THE ATAXIA-TELANGIECTASIA MUTATED (ATM) GENE PLAYS A PIVOTAL ROLE IN THE REPAIR OF DNA IN CASES OF MYELODYSPLASTIC NEOPLASM

Abstract

Myelodysplastic Neoplasm (MDS) is an age-associated neoplasia characterized by frequent epigenetic abnormalities being hypermethylation the most common, frequently associated with transformation to acute myeloid leukemia (AML). DNA repair genes have been constantly downregulated in MDS during progression to AML, but these genes have not been evaluated regarding methylation status or mutational profile. Using three different cohorts of MDS (Cohort A- 56 patients; Cohort B- 100 patients; Cohort C- 76 patients) in three sequential steps we evaluated with complementary methods (pyrosequencing, real-time PCR, immunohistochemistry, cytogenetics and search of mutations) the DNA repair genes for the following analyses: methylation of single-strand (XPA, XPC, XPG-ERCC5, CSA-ERCC8, and CSB-ERCC6) and double-strand (ATM, BRCA1, BRCA2, LIG4, and RAD51) DNA repair genes by pyrosequencing; gene expression by RT-qPCR of the ATM gene; and protein expression by immunohistochemistry of ATM, 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5-hmC). Additionally, we used cBioPortal to search for mutations in single-strand and double-strand DNA repair genes. Regarding methylation of single strand repair genes, we detected XPA with higher methylation for low risk MDS than high risk MDS (p < 0.0001) while for double-strand repair pathway, only ATM showed higher methylation for patients who transformed to AML compared to cases who did not transform into AML (p = 0.016). In a second step, we evaluated the gene expression of ATM, showing downregulation in MDS compared to controls (p = 0.042). In a third step, when we divided patients according to WHO classification of 2022, we detected a progressive reduction of ATM expression from subtypes considered as low risk disease (Hypoplastic MDS, MDS with Ring Sideroblasts, MDS with deletion (5q)) compared to high risk MDS (increased of blasts 1 and 2) and AML. We also detected patients who transformed into AML showed a higher ratio of 5mc/5hmC than cases who did not transform (p = 0.045). In addition, we observed a higher tissue methylation score (M-score of 5mC%) in patients classified as poor cytogenetic risk by the IPSS-R than patients classified as good cytogenetic risk (p = 0.035). Finally, using the most recent platform CBioportal (including the cases of Molecular IPSS with more than 7000 patients), we detected ATM as the most mutated, among the DNA repair genes, with the greater variety of mutations such as frameshift, missense and splice compared to others and only ATM mutations were classified as oncogenic according to the standards for the classification of pathogenicity of somatic variations in cancer (SOP 2022). We believe all the results presented herein suggest that ATM is silenced or down regulated in MDS by methylation or mutations, ultimately increasing the chance of AML transformation.