Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled assessment of single-cell mutational co-occurrence. We discovered changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare, but present, mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications.

Clonal hematopoiesis (CH) is an age-associated phenomenon that increases risk for hematologic malignancy and cardiovascular disease. CH is thought to increase cardiovascular disease risk through inflammation in the peripheral blood1. Here, we profile peripheral blood gene expression in 104,566 single cells from a cohort of 17 CH patients and 6 controls. We discovered that patients harboring DNMT3A and TET2 CH mutations at baseline and in response to IL-6 stimulation confer a pro-inflammatory profile to CD14+ monocytes through previously unrecognized pathways including Macrophage Inhibitory Factor (MIF). A germline genetic variant in MIF modifies TET2 CH cardiovascular disease risk. Using mitochondrial lineage tracing, we used a novel method to compare gene expression between mutated and non-mutated cells within individual CH patients. We found that the mutated CH monocytes, but not non-mutated monocytes are pro-inflammatory, explaining why patients with larger CH clones have increased cardiovascular disease risk.

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) results from a clonal expansion of hematopoietic stem cells due to somatic mutations in genes such as DNMT3A or TET2. Patients with CHIP have poor cardiovascular outcomes. TET2 CHIP clones that make up >20% of blood increases risk. Whether CHIP mutated cells confer risk directly or through polarizing other cells is presently unknown as prior work has been unable to simultaneously resolve mutational status and transcriptome of an individual cell. Methods: We performed single cell RNA-seq on blood from patients with DNMT3A CHIP (N=9), TET2 CHIP (N=8), and controls (N=6). We identified individual cell CHIP mutational status with a novel method that leverages mitochondrial mutations to identify cell lineage. Results: Across 104,556 single cells, CHIP patients had a higher proportion of CD14 monocytes (32% vs. 18%). Within individuals, CHIP mutated cells exhibited a strong myeloid bias. At the extreme, 93% of TET2 mutated cells were monocytes in one patient. Comparing TET2 CHIP monocytes to controls identified a hyper-inflammatory phenotype with increased expression of inflammatory cytokines and their downstream targets, and elevated expression of adhesion molecules. Comparing TET2-mutated CD14 monocytes to unmutated monocytes within the same individual localized the inflammatory phenotype to the mutated cells. Conversely, this phenotype was not observed in DNMT3A CHIP monocytes either in aggregate or within the same individual. Conclusions: Simultaneous, high-throughput, single-cell resolution of DNA mutation status and RNA transcriptomes identifies aberrant cellular programs in TET2-mutated CD14 monocytes. Our data support a simple mechanistic model for CHIP cardiovascular disease risk that is proportional to the number of mutated cells present. Differences observed between TET2 and DNMT3A provide a potential explanation for the differential pathogenicity of these two mutations.

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