S71 EXABS-169-MDS Update on the Biology of MDS Simona Colla, PhD1,* 1The University of Texas, MD Anderson Cancer Center, Houston, 1515 Holcombe Blvd, 77030, USA *Corresponding author: scolla@mdanderson.org Keywords Myelodysplastic syndromes, clonal cytopenia of undetermined significance, hematopoietic stem and progenitor cells, natural killer cells, single-cell technologies Introduction Myelodysplastic syndromes (MDS) arise from a small population of disease-initiating hematopoietic stem cells (HSCs) that persist and expand during conventional therapy and are major contributors to disease progression.1–4 In the last few years, single-cell technologies coupled with mouse functional studies have greatly improved our understanding of the molecular mechanisms driving MDS pathogenesis. These studies have revealed that MDS are driven by multistep processes that affect a recurrent set of genes or cytogenetic aberrations, leading to the clonal expansion of mutant HSCs over their normal counterparts.5 However, nearly all hematopoietic stem and progenitor cells (HSPCs) harbor pathogenic genetic alterations at the onset of MDS,6 which suggests that, except for allogeneic HSC transplantation, no therapy can cure the disease. Indeed, hypomethylating agent (HMA)-based therapy, the standard of care for MDS patients, can enhance the differentiation of cells downstream of HSPCs without affecting the clonal mutational burden6. The prognosis of patients with HMA therapy failure is dismal owing to a lack of effective front- or second-line treatment options. Thus, therapeutic strategies targeting the biological mechanisms underlying the pre-leukemic stage of MDS, clonal cytopenia of undetermined significance (CCUS), when the mutation burden is low and symptoms are minimal, can improve the dismal outcomes of these patients. CCUS is an unexplained cytopenia that arises in the context of myeloid-associated somatic mutations in the HSPC compartment yet does not meet diagnostic criteria for MDS because of a lack of dysplasia.7 The two most frequently mutated genes in CCUS, DNMT3A and TET2, which both encode epigenetic regulators, are pathogenic initiating events in MDS.5 In contrast to clonal hematopoiesis of indeterminate potential, a common age-related condition that has a risk of progression to a myeloid malignancy of less than 1% per year,8 CCUS confers a 10-year cumulative probability of progression to MDS of 82%; this probability exceeds 95% if the CCUS clone has a variant allele frequency greater than 20%.9,10 Currently, there is no standard of care for patients with CCUS other than supportive care, even though many of these patients have transfusion needs. Thus, there is an unmet need to develop interventional strategies to prevent or delay the evolution of CCUS to MDS in patients with a higher risk of progression. Results To develop prevention strategies that arrest CCUS before the disease progresses to MDS and recognizing that CCUS is an aging-related disease, we dissected at the single-cell level the ways in which CCUS mutations affect the transcriptional and epigenetic profile of the aging HSPCs and bone marrow (BM) mononuclear cells (MNCs). Our single-cell RNA sequencing (scRNA-seq) analysis of Lin–CD34+ HSPCs from CCUS patients with DNMT3A and/or TET2 mutations and from age-matched healthy donors (HDs) revealed that CCUS mutations induce myeloid differentiation and maintain HSPCs in a state of persistent metabolic activation, which was also confirmed by metabolomic analysis. These results suggest that HSCs, which in homeostatic conditions rely on anaerobic glycolysis to maintain their quiescent state,11 switch to mitochondrial oxidative metabolism in CCUS to meet the high-energy demands necessary to sustain activated myeloid differentiation.12 Further differential transcriptomic analysis of eHD and CCUS HSCs showed that the pro-inflammatory cytokine IL-1b was significantly upregulated in CCUS HSCs (P adj = 1.70×10–71). In light of previous findings in mice showing that chronic IL-1b exposure drives HSC differentiation towards myelopoiesis at the expense of erythropoiesis and lymphopoiesis,13 these results may explain why CCUS HSCs are myeloid-primed and metabolically activated. Interestingly, the expression of other cytokines, such as CXCL2, CXCL3, and MIF, which play a role in the pathogenesis of cardiovascular diseases,14 were also significantly increased in CCUS HSCs. To evaluate whether a dysfunctional immune phenotype could contribute to the maintenance of CCUS HSPCs, we performed scRNA-seq analysis of HD and CCUS BM MNCs and dissected the intercellular crosstalk between each BM population. An analysis of cellular communication networks revealed that the predominant interactions present in CCUS BM samples but not HD BM samples were from natural killer (NK) cells to CD8+ T cells, and these interactions were predicted to induce immune tolerance. Further scRNA-seq analyses revealed that NK cells from CCUS patients were in a state of advanced differentiation compared with those from HDs but exhibited a reduced capability of producing interferon-g, which underscores their limited activity. Compared with HD NK cells, CCUS NK cells had a significantly
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