Abstract
Non-small cell lung cancer (NSCLC) is one of the most commonly diagnosed cancers in New Zealand. Despite advancements in treatments such as targeted therapies, drug resistance and relapse remain significant challenges. Drug resistance can occur through pre-existing mutations or adaptive mutability, where drug-tolerant persister cells (DTPs) increase their mutation rate to acquire resistance conferring mutations. This response arises in drug-tolerant persister (DTP) cells, which enter a dormant, slow-cycling, stem-cell-like state to withstand initial treatment, eventually acting as a reservoir from which stable genetic resistance can arise. Low-fidelity DNA polymerases, which introduce new mutations during DNA repair, are key to the adaptive mutability response. Additionally, apoptotic cancer cells may use paracrine signalling, such as the Phoenix Rising pathway, to stimulate proliferation and acquired drug resistance in neighbouring cells. We hypothesized that inhibiting apoptotic signalling and low-fidelity DNA polymerases in NSCLC cells would limit the adaptive mutability response in DTPs and prevent drug resistance.
We treated EGFR-mutant PC9 cells with EGFR-targeting tyrosine-kinase inhibitors (TKIs), gefitinib or osimertinib, to induce DTPs. To investigate whether apoptotic cell signals released from KRAS-mutant H358 cells would induce the adaptive mutability response in DTP PC9 cells, we conducted a conditioned media experiment. At 24 hours of treatment with conditioned media from untreated or sotorasib-treated H358 cells, we found adaptive mutability markers to be significantly downregulated. Conversely, after 48 hours of treatment with conditioned media from untreated or sotorasib-treated H358 cells, adaptive mutability markers were significantly upregulated. Adaptive mutability is characterised by downregulated high-fidelity DNA polymerase, homologous recombination (HR), and mis-match repair (MMR) genes, and upregulated low-fidelity DNA polymerase genes. Our findings suggest exposing PC9 cells to conditioned media for 24 or 48 hours does not induce the adaptive mutability response. Nonetheless, the conditioned media had an effect on the expression of DNA polymerase and DNA repair genes. We found no difference in PGE2 concentration in the media harvested from untreated or treated H358 or PC9 cells, suggesting it is unlikely that PGE2 was the mediator for these gene expression changes.
Long-term treatments combining osimertinib with either candesartan cilexetil (CC), a low- fidelity DNA polymerase inhibitor, or celecoxib, a Non-Steroidal Anti-Inflammatory Drug (NSAID), showed that osimertinib and CC had no significant effect on cell growth or morphology, while the combination of osimertinib and celecoxib delayed cell growth for four weeks and induced senescence-like morphology. To identify the mechanism for these growth and morphology changes, we investigated changes in the expression of adaptive mutability markers in PC9 cells through qPCR. We found cells treated with the combination of osimertinib and celecoxib had no significant changes to adaptive mutability marker expression in comparison to cells treated with osimertinib alone, apart from increased POLA and MLH1 expression. Furthermore, a flow cytometry-based homologous recombination (HR) assay showed significantly reduced HR capacity in PC9 cells treated with osimertinib in combination with celecoxib in comparison to cells treated with either agent alone.
The connection between cell proliferation and changes to the adaptive mutability response was also investigated by measuring cell growth and key adaptive mutability markers in serum- deprived H358 or PC9 cells, finding cell growth decreased proportionally to the amount of serum present in cell culture media. Adaptive mutability marker expression in PC9 cells also decreased in proportion to the amount of serum present in cell culture media, but the same relationship was not evident in H358 cells. Lastly, we observed that reduced serum concentration in cell culture media may inhibit cell growth through effects on the ERK/pERK signalling pathway.
Overall, we identified that combining osimertinib and celecoxib is a promising strategy to delay cell growth in NSCLC. Further research into the complex relationship between apoptotic cell signalling, adaptive mutability, and acquired drug resistance may lead to the identification of new treatment strategies that improve patient outcomes and therapeutic efficacy.