Mesodermal Development From Reprogrammed Fanconi Anemia Cells Is Affected by ALDH2 Enzymatic Activity

Fanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination.

Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes.

It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients.

In FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study.

To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway.

To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues.

Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage.

No relevant conflicts of interest to declare.