Targeting ephrin A3 in the bone marrow niche offers a promising therapeutic strategy to impede hematopoietic stem cell maintenance in MDS patients

Abstract Myelodysplastic syndrome (MDS) comprises a group of clonal stem cell disorders characterized by ineffective haematopoiesis. Using humanized bone marrow (BM) niches within immunodeficient mice, we have previously demonstrated a critical dependence of MDS hematopoietic stem and progenitor cells (HSPCs) on the human BM niche (Leukemia 2017, BCD 2021). To further investigate this niche-specific dependency, we coupled high resolution omics to a dual approach combining an in-vivo humanized xenograft model and an ex-vivo co-culture system. HSPCs from MDS patients (n=11) and age-matched healthy donors (HD, n=8) were cultured with mesenchymal stromal cells (MSCs) derived from either MDS or HD donors, under both direct-contact and trans-well conditions. Bulk RNA sequencing and single-cell RNA sequencing allowed us to map the HSPC–MSC interactome and to target candidate genes via a large-scale siRNA knockdown approach in primary human MSCs. Notably, MDS HSPCs proliferated ~4 times more in direct contact with MSCs than in trans-well conditions (P<0.01). A similar proliferation advantage was observed when MDS HSPCs were co-cultured with HD MSCs. Interestingly, CD34⁺CD38⁻ HSCs from HDs exhibited significantly higher proliferation when cultured with HD MSCs compared to MDS MSCs (11.5% vs. 5.6%; P<0.03). Unsupervised clustering of bulk RNA sequencing data from MSCs revealed that MDS HSPCs could reprogram HD MSCs toward a ‘disease-like transcriptomic‘ profile, but only under direct-contact conditions. In contrast, MDS MSC co-cultured with HD HSPCs closely resembled the HD MSCs co-cultured with HD HSPCs. Further analysis of the bulk MSC RNA transcriptome revealed a group of differentially expressed genes (n≈1000, adjPvalue<0.05). Meta-enrichment analysis on the differentially expressed genes showed enrichment of gene sets related to cell-cell signalling, stress response, such as regulation of cellular activation/response and myeloid leukocyte activation. Using an unbiased systems biology approach Niche-net algorithm (Browaeys, 2020), we then identified a novel complex network of ligand-receptor interactions (n=84, P<0.05) between the HSPCs and MSCs, that could be responsible for rewiring of the intrinsic core pathways of the MSCs.Using an siRNA screening approach on primary MSC, we targeted these receptor genes (n=46) along with additional targets that were identified from our analysis. Notably, siRNA-mediated knockdown of a significant number of these genes led to growth arrest and apoptosis in MSCs, highlighting their critical role in maintaining MSC biology. Among the most promising candidates, Ephrin type-A receptor 3 (EPHA3) emerged as a key gene with minimal impact on proliferation of HD MSCs upon siRNA knockdown. Our interaction analysis from bulk-RNA sequencing revealed that EPHA3 receptor on MSCs is highly regulated by IL10 expressed by HSPCs. Single-cell RNA sequencing of humanized scaffolds seeded with patients MDS HSPC and MSC confirmed EPHA3 was specifically expressed in MSCs, while as IL10 was restricted to myeloid cells. These findings were further corroborated by publicly available human BM single-cell RNA sequencing dataset. Stimulation of HD MSCs (donors, n=7) with recombinant human IL-10 induced a dose-dependent increase in EPHA3 expression at both mRNA and protein levels. To functionally validate this further, we used OCI-AML3 cells (known to express and secrete IL10). Knockdown of IL10 in the OCI-AML3 cells, followed by co-culture with HD MSCs, abolished EPHA3 upregulation in MSCs. To understand the biological implication of lack of EPHA3-IL10 interactions on MDS HSPCs, we generated a knockdown of EPHA3 in BM MSCs. Long-term co-culture of BM CD34+ HSPCs from healthy donors (n=2) and MDS patients (n=2) resulted in up to 14-fold reduction in MDS HSPCs, with little impact on healthy donor HSPCs. Experiments are currently underway to better understand the biological impact of EPHA3 inhibitor on MSCs and HSPCs in MDS. In conclusion, our integrative and unbiased analysis of the MDS bone marrow niche reveals novel insights into the intrinsic dysregulation of MDS MSCs. We identify a complex network of ligand–receptor interactions driven primarily by MDS-HSPCs that modulate MSC behaviour, contributing to disease maintenance. Notably, we describe a novel IL10–EPHA3 HSPC and niche interaction that selectively supports MDS clones, offering a promising therapeutic target for disrupting the pathological HSPC–MSC crosstalk in MDS.