ATM function and its relationship with ATM gene mutations in chronic lymphocytic leukemia with the recurrent deletion (11q22.3-23.2).

Y Jiang,W Wierda,M J Keating,P A Thompson,K-A Do,X Liu,X Su,W Plunkett,H-C Chen

doi:10.1038/bcj.2016.69

Abstract

Approximately 10–20% of chronic lymphocytic leukemia (CLL) patients exhibit del(11q22–23) before treatment, this cohort increases to over 40% upon progression following chemoimmunotherapy. The coding sequence of the DNA damage response gene, ataxia-telangiectasia-mutated (ATM), is contained in this deletion. The residual ATM allele is frequently mutated, suggesting a relationship between gene function and clinical response. To investigate this possibility, we sought to develop and validate an assay for the function of ATM protein in these patients. SMC1 (structural maintenance of chromosomes 1) and KAP1 (KRAB-associated protein 1) were found to be unique substrates of ATM kinase by immunoblot detection following ionizing radiation. Using a pool of eight fluorescence in situ hybridization-negative CLL samples as a standard, the phosphorylation of SMC1 and KAP1 from 46 del (11q22–23) samples was analyzed using normal mixture model-based clustering. This identified 13 samples (28%) that were deficient in ATM function. Targeted sequencing of the ATM gene of these samples, with reference to genomic DNA, revealed 12 somatic mutations and 15 germline mutations in these samples. No strong correlation was observed between ATM mutation and function. Therefore, mutation status may not be taken as an indicator of ATM function. Rather, a direct assay of the kinase activity should be used in the development of therapies.

Highlights

Recurrent cytogenetic abnormalities occur frequently in chronic lymphocytic leukemia (CLL), ~ 70–80% of cases exhibit recurrent chromosomal abnormalities that can be identified by fluorescence in situ hybridization (FISH)
The ATM protein is an important regulator of the DNA damage response pathway; it is notable that deletion of 11q has been associated with resistance to DNA-damaging chemotherapy.[4]
Peripheral blood mononuclear cells were isolated from leukemic-phase blood as described previously,[13] and the cells were maintained at 107 cells per ml concentration at 37 °C overnight or 1 h before 10 Gy irradiation with Gamma Cell 1000 irradiator (Best Theratronics, Ottawa, ON, Canada)

Summary

Introduction

Recurrent cytogenetic abnormalities occur frequently in chronic lymphocytic leukemia (CLL), ~ 70–80% of cases exhibit recurrent chromosomal abnormalities that can be identified by fluorescence in situ hybridization (FISH). To determine whether the function of ATM in a cell population could be quantitated based on the phosphorylation of KAP1 and SMC1 induced by other DNA-damaging agents, we investigated the effects on SMC1 and KAP1 phosphorylation after exposure of cells to neocarzinostatin, Targeted sequencing of the ATM gene etoposide and doxorubicin, all of which induce double-strand DNA breaks.[9,10,20,21] As described above, A–T cell mixtures were

Results

Conclusion