Radiation sensitizing

Radiation sensitizing through inhibition of NHEJ by the novel small molecule inhibitor RK-33 in lung cancer.

Field of research:
Oncology

Introduction:
Lung cancer is the leading cause of cancer mortality worldwide. More importantly, only about 30% of non-small-cell lung cancer patients respond to current combination therapy, which includes radiation and platinum based compounds.
Radiation therapy works by causing DNA damage. The two most important repair mechanisms of DNA double stranded breaks are non-homologous end joining (NHEJ) and homologous recombination (HR). Impaired NHEJ increases radiation sensitivity hence pharmaceutically inhibiting NHEJ would be an excellent way to improve radiation therapy.
We have designed a NHEJ inhibitor; RK-33, to fit into the ATP binding domain of DDX3. In this project we demonstrate how RK-33 increases radiation efficacy via inhibiting NHEJ.

Material & methods:
To determine viability of different lung cancer cell lines we performed colony forming assays after treatment with RK-33 and radiation. DNA break repair was analyzed by immunofluorescent staining of A549 lung cancer cells with γH2AX and 53BP1 antibodies. Cyclin D1 protein expression was determined by western blotting and FACS analysis was used to examine the cell cycle after treatment with RK-33. With a HR and a NHEJ reporter assay, we determined the extent of inhibition of HR and NHEJ by RK-33.
For in vivo studies, we used a lung cancer model of CCSP-Twist1-Kras induced transgenic mice. RK-33 was injected in the intra-peritoneal cavity 3 times a week. A dedicated small animal radiation platform was used to treat mice with 15Gy of stereotactic radiation. Finally, tumor volume was determined by micro-CT. All experiments were done in replicates.

Results:
RK-33 in combination with radiation reduces the ability of lung cancer cells to form colonies. RK-33 impairs DNA break repair and causes a cell cycle arrest in the G1 phase of lung cancer cells. RK-33 did not decrease HR but did decrease NHEJ by about 40%.
A 72% reduction of tumor load was accomplished after treatment with RK-33 and radiation compared to 28% tumor volume reduction in the control group who received radiation alone. No evident toxicity became apparent during the treatment with RK-33.

Conclusion:
These in vitro and in vivo results indicate that RK-33 is a promising and safe radiosensitizer, via inhibition of NHEJ, in lung cancer.