Mechanism of Action of Azacytidine in Myelodysplastic Syndromes (MDS)

Introduction: Myelodysplastic syndromes (MDS) have historically been classified as a set of heterogeneous hematopoietic stem cell (HSC) disorders, which are characterized clinically by abnormalities in the hematopoietic system. However, several recent landmark studies have now demonstrated that the pathogenesis of MDS is not confined to HSCs, and mesenchymal stromal cells (MSCs) in the bone marrow also play important contributing roles in sustaining the disorder. Treatment for MDS using hypomethylating agents such as azacytidine is effective, with patients showing recovery of blood counts and long-term restoration of normal hematopoiesis - an outcome that is plausibly brought about only by the reversal of abnormalities the bone marrow stem cell niches. In this work, we investigate the use of azacytidine in both HSCs and MSCs of MDS patients in order to better understand its therapeutic mechanism on stem cell niches, as well as to inform strategies for the development of future therapies for similar hematopoietic disorders.

Methods: Cryopreserved BM MDS samples (n=20) were obtained from the Department of Hematology repository at Singapore General Hospital. Healthy MSCs were derived from bone marrow aspirates of healthy donors, obtained at Singapore General Hospital. Healthy CD34+ HSCs were purchased from Lonza. Osteogeneic and adipogeneic differentiation capabilities and proliferation capacities were performed on MSCs. Proliferation, cell cycling and apoptosis in HSCs were analysed. Gene expression profiling for MDS candidate genes was performed by quantitative PCR on both MSCs and HSCs. Co-culture experiments with healthy CD34+ cells on MDS MSCs were investigated. All assays were performed on both MSCs and HSCs, before and after azacytidine treatment.

Results: MDS MSCs have significantly reduced proliferative capacities (p=0.02) and osteogeneic differentiation potentials (p=0.0006) compared to healthy MSCs. Gene expression profiling of MDS MSCs showed a 4.6-fold (n=17; p=0.0002) and 6.2-fold (n=15; p=0.0002) reduction in osteogeneic markers like Runx2 and Osterix respectively. Hematopoietic growth factors and chemokines such as IGF1, IL-8 and Angiopoietin-1 are 5.35-fold (n=17; p<0.0001), 3.36-fold (n=20; p=0.02) and 1.45-fold (n=15; p=0.2) lower than healthy controls. After treatment with azacytidine, MDS MSCs demonstrated significant increased proliferative capacities (n=4; p<0.0001) and differentiation potentials (n=3; p<0.0001) in comparison to healthy MSCs. Significant increase in gene expression of Osterix (n=5; p<0.0001) was seen in comparison to healthy controls.

In MDS HSCs, expression of hematopoietic, cell cycling and apoptosis genes such as CXCR4, CCL3, Cyclin D1 and BCL2 are significantly different from healthy HSC - 13 fold (n=15; p=0.1005), 6.8 fold (n=15; p=0.014), 20 fold (n=19; p=0.2673) and 5.26 fold (n=19; p=0.0478) lower than healthy HSCs, respectively. Proliferation of MDS HSCs in culture was 3.3 fold higher than healthy HSCs but treatment with azacytidine of 1µM and 5µM reduced the growth advantage of MDS HSCs to 3 fold and 4.2 fold in comparison with similarly treated healthy controls.

Co-culture experiments of healthy CD34+ cells on MDS MSCs, induced a gene expression profile in healthy HSCs similar to MDS HSC. After treatment of MDS MSCs with azacytidine, the gene expression of expanded healthy CD34+ cells was normal.

Conclusion: MDS stromal cells are functionally abnormal and have the ability to instruct healthy HSCs to adopt genetic features that resemble MDS HSCs. Treatment with azacytidine restores normal function to MDS MSCs while conferring a growth disadvantage to MDS HSCs but not healthy HSCs. These observations help elucidate for the first time a possible mechanism of action by azacytidine on stromal cells in the treatment of MDS and further suggest that therapies which also target stromal elements in bone marrow niches may be necessary in achieving more favorable outcomes for hematopoieic disorders such as MDS.