Dysregulated Proteostasis Restrains the Developmental HSC Expansion in Fanconi Anemia

Hematopoietic stem cells (HSCs) require precise regulation of protein synthesis, processing, and degradation. Protein homeostasis (Proteostasis) is particularly critical during development, relying on chaperone proteins to maintain folding capacity in a highly proliferative HSC pool. Even modest changes in synthesis or degradation lead to the accumulation of unfolded proteins and compromise fetal liver HSC pool formation. Fanconi anemia (FA) is a multisystem disorder that manifests with bone marrow failure (BMF) early in life. Previous reports from our lab and others suggest that pre-natal HSC depletion in FA is a critical factor for the early onset of clinical BMF. To identify the cellular and molecular mechanisms underlying prenatal HSC defects in FA, we performed single-cell transcriptome profiling in both Fancd2 ^+/+and Fancd2 ^-/- FL samples. Gene set enrichment analysis (GSEA) of differentially expressed genes revealed significantly increased expression of c-Myc target genes in Fancd2 ^-/- HSCs (normalized enrichment score, NES: 2.04, p<10 ^-5 FDR<10 ^-5), matched by an increase in c-Myc in Fancd2 ^-/- LT-HSC (CD150 ⁺ CD48 ^- Lineage ^- Sca-1 ⁺ c-KIT ⁺) at the protein level (1.7-fold (p<0.05). Our sc-RNA Seq data further showed an increase in ribosome biogenesis (NES: 1.8, p<10 ^-5, FDR<10 ^-3) and translation (NES: 2.2, p<10 ^-5, FDR<10 ^-5) in Fancd2 ^-/- HSC, functionally corroborated by increased protein synthesis rates across Fancd2 ^-/- FL HSPC subpopulations (1.3-fold in LT- HSCs, p<0.01) using the in vivo O-Propargyl-puromycin (OPP) assay. Not surprisingly, the increased protein synthesis rates exceed the physiologic protein folding capacity, leading to elevated levels of unfolded proteins in Fancd2 ^-/- FL HSPCs by Tetraphenylethene maleimide (TPE-MI) stain. The aggregated misfolded proteins (aggresomes) in Fancd2 ^-/- FL HSPCs were also observed by immunofluorescence microscopy. Revisiting our scRNA Seq data, the observed proteotoxic stress was also reflected in the GSEA enrichment for unfolded protein response (UPR; NES: 1.30, p=0.01, FDR=0.01) as well as heat shock stress response (NES:1.7,p<0.0001, FDR=0.09), and coincided with upregulation of response to heat shock factor-1 ( Hsf-1; NES:1.86,p<0.0001, FDR=0.02) in Fancd2 ^-/- FL HSCs. Because chaperone supplementation has been shown to enhance HSC protein folding capacity, we serially injected the bile acid TUDCA (a chemical chaperone) into the pregnant dam. This not only significantly reduced unfolded protein levels, but indeed rescued FL HSC numbers in Fancd2^-/- littermates. Our observations in aggregate indicate that dysregulated proteostasis is a previously unrecognized pathogenic process constraining fetal Fancd2 ^-/- hematopoiesis. Protection of the HSPC proteome appears to be a new, non-canonical function of FANCD2, without immediate connection to the FA pathway and DNA crosslink repair. Importantly, we demonstrate the successful pharmacological rescue of the fetal Fancd2^-/- HSPC pool.