Single cell multiomics reveal clonal and functional dynamics of MDS stem/progenitor cells during hypomethylating therapy

Progressive somatic mutations in hematopoietic stem cells (HSCs) drive the development of myelodysplastic neoplasms (MDS). Hypomethylating agents such as azacitidine (AZA) can improve blood counts and reduce blasts, although responses are rarely durable. Determinants of AZA response are complex and incompletely understood, although accumulating evidence suggests that epigenetic rewiring of mutated HSCs underlies improved hematopoietic output. Using single cell multiomics on longitudinal bone marrow samples, we show that AZA responsiveness involves expansion of cells with transcriptomic profiles shared with hematopoietic stem and progenitor cells (HSPCs) from healthy donors. These regenerating cells are depleted of copy number variations and of TP53 mutations. We also identify patient-restricted cell populations, some of which recede through transcriptional restoration or AZA cytotoxicity, and others which expand, regardless of initial clinical response, and dominate at progression. Individual patients carried multiple patient-restricted populations which had unique surface immunophenotypes and were genetically distinct. Strikingly, sorted cells from in vivo progression clones that were AZA-refractive in patients regained AZA-sensitivity when cultured in vitro, suggesting that lack of AZA response at the cellular level can be modulated by cell-extrinsic factors in vivo. Overall, we find that AZA response involves partial hematopoietic regeneration via functional differentiation of mutated, but not cytogenetically abnormal HSPCs, and that persistence of AZA-refractive sub-populations contributes to eventual disease progression.