Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single-Cell Multi-Omics Sequencing

Background

Despite the large impact of age-related clonal hematopoiesis (CH) on leukemogenesis, the mechanism by which CH clones are selected in the aged individuals is still not fully understood, because a few studies have investigated functional impacts of CH-mutations using primary human materials with most studies being performed using mouse models. A major challenge in analyzing human CH is the small size of mutant clones and the lack of robust methods to analyze mutant cells separately from wild-type (WT) counterparts.

Methods

We developed a novel single-cell sequencing platform that enables simultaneous detection of mutations and gene expression, which was used for the analysis of gene expression profiles of hematopoietic stem and progenitor cells (HSPCs) harboring CH-related driver mutations. These profiles were compared to those of wild-type (WT) cells derived from both CH(+) and CH(−) samples. Whole bone marrow (BM) cells from these cases were also analyzed using single-cell RNA sequencing (scRNAseq). For mouse competitive bone marrow transplantation (BMT) assays, BM from Ly5.2/Ly5.2 Tet2; Mx1-Cre+ conditional heterozygous knockout (cKO) mice donors or their littermate control mice donors were transplanted with BM from competitor Ly5.1/Ly5.2 WT mice into lethally irradiated Ly5.1/5.1 WT mice at the ratio of 1:99. After the cKO allele was deleted by injecting pIpC, BM cells were harvested and subjected to scRNAseq with surface protein detection. Donor cells and competitor cells were distinguished based on CD45.1/CD45.2 protein expression levels.

Results

We first analysed CH(+) cases carrying TET2, DNMT3A, SF3B1, SRSF2, IDH1 and IDH2 mutations using our scRNAseqplatform. Regardless of mutation type, mutant cells showed an upregulation of genes implicated in an enhanced cell proliferation compared to the endogenous WT cells in the same cases. Of interest, we observed that inflammatory response-related genes were significantly downregulated in mutant cells, while they were generally upregulated in BM cells from aged CH(−) donors, suggesting the role of an inflammatory BM environment associated with aging in the positive selection of mutant cells.

Based on these observations, we further investigated the role of the BM microenvironment in the development of CH by comparing the phenotype of WT cells in CH(+) and CH(−) cases. Strikingly, WT cells from CH(+) cases exhibited increased expression of genes related to interferon gamma response and ‘immune system’ compared to those from CH(−) cases, suggesting that the BM environment in CH(+) cases underwent enhanced inflammatory changes. Among CH-related mutations, those affecting TET2 were associated with the highest upregulation of genes related to interferon alpha response and innate immune system in the endogenous WT cells, compared to those from age-matched CH(−) cases.

To investigate whether the altered BM environment is ascribed to the presence of mutant cells, we performed competitive BMT assays using 1% of Tet2 heterozygous mutant or WT competitor (Ly5.2/Ly5.2) to mimic the human TET2-mutated CH cases (see Methods), in which the phenotype of WT cells (Ly5.1/Ly5.2) were compared between both transplants with and without mutant competitors. Strikingly, the WT BM cells co-transplanted with Tet2 cKO mice exhibited an enhanced expression of genes related to interferon responses and innate immune system, compared to the WT BM cells co-transplanted with WT littermate controls. The mouse WT BM cells co-transplanted with Tet2 cKO mice and human WT cells in TET2-CH(+) cases showed significant overlaps in enhanced hallmark pathways (p-value:8.9x10^-4, odds ratio: 21.5). Interestingly, Tet2 cKO granulocytes showed an enhanced expression of genes related to interferon response, immune system, and pro-inflammatory cytokine response such Il6 and Tnf, compared to WT competitor cells. These results suggest that an altered immune environment might have affected the phenotype of WT cells in Tet2 cKO-transplanted mice, which is thought to be caused by non-cell autonomous effects of Tet2 mutated cells mediated by proinflammatory cytokine networks.

Conclusions

Our results suggest that mutant cells in CH(+) BM may exert non-cell autonomous effects on WT cells, which along with aged BM environments, may contribute to the positive selection of CH clones, playing a pivotal role in the pathogenesis of CH.

Inagaki:Ono Pharmaceutical: Current Employment. Nannya:Pfizer: Speakers Bureau; Novartis: Speakers Bureau; Otsuka Pharmacutical: Consultancy; Daiichi Sankyo: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; KyowaHakko Kirin: Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Bristol Meyer Squibb: Consultancy; Otsuka Pharmaceutical: Speakers Bureau; Astra-Zeneca: Speakers Bureau; Bristol Meyer Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Yoda:Chordia Therapeutics inc.: Research Funding. Kakiuchi:Sumitomo Pharma: Research Funding. Makishima:Nippon Shinyaku: Research Funding. Ogawa:Nanpuh Hospital: Other: Endowed chair; Nihonshinyaku Co., Ltd.: Other: Donation; Nakatani Foundation: Speakers Bureau; The Mitsubishi Foundation: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Inc: Current equity holder in publicly-traded company; Chordia Therapeutics Inc.: Consultancy, Other: Endowed chair, Research Funding; Eisai Co., Ltd.: Consultancy, Research Funding.