBCL latency.
Flow cytometry, histopathology and immunohistochemistry
5
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At necropsy, involved tissues were collected for cellular, histological, and molecular
analysis. For analysis of CSR from IgM to IgG1, splenic B cells were activated ex vivo
with LPS, 20 µg/mL and IL4, 10 ng/mL for ~72 hours. For immunophenotyping, cells
were stained with fluorochrome-conjugated antibodies against CD3, B220, IgM, CD95,
CD138, and IgG1 (BD Pharmingen). For γH2AX analysis, activated B cells were fixed
in 70% ethanol then incubated with rabbit anti-γH2AX antibody (Abcam 81299) followed
by Alexa Fluor 647-conjugated goat-anti-rabbit secondary antibody (Abcam). After
washing, cells were incubated with 1 µg/ml DAPI to stain DNA. Data were acquired on
a FACSCalibur or a Stratedigm S1000 flow cytometer and analyzed with FlowJo
software. For histopathology, formalin-fixed paraffin-embedded sections were stained
with H&E and biotinylated PNA (Vector, B-1075) by standard methods.
Clonality and mutation analysis
Splenic and Peyer’s patch B cells from healthy IµHABcl6 and IµHABcl6 Ung-/- Msh2-/mice were used as controls. All oligonucleotide primer sequences are listed in
supplemental Table 1. To assess clonality, the rearranged VH sequence was amplified
from genomic DNA using a mixture of forward primers designed to represent most
mouse VH gene families and a reverse primer from the JH4 intron as previously
described.36 Using this protocol, four major bands corresponding to rearranged JH1,
JH2, JH3 and JH4 segments can be detected from a normal B cell population while only
one major band will arise from clonal malignant B cells.40 Mutation analysis of Ig (JH4
intron, IgH 5'Sµ, and core IgH Sµ) and non-Ig (cMyc, Pim1, RhoH, Cd79a, CD79b,
H2afx, Pax5, and Cd83) AID target loci was carried out by PCR amplification using
high-fidelity Phusion polymerase (New England Biolabs) followed by sequencing of
amplification and TA cloned products. Sequences were compared with NCBI
references and with sequences from healthy littermates to identify both clonal and nonclonal mutations. Sequence analysis and statistical comparisons were performed as
previously described.9 Clonal and subclonal mutations were only counted once toward
the mutation frequency. Deletion frequency was calculated as number of events per
cloned sequence. Statistical significance of mutation frequencies was assessed by
Pearson’s chi-squared test. Fold-enrichment of mutations in AID hotspots
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(DGYW/WRCH) was calculated as the ratio of observed-to-expected frequency of
hotspot C/G to T/A mutations.
Gene expression profiling
Total RNA from tumors was purified using the RNeasy kit (Qiagen). cRNA labeling,
hybridization to Illumina MouseWG-6 v2.0 BeadChips and scanning were performed
according to the manufacturer’s instructions (Illumina). Analysis was performed using
the lumi and limma in Bioconductor R package. The expression data reported in this
paper have been deposited in the NCBI Gene Expression Omnibus database under
series accession number GSE48304.
Spectral karyotyping
Metaphase slides were prepared from lymphoma cells grown in culture according to
standard cytogenetic procedures. Mouse SkyPaint probe (Applied Spectral Imaging,
ASI) was added to each slide according to standard ASI protocol. Image acquisition
was performed with a COOL-1300 SpectraCube camera (ASI) mounted on an Olympus
BX43 microscope using a SKY optical filter (ASI). For each sample, a minimum of 20
metaphases was analyzed for chromosome abnormalities using the HiSKY v6.0
software (ASI).
Results
BCL6-driven lymphomas can arise in the combined absence of BER and MMR
BCL6 is considered the master regulator of the GC response where its physiologic role
in B cells is to establish a molecular environment that permits the accumulation of
mutations through transcriptional repression of DNA damage response, cell cycle arrest,
and B cell maturation.41 BCL6 is often constitutively expressed in human DLBCL,
typically as a result of a chromosome translocation with an Ig locus. Similarly, through
deregulated expression of BCL6, IµHABcl6 mice spontaneously develop a clonal GCderived lymphoma that emulates human DLBCL.36 In these mice, enforced B cell
specific expression of BCL6 is achieved through the insertion of a full-length HA-tagged
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murine Bcl6 coding sequence downstream of the IgH Iµ promoter. In the absence of
AID, tumor incidence in these mice is markedly reduced and phenotype is restricted to
marginal zone lymphoproliferations, supporting the notion that AID is required for GCderived lymphomagenesis.24 In non-malignant B cells that are deficient in both UNG
and MSH2, U-G mismatches are not recognized and are simply replicated, revealing the
footprint of AID by yielding C/G to T/A transitions.9,26,28,29 Thus, to investigate the role of
AID-associated BER and MMR in the pathogenesis of GC lymphoma, we crossed
IµHABcl6 mice onto a background deficient in both UNG (Ung-/-)39 and MSH2 (Msh2-/).38
Young (<3 months) healthy IµHABcl6 and IµHABcl6 Ung-/- Msh2-/- mice displayed similar
GC architecture (supplemental Figure 1) and a normal distribution of B and T cells
(Figure 1). Consistent with previous studies,24,36 6 of 22 (27.3%) IµHABcl6 mice
became sick starting at ~12 months of age. However, 29 of 33 (87.9%) IµHABcl6 Ung-/Msh2-/- mice became sick rapidly with an onset as early as 3 months and a median
survival of 6.8 months (Figure 2A). Sick mice in both groups were found to be
moribund with enlarged spleens and variable nodal and thymic involvement. All
IµHABcl6 tumors analyzed were derived from mature B220+ IgM+ CD138- B cells
(Figure 1). Of 19 IµHABcl6 Ung-/- Msh2-/- tumors available for characterization,
immunophenotyping demonstrated 8 mature B220+ IgM+ CD138- B cell lymphomas
(Figure 1), 3 pre-B cell lymphomas, 7 T cell lymphomas, and 1 histiocytic sarcoma
(Figure 2B). UNG-deficient mice have been shown to develop a FL while MSH2deficient mice develop numerous malignancies but predominantly pre-B cell and T cell
lymphomas.37,38,42,43 As a central component of MMR, MSH2 provides global protection
against genomic instability.44,45 Indeed, among 18 Ung-/- Msh2-/- control mice, median
survival was 7 months with 3 pre-B cell lymphomas and 5 T cell lymphomas but no
mature B cell lymphomas (Figure 2A-B). Thus, the development of pre-B cell and T
cell malignancies in IµHABcl6 Ung-/- Msh2-/- mice is independent of BCL6 and is likely a
reflection of MSH2 deficiency. Moreover, this data demonstrates that mature BCL6driven B cell lymphomas can arise independent of AID-associated BER and MMR.
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BER and MMR regulate the latency of BCL6-driven lymphomagenesis
Focused examination of the mature B cell lymphomas from both genotypic groups
showed clonal IgH gene rearrangements (Figure 3A), expression of GC markers
(Fas/CD95, PNA) (Figures 1, 4), and disruption of lymphoid architecture with infiltration
by large lymphoid cells consistent with GC-derived DLBCL (Figure 4). Analysis of gene
expression profiles of representative tumors did not reveal any consistent differences
between DLBCLs from each genotype (Figure 3B) and clearly distinguished the
DLBCLs from pre-B cell lymphomas (Figure 3C, supplemental Table 2). Although the
background effect of MSH2 deficiency on the development of other malignancies
precludes an accurate comparison of the true incidence of DLBCL between genotypes,
the median time to development of disease was 2.5-fold shorter in IµHABcl6 Ung-/Msh2-/- mice compared with IµHABcl6 mice (Figure 3D). This led to a significantly
shortened overall survival for mice with DLBCL in the IµHABcl6 Ung-/- Msh2-/- group
(Figure 3E). Thus, BER and MMR pathways exert a combined protective effect by
increasing the latency period to BCL6-driven lymphomagenesis.
Absence of UNG prevents development of BCL6-driven DLBCL
To investigate the contribution of individual BER and MMR pathways to the
pathogenesis of GC lymphoma, we generated IµHABcl6 Ung-/- and IµHABcl6 Msh2-/single-deficient mice. The majority of IµHABcl6 Ung-/- mice remained healthy beyond
20 months with only 3 of 22 (13.6%) mice becoming sick starting at ~16 months (P =
0.018; Figure 2A-B). These mice were found to have clonal splenic lymphomas
comprised of mature B220+ IgM+ CD138- B cells (data not shown). Similar to the low
penetrant FLs which arise after a prolonged latency in the absence of UNG,37 these
lymphomas had expanded follicles with a population of small lymphocytes that were
negative or only weakly positive for PNA (Figure 4). Thus, in the setting of deregulated
BCL6, loss of UNG confers a significant survival advantage by preventing GC B cells
from evolving into DLBCL. Conversely, this also suggests that UNG might play an
active role in facilitating events that lead to GC B cell transformation.
Absence of MSH2 promotes development of BCL6-driven DLBCL
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In contrast to IµHABcl6 Ung-/- mice, 20 of 22 (90.9%) IµHABcl6 Msh2-/- mice rapidly
succumbed to malignancy starting at ~3 months and had a median survival of 6 months
(Figure 2A). Of 16 tumors available for analysis, there were 3 DLBCLs, 7 pre-B cell
lymphomas (Figure 4), 4 T cell lymphomas, 1 histiocytic sarcoma, and 1 squamous cell
carcinoma (Figure 2B). Similar to the IµHABcl6 Ung-/- Msh2-/- mice, the development of
non-GC B cell malignancies in the IµHABcl6 Msh2-/- mice is likely a reflection of MSH2
deficiency that occurs independent of BCL6. Analysis of gene expression profiles
clustered the IµHABcl6 Msh2-/- DLBCLs with DLBCLs from IµHABcl6 and IµHABcl6
Ung-/- Msh2-/- mice (Figure 3B-C). In addition, independent analysis of latency and
overall survival specifically from DLBCL indicated a trend that was similar to that of
IµHABcl6 Ung-/- Msh2-/- mice (Figure 3D-E). Although the technical challenge of also
deleting AID from our model limited our ability to fully assess its contribution to UNG
and MSH2-dependent lymphomagenesis, the development of other malignancies from
cell types that do not express AID exposes a critical role for MSH2 that extends beyond
immune diversification. Altogether, these results demonstrate that IµHABcl6, IµHABcl6
Ung-/- Msh2-/- and IµHABcl6 Msh2-/- mice develop the same GC B cell lymphoma but
have the most favorable survival when MSH2 is present. This indicates that in contrast
to UNG-mediated BER, MMR provides a protective advantage against BCL6-driven
DLBCL.
BER and MMR protect against widespread cytidine deamination
The impact of UNG and MSH2 single deficiency on the development of BCL6-driven
DLBCL highlights the presence of opposing pathways to lymphomagenesis. However,
the rapid development of DLBCL in the absence of both UNG and MSH2 suggests the
existence of a complex and unbalanced interplay between BER and MER whereby the
tumor promoting potential of MSH2 deficiency is dominant over the protective effect of
UNG deficiency. To gain insight into these mutagenic mechanisms, we sequenced AID
target loci in B cell tumors from selected genotypes.
Analysis of the IgH JH4 intronic region from IµHABcl6 and IµHABcl6 Ung-/- Msh2-/DLBCLs revealed similar frequencies of both clonal and non-clonal mutations while only
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non-clonal mutations were detected in IµHABcl6 Ung-/- FLs and IµHABcl6 Msh2-/DLBCLs (Figure 5A). As expected, the majority of IgH JH4 intronic mutations in
healthy IµHABcl6 Ung-/-Msh2-/- GC B cells and IµHABcl6 Ung-/-Msh2-/- DLBCLs were
C/G to T/A transitions (Figure 5B). However, in contrast to healthy IµHABcl6 GC B
cells, the majority of mutations in the remaining lymphomas were also C/G to T/A
transitions (Figure 5B). Overall mutation frequencies within the IgH JH4 intronic region
were 4 to 24-fold lower in tumors compared with healthy IµHABcl6 GC B cells (Figure
5B) yet analysis of the IgH 5'Sµ region did not demonstrate any significant differences
in mutation frequencies between ex vivo activated B cells from healthy mice and mature
B cell lymphomas (Figure 5C). Nonetheless, much like many human lymphomas,46
these tumors remained IgM+ and do not undergo CSR, suggesting an underlying defect
in downstream processing of AID-generated U-G mismatches (Figure 5C). Additional
sequencing of eight non-Ig AID target genes (cMyc, Pim1, RhoH, Cd79a, CD79b, H2afx,
Pax5, and Cd83)9 in 12 B cell tumors (7 DLBCL, 3 FL, 2 pre-B cell) revealed six unique
clonal mutations within Pim1 (3 mutations), Pax5 (2 mutations), and RhoH (1 mutation)
(Figures 5A, D). These mutations were found only in DLBCLs from IµHABcl6 Ung-/Msh2-/- mice and were all C/G to T/A transitions. Further analysis of Pim1 and RhoH
also revealed non-clonal mutation frequencies in IµHABcl6 Ung-/- Msh2-/- DLBCLs that
were significantly higher than in the other lymphomas (Figure 5A). Collectively, the
presence of clonal and non-clonal C/G to T/A transitions indicates that ongoing AID
activity targets Ig and non-Ig loci in these lymphomas. In addition, although the IgH JH4
intron appears to be targeted less frequently by AID in these lymphomas, the higher
frequency of clonal and non-clonal mutations in non-Ig genes in the absence of UNG
and MSH2 supports the notion that the net effect of BER and MMR might restrict
lymphomagenesis by preventing widespread accumulation of C/G to T/A transition
mutations.
Absence of MMR reveals UNG-dependent and independent genomic instability
During our non-Ig gene mutational analysis, we also identified numerous deletions
within RhoH. This locus contains two microsatellite repeats within the 1-kb region
downstream of the transcriptional start site: one [AG]9 dinucleotide repeat and one [A]10
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mononucleotide repeat (Figure 5D). Mono- and dinucleotide deletions within these
microsatellite repeats were present in DLBCLs from all genotypes but were most
prominent in the IµHABcl6 Msh2-/- DLBCLs (Figure 6A). Deletion frequency in the
IµHABcl6 Ung-/- Msh2-/- DLBCLs was lower than the IµHABcl6 Msh2-/- DLBCLs and
further decreased in the MSH2-sufficient genotypes. This indicates that a subset of
these deletions is dependent on the presence of UNG. In addition to their roles in
immune diversification, MSH2 is required for maintaining stability of microsatellite repeat
sequences and UNG regulates genomic uracil load incorporated during DNA
replication.45,47 In the absence of UNG, aberrant A/U base pairs within microsatellite
repeats would simply be replicated. However, in the absence of MSH2, UNG-generated
abasic sites can be processed into strand lesions with an imbalance in BER leading to
microsatellite instability.47,48 A similar analysis of healthy ex vivo activated B cells also
demonstrated a high frequency of UNG-dependent deletion and intra-Sµ recombination
events involving both AID hotspot motifs and repetitive DNA sequences within the core
IgH Sµ region (Figure 6B and supplemental Figure 2). Altogether, these findings are
consistent with the presence of both UNG-dependent and independent mechanisms for
initiating genomic instability in GC B cells that are normally offset by the actions of
MSH2.
As part of the DNA damage signaling response, histone H2AX is phosphorylated to
γH2AX and forms foci at sites of DSBs and SSBs.49 To further investigate the opposing
roles of BER and MMR in regulating genomic stability in B cells, we measured γH2AX
formation in healthy ex vivo activated B cells from mice deficient in UNG, MSH2 and
both. Although no obvious differences in γH2AX formation could be detected by
immunoblotting (not shown), analysis of single cells by flow cytometry demonstrated a
small but consistent and statistically significant increase in γH2AX+ B cells from Msh2-/mice compared with B cells from wild type and Ung-/- mice (Figure 6C-D and
supplemental Table 3). In addition, γH2AX formation was significantly higher in Ung-/Msh2-/- B cells than wild type B cells but lower than that seen in Msh2-/- B cells. This
provides additional evidence of interplay between BER and MER whereby DNA strand
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lesions can arise in activated B cells in an UNG-dependent manner but are offset by the
actions of MSH2.
Consistent with previous findings, spectral karyotyping of IµHABcl6 DLBCLs
demonstrated complex chromosome abnormalities.36 We also identified numerous
clonal and sub-clonal chromosome abnormalities in IµHABcl6 Ung-/- Msh2-/- DLBCLs
and IµHABcl6 Ung-/- FLs (Table 1 and supplemental Figure 2). Although there were no
recurrent translocations or deletions, chromosome 15 (location of cMyc) was involved in
an aneuploidy in 7 of 9 karyotypes analyzed. Thus, genomic instability can develop in
GC B cell lymphomas through mechanisms independent of AID-associated BER and
MMR pathways. A specific cause for this is not clear but it is likely multifactorial, with
our findings supporting previous reports implicating a role for microsatellite instability
when MMR is impaired, along with the added stress to DNA damage response
mechanisms in the setting of an imbalance in genomic uracil homeostasis.44,50-52
Discussion
Immune diversification in GC B cells occurs in two phases: AID-mediated cytidine
deamination during phase one followed by BER and MMR of resulting U-G mismatches
during phase two. While this adaptation facilitates SHM and CSR of the Ig genes, AID
binds to ~12,000 sites across the genome and off-target events are associated with GC
and post-GC B cell malignancies.2,12 Although evidence suggests that AID may initiate
the process of lymphomagenesis,10,22-24 we demonstrate here that downstream BER
and MMR pathways ultimately regulate the malignant fate and timing of GC B cell
transformation. We propose that lymphoma may arise as a result of events that take
place in both phases of immune diversification. When phase one is left unchecked, as
occurs in the absence of both UNG and MSH2, widespread production of U-G
mismatches is associated with the accumulation of somatic C/G to T/A transition
mutations within non-Ig genes and a significantly shorter latency to the development of
BCL6-driven DLBCL. Moreover, a critical role for MSH2 in general genomic
maintenance is made evident by the additional development of other malignancies
independent of BCL6. Thus, while the combined actions of UNG and MSH2 prevent the
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accumulation of potentially lymphomagenic C/G to T/A transition mutations, these
findings also suggest that MMR imparts an additional and important layer of genomic
protection to GC B cells.
During phase two of immune diversification, UNG targets areas of high uracil density to
create the majority of AID-initiated DNA breaks, with MSH2 acting downstream of UNG
either independently or as a back-up mechanism for resecting distantly separated U-G
mismatches into strand lesions.53 Absence of MSH2 or MSH6 causes ~50-80%
reductions in CSR while absence of UNG causes >95% reductions in CSR in mice and
humans.26-29,54,55 In addition, UNG deficiency in mice impairs formation of
translocations between IgH and cMyc and reduces the incidence of post-GC
plasmacytomas in Bcl-xL transgenic mice.56,57 Consistent with the notion that absence
of UNG might limit the formation of lymphomagenic DNA lesions, our findings
demonstrate that UNG deficiency prevents the development of BCL6-driven DLBCL.
This phenotype resembles the protective effect of AID-deficiency in IµHABcl6 mice,24
indicating that under physiologic conditions, downstream UNG-mediated processing of
U-G mismatches is an essential step in AID-initiated lymphomagenesis.
In addition to forming MutSα with MSH6, MSH2 heterodimerizes with MSH3 to form
MutSβ. Both MMR complexes maintain genomic stability by repairing small insertion
and deletion loops generated within microsatellite repeats during DNA replication,45 but
the ability of MutSα to repair nucleotide mismatches imparts a distinct role in antibody
diversification. Despite this mutagenic role and consistent with the reduced capacity of
MSH2 to facilitate the formation of AID-initiated strand lesions in the absence of UNG,
the lack of DLBCL development in IµHABcl6 Ung-/- mice illustrates that MMR alone
cannot promote BCL6-driven lymphomagenesis. Conversely, in the absence of MSH2,
GC B cells exhibit extreme microsatellite instability,52 with our findings demonstrating
increased formation of UNG-dependent γH2AX foci in activated B cells prior to the
development of lymphoma and an associated rise in microsatellite instability in MSH2deficient DLBCLs. While we cannot determine the extent of AID-dependence, this is
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consistent with a dual protective role for MSH2 in GC B cells in the maintenance of
microsatellite stability and the restriction of UNG-dependent lesions.
It is unknown how AID-associated BER and MMR pathways compete for processing of
U-G mismatches or what factors determine whether repair will occur in a high- or lowfidelity manner. Nonetheless, our findings support a model whereby the actions of UNG
and MSH2 contribute to SHM and CSR of the Ig genes while simultaneously attenuating
malignant transformation by limiting the accompanying genome-wide AID-generated
uracil load and maintaining genomic integrity. This combined effort is comprised of a
complex interplay between BER and MMR which individually manifest as opposing roles
with UNG promoting the formation of potentially deleterious intermediates including
abasic sites, SSBs, DSBs and deletions while MSH2 restricts the formation of UNGdependent lesions outside the Ig loci and provides an additional layer of general
protection from genomic instability. The end result is a net protective effect that delays
lymphomagenesis in the setting of an initial genetic hit (in this case, deregulated
expression of BCL6) but any breakdown in this balance of repair might subsequently
permit the accumulation of lymphomagenic mutations. Consistent with this model, it is
not surprising that germline and somatic mutations of genes involved in MMR (including
MSH2, MSH6, and EXO1) have been found in up to 30% of human DLBCL while
mutations of BER genes occur with much less frequency.58,59 Additional features of the
IµHABcl6 DLBCL model that resemble human disease include lack of CSR and a
predominance of mutations at C/G base pairs. Functional impairment of CSR in human
activated B cell DLBCL is strongly associated with a predisposition to chromosome
translocations and thought to be due to acquired defects in DNA repair.46 Similarly, C/G
mutation bias within Ig and non-Ig AID target loci in human DLBCL and lymphoma cell
lines is a common finding and is consistent with impairment of MMR.13 Altogether, this
suggests that disruption of the balance of AID-associated BER and MMR may be a
recurrent event in GC B cell malignancy that provides a selective advantage and
contributes to the pathogenesis as well as heterogeneity of these diseases.
15
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Acknowledgements
We thank J. Koeman and the Cytogenetics Core at the Van Andel Research Institute for
cytogenetic analysis; the Keck Biotechnology Facility at Yale University for DNA
sequencing and microarray hybridization; Michael Schadt, Yale Mouse Research
Pathology for histology; Amos Brooks, Yale Research Histology Lab for
immunohistochemistry; and Riccardo Dalla-Favera at Columbia University for providing
the IµHABcl6 mice. We also thank David Schatz and Velizar Shivarov for thoughtful
discussions and critical review of the manuscript. This work was supported by the
National Institutes of Health, National Cancer Institute (NCI K08CA140718) (M.P.S.), a
Scholar Award from the American Society of Hematology (M.P.S.), and the Concern
Foundation (M.P.S.).
Authorship
X.G. performed the experiments, analyzed data, and co-wrote the manuscript. C.J.B.
reviewed the histology and immunohistochemistry. Z.L. analyzed and interpreted gene
expression profiling data. M.P.S. coordinated the study, designed and performed
experiments, analyzed data, and wrote the manuscript. All authors reviewed the
manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
16
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20
Table 1. Composite karyotypes from representative tumor samples
Genotype
Pathology
JH4 intron
Spectral karyotype
340
IµHABcl6
DLBCL
M
40~43<2n>,XX,+8[11],+8x2[6],Der(10)T(10A4;13?A)T(13?B;17D)[3],+15[19],Der(17)
T(10A4;17D)[3][cp21] / 84~88<4n>,XXXX,+8x2~6[4],+15x2[4][cp4]
379
IµHABcl6
DLBCL
M
42~44<2n>,X,X[14],Del(X?A6)[5],Dp(5)[14],Der(10)[3],+13[7],+13x2[2],+15[17],+15x2[2],+16[12],+16x2[2],+19
[19][cp19] / 84~89<4n>,XX or XXX,-X[2],Xx2[4],Dp(5)x2[6],+15x2[3],+15x3[3],+16x2[5],+16x4[1],+19x2[6][cp6]
438
IµHABcl6
DLBCL
UM
40~42<2n>,XY,Del(2G)[3],Der(4)T(4E2;13A3)[3],+13[1],+15[3],+16[4],+17[3][cp24] /
84<4n>,XXYY,Der(4)T(4E2;13A3)x2,+13,+15,+16,+16[cp1]
167
IµHABcl6 Ung-/-Msh2-/-
DLBCL
M
40~41,XX,T(2C2;17A2)[21],+15[2],+19[10][cp30]
375
IµHABcl6 Ung-/-Msh2-/-
DLBCL
M
40,XY,Dp(1E-F)[2][cp30]
414
IµHABcl6 Ung-/-Msh2-/-
DLBCL
M
40,XX[5] / 40~42,XX,T(8E1;13A3)[2],Der(10)?Del(10D)Dp(10A-C)[3],Der(10)Dp(10AC)T(10C;16C2)[22],+15[5],+Der(15)T(10D;15D1)[21][cp34] /
79~83<4n>,XXXX,Der(10)?Del(10D)Dp(10A-C)x2[1],Der(10)Dp(10AC)T(10C;16C2)[1],+15x2[2],+Der(15)T(10D;15D1)x2[1][cp6]
491
IµHABcl6 Ung-/-Msh2-/-
DLBCL
M
40,XX,+Rb(XA1.7A1)[30][cp30]
448
IµHABcl6 Ung-/-
FL
M
41<2n>,X,-X[30],+15[30],+17[30][cp30]
496
IµHABcl6 Ung-/-
FL
M
40~43<2n>,XY,+Y[2],+2[6],+5[2],Del(6B3-E1)[24],+15[20],+17[11],+mar[3][cp30]
Abbreviations: DBLCL, diffuse large B cell lymphoma; FL, follicular lymphoma; M, mutated; UM unmutated; [ ], cell number analyzed;
Der, Derivative chromosome; cp, composite; Del, Deletion; Dp, Duplication; T, Reciprocal translocation; Der(N)T, Non-reciprocal
translocation; Rb, Robertsonian translocation; mar, marker chromosome.
21
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Mouse ID
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Figure Legends
Figure 1. IµHABcl6 and IµHABcl6 Ung-/- Msh2-/- mice have similar lymphocyte
development and develop similar lymphomas. Representative flow cytometric
analysis of splenocytes from healthy (n = 3 for each genotype) and sick (n = 6 IµHABcl6
and n = 19 IµHABcl6 Ung-/- Msh2-/-) mice. Cells were stained with T cell (CD3), mature
B cell (B220, IgM), GC B cell (CD95) and plasma cell (CD138) markers as indicated.
For healthy spleens, only the lymphocyte gate is shown. For diseased spleens, cells
were ungated.
Figure 2. Absence of UNG and/or MSH2 influences lymphomagenesis in IµHABcl6
mice. (A) Kaplan-Meier overall survival curves for IµHABcl6 mice with indicated
genotypes. Median survival for IµHABcl6 and IµHABcl6 Ung-/- mice was not reached. P
values were calculated using log-rank (Mantel-Cox) tests. (B) Distribution of tumor
types among the different genotypes (DLBCL, diffuse large B cell lymphoma; FL,
follicular lymphoma; other, histiocytic sarcoma and squamous cell carcinoma).
Figure 3. IµHABcl6 Ung-/-Msh2-/- IµHABcl6 Msh2-/- mice succumb to early onset
DLBCL. (A) Clonality analysis of mature B cell lymphomas was carried out on all B cell
tumors using Ig variable region amplification. Four major PCR fragments corresponding
to respective JH1, JH2, JH3, and JH4 arrangement are labeled on the left. Multiple
bands are present in a polyclonal population of healthy B cells (lane 1) while a single or
dominant band indicates a clonal population. Representative specimens are shown.
Lanes 2-4: DLBCLs from IµHABcl6 mice. Lanes 5-7: FLs from IµHABcl6 Ung-/- mice.
Lanes 8, 9: DLBCLs from IµHABcl6 Msh2-/- mice. Lanes 10-12: DLBCLs from
IµHABcl6 Ung-/- Msh2-/- mice. M, 1-kb DNA marker. (B) Unsupervised hierarchal
clustering of gene expression profiles obtained from Illumina Mouse WG-6 v2.0
microarrays of representative B220+ IgM+ CD138- IµHABcl6 (n = 3), IµHABcl6 Ung-/Msh2-/- (n = 4), and IµHABcl6 Msh2-/- (n = 2) mature GC B cell lymphomas (color coded).
Rows represent different gene probes and columns denote individual samples. The
scale indicates relative changes in gene expression normalized by the standard
22
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deviation (-2 to 2 in log2 units, 0 represents the mean expression level of a given gene
across samples). (C) Supervised hierarchical clustering of gene expression data from
representative IµHABcl6 (n =3), IµHABcl6 Ung-/- Msh2-/- (n = 6), and IµHABcl6 Msh2-/- (n
= 5) B cell lymphomas. The pre-B cell lymphomas and DLBCLs (defined by
immunophenotyping and histology) are noted on the bottom and have distinct gene
expression profiles (supplemental Table 2). (D) Median time to development of DLBCL
in IµHABcl6 mice (squares) was 16.2 months compared with 6.5 months in the
IµHABcl6 Ung-/- Msh2-/- mice (circles) and 5.8 months in IµHABcl6 Msh2-/- mice
(triangles). Bars denote the median. P values were calculated using the one-tailed
Mann-Whitney test. (E) Kaplan-Meier overall survival curves for IµHABcl6 mice with
indicated genotypes that developed DLBCLs. P values were calculated using the logrank (Mantel-Cox) test.
Figure 4. Histologic analysis of B cell tumor types.
Representative H&E and PNA-stained sections of mouse spleen from wild type and
IµHABcl6 mice with lymphoma from indicated genotypes. Wild type mouse spleen is
comprised of white pulp with follicles (*) and a distinct marginal zone (arrow heads)
separated by distinct areas of red pulp (**) with scattered PNA+ cells within follicles and
the red pulp. In contrast, mice with lymphoma have marked expansion of the white pulp
(*) with little or no visible red pulp (**). Neoplastic lymphocytes from IµHABcl6 and
IµHABcl6 Ung-/- Msh2-/- mice are large and pleomorphic with abundant cytoplasm and
are frequently PNA+ (arrows), consistent with DLBCL. Lymphomas from IµHABcl6 Ung/-
mice have expanded follicles comprised of small monotypic lymphocytes that are
predominantly PNA-, consistent with FL. Pre-B cell lymphomas, shown here from
IµHABcl6 Msh2-/- mice, are comprised of medium-sized monotypic PNA- lymphocytes.
Images were obtained on a Zeiss Axio Imager A1 microscope. Short scale bars are 500
µM and long scale bars are 50 µM.
Figure 5. Ig and non-Ig gene mutation patterns in IµHABcl6 GC B cell
lymphomas. (A) Sequence analysis of AID target genes was performed on genomic
DNA from mature B cell tumors that developed in IµHABcl6 (n = 3), IµHABcl6 Ung-/23
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Msh2-/- (n = 4), IµHABcl6 Ung-/- (n = 3), and IµHABcl6 Msh2-/- (n = 2) mice. With
exception of the IµHABcl6 Ung-/- FLs, all tumors in this graph are DLBCLs. A total of
~632-kb of sequencing data were obtained. No mutations were detected in any pre-B
cell lymphomas, consistent with their AID-independent stage of development (not
shown). Mutation frequencies for JH4, Pim1 and RhoH are shown as indicated. Bars
denote mutation frequency on the y-axis with black and white fill indicating clonal and
non-clonal mutations, respectively. Statistical significance was assessed by Pearson’s
chi-squared test. NS, not significant; *, P < 0.05; **, P < 0.01. (B) The pattern of
nucleotide substitutions detected in the IgH JH4 regions from mature B cell tumors with
the indicated genotypes was compared with that of Peyer’s patch GC B cells from
healthy 4-month-old IµHABcl6 and IµHABcl6 Ung-/- Msh2-/- mice. Nucleotides in the left
column are mutated to the nucleotides in the top row. If the same nucleotide substitution
occurred at the same site in multiple clones, it was counted only once. Percentage of
mutations occurring at a specific nucleotide base is calculated in the last column. Total
mutation frequency and hotspot fold over random (calculated as the ratio of observedto-expected frequency of hotspot C/G to T/A mutations) is below each box. (C) Analysis
of CSR and subsequent mutational analysis of Ig 5'Sµ region was performed on ex vivo
activated healthy B cells (white bars) and mature B cell lymphomas (black bars) from
the indicated genotypes. Numbers on the x-axis denote the percentage of cells that
underwent CSR from IgM to IgG1 (determined by flow cytometry). (D) Clonal mutation
plots of Pim1, Pax5 and RhoH. Genomic loci are shown with untranslated (open boxes)
and coding (filled boxes) regions. A ~1-kb region (brackets and dashed lines)
downstream of the major transcriptional start site (arrows) was sequenced. Analysis of
~96-kb of total sequencing data from 12 B cell tumors (7 DLBCL, 3 FL, 2 pre-B cell)
revealed six unique clonal mutations within Pim1, Pax5, and RhoH. All mutations were
found in DLBCLs from IµHABcl6 Ung-/- Msh2-/- mice. Mutations (underlined) and
surrounding nucleotides are shown. Bolded sequences indicate an AID hotspot motif.
The [AG]9 and [A]10 microsatellite repeats within the RhoH locus are indicated.
Figure 6. Deletions within RhoH and DNA DSBs are increased in an UNG
dependent manner in the absence of MSH2. (A) Sequence analysis of the RhoH
24
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locus identified mono- and dinucleotide deletions within [A]10 (white bar) and [AG]9
(black bar) microsatellite repeats. A total of 30 deletions were identified in 254
sequences representing ~450-kb of sequencing data. Deletion frequency is
represented as a percentage of the number of cloned sequences that contained a
unique deletion. Statistical significance was calculated using Pearson’s chi-squared
test. (B) Sequence analysis of the core IgH Sµ region identified unique intra-Sµ
recombination events (white bar) and deletions (black bar) within AID hotspot motifs and
repetitive sequences. A total of 25 events were identified in 228 sequences
representing ~168-kb of sequencing data. (C) Splenic B cells were obtained from wild
type mice and healthy mice deficient in UNG, MSH2 and both. Cells were activated ex
vivo with LPS and IL4 for 48 hours followed by measurement of γH2AX formation by
flow cytometry. The graph depicts the mean and standard deviation of % γH2AX+ cells
from 4-6 different mice of each genotype. Statistical significance was calculated by twotailed t-test. (D) Representative flow cytometric analysis of γH2AX formation (y-axis)
from each genotype. DAPI staining for DNA content is shown on the x-axis. Wild type
B cells treated with 20 μM etoposide for 15 minutes at the end of activation were used
as a positive control for γH2AX formation.
25
Healthy
Wild type
I HABcl6
DLBCL
I HABcl6
Ung-/- Msh2-/-
I HABcl6
I HABcl6
Ung-/- Msh2-/-
24.7
2.77
21.7
2.09
23.7
0.66
3.34
4.87
0.37
3.28
13.7
58.8
16.8
59.5
15.8
59.8
8.53
83.3
0.92
95.4
2.91
56.9
2.67
53.5
2.21
50.3
4.64
85.6
0.71
84.5
28.3
11.9
31.8
12.1
34.2
13.3
8.31
1.44
0.67
14.1
29.8
12.3
31.8
13.2
37.4
10.1
8.96
71.0
1.15
71.9
7.49
50.4
4.04
50.9
0.64
51.8
1.83
18.2
0.13
26.8
0.90
11.4
1.18
6.90
0.55
7.35
0.30
1.69
0.55
13.9
33.4
54.3
33.2
56.7
36.5
55.6
14.2
83.8
0.50
85.1
CD3
IgM
CD95
CD138
B220
Figure 1
Figure 2
A
Overall survival (%)
100
75
P = 0.018
50
P < 0.0001
25
0
0
10
20
30
Months
IμHABcl6 (n = 22)
IμHABcl6 Ung-/- Msh2-/- (n = 33)
IμHABcl6 Ung-/- (n = 22)
IμHABcl6 Msh2-/- (n = 22)
Ung-/- Msh2-/- (n = 18)
100
80
Healthy
DLBCL
Pre-B cell
FL
T cell
Other
60
40
20
(n
=
2 -/- 22)
c
(
l6
n
Iµ
=
H
U
AB
23
ng
-/)
cl
6
(n
M
=
s
U
22
h2
ng
-/)
-/(n
M
=
sh
2 -/- 18)
(n
=
18
)
0
sh
M
H
AB
-/-
ng
U
6
cl
AB
H
Iµ
Iµ
Iµ
H
AB
cl
6
Distribution of phenotypes (%)
B
Figure 3
A
1
2 3 4
5 6
IµHABcl6
7 8 9 10 11 12 M
JH1
JH2
JH3
JH4
IµHABcl6 Ung-/- Msh2-/IµHABcl6 Msh2-/<-2
B
0
>2
C
pre-B
- cell
P = 0.0119
P = 0.0003
15
10
5
100
P = 0.002
75
50
P < 0.0001
25
0
0
H
Iu
H
AB
AB
cl
cl
6
6
U
ng
-/M
sh
Iu
2 -/H
AB
cl
6
M
sh
2 -/-
0
Iu
DLBCL Latency (months)
20
E
Overall survival, DLBCL (%)
D
DLBCL
10
20
Months
IμHABcl6 (n = 22)
IμHABcl6 Ung-/- Msh2-/- (n = 12)
IμHABcl6 Ung-/- (n = 19)
IμHABcl6 Msh2-/- (n = 8)
30
Figure 4
HE
PNA
*
**
Wild type
*
**
*
IµHABcl6
IµHABcl6
Ung-/Msh2-/-
*
**
IµHABcl6
Ung-/-
IµHABcl6
Msh2-/-
*
**
*
A
D
*
60
Mutation frequency
(x10-5 mutations per bp)
Pim1
*
T
CGCC
Non-clonal
Clonal
50
40
0
200
30
0
200
RhoH
-/-
Pim1
200
[AG]9
131-147
cl
0
Iµ
H
AB
4
G
5
C
2
4
T
4
4
3
5
5
20
A
4
23
G
11
14
32
C
0
1
25
T
0
0
7
0
0
0
C
IgH 5'Sμ Mutation frequency
(x10-5 mutations per bp)
300
Non-clonal
Clonal
250
200
150
100
50
0
40 0.4 1.4 18
0
0
0
0
Lymphoma cells
AB
cl
6
-/-
I
U µH
Iµ ng -/- AB
c
H
Iµ AB Ms l6
H c h2
A B l6
-/c l Un
6
g
M -/Iµ
sh
H
AB
2 -/cl
6 Iµ
U H
Iµ ng -/- AB
c
H
Iµ AB Ms l6
H c h2
A B l6
-/c l Un
6
g
/M
sh
2
Healthy B cells
I HABcl6
DLBCL (n = 3)
A G C
T
0
0
A
0
41
G
11
15
59
C
2
0
0
T
0
0
0
400
600
800
1000
T
AGCT
I HABcl6 Ung-/- Msh2-/Peyer’s patch B cells (n = 1)
A G C
T %
Mutation frequency = 288 x 10-5 /bp
227 x 10-5
Hotspot fold over random = 3.4 (P < 0.0001) 3.2 (P < 0.0001)
H
1000
RhoH
6 IµH
U
n A
Iµ g -/- Bc
H
M l6
A
Iµ B s h
H cl 2 AB 6
/c l Un
6
g -/M
Iµ
sh
H
2 -/AB
cl
6 IµH
U
n A
Iµ g -/- Bc
H
M l6
A
Iµ B s h
H cl 2 AB 6
/c l Un
6
g -/M
Iµ
sh
H
2 -/AB
cl
6 IµH
U
n A
Iµ g -/- Bc
H
M l6
A
Iµ B s h
H cl 2 AB 6
/c l Un
6
g -/M
sh
2
JH4
A
Iµ
800
T
T
TGCC AGCT
**
I HABcl6 Peyer’s patch
B cells (n = 2)
A G C
T %
Figure 5
600
*
20
0
%CSR
400
Pax5
10
B
T
TGCT
T
GACT
0
0
0
%
400
A
0
50
G
13
8
45
C
0
0
5
T
0
0
1
55 x 10-5
3.6 (P < 0.0001)
800
[A]10
776-785
I HABcl6 Ung-/- Msh2-/DLBCL (n = 4)
A G C
T %
0
0
600
0
0
1
1000
I HABcl6 Ung-/FL (n = 3)
A G C
T
7
A
0
50
G
6
12
43
C
0
0
0
T
0
0
2
0
46 x 10-5
3.1 (P < 0.0001)
0
0
0
%
1
8
0
46
6
46
0
22 x 10-5
2.9 (P < 0.0098)
0
Figure 6
B
[A]10
C
Intra-Sμ recombination
[AG]9
Nucleotide deletions
P = 0.014
P = 0.004
W
2 -/sh
-/-
ng
M
ng
sh
U
pe
ty
ild
sh
sh
-/-
M
ild
ng
Iµ
H
AB
U
U
W
2 -/-
0.0
-/-
0
2 -/-
% H2AX formation
1.0
0.5
ty
Iµ
H
cl
AB
6
cl
U
6
ng
-/M
Iµ
sh
H
2 -/AB
cl
6
Iµ
U
H
ng
AB
-/cl
6
M
sh
2 -/-
P = 0.038
1.5
5
pe
0
10
ng
10
15
M
20
2.0
20
2 -/-
30
U
Core IgH Sµ
deletion frequency (%)
RhoH deletion frequency (%)
40
2.5
25
M
P = 0.03
-/-
A
D
Wild type
+etoposide
H2AX
12.8
DAPI
Wild type
0.61
Ung-/- Msh2-/0.93
Ung-/0.48
Msh2-/1.4
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Prepublished online September 18, 2015;
doi:10.1182/blood-2015-02-628164
AID-associated DNA repair pathways regulate malignant transformation in
a murine model of BCL6-driven diffuse large B cell lymphoma
Xiwen Gu, Carmen J. Booth, Zongzhi Liu and Matthew P. Strout
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