Mechanisms that can promote peripheral B-cell lymphoma in ATM-deficient mice

Abstract

The Ataxia Telangiectasia-mutated (ATM) kinase senses DNA double-strand breaks (DSB) and facilitates their repair. In humans, ATM deficiency predisposes to B- and T-cell lymphomas, but in mice it leads only to thymic lymphomas. We tested the hypothesis that increased DSB frequency at a cellular oncogene could promote B-cell lymphoma by generating ATM-deficient mice with a V(D)J recombination target (DJβ cassette) within c-myc intron 1 ("DA" mice). We also generated ATM-deficient mice carrying an Eμ-Bcl-2 transgene (AB mice) to test whether enhanced cellular survival could promote B-cell lymphomas. About 30% of DA or AB mice and nearly 100% of mice harboring the combined genotypes (DAB mice) developed mature B-cell lymphomas. In all genotypes, B-cell tumors harbored oncogenic c-myc amplification generated by breakage-fusion-bridge (BFB) from dicentric chromosomes formed through fusion of IgH V(D)J recombination-associated DSBs on chromosome 12 to sequences downstream of c-myc on chromosome 15. AB tumors demonstrate that B lineage cells harboring spontaneous DSBs leading to IgH/c-myc dicentrics are blocked from progressing to B-cell lymphomas by cellular apoptotic responses. DA and DAB tumor translocations were strictly linked to the cassette, but occurred downstream, frequently in a 6-kb region adjacent to c-myc that harbors multiple cryptic V(D)J recombination targets, suggesting that bona fide V(D)J target sequences may activate linked cryptic targets. Our findings indicate that ATM deficiency allows IgH V(D)J recombination DSBs in developing B cells to generate dicentric translocations that, via BFB cycles, lead to c-myc-activating oncogenic translocations and amplifications in mature B cells.

Figure 1. DA and AB Mice Develop B-Cell Lymphoma at an Early Age

Kaplan-Meier curves showing survival of mice within the DA (A) and AB (B) cohorts. Arrowheads indicate mice that developed B-cell lymphoma.

Figure 2. DA, AB, and DAB Tumors have Clonal IgH Rearrangements and c-myc Amplification

Southern blot analysis on EcoRI-digested genomic DNA of tumor samples from DA, AB, and DAB cohorts. Upper diagrams represent IgH (left) and c-myc (right) loci with position of EcoRI (RI) sites and probes used. For each Southern blot panel, molecular weight markers and probes are indicated on the left, and the running position of the germ-line (gl) band is indicated on the right. In the J_H4–3 panel, dots indicate clonal V(D)J rearrangements, triangles indicate bands corresponding to the pre-rearranged V_HB1–8 IgH locus present in some of the mice, and stars indicate the cross-reacting band from the Eμ-Bcl-2 transgene in DAB and AB mice. Red boxes indicate co-migrating rearranged c-myc and J_H bands. Lack of amplified J_H or c-myc band in some of the tumors is due of deletion of the region recognized by the probes upon translocation (details in Supplementary Tables S2, 4, 6).

Figure 3. ATM-deficient B-cell lymphomas harbor Clonal IgH/c-myc Complicons

(A) Diagrams represent normal chromosomes 12 (left panel) and 15 (right panel) with position of either IgH or c-myc BAC probes. (B) FISH analysis showing two types of complicons (“C”); only relevant chromosomes (chrs) are shown. Tumors with chr12-based complicon (e.g. DAB601, left) always have one normal chromosome 12 (n12) and two normal chromosome 15s (n15); tumors with chr15-based complicons (e.g. DA64, right) also contain a T(12;15) juxtaposing IgH to sequences downstream of c-myc (details in Supplementary Tables S2, 4, 6). The same metaphase was sequentially hybridized with whole chromosome paints (top) and locus-specific probes (bottom). Probes used are indicated on the left. (C) Schematic showing location of sequenced chromosome 12 and chromosome 15 translocation breakpoints in DAB (blue) and DA (red) tumor samples. Corresponding numbers on the chromosomes diagrams denote the locations of the two break-points joined in a translocation in given tumors (listed on the right). Black arrows show the length of the indicated regions. On chromosome 15, c-myc exons 1, 2, and 3 are indicated. The DJβ cassette is indicated in purple between exon1 and exon 2 of c-myc.

Figure 4. Chromosome 15 Translocations in DAB and DA Tumors Involve the c-myc^DJβ Allele

(A) Map of c-myc^WT and c-myc^DJβ alleles showing SacI (SI) and KpnI (KI) restriction sites and location of probes. (B and C) Southern blot analysis of SacI- (B) or KpnI-digested (C) genomic DNA from DA and DAB tumors with indicated probes. Molecular weight markers and running position of WT c-myc (c-myc^WT), non-rearranged c-myc^DJβ (c-myc^DJβNR) and rearranged c-myc^DJβ (c-myc^DJβR) alleles are indicated. DNA from 129/Sve spleen (WT SPL) and c-myc^DJβ/DJβ spleen (DJβ SPL)and thymus (DJβ THY) were used as controls. In the MycD blot (B), rearrangements of the c-myc^DJβ allele are compatible with precise (496) or aberrant (other tumors) V(D)J recombination joins within the DJβ cassette in the absence of ATM. In the Myc3’ blot (C), tumor 538 shows an amplified and rearranged KpnI fragment derived from the c-myc^DJβ allele, as expected from the cloned translocation breakpoint (Fig. 4 and Supplementary Table S6). Tumors 538 and 806 shows amplification with Myc3’ probe but not MycD probe, consistent with data shown in Fig. 2 and Supplementary Fig. S1. (D) Schematic showing 1he positions of tumor translocation junctions (top) and cryptic RSSs (bottom) in the region from c-myc exon one to 6 kb downstream and two other short regions more than 100 kb downstream of c-myc. Prediction of cryptic RSSs within this 6kb downstream region was generated with the program described at: http://www.itb.cnr.it/rss/index.html. Note that no strong cryptic RSS was predicted to lie in the 6.4 kb region, spanning the c-myc gene, upstream of the first left cryptic RSS. Blue boxes represent c-myc exons; yellow box represents the DJβ cassette. Red triangles indicate junctions from 1: DAB361, 2: DAB494, 3: DAB496, 4: DAB601, 5: DAB538, 6: DA473, 7: DA64, 8: DA360 tumors. Cryptic 23RSSs are showed in green, while 12RSSs in black; arrowheads indicate RSSs in (−) orientation if pointing to left, RSSs in (+) orientation if pointing to the right.

Figure 5. A Proposed BFB Mechanism for the Generation of c-myc Complicons Found in ATM-deficient B-cell Lymphomas

In ATM-deficient pro-B cells, RAG-initiated DSBs generated during D_H-to-J_H recombination at IgH, are not properly repaired and can be jointed to DSB downstream of c-myc locus, leading to the generation of 12;15 dicentric chromosomes. Dicentrics are unstable and can be broken again during chromosome segregation. In the absence of ATM, these unrepaired chromosomes can persist and due to lack of a G1 checkpoint can be replicated to generate dicentrics. Repeated breakage-fusion-bridge (BFB) cycles result in further amplification of c-myc. Finally, a complicon is formed when the dicentric is stabilized by recombination with a third chromosome via telomere capture. More details of this general model as it applies to NHEJ/p53 double deficient pro-B-cell lymphomas can be found in ref . Chr12 is showed in blue, chr15 in green, and the third chromosome participating to the complicon in purple. The black bar represents Eμ enhancer and the yellow box represents c-myc. The red arrows indicate c-myc transcriptional orientation.