Mechanisms of Resistance to Reprogramming of Cells Defective In the Fanconi Anemia DNA Repair Pathway

Abstract 196

Fanconi anemia (FA), the most common inherited bone marrow failure syndrome, is characterized by progressive loss of hematopoietic stem cells, aplastic anemia, genomic instability and cancer predisposition. Induced pluripotent stem (iPS) cells are a promising source of cells for disease-specific investigations and genetic correction. It has been reported that human FA dermal fibroblasts are resistant to direct reprogramming without prior genetic correction (Raya et al., Nature, 2009), but the mechanism remains unclear. In this study we aimed to define the role of the FA pathway during the transition from fibroblasts to iPS cells in murine cells. We transduced Fanca-/- and wild type (wt) tail-tip fibroblasts with four defined factors (Oct3/4, Klf4, Sox2, c-Myc) and enumerated the number of iPS colonies that were derived from 1×10⁵ cells. We noted a >10-fold decrease in the reprogramming efficiency of Fanca-/- cells compared to wt controls [median (range): wt 132 (0 to 1296) colonies, efficiency 0.328%, n=17; Fanca-/- 4 (0 to 80) colonies, efficiency 0.019%, n=10, p<0.0001, Wilcoxon test]. Despite the markedly reduced reprogramming efficiency, Fanca-/- fibroblasts yielded iPS cells that expressed pluripotency markers (Oct3, Nanog, SSEA-1), gave rise to mature teratomas, and were able to generate chimeric mice. Given the defective DNA repair phenotype of FA cells, we compared baseline and reprogramming-induced DNA damage and senescence in wt and Fanca-/- cells. We observed that double strand DNA (dsDNA) breaks (γH2AX foci) and senescence were significantly increased in Fanca-/- cells four days following the transduction with the reprogramming viruses as compared to wt cells (p<0.0001 and p=0.0012, respectively; Table 1). Because reactive oxygen species (ROS) have been implicated as cellular mediators of DNA damage, senescence and genomic instability in FA cells, we measured the ROS levels during reprogramming and observed a 1.7-fold ROS induction in Fanca-/- fibroblasts. Addition of the ROS scavenger N-acetylcysteine (NAC, 100 μ M) reduced the number of dsDNA breaks in the Fanca-/- cells but failed to increase the reprogramming efficiency, possibly due to off-target toxicity. Therefore, to further assess the impact of oxidative DNA damage on the reprogramming of FA iPS cells, we compared the reprogramming efficiency of Fanca-/- and wt fibroblast that were derived concurrently in normoxic (21% O₂) or hypoxic (5% O₂) conditions. In both Fanca-/- and wt cells, we observed a significant increase of the reprogramming efficiency under hypoxic conditions (p=0.0098 and p=0.0462, respectively). Complementation of Fanca-/- fibroblasts with the FANCA gene in combination with reprogramming under hypoxic conditions led to a significant rescue of the reprogramming efficiency (p=0.0109, Table 2). This correlated with a significant reduction in senescence and a trend towards decreased dsDNA breaks. These data indicate that oxidative DNA damage engages the FA signaling pathway during the reprogramming process, and acts as a negative physiologic regulator of reprogramming in Fanca-/- cells. Our study implicates the FA pathway as essential to the repair of dsDNA breaks that are induced during the reprogramming process, and provides a plausible mechanism for the reduced efficiency of reprogramming in Fanca-/- cells.

Table 1:

Increased dsDNA breaks (γH2AX foci) and senescence (β-galactosidase) in Fanca-/- fibroblasts four days post infection with reprogramming viruses.

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