Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Background Clonal hematopoiesis (CH) in aged populations has been drawing an increasing attention of recent years due to its implication in the risk of hematopoietic malignancies and cardiovascular complications. However, our knowledge about CH has been largely based on genetic studies, while few functional analyses have been performed using human materials with most studies being confined to artificial models using mice.

Methods To investigate the phenotypes of mutated and wild-type (WT) cells separately in CH, we developed a Fluidigm C1-based single cell (SC)-sequencing(seq) platform that enables a simultaneous analysis of gene mutations and expression. We analyzed 10,178 hematopoietic stem/progenitors (HSPCs) derived from bone marrow (BM) of patients with (n=11) and without (n=17) CH with this platform. SCRNAseq of whole BM (183,371) from young (54-71 y.o.) and elderly (78-84 y.o.) patients with (n=5) or without (n=16) TET2 CH were also performed.

Results In the analysis of HSPCs from CH(−) elderly individuals, we found a significant positive correlation between age and expression of gene sets implicated in inflammatory responses including TGFβ and IL6 signalling. We next analysed CH(+) samples from the individuals carrying TET2, DNMT3A, SF3B1 and IDH1 mutations using the modified C1 platform. Regardless of mutation type, mutant cells showed an upregulation of genes implicated in an enhanced cell proliferation compared to WT cells, whereas genes related to inflammatory responses were downregulated in mutant cells. These results suggest that mutant HSPCs show an enhanced cell proliferation and an attenuated response to an inflammatory microenvironment in aged BM, compared to endogenous WT counterparts.

Based on these observations, we further investigated the role of the BM microenvironment in CH, in which we compared the phenotype of WT cells in CH(+) and CH(−) cases. We first investigated a possible effect of mutant cells on endogenous WT cells. In a case with IDH1-mutated CH, IDH1-mutated cells (Mut^IDH1) showed a gene expression profile suggestive of an enhanced cell proliferation compared to endogenous IDH1-WT cells (WT^IDH1). However, when compared to WT cells from CH(−) cases (WT^cont), WT^IDH1 showed significantly suppressed cell proliferation, suggesting that the enhanced proliferation of Mut^IDH1 might be relative to the decreased proliferation of WT^IDH1 through non-cell-autonomous effects of Mut^IDH1. In agreement with this, mouse BM cells treated with 2-hydroxyglutalate (2HG), an oncometabolite produced by IDH1-mutant cells, mimicked WT^IDH1, including downregulated E2F target genes and upregulated inflammation-related genes, compared to WT^cont.

Non-cell autonomous effects of mutant cells were also seen in three cases with TET2-mutated CH. Endogenous TET2-WT cells (WT^TET2) showed an upregulation of genes involved in interferon signaling, DNA repair and cell proliferation compared to those from CH(−) cases. In fact, we mimicked TET2-CH using competitive transplantation of bone marrow (BM) cells from conditional Tet2^+/- or their littermates (Ly5.2), and Tet2^+/+ (Ly5.1/5.2) mice into lethally irradiated mice (Ly5.1). In line with the observation in human CH, the WT BM cells co-transplanted with Tet2^+/- competitors (WT^Tet2-comp) also exhibited enhanced gene expression of these pathways, compared to those co-transplanted with Tet2^+/+ WT competitors (WT^cont-comp). We next performed single-cell gene expression analysis of whole BM cells from patients with and without TET2-mutated CH. Aberrant expression of RIG-I like receptors, such as IFIH1 and DDX58, were found in TET2-CH(+) elderly compared to CH(−) elderly, which might contribute to the activated interferon response in TET2-CH(+) elderly. Intriguingly, elderly with TET2-mutated CH also showed decreased expression of genes involved in heme metabolism including HBB in mature erythroid cells, compared to CH(−) elderly, suggesting defected erythroid cell maturation.

Conclusions Our results suggest that mutated cells in CH(+) BM are less sensitive to inflammatory BM microenvironments characteristic of aged individuals than WT cells, which might confer a selective fitness to mutant cells. Non-cell autonomous effects of mutant cells on endogenous WT cells might also favor positive selection of CH-clones, and further contribute to inflammatory BM environments, contributing to the pathogenesis of CH.

Nakagawa:Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Nannya:Daiichi Sankyo Co., Ltd: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sumitomo Pharma: Speakers Bureau; Astrazeneca: Speakers Bureau; Takeda Pharmaceutical Company: Speakers Bureau; Kyowa-Kirin: Speakers Bureau; Fuji Pharma: Honoraria; Filgen: Speakers Bureau; Otsuka Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Asahi Kasei Pharma: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Janssen Pharmaceutical: Speakers Bureau; Pfizer: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Daiichi Sankyo RD Novare: Research Funding. Yoda:Chordia Therapeutics inc.: Research Funding. Ogawa:Pfaizer: Speakers Bureau; Astellas: Speakers Bureau; The Mitsubishi foundation: Honoraria; DaiichiSankyo: Speakers Bureau; Otsuka Pharmatheutical: Research Funding; The Chemo-Sero-Therapeutic Research Institute: Speakers Bureau; Chordia Threapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding; 2014-191287: Patents & Royalties; 62/187386 (US01): Patents & Royalties; 15/353395 (US03): Patents & Royalties; PCT/JP2014/062112 (WO01): Patents & Royalties; ASAHI Genomics: Current equity holder in publicly-traded company; Esai Pharmatheutical: Consultancy; 2013-526957 (JP02): Patents & Royalties; Sysmex: Honoraria; Astrazeneca: Speakers Bureau; 2015-239547: Patents & Royalties; Nanpu Hospital: Research Funding; 2013-096582 (JP01): Patents & Royalties; Novartis: Honoraria, Speakers Bureau; MSD: Speakers Bureau; Clinical Research Support Center Kyushu: Research Funding; Kirin/Chugai: Speakers Bureau.