Since the development of small-molecule inhibitors targeting mutated EGFR, lung cancer patients’ outcomes have significantly improved. Nonetheless, tumors eventually develop resistance to these inhibitors, resulting in ineffective therapy and relapse. A major mechanism behind therapy resistance is the emergence of new mutations leading to amino acid substitutions in EGFR, such as T790M and C797S, which evade the inhibition. Although it is clear that EGFR-inhibitors cause an increased mutagenesis, the DNA damage mechanism behind the emergence of specific resistance mutations remain poorly understood. Aim: In this project, we study the role of low-fidelty polymerases (TLS) in the emergence of de novo resistance mutations in NSCLC.

Frequency of resistance mutations was determined by digital droplet PCR (ddPCR); NSCLC cell lines were treated for 10 or 28 days with EGFR-TKI alone or combined with other pharmacological inhibitors. At appropriate time points, gDNA was harvested and subjected to ddPCR for T790M or C797S quantification using Taqman probes (BioRad). To determined cell viability, cells were incubated for 72h with the appropriate drug treatment, and cell viability was assessed by MTT assay. Gene expression levels were determined by RT-PCR. REV3L knock down was achieved by 2nd generation lentiviral transduction.

We established an in vitro ddPCR assay to detect the mutations in a highly sensitive way, following their emergence over a 28-day time frame. As C797S was detected in several NSCLC cell lines prior to drug treatment, we are currently performing a fluctuation assay to distinguish colonies derived from pre-existing resistant clones, and de novo induced resistance mutations. Following TKI-exposure, REV3L, REV1 and other TLS polymerases were upregulated in PC9 cells. JH-RE06, a REV1-REV7 inhibitor that previously showed superiority in melanoma and colorectal cancer, did not affect the sensitivity of PC9 cells to erlotinib or osimertinib (MTT assay), however might prevent the emergence of resistance mutations. Similarly, REV3L and REV1 knockdown cell lines have been established, and will be assessed for their mutability in response to EGFR-TKIs in vitro.

If our promising preliminary results of TLS inhibition preventing resistance mutations will be confirmed, in vivo PDX studies will be performed to further assess the efficiency of JH-RE06 to prevent relapses in NSCLC. This work will pinpoint new opportunities for drug development to overcome the current major drawback of anti-EGFR therapy and will overall aid in improving lung cancer patients’ survival outcomes.