Azacitidine Directly Acts on the Mesenchymal Stromal Cell Compartment in Myelodysplastic Syndromes to Alter Niche Function and Suppress Malignant Hematopoeitic Stem/Progenitor Cells

Background: Hematopoietic stem/progenitor cells (HSPC) are highly dependent on interaction with the specific bone marrow microenvironment (niche) which supports their survival and directs their differentiation. Increasing evidence suggests that the niche plays a central role in the development of clonal stem cell disorders such as myelodysplastic syndromes (MDS). Malignant clonal HSPC in MDS have been shown to reprogram mesenchymal stromal cells (MSC) to promote disease progression. In turn, the altered stromal microenvironment in MDS may directly impair healthy hematopoiesis. Accordingly, MSC isolated from MDS patients have shown phenotypical and functional abnormalities, which were not observed in patients successfully treated with azacitidine (AZA), currently the standard of care for higher-risk MDS patients. Although AZA is a potent inhibitor of DNA methyltransferases and is thus considered a hypomethylating agent, the exact anti-leukemic mechanisms are not understood and a clear correlation between DNA demethylation and response to AZA has not been demonstrated thus far. Hence, we looked beyond the current focus of AZA effects on clonal hematopoiesis and hypothesized that AZA may also act through direct modulation of the MSC compartment. To answer this question, we examined primary MSC treated in vitro with AZA as well as the functional interaction between MSC and HSPC after AZA treatment.

Methods: Primary MSC were isolated from bone marrow of untreated MDS patients or age-matched healthy controls, expanded by plastic adherence, cultured until confluency under defined conditions and used for up to 4 passages. Identity of MSC was confirmed by presence of characteristic cell surface markers CD73, CD271, CD105 and CD90 and absence of CD45 and CD34 by flow cytometry. MSC were differentiated into the adipogenic or osteogenic lineage after in vitro treatment with AZA (10 uM). Co-culture experiments with CD34⁺ HSPC from MDS patients or healthy controls were performed on AZA-treated or untreated MDS-MSC or healthy MSC, respectively. After four days, CD34⁺ cells were harvested and assessed by colony-forming unit assays in methylcellulose. Single colonies were genotyped for MDS-associated mutations known to be present in the bone marrow sample. Each colony was genotyped for one or two known MDS-associated mutations and an input patient specimen was used as a control. Finally, RNA sequencing was performed on a mesenchymal stromal cell line (EL08-1D2), either untreated or treated with AZA for 4 days in vitro to uncover pathways affected by AZA in stromal cells.

Results: MDS-MSC showed a decreased proliferative capacity as well as markedly impaired differentiation into the osteoblastic lineage compared to healthy MSC. Treatment with AZA had no effect on cell surface marker expression or viability of either healthy or MDS-MSC. AZA treatment significantly reduced the ability of MDS-MSC to differentiate towards the adipogenic lineage while osteogenic differentiation was severely impaired with or without AZA treatment. In contrast, osteogenic differentiation of healthy MSC was enhanced after AZA treatment. In co-culture experiments pretreatment with AZA significantly improved stromal support of healthy CD34+ HSPC while malignant MDS-HSPC were suppressed. RNA sequencing analysis revealed differential gene expression in AZA-treated vs untreated stroma with significantly more genes upregulated in AZA-treated samples. Specific changes in pathways involved in ECM-receptor interaction, cell adhesion molecules (CAMs), cytokine-cytokine receptor interaction and metabolic pathways in AZA treated EL08-1D2 stromal cells were determined and validated in primary MDS MSC samples.

Conclusion: We show here that AZA directly modulates altered MSC function in MDS, thus influencing interaction with HSPC and leading to suppression of malignant hematopoiesis. Our data suggest an additional mode of action by which AZA exerts its therapeutic activity. To our knowledge this analysis is the first to examine the direct effect of AZA treatment on MSC.