Loss of nr4a1 abrogates Fitness of asxl1-mutant Hematopoietic Clones
Authors: Avagyan Serine , Henninger Jonathan E , Mannherz William P. , Mistry Meeta , Yang Song , Weber Margaret C. , Moore Jessica , Zon Leonard I. 
Published: November 2021

Clonal fitness of mutant hematopoietic stem and progenitor cells (HSPCs) underlies clonal hematopoiesis (CH), a state of clonal expansion associated with increased risk of blood malignancies and cardiovascular disease. Mechanisms by which acquired mutations lead to clonal fitness are not known. We used a zebrafish model to study the effect of acquired asxl1 mutations on HSPC clonality with TWISTR (Tissue editing With Inducible Stem cell Tagging via Recombination) that combined mosaic CRISPR-Cas9 mutagenesis with color labeling of HSPC clones. TWISTR asxl1 mutants showed clonal dominant states with the expansion of single-colored clones contributing to over 30% of myelopoiesis. These zebrafish had normal hematopoietic output and no major lineage skewing. Single-cell RNA sequencing analysis of TWISTR mutant marrow cells harboring asxl1 mutations showed &gt;10-fold upregulation of inflammatory cytokines in mutant mature myeloid cells and &gt;30-fold upregulation of anti-inflammatory modulators in immature progenitors. Increased inflammation has been widely documented in persons with CH with acquired mutations in DNMT3A, TET2, ASXL1 and other genes. Moreover, chronic inflammation due to infection was shown to promote relative clonal fitness in Dnmt3a mutant mice. Based on our results, we proposed the hypothesis that upregulation of the anti-inflammatory genes, including nr4a1, served as a mechanism of resistance to chronic inflammation created by the mutant HSPC progeny, resulting in a self-perpetuating cycle of clonal fitness in that environment. To test this, we used TWISTR to generate mosaic mutants of asxl1 and nr4a1 by injecting zebrafish with guide RNAs targeting exon 12 of asxl1 and exon 3 of nr4a1 together. Our model would predict that abrogation of nr4a1 expression in asxl1-mutant clones would weaken their fitness relative to clones that maintained nr4a1 expression. We sorted over 300 clones of various sizes from this cohort of zebrafish and sequenced the two targeted genes. We found that clones with frameshift mutations in asxl1 with either no nr4a1 mutations or heterozygous nr4a1 mutations had a clone size of 20%±14% or 19.7%±15% in myeloid cells, respectively. Asxl1-mutant clones with biallelic frameshift mutations in nr4a1 were significantly smaller with a clone size of 13.8±11.5% (p &lt; 0.015). Clones with intact asxl1 did not differ in their clone size independent of nr4a1 genetic status (11.9% wildtype nr4a1 vs 11.5% homozygous nr4a1 mutant). Chemical inhibition of nr4a1 over 3 months resulted in reduced change of edited clones in asxl1-mutant zebrafish compared to vehicle-treated zebrafish, with median change in allelic fraction of 3%±4.8% vs 5.3%±7.5%, respectively. This suggested that upregulation of nr4a1 in asxl1-mutant clones maintains their fitness in inflammatory conditions, potentially by limiting HSPC exhaustion. We successfully used TWISTR to study asxl1 induced CH in zebrafish and identified nr4a1 upregulation as a critical pathway engaged for establishing clonal fitness.

Disclosures

Zon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Amagma Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Branch Biosciences: Current holder of individual stocks in a privately-held company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.