Exploiting Conditional Vulnerabilities Caused by the Systems Level Effects of Tumor Treating Fields

Abstract

Tumor Treating Fields (TTFields) delivers low-intensity, intermediate frequency, alternating electric fields non-invasively to a tumor. Optune, a TTFields delivery device, has been approved for glioblastoma and clinical trials are ongoing for other cancers. Other mechanisms of action besides interference with mitosis have been identified and suggest alternative clinical strategies to the use of TTFields. Human NSCLC and pancreatic cell lines were used to examine TTFields effects in vitro while an tumor explant system was used for ex vivo studies. The Inovitro system was used to generate TTFields. Quantification of DNA damage was done by using 53BP1 and γ-H2AX foci. The DNA fiber assay was used to quantify newly synthesized DNA to examine replication fork dynamics. R loop detection was done by dot-blot and immunofluorescence using the S9.6 antibody. Clonogenic cell survival was performed with TTFields combined with specific agents (ionizing radiation (IR), cisplatin, olaparib, IR plus cisplatin, IR plus olaparib). We have developed an ex vivo model for lung tumors that are exposed to to TTFields. TTFields exposure decreased the expression of FANC/BRCA1 pathway genes resulting in the downregulation of DNA DSB repair of IR-induced DSBs. Most importantly, TTFields alone increased the number of γH2AX foci and the incidence of chromatid aberrations as a function of TTFields exposure time. Furthermore, TTFields exposure decreased the length of newly replicated DNA and increase R-loop formation as a function of exposure time, suggesting that TTFields induce replication stress. We hypothesized that by applying TTFields, a systems level conditional vulnerability develops (inhibition of DNA repair, increased replication stress, production of DSBs, reduced mitophagy, and other stresses) rendering cells more susceptible to agents that cause DNA damage or replication stress or interfere with its repair. In agreement with our hypothesis, NSCLC cell susceptibility to radiation increased when cells were exposed to TTFields prior to IR treatment compared to IR treatment followed by TTFields. TTFields also enhanced cisplatin toxicity in a synergistic manner. Furthermore, TTFields exposure of NSCLC and pancreatic tumor cells concomitant with the PARP inhibitor Olaparib followed by radiation resulted in synergistic cell killing compared to radiation or Olaparib alone or in combination. Our initial results of ex vivo tumor explants exposed to TTFields supports our in vitro results in that we see the down-regulation of key DNA repair pathway proteins as was seen in our in vitro studies. Our data identifies systems level perturbations to key molecular and biochemical pathways that render tumor cells vulnerable to a number of different agents. We suggest that TTFields would serve well as neoadjuvant therapy prior to radiation treatment and either prior to or concomitant with chemotherapy agents.