Figure 5.

In DLL4-expressing T-ALL, demcizumab treatment showed the same therapeutic effect as γ-secretase inhibition. A) Normalized TPM expression levels of DLL4 in 264 T-ALL patient samples (Mullighan dataset) in decreasing order. Samples with the highest expression level (z score > 2) are highlighted in red (top 2.6%). The dashed blue horizontal line indicates the mean expression level, and the solid blue horizontal line indicates the median expression level across all samples. B) Analysis of DLL4 expression by western blotting in four different patient-derived T-ALL (PDTALL). C) The spleens of moribund NRG mice harboring different PDTALL cells were homogenized and stained. Cells were gated negatively (DAPI) and positively for CD45 expression, and analyzed for the expression of human DLL4 or the isotype IgG control. PDTALLs were expanded in mice before performing the in vitro assays presented in (D), (E) and (F). D) Analysis of HES1 and DTX1 mRNA expression in the indicated PDTALL cells incubated with vehicle (control), 20 µg/ml of murine anti-DLL4 antibody, 20 µg/ml of human anti-DLL4 antibody (demcizumab), or 500 nM of DBZ for 48 h. Data (treatments vs control) were compared by one-way ANOVA for each PDTALL, n = 3 each condition. E) Viability of PDTALL9 (upper), 13 (middle) and 19 (low) cells incubated with the indicated doses of murine anti-DLL4 antibody, human anti-DLL4 antibody (demcizumab), or DBZ for 1, 2 and 3 days. Cells were gated using a human anti-CD45 antibody, and cell death analyzed by DAPI staining. Data (treatments vs control) were compared by two-way ANOVA for each day; n = 2 per condition. F) Apoptosis analyses of PDTALL9 (upper), 13 (middle) and 19 cells (lower) performed by flow cytometry after staining with AnnexinV and DAPI following incubation with vehicle (control), murine anti-DLL4 antibody, human anti-DLL4 antibody, or DBZ as in (D). Data (treatments vs control) were compared by two-way ANOVA, n = 3 per condition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.