Selective advantage of mutant stem cells in human clonal hematopoiesis is associated with attenuated response to inflammation and aging.

Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire mutations, most frequently in the DNMT3A and TET2 genes, conferring a competitive advantage through mechanisms that remain unclear. To gain insight into how CH mutations enable gradual clonal expansion, we used single-cell multi-omics with high-fidelity genotyping on human CH bone marrow (BM) samples. Most of the selective advantage of mutant cells occurs within HSCs. DNMT3A- and TET2-mutant clones expand further in early progenitors, while TET2 mutations accelerate myeloid maturation in a dose-dependent manner. Unexpectedly, both mutant and non-mutant HSCs from CH samples are enriched for inflammatory and aging transcriptomic signatures, compared with HSCs from non-CH samples, revealing a non-cell-autonomous effect. However, DNMT3A- and TET2-mutant HSCs have an attenuated inflammatory response relative to wild-type HSCs within the same sample. Our data support a model whereby CH clones are gradually selected because they are resistant to the deleterious impact of inflammation and aging.

Precise single-cell transcriptomic mapping of normal and leukemic cell states reveals unconventional lineage priming in acute myeloid leukemia.

Initial classification of acute leukemia involves the assignment of blasts to cell states within the hematopoietic hierarchy based on morphological and immunophenotypic features. Yet, these traditional classification approaches lack precision, especially at the level of immature blasts. Single-cell RNA-sequencing (scRNA-seq) enables precise determination of cell state using thousands of markers, thus providing an opportunity to re-examine present-day classification schemes of acute leukemia. Here, we developed a detailed reference map of human bone marrow hematopoiesis from 263,519 single-cell transcriptomes spanning 55 cellular states. Cell state annotations were benchmarked against purified cell populations, and in-depth characterization of gene expression programs underlying hematopoietic differentiation was undertaken. Projection of single-cell transcriptomes from 175 samples spanning acute myeloid leukemia (AML), mixed phenotype acute leukemia (MPAL), and acute erythroid leukemia (AEL) revealed 11 subtypes involving distinct stages of hematopoietic differentiation. These included AML subtypes with notable lymphoid or erythroid lineage priming, challenging traditional diagnostic boundaries between AML, MPAL, and AEL. Quantification of lineage priming in bulk patient cohorts revealed specific genetic alterations associated with this unconventional lineage priming. Integration of transcriptional and genetic information at the single-cell level revealed how genetic subclones can induce lineage restriction, differentiation blocks, or expansion of mature myeloid cells. Furthermore, we demonstrate that distinct cellular hierarchies can co-exist within individual patients, providing insight into AML evolution in response to varying selection pressures. Together, precise mapping of hematopoietic cell states can serve as a foundation for refining disease classification in acute leukemia and understanding response or resistance to emerging therapies.

SINGLE CELL DISSECTION OF DEVELOPMENTAL ORIGINS AND TRANSCRIPTIONAL HETEROGENEITY IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA.

Sequencing of bulk tumor populations has improved genetic classification and risk assessment of B-ALL, but does not directly examine intratumor heterogeneity or infer leukemia cellular origins. We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) and compared them to a reference map of normal human B-cell development established using both functional and molecular assays. Intra-sample heterogeneity was driven by cell cycle, metabolism, differentiation, and inflammation transcriptional programs. By inference of B lineage developmental state composition, nearly all samples possessed a high abundance of pro-B cells, with variation between samples mainly driven by sub-populations. However, ZNF384- r and DUX4- r B-ALL showed composition enrichment of hematopoietic stem cells, BCR::ABL1 and KMT2A -r ALL of Early Lymphoid progenitors, MEF2D -r and TCF3::PBX1 of Pre-B cells. Enrichment of Early Lymphoid progenitors correlated with high-risk clinical features. Understanding variation in transcriptional programs and developmental states of B-ALL by scRNA-seq refines existing clinical and genomic classifications and improves prediction of treatment outcome.