The mature sporadic T-cell malignancy, T-cell prolymphocytic leukemia (T-PLL) is remarkable for frequently harbouring somatic mutations of the Ataxia Telangiectasia (A-T) gene, ATM. Because some data suggest ATM is frequently rearranged in T-PLL, it was decided to investigate such rearrangements in detail by cloning breakpoints. Among 17 T-PLL tumour samples, three rearrangements were detected by Southern blotting. Two cases harboured a unique type of intragenic duplication in which breakpoints arose at the consensus sequence RGYW/WRCY. The third case harboured a large deletion terminating within the ATM gene. Also, 13 T-cell acute lymphoblastic leukemia (T-ALL) samples were examined and one sample harboured a deletion– insertion with the RGYW motif at the breakpoint in ATM. This is the first known deleterious mutation detected in ATM in T-ALL. Interestingly, the RGYW motif is the signal for a cell-cycle regulated DNA double strand break (DSB) that initiates somatic hypermutation of immunoglobulin and, probably, T-cell receptor genes. The structures of the ATM duplications suggest they may arise from an error in somatic hypermutation. We suggest that aberrant components of somatic hypermutation may contribute to the defective DSB repair characteristic of cancer.
Defective DNA double strand break (DSB) repair and increased incidence of chronic and acute T-cell leukemias are notable features of Ataxia Telangiectasia (A-T) (Hecht and Hecht, 1990; Taylor et al., 1996) and it has been proposed that the product of the A-T gene, Atm protein, surveys the genome for DSBs (Meyn, 1999). In sporadic T-cell leukaemias, the chronic form (T-cell prolymphocytic leukaemia (TPLL)) harbours nucleotide changes or rearrangements in the A-T gene, ATM, in 50% of cases (Vorechovsky et al., 1997; Stilgenbauer et al., 1997; Yuille et al., 1998; Stoppa-Lyonnet et al., 2000; Stankovic et al., 2001). By contrast, no ATM mutations have been found in acute T-cell leukaemia (T-cell acute lymphoblastic leukaemia (T-ALL)), (Takeuchi et al., 1998; Haidar et al., 2000; Luo et al., 1998). In T-PLL, Southern blotting showed that one in five cases harboured a rearrangement within ATM (the less robust method of examining stained DNA fibres over-estimated this proportion) (Yuille et al., 1998). By contrast, constitutional ATM rearrangements arise in less than 1% of A-T patients (see A-T database at www.vmresearch.org). This difference suggested that the ATM gene might be susceptible to rearrangement during T-cell leukemogenesis. To investigate this possibility, an examination was undertaken of ATM breakpoints in T-PLL and T-ALL.
Restricted tumour DNA from 17 cases of T-PLL and 13 cases of T-ALL (Table 1) was examined with ATM cDNA and exon-specific probes. Rearrangements were found in three T-PLL cases and one T-ALL case. Three of the breakpoints (in T-PLL17, T-PLL16 and T-ALL10) were cloned by long-distance PCR. Total RNA was subjected to analysis by RT–PCR using primer pairs spanning the ATM coding sequence. The results are presented below and are summarized in Figure 1.
In T-PLL17, (Figure 1, panel 1) RT–PCR with primers in ATM exons 55 to 62 yielded a product of 972 bp (117 bp longer than normal) (Figure 1, panel 1b). This product harboured two consecutive copies of ATM exon 58, suggesting there had been a duplication involving this exon. This is predicted to result in insertion of 39 amino acids. Long-distance PCR on T-PLL17 DNA using primers within ATM exons 57 and 59 gave the expected 8.3 Kb product and a 8.6 Kb product. PCR with primers within ATM introns 57 and 58 gave the expected 2.3 Kb product and a 2.6 Kb product that harboured two adjacent copies of ATM exon 58 (Figure 1, panel 1c and Figure 2). This structure was consistent with Southern blotting data (Figure 1, panel 1a). Additional rearranged bands were consistent with a deletion event on the second allele between ATM introns 54 and 56. RT–PCR analysis indicated that the deletion abrogated ATM transcription (Figure 1, panel 1b and data not shown).
In T-PLL16 (Figure 1, panel 2), RT–PCR with primers in ATM exons 28 and 33 yielded a normal 857 bp product and a 1051 bp product which harboured a duplication of ATM exon 30, a partial duplication of ATM exon 31 and a segment of ATM intron 29 (Figure 1, panel 2b). This results in a stop codon in the 3′ copy of exon 30. Long-distance PCR on T-PLL16 DNA using primers within ATM exons 29 and 32 yielded a normal 5.1 Kb product and a 6.3 Kb product (Figure 1, panel 2c). PCR on T-PLL16 DNA using a forward primer in ATM exon 31 and a reverse primer in ATM exon 30 gave a single product of 864 bp (not shown) whose sequence identified a breakpoint within ATM exon 31 juxtaposed to sequence within intron 29 (Figure 1, panel 2c and Figure 2). This duplication was consistent with Southern blotting data (Figure 1, panel 2a).
Furthermore, RT–PCR on T-PLL16 RNA with primers in ATM exons 38 and 45 gave rise to a normal 893 bp product and a 806 bp product which lacked ATM exon 40 (87 bp) presumably because of the splice site mutation at IVS40 +1 G>C (Table 2). The net effect is loss of 29 amino acids. RT–PCR on T-PLL16 RNA with primers from ATM exons 29 and 41 showed that this splice site mutation was not on the same allele as the duplication of ATM exons 30 and 31.
In T-ALL10 (Figure 1, panel 3), Southern blotting identified a rearrangement between ATM introns 22 and 24 (Figure 1, panel 3a). RT–PCR on T-ALL10 RNA with primers from ATM exons 19 and 25 yielded normal 771 bp product and a 564 bp product which harboured a deletion of ATM exons 23 and 24 (Figure 1, panel 3b). This is predicted to result in deletion of 69 amino acids. Long-distance PCR on DNA from T-ALL10 using primers from ATM introns 20 and 24 yielded a product of 2617 bp (Figure 1, panel 3c) which harboured a deletion within ATM intron 22 to within ATM intron 24. At the intron 22 breakpoint there was an insertion of a simple sequence repeat (TAAA) which abutted part of a L1P LINE1 sequence (Figure 1, panel 3c and Figure 2). This L1P LINE sequence had recombined with an L1P3 LINE1 sequence in intron 24 but the precise site of recombination could not be determined because of the similarity of the two sequences. Tumour T-ALL10 arose in a four-year-old girl (Ravid et al., 1980).
In T-PLL15, ATM exon 29 and 30 probes identified a new band. Analysis suggested a deletion extending toward the centromere from within ATM intron 28 to an unknown locus and this was supported by gene dosage experiments (data not shown).
The T-PLL and T-ALL samples were also subjected to exon-scanning to identify ATM nucleotide changes. DNA was available from mouthwash samples from some T-PLL patients. The results of this analysis are summarized in Table 2. One a T-PLL patient harboured a non-conservative ATM nucleotide change that was germline. This finding contrasts with the tenet that up to half of T-PLL is in ATM heterozygotes (Vanasse et al., 1999) and with data from this lab and elsewhere suggesting germline ATM mutations might be absent in sporadic T-PLL (Stoppa-Lyonnet et al., 2000; Stankovic et al., 2001). Non-conservative nucleotide changes were detected in seven T-PLL samples but not in T-ALL, consistent with previous findings.
No recombination signal sequences were identified in the flanks of the breakpoints in T-PLL16, T-PLL17 or T-ALL10. However, the consensus sequence RGYW or its complement WRCY abutted the breakpoints in all three cases (see boxed sequences in Figure 2). This motif is of interest since somatic hypermutation in B-cells is initiated by a DSB at RGYW/WRCY in immunoglobulin variable region (IgV) genes (Papavasiliou and Schatz, 2000). Data on T-cells indicates that somatic hypermutation at T-cell receptor alpha (TCRA) genes arises by the same mechanism as in B-cells (Zheng et al., 1994). The finding that ATM breakpoints are at RGYW in T-PLL and T-ALL provides evidence that, during T-cell leukemogenesis, ATM rearrangements are mediated at least in part by the somatic hypermutation mechanism. ATM is likely cleaved at RGYW and this DSB is then misrepaired to yield a duplication or insertion/deletion. Papavasiliou et al. (2000) have proposed a model, outlined in Figure 3a, for somatic hypermutation following an initial DSB at RGYW. A variant of this model (Figure 3b) leads to ATM exon duplications (or deletions) commencing at RGYW.
The detailed analysis of rearrangements presented here provides evidence that a specific mechanism of DNA cleavage initiates inactivation of a specific tumor suppressor gene during tumorigenesis. Other workers have provided evidence suggesting that RGYW motifs represent frequent targets for somatic hypermutation for oncogenes such as BCL-6 in normal germinal centre-derived B-cells and in their neoplastic counterparts (Shen et al., 1998; Pasqualucci et al., 1998, 2000). The motif has been suggested as a target for reciprocal translocation activating BCL-1, BCL-2 and MYC oncogenes because of the absence of classic recombination signal sequences (Klein et al., 1998). We now extend this suggestion: RGYW/WRCY motifs may also represent during tumorigenesis targets for tumour suppressor gene disruption and for other non-reciprocal rearrangements. Indeed the ATM deletion-insertion in T-ALL10, taken with the absence of pathogenic nucleotide changes in T-ALL, provides initial evidence that during leukemogenesis in early thymic cells, RGYW/WRCY motifs in ATM may be more susceptible to rearrangement than the gene is to nucleotide changes. Finally, we suggest that aberrant components of somatic hypermutation may contribute to the defective DSB repair characteristic of cancer.
- ATM :
Ataxia Telangiectasia Mutated gene
T-cell prolymphocytic leukemia
T-cell acute lymphoblastic leukemia
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We thank Ray Powles (Royal Marsden NHS Trust Hospital) for samples. PS Bradshaw is a Gordon Piller Scholar of the Leukemia Research Fund. We acknowledge support from the Kay Kendall Leukemia Trust.
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Leukemia & Lymphoma (2012)
Expert Opinion on Investigational Drugs (2012)