Abstract
IntroductionT cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy that originates from malignant transformation of T cell progenitors. It accounts for 15% of paediatric and 25% of adult ALLs. Most frequent genetic alterations associated with this neoplasia are activating mutations in NOTCH1 and loss of function mutation for FBXW7 and PTEN. Current chemotherapeutic approaches demonstrate good cure and survival rates in patients. However prognosis of relapsed patients is still dismal. Therefore there is an urgent need to identify new targets for the development of more effective therapeutic compounds.Material and methodsCRISPR/Cas9 editing technology was used to generate mosaic mutant animals in Xenopus tropicalis, a true diploid amphibian. sgRNAs were designed to induce activating (i.e. truncating) mutations in notch1* and loss-of-function mutations in fbxw7 and pten. Cas9 recombinant protein and different sgRNA combinations were injected in 4 cell stage embryos. Animals were raised for 6 weeks. Liver, spleen, kidneys and thymus were dissected and further processed.Results and discussionsCombined injections of sgRNAs that targeted notch1 and pten resulted in mosaic mutant animals that exhibited pale appearance, enlarged spleen and haemorrhagic hindlimbs. Furthermore, sudden unexplained full penetrant mortality was observed around the time of metamorphosis. From diseased animals, genomic DNA was extracted from dissected thymi and the targeted gene loci were PCR-amplified and deep sequenced. Interestingly, several thymi showed clonal enrichment of a single cell population with a unique INDEL mutation pattern in one notch1 allele and two pten alleles, indicative of a leukemic cell population in the mosaic mutant animals. Exploiting a concept of clonal enrichment and negative selection for mutations in genes essential for T-ALL formation, we are using multiplexed gene inactivation to identify T-ALL dependency factors and hence explore novel routes for targeted therapy. We are currently verifying the feasibility of this experimental concept by multiplexed injections of pten and notch1 together with myc. Furthermore, we are validating our model by conducting flow cytometric and immunohistochemical analysis of the blood.ConclusionWe generated a genetic T–ALL model in X. tropicalis by co-targeting of notch1* and pten. We expect this model to provide new opportunities for identification and validation of novel driver genes and dependency factors for T-ALL.
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