Figure 3.
MCL1 inhibition (S63845) cannot eliminate BAX-deficient VEN-resistant cells. (A) Top: mean IC50 for S63845 of 9 VEN-sensitive (blue) and VEN-resistant cell lines (red) determined by flow cytometry after 48 hours. N ≥3. Lower part: heat map with relative BAX and MCL1 protein level in VEN-resistant cell lines. (B) Validation of BAX KO in OSU cells by immunoblot. N = 3. (C) Sensitivity of OSU KO cells toward VEN determined by flow cytometry after VEN treatment for 48 hours. Mean ± SD of 4 experiments. (D) Allelic fraction of TP53 (c.515T>A) and BAX (c.361del) before and after VEN treatment (24 hours, 5 nM) in a high-risk CLL patient. Mean plus SD, 3 technical replicates; P = .0061 (E) Viability of purified, malignant splenic B cells of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red) and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple) mice treated with VEN, S63845 (24 hours), or fludarabine (48 hours), determined by MTT assays. Mean ± SD of 2 experiments; 3 technical replicates each. (F) Immunophenotyping of splenocytes of a 50-week-old Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl mouse. Gating strategy of viable, single cells on FSC/SSC dot plot and FSC-area/FSC-height dot plot. Analysis for Cd45, Cd19, IgM, and IgD expression. (G) Spleen weight and length of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue; n = 6), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red; n = 6), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple; n = 5) mice. Mean ± SD. ∗P < .05, compared with Bax wild-type mice, Student t test. (H) Determination of the amount of Cd19+/B220dim/neg-positive cells in the blood (left panel; 32-week-old animals; n = 5, n = 8, and n = 2, respectively) and splenocytes (right panel; 44 weeks old animals; n = 4, n = 5, and n = 4, respectively) of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple) mice. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001, compared with Bax wild-type mice, Student t test. (I) Kaplan-Meier curves of overall survival of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue; 48 weeks; n = 14), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red; 56 weeks; n = 7), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple; 59.5 weeks; n = 6) mice. Survival of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple) compared with the respective controls (log-rank test; P = .0914). FS, forward scatter integral; FSC, forward scatter; IgM, immunoglobulin M; INT, integral; KO, knockout; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid; ns, not significant; SSC, side scatter integral.

MCL1 inhibition (S63845) cannot eliminate BAX-deficient VEN-resistant cells. (A) Top: mean IC50 for S63845 of 9 VEN-sensitive (blue) and VEN-resistant cell lines (red) determined by flow cytometry after 48 hours. N ≥3. Lower part: heat map with relative BAX and MCL1 protein level in VEN-resistant cell lines. (B) Validation of BAX KO in OSU cells by immunoblot. N = 3. (C) Sensitivity of OSU KO cells toward VEN determined by flow cytometry after VEN treatment for 48 hours. Mean ± SD of 4 experiments. (D) Allelic fraction of TP53 (c.515T>A) and BAX (c.361del) before and after VEN treatment (24 hours, 5 nM) in a high-risk CLL patient. Mean plus SD, 3 technical replicates; P = .0061 (E) Viability of purified, malignant splenic B cells of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red) and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple) mice treated with VEN, S63845 (24 hours), or fludarabine (48 hours), determined by MTT assays. Mean ± SD of 2 experiments; 3 technical replicates each. (F) Immunophenotyping of splenocytes of a 50-week-old Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl mouse. Gating strategy of viable, single cells on FSC/SSC dot plot and FSC-area/FSC-height dot plot. Analysis for Cd45, Cd19, IgM, and IgD expression. (G) Spleen weight and length of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue; n = 6), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red; n = 6), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple; n = 5) mice. Mean ± SD. ∗P < .05, compared with Bax wild-type mice, Student t test. (H) Determination of the amount of Cd19+/B220dim/neg-positive cells in the blood (left panel; 32-week-old animals; n = 5, n = 8, and n = 2, respectively) and splenocytes (right panel; 44 weeks old animals; n = 4, n = 5, and n = 4, respectively) of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple) mice. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001, compared with Bax wild-type mice, Student t test. (I) Kaplan-Meier curves of overall survival of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxwt/wt (blue; 48 weeks; n = 14), Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red; 56 weeks; n = 7), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple; 59.5 weeks; n = 6) mice. Survival of Eμ-TCL1tg/wt; Cd19Cre+/wt; Baxfl/wt (red), and Eμ-TCL1tg; Cd19Cre+/wt; Baxfl/fl (purple) compared with the respective controls (log-rank test; P = .0914). FS, forward scatter integral; FSC, forward scatter; IgM, immunoglobulin M; INT, integral; KO, knockout; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid; ns, not significant; SSC, side scatter integral.

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