Successful cancer treatment with radiation depends heavily on whether a therapeutic gain can be achieved. Sophisticated radiation delivery instrumentation can minimize the normal tissue included in the radiation field; however, invariably normal tissues are included necessitating a need to identify agents that might differentially radiosensitize tumor as opposed to normal tissues. Cytotoxic chemotherapy combined with radiation is currently used to enhance local tumor control at the expense of increasing normal tissue toxicity (17
). Ideally what is needed are approaches that result in selective tumor radiosensitization.
The current findings suggest that AZD7762-mediated Chk1/2 inhibition may offer considerable selective tumor radiosensitization. AZD7762 did not exert appreciable cytotoxicity alone both in vitro and in vivo. In addition, the normal human fibroblast cell line 1522 was not radiosensitized by AZD7762, suggesting that other normal tissues would not be radiosensitized by AZD7762. In general there was a relationship between AZD7762-mediated radiation sensitization and the p53 status of the cell line. Cell lines that carried p53 mutations were enhanced to a greater extent than p53 WT lines. This was particularly apparent in the H460 cell line pair, where the only difference between the cell lines was the p53 status. Consistent with the in vitro data for HT29 cells, when AZD7762 and fractionated radiation treatment were evaluated in a HT29 xenograft tumor model, significant enhancement in radiation-induced tumor regrowth delay was observed. It should be noted that AZD7762-mediated enhancement of tumor regrowth delay required two daily doses of AZD7762 separated by 8 hr after each radiation fraction consistent with the prolonged radiation-induced activation of pChk1 ().
Inhibition of Chk1/2 by AZD7762 has been shown to enhance the cytotoxicity of DNA damaging chemotherapy drugs in part by abrogation of the G2 checkpoint. The enhancement was greater in cell lines with compromised p53 status (9
). In the current study, AZD7762 treatment resulted in abrogation of the radiation-induced G2 delay for every cell line tested (, Supplementary Fig. 6A-B
), yet normal 1522 cells were not radiosensitized by AZD7762. Thus, abrogation of the radiation-induced G2 checkpoint by AZD7762 was insufficient to explain the mechanism of radiosensitization. Like AZD7762, the mechanism for caffeine-mediated radiosensitization has been largely attributed to abrogation of the G2 checkpoint (18
). However, there are reports, which show no relationship between radiation-induced G2 abrogation with caffeine and radiosensitization (19
). Other mechanisms identified in the current study that may be more pertinent include the effects of AZD7762 on radiation-induced repair. It has been proposed that Chk1 is required of homologous recombination repair (HRR) (20
), which normally occurs in the S and G2 (21
). Likewise, another major repair pathway is the non-homologous end joining (NHEJ), which predominantly occurs in G1 (22
). Since p53 mutated cells lack a G1 checkpoint, they may be more dependent on HRR as opposed to NHEJ. Wild type p53 cells, expressing both a G1 and G2 checkpoint following radiation treatment, should be capable of utilizing both types of repair. Thus, it would be anticipated that Chk1/2 inhibition would predominantly impact HRR in p53 mutated cells (20
). Consistent with this was our findings that AZD7762 inhibited the repair of radiation-induced damage (γ-H2AX) and enhanced mitotic catastrophe which led to greater radiosensitization in p53 mutated cells. Further support for inhibition of HRR by Chk1/2 inhibition comes from plateau phase HT29 cells, which were not radiosensitized by AZD7762 (Supplementary Fig. S3
). Plateau phase HT29 cells were predominantly in G1 (86%) during the radiation and AZD7762 treatment. It is interesting to speculate that repair of radiation damage in plateau phase cells would be through and not affected by Chk1/2 inhibition. Studies are ongoing to test this hypothesis.
Several protein biomarkers from xenograft studies were identified as potential surrogates to guide clinical trials with AZD7762 and radiation. As was seen for in vitro studies, AZD7762 alone and radiation activated γ-H2AX levels. AZD7762 combined with radiation inhibited the return of γ-H2AX to normal levels. The reason for the AZD7762 induction of γ-H2AX is not clear; however, it may be activated as a result of replication stress (23
). Interestingly, pChk1 was activated by both radiation and AZD7762 alone. The latter might be indicative of a DNA damage response associated with γ-H2AX activation. Lastly, radiation was shown to induce cyclin B and AZD7762 markedly inhibited its induction, consistent with the radiation-induced G2 abrogation observed in in vitro studies. Collectively, various combinations of these markers may give indication that AZD7762 is targeting necessary pathways to illicit tumor radiosensitization in clinical trials.