ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin
Veronika Navrkalova,
Ludmila Sebejova,
Jana Zemanova,
Jana Kminkova,
Blanka Kubesova,
Jitka Malcikova,
Marek Mraz,
Jana Smardova,
Sarka Pavlova,
Michael Doubek,
Yvona Brychtova,
David Potesil,
Veronika Nemethova,
Jiri Mayer,
Sarka Pospisilova,
Martin Trbusek
Affiliations
Veronika Navrkalova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Ludmila Sebejova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Jana Zemanova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Jana Kminkova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Blanka Kubesova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno
Jitka Malcikova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Marek Mraz
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Jana Smardova
Department of Pathology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Sarka Pavlova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Michael Doubek
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Yvona Brychtova
Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
David Potesil
Core Facility – Proteomics, CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic
Veronika Nemethova
Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Jiri Mayer
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Sarka Pospisilova
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
Martin Trbusek
Department of Molecular Medicine, CEITEC - Central European Institute of Technology, Masaryk University, Brno;Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno
ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.
