Focused chemical genomics using zebrafish xenotransplantation as a pre-clinical therapeutic platform for T-cell acute lymphoblastic leukemia

Abstract

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient's cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to γ-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time.

Figure 1.

T-cell acute lymphoblastic leukemia cell lines harboring defined mutation in NOTCH and PTEN have differential responses in vitro and in vivo to inhibition of Notch (Compound E), AKT (triciribine), and mTOR (rapamycin). (A) In vitro cell viability assay. Cell lines were treated with drug or vehicle (0.01% DMSO) for 72 h, and cell viability determined (via alamarBlue) as a percentage of vehicle control. (B) Schematic of the in vivo zebrafish XT cell proliferation assay. (C) Representative brightfield and fluorescent and magnified fluorescent images (from left to right) of zebrafish embryos transplanted with CM-DiI-labeled T-ALL cell lines at 0 hpt (baseline = 48 hpi = 96 hpf) and 48 hpt (96 hpi = 144 hpf) with or without drug. (D) In vivo proliferation of T-ALL cell lines in the zebrafish XT model. Groups of 15–20 embryos were used for each time point and treatment, 50–100 cells were injected per fish, and the number of fluorescent cells was enumerated as described in Corkery et al. Base-line number of cells was determined at 0 hpt and all drug treatments (at 48 hpt) are shown as a fold change of the baseline. All cell lines engrafted and proliferated in the zebrafish, as represented by the significant increase in number of leukemia cells from baseline to 48hpt with vehicle. Means+SEM; n=3; P*<0.05, P**<0.01, P***<0.001 for significant decrease in number of cells determined using one-way ANOVA followed by Dunnett’s multiple comparison test. N: number of independent experiments, with 15–20 embryos per group per experiment. Hpt: hours post treatment; hpi: hours post injection. Scale bars are 500 μM.

Figure 2.

T-cell acute lymphoblastic leukemia patient Sample 1 responds in vivo to Notch inhibition with compound E, but not to mTOR inhibition with rapamycin. (A) Representative brightfield and fluorescent and magnified fluorescent images (from left to right) of zebrafish embryos transplanted with patient Samples 1 and 2 at 0 hpt (baseline) and 72 hpt with or without drug. Each embryo was xenotransplanted with approximately 500 cells. (B) Schematic of in vivo zebrafish XT cell proliferation assay. (C) Quantification of patient sample engraftment fold change with or without compound E treatment. Patient Sample 1 and 2 engrafted and proliferated in the zebrafish XT model (indicated by the significant increase in number of leukemia cells from baseline to 48 hpt with vehicle). Patient Sample 1 responded significantly to compound E treatment. Engraftment fold change was determined by sacrificing embryos at 0 hpt and 48 hpt and performing cytospins with dissociated embryos and immunohistochemistry for PML bodies (only present in human cells, not in zebrafish cells). Means+SEM; N: 1; P*<0.05, P**<0.01, P***<0.001 for significant decrease in number of cells determined using unpaired 2-tailed Student’s t-test. Groups of 15–20 embryos were used for each time point and drug treatment. Scale bars are 500 μM.

Figure 3.

Patient Sample 1 harbors a rare NOTCH1 mutation. (A) Results from Sanger sequencing of patient samples. Patient Sample 1 (PS 1) had a heterozygous mutation in the heterodimerization (HD) domain of the NOTCH1 gene (p.A1696D). Patient Sample 2 (PS 2) did not have any mutations in the genes sequenced. (B) p.A1696D mutated NOTCH1 up-regulates luciferase reporter activity. HeLa cells transfected with JH23a Notch reporter plasmid, Renilla, and+Notch with A1696A/D or the PEST mutation. P.A1696D mutated Notch plasmid up-regulated luciferase reporter activity to a similar degree as constitutively active Notch (with PEST mutation). Means+SEM; N=3; N: biological replicates.