Abstract 4371: PHI-501 as a potent pan-RAF/DDRs inhibitor suppresses lung cancer cell proliferation and overcomes KRAS G12C inhibitor resistance

Abstract Background: Mutations of the KRAS are one of the most commonly mutated oncogenes in cancer. Oncogenic mutations in KRAS typically occur at hotspots in the protein, increasing the steady-state levels of KRAS proteins in the GTP-bound state that are capable of driving oncogenic signaling through downstream effector pathways, such as the MAPK and PI3K pathways. The recent development of direct KRASG12C inhibitors(G12Ci) has improved outcomes in KRAS mutant cancer. Mechanisms of acquired resistance to G12Ci are heterogeneous including both on-target and off-target resistance. On-target acquired resistance includes secondary codon 12 KRAS mutations, but also acquired codon 13 and codon 61 alterations, and mutations at drug binding sites. Off-target acquired resistance can derive from activating mutations in KRAS downstream pathways including NRASQ61K and BRAFV600E, acquired oncogenic fusions, gene level copy gain, or oncogenic alterations in other pro-proliferative and anti-apoptotic pathways. PHI-501 is a potent and dual kinase inhibitor for pan-RAF and discoidin domain receptor (DDR). In this study, we investigated the anti-tumor effect of PHI-501 as monotherapy and addressed the molecular mechanism underlying overcoming G12Ci resistance. Methods: RAF family kinase profiling was performed. Cell viability was measured using the WST assay in human lung cancer cell lines and Ba/F3 cell lines expressing BRAFV600E, DDR1 and BRAFV600E/DDR1. Western blot was used to investigate the effects of down-stream signals. In vivo anti-tumor activities of PHI-501 were evaluated in the A549 mouse xenograft model. Results: In Ba/F3 cells carrying BRAFV600E/DDR1, PHI-501 displayed 70-fold higher cellular potency (IC50 0.012 µM) over control cells. PHI-501 demonstrated potent anti-proliferative activity (IC50 < 0.2 µM) in 7 lung cancer cell lines harboring KRAS wild-type, KRAS or NRAS mutation. PHI-501 profoundly inhibited the phosphorylation of MEK, ERK, and AKT proteins as the downstream signaling of EGFR and DDR1 in KRAS mutant lung cancer cells. The growth inhibitory activity of PHI-501 in KRASG12V, KRASG12S, KRASQ61H and NRASQ61K lung cancer cells was superior to that of G12Ci (sotorasib and adagrasib) and BRAF inhibitors (encorafenib and avutometinib). PHI-501 considerably suppressed the reactivation of both pERK and pAKT by inhibition of pDDR1 expression in lung cancer cells. Consistently, treatment of A549 (KRASG12S) with PHI-501 in mice resulted in dramatic tumor regression superior to control groups. Conclusion: The treatment of PHI-501 alone showed robust growth inhibition in lung cancer cells harboring KRAS wild-type or KRAS and NRAS mutation. Our results suggest that PHI-501 as the pan-RAF/DDRs dual inhibitor is beneficial for the treatment of lung cancer and even helps overcome the resistance of previous G12Ci treatments. Citation Format: An-Na Moon, Ji Young Kim, Hee-sun Hwang, Gi-Jun Sung, Jinsun Kwon, Ky-Youb Nam, Kyu-Tae Kim, June Han, Jeong Hyeok Yoon, Ki-Young Lee. PHI-501 as a potent pan-RAF/DDRs inhibitor suppresses lung cancer cell proliferation and overcomes KRAS G12C inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4371.