ISSCR 2022

A CHEMICAL INHIBITOR OF NR4A1 ALTERS ASXL1-MUTANT BLOOD STEM CELL CLONAL DYNAMICS

Clonal expansion of hematopoietic stem and progenitor cells (HSPCs) with acquired mutations underlies clonal hematopoiesis (CH). Abating this expansion may reduce hematologic and cardiovascular risks associated with CH. Here, we demonstrate a chemical that can alter mutant clonal dynamics in vivo. We recently published a zebrafish model to study CH, called TWISTR, combining mosaic CRISPR-Cas9 mutagenesis with color labeling of HSPC clones. We identified the critical role of inflammation in promoting the fitness of mutant clones. With heightened inflammatory state in the marrow, mutant progenitors had upregulation of anti-inflammatory genes, including nr4a1, as a means of resistance to inflammation. Using CH with asxl1 mutations, we showed that genetic abrogation of nr4a1 prevented the expansion asxl1 clones. Here, we tested the effect of pharmacologic inhibition of nr4a1 in adult zebrafish with asxl1-mutant CH. Zebrafish were treated with the inhibitor with a 5-days-on-2-days-off schedule, and clonal dynamics was assessed by peripheral blood sampling. In vehicle-treated zebrafish, asxl1 clones with frameshift insertion-deletions (indels) expanded with an average clone size increase of +59.4% [-69.3% to +795.9%] from baseline after 3 months (p < 0.003, Wilcoxon rank test). Inhibition of nr4a1 was associated with significantly reduced, non-significant expansion of asxl1-mutant clones with frameshift indels, with an average increase of +13.4% [-91.1% to +391.9%]. No significant changes were observed in clones with in-frame indels in either cohort. Examination of mutant clonal dynamics revealed several important findings. In vehicle-treated mutant zebrafish, small clones of <10% were more likely to expand than large clones of >10% (+106.3% vs +5.8%, p < 0.02). Clones with in-frame indels largely remained constant. However, in zebrafish with a significant growth of a clone with a frameshift indel, we observed a concurrent diminishing of another clone with an in-frame indel. This suggested an interclonal regulation of HSPC clone size in CH. Mutant clones in the experimental cohort halted their growth but did not diminish with treatment. We successfully used TWISTR to study the clonal dynamics of asxl1 induced CH, and showed that pharmacologic inhibition of a pro-survival pathway could be used for the treatment of CH.