Figure 1.
Heat map summarizing Bliss independence (BI) values for the strongest radiosensitizers.
BI values correspond to (FCexp - FCobs ) x 100 and were calculated based on the raw data from the screen depicted in Figure S4. Drug concentrations ranged from 10nM to 1.25µM. Radiosensitization was not considered if the surviving fraction (SF) following drug treatment alone was less than 0.125, since a synergistic effect of the combination with IR may not be detectable. Although CI-1040, Raf265, and GDC-0980 were slightly radiosensitizing, they were not included in follow-up studies since other inhibitors of the same pathways (AZD6244 and BEZ235) exhibited more potent synergy.
Figure 2.
All rectal cancer but only some pancreatic cancer cell lines are radiosensitized by BEZ235 and AZD7762.
Effects of BEZ235 (A) or 250nM AZD7762 (B) 1 week post-IR. Different concentrations of BEZ235 were used depending on the sensitivity of each cell line to BEZ235 treatment in the absence of IR (the pancreatic lines were more sensitive to BEZ235): 250nM for SW837, RCM-1, and SW1463 cells, 50nM for CaR-1 and Capan-1 cells, 25nM for PANC-1 cells, and 1nM for PATU8902 and PATU8988T cells. The HTA was used for the rectal cancer cell lines and Capan-1 cells, and cell counting was used for the remaining lines. Left, Data are normalized to vehicle plus sham IR treatment (effects of the SMIs in the absence of IR). Right, Data are normalized to corresponding non-irradiated controls. p-values ≤ 0.05 for IR plus SMI treatment compared to IR plus vehicle control are indicated by asterisks.
Figure 3.
Pancreatic cancer cell lines exhibit variability in response to combination treatment of 250nM AZD7762 and IR.
The CellTiter-Glo assay was performed 1 week post-IR. Left, Data are normalized to vehicle plus sham IR treatment (effects of the SMIs in the absence of IR). Right, Data are normalized to corresponding non-irradiated controls; solid bars represent the effects of IR alone. p-values ≤ 0.05 for IR plus SMI treatment compared to IR plus vehicle control are indicated by asterisks.
Figure 4.
The chemotherapeutic agent 5-FU synergizes with AZD7762 and enhances radiosensitization by AZD7762.
HTA results for SW837 cells are shown. See Figure S10 for data on RCM-1 cells and BEZ235 treatment. (A) Data are normalized to vehicle plus sham IR. (B) Data are normalized to corresponding non-irradiated controls. p-values ≤ 0.05 for IR plus SMI treatment compared to IR plus vehicle control are indicated by asterisks.
Figure 5.
Combination treatment of AZD7762 and IR inhibits DNA repair and induces apoptosis.
Western blotting results for RCM-1 cells are shown. See Figure S14 for the quantification and data on SW837 and SW1463 cells. The number of days post-IR is indicated. (A) DNA DSBs as indicated by γH2AX levels. (B) Apoptosis as indicated by cleaved PARP levels.
Figure 6.
AZD7762 is a more potent radiosensitizer than the ATM inhibitor KU-55933.
The CellTiter-Glo assay was performed with SW837 cells 1 week post-IR. Data are normalized to corresponding non-irradiated controls. p-values ≤ 0.05 for IR plus SMI treatment compared to IR plus vehicle control are indicated by asterisks.
Figure 7.
AZD7762 radiosensitizes and inhibits Chk1 activity at a 10-fold lower concentration than two Chk1-specific inhibitors.
(A) The CyQUANT assay was performed with SW837 cells 1 week post-IR. SCH900776 and LY2603618 are Chk1 inhibitors. Data are normalized to corresponding non-irradiated controls. p-values ≤ 0.05 for IR plus SMI treatment compared to IR plus vehicle control are indicated by asterisks. (B) SW837 cells were treated with vehicle or SMI two hours prior to IR, and protein lysates were collected three hours post-IR. Chk1 and Chk2 autophosphorylation sites were measured by Western blotting.