Figure 2.

Silmitasertib enhances venetoclax-induced apoptosis via the GSK3B-MCL1 axis, and combined treatment is effective in a zebrafish xenograft model. (A) The association of MCL1 dependence assessed by basal BH3 profiling with venetoclax sensitivity. Patient-derived xenograft BCP-ALL samples were exposed ex vivo to the MS1 peptide specifically bound to MCL1, to assess MCL1 dependence (basal BH3 profiling). Spearman’s correlation was used to analyze correlation between venetoclax sensitivity and priming for MS1 (MCL1 dependence). Linear regression and 95% confidence intervals are shown. (B) BCP-ALL cell lines showing low or high venetoclax sensitivity were exposed (4 hours) to venetoclax at their corresponding EC₁₀ concentrations (697, 27 nM; venetoclax/HAL-01, 2 nM venetoclax) followed by exposure to increasing concentrations of the MCL1-specific inhibitor peptide MS1 or vehicle control (1 hour) before cytochrome C release was measured by fluorescence-activated cell sorting (dynamic BH3 profiling). The difference of cytochrome C release between venetoclax and vehicle treatment (Delta priming) is shown for the MS1 peptide. Supplemental Figure 5 depicts raw values for this experiment. Means ± standard deviation (SD) from 3 independent experiments performed in triplicate are shown. ***P < .001. (C) Western blot analysis performed with the indicated antibodies, on lysates of 697 and SEM cells treated for 6 hours with vehicle (dimethylsulfoxide), 5 µM silmitasertib, and/or 0.05 µM venetoclax. HSP60 was used as the loading control. MCL1 band intensity is quantified in supplemental Figure 6A. Further data on time and concentration dependent changes in MCL1 are shown in Supplemental Figures 6B-D and 7A-B. (D) Combination effects on viability of NALM-6 wild-type cells, NALM-6 cells transduced with a lentiviral human MCL1 expression vector, and NALM-6 cells with complete knockout of GSK3B induced by CRISPR/Cas9 genome editing. Cells were analyzed with a WST-1 assay after a 48-hour treatment with serial dilutions of single and combined compounds, similar to Figure 1B. Combination effects were determined by a Loewe synergy model (Combenefit software) integrating the means of duplicates from 3 independent experiments for NALM-6 wild-type and MCL1-overexpressing cells and integrating data from 4 single-cell clones in NALM-6 GSK3B KO cells (clones c.1, c.2, c.5, and c.6; supplemental Figure 9B). NALM-6 wild-type cells showed 31 synergistic treatment combinations and a sum of synergy scores of 96.4, compared with 9 synergistic treatment combinations, in both MCL1 and GSK3B KO clones (corresponding synergy scores: 17.9 and 19.6). Each data point represents 1 drug or combination. (E) NALM-6 cells (stably expressing eGFP) were transduced with a lentiviral human MCL1 expression vector or empty vector (negative control) before treatment with 5 µM silmitasertib and/or 0.5 µM venetoclax for 48 hours. ANXA5 staining was assessed by flow cytometry to determine apoptosis. Data are shown as the mean ± SD. P values from 5 independent experiments were determined by Mann-Whitney U test. (F) SEM cells were engrafted into the pericardium in 2-day immunosuppressed zebrafish embryos, which were bathed in 1 µM venetoclax and/or 1 µM silmitasertib (tolerated dose) with 5 µM silmitasertib injected to the pericardium. Apoptosis was assessed after 72 hours. Each experiment (n = 5) was measured as the mean of a pool of 12 PDX embryos. Bars represent means of each experiment ± SD. Mann-Whitney U test. (G) Proposed mechanism by which silmitasertib sensitizes ALL cells to venetoclax. *P < .05; **P < .01; ***P < .001.