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Brief Report IMMUNOBIOLOGY
Aberrant TCRd rearrangement underlies the T-cell lymphocytopenia and t(12;14) translocation associated with ATM deficiency Wenxia Jiang,1,2 Brian J. Lee,1,2 Chen Li,1,2 Richard L. Dubois,1,2 Monica Gostissa,3 Frederick W. Alt,3 and Shan Zha1,2,4 1
Department of Pathology and Cell Biology, Institute for Cancer Genetics, and 2Herbert Irving Comprehensive Cancer Research Center, Columbia University Medical Center, New York, NY; 3Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, and Department of Genetics, Harvard Medical School, Boston, MA; and 4Division of Hematology, Oncology, and Stem Cell Transplantation, Department of Pediatrics, College for Physicians and Surgeons, Columbia University Medical Center, New York, NY
Key Points • ATM-deficient T-cell lymphocytopenia is in part caused by defects in TCRd rearrangements. • Aberrant TCRd arrangement is required for the recurrent t(12;14) translocations, but not chromosome 14 amplification, in ATMdeficient thymic lymphomas.
Ataxia telangiectasia mutated (ATM) is a protein kinase and a master regulator of DNAdamage responses. Germline ATM inactivation causes ataxia-telangiectasia (A-T) syndrome with severe lymphocytopenia and greatly increased risk for T-cell lymphomas/ leukemia. Both A-T and T-cell prolymphoblastic leukemia patients with somatic mutations of ATM frequently carry inv(14;14) between the T-cell receptor a/d (TCRa/d) and immunoglobulin H loci, but the molecular origin of this translocation remains elusive. ATM2/2 mice recapitulate lymphocytopenia of A-T patients and routinely succumb to thymic lymphomas with t(12;14) translocation, syntenic to inv(14;14) in humans. Here we report that deletion of the TCRd enhancer (Ed), which initiates TCRd rearrangement, significantly improves ab T cell output and effectively prevents t(12;14) translocations in ATM2/2 mice. These findings identify the genomic instability associated with V(D)J recombination at the TCRd locus as the molecular origin of both lymphocytopenia and the signature t(12;14) translocations associated with ATM deficiency. (Blood. 2015;125(17):2665-2668)
Introduction translocation patterns of ATM-deficient thymic lymphomas,3 suggesting that TCRd might be involved. In this context, TCRd-related translocations are found in the majority of T-ALL and .90% of TAL1 translocated cases.8 Using ATM2/2Ed2/2 mice, here we identified aberrant TCRd rearrangements as the origin for both ab T-cell development defects and the recurrent t(12;14) translocations in ATM-deficient mice.
As a master regulator of the DNA damage response, ataxia telangiectasia mutated (ATM) kinase is rapidly activated by DNA doublestrand breaks and phosphorylates substrates involved in both DNA repair and checkpoint control. Loss of ATM causes ataxiatelangiectasia (A-T) syndrome characterized by primary immunodeficiency and greatly increased risk for lymphoid malignancies, especially of T-cell lineage.1 Moreover, .50% of T-cell prolymphoblastic leukemia (T-PLL) patients carry a somatic mutation of ATM.2 Both T-PLL and A-T patients carry inv(14;14) that involves the T-cell receptor a/d (TCRa/d) and immunoglobulin H loci on human chromosome 14. However, the molecular origin of the seemingly T-cell–specific lymphocytopenia and inv(14;14) remains unknown. ATM2/2 mice recapitulate the T-cell lymphocytopenia of A-T patients and succumb to thymic lymphomas with TCRa/d-related chromosome 14 amplification and t(12;14) translocations, syntenic with inv(14;14) in humans.3 Reduced ab T-cell numbers and blockade at the double-positive (DP) to single-positive (SP) transition is characteristic of ATM-deficient T cells.4,5 Both human and mouse TCRd loci reside between Va and Ja segments.6 As such, TCRa rearrangement deletes the TCRd gene, leading to irreversible commitment to the ab T-cell lineage. This stage-specific rearrangement of the TCRd in double-negative (DN) and TCRa in DP T cells is driven by the loci-specific enhancer elements Ed and Ea, respectively.6,7 However, Ea deletion does not affect the kinetics or
All animal work was conducted with proof from the institutional Animal Care and Use Committee of Columbia University. ATM2/29 and Ed2/210 mice were described previously. T-cell development was characterized in 4- to 6-week-old mice as previously described.11 Briefly, single-cell suspensions of thymus and spleen were counted and stained with fluorophore-conjugated antibodies against CD4, CD8, CD3, TCRb, or TCRg. For DN staining, thymocytes were counterstained with phycoerythrin-conjugated anti-CD4, CD8, B220, TCRgd, Ter119 and CD19 antibodies, then with fluorophoreconjugated anti-CD44 and anti-CD25 antibodies. Flow cytometry data were collected on FACSCalibur (BD) and analyzed with FlowJo. For cytogenetic analyses, single cell suspensions were incubated with colcemid (10 mg/mL) for 4 to 10 hours and fixed and stained3 using ASI applied spectral imaging paints. Images were acquired and analyzed with Isis and Metafer 4 (MetaSystems, Newton, MA). Comparative genomic hybridization (CGH)
Submitted January 13, 2015; accepted February 21, 2015. Prepublished online as Blood First Edition paper, February 26, 2015; DOI 10.1182/blood2015-01-622621.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2015 by The American Society of Hematology
BLOOD, 23 APRIL 2015 x VOLUME 125, NUMBER 17
Study design
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Figure 1. Deletion of Ed partially rescues lymphocytopenia in ATM-deficient mice. (A) Representative flow cytometric analyses of premalignant thymocytes from wild-type (WT), ATM2/2, Ed2/2, and ATM2/2Ed2/2 mice (4-6 weeks). (B) Total thymic gd cell number counts (310 6 ). (C) Total number of thymocytes and DP cells (3106). (D) SP cell number counts (3106). Data represent the average 6 standard deviation from at least 5 independent mice of each genotype (see supplemental Figure 1A for details). The P values between 2 genotypes were calculated based on the 2-tailed Student t test assuming unequal variance. Supplemental Figure 1B summarizes the P value from pairwise comparison for total, DP, and SP thymocytes between different genotypes.
was performed with the Agilent 244K mouse array. Reverse-transcription polymerase chain reaction (PCR) methods are detailed in Figure 2F and supplemental Table 1 (available on the Blood Web site).
Results and discussion ATM2/2Ed2/2 mice were born at the expected ratio and undistinguishable from ATM2/2 littermates. In thymuses of ATM2/2 Ed2/2 mice, gd T-cell frequency was reduced, but not abolished, consistent with the role of Ea in residual gd T-cell development10 (Figure 1A-B). Although thymus cellularity was reduced by ;50% in ATM2/2 mice, both thymus cellularity and the number of mature SP T cells were significantly rescued in ATM2/2Ed2/2 mice (Figure 1C-D). This is unexpected, given that .90% of thymocytes are ab T cells. Further analyses showed that this rescue was mostly due to moderate increases of DNIV (CD252CD442) percentage and marked increases of DP thymocyte number without changes in the SP to DP ratio (Figure 1C-D and supplemental Figure 1A-B). TCRd undergoes V(D)J recombination in DNII-DNIII (CD251).10 ATM is particularly important for inversional V(D)J recombination,12 and Vd5 is located downstream of Cd and requires inversional rearrangement.13 The configuration of the TCRa/d locus also determines that if DNA breaks in the TCRd loci are unrepaired, then Ea would separate from Vas and disable TCRa V(D)J recombination
(Figure 2C). These findings reveal an important role of genomic instability at the TCRd locus in ab T-cell development and T-cell lymphocytopenia associated with ATM deficiency, and they suggest that suppression of TCRd rearrangement might improve T-cell counts in A-T patients. Despite improved T-cell counts, the kinetics of succumbing to thymic lymphomas was similar in ATM2/2Ed2/2 mice and ATM2/2 controls (P 5 .1), with a median survival of 112 days (vs 99 for ATM2/2 controls) (Figure 2A). ATM2/2Ed2/2 thymic lymphomas consisted of immature (surface TCRblow) DP T cells similar to ATM2/2 controls (supplemental Figure 1C). Southern blot identified clonal TCRb rearrangements and focal amplification centromeric to the TCRa/d loci (chromosome 14) in all ATM2/2Ed2/2 (or ATM2/2 Ed1/2) lymphomas as documented for ATM2/2 lymphomas (supplemental Figures 1D and 2A).3 ATM2/2 thymic lymphomas were derived from immature T cells that had not yet undergone TCRa arrangement, evidenced by retention of the Cd.3 All ATM2/2Ed1/2 and 2 of 5 ATM2/2Ed2/2 lymphomas (545 and 2280) retained at least 1 Cd, suggesting that the chromosome 14 amplifications most likely occurred during TCRd rearrangement in those tumors, consistent with the ability for Ea to drive residual TCRd rearrangements in the absence of Ed.10 Notably, the other 3 ATM2/2Ed2/2 lymphomas showed homozygous deletion of Cd, indicative of biallelic TCRa rearrangements (Figure 2C). Southern blot further confirmed that all 3 tumors (497, 713, and 745) rearranged the proximal Ja segments (Figure 2C). Thus, TCRd rearrangement is not required for
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Figure 2. Aberrant TCRd rearrangement is required for t(12;14), but not chromosomal 14 amplifications, in murine ATM-deficient thymic lymphomas. (A) Survival curve for ATM2/2 (n 5 10), ATM2/2Ed2/2 (n 5 11), and ATM2/2Ed1/2 (n 5 8) mice with median survival at 99, 112, and 137 days, respectively. P values were calculated using the log-rank test. (B) Representative chromosome 12 (green) and chromosome 14 (red) paint analyses of a metaphase from no. 745 ATM2/2Ed2/2 lymphomas (without t(12;14)) and no. 900 ATM2/2Ed1/2 lymphomas with t(12;14) translocation. More than 20 metaphases were analyzed for each lymphoma, and t(12;14) in .50% metaphases was used as the criterion for clonal translocations. The results are summarized in panel E. (C) Southern blot mapping the rearrangements of Cd and Ja. The diagram shows the TCRa/d locus, EcoRI (black arrows), and probe locations (black dashed line for Southern probes and red dashes for adjacent CGH probes). A total of 10 mg EcoRIdigested genomic DNA from either tumor (T) or the corresponding kidney (K) was analyzed on each lane. Loading control probe detected a fragment of the single-copy DNAPKcs gene. (D) CGH analyses of ATM2/2Ed2/2 thymic lymphomas 745 and 545. Each tumor DNA was analyzed with matched kidney control DNA on a 244K mouse CGH array from Agilent. The log ratio of tumor vs kidney control at each probe location is displayed in centromere-to-telomere orientation for each chromosome (from 1 to 19 then X/Y, from left to right). The red arrows and labels indicate recurrent amplification of Notch1, chromosome 14 upstream of TCRa/d locus, and trisomy 15. Green arrows and labels indicate recurrent deletion of the telomeric portion of chromosome 12 and the Pten locus, as well as deletional rearrangement in TCR loci. (E) Summary of chromosome 12/14 paint analyses of ATM2/2Ed2/2 and ATM2/2Ed1/2 lymphomas. (F) Quantitative PCR results that measured the expression of IL7R, Jak1, Notch1, Pten, Rb1, Bcl11b, and Psmc6 genes from ATM2/2Ed1/1 (n 5 3), ATM2/2Ed1/2 (n 5 4), ATM2/2Ed2/2 (n 5 5) lymphomas. Psmc6 locates at the amplification area within chromosome 14 and is used as a marker for the amplification here.3 Bcl11b is within the region that is hemizygously deleted in chromosome 12.3 Briefly, total RNA was extracted using Trizol (Invitrogen, CA) from a single-cell suspension derived from ATM2/2 and ATM2/2Ed2/2 thymic lymphomas as well as wild-type total thymus, and dissolved in 100 mL DEPCH2O and verified to have A260 nm/280 nm between 1.8 and 2. Approximately 5 mg of total RNA was treated with RNase-free DNase I (Roche). The DNaseI activity was abolished by the addition of 2 mL of 25-mM EDTA and incubation at 65°C for 15 minutes. Reverse transcription was performed using SuperScript III reverse transcriptase (Invitrogen) and Oligod(T)18 (New England Biolabs) according to the manufacturer’s instructions. The resulted complementary DNA was dissolved in TE and quantified with NanoDrop (Thermo Scientific). Quantitative PCR was then performed with gene-specific primers (supplemental Table 1) for Notch1 (NM_008714.3, 138 bp across exons 25-26), Jak I (NM_146145.2, 162 bp across exons 18-20), IL7R (NM_008372.4, 149 bp across exons 5-7), Rb1 (NM_009029.2, 144 bp across exons 23-25), Bcl11b (NM_001079883.1, 126 bp across exons 3-4, within chromosome 12 telomeric deletion), Pten (NM_008960.2, 144 bp across exons 7-8), Psmc6 (NM_025959.3, 196 bp across exons 7-9, within chromosome 14 amplification), and b-actin (actb; NM_007393.3, 154 bp across exons 1-2). Reaction setups include 10 mL Sybr Select Master Mix (Applied Biosystems), 250 nM each primer, 10 ng complementary DNA, and water to a total of 20 mL. The PCR condition was 50°C (2 minutes), 95°C (10 minutes), and [95°C (15 seconds), 60°C (1 minute)] 3 40, followed by the melt curve program provided by 7500 Real Time PCR System (Applied Biosystems). The results were analyzed with 7500 software package (v 2.0.6) and the DDCT method (included in 7500 software) with the expression of b-actin as the reference and the expression of the specific gene in the wild-type samples as controls for normalization. Each gene in each sample was analyzed in triplicate together with H2O control. The H2O control samples always had CT beyond the range (.40) in all experiments.
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chromosome 14 amplification and T-cell lymphomagenesis in ATM-deficient mice. In the absence of the TCRd rearrangement, aberrant TCRa rearrangement is able to promote chromosome 14 amplifications and oncogenic transformation of ATM-deficient T cells. We next investigated the origin of the t(12;14) translocations characteristic of ATM deficiency.3 Chromosome paint identified clonal t(12;14) translocations in all ATM2/2Ed1/2 lymphomas and the 2 Cd-positive ATM2/2Ed2/2 lymphomas (545 and 2280), but not in the 3 ATM2/2Ed2/2 lymphomas (497, 713, and 745) with biallelic TCRa rearrangements (Figure 2B,E). CGH analyses of 2 Cd-positive (2280 and 545) and 2 Cd-negative (497 and 745) ATM2/2 Ed2/2 lymphomas confirmed focal amplification upstream of the TCRa/d locus (Figure 2D and supplemental Figure 2B) and also revealed a unique hemizygous deletion of chromosome 14 downstream of the TCRa/d locus only in Cd-negative (497 and 745) tumors (Figure 2D and supplemental Figure 2B). CGH probe mapping showed that the deletion started at the proximal Ja (Figure 2C-D). Together with the absence of the t(12;14) translocation, this result suggests that when chromosome 14 amplification occurs in DP T cells during TCRa arrangement, the break in chromosome 12 is no longer available to form the t(12;14) translocation, causing the telomeric portion of chromosome 14 to be lost and pointing to concurrent immunoglobulin H and TCRd rearrangement in DN T cells as the molecular origin for t(12;14). This further implied that t(12;14) and the syntenic inv(14,14) are not essential for T-cell transformation. Despite the lack of t(12;14), both Cd-negative (497 and 745) tumors displayed hemizygous deletion of chromosome 12 telomeric regions, supporting the existence of potential tumor-suppressor genes (eg, Bcl11b).3,14-16 In this context, SKY analyses identified t(12;15) translocations involving chromosome 15 in tumor 745 (Figure 2B and supplemental Figure 2C). Finally, CGH also indicated that murine ATM2/2 thymic lymphomas, regardless of Ed status, display genetic changes that have been associated with human T-cell acute lymphoblastic leukemia (T-ALL), including trisomy of chromosome 15 containing c-Myc, amplification and overexpression
of Notch1, deletion of Pten, and overexpression of IL7R (Figure 2F), highlighting ATM-deficient murine thymic lymphomas as a bona fide animal model for human T-ALL. In summary, our study identifies aberrant TCRd rearrangement in the absence of ATM together with the unique configurations of the TCRa/d locus as one of the major causes to both T-cell–specific lymphocytopenia and the t(12;14) and related inv(14;14) associated with ATM deficiency and potentially in other T-ALL.
Acknowledgments This work was supported in part by National Institutes of Health (NIH) National Cancer Institute grants 5R01CA158073, 1R01CA184187, and 1P01CA174653-01 and American Cancer Society Research Scholar Grant RSG-13-038-01 DMC (S.Z.) and by NIH National Cancer Institute grant P01CA109901 (F.W.A.). S.Z. was a St Baldrick’s Scholar for Pediatric Cancer and is a Leukemia Lymphoma Society Scholar. W.J. was supported in part by NIH National Cancer Institute grant T32-CA09503. F.W.A. is a Howard Hughes Medical Institute investigator.
Authorship Contributions: W.J., F.W.A., and S.Z. designed experiments; S.Z. wrote the paper; M.G. performed the SKY analyses for tumor 745; and W.J., B.J.L., C.L., R.L.D., and S.Z. performed the rest of the experiments. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Shan Zha, Institute for Cancer Genetics, Columbia University Medical Center, 1130 St. Nicholas Ave, Room 503B, New York, NY 10032; e-mail: [email protected].
References 1. Lavin MF. Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol. 2008;9(10): 759-769.
7. Sleckman BP, Bardon CG, Ferrini R, Davidson L, Alt FW. Function of the TCR alpha enhancer in alphabeta and gammadelta T cells. Immunity. 1997;7(4):505-515.
12. Bredemeyer AL, Sharma GG, Huang CY, et al. ATM stabilizes DNA double-strand-break complexes during V(D)J recombination. Nature. 2006;442(7101):466-470.
2. Stankovic T, Stewart GS, Byrd P, Fegan C, Moss PA, Taylor AM. ATM mutations in sporadic lymphoid tumours. Leuk Lymphoma. 2002;43(8): 1563-1571.
8. Larmonie NS, Dik WA, Meijerink JP, Homminga I, van Dongen JJ, Langerak AW. Breakpoint sites disclose the role of the V(D)J recombination machinery in the formation of T-cell receptor (TCR) and non-TCR associated aberrations in T-cell acute lymphoblastic leukemia. Haematologica. 2013;98(8):1173-1184.
13. Krangel MS, Carabana J, Abbarategui I, Schlimgen R, Hawwari A. Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus. Immunol Rev. 2004;200:224-232.
3. Zha S, Bassing CH, Sanda T, et al. ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification. J Exp Med. 2010;207(7):1369-1380. 4. Borghesani PR, Alt FW, Bottaro A, et al. Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice. Proc Natl Acad Sci USA. 2000; 97(7):3336-3341.
9. Zha S, Sekiguchi J, Brush JW, Bassing CH, Alt FW. Complementary functions of ATM and H2AX in development and suppression of genomic instability. Proc Natl Acad Sci USA. 2008;105(27): 9302-9306.
5. Barlow C, Hirotsune S, Paylor R, et al. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell. 1996;86(1):159-171.
10. Monroe RJ, Sleckman BP, Monroe BC, et al. Developmental regulation of TCR delta locus accessibility and expression by the TCR delta enhancer. Immunity. 1999;10(5):503-513.
6. Sleckman BP, Bassing CH, Bardon CG, et al. Accessibility control of variable region gene assembly during T-cell development. Immunol Rev. 1998;165:121-130.
11. Li G, Alt FW, Cheng HL, et al. Lymphocytespecific compensation for XLF/cernunnos endjoining functions in V(D)J recombination. Mol Cell. 2008;31(5):631-640.
14. Gutierrez A, Kentsis A, Sanda T, et al. The BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia. Blood. 2011;118(15): 4169-4173. 15. De Keersmaecker K, Real PJ, Gatta GD, et al. The TLX1 oncogene drives aneuploidy in T cell transformation. Nat Med. 2010;16(11):1321-1327. 16. Ehrlich LA, Yang-Iott K, Bassing CH. Tcrd translocations that delete the Bcl11b haploinsufficient tumor suppressor gene promote atm-deficient T cell acute lymphoblastic leukemia. Cell Cycle. 2014;13(19):3076-3082.
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2015 125: 2665-2668 doi:10.1182/blood-2015-01-622621 originally published online February 26, 2015
Aberrant TCRδ rearrangement underlies the T-cell lymphocytopenia and t(12;14) translocation associated with ATM deficiency Wenxia Jiang, Brian J. Lee, Chen Li, Richard L. Dubois, Monica Gostissa, Frederick W. Alt and Shan Zha
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Brief Report IMMUNOBIOLOGY
Aberrant TCRd rearrangement underlies the T-cell lymphocytopenia and t(12;14) translocation associated with ATM deficiency Wenxia Jiang,1,2 Brian J. Lee,1,2 Chen Li,1,2 Richard L. Dubois,1,2 Monica Gostissa,3 Frederick W. Alt,3 and Shan Zha1,2,4 1
Department of Pathology and Cell Biology, Institute for Cancer Genetics, and 2Herbert Irving Comprehensive Cancer Research Center, Columbia University Medical Center, New York, NY; 3Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, and Department of Genetics, Harvard Medical School, Boston, MA; and 4Division of Hematology, Oncology, and Stem Cell Transplantation, Department of Pediatrics, College for Physicians and Surgeons, Columbia University Medical Center, New York, NY
Key Points • ATM-deficient T-cell lymphocytopenia is in part caused by defects in TCRd rearrangements. • Aberrant TCRd arrangement is required for the recurrent t(12;14) translocations, but not chromosome 14 amplification, in ATMdeficient thymic lymphomas.
Ataxia telangiectasia mutated (ATM) is a protein kinase and a master regulator of DNAdamage responses. Germline ATM inactivation causes ataxia-telangiectasia (A-T) syndrome with severe lymphocytopenia and greatly increased risk for T-cell lymphomas/ leukemia. Both A-T and T-cell prolymphoblastic leukemia patients with somatic mutations of ATM frequently carry inv(14;14) between the T-cell receptor a/d (TCRa/d) and immunoglobulin H loci, but the molecular origin of this translocation remains elusive. ATM2/2 mice recapitulate lymphocytopenia of A-T patients and routinely succumb to thymic lymphomas with t(12;14) translocation, syntenic to inv(14;14) in humans. Here we report that deletion of the TCRd enhancer (Ed), which initiates TCRd rearrangement, significantly improves ab T cell output and effectively prevents t(12;14) translocations in ATM2/2 mice. These findings identify the genomic instability associated with V(D)J recombination at the TCRd locus as the molecular origin of both lymphocytopenia and the signature t(12;14) translocations associated with ATM deficiency. (Blood. 2015;125(17):2665-2668)
Introduction translocation patterns of ATM-deficient thymic lymphomas,3 suggesting that TCRd might be involved. In this context, TCRd-related translocations are found in the majority of T-ALL and .90% of TAL1 translocated cases.8 Using ATM2/2Ed2/2 mice, here we identified aberrant TCRd rearrangements as the origin for both ab T-cell development defects and the recurrent t(12;14) translocations in ATM-deficient mice.
As a master regulator of the DNA damage response, ataxia telangiectasia mutated (ATM) kinase is rapidly activated by DNA doublestrand breaks and phosphorylates substrates involved in both DNA repair and checkpoint control. Loss of ATM causes ataxiatelangiectasia (A-T) syndrome characterized by primary immunodeficiency and greatly increased risk for lymphoid malignancies, especially of T-cell lineage.1 Moreover, .50% of T-cell prolymphoblastic leukemia (T-PLL) patients carry a somatic mutation of ATM.2 Both T-PLL and A-T patients carry inv(14;14) that involves the T-cell receptor a/d (TCRa/d) and immunoglobulin H loci on human chromosome 14. However, the molecular origin of the seemingly T-cell–specific lymphocytopenia and inv(14;14) remains unknown. ATM2/2 mice recapitulate the T-cell lymphocytopenia of A-T patients and succumb to thymic lymphomas with TCRa/d-related chromosome 14 amplification and t(12;14) translocations, syntenic with inv(14;14) in humans.3 Reduced ab T-cell numbers and blockade at the double-positive (DP) to single-positive (SP) transition is characteristic of ATM-deficient T cells.4,5 Both human and mouse TCRd loci reside between Va and Ja segments.6 As such, TCRa rearrangement deletes the TCRd gene, leading to irreversible commitment to the ab T-cell lineage. This stage-specific rearrangement of the TCRd in double-negative (DN) and TCRa in DP T cells is driven by the loci-specific enhancer elements Ed and Ea, respectively.6,7 However, Ea deletion does not affect the kinetics or
All animal work was conducted with proof from the institutional Animal Care and Use Committee of Columbia University. ATM2/29 and Ed2/210 mice were described previously. T-cell development was characterized in 4- to 6-week-old mice as previously described.11 Briefly, single-cell suspensions of thymus and spleen were counted and stained with fluorophore-conjugated antibodies against CD4, CD8, CD3, TCRb, or TCRg. For DN staining, thymocytes were counterstained with phycoerythrin-conjugated anti-CD4, CD8, B220, TCRgd, Ter119 and CD19 antibodies, then with fluorophoreconjugated anti-CD44 and anti-CD25 antibodies. Flow cytometry data were collected on FACSCalibur (BD) and analyzed with FlowJo. For cytogenetic analyses, single cell suspensions were incubated with colcemid (10 mg/mL) for 4 to 10 hours and fixed and stained3 using ASI applied spectral imaging paints. Images were acquired and analyzed with Isis and Metafer 4 (MetaSystems, Newton, MA). Comparative genomic hybridization (CGH)
Submitted January 13, 2015; accepted February 21, 2015. Prepublished online as Blood First Edition paper, February 26, 2015; DOI 10.1182/blood2015-01-622621.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2015 by The American Society of Hematology
BLOOD, 23 APRIL 2015 x VOLUME 125, NUMBER 17
Study design
2665
From www.bloodjournal.org by guest on September 10, 2016. For personal use only. 2666
JIANG et al
BLOOD, 23 APRIL 2015 x VOLUME 125, NUMBER 17
Figure 1. Deletion of Ed partially rescues lymphocytopenia in ATM-deficient mice. (A) Representative flow cytometric analyses of premalignant thymocytes from wild-type (WT), ATM2/2, Ed2/2, and ATM2/2Ed2/2 mice (4-6 weeks). (B) Total thymic gd cell number counts (310 6 ). (C) Total number of thymocytes and DP cells (3106). (D) SP cell number counts (3106). Data represent the average 6 standard deviation from at least 5 independent mice of each genotype (see supplemental Figure 1A for details). The P values between 2 genotypes were calculated based on the 2-tailed Student t test assuming unequal variance. Supplemental Figure 1B summarizes the P value from pairwise comparison for total, DP, and SP thymocytes between different genotypes.
was performed with the Agilent 244K mouse array. Reverse-transcription polymerase chain reaction (PCR) methods are detailed in Figure 2F and supplemental Table 1 (available on the Blood Web site).
Results and discussion ATM2/2Ed2/2 mice were born at the expected ratio and undistinguishable from ATM2/2 littermates. In thymuses of ATM2/2 Ed2/2 mice, gd T-cell frequency was reduced, but not abolished, consistent with the role of Ea in residual gd T-cell development10 (Figure 1A-B). Although thymus cellularity was reduced by ;50% in ATM2/2 mice, both thymus cellularity and the number of mature SP T cells were significantly rescued in ATM2/2Ed2/2 mice (Figure 1C-D). This is unexpected, given that .90% of thymocytes are ab T cells. Further analyses showed that this rescue was mostly due to moderate increases of DNIV (CD252CD442) percentage and marked increases of DP thymocyte number without changes in the SP to DP ratio (Figure 1C-D and supplemental Figure 1A-B). TCRd undergoes V(D)J recombination in DNII-DNIII (CD251).10 ATM is particularly important for inversional V(D)J recombination,12 and Vd5 is located downstream of Cd and requires inversional rearrangement.13 The configuration of the TCRa/d locus also determines that if DNA breaks in the TCRd loci are unrepaired, then Ea would separate from Vas and disable TCRa V(D)J recombination
(Figure 2C). These findings reveal an important role of genomic instability at the TCRd locus in ab T-cell development and T-cell lymphocytopenia associated with ATM deficiency, and they suggest that suppression of TCRd rearrangement might improve T-cell counts in A-T patients. Despite improved T-cell counts, the kinetics of succumbing to thymic lymphomas was similar in ATM2/2Ed2/2 mice and ATM2/2 controls (P 5 .1), with a median survival of 112 days (vs 99 for ATM2/2 controls) (Figure 2A). ATM2/2Ed2/2 thymic lymphomas consisted of immature (surface TCRblow) DP T cells similar to ATM2/2 controls (supplemental Figure 1C). Southern blot identified clonal TCRb rearrangements and focal amplification centromeric to the TCRa/d loci (chromosome 14) in all ATM2/2Ed2/2 (or ATM2/2 Ed1/2) lymphomas as documented for ATM2/2 lymphomas (supplemental Figures 1D and 2A).3 ATM2/2 thymic lymphomas were derived from immature T cells that had not yet undergone TCRa arrangement, evidenced by retention of the Cd.3 All ATM2/2Ed1/2 and 2 of 5 ATM2/2Ed2/2 lymphomas (545 and 2280) retained at least 1 Cd, suggesting that the chromosome 14 amplifications most likely occurred during TCRd rearrangement in those tumors, consistent with the ability for Ea to drive residual TCRd rearrangements in the absence of Ed.10 Notably, the other 3 ATM2/2Ed2/2 lymphomas showed homozygous deletion of Cd, indicative of biallelic TCRa rearrangements (Figure 2C). Southern blot further confirmed that all 3 tumors (497, 713, and 745) rearranged the proximal Ja segments (Figure 2C). Thus, TCRd rearrangement is not required for
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Figure 2. Aberrant TCRd rearrangement is required for t(12;14), but not chromosomal 14 amplifications, in murine ATM-deficient thymic lymphomas. (A) Survival curve for ATM2/2 (n 5 10), ATM2/2Ed2/2 (n 5 11), and ATM2/2Ed1/2 (n 5 8) mice with median survival at 99, 112, and 137 days, respectively. P values were calculated using the log-rank test. (B) Representative chromosome 12 (green) and chromosome 14 (red) paint analyses of a metaphase from no. 745 ATM2/2Ed2/2 lymphomas (without t(12;14)) and no. 900 ATM2/2Ed1/2 lymphomas with t(12;14) translocation. More than 20 metaphases were analyzed for each lymphoma, and t(12;14) in .50% metaphases was used as the criterion for clonal translocations. The results are summarized in panel E. (C) Southern blot mapping the rearrangements of Cd and Ja. The diagram shows the TCRa/d locus, EcoRI (black arrows), and probe locations (black dashed line for Southern probes and red dashes for adjacent CGH probes). A total of 10 mg EcoRIdigested genomic DNA from either tumor (T) or the corresponding kidney (K) was analyzed on each lane. Loading control probe detected a fragment of the single-copy DNAPKcs gene. (D) CGH analyses of ATM2/2Ed2/2 thymic lymphomas 745 and 545. Each tumor DNA was analyzed with matched kidney control DNA on a 244K mouse CGH array from Agilent. The log ratio of tumor vs kidney control at each probe location is displayed in centromere-to-telomere orientation for each chromosome (from 1 to 19 then X/Y, from left to right). The red arrows and labels indicate recurrent amplification of Notch1, chromosome 14 upstream of TCRa/d locus, and trisomy 15. Green arrows and labels indicate recurrent deletion of the telomeric portion of chromosome 12 and the Pten locus, as well as deletional rearrangement in TCR loci. (E) Summary of chromosome 12/14 paint analyses of ATM2/2Ed2/2 and ATM2/2Ed1/2 lymphomas. (F) Quantitative PCR results that measured the expression of IL7R, Jak1, Notch1, Pten, Rb1, Bcl11b, and Psmc6 genes from ATM2/2Ed1/1 (n 5 3), ATM2/2Ed1/2 (n 5 4), ATM2/2Ed2/2 (n 5 5) lymphomas. Psmc6 locates at the amplification area within chromosome 14 and is used as a marker for the amplification here.3 Bcl11b is within the region that is hemizygously deleted in chromosome 12.3 Briefly, total RNA was extracted using Trizol (Invitrogen, CA) from a single-cell suspension derived from ATM2/2 and ATM2/2Ed2/2 thymic lymphomas as well as wild-type total thymus, and dissolved in 100 mL DEPCH2O and verified to have A260 nm/280 nm between 1.8 and 2. Approximately 5 mg of total RNA was treated with RNase-free DNase I (Roche). The DNaseI activity was abolished by the addition of 2 mL of 25-mM EDTA and incubation at 65°C for 15 minutes. Reverse transcription was performed using SuperScript III reverse transcriptase (Invitrogen) and Oligod(T)18 (New England Biolabs) according to the manufacturer’s instructions. The resulted complementary DNA was dissolved in TE and quantified with NanoDrop (Thermo Scientific). Quantitative PCR was then performed with gene-specific primers (supplemental Table 1) for Notch1 (NM_008714.3, 138 bp across exons 25-26), Jak I (NM_146145.2, 162 bp across exons 18-20), IL7R (NM_008372.4, 149 bp across exons 5-7), Rb1 (NM_009029.2, 144 bp across exons 23-25), Bcl11b (NM_001079883.1, 126 bp across exons 3-4, within chromosome 12 telomeric deletion), Pten (NM_008960.2, 144 bp across exons 7-8), Psmc6 (NM_025959.3, 196 bp across exons 7-9, within chromosome 14 amplification), and b-actin (actb; NM_007393.3, 154 bp across exons 1-2). Reaction setups include 10 mL Sybr Select Master Mix (Applied Biosystems), 250 nM each primer, 10 ng complementary DNA, and water to a total of 20 mL. The PCR condition was 50°C (2 minutes), 95°C (10 minutes), and [95°C (15 seconds), 60°C (1 minute)] 3 40, followed by the melt curve program provided by 7500 Real Time PCR System (Applied Biosystems). The results were analyzed with 7500 software package (v 2.0.6) and the DDCT method (included in 7500 software) with the expression of b-actin as the reference and the expression of the specific gene in the wild-type samples as controls for normalization. Each gene in each sample was analyzed in triplicate together with H2O control. The H2O control samples always had CT beyond the range (.40) in all experiments.
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chromosome 14 amplification and T-cell lymphomagenesis in ATM-deficient mice. In the absence of the TCRd rearrangement, aberrant TCRa rearrangement is able to promote chromosome 14 amplifications and oncogenic transformation of ATM-deficient T cells. We next investigated the origin of the t(12;14) translocations characteristic of ATM deficiency.3 Chromosome paint identified clonal t(12;14) translocations in all ATM2/2Ed1/2 lymphomas and the 2 Cd-positive ATM2/2Ed2/2 lymphomas (545 and 2280), but not in the 3 ATM2/2Ed2/2 lymphomas (497, 713, and 745) with biallelic TCRa rearrangements (Figure 2B,E). CGH analyses of 2 Cd-positive (2280 and 545) and 2 Cd-negative (497 and 745) ATM2/2 Ed2/2 lymphomas confirmed focal amplification upstream of the TCRa/d locus (Figure 2D and supplemental Figure 2B) and also revealed a unique hemizygous deletion of chromosome 14 downstream of the TCRa/d locus only in Cd-negative (497 and 745) tumors (Figure 2D and supplemental Figure 2B). CGH probe mapping showed that the deletion started at the proximal Ja (Figure 2C-D). Together with the absence of the t(12;14) translocation, this result suggests that when chromosome 14 amplification occurs in DP T cells during TCRa arrangement, the break in chromosome 12 is no longer available to form the t(12;14) translocation, causing the telomeric portion of chromosome 14 to be lost and pointing to concurrent immunoglobulin H and TCRd rearrangement in DN T cells as the molecular origin for t(12;14). This further implied that t(12;14) and the syntenic inv(14,14) are not essential for T-cell transformation. Despite the lack of t(12;14), both Cd-negative (497 and 745) tumors displayed hemizygous deletion of chromosome 12 telomeric regions, supporting the existence of potential tumor-suppressor genes (eg, Bcl11b).3,14-16 In this context, SKY analyses identified t(12;15) translocations involving chromosome 15 in tumor 745 (Figure 2B and supplemental Figure 2C). Finally, CGH also indicated that murine ATM2/2 thymic lymphomas, regardless of Ed status, display genetic changes that have been associated with human T-cell acute lymphoblastic leukemia (T-ALL), including trisomy of chromosome 15 containing c-Myc, amplification and overexpression
of Notch1, deletion of Pten, and overexpression of IL7R (Figure 2F), highlighting ATM-deficient murine thymic lymphomas as a bona fide animal model for human T-ALL. In summary, our study identifies aberrant TCRd rearrangement in the absence of ATM together with the unique configurations of the TCRa/d locus as one of the major causes to both T-cell–specific lymphocytopenia and the t(12;14) and related inv(14;14) associated with ATM deficiency and potentially in other T-ALL.
Acknowledgments This work was supported in part by National Institutes of Health (NIH) National Cancer Institute grants 5R01CA158073, 1R01CA184187, and 1P01CA174653-01 and American Cancer Society Research Scholar Grant RSG-13-038-01 DMC (S.Z.) and by NIH National Cancer Institute grant P01CA109901 (F.W.A.). S.Z. was a St Baldrick’s Scholar for Pediatric Cancer and is a Leukemia Lymphoma Society Scholar. W.J. was supported in part by NIH National Cancer Institute grant T32-CA09503. F.W.A. is a Howard Hughes Medical Institute investigator.
Authorship Contributions: W.J., F.W.A., and S.Z. designed experiments; S.Z. wrote the paper; M.G. performed the SKY analyses for tumor 745; and W.J., B.J.L., C.L., R.L.D., and S.Z. performed the rest of the experiments. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Shan Zha, Institute for Cancer Genetics, Columbia University Medical Center, 1130 St. Nicholas Ave, Room 503B, New York, NY 10032; e-mail: [email protected].
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2015 125: 2665-2668 doi:10.1182/blood-2015-01-622621 originally published online February 26, 2015
Aberrant TCRδ rearrangement underlies the T-cell lymphocytopenia and t(12;14) translocation associated with ATM deficiency Wenxia Jiang, Brian J. Lee, Chen Li, Richard L. Dubois, Monica Gostissa, Frederick W. Alt and Shan Zha
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