Article Figures & Data
Figures
- Figure 1.
PDAC sensitivity to AZD6738 monotherapy. A, Frequency of alterations in genes reported to confer sensitivity to ATR inhibition, from the Cancer Genome Atlas cohort of PDAC samples (http://www.cbioportal.org). B and C, Human (B) and mouse (C) PDAC cell lines were grown in increasing concentrations of AZD6738 for 3 days to generate dose–response curves using the SRB assay. The GR metrics R package (developed by Clarke and colleagues, 2017) was used to generate normalized growth rate inhibition (GR) values, thus controlling for the different doubling times of the cell lines. Drug concentrations that bring the GR value to zero are cytostatic, whereas negative GR values represent cytotoxicity. Each point represents the mean of three independent experiments ± SEM. D and E, Comparison of GR50s for AZD6738 alone or in combination with low dose of gemcitabine [5 nmol/L for the most sensitive mouse PDAC lines (D) or 10 nmol/L for KPCFT79653 and human lines (E)]. Data are represented as mean ± SEM. Multiple t tests were performed and statistical significance determined using the Holm–Sidak method; **, P ≤ 0.01; ***, P ≤ 0.001, and ns, P > 0.05.
- Figure 2.
AZD6738 abrogates the gemcitabine-induced checkpoint activation. A, Western blotting for K8484 cells treated as indicated for 7 hours. B, Western blotting for MiaPaCa-2 cells treated as indicated for 24 hours. C, Western blotting for MiaPaCa-2 and Panc-1 cells treated as indicated for 30 hours. D, MiaPaCa-2 cells were treated with scramble or ATM-specific siRNA. After 24 hours, cells were treated as indicated for 24 hours, and lysates were immunoblotted. E, Western blotting for MiaPaCa-2 and Panc-1 cells treated as indicated for 30 hours. Arrowheads indicate the bands of interest. Note that the upper band in the pChk1-S345 blots appearing in lysates of MiaPaCa-2 treated with the combination is most likely the result of a cross-reaction with p-Chk2.
- Figure 3.
AZD6738 and gemcitabine synergistically inhibit cell growth in a panel of PDAC cell lines. A, MiaPaCa-2 and Panc-1 cells were treated with AZD6738 and gemcitabine in an 8 × 8 concentration grid for 72 hours. Cell viability was determined by measuring the total protein content using the sulforhodamine B assay. The experimental data (left, values are percentage growth inhibition compared with control) were analyzed independently with the two synergy models (Bliss and Loewe) using the Combenefit software. Data, mean ± SD, n = 3. B and C, MiaPaCa-2 cells were treated with AZD6738 and gemcitabine in a 6 × 8 concentration grid for 24 hours. The drugs were washed out and replaced with fresh medium containing the YOYO-3 Iodide cell-impermeant dye to follow cell death. Three random fields per sample were imaged by time lapse microscopy every 3 hours for 4 days. Cell growth was analyzed using cell confluency data and expressed as percentage of growth inhibition compared with control (B, left plot) and Combenefit software for synergy analyses (B, 2 right plots). Growth curves (C, left plot) and cell death accumulation (C, right plot) from control cells or cells treated with indicated concentrations of AZD6738 and gemcitabine. Data, mean ± SEM, and n = 2. D, Clonogenic survival of Panc-1 and MiaPaCa-2 cells plated at very low density and exposed to the indicated drug combinations for 24 hours before washout. Cells were left to grow for 8 days after washout. Data, mean ± SD, n = 3.
- Figure 4.
Scheduling of the combination of AZD6738 with gemcitabine. A, Four cell lines from the KPC mouse model were plated at low density and treated with the first condition for 16 hours, then the medium was replaced with the second condition and cell growth was monitored by time lapse microscopy every 3 hours for 4 days. Drugs were used at the GI50 (72 hours) concentrations. B, Cells were incubated with 10 nmol/L gemcitabine for 16 hours and then fixed at the time point shown after washout into fresh medium or with 300 nmol/L ATRi. Immunofluorescence followed by quantitative microscopy was realized to obtain the percentage of cells positive for γH2AX. C, Dose-schedule schematic (left) of the in silico modeling of AZD6738 mouse PK for the gut, circulating, and peripheral compartments (right).
- Figure 5.
The combination of AZD6738 + gemcitabine achieves antitumor effect and improves survival, in a subcutaneous allograft of a KPC cancer cell line. A, Tumor volume of K8484 allografts. Ten mice per group were treated as indicated in the legend (middle of the figure) for 3 consecutive weekly cycles (see dose/schedule schematic in Fig. 4C). After a 1-week break, treatment was resumed for another 3 weeks. Data are represented as mean ± SEM. +, Culled animals. B, Changes in individual tumor volume between D14 and D25 (when all mice were still alive). C, Changes in individual tumor volume between D46 and D60 (only 2 mice remaining in the gemcitabine alone group). D, Kaplan–Meier survival curves. E, Body weight change relative to pretreatment body weight. Data are represented as mean ± SEM. F, Quantification of γH2AX-positive cells from tumors IHC. Data are represented as mean ± SD. A one-way ANOVA analysis with Tukey pairwise comparisons was performed; **, P ≤ 0.01; ****, P ≤ 0.0001, and ns, P > 0.05.
- Figure 6.
The combination of AZD6738 + gemcitabine induces tumor regression in a subgroup of KPC tumors. A, Schematic of the study and dosing schedule. B, Changes in individual tumor long diameter between day –1 and day 12. Dotted lines represent the mean of each group. A one-way ANOVA analysis was performed with Tukey pairwise comparisons; **, P ≤ 0.01 and ns, P > 0.05. During the 12-day study, in the gemcitabine-alone group, 2 mice had a “stable” (<10% diameter change) disease, whereas 16 had a “progressive” (>10% diameter change) disease. In the combination group, 4 mice had a stable disease, whereas 6 had a progressive disease. If the probability to have a stable disease in the combination group is the same as in the gemcitabine alone group (i.e., 2/18), we would expect to observe 4 or more stable disease out of 10 animals only 1.84% of the time (P = 0.018). C, Quantification of γH2AX-positive cells from tumors IHC. Data are represented as mean ± SD. A one-way ANOVA analysis with Tukey pairwise comparisons was performed; ***, P ≤ 0.001.
Tables
- Table 1.
Summary of AZD6738 sensitivity and synergy with gemcitabine across the panel of PDAC cell lines
AZD6738 GR50 SUM_SYN_ANT SUM_SYN_ANT_WEIGHTED SYN_MAX SYN_SPREAD Without gem With gem LOEWE BLISS AVG LOEWE BLISS AVG LOEWE BLISS AVG LOEWE BLISS AVG MOUSE LINES K8484 536 158 Gem 5 nmol/L 5 11 8 4 8 6 11 32 21 0.80 0.62 0.71 TB32048 710 355 11 22 17 9 15 12 23 37 30 0.80 0.79 0.79 DT8082 763 395 0 8 4 0 5 3 23 23 23 0.52 0.66 0.59 TB31456 771 359 13 31 22 9 19 14 29 66 47 0.69 0.70 0.69 KPCFT79653 2609 1346 Gem 10 nmol/L 15 29 22 6 13 9 36 55 45 0.68 0.73 0.71 HUMAN LINES SW1990 935 14 56 13 35 23 2 13 23 24 24 1.61 1.27 1.44 Capan-1 1572 21 65 31 48 36 16 26 35 35 35 1.52 1.28 1.40 ASPC1 1983 691 51 31 41 24 13 18 29 26 27 1.43 1.32 1.38 HPAFII 3239 427 90 39 64 39 12 25 38 21 29 1.62 1.57 1.60 Capan-2 4775 2208 62 57 60 12 10 11 33 33 33 1.52 1.48 1.50 MiaPaCa-2 9844 854 91 85 88 57 49 53 52 60 56 1.37 1.23 1.30 Panc-1 >30000 3496 166 135 151 96 74 85 61 57 59 1.76 1.65 1.71 NOTE: GR50 for all cell lines was obtained using the GR metrics R package (developed by Clarke and colleagues, 2017). Mouse and human lines are ranked by increasing GR50 for AZD6738. Summing of synergy and antagonism was used to compare combinations using Metrics obtained from Combenefit. Both Bliss, Loewe models, and the average (AVG) are shown. SUM_SYN_ANT is the sum of synergy and antagonism observed in concentration logarithmic space. For instance, an integrated synergy of 50 is equivalent to an extrasynergistic effect of 50%, which is spread over a square of 1 log x 1 log in the 2-d log-concentration space. Both antagonistic and synergistic effects are considered in this metric. The integrated weighted synergy SUM_SYN_ANT_WEIGHTED incorporates a weight based on the dose response, which biases the total score toward synergy achieving highest effect. Hence, a synergy of 50% leading to a combined full effect (100%) will have more weight than if the corresponding effect was only 20% or 30%. Both antagonistic and synergistic effects are considered in this metric. SYN_MAX is the maximum level of synergy observed. SYN_SPREAD is a measure of synergy spread in the logarithmic concentration space.
Volume 17, Issue 8
