Disruption Of ATM Research Articles

Phase II Trial to Evaluate the Efficacy and Safety of the Combination of Obinutuzumab and Bendamustine Treatment in Patients (Pts) with Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL)

Introduction: The use of combined rituximab (R)-bendamustine(B) has shown to be effective in terms of overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) in pts with relapsed/refractory CLL. Based on the enhanced in vitro and in vivo anti-CLL activity of obinutuzumab compared to rituximab, we designed this phase II study to evaluate the efficacy of obinutuzumab(Obi) with B in patients (pts) with relapsed or refractory CLL.Methods Between April 2014 and April 2017, 72 adult pts with relapsed or refractory CD20+ CLL (according to NCI criteria) and active disease (IWCLL 2008) were included in this prospective, multicenter, non-comparative, phase II study. Pts received up to six-28-day cycles of treatment consisting of obinutuzumab (1000 mg) given by intravenous (IV) infusion on Days (D) 1, 8, and 15 of Cycle 1 and on D1 of subsequent cycles, and B (70 mg/m2) by IV infusion on D2 and 3 of Cycle 1 and on D1 and 2 of subsequent cycles. The primary endpoint was ORR (by IWCLL 2008); secondary endpoints included best RR, OS and PFS, duration of response and minimal residual disease (MRD) rate detected by multicolor flow cytometry (sensitivity: 1 CLL cell in 10-4). We present the results of an interim analysis at 6 months of last Obi-B cycle with the data cut-off date of 25th of April of 2017.Results. Pts were mostly male (66.7%), with a median age of 67.9 (61.6-72.7) years and ECOG 0 in 62.5% of them. Rai clinical stage was low/intermediate (0-II) in 52.8% pts and high-risk (III-IV) in 47.2% pts. Forty eight pts relapsed after an FC+/-R (58.3%) or B-R (8.3%) therapy and 7(9.7%) pts were refractory to last immunochemotherapy. Expression of CD38 (>30%), CD49D (>20%), ZAP-70 was seen in 15.3%, 55.6%, and 39.4% of pts respectively, and unmutated immunoglobulin heavy chain gene (IGHV) status in 75.0% of pts. Genetic abnormalities detected by FISH and Sanger sequencing were: del13q(44.4%), trisomy 12(18.05%), del11q/ATMmutation(43.05%), del17p/p53mutation (26.3%), mutations of BIRC3(4.1%), SF3B1(9.7%) and NOTCH1(23.6%). Out of 72 enrolled pts, 80.6% and 79.2% completed 6 cycles of treatment with Obi and B respectively. Median (range) treatment duration was 4.9 (4.6-5.1) months. Obi doses were modified in 75% of pts (premature discontinuations: 4%; infusion rate reduction: 15.9%; temporarily interruptions: 77.8%). B was modified in 52.8% of pts (premature discontinuations: 7.2%; temporarily interruptions: 55.4%; dose reductions: 30.1; dose reduction rate: 2.4%). On an intent to treat analysis, 70(97.2%) pts were evaluable for efficacy according to protocol, the ORR was 81.4 % (CR 35.7%, CRi 7.1% and PR 38.6%), PD (2.9%) and SD (5.7%). In the 7 pts with refractory CLL, the ORR was 71.4% (CR 42.9%, CRi 14.3% and PR 14.3%) and SD 28.6%. After a median follow-up of 13.7 months, the median PFS was 27.0 months (figure 1). The median time to retreatment 33.8 months (figure 2). Significant differences were found in the PFS according to MRD status in Bone Marrow (Category: Cat.0: MRD level<0.01, 30.2 months, Cat. 1: MRD between 0.01-0.1: 21.6 months and Cat. 2:MRD>0.1: 12.1 months; p<0.001) and in the PFS regarding the IGHV mutation status (unmutated, 24.1 months vs mutated, median non-reached; p<0.05). The presence of p53 and ATM disruption did not have a significant impact on response, perhaps because of the high RR (81.4%). Sixty-three pts (87.5%) reported at least 1 toxicity, being the most common grade 3/4 neutropenia (51.4%) and thrombocytopenia (19.5%). In total, 82 SAEs or AESI were reported in 34(53.9%) pts. The discontinuation rate due to AEs was 18.1%. Two deaths occurred due to toxicities, acute myeloid leukemia and septic shock.Conclusions This interim analysis anticipates that the combination of obinutuzumab with Bendamustine is feasible, effective with an ORR of 81.4% and manageable in relapsed/refractory CLL. Achievement of MRD negativity in the bone marrow of pts treated with Obinutuzumab+Bendamustine seems associated with a longer PFS regardless of genetic abnormalities. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeted Next-Generation Sequencing of TP53, BIRC3, NOTCH1, SF3B1, and ATM Identifies a Significant Proportion of Lower-Risk Chronic Lymphocytic Leukemia with Unfavorable Prognostic Indicators

IntroductionThe clinical course of chronic lymphocytic leukemia (CLL) is heterogeneous, ranging from indolent disease with essentially normal life expectancy to rapidly progressive disease with significantly decreased survival. Traditional risk stratification is dependent in large part on cytogenetic analysis by fluorescence in situ hybridization (FISH), with del(13q) as a sole abnormality associated with a favorable prognosis, trisomy 12 or normal cytogenetics associated with an intermediate prognosis, and del(11q) or del(17p) associated with an unfavorable prognosis.1 In the last few years, recurrent mutations in key genes including TP53, BIRC3, NOTCH1, SF3B1, and ATM have also been identified as unfavorable prognostic indicators in CLL.2-4 In a study by Rossi et al, integration of mutations and cytogenetic abnormalities was found to significantly improve the accuracy of survival prediction in CLL beyond that of FISH alone.2 The aim of this study is to evaluate the frequency of these unfavorable mutations in an unselected cohort of CLL patients seen in the community setting, and to determine the percentage of lower-risk patients with a worse than expected prognosis who may potentially benefit from earlier and/or novel therapy.MethodsAn unselected cohort of 633 CLL patients seen in the community setting was included in this study. Demographic data included a mean age of 69 years, male to female ratio of 1.8:1, and mean WBC count of 48 g/dL. DNA sequences of key exons in TP53, BIRC3, NOTCH1, SF3B1, and ATM were determined from peripheral blood or bone marrow aspirates using a clinically validated next-generation sequencing (NGS) assay with a 5% lower limit of detection for single nucleotide variants. Results were compared between duplicate samples and annotated using software that queried databases containing known somatic mutations and germline variants. FISH analysis utilized probes specific for 11q22.3 (ATM), chromosome 12, 13q14.3/13q34, and 17p13.1 (TP53). CLL diagnoses were confirmed by flow cytometry, with FISH for t(11;14)(IGH-CCND1) also performed to exclude mantle cell lymphoma. The percentage of CLL cells by flow cytometry ranged from 18-98%, with a mean of 69%.ResultsCytogenetic abnormalities were detected in 85% of CLL patients in this study, with del(13q) the most frequent (65%), followed by trisomy 12 (19%), del(11q) (17%), and del(17p) (10%). Mutations in any one of the 5 genes were detected in 37% of patients, with TP53 mutation the most frequent (12%), followed by mutations in NOTCH1 (11%), SF3B1 (10%), ATM (7%), and BIRC3 (3%). TP53 disruption as a whole was found in 14% of patients, consisting of del(17p) only (2%), TP53 mutation only (5%), and biallelic TP53 disruption (7%). ATM disruption as a whole was found in 21% of patients, consisting of del(11q) only (14%), ATM mutation only (4%), and biallelic ATM disruption (4%). Lower-risk patients, as defined by FISH showing either isolated del(13q), trisomy 12, or normal results, comprised 75% of patients, with 29% showing one or more unfavorable mutation: NOTCH1 (12%), SF3B1 (8%), TP53 (6%), ATM (4%), and BIRC3 (3%).ConclusionsMutations in TP53, BIRC3, NOTCH1, SF3B1, and ATM have previously been shown to be indicators of unfavorable prognosis in CLL. Using a clinically validated NGS assay, 37% of the CLL patients in our cohort were shown to have a mutation in any one of the 5 genes. In patients with lower-risk disease as defined by FISH, 29% showed one or more unfavorable mutation, including 6% with a TP53 mutation. Mutational profiling using NGS can thus help identify a significant proportion of lower-risk CLL patients with a worse than expected prognosis who may potentially benefit from earlier and/or novel therapy.ReferencesDohner H, et al. N Engl J Med 2000;343:1910-1916Rossi D, et al. Blood 2013;121:1403-1412Austen B, et al. J Clin Oncol 2007;25:5448-5457Skowronska A, et al. J Clin Oncol 2012;30:4524-32 DisclosuresNo relevant conflicts of interest to declare.

Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1) with DNA damage response genes.

Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

ATM Mutations in Major Stereotyped CLL Subsets: Enrichment in Subset #2 is Associated with Unfavourable Outcome

Chronic lymphocytic leukemia (CLL) is a paradigmatic malignancy where the interplay of cell-extrinsic and cell-intrinsic factors has a major impact on disease evolution. Indeed, extrinsic triggering, e.g. antigenic stimulation through the B-cell receptor (BcR), together with intrinsic aberrations, e.g. accumulation of genetic defects, play a major role throughout the natural history of CLL. The importance of antigen involvement is underscored by the existence of 'stereotyped' BcR in up to 30% of CLL patients. Notably, CLL patients with stereotyped BcR can be grouped into different subsets, each with a subset-biased biological and clinical profile. For instance, while the clinically aggressive subset #2 (IGHV3-21/IGLV3-21, comprising both mutated (M-CLL) and unmutated (U-CLL) IGHV genes) displays a remarkably high frequency of SF3B1 mutations, subset #8, a subset with the highest risk of Richter transformation, shows a strong association with trisomy 12 and NOTCH1 mutations. ATM defects are implicated in the evolution of CLL and are associated with a dismal prognosis, however the extent to which they contribute to the genetic landscape in stereotyped subsets remains unexplored.To gain insight into this issue, we assessed the frequency of ATM mutations in 249 well-characterized CLL patients assigned to major subsets #1-8. The entire coding region of ATM (62 exons) was investigated with two different targeted deep-sequencing approaches, i.e. Haloplex technology (HiSeq, coverage ~1500X) or the Nextera XT kit (MiSeq, coverage ~4000X). A conservative variant allele frequency cut-off of 10% was selected, and mutations were validated by Sanger sequencing.Altogether, we identified 61 ATM mutations in 47/249 (19%) patients across all major subsets (Fig. 1). As expected, the majority of identified ATM mutations (n=43, 70%) have not yet been reported while the remaining 30% were listed in the HGMD or COSMIC mutation databases. The spectrum of ATM mutations included missense (n=31), nonsense (n=9), splicing (n=6), and frame-shift (n=14) mutations, and one in-frame deletion. Missense substitutions were distributed along the entire gene without any 'hotspot' region or preferred domain. The highest mutation frequency was detected in subset #2 (26%), with a significant enrichment in U-CLL vs. M-CLL cases, (13/33 vs. 8/48 subset #2 cases, respectively; p=0.021). Within poor-prognostic U-CLL subsets, ATM mutations were also frequent in subsets #6 (25%) and #7 (23%), while subsets #3, #5, #1, and #8 showed lower frequencies (17%, 17%, 13%, and 7%, respectively). The favorable prognostic M-CLL subset #4 exhibited a low frequency (7%) of ATM mutations. Notably, when comparing the two most populated subsets, i.e. #1 and #2, ATM mutations were overrepresented in the latter with a borderline significance value (p=0.086); when restricting the analysis to U-CLL #2 cases a significantly higher frequency was observed compared to #1 (13/33 vs 9/68; p=0.0045).Regarding the clinical impact of ATM defects in subset #2, we divided patients into subgroups with biallelic inactivation (def-ATM), sole 11q-, sole ATM -mutation, TP53 -aberrations and WT. While both groups with mono-allelic ATM disruption showed a significantly reduced overall survival compared to WT (median survival sole ATM -mutation, 71 months, sole 11q-, 40 months, vs. 123 months in the WT group; p=0.002 and 0.02, respectively), a non-significant reduction of overall survival was observed for patients with bi-allelic ATM aberrations (70 months, p=0.29) (Fig. 2). The few subset #2 patients with TP53 defects showed a similar survival as WT group, underscoring previous observations that TP53 dysfunction per se plays a minor role in this subset.In summary, we demonstrate that ATM mutations can be added to the list of genetic defects with a biased distribution in stereotyped subsets. The enrichment of ATM defects in subset #2 was associated with a negative impact on overall survival, suggesting a role for ATM inactivation in shaping the aggressive phenotype of this subset. This study further supports the recent suggestion that CLL development is driven by antigenic selection, coupled with preferential acquisition of specific genetic defects. The work was supported by the projects MSMT CR CZ.1.05/1.1.00/02.0068, IGA NT13493-4/2012 and TACR TE02000058. [Display omitted] [Display omitted] DisclosuresLangerak:Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. . Strefford:Roche: Research Funding. Stamatopoulos:Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding.

Towards Response Prediction Using Integrated Genomics in Chronic Lymphocytic Leukaemia: Results on 250 First-Line FCR Treated Patients from UK Clinical Trials

Background: Major progress has been made in understanding disease biology and therapeutic options for patients with chronic lymphocytic leukaemia (CLL). Recurrent mutations have been discovered using next generation sequencing, but with the exception of TP53 disruption their potential impact on response to treatment is unknown. In order to address this question, we characterised the genomic landscape of 250 first-line chemo-immunotherapy treated CLL patients within UK clinical trials using targeted resequencing and whole-genome SNP array.Methods: We studied patients from two UK-based Phase II randomised controlled trials (AdMIRe and ARCTIC) receiving FCR-based treatment in a first-line treatment setting. A TruSeq Custom Amplicon panel (TSCA, Illumina) was designed targeting 10 genes recurrently mutated in CLL based on recent publications.Average sequencing depth was 2260X. The cumulated length of targets sequenced was 7.87 kb from 330 amplicons covering 160 exons. Alignment and variant calling included a combination of three pipelines to confidently detect SNVs, indels and low level frequency mutations. SNP array testing was performed using HumanOmni2.5-8 BeadChips, (Illumina) and data analysed using Nexus 6.1 Discovery Edition, Biodiscovery. We performed targeted resequencing and genome-wide SNP arrays using selected samples’ germline material to confirm somatic mutations (n=40). Univariate and multivariate analyses using minimal residual disease (MRD) as the outcome measure were performed for 220 of the 250 patients.Results: Pathogenic mutations were identified in 165 (66%) patients, totalling 268 mutations in 10 genes. ATM was the most frequently mutated gene affecting 67 patients (29%) followed by SF3B1 (n=56, 24%), NOTCH1 (n= 32, 14%), TP53 (n= 21, 9%), BIRC3 (n= 17, 7%) and XPO1 (n=14, 6%). Less frequently recurrent mutations were seen in SAMHD1 (n=8, 3%), MYD88 (n= 4, 2%), MED12 (n=7, 3%) and ZFPM2 (n=5, 2%). Integrating sequencing and array results increased the patients with one or more CLL driver mutation from 66% to 94%. As previously reported del17p and TP53 mutations are co-occurring and associate with MRD positivity in all cases (n=15, p=0.0002). We report on minor TP53 subclones in 11 patients (VAF 1-5%), 8 of whom have MRD data available and were also associated with MRD positivity. Deletions of 11q were present in 44 patients. These lesions always included ATM but not always BIRC3. Bialleleic disruption was present in ATM for 27 patients (significantly associated with MRD positivity) and in BIRC3 for 4 patients. Rather surprisingly, trisomy 12 (n=33) and NOTCH1 mutations (n=28) were associated with MRD negativity (p=0.006 and 0.097, respectively). Analysing clonal and subclonal mutations per gene revealed the majority of mutations in SF3B1 and BIRC3 were subclonal (65% and 87% respectively). In contrast almost all SAMHD1 and MYD88 mutations were clonally distributed. There was an association between NOTCH1 subclonal mutations and MRD negativity, compared to clonal mutations, but this difference was not seen in the remaining mutated genes. From our copy number data, the presence of subclones was associated with MRD positivity (p=0.05). Combining important lesions in a multiple logistic regression analysis to predict MRD positivity, bialleleic ATM disruption, together with TP53 disruption, were the strongest predictors, followed by SAMHD1, whereas BIRC3 monoalleleic mutations were a medium predictor for MRD negativity.Conclusion: This is the first integrated genome-wide analysis of the distribution and associations of CLL drivers, using targeted deep resequencing and whole genome SNP arrays in an FCR-based first-line treatment setting. We have shown subclonal and clonal mutation profiles in all patients. For patients with two or more CLL-associated mutations we have begun to unravel clonal hierarchies. We have developed a comprehensive model using MRD as an outcome measure and have found bialleleic ATM mutations and SAMHD1 disruption to strongly predict for MRD positivity. Using MRD status as a robust proxy for PFS not only enables us to confirm results of previous studies, but is advantageous also in considerably reducing the timeframe for results. Indeed, we suggest that MRD status should be assessed routinely in future studies to complement modern integrated genomics approaches. DisclosuresHillmen:Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding.

Clinical implications of the molecular genetics of chronic lymphocytic leukemia.

Genetics and molecular genetics have contributed to clarify the biological bases of the clinical heterogeneity of chronic lymphocytic leukemia. In recent years, our knowledge of the molecular genetics of chronic lymphocytic leukemia has significantly broadened, offering potential new clinical implications. Mutations of TP53 and ATM add prognostic information independently of fluorescence in situ hybridization cytogenetic stratification. In addition, next generation sequencing technologies have allowed previously unknown genomic alterations in chronic lymphocytic leukemia to be identified. Mutations of NOTCH1, SF3B1 and BIRC3 have been associated with short time to progression and survival. Each of these lesions recognizes a different distribution across different clinical phases and biological subgroups of the disease. The clinical implications of these molecular lesions are in some instances well established, such as in the case of patients with TP53 disruption, who should be considered for alternative therapies/allogeneic stem cell transplant upfront, or in patients with ATM disruption, who are candidates to rituximab-based immunochemotherapy. On the contrary, NOTCH1, SF3B1 and BIRC3 mutations appear to have a specific significance, the clinical value of which is currently being validated, i.e. association to Richter syndrome transformation for NOTCH1 mutations, and short progression-free survival after treatment for SF3B1 mutations. Certainly, these new lesions have helped clarify the molecular bases of chronic lymphocytic leukemia aggressiveness beside TP53 disruption. This review covers the recent advancements in our understanding of the molecular genetics of chronic lymphocytic leukemia and discusses how they are going to translate into clinical implications for patient management.

24 TARGETED DISRUPTION OF ATAXIA-TELANGIECTASIA MUTATED GENE IN MINIATURE PIGS BY SOMATIC CELL NUCLEAR TRANSFER

Ataxia-telangiectasia (A-T) is a genetic disease caused by germline biallelic mutation in the ataxia-telangiectasia mutated gene (ATM) that results in partial or complete loss of ATM expression or activity. A diverse phenotype of the disease includes progressive cerebellar ataxia, oculocutaneous telangiectasias, radiation hypersensitivity, increased cancer incidence, immunodeficiency and chromosomal instability. The frequency of A-T in the United States and United Kingdom has been estimated to be 1:40 000. Heterozygous carriers may also have predisposition to diverse cancers. Although ATM-deficient mice have been produced, none reflects the extent of neurological abnormalities involving the loss of Purkinje cells of the cerebellum seen in patients. Hence, miniature pigs, which are anatomically and physiologically closer to humans, might serve as an alternative model for human A-T. In the present study, we attempted a targeted disruption of ATM in miniature pigs by somatic cell nuclear transfer. Most A-T patients possess mutated ATM with truncation, resulting in prematurely terminated ATM proteins that are highly unstable. To induce the truncation of ATM, we disrupted exon 59 region of ATM that has been known to be critical for ATM kinase activity. Miniature pig fetal fibroblasts were transfected with ATM-targeting vector and treated by neomycin for 2 weeks. A total of 139 colonies were screened by PCR and 3 among them were identified as homologous recombinants with monoallelic disruption of ATM (targeting efficiency = 2.1%). One of the 3 colonies was chosen and used for subsequent nuclear transfer. In total, 611 nuclear transfer embryos reconstructed with ATM-targeted fetal fibroblasts were transferred into 5 surrogate gilts. Two gilts became pregnant and developed to term with a total of 5 live piglets delivered. Being analysed by PCR, all piglets born were found to be ATM gene-targeted. Because conventional murine models of human A-T have provided only limited insights into therapies and pharmacological treatments, the generation of miniature pigs with disrupted ATM will allow new opportunities to more precisely understand A-T and to accelerate discovery of strategies for prevention and treatment of the disease. In addition, such animals may be utilised in studying cancer with respect to the involvement of ATM in cell cycle arrest, DNA repair and apoptosis.

Meiotic Checkpoints: Repair or Removal?

Defects in meiosis can produce different checkpoint responses in female and male animals, suggesting that meiotic checkpoints exhibit sexual dimorphism. A recent study in Caenorhabditis elegans indicates that meiotic checkpoint activation is similar between the sexes and the primary difference lies in the downstream consequences.

ATM Limits Incorrect End Utilization during Non-Homologous End Joining of Multiple Chromosome Breaks

Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ) repair of multiple chromosomal double-strand breaks (DSBs). We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ) or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs). In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI–resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ) factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ.

Pot1 and cell cycle progression cooperate in telomere length regulation

Removal of the vertebrate telomere protein Pot1 results in a DNA damage response and cell cycle arrest. Here we show that loss of chicken Pot1 causes Chk1 activation, and inhibition of Chk1 signaling prevents the cell cycle arrest. However, arrest still occurs after disruption of ATM, which encodes another DNA damage response protein. These results indicate that Pot1 is required to prevent a telomere checkpoint mediated by another such protein, ATR, that is most likely triggered by the G-overhang. We also show that removal of Pot1 causes exceptionally rapid telomere growth upon arrest in late S/G2 of the cell cycle. However, release of the arrest slows both telomere growth and G-overhang elongation. Thus, Pot1 seems to regulate telomere length and G-overhang processing both through direct interaction with the telomere and by preventing a late S/G2 delay in the cell cycle. Our results reveal that cell cycle progression is an important component of telomere length regulation.

Effect of Atm disruption on spontaneously arising and radiation-induced deletion mutations in mouse liver.

Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse "gpt-delta" system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi- (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm+/+ genotype showed a dose-dependent increase in the Spi- mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8x10(-6). The livers from Atm-/- mice yielded a virtually identical dose-response curve for X rays with a background fraction of 2.4x10(-6). Structural analyses revealed no significant difference in the proportion of -1 frameshifts and larger deletions between Atm+/+ and Atm-/- mice, although larger deletions prevailed in X-ray-induced Spi- mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.

Telomeric associations and chromosome instability in ataxia telangiectasia T cells characterized by TCL1 expression

T-cell tumors in ataxia telangiectasia (AT), such as T-PLL/T-CLL, are first preceded by the development of a large clone of T-lymphocytes, characterized by chromosomal rearrangements, which usually involve specific regions such as the 14q11 region. Malignancy develops years later, after additional chromosomal changes resulting from the genomic instability consequent to ATM disruption and to the activation of the TCL1 oncogene. Here we report the results of a cytogenetic follow-up of an AT patient (AT94-1), still without signs of hamatological abnormalities, bearing a T-lymphocyte clone characterized by the t(14;14)(q11;q32) rearrangement and having TCL1 expression. We demonstrated that in clonal cells TCL1 expression correlates with increasing genomic instability and in time this mainly induces chromosomal rearrangements and telomeric associations ( tas). Chromosome 21 is not randomly involved; in particular, an i(21q) indicates that it is a subclone prone to additional genetic changes and could represent an early chromosomal rearrangement involved in tumorigenesis. With regard to the increase in tas, we observed that: (i) it is inversely correlated with the proliferative ability of AT94-1 lymphocytes in PHA-stimulated short-term cultures (cell aging in vitro); (ii) this increase is not due to changes either in cell radiosensitivity (measured as bleomycin (BML)-sensitivity) or due to an illegitimate recombination (measured as adriamycin-sensitivity), which may not be sufficient for tumor development.

Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation.

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM-/- DT40 cells displayed retarded cellular proliferation, defective G2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM-/- DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM-/- DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.

Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects, and thymic lymphoma.

ATM, the gene mutated in the inherited human disease ataxia-telangiectasia, is a member of a family of kinases involved in DNA metabolism and cell-cycle checkpoint control. To help clarify the physiological roles of the ATM protein, we disrupted the ATM gene in mice through homologous recombination. Initial evaluation of the ATM knockout animals indicates that inactivation of the mouse ATM gene recreates much of the phenotype of ataxia-telangiectasia. The homozygous mutant (ATM-/-) mice are viable, growth-retarded, and infertile. The infertility of ATM-/- mice results from meiotic failure. Meiosis is arrested at the zygotene/pachytene stage of prophase I as a result of abnormal chromosomal synapsis and subsequent chromosome fragmentation. Immune defects also are evident in ATM-/- mice, including reduced numbers of B220+CD43- pre-B cells, thymocytes, and peripheral T cells, as well as functional impairment of T-cell-dependent immune responses. The cerebella of ATM-/- mice appear normal by histologic examination at 3 to 4 months and the mice have no gross behavioral abnormalities. The majority of mutant mice rapidly develop thymic lymphomas and die before 4 months of age. These findings indicate that the ATM gene product plays an essential role in a diverse group of cellular processes, including meiosis, the normal growth of somatic tissues, immune development, and tumor suppression.