A Novel Source of Endogenous DNA Damage That Requires Repair By the Fanconi Anemia Pathway

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome. Impaired DNA interstrand crosslink (ICL) repair is the underlying mechanism for BMF in FA. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage are most likely to play an important role in the pathogenesis of FA. Metabolic by-products, such as reactive aldehydes, have been implicated in the acceleration of BMF or leukemia in both humans and mice lacking the ICL repair pathway. However, the potential contribution of other detoxifying metabolic enzymes to genome maintenance has not been systematically investigated. Identification of all sources of endogenous DNA damage will allow us to develop novel strategies to prevent DNA damage from arising, and possibly prevent BMF and leukemia in FA as well as other BMF syndromes.

To determine whether detoxifying enzymes other than ALDH2 or ADH5 play a role in the protection of HSPC, we performed a metabolism-focused CRISPR/Cas9 synthetic lethality screen (3000 metabolism genes, 10 sgRNA per gene), using wild-type and FANCD2-/- Jurkat cells. From the screen, we identified ALDH9A1 as the most significantly depleted gene in FANCD2-/- Jurkat cells compared with wild-type. Eight out of ten sgALDH9A1 were significantly depleted in FANCD2-/- Jurkat cells, indicating robust effect of the ALDH9A1 knockout. ADH5, a known synthetic lethal gene with FA, was also depleted in FANCD2-/- Jurkat cells, but to a lesser degree. In vitro fluorescence-based competition assay confirmed synthetic lethal interaction between the two genes, in two independent FANCD2-/- Jurkat clones.

To determine whether ALDH9A1 deficiency also caused cell death in FA-deficient human hematopoietic stem progenitor cells (HSPCs), we performed an in vitro validation assay using human umbilical cord blood (UCB). UCB CD34+ cells were edited by ribonucleoprotein delivery of Cas9 and sgRNA, in which either of sgCTRL, sgFANCD2 or sgALDH9A1, or both sgFANCD2 and sgALDH9A1 were used. Edited cells were grown on methylcellulose for 10 to 14 days, after which individual colonies were scored and harvested for sequencing. While CD34+ cells that were targeted by both sgFANCD2 and sgALDH9A1 (double KO) achieved lower editing efficiency for each gene compared with cells targeted by single guides, they produced the fewest hematopoietic colonies and the lowest frequency of GEMM (Granulocyte, Erythrocyte, Macrophage and Megakaryocyte) colonies. We observed fewer colonies targeted for both genes (biallelic double KO; observed to expected ratio 0.33) as compared to either single gene KO. These results suggest that loss of ALDH9A1 is deleterious in FANCD2-deficient HSPC.

Lastly, we generated a Fanca-/-Aldh9a1-/- mouse model to examine the in vivo hematopoietic phenotype due to increased endogenous aldehydes. These mice were born at the Mendelian ratio without significant anomalies except rare cases of eye abnormalities. At three months of life, Fanca-/-Aldh9a1-/- mice had lower platelet counts than wild-type, Fanca-/- or Aldh9a1-/- control mice, but total white blood counts and hemoglobin levels were similar between groups. A follow-up result of this mouse model will be presented at the meeting.

In conclusion, we identified that cells with ALDH9A1 deficiency require the FA pathway for survival. ALDH9A1 may protect human and mouse HSPC that are deficient in the FA pathway from DNA damage and cell death.