Abstract
Fanconi anemia (FA), is a rare DNA repair-deficiency syndrome that confers cancer predisposition and progressive bone marrow failure (BMF) in most FA patients. Studies by several groups, including ours, have revealed that the underlying hematopoietic stem cell (HSC) deficits have their origin in utero, well before the clinical onset of BMF in patients during early childhood. We previously reported in a murine model of FA (Fancd2-/-) that replication stress and ATR checkpoint activation specifically limit the physiologic HSC pool expansion in the fetal liver. To identify the molecular determinants, we performed single-cell transcriptome profiling in E13.5 and littermates in both HSPCs and non-hematopoietic, fetal liver niche cells. Results show that cell composition and developmental trajectories among hematopoietic stem and progenitor cells (HSPCs) and fetal liver niche cells are broadly similar between the two genotypes. Gene set enrichment analysis (GSEA), revealed that the loss of FANCD2 in HSC leads to significantly increased expression of C-MYC target genes (normalized enrichment score, NES: 2.04, p<10-5 FDR<10-5), also reflected in 1.7-fold (p<0.05) increased levels of phosphorylated C-MYC protein in Fancd2-/- LT-HSC (CD150+ CD48- Lineage- Sca-1+ c-KIT+). This, in turn, promotes ribosome biogenesis (NES: 1.8, p<10-5, FDR<10-3) and translation (NES: 2.2, p<10-5, FDR<10-5) and elevated protein synthesis, measured in vivo by O-Propargyl-puromycin (OPP) assay, in all Fancd2-/- fetal liver HSPC subpopulations (LT- HSCs 1.3-fold p<0.01). Proteostasis is a tightly regulated process, whereby even small changes impact HSC self-renewal capacity. With translation as a highly error-prone process, we reasoned that an increased rate of protein synthesis would overwhelm the HSCs protein folding capacity. Usingan adapted Tetraphenylethene maleimide (TPE-MI) assay, we found 2.5-fold increase in misfolded proteins (p=0.01) in Fancd2-/- compared to WT fetal liver LT-HSC. This observation is consistent with a transcriptionally upregulated unfolded protein response (UPR) GSEA pathway (NES: 1.30, p<0.01, FDR=0.01) in our scRNA-Seq analysis.
Altogether, we propose a model whereby FA pathway disruption during development initiates a self-reinforcing loop of replication stress and forced MYC activity that dysregulates proteostasis, with immediate consequences for self-renewal and long term impact on HSC pool size.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
