The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.

] Department of Hematology Academic Medical Center Amsterdam The Netherlands [2] Laboratory of Experimental Immunology Academic Medical Center Amsterdam The Netherlands

] Department of Hematology Academic Medical Center Amsterdam The Netherlands [2] Laboratory of Experimental Immunology Academic Medical Center Amsterdam The Netherlands [3] LYMMCARE Amsterdam The Netherlands

] Department of Hematology Academic Medical Center Amsterdam The Netherlands [2] LYMMCARE Amsterdam The Netherlands

] Laboratory of Experimental Immunology Academic Medical Center Amsterdam The Netherlands [2] LYMMCARE Amsterdam The Netherlands

Bioinformatics Laboratory Department of Clinical Epidemiology Biostatistics and Bioinformatics Academic Medical Center Amsterdam The Netherlands

Department of Hematology Medical Center Leeuwarden Leeuwarden The Netherlands

Department of Molecular Medicine Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Translational Oncology National Center for Tumor Diseases and Department of Medicine 5 University Hospital Heidelberg Heidelberg Germany

Laboratory of Experimental Immunology Academic Medical Center Amsterdam The Netherlands

MRC Holland Amsterdam The Netherlands

School of Cancer Sciences University of Birmingham Birmingham UK

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