Leukemia (2001) 15, 1448–1450  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Investigation on the role of the ATM gene in chronic myeloid leukaemia JV Melo1, A Kumberova1, AG van Dijk2, JM Goldman1 and MR Yuille2 1 Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London; UK and 2Academic Department of Haematology and Cytogenetics, Institute of Cancer Research, Sutton, UK Chronic myeloid leukaemia (CML) is characterised by an indolent, chronic phase (CP) preceding an acute transformation to blast crisis (BC). While the BCR-ABL fusion oncogene is strongly implicated in the CP, the molecular changes underlying BC are largely unknown. The ataxia telangiectasia gene, ATM, is a candidate gene for this transformation because the complex karyotypes associated with BC of CML suggest that DNA double-strand break repair is defective and because the ABL pathway involves the interaction between the Abl and the Atm proteins. We performed a mutational analysis for ATM in CML using genomic DNA from 14 CML cell lines and 59 CML patients in BC. No clearly deleterious nucleotide changes were observed. A new polymorphism C4138T was discovered which results in a non-conservative amino acid substitution (H1380Y). This variant lies in the Atm recognition motif for the Abl protein. While ATM is unlikely to contribute substantially to CML, further investigation of the H1380Y substitution should clarify whether it has any functional effect. Leukemia (2001) 15, 1448–1450. Keywords: chronic myeloid leukaemia; ataxia telangiectasia; blast crisis; mutation; genomic instability Introduction Chronic myeloid leukaemia (CML) is a myeloproliferative disorder that classically evolves in three clinical stages: a chronic phase (CP) with a relatively benign phenotype, an accelerated phase (AP) characterised by increasing refractoriness to therapy, and a final acute transformation or blast crisis (BC) which is usually unresponsive to treatment and invariably fatal. The onset of CML is caused by the generation of the BCR-ABL oncogene in a t(9;22)(q34;q11) chromosomal translocation. The leukaemogenic nature of the encoded Bcr-Abl fusion protein relies on the fact that it has a constitutively activated tyrosine kinase which ultimately leads to a deregulated proliferation, decreased cell adherence and reduced apoptotic response to mutagenic stimuli.1 The latter feature is believed to underlie the invariable evolution of the disease to BC, by facilitating the emergence of additional deleterious mutations. However, the known abnormalities in RAS, p53, RB and p16,2–5 account for less than 30% of the BC.3 The search for other possible causative mutations is naturally focused on genes that are relevant to the control of cell cycle, differentiation or apoptosis, and which have been associated with the genesis of other malignancies.6,7 Ataxia telangiectasia, a recessive disorder due to mutations in the ATM gene, is characterised by genomic instability and by early onset of solid and haematological malignancies.8,9 After X-irradiation, the Atm protein directly interacts with the Abl protein via 10 amino acids encoded by sequence within exon 3010 and then phosphorylates Abl at Ser 465.11 At the same time, the Atm protein interacts with and phosphorylates Correspondence: JV Melo, Dept of Haematology – ICSM, Hammersmith Hospital, Ducane Road, London W12 0NN, UK; Fax: + 44 (0)20 8742 9335 Received 26 February 2001; accepted 18 May 2001 p53 Ser15 via residues encoded within ATM exons 61 to 65.12,13 This Atm protein kinase activity also has Chk2 as a substrate.14 The last 10 exons encode a region called the PIKlike domain that shows extensive homology to phosphatidylinositol 3- and 4-kinases.15 Sporadic forms of some of the haematological malignancies that are prevalent in A-T patients are mutated at ATM. In T cell prolymphocytic leukemia (T-PLL) half of cases acquire mutations and there is a clustering of missense mutations in exons 50 to 65.16 ATM is also mutated in a B cell non-Hodgkin’s lymphoma subtype and B cell chronic lymphocytic leukemia.17–19 The number of mutations reported so far is small but consistent with clustering in exons 50 to 65. ATM is a candidate gene in CML because the complex karyotypes associated with BC of CML suggest that there is genomic instability and because the ABL pathway is strongly implicated in CML and this pathway involves the interaction between the Abl and the Atm proteins. We therefore performed a mutation analysis of two regions of the ATM gene in CML cases and cell lines: the region involved in the interaction with Abl and the mutational cluster region comprising the last 15 exons of ATM. Materials and methods Study population Fourteen cell lines established from the blast crisis (BC) of CML were investigated: K562, KCL22, KYO1, KU812, LAMA84, BV173, EM3, EM2, NALM1, CML-T1, AR230, SD1, TOM1 and MEG-01. These lines20 were either purchased from cell repository banks (American Type Culture Collection, Rockville, MD, USA; European Collection of Cell Cultures, Winchester, UK; or German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) or kindly donated by their originators. Cells were grown in RPMI1640 (GibcoBRL, Paisley, UK) supplemented with penicillin, streptomycin, L-glutamine and 10% fetal calf serum. Peripheral blood leucocytes from 59 patients with CML in BC and 83 healthy adult volunteers were obtained by red cell lysis. For some of the latter group, buccal epithelial cells from saline mouthwash were used instead of or in addition to blood leucocytes. The BC leucocyte suspensions were further fractionated by density centrifugation and the mononuclear cells containing 85–95% of blasts were isolated for the mutation analysis. All samples were obtained after informed consent. Mutation detection DNA was extracted by conventional methods and 50 ng was used in polymerase chain reaction (PCR) amplification using primers designed for exons 27 to 33 and exons 50 to 65. Exons are numbered according to Uziel et al.21 Primer sequences were in the flanking introns (except for reverse primer for exon CML and the ATM gene JV Melo et al 65), and their design was based on the sequence deposited as GenBank accession number U82828. Forward and reverse primers (5⬘–3⬘) were as follows: exon 27 = CTT AAC ACA TTG ACT TTT TGG and GTA TGT GTG TTG CTG GTG AG; exon 28 = GCT GAT GGT ATT AAA ACA GTT T and GTT ATA TCT CAT ATC ATT CAG G; exon 29 = TGC CTT TTG AGC TGT CTT GA and AGA CAT TGA AGG TGT CAA CCA A; exon 30 = TGA ACA AAA CTT TTT AAA ACG ATG AC and AGA AGG AAT GTT CTA TTA TTA AAC TCA; exon 31 = CCG AGT ATC TAA TTA AAC AAG and CAG GAT AGA AAG ACT GCT TAT; exon 32 = CCA GAA CTT ACT GGT TGT TGT TG and AAA ACA CTC AAA TCC TTC TAA CAA T; exon 33 = TTC GCA ACG TTA TGG TGG TAT and TGC TAG AGC ATT ACA GAT TTT TG; exon 50 = GGG CAG TTG GGT ACA GTC AT and GTA ACA ATG TTT CAC TCC ACC C; exon 51 = CGT GGG TTG GAC AAG TTT G and TAA GCC GAC CTT TAG AGC TCC; exon 52 = TTT CCC TGG GAT AAA AAC CC and TAC ACG ATT CCT GAC ATC AAG G; exon 53 = CCA CTT GTG CTA ATA GAG GAG C and TTC CAT TTC TTA GAG GGA ATG G; exon 54 = TGC AGG CAT ACA CGC TCT AC and CCA GCC TTG AAC CGA TTT TA; exon 55 = AAA GGC ACC TAA GTC ATT GAC G and GGG AAT GTT GAA GCC ATC AG; exon 56 = CTT GAC CTT CAA TGC TGT TCC and TGC CAA TAT TTA GCC AAT TTT G; exon 57 = CAC ATC GCA TTT GTT TCT CTG and CAA AAT CCC AAA TAA AGC AGA A; exon 58 = ATT GGT TTG AGT GCC CTT TG and ATT ATG AAT ATG GGC ATG AGC C; exon 59 = AGG TCA ACG GAT CAT CAA ATG and AGC TGT CAG CTT TAA TAA GCC A; exon 60 = ATC CTG TTC ATC TTT ATT GCC C and CAA AAA TAA AAC CTG CCA AAC A; exon 61 = CTC AAC ATG GCC GGT TAT G and CAA ACA ACA TTC CAT GAT GAC C; exon 62 = TGA GGA AGG CAG CCA GAG and GTG CAA AGA ACC ATG CCC; exon 63 = TTG ACA ACT TGG TGT GTA ACG and GCC ACA TCC CCC TAT GTT AA; exon 64 = TCC CCC ATC AAC TAC CAT GT and GAA CAG TTT AAA GGC CTT GGG; exon 65 = CAA GGC CTT TAA ACT GTT CAC C and TTG GCA GGT TAA AAA TAA AGG C. The PCR volume was 10 ␮l, with 0.1 ␮Ci P-32dCTP in a 96-well plate, using the Hybaid Touchdown sub-ambient apparatus (Hybaid, Ashford, UK). Where a PCR product was longer than 250 bp, it was digested with an appropriate restriction enzyme to yield two or three fragments prior to single-strand conformation polymorphism (SSCP). PCR product in 50% formamide was heat-denatured, snapchilled and fractionated for 6 h at room temperature at 15 W on a 6% polyacrylamide gel containing 10% glycerol. The gel was dried down on a sheet of 3MM paper and visualised by autoradiography. Some samples gave rise to abnormal bands. These samples were re-amplified with the original primers, subcloned in the TOPO-TA vector (Invitrogen, Groningen, The Netherlands) following the manufacturer’s protocol, and sequenced with M13 primers and the Big Dye kit (Applied Biosystems Warrington, UK) on an ABI Prism 377 DNA Sequencer (Applied Biosystems). Nucleotides (nt) are identified by counting from the first base of the first codon of ATM cDNA (GenBank accession number U33841, nucleotide 190). 1449 Figure 1 Autoradiogram of SSCP mutation detection for ATM exon 31. Labelled PCR products were digested with EcoRI and fractionated by SSCP. The arrow indicates an extra band seen in one CML patient in blast crisis and in chronic phase (BC1 and CP1, respectively) and in a second CML patient in blast crisis. Figure 2 Analysis of ATM C4138T by MlnI digestion of exon 30 PCR product. Lane 1: 123 bp DNA ladder (Gibco BRL, Paisley, UK). Lanes 2 and 3: undigested products. Lanes 4–13: products digested with MlnI. Vertical arrow (lane 10) indicates a heterozygote. one of these two patients a DNA sample from diagnosis at CP was shown to have the same additional band. Sequencing of the abnormal PCR products identified C4138T (H1380Y) in NALM1, and C4258T (L1420F) in the two patients. C4138T results in the loss of an MnlI site and so can be detected by its resistance to restriction (Figure 2). Screening of 78 healthy persons identified one heterozygote with C4138T. SSCP analysis of 83 healthy persons for exon 31 identified one individual heterozygous for C4258T (Table 1). Discussion This study was undertaken to assess the contribution of ATM to the blastic transformation of CML by mutation detection analysis of two key regions of ATM. DNA from 73 samples was examined for mutations in ATM exons encompassing the Table 1 Exon Polymorphisms in the Abl-binding region of ATM in CML Nucleotide and amino acid change Results DNA was prepared from 14 CML cell lines and 59 patients in BC of CML, amplified with primers for ATM exons 27–33 and exons 50–65 and subjected to SSCP gel fractionation. Additional bands abnormal in position were detected in exon 30 for NALM1 and in exon 31 in two patients (Figure 1). In Allelic counts (%) CML General population 30 C4138T H1380Y C = 145 (99.3) T = 1 (0.7) C = 165 (99.4) T = 1 (0.6) 31 C4258T L1420F C = 144 (98.6) T = 2 (1.4) C = 165 (99.4) T = 1 (0.6) Leukemia CML and the ATM gene JV Melo et al 1450 Abl-binding region and the region most strongly implicated in sporadic haematological malignancy. No clearly deleterious nucleotide changes were detected in the CML samples. However, a non-conservative amino acid change H1380Y (C4138T) was discovered that lies directly within the Abl-binding motif DPAPNPPHFP in exon 30 and two samples had a nearby rare polymorphism, C4258T, causing a non-conservative amino acid change (L1420F) that has been reported previously.22,23 Both nucleotide changes detected in the CML samples appeared to have arisen on one allele. It is likely that C4138T is a germline variant since the same variant was detected in a panel of healthy persons. However, there is evidence that some ATM germline variants recur as somatic mutations.24 It will be of interest to determine if either H1380Y or L1420F affect the interaction of the Abl with the Atm protein. It will also be of interest to test the frequency of these probable polymorphisms in a fully matched population. In conclusion, this study has provided evidence that tends to exclude mutations at ATM from playing any role in CML or its progression. These results were obtained by SSCP mutation detection, a technique that was validated by the identification of known and novel rare polymorphisms in both the CML and the normal populations studied. Moreover, the same assay has been used previously to identify frequent sequence changes in T-PLL25,26 and some B cell malignancies.27 The molecular basis for the complex karyotypes detected in BC of CML remains unexplained. Future candidates for investigation should include genes which when mutated are associated with genomic instability and elevated risk of myeloid malignancy. Acknowledgements We acknowledge support from the Kay Kendall Leukaemia Trust and the Leukaemia Research Fund. AGvD was a student on placement from the University of Manchester. We thank PS Bradshaw for critically reading the manuscript. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 References 1 Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000; 96: 3343–3356. 2 LeMaistre A, Lee MS, Talpaz M, Kantarjian HM, Freireich EJ, Deisseroth AB, Trujillo JM, Stass SA. Ras oncogene mutations are rare late stage events in chronic myelogenous leukemia. Blood 1989; 73: 889–891. 3 Feinstein E, Cimino G, Gale RP, Alimena G, Berthier R, Kishi K, Goldman J, Zaccaria A, Berrebi A, Canaani E. p53 in chronic myelogenous leukemia in acute phase. 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