Abstract
To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. After ENU mutagenesis, genetic lesions associated with failure of T cell development were identified by meiotic recombination mapping, positional cloning, and whole genome sequencing. Recessive genetic variants in 33 genes were identified and confirmed as causative by additional experiments. The mutations affected T cell development but did not perturb the development of an unrelated cell type, growth hormone-expressing somatotrophs, providing an important measure of cell-type specificity of the genetic variants. The structure of the genetic network encompassing the identified components was established by a subsequent genetic interaction analysis, which identified many instances of positive (alleviating) and negative (synthetic) genetic interactions. Several examples of synthetic lethality were subsequently phenocopied using combinations of small molecule inhibitors. These drugs not only interfered with normal T cell development, but also elicited remission in a model of T cell acute lymphoblastic leukaemia. Our findings illustrate how genetic interaction data obtained in the context of entire organisms can be exploited for targeted interference with specific cell types and their malignant derivatives.
Highlights
To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis
Inhibiting the activities of genes highly expressed during early T cell differentiation, such as BCL-2 or JAK1/JAK2, significantly reduced leukaemic burden in mouse T-cell acute lymphoblastic leukaemia (T-ALL) xenografts, regardless of their mutation status[15,16,17], suggesting that interference with the function of genes expressed in a tissue-specific or tissue-restricted fashion offers an opportunity for targeted tumour therapy
To establish the key nodes in the genetic network regulating the differentiation of immature T cells, we conducted two forward ENU mutagenesis screens[18,19] to identify recessive genes regulating developing T cells in the thymus of zebrafish in an unbiased manner
Summary
To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. Genetic interaction is said to occur when the phenotype of a double-mutant organism deviates from the expected neutral phenotype It can be positive (alleviating), when the phenotype is less severe than expected, or negative (synthetic) when the combination of two individually benign gene mutations into a single genetic background results in a more severe phenotype, such as loss of cell viability[10]. The latter outcome is attractive from the viewpoint of cancer therapy In this context, synthetic lethality screens seek to identify and perturb genes that are required for the survival of a target cancer cell carrying a specific oncogenic mutation, as exemplified by the success of small molecule PARP inhibitors in patients with BRCA1-deficient tumours[11]. We delineated the genetic network underlying the development of T cells in zebrafish, established the nature of synthetic lethal interactions, and exploited this information for combinatory inhibitor treatments that proved effective in preventing tumour progression in an in vivo model of T-ALL
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