A Model of Clonal Evolution and Myelodysplasia (MDS) on Mice with Genetic Disruption of Both Fancc and Fancg.
Authors: Ciccone Samantha , Pulliam Anna , Li Xiaohong , Si Yue , Orazi Attilio , Bagby Grover C. , Clapp D. Wade 
Published: October 2019

Abstract
Fanconi anemia (FA) is a rare inherited chromosomal instability syndrome characterized by bone marrow failure and a high relative risk of MDS. Eight FA proteins associate in a core nuclear complex and function at least in part to catalyze the monoubiquitination of the downstream target protein, FANCD2 in response to DNA damage. In this nuclear pathway the FA proteins are epistatic in the activation of FANCD2 since inactivation of any one of the eight FA proteins results in failure of FANCD2 monoubiquitination and hypersensitivity to cross-linking agents. Although biochemical studies have attributed additional survival signaling functions to the FA proteins, these functions have not been evaluated using a genetic model. Murine models of FA have been established using homologous recombination for gene disruption. Although all strains of knockout mice are hypersensitive to mitomycin c, none of the single gene knockout mice display bone marrow failure, MDS, or myeloid leukemia. Seeking to develop such a model, we utilized a genetic intercross to generate mice that harbor disruptions in both Fancc and Fancg. Genetic disruption of both Fancc and Fancg predispose Fancc−/−;Fancg −/− mice or recipients adoptively transferred with Fancc −/−; Fancg −/− hematopoietic stem cells to MDS analogous to the disease phenotype in FA patients as defined histologically and by cytogenetic analysis. Genome wide transcriptomal analysis and hierarchical clustering by genotypic group of bone marrow cells from wild type, single knockout, and double knockout mice (n=3 each) confirmed substantial differences between hematopoietic cells of Fancc, Fancg and double knockout (DKO) mice. Serial pairwise analysis and gene pattern analyses (GeneSifter) showed that of the 1190 genes expressed differentially (by a factor of >1.5, FDR adjusted p<0.05) in Fancc−/− marrow cells only 134 were differentially expressed in Fancg−/− cells. Of the 524 genes expressed differentially in Fancg−/− marrow compared to WT, 277 were not expressed differentially in Fancc−/− marrow compared to WT. In pairwise analysis of Fancc−/− vs. Fancg−/− gene expression, ontologies of those genes more highly expressed in Fancc −/− cells included responses to biotic stress, defense and immune response. The most over-represented ontological category of those genes more highly expressed in Fancg−/− cells was response to oxidative stress. Since these genes are not epistatic in regards to the hematopoietic phenotype, and the transcriptomal consequences of their loss-of-function in marrow cells are significantly different, this genetic model confirms that the Fancc and Fancg proteins are multi-functional. Transcriptosomal analyses were conducted on DKO mice that contained MDS and DKO mice with no overt disease. The transcriptome of DKO marrow cells was unique in that 152 suppressed and 687 activated gene products relative to WT samples were not found in either Fancc−/− or Fancg−/− samples. Furthermore, there are distinct transcriptomal differences between the DKO mice with MDS and those that do not have MDS. These data suggest that some of these changes may be adaptive and involved in the molecular pathogenesis of MDS. The DKO model provides the first preclinical platform to systematically evaluate the molecular pathogenesis of bone marrow failure and myelodysplasia in the setting of Fanconi anemia.