Mosaic Mutagenesis In Vivo Reveals Mutant Blood Stem Cells Intrinsically Resistant to Inflammatory Mediators in Clonal Hematopoiesis

Clonal hematopoiesis (CH) is a state of clonal dominance of a mutant hematopoietic stem and progenitor cell (HSPC) promoted by unknown mechanisms. To study the competitive behavior and the mechanism of such expansion in a native environment, we used a combinatorial approach of HSPCs labeling and mutagenesis in vivo in a technique we called Tissue editing With Inducible Stem cell Tagging via Recombination (TWISTR). TWISTR utilizes Zebrabow zebrafish to fluorescently label endogenous HSPCs, and allows following of the clonal activity and sorting of labeled HSPCs within the hematopoietic system. CRISPR/Cas9 mutagenesis was used to simultaneously induce mosaic insertions/deletions (indels) in zebrafish orthologs of 12 human CH genes during development, and zebrafish were followed for 8 months. As such, mutant stem cells were directly competed against endogenous wildtype stem cells. We achieved a high degree of mosaic mutagenesis with heterozygous edits, with a median of 5 targeted genes per zebrafish in total marrow cells at variant allele frequency (VAF) of 5% or greater. Serial sampling and sequencing of peripheral blood cells showed selective expansion of clones with frameshift indels in exon 12 of asxl1, while clones with other CH mutations did not show a significant change over the 4 months period evaluated. No changes were noted in control gene indels or in the CH gene indels in non-hematopoietic tissue in the same time period. We identified significant enrichment of frameshift indels in asxl1 in sorted dominant clones compared to sorted smaller clones in the same zebrafish (p<0.01) at 8 months, while indels in DNMT3A ortholog dnmt8 were present in clones of various competitive nature. Introduction of asxl1 mutations singly in a mosaic fashion also resulted in clonally dominant states. To identify gene expression signatures associated with the clonal expansion, we used single cell RNA sequencing of marrow cells in zebrafish with a dominant clone with unique sets of mutations. We identified increased expression of inflammatory signaling genes in these zebrafish compared to controls, consistent with recent findings in other organisms. Within sorted dominant clones, mutant neutrophils and macrophages exhibited more than 4-fold increased expression of cytokines, such as il1b and tnfb. In mutant HSPCs and myeloid-biased progenitor cells, we observed elevated levels of genes involved in suppressing responses to cytokines and inflammatory lipids, like socs3a, nr4a1, atf3 and ier2a. This supports a model in which the mutant HSPCs of the dominant clone express anti-inflammatory genes that dampen their response to the inflammatory mediators produced by mature cells of the same clone, limiting the inflammation-induced HSPC exhaustion and providing a selective mechanism for the mutant stem cells to expand over time. We successfully used the Zebrabow color labeling system and prospective endogenous mosaic mutagenesis to show that mutant asxl1 is a potent inducer of CH, and that CH gene mutations lead to clonal dominance by modifying the response of stem cells to inflammatory cues.