Understanding endogenous oncogenic KRAS mutations in isogenic near-diploid human cell lines derived from hTERT RPE-1

The KRAS gene is a frequently mutated oncogene in human cancers, with around 17% of cancers exhibiting KRAS mutations according to COSMIC v98. Efforts to target oncogenic KRAS and its signaling pathways have been ongoing for over four decades since its discovery in 1982. Despite extensive research, targeting oncogenic KRAS signaling has proven challenging. The recent approval of first-generation RAS inhibitors specific for the G12C oncogenic mutation marks a significant milestone in cancer research, although issues with drug efficacy and acquired resistance persist.

To advance our understanding of oncogenic RAS signaling, it is essential to develop a simple and reliable experimental system to assess the physiological impact of oncogenic KRAS mutations. To address this need, we generated a series of near-diploid human hTERT RPE-1 cell lines with endogenous oncogenic KRAS mutations at glycine 12 (KRASG12V/+, KRASG12C/+, KRASG12D/+, or KRASG12G(Wild-type)/+).

In a proof-of-concept study, we examined cancer-related phenotypes of these cell lines. Cells with KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotypes, along with WT KRASG12G(WT)/+ cells, demonstrated that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility, with reduced focal adhesions in KRASG12V/+ cells. Epidermal growth factor-induced phosphorylation of ERK and AKT was comparable among KRASG12V/+, KRASG12C/+, KRASG12D/+ and KRASG12G(WT)/+ cells. Notably, KRASG12X/+ cells showed varying responses to different inhibitors, with KRASG12V/+ and KRASG12D/+ cells being more sensitive to hydroxyurea and MEK inhibitors (U0126 and trametinib) but more resistant to the PI3K inhibitor PIK-90 compared to KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 exhibited an additive inhibition on growth rate, which was more pronounced in KRASG12V/+ cells than in WT cells. These cell lines will be invaluable for studying oncogenic RAS signaling and developing effective anti-KRAS agents with minimal cytotoxicity to WT cells.