Bone marrow environmental alterations underlie proliferative clonal expansion in clonal hematopoiesis Free

Background Age-related clonal hematopoiesis (CH) is common in the elderly and associated with increased risk of hematologic malignancies and other age-related non-neoplastic disorders. Despite its clinical relevance, the mechanisms underlying the selection and expansion of mutant CH clones in aging remain unclear. In particular, studies using human bone marrow (BM) are limited by the small size of mutant clones and the lack of robust platforms to distinguish mutant from wild-type (WT) cells.

Methods To address these questions, we established a single-cell multi-omics platform enabling simultaneous detection of somatic mutations and gene expression in human hematopoietic stem and progenitor cells (HSPCs). Using this platform, we analyzed Lineage⁻ CD34⁺ HSPCs from CH(+) individuals (7,116 cells from 16 cases) and age-matched CH(−) controls (3,898 cells from 16 cases). In parallel, we also analyzed whole BM cells from CH(+) (97,459 cells from 11 cases) and CH(−) cases (145,283 cells from 18 cases) using Chromium-based single-cell RNA sequencing to characterize BM microenvironment, including stromal cells. To investigate the functional impact of mutant cells on WT cells, we performed competitive BM transplantation (BMT) assays in mice. BM from Ly5.2/Ly5.2 Tet2⁺^/− Mx1-Cre conditional heterozygous knockout (cKO) mice or their WT littermates was mixed with Ly5.1/Ly5.2 WT competitor BM (1–5% mutant to 95–99% competitor), modeling the small clone sizes observed in human CH. Following Cre induction by pIpC, BM cells were harvested for single-cell RNA sequencing and surface protein profiling. Donor-derived cKO and WT cells were distinguished based on CD45.1/CD45.2 expression.

Results Single-cell multi-omics of CH(+) HSPCs carrying TET2, DNMT3A, SF3B1, SRSF2, IDH1, and IDH2 mutations revealed consistent upregulation of proliferation-associated genes compared with WT cells from the same individuals. Mutant cells also exhibited markedly attenuated transcriptional responses to proinflammatory cytokines such as TNFA, in contrast to strongly upregulated proinflammatory cytokine signaling in both hematopoietic and stromal cells in aged CH(−) BM. These results suggest that attenuated response to age-associated proinflammatory stimuli in the aged BM may contribute to the selective expansion of CH clones.

Further analysis of the BM microenvironment in CH(+) individuals revealed broadly remodeled BM immune microenvironment, which may support clonal expansion. Compared with WT cells from CH(−) individuals, those from CH(+) marrow showed upregulation of cell cycle- and interferon-related genes. CH(+) BM also exhibited an altered immune composition, characterized by increased T cells and decreased B cells, together with shifts in myeloid and erythroid progenitors. Consistently, even older TET2-CH(+) cases with relatively small clone sizes (variant allele frequency [VAF] 4.6–8.1%) showed marked upregulation of interferon response genes in endogenous WT cells, suggesting that mutant clones may influence the surrounding BM environment even at low levels.

To directly test whether these BM environmental changes could be attributed to the presence of mutant cells, we performed competitive BMT assays using 1–5% Tet2 heterozygous mutant donor cells mixed with WT competitors to model the low-level chimerism observed in human TET2-mutated CH. Strikingly, WT HSPCs co-transplanted with Tet2-mutant cells exhibited a gene expression profile closely resembling that of WT cells from human TET2-CH(+) marrow, including enhanced interferon signaling. By contrast, WT cells co-transplanted with Tet2-WT competitors did not. These results provide direct evidence that mutant cells in CH(+) marrow can exert non–cell-autonomous effects on WT cells, thereby altering their transcriptional state.

Conclusions CH-associated mutant cells not only display an intrinsically proliferative phenotype but also induce non-cell-autonomous effects on WT cells within the BM environment. In the context of the proinflammatory milieu associated with aging, such environmental alterations may promote the positive selection and expansion of mutant clones, thereby shaping the pathogenesis of CH.