Increased radiation toxicity with germline ATM variant of uncertain clinical significance

BackgroundWhile patients with ataxia telangiectasia are known to have increased radiation sensitivity, patients with germline heterozygous ataxia telangiectasia mutated (ATM) mutations can have widely varying functional and clinical effects, which can make management decisions difficult. With an increased prevalence of gene panel-based testing for breast cancer patients, radiation oncologists are increasingly confronted with patients who carry germline ATM variants of uncertain clinical significance. This study describes the clinical courses and outcomes of 5 breast cancer patients with varying germline heterozygous ATM mutations undergoing radiation therapy at our institution in order to provide additional knowledge of the varying clinical effects to aid future decision making.Case SeriesWe identified 5 patients with breast cancer and varying germline heterozygous ATM mutations treated at the University of North Carolina Hospitals between 2015 and 2017. The median age at breast cancer diagnosis for the patient series was 46. Clinical effects of radiation treatment varied amongst the 5 patients. The one patient with a pathogenic ATM mutation had no increased radiation related toxicity. Of the 4 patients with ATM variants of uncertain significance, one patient had increased radiation sensitivity with Grade 3 dermatitis. All patients have remained recurrence free with a median duration of 18 months.ConclusionOur data illustrates that patients with germline heterozygous ATM mutations can have widely varying clinical effects with radiation therapy. Given the possibility of unpredictable deleterious effects, our study highlights the importance of caution and careful consideration when devising the multi-modality management strategy in these patients.     

Metastasis of neuroendocrine tumors are characterized by increased cell proliferation and reduced expression of the ATM gene

Purpose

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare group of tumors with a wide spectrum of clinical behavior. However, there are no known clinically relevant biomarkers to predict metastasis.

Experimental Design

To investigate differential gene expression signatures of metastatic vs non-metastatic NETs, we studied cell cycle regulatory genes in 19 metastatic and 22 non-metastatic colorectal NETs by PCR arrays. Immunohistochemistry (IHC) and quantitative real-time RT-PCR were performed to verify the results and another set of 38 GEP-NETs were further studied for validation.

Results

We first delineated six candidate genes for metastasis including ATM, CCND2, RBL2, CDKN3, CCNB1, and GTSE1. ATM was negatively correlated with metastatic NETs (p<0.001) with more than 2-fold change compared to non-metastatic NETs. Overexpression of ATM protein by IHC was strongly correlated with high ATM mRNA levels and low Ki-67 labeling index. Patients with ATM-negativity by IHC showed significantly decreased overall survival than patients with ATM-positivity (median OS, metastatic vs non-metastatic NETs; 2.7 years vs not reached; p = 0.003) and 85.7% of metastatic NETs were ATM-negative. In another validation set of GEP-NETs, decreased mRNA of ATM gene was associated with metastasis and remained significant (p = 0.023).

Conclusions

ATM down-regulation was strongly associated with metastatic NETs when compared with non-metastatic NETs and ATM may be a potential predictive marker for metastasis as well as a novel target in metastatic GEP-NETs.     

ATM haplotypes and associated mutations in Iranian patients with ataxia–telangiectasia: recurring homozygosity without a founder haplotype

Ataxia–telangiectasia (A–T) is an autosomal recessive disorder caused by mutations in the ATM gene. The ATM gene spans more than 150 kb at chromosomal region 11q23.1 and encodes a product of 3,056 amino acids. The ATM protein is a serine/threonine protein kinase and is involved in oxidative stress, cell cycle control, and DNA repair. We analyzed the 11q22-23 haplotypes and associated mutations of 16 Iranian families. We utilized standardized short tandem repeat (STR) haplotypes to enhance mutation identification. In addition to the STR markers, single-nucleotide polymorphism haplotypes were determined, using three critical polymorphisms. The entire gene was screened sequentially by protein truncation testing, single-strand conformation polymorphism, and denaturing high-performance liquid chromatography to identify the disease-causing mutations. Of the expected 32 mutations, 25 (78%) were identified. All but two mutations led to a truncated or null form of the ATM protein (nonsense, splice site, or frameshift). Twelve mutations were identified for 15 haplotypes. Five mutations were novel. Mutations were located throughout the entire gene, with no clustering. Despite the absence of an Iranian founder mutation, three-fourths of the families were homozygous, suggesting that many undetected ATM mutations still exist in Iran. This study establishes a database for Iranian A–T families, and extends the global spectrum of ATM mutations.     

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.     

Mutations of the ATM gene detected in Japanese ataxia-telangiectasia patients: possible preponderance of the two founder mutations 4612del165 and 7883del5

The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method. As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5 in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T→A substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant among Japanese ATM mutant alleles. Received: 15 September 1997 / Accepted: 12 January 1998     

Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences.

Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations.     

Inhibition of ATM kinase activity does not phenocopy ATM protein disruption: Implications for the clinical utility of ATM kinase inhibitors

Biallelic mutations in ataxiatelangiectasia mutated (ATM), which encodes for a protein kinase, cause ataxia telangiectasia (A–T). A–T is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation (IR). A–T patients typically lack both detectable ATM protein and ATM kinase activity, and small molecule inhibitors of ATM kinase activity have been developed as strategies to improve radiotherapy for the treatment of cancers. As predicted, inhibition of ATM kinase activity is sufficient to radiosensitize cells. However, we recently showed that inhibition of ATM kinase activity disrupts DNA damage-induced sister chromatid exchange (SCE). This result was unanticipated since SCE is normal in A–T cells that lack detectable ATM protein. In these studies, we showed, for the first time, that the consequences of inhibition of ATM kinase activity and adaptation to ATM protein disruption are distinct. Here, we discuss the mechanistic implications of this finding for the function of ATM at the replication fork and the clinical utility of ATM kinase inhibitors.Key words: ataxia telangiectasia, ATM, KU55933, KU60019, double-strand break repair, homologous recombination, sister chromatid exchange     

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.     

ATM pathway is essential for ionizing radiation-induced autophagy

BackgroundATM plays an important role in response to DNA damage, while the roles of ATM in radiation-induced autophagy are still unclear in cervical cancer cells.MethodsHuman cervical cancer cells, Hela, were used, and cell models with ATM^?/? and MAPK14^?/? were established by gene engineering. Western blot was implemented to detect protein expression. MDC staining and GFP-LC3 relocalization were used to detect autophagy. CCK-8 was used to detect cell viability. Radiosensitivity was analyzed by colony formation assays. Co-immunoprecipitation was used to detect the interaction between different proteins, and apoptosis was detected by flow cytometry.ResultsAfter radiation autophagy was induced, illustrated by the increase of MAPLC3-II/MAPLC3-I ratio and decrease of p62, and phosphorylation of ATM simultaneously increased. ATM^?/? cells displayed hypersensitivity but had no influence on IR-induced apoptosis. Then inhibitor of ATM, KU55933, ATM and MAPK14 silencing were used, and autophagy was induced by IR more than 200% in control, and only by 35.72%, 53.18% and 24.76% in KU55933-treated cells, ATM^?/? and MAPK14^?/? cells, respectively. KU55933 inhibited IR-induced autophagy by activating mTOR pathways. ATM silencing decreased the expression of MAPK14 and mTOR signals significantly. Beclin's bond to PI3KIII and their interaction increased after IR, while in ATM^?/? and MAPK14^?/? cells this interaction decreased after IR. Both ATM and MAPK14 interacted with Beclin, while ATM^?/? and MAPK14^?/? cells showed no interaction.ConclusionsATM could promote IR-induced autophagy via the MAPK14 pathway, the mTOR pathway, and Beclin/PI3KIII complexes, which contributed to the effect of ATM on radiosensitivity.     

TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin,microsatellite instability and KRAS mutations

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4–9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6 % of the patients (28/47) including 20 (71.4 %) missense mutations, 7 nonsense mutations (25 %) and 1 (3.6 %) frameshift mutation. 89.3 % (25/28) of the detected mutations were in exons 5–8, whereas 10.7 % (3/28) were in exon 4. Among the 27 non frameshift mutations, 89 % (24/27) were transitions and 11 % (3/27) were transversions. 64.3 % (18/27) of the altered amino acids corresponded to arginine. 74 % (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25 % (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.     

Ataxia Telangiectasia Mutated (ATM) Is Dispensable for Endonuclease I-SceI-induced Homologous Recombination in Mouse Embryonic Stem Cells

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.     

A double missense mutation in the ATM gene of a Dutch family with ataxia telangiectasia

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar ataxia, telangiectasia, immunodeficiency, elevated α-fetoprotein levels, chromosomal instability, predisposition to cancer, and radiation sensitivity. We report the identification of a new, double missense mutation in the ataxia telangiectasia gene (ATM) of a Dutch family. This homozygous mutation consists of two consecutive base substitutions in exon 55: a T→G transversion at position 7875 of the ATM cDNA and a G→C transversion at position 7876. These transversions were confirmed by polymerase chain reaction/primer-induced restriction analysis with CelII. The double base substitution results in an amino acid change of an aspartic acid to a glutamic acid at codon 2625 and of an alanine to a proline at codon 2626 of the ATM protein. Both amino acids are conserved between the ATM protein and its functional homolog, the Atm gene product in the mouse. Furthermore, the Chou-Fasman and Robson predictions both demonstrate a change in the secondary structure of the ATM protein carrying the D2625E/A2626P mutation. These findings suggest that the double base substitution in the ATM gene is a disease-causing mutation. Received: 6 October 1997 / Accepted: 5 November 1997