Abstract
A increased oxidative stress is one of the hallmarks of chronic myeloid leukemia (CML). Manganese Superoxide dismutase 2 (SOD2) is a key scavenger enzyme against reactive oxygen species (ROS) in eukaryotic cells, playing a crucial role in antioxidant defense but its potential implication in the CML pathophysiology is not determined. We have analyzed the expression of SOD2 at the RNA level in a large cohort of CML patients (n=49) compared to a cohort of controls with non-malignant hyperleucocytosis (n= 19). This analysis showed a major decrease of SOD2 mRNA, inversely correlated with leucocytosis (p< 0.0001) and with Sokal Score, high-risk patients showing a lower expression of SOD2 (p= 0.019). To determine the effects of the silencing of SOD2 gene in an experimental CML model, we analyzed SOD2 expression in the human UT7 cell line expressing either native or BCR-ABL carrying T315I mutation. SOD2 protein levels in BCR-ABL expressing cells were similar to parental UT7 cells, suggesting that BCR-ABL has no direct effect on SOD2 expression. We have then silenced SOD2 expression in UT7 cells as well as UT7-BCR-ABL cells. Interestingly, the expression of H2O2 levels measured with flow cytometry was not changed after SOD2 inhibition, suggesting activation of compensatory anti-oxidant pathways. A global analysis of 27 anti-oxidant gene expression showed a reduction of PRDX2 only in BCR-ABL-expressing cells. To analyze same cells at the genomic level, we have performed CGH arrays in SOD2 silenced UT7, UT7-BCR-ABL and UT7-T315I cells as compared to their counterparts expressing SOD2. These experiments showed a major genetic instability with deletions and duplications in several chromosomal loci only in native or T315I-mutated BCR-ABL expressing cells silenced for SOD2. The 13q31.3-q34 locus, containing GPC5 and GPC6 genes, was found to be duplicated in non-mutated BCR-ABL cells deficient in SOD2 expression. GPC5 and GPC5 belong to the Glypican family, which are cell surface heparan sulfate proteoglycans anchored to the cell membrane via glycosyl-phosphatidyl-inositol-structures. The expression of several GPC is increased in some malignancies, including glioma, pancreatic carcinoma, breast cancer (GPC1), hepatocellular carcinoma (GPC3) and rhabdomyosarcoma (GPC5). Furthermore, the 13.q31.3 region, duplicated in BCR-ABL-expressing cells upon SOD2 deletion, contains the polycistronic mir-17-92 cluster (mir-17, mir-18a, mir19a, mir-20a, mir-19b1, mir-92a1), which is highly increased in CML CD34+ cells. Interestingly, the inhibition of SOD2 expression did not lead to the same genomic modifications detected by CGH arrays; the duplication of the 13q31.3 and 13q31.3q24 regions was not found in UT7 BCR-ABL cells harboring the T315I mutation. T315I cells silenced for SOD2 expression exhibited multiple abnormalities among which a large deletion of the 20q11.21 region which encodes the defensin gene family (DEFB115, DEFB116, DEFB118, DEFB119, DEFB121, DEFB122, DEFB123, DEFB124; REM1, DUSP15). Beta-defensins are antimicrobial peptides, which are part of the innate immunity against pathogens. The loss of beta-defensin 1 gene has already been reported in solid tumors. In conclusion, we demonstrate in a large cohort of CML patients, a major decrease of SOD2 expression, involved in antioxidant defense. We further show for the first time a link between loss of SOD2 expression and genetic instability in BCR-ABL expressing cells as demonstrated by CGH arrays. Genetic consequences of reduced SOD2 expression in CML should be further analyzed in prospective studies as patients with low SOD2 expression could be more prone to develop a mutator phenotype under TKI therapies.
Disclosures:
Guilhot: Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:Bristol Myers Squibb, Novartis: Consultancy, Honoraria.
Genetic instability is prevented by Mrc1‐dependent spatio‐temporal separation of replicative and repair activities of homologous recombination
