PF345 THE INVOLVEMENT OF OXIDATIVE STRESS, INFLAMMATION, AND DNA DAMAGE RESPONSE IN PATHOPHYSIOLOGY OF DIAMOND‐BLACKFAN ANEMIA
Authors: Kapralova K. , Saxova Z. , Kralova B. , Lanikova L. , Pospisilova D. , Divoky V. , Horvathova M. 
Published: June 2019

Background:Diamond‐Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by erythroid aplasia mostly caused by different pathogenic variants in ribosomal protein (RP) genes. The ribosomal stress and subsequent activation of the p53 tumor suppressor pathway result in increased apoptosis of erythroid precursors and/or induction of cell cycle arrest.Aims:We studied the contribution of p53 pathway, oxidative stress, and inflammation to the DBA pathophysiology.Methods:We analyzed the bone marrow and peripheral blood samples from DBA patients listed in the Czech DBA Registry. We also prepared the RPL5‐ and RPS19‐deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. Activation of p53 pathway, oxidative DNA damage, and pro‐inflammatory cytokines were assessed in undifferentiated and differentiated RPL5‐ and RPS19‐deficient cells; the differentiation was induced by dimethyl sulfoxide (DMSO) for at least 48 hours.Results:Created RP‐deficient MEL cells presented the main phenotypic features of DBA patients’ erythroid cells: decreased levels of RPL5 or RPS19 proteins, decreased proliferation capacity, increased apoptosis, and abnormal GATA1 expression (2‐times lower than in control cells) upon differentiation. The increased p53 activation was also confirmed in RPL5‐ and RPS19‐deficient cells consistent with increased p53 expression in the bone marrow of DBA patients (p53+ cells: 8.4% in RPL5‐ and 7.3% in RPS19‐mutant patient vs. 0.4% in normal controls). One of the known inducer of p53 is oxidative stress causing oxidative DNA damage. Indeed, elevated positivity for 8‐oxoguanine (8‐oxoG), a marker of oxidative DNA damage, was detected in differentiated RP‐deficient MEL clones and in DBA patients’ samples in contrast to control samples. It is known, that reactive oxygen species (ROS) are produced under inflammation resulting in ROS‐mediated DNA damage response (DDR). The upregulation of pro‐inflammatory cytokines TNF‐α, IL6, and IL1b was found in RPL5‐ and RPS19‐deficient cells and also in the serum from DBA patients along with the upregulation of other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF‐γ, and GM‐CSF). Moreover, elevated pro‐inflammatory cytokines play a role in cellular senescence which was confirmed in RP‐deficient MEL clones by significantly elevated β‐galactosidase activity. In addition, increased ROS levels were detected in all differentiated RPL5‐ and RPS19‐deficient cells and in the serum of all DBA patients with elevated inflammatory cytokines. Finally, the DNA double‐strand breaks can occur as an outcome of ROS production responding by phosphorylation of Ser‐139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RP‐deficient MEL clones in comparison with control cells (mean intensity of fluorescence for RPL5‐deficient cells: 2.63 ± 0.34; RPS19‐deficient cells: 3.29 ± 0.41; control cells: 1.02 ± 0.22).Summary/Conclusion:The defective ribosomal biogenesis is associated with abnormal GATA1 expression and augmented apoptosis of erythroid cells. Our results show that the upregulation of inflammatory cytokines, senescence, oxidative stress, increased DNA damage, and activation of DDR signaling are also playing an important role in DBA pathogenesis. Our results also suggest the possibility of using the anti‐oxidative and anti‐inflammation therapy in DBA patients as the additive treatment for moderating DBA phenotype.Grants support: AZV 16–32105A, IGA_LF_2019_006, and by LTAUSA17142 (BK,LL).