ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin

Abstract

ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n . only part of this activity is transferred to the p53 pathway, leading to pro-apoptotic signaling. Monitoring of ATM function may be a feasible tool for disclosing ATM mutations. Several slightly modified tests have been suggested, based on monitoring p53 and p21 accumulation after cell exposure to ionizing radiation (IR). 17,[24][25][26] An alternative approach utilizing etoposide and nutlin-3a was also used which enabled efficient differentiation of TP53 and ATM defects. 27Despite undeniable progress, ATM mutation identification in CLL remains challenging due to: i) an extreme gene size (62 coding exons) with lack of well-characterized (hot-spot) mutations; ii) the difficult interpretation of polymorphisms and pathogenic mutations resulting from only vague information about their functional consequences. Therefore, relevant information is lacking about the clinical impact of ATM mutations, including the response of affected patients to chemoimmunotherapy.In our CLL patient cohort, we found that all ATM defects involving mutation(s) resulted in disruption of ATM activity towards p53 pathway activation. In addition, we present a novel functional test based on monitoring p21 induction after parallel treatment of CLL cells with doxorubicin and fludarabine that have different DNA damage mechanisms. This test proved to be an effective means to search for ATM mutations, which had been selected in a dominant proportion of leukemic cells. Table 1. The cohort consisted of predominantly high-risk CLL patients (harboring TP53 defect and/or 11q-and/or unmutated IGHV), and 45% of patients were treated with various regimens before ATM mutation analysis (Online Supplementary Table S1). Design and Methods Patients' samples ATM mutation analysisCustom resequencing microarray (Affymetrix, CA, USA) was used to detect 1-nt substitutions in all 62 coding exons and splicing sites of the ATM gene. The resequencing procedure was carried out according to the manufacturer's protocol (Affymetrix GeneChip Custom Resequencing Array Protocol). The resequencing principle is based on allele-specific hybridization. The hybridization of fluorescently labeled DNA fragments to particular positions determined the nucleotides in sequence with the ability to distinguish between homozygous and heterozygous state. Final sequence data was acquired using The GeneChip Sequence Analysis Software (GSEQ) processing fluorescence intensity files.Direct Sanger sequencing (3130xl Genetic Analyzer, Applied Biosystems) was used to: i) confirm ATM alterations detected by microarray analysis; ii) identify other mutations indicated by functional test and/or Western blot (WB) through screening of all 62 coding exons and splicing sites. A search was made for all identified sequence variations in appropriate databases of single nucleotide polymorphisms (SNPs) and mutations.Variations detected by the resequencing array but not confirmed by Sanger sequencing were evaluated by next-generation deep sequencing techno...

ATM Mutations in Cancer: Therapeutic Implications

“…As referenced above, heterozygous germ-line ATM mutations appear to impart some vulnerability to chemotherapy and radiotherapy toxicity. To this end, many groups have designed assays to measure ATM protein function (36,54,63,64).…”

Discussion of Pathogen and Potential Therapeutic Strategy for Triple Negative Breast Cancer Based on a Rare Case Study

“…ATM is another gene mediating DNA methylation [8]. ATM inactivating mutant had been shown its resistant to those DNA damaging drugs probably through the immune system, which affected efficacy of chemotherapy to leukemia [9]. Taken together, we believe that Immunomodulating drugs maybe the promising treatment for TNBC patients.…”

Assessment of TP53 functionality in chronic lymphocytic leukaemia by different assays; an ERIC‐wide approach

“…Deletions of chromosome 17p (TP53) and 11q (ATM) coincide with TP53 and ATM mutations respectively, however with markedly different frequencies (80% and 40% respectively; Malcikova et al, 2009;Navrkalova et al, 2013). Sole TP53 mutations and deletions usually lead to TP53 (p53) dysfunction (Mohr et al, 2011) and, in case of deletions in ATM, a mutation of the residual ATM allele determines whether there is complete ATM inactivation resulting in TP53-dysfunction (Navrkalova et al, 2013). Therefore, analysis of mutations in TP53 and ATM genes in addition to fluorescent in situ hybridization (FISH) analysis is of additional clinical value (Skowronska et al, 2012).…”