P705: azacytidine therapy reverts mds-associated changes in msc-derived extracellular matrix

Background: Myelodysplastic neoplasms (MDS) are characterized by ineffective marrow hematopoiesis and peripheral blood cytopenia in one or more hematopoietic lineages. The disease is driven by genetic and epigenetic lesions in the hematopoietic clone, but also in stromal cells of the bone marrow microenvironment. The mesenchymal stromal cell (MSC) derived extracellular matrix (ECM) plays a key role in coordinating both physical and biochemical interactions among the cells in hematopoietic bone marrow. However, the involvement of ECM in bone marrow failure is underexplored. We have previously described disease-associated changes in biophysical structure and glycosaminoglycan (GAG) content of MSC-derived ECM and demonstrated an impact on hematopoietic behavior (Bains et al., 2022). Aims: Based on a published observation of changes in ECM-associated gene expression in cultured MSC treated ex vivo with azacytidine (Wenk et al., 2018), we aim to examine heritable changes in MSC-derived ECM in a sequential study of a small number of HR-MDS patients sampled before and after azacytidine therapy. Methods: MSC from healthy donors (HD), low-risk (LR-) and high-risk (HR-) MDS patients prior to treatment and from HR-MDS patients at screening, after 4 cycles of azacytidine were cultured for 10 days on glass slides pre-coated sequentially with poly-octadecene-alt-maleic anhydride and fibronectin. The monolayers were decellularized by washing with 20 mM ammonia solution, and then subject to DNase treatment. Matrix mechanical properties were analysed by atomic force microscopy, GAG content by histochemical staining with peanut and wheat germ agglutinin and by staining of electrophoresed proteins using the metachromatic dye “stains all”. Total collagen content was quantified by Sircol assay while specific levels of collagens 1 and 4 were analysed by immunostaining. Hematopoietic support was assessed by culturing hematopoietic stem and progenitor cells (HSPC) on ECM for 7 days followed by direct transfer into colony-forming assays. HSPC morphology was analysed by immunostaining. Results: Compared to ECM from healthy donor MSCs, ECM produced by HR-MDS derived MSCs had a decreased stiffness, a higher collagen content and a specific increase in collagens I and IV. MDS-derived ECM (both LR- and HR-) had a reduced ability to support HSPC, as revealed by a loss of both polar morphology and of subsequent colony forming potential. In contrast, MSCs isolated following azacytidine therapy generated ECM with increased stiffness and reduced collagen content but no changes in the GAG profile. This post-therapy ECM also had an improved capacity for HSPC support, maintaining a higher colony forming activity and a polarised HSPC morphology similar to that seen on ECM from healthy donor. Conclusion MDS-associated changes in the composition, physical and functional properties of MSC-derived ECM suggest a role in the pathogenesis of MDS and identify ECM as a potential therapeutic target. Azacitidine therapy in vivo results in heritable reversal of MDS-associated changes in ECM and hematopoietic support that are maintained during subsequent ex-vivo culture of MSC in the absence of azacytidine. References Bains, A.K. et al. (2022) Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes. Front. Oncol., 12, 961473. Wenk, C. et al. (2018) Direct modulation of the bone marrow mesenchymal stromal cell compartment by azacitidine enhances healthy hematopoiesis. Blood Adv, 2, 3447–3461. Keywords: Myelodysplastic syndrome, Azacitidine, Bone marrow failure, Mesenchymal cells