Abstract
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.
Highlights
Chronic lymphocytic leukemia (CLL), the most common leukemia in the Western world, is characterized by an extremely variable clinical course
The reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) assay was performed on all (n = 30) samples from the training cohort and showed upregulation of cluster I genes following ionizing irradiation (IR) in wild type (WT) samples and impaired upregulation in tumor protein p53 (TP53)/ataxia telangiectasia mutated (ATM)-defective CLL samples following IR, confirming earlier results from Stankovic et al.[9]
Defects of the ATM-p53 pathway can be caused by other mechanisms, such as polymorphisms in MDM217,18 and cyclindependent kinase N1A (CDKN1A),19 hypermethylation of the TP53 promotor[20] or by novel recurrent mutations, such as those described recently for the SAMHD1 gene.[21]
Summary
Chronic lymphocytic leukemia (CLL), the most common leukemia in the Western world, is characterized by an extremely variable clinical course. 8% and 18% of CLL patients requiring frontline therapy harbor defects of TP53 or ATM, respectively.[3,6] These frequencies increase when the disease progresses following initial therapies. TP53 and ATM aberrations both lead to p53 dysfunction, there are substantial differences both at the clinical and at the cellular level that distinguish TP53-defective from ATM-defective CLL. TP53-disruptive CLL exhibits a complete absence of DNA-damage-induced apoptosis in vitro, whereas ATM-disruptive CLL retains a capacity for apoptosis after in vitro-induced DNA damage, though at a reduced level.[7,8] In addition, microarray analyses revealed that TP53- and ATM-mutant CLL share a defect in activating proapoptotic responses after DNA damage but are distinguished by major differences in activating prosurvival responses.[9].
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