Chemical Modulation of ELF1 Binding to RUNX1 Target Genes Leads to Increased Hematopoietic Stem Cell Self-Renewal

Hematopoietic stem cell (HSC) expansion and self-renewal are important for stem cell therapy. Expanding HSCs ex vivo remains challenging. To identify novel compounds that increase HSCs, we screened 3,840 small molecules using a zebrafish blastomere culture screening system with a HSC specific Runx1+23:GFP reporter and identified a RUNX1/CBFβ modulator, Ro5-3335, which increases Runx1+ cells. Using spinning disk confocal time-lapse imaging in live zebrafish embryos, we found that Ro5-3335 treatment increases HSC number as much as 1.79 fold (p<0.0001) and significantly increases HSC divisions (5 vs 3.5 divisions, p=0.009) in the stem cell niche during early hematopoiesis. Transplantation of Ro5-3335 treated Runx1+ cells into zebrafish embryos revealed enhanced engraftment by 1.60 fold (p<0.001). To determine whether the increased HSC production during development increases HSC clones in adulthood, we used a brainbow color barcoding system to lineage trace each HSC and its blood progenies. Treatment of Ro5-3335 during definitive hematopoiesis significantly increases HSC clones in adulthood (23 vs 18.6 clones, p<0.0001). To determine whether the effect of Ro5-3335 on HSC expansion is conserved in mammalian systems, we cultured human CD34+ HSPCs and found that Ro5-3335 promotes CD34+CD38-CD45RA-CD90+ HSC expansion in 6-day ex vivo culture (20.3 vs 15.8 fold (p=0.0157)). RUNX1 is an important transcription factor for HSC proliferation and differentiation. Chromatin immunoprecipitation sequencing (ChIP-seq) of RUNX1 showed that Ro5-3335 treatment increased RUNX1 binding to target genes in human CD34+ HSPCs, independent of CBFβ binding. Motif analysis of the genes with increased RUNX1 binding and increased transcription suggested binding of ELF family transcription factors to RUNX1 target genes upon Ro5-3335 treatment. ELF1 ChIP-seq confirmed that ELF1 has increased binding to RUNX1 targets, including cell cycle genes CDC45, CDC37L, CCND2, and CCND3 upon Ro5-3335 treatment. Knocking down Elf2b in zebrafish embryos abolished the effect of Ro5-3335 both on HSC expansion and HSC clonality. Together, our studies provide the first evidence to show that it is possible to pharmacologically increase stem cell clonality in vivo.

Choudhuri:Fulcrum Therapeutics: Current Employment. Kubaczka:ElevateBio: Research Funding. Mandelbaum:Bristol-Myers Squibb: Current Employment. Zon:Fate Therapeutics: Current holder of stock options in a privately-held company; Scholar Rock: Current holder of stock options in a privately-held company; CAMP4 Therapeutics: Current holder of stock options in a privately-held company; Triveni Bio: Current holder of stock options in a privately-held company.