LAUSR

Background: PHI-501 has been developed as a novel inhibitor of NRAS mutated acute myeloid leukemia. Big data and artificial intelligence (AI)-based drug discovery platform and cell-based investigation identified PHI-501 repurposable against melanoma as a novel pan-RAF inhibitor. BRAF activated by the common V600E and other mutations, as well as by upstream NRAS mutation are frequent in malignant melanoma. Regardless of the therapeutic effect of class I BRAF inhibitors such as vemurafenib, dabrafenib and encorafenib, melanoma patients with BRAF non-V600/RAS mutations remain limited response. In this study, we evaluated PHI-501 to potently inhibit the tumor cell growth and overcome the limited response in melanoma. Methods: The growth inhibitory activity of PHI-501 was determined using CellTiter-Glo luminescent assay in a panel of cancer cell lines. The effect of PHI-501 on anchorage independent growth and cell proliferation compared to vemurafenib or belvarafenib (pan-RAF inhibitor) were tested in A375 (BRAFV600E), C8161 (BRAFG464E) and SK-MEL-2 (NRASQ61R) melanoma cell lines by soft agar assay, western blot and FACS analysis. To measure cell motility in a controlled environment, cells were cultured in monolayer followed by a wound-healing assay in a treatment with 0.1 µM of each compound for 12 hours. Anti-tumor activities of PHI-501 were evaluated in A375 or C8161 xenograft mouse in vivo model. Results: PHI-501 showed 3- to 119-fold more effective growth inhibition than other RAF inhibitors, vemurafenib and belvarafenib, in SK-MEL-2 (GI50: 0.15 µM for PHI-501 vs 7.13 µM and 0.37 µM, respectively) and C8161 (GI50: 0.33 µM for PHI-501 vs 39.56 µM and 2.10 µM, respectively) melanoma cell lines, and did not show a potential to paradoxical activation. PHI-501 induced melanoma cell apoptosis more efficiently than other RAF inhibitors, as shown in increased G0/G1-arrested and annexin V-positive cell population and abundant cleavage of PARP1 after drug treatment. In the cell wound-healing assay, PHI-501 exhibited promising antimetastatic potential via inhibiting migration of all tested melanoma cells by up to 70%, unlike other RAF inhibitors. Anti-tumor activity of PHI-501 was dose-proportional in A375 xenograft model. Tumor growth was significantly reduced in C8161 xenograft mice treated with PHI-501 (TGI: 75.0%), compared with vehicle and vemurafenib (TGI: 12.3%). Conclusion: PHI-501, a novel pan-RAF inhibitor, has potent oral anti-tumor activity. Melanoma cells harboring BRAF non-V600/NRAS or BRAF common V600E mutations exhibited significantly reduced proliferation, increased apoptosis and inhibited migration upon treatment with PHI-501. The results of this study suggest that PHI-501 has a potential to overcome the limited response in the treatment of melanoma, and warrant evaluation of PHI-501 as a single agent to treat both BRAF-and NRAS-mutated metastatic melanoma. Citation Format: Namkyeong Kim, Jung Hee Park, Ky-Youb Nam, June H-J Han, Kyu-Tae Kim, Sandip Sengupta, Jeong Hyeok Yoon, Taebo Sim. PHI-501, a novel and potent pan-RAF inhibitor in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1627.