American Society of Hematology

Figure 6

$Figure 6. ATR inhibition synergizes with existing therapeutic agents both in ATM-defective and TP53-defective primary CLL cells. CFSE-labeled primary CLL cells with ATM defect (CLL22, CLL23, CLL24) (A) or TP53 defect (CLL29, CLL31, CLL32) (B) cocultured with CD40L/IL-21 were treated with chlorambucil, fludarabine, 4HC, or ibrutinib with or without coadministration of AZD6738 (1 μM). Viability was assessed after 96 hours by propidium iodide exclusion of the proliferating cell population as identified by reduction in CFSE fluorescence intensity. Surviving fraction is expressed relative to untreated controls for chemotherapy treatment alone (no AZD6738) and relative to 1 μM AZD6738 monotherapy for the cotreated samples. Addition of AZD6738 significantly enhanced sensitivity of ATM-defective primary CLL samples to chlorambucil and 4HC, and TP53-defective primary CLL samples to these therapies and also to fludarabine and ibrutinib at ≥1 dose combination. Data are displayed as mean ± SEM. Statistical significance was determined using 2-way ANOVA with Bonferroni post hoc analysis. Statistical significance vs no AZD6738 is indicated by *P < .05, **P < .01, and ***P < .001. (C-D) AZD6738 is synergistic with chlorambucil, fludarabine, 4HC, and ibrutinib in primary CLL samples with ATM (C) or TP53 (D) defect across a range of effective drug doses. CI values were calculated using the median-effect method. Each point represents the mean CI value of 3 samples plotted against the corresponding mean affected fraction that is expressed relative to untreated controls. CI <0.9 represents synergism, CI = 0.9-1.1 represents additive effect, and CI >1.1 represents antagonism. The actual values are presented in Table 2. (E-F) A primary CLL xenograft (CLL25) with a biallelic ATM defect (del(11q) and 1407I>T ATM mutation) was randomized into 4 treatment arms (n = 5 each): AZD6738, chlorambucil, AZD6738-chlorambucil cotreatment, and vehicle. AZD6738 treatment alone or in combination with chlorambucil significantly reduced tumor load relative to vehicle, and the addition of AZD6738 to chlorambucil led to a significantly greater reduction in tumor load relative to chlorambucil monotherapy. The relative number of CLL cells in panel F was normalized to vehicle-treated controls. Data are displayed as mean ± SEM. Statistical significance was determined using 2-way ANOVA with Bonferroni post hoc analysis and is indicated by *P < .05, **P < .01, and ***P < .001.$

ATR inhibition synergizes with existing therapeutic agents both in ATM-defective and TP53-defective primary CLL cells. CFSE-labeled primary CLL cells with ATM defect (CLL22, CLL23, CLL24) (A) or TP53 defect (CLL29, CLL31, CLL32) (B) cocultured with CD40L/IL-21 were treated with chlorambucil, fludarabine, 4HC, or ibrutinib with or without coadministration of AZD6738 (1 μM). Viability was assessed after 96 hours by propidium iodide exclusion of the proliferating cell population as identified by reduction in CFSE fluorescence intensity. Surviving fraction is expressed relative to untreated controls for chemotherapy treatment alone (no AZD6738) and relative to 1 μM AZD6738 monotherapy for the cotreated samples. Addition of AZD6738 significantly enhanced sensitivity of ATM-defective primary CLL samples to chlorambucil and 4HC, and TP53-defective primary CLL samples to these therapies and also to fludarabine and ibrutinib at ≥1 dose combination. Data are displayed as mean ± SEM. Statistical significance was determined using 2-way ANOVA with Bonferroni post hoc analysis. Statistical significance vs no AZD6738 is indicated by *P < .05, **P < .01, and ***P < .001. (C-D) AZD6738 is synergistic with chlorambucil, fludarabine, 4HC, and ibrutinib in primary CLL samples with ATM (C) or TP53 (D) defect across a range of effective drug doses. CI values were calculated using the median-effect method. Each point represents the mean CI value of 3 samples plotted against the corresponding mean affected fraction that is expressed relative to untreated controls. CI <0.9 represents synergism, CI = 0.9-1.1 represents additive effect, and CI >1.1 represents antagonism. The actual values are presented in Table 2. (E-F) A primary CLL xenograft (CLL25) with a biallelic ATM defect (del(11q) and 1407I>T ATM mutation) was randomized into 4 treatment arms (n = 5 each): AZD6738, chlorambucil, AZD6738-chlorambucil cotreatment, and vehicle. AZD6738 treatment alone or in combination with chlorambucil significantly reduced tumor load relative to vehicle, and the addition of AZD6738 to chlorambucil led to a significantly greater reduction in tumor load relative to chlorambucil monotherapy. The relative number of CLL cells in panel F was normalized to vehicle-treated controls. Data are displayed as mean ± SEM. Statistical significance was determined using 2-way ANOVA with Bonferroni post hoc analysis and is indicated by *P < .05, **P < .01, and ***P < .001.

This Feature Is Available To Subscribers Only