Oxidative Stress in Stem / Progenitor Cells and Specific Antioxidant Response in Bone Marrow from Myelodysplastic Syndrome Patients

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous group of blood diseases with varying degrees of severity, which can be classified into one of several subgroups based on features of the abnormal cells. The progression of MDS to secondary AML (sAML) is a good example of the multi-step theory of leukemogenesis in which a series of mutations occur in an initially normal cell and transform it into a cancer cell. Reactive Oxygen Species (ROS) are highly involved in normal hematopoiesis, a low amount being associated with quiescence and self-renewal, while a higher level is requested for ROS signaling pathways. The level of intracellular ROS is finely tuned by the expression of ROS-related enzymes that are suggested as major players in human MDS / AML. Altogether, these elements highlight the potential implication of redox metabolism in the pathophysiology of MDS / sAML.

Methods: This study focused on ROS level and antioxidant gene expression in bone marrow (BM) cells from 97 patients compared to 25 healthy controls. All subjects were informed and consenting following a procedure approved by the ethical committee and provided a written informed consent. sAML patients were characterized at diagnosis by proliferation of blasts (>20%) and dysplasia in BM samples. The sternal BM samples were obtained at the time of diagnosis (University hospital of Tours and Cochin hospital in Paris), and were compared with BM from aged-matched healthy donors. Flow cytometry analyses and RNA extraction were processed within the first hour following BM aspiration. We quantified: (i) the level of ROS by flow cytometry (DCFDA staining) in the subpopulations of BM cells identified with anti-CD45-APC-H7, anti-CD34-PE-Cy7 and anti-CD38-APC antibodies; (ii) the expression level of 28 transcripts encoding for major enzymes implicated in the antioxidant cellular response by qRT-PCR using Universal Probe Library technology. All statistical analyses were conducted using R v3.2.2 software. Differences in ROS levels between pathologies was tested using Kruskall-Wallis test followed by Dunn's post hoc test. Differences in expression data between patients and controls were tested using Mann-Whitney test on ΔCt and RQ. Principal component analyses were performed using FactoMineR package on ΔCt values. Significant differences were identified on a change of at least 2-fold and P value less than .05.

Results: The results highlight increased ROS level in BM non-lymphoid cells, especially in CD34^high CD38^low stem and progenitor cells. This increase is also much important in case of sAML compared to low grade (MDS-SLD and MDS-MLD) or high grade MDS (MDS-EB1 and MDS-EB2). Interestingly and as expected, the most important oxidative stress was found in erythroblast subpopulation in MDS-SLD-RS and MDS-MLD-RS, enriched in ring sideroblasts. Moreover, we identified a specific antioxidant signature in the different MDS / sAML bone marrow samples. More precisely, the progression of the disease was characterized by 3-step antioxidant cellular response: (i) overexpression of enzymes reducing disulfides bonds in protein (low-grade MDS - GLRX family), then (ii) enzymes detoxifying H₂O₂ (high-grade MDS - PRDX and GPX families) and finally (iii) decreased expression of these enzymes in sAML.

Conclusion: We report for the first-time progressive increase in ROS level in CD34^posCD38^low stem andprogenitor cells from MDS patients compared to healthy controls, and higher level in sAML compared to MDS patients. In the light of these findings, antioxidant treatments should be explored in MDS with low blast counts, to avoid the risk of promoting transformation. Concomitantly, MDS BM cells initiate antioxidant program by overexpressing enzymes reducing disulfide bonds in proteins (GLRX family) then reinforced by H₂O₂ detoxifying enzymes (PRDX and GPX families), these enzymes being dramatically decreased when transformation in sAML. This specific molecular antioxidant response could be considered as biomarkers useful for diagnosis and follow-up of the disease.