Tumor Treating Fields Elicit a Conditional Vulnerability in Non-Small Cell Lung Cancer Cell Lines Through the Down-Regulation of Key DNA Repair and Replication Stress Pathways that When Targeted with Chemoradiation Results in Synergistic Cell Killing

Abstract

Tumor treating fields (TTFields) is a new physical modality of cancer therapy composed of low-intensity, intermediate frequency, alternating electric fields non-invasively applied to the region of the tumor. TTFields have revolutionized the treatment of recurrent and newly diagnosed glioblastoma with clinical trials ongoing for other cancers. Initial experiments revealed that a major mechanism by which TTFields kill cancer cells is through the disruption of mitosis. However, other mechanisms have now been identified. Based upon these newly described mechanisms we have examined the combination of TTFields with either cisplatin or a PARP inhibitor (Olaparib), either alone or in combination with radiation. A panel of 5 non-small cell lung cancer cell lines were exposed to TTFields for 24, 48 or 72h. Gene expression analysis, western analysis, DNA repair, DNA replication and cell survival were assessed as a function of time using combinations of TTFields, radiation, cisplatin or a PARP inhibitor. We found that the expression of the BRCA1 DNA damage repair pathway genes were significantly downregulated (P < 0.05) upon TTFields treatment which was confirmed at the protein level by western blot. TTFields treatment also slowed the repair of ionizing radiation-induced DNA damage compared to radiation alone which was evident by an increased number of DNA double strand break repair foci at any given time. TTFields alone caused the number of γH2AX foci to increase with time of exposure. The length of newly replicated DNA was reduced as a function of TTFields exposure time while the number and intensity of R-loop formations (DNA:RNA hybrid structures) increased, suggesting increased replication stress. Based upon these newly identified mechanisms of TTFields action, we hypothesized that by applying TTFields first, a conditionally vulnerable environment would develop rendering cells more susceptible to radiation, cisplatin or other agents such as PARP inhibitors that caused DNA damage, interfered with DNA repair or inhibited the resolution of stalled replication forks. We found that the effect of TTFields exposure concomitant with the PARP inhibitor Olaparib followed by radiation was synergistic compared to radiation or olaparib alone or in combination, although the degree of sensitization and synergy varied across the cell lines. We suggest that new strategies for TTFields application be considered based upon these newly identified mechanisms of action for TTFields. For example, the use of TTFields prior to radiation treatment (TTFields loading), and either prior to or concomitant with, chemotherapy agents that cause DNA damage or interfere with DNA repair or DNA replication fork resolution may result in more favorable clinical responses.