Single-cell multi-omics profiling reveals distinct patterns of clonal expansion and microenvironmental shifts associated with disease evolution in patients with Myelodysplastic Syndromes

Abstract

Abstract Background. Myelodysplastic Syndromes (MDS) present an increased risk of progression to Acute Myeloid Leukemia (AML). The complex interactions between neoplastic clone, bone marrow (BM) microenvironment and immune cells during disease evolution remain poorly understood. We used multi-omics single-cell approach to define patterns of clonal expansion and microenvironment shifts associated with MDS disease progression. Methods. We analyzed paired BM samples at diagnosis and at time of AML transformation from 20 MDS patients who had not received disease-modifying treatments before progression. Single-cell analysis was performed by CITE-seq, integrating transcriptomic and protein expression data from hematopoietic stem and progenitor cells (HSPC), myeloid, T and NK cells, in combination with single-cell genotyping (TAPESTRI). To study longitudinal dynamics of cell states, we projected each cell into gene expression space and quantified the fold-enrichment of transcriptionally similar cells between diagnosis and AML by k-nearest neighbor analysis. Differential gene/protein expression analyses were performed by linear mixed-effects models accounting for inter-patient variability. Results. We identified two evolution patterns in HSPC compartment. In 9 patients (pts), progression was marked by emergence of novel HSPC clusters with leukemic stem cell (LSC)-like phenotype (absent/minimally detectable at diagnosis), showing upregulation of LSC markers (CD99, CD44) and immune evasion proteins (CD47, CD276) and downregulation of TGF-β and interferon (INF) response programs. In the remaining pts, progression was associated with expansion of a multipotent progenitor (MPP)-like population (already present at diagnosis). MPP-like cells exhibited increased activity of proliferative and INF-related inflammatory pathways, as well as downregulation of HLA molecules, suggesting the involvement of distinct immune escape mechanisms. Patients with NPM1, RUNX1, or TP53 mutations were more likely to show emergence of LSC-like clusters, whereas MDS with spliceosome gene mutations had heterogeneous patterns of HSPC evolution. Notably, pts showing LSC-like cluster emergence progressed more rapidly to AML (p=0.01). Considering BM microenvironment, across all pts, disease progression was associated with increased inflammatory monocytes (CD14⁺ CD86⁺ and high expression of INF-related genes) and neutrophils, suggesting that mature myeloid cells contribute to shape a pro-inflammatory marrow niche. Longitudinal analysis of immune cell states in all pts revealed widespread remodeling from diagnosis to evolution: 1) NK cells reduced their cytotoxic activity (GZMK/B-, PRF1-) and upregulated pro-inflammatory programs (NF-kb, IFN-γ); 2) T-regs acquired a highly immunosuppressive phenotype, with increased ICOS expression, downregulation of BACH2, and a switch to CD45RO⁺; 3) CD4⁺ effector memory T cells showed lower cytotoxic potential reducing GZMB/GZMK expression. Notably, in a subset of pts, small populations of these dysfunctional immune subsets—particularly highly suppressive T-regs—were already detectable at diagnosis and were associated with a shorter time to progression (p = 0.02). When comparing immunological changes based on the type of HSPC expansion, pts with LSC-like cluster displayed a more exhausted immune microenvironment, characterized by reduced frequencies of naïve T cells and increased terminally differentiated effector memory T cells, potentially supporting the selective advantage of LSC-like clones. Conversely, pts with MPP-like expansion showed increased IFN signaling across multiple immune cell populations. TP53-mutated MDS exhibited a distinct inflammatory signature, independent of IFN signaling, in both mature myeloid cells and T-regs. These myeloid cells showed HLA downregulation, while T-regs were enriched for a CD161⁺ subset with enhanced suppressive function—indicating a specific pattern of immune dysregulation driven by myeloid inflammation and impaired antigen presentation. Conclusion. MDS follow distinct evolutionary trajectories within the HSPC compartment. Consistent alterations in the BM microenvironment emerged as a potential common driver of disease progression. Early detection of rare, aberrant myeloid and immune cell populations at diagnosis may help identify pts at higher risk of rapid transformation to AML. TP53-mutated MDS exhibited a unique immunosuppressive profile, which may be a driver of their poor prognosis.