Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

Common and overlapping oncogenic pathways contribute to the evolution of acute myeloid leukemias.

Fusion oncogenes in acute myeloid leukemia (AML) promote self-renewal from committed progenitors, thereby linking transformation and self-renewal pathways. Like most cancers, AML is a genetically and biologically heterogeneous disease, but it is unclear whether transformation results from common or overlapping genetic programs acting downstream of multiple mutations or by the engagement of unique genetic programs acting cooperatively downstream of individual mutations. This distinction is important, because the involvement of common programs would imply the existence of common molecular targets to treat AML, no matter which oncogenes are involved. Here we show that the ability to promote self-renewal is a generalized property of leukemia-associated oncogenes. Disparate oncogenes initiated overlapping transformation and self-renewal gene expression programs, the common elements of which were defined in established leukemic stem cells from an animal model as well as from a large cohort of patients with differing AML subtypes, where they strongly predicted pathobiological character. Notably, individual genes commonly activated in these programs could partially phenocopy the self-renewal function of leukemia-associated oncogenes in committed murine progenitors. Furthermore, they could generate AML following expression in murine bone marrow. In summary, our findings reveal the operation of common programs of self-renewal and transformation downstream of leukemia-associated oncogenes, suggesting that mechanistically common therapeutic approaches to AML are likely to be possible, regardless of the identity of the driver oncogene involved.

Germ line gain of function with SOS1 mutation in hereditary gingival fibromatosis.

Mutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G(1) to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans.