HSC loss in FA is due to failure to resolve DNA inter-strand crosslinks (ICL), which can be induced by reactive aldehydes, radiation, or other clastogenic agents. Aldehyde exposure may occur through environmental sources, e.g. ingestion, absorption, and inhalation, or endogenously as a byproduct of cellular metabolism. The ALDH2*2 genotype, a dominant-negative point mutation in the aldehyde dehydrogenase 2 (ALDH2) gene, causes the "Asian flushing syndrome" when ethanol (EtOH) is ingested, due to decreased metabolism of small aldehydes, particularly acetaldehyde. ALDH2*2 is a disease modifier in FA, causing more rapid bone marrow failure and earlier leukemia onset in doubly affected children. Similarly, mice experimentally doubly knocked out for FANCD2 and ALDH2 demonstrate increased HSC loss, which is accelerated by EtOH exposure. To reduce aldehyde exposure, we developed a small molecule ALDH activator, Alda-1, which increases the enzymatic activity of both wild type (WT) and mutant ALDH2.

We hypothesized that DNA damage and HSC loss in FA would be prevented by reduction of the aldehyde load. To test the effects of Alda-1 mediated ALDH2 activation, we generated a novel murine FA model with FANCD2 KO and knock-in of the ALDH2*2 mutation into the murine locus. The FANCD2-/- ALDH2*1/*2 genetic model and parental controls were then tested after exogenous aldehydic challenge and/or therapeutic intervention with Alda-1. Increased aldehydic load was experimentally induced by EtOH administration 10 mg/kg/day IP, while Alda-1 10 ug/kg/day was continuously administered via osmotic pump. For each of these conditions, marrow was analyzed for HSC and progenitor cell (HSPC) number, HSC gene expression, and function.

The importance of the altered aldehyde metabolism due to ALDH2*2 genotype was demonstrated by progressive loss of HSPC in ALDH2*2/*2 and FANCD2-/- ALDH2*1/*2 mice, e.g., 5-fold and 2-fold decline in long-term HSC (LT-HSC), respectively, by 36 weeks. Experimental EtOH challenge to increase the aldehyde load precipitously decreased HSC numbers of all genotypes. After 5 weeks of EtOH challenge, LT-HSC decreased 35-fold in FANCD2-/- ALDH2*1/*2, 12.5-fold in FANCD2-/-ALDH2*1/*1, and 10.5-fold in WT mice. Long-term Alda-1 treatment to decrease aldehydic load rescued FA mice from HSC loss. After 7 months of Alda-1 treatment, LT-HSC numbers in FANCD2-/-ALDH2*1/*2 and FANCD2-/-ALDH2*1/*1 were approximately 10-fold higher than untreated controls. There were no clinically observed adverse effects. Aldehyde exposure and Alda-1 treatment altered gene expression of HSC. Principal component analysis and clustering of HSC gene expression showed that the first principal component representing 40% of the variation in gene expression could be attributed to increased aldehydic load, either genetically (ALDH2*2 genotype) or environmentally (EtOH administration) induced, while Alda-1 treatment obviated these effects. HSC from Alda-1 treated mice clustered with those from control WT mice. To test whether Alda-1 improved HSC function as well as phenotypic number, engraftment potential was assessed with competitive repopulation assays of sorted HSC from congenic untreated donors vs short-term (3 weeks) Alda-1 treated donors. HSC from Alda-1 treated mice had 2-4 fold greater granulocyte repopulating capacity than those from the untreated donors.

Our results demonstrate that Alda-1 treatment rescues HSC from aldehyde induced loss, whether from genetic variation (FANCD2- and/or ALDH2*2) or experimental challenge (EtOH administration). Furthermore, Alda-1 treatment prevents the abnormal HSC gene expression induced by increased aldehydic load. HSC function is improved by Alda-1 with greater repopulating capacity observed even after short-term treatment. These preclinical experiments provide compelling proof-of-concept that sustained activation of ALDH2 can prevent HSC loss in FA. Potential applications include long-term administration to prevent the development of marrow failure or leukemia, and HSC expansion to increase the number of cells available for gene therapy with autologous HSC. Our results suggest that a clinical trial of ALDH2 activation in FA patients to prevent marrow failure is warranted.

Disclosures

Van Wassenhove:U.S. Patent Office: Patents & Royalties: patent pending - submitted for ALDH2 activators to expand hematopoietic stem cells. Chen:Foresee Pharmaceuticals: Patents & Royalties: patents licensed to Foresee related to compounds that activate aldehyde dehydrogenase 2 (ALDH2) and correct the dysfunction in ALDH2*2; U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Mochly-Rosen:Foresee Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: patents licensed to Foresee related to compounds that activate aldehyde dehydrogenase 2 (ALDH2) and correct the dysfunction in ALDH2*2; U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Weinberg:U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells.

Author notes

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Asterisk with author names denotes non-ASH members.

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