Figure 6.
Evaluation of activity of BCR and CDK4/6 inhibitors against Eμ-TCL1 leukemia cells with combined TP53/CDKN2A/2B deficiency. (A) Effects of ibrutinib, R406, and idelalisib on proliferation of unstimulated or CpG-stimulated TCL1-355 leukemia cells with biallelic TP53/CDKN2A/2B disruption. Cells were cultured for 6 hours with BrdU prior to harvesting for flow cytometry analysis. All inhibitors were used at 1 μM concentration. Statistical analysis was performed using 1-way repeated measures analysis of variance (ANOVA) with the Holm-Šidak test for multiple comparisons. (B) Immunoblotting analysis of CDK4 and CDK6 protein expression in 3 normal splenic B-cell samples (NB1-NB3), the 3 CDKN2A/2B/TP53-ko leukemias (TCL1-355-TKO, TCL1-863-TKO, and TCL1-699-TKO), and their non-CDKN2A/2B/TP53–aberrated counterparts (TCL1-355, TCL1-863, TCL1-699). (C) Effects of palbociclib (Pal.) on proliferation of unstimulated or CpG-stimulated TCL1-355 leukemia cells with biallelic TP53/CDKN2A/2B disruption. Cells were cultured with BrdU for 20 hours prior to harvesting. Statistical analysis was performed using the paired Student t test. (D) Analysis of effects of palbociclib in combination with ibrutinib or idelalisib on proliferation of CpG-stimulated TCL1-355 leukemia cells with biallelic TP53/CDKN2A/2B disruption. The combination index (CI) was calculated using CompuSyn software (Paramus, NJ) according to the Chou-Talalay method.61 Mean values from 3 technical replicates were used in the equation. CI values below 1.0 indicate synergistic activity. (E) A total of 1 × 107 TCL1-355 TP53/CDKN2A/2B-ko cells were engrafted in C57BL/6 mice via intraperitoneal injection and randomly assigned to each of the 4 experimental cohorts. Treatment with palbociclib, ibrutinib, or palbociclib plus ibrutinib was started 3 days later. The number of leukemic cells in peripheral blood after 10 days of treatment is shown. (F) Survival analysis of mice treated with palbociclib, ibrutinib, or palbociclib plus ibrutinib. The x-axis indicates days from adoptive transfer. Survival curves were estimated using the Kaplan-Meier method and curve differences among groups were assessed using the log-rank test and the Holm-Šidak method for multiple comparisons. Ibr, ibrutinib; Idel, idelalisib; Unt, untreated.

Evaluation of activity of BCR and CDK4/6 inhibitors against Eμ-TCL1 leukemia cells with combined TP53/CDKN2A/2B deficiency. (A) Effects of ibrutinib, R406, and idelalisib on proliferation of unstimulated or CpG-stimulated TCL1-355 leukemia cells with biallelic TP53/CDKN2A/2B disruption. Cells were cultured for 6 hours with BrdU prior to harvesting for flow cytometry analysis. All inhibitors were used at 1 μM concentration. Statistical analysis was performed using 1-way repeated measures analysis of variance (ANOVA) with the Holm-Šidak test for multiple comparisons. (B) Immunoblotting analysis of CDK4 and CDK6 protein expression in 3 normal splenic B-cell samples (NB1-NB3), the 3 CDKN2A/2B/TP53-ko leukemias (TCL1-355-TKO, TCL1-863-TKO, and TCL1-699-TKO), and their non-CDKN2A/2B/TP53–aberrated counterparts (TCL1-355, TCL1-863, TCL1-699). (C) Effects of palbociclib (Pal.) on proliferation of unstimulated or CpG-stimulated TCL1-355 leukemia cells with biallelic TP53/CDKN2A/2B disruption. Cells were cultured with BrdU for 20 hours prior to harvesting. Statistical analysis was performed using the paired Student t test. (D) Analysis of effects of palbociclib in combination with ibrutinib or idelalisib on proliferation of CpG-stimulated TCL1-355 leukemia cells with biallelic TP53/CDKN2A/2B disruption. The combination index (CI) was calculated using CompuSyn software (Paramus, NJ) according to the Chou-Talalay method.61 Mean values from 3 technical replicates were used in the equation. CI values below 1.0 indicate synergistic activity. (E) A total of 1 × 107 TCL1-355 TP53/CDKN2A/2B-ko cells were engrafted in C57BL/6 mice via intraperitoneal injection and randomly assigned to each of the 4 experimental cohorts. Treatment with palbociclib, ibrutinib, or palbociclib plus ibrutinib was started 3 days later. The number of leukemic cells in peripheral blood after 10 days of treatment is shown. (F) Survival analysis of mice treated with palbociclib, ibrutinib, or palbociclib plus ibrutinib. The x-axis indicates days from adoptive transfer. Survival curves were estimated using the Kaplan-Meier method and curve differences among groups were assessed using the log-rank test and the Holm-Šidak method for multiple comparisons. Ibr, ibrutinib; Idel, idelalisib; Unt, untreated.

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