Abstract 4440: The efficacy of NST-628, a non-degrading pan-RAF/MEK molecular glue, and MEK inhibitors for overcoming acquired osimertinib resistance mediated by TRIM24-BRAF fusions in EGFR mutant NSCLC

Abstract In patients with EGFR-mutant non-small cell lung cancer (NSCLC), acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) can be driven by novel gene fusions. Among these, acquired fusions in the BRAF gene occur in approximately 2% of osimertinib resistant cases. Based on paired molecular profiling of biopsies from a patient with classical EGFR exon 19 deletion (del19) NSCLC, taken pre and post osimertinib treatment, we identified a novel BRAF fusion, TRIM24-BRAF, and hypothesized that this aberration played a causal role in osimertinib resistance. To test this hypothesis, we transfected HCC827 and HCC4006 EGFR mutant NSCLC cell lines to express TRIM24-BRAF fusion variants using lentivirus vectors and performed a 3 day cell viability assay to assess the impact of these fusions on sensitivity to osimertinib. Expression of TRIM24-BRAF fusions resulted in an IC50 of at least 100 times higher as compared to the parental cell lines, confirming that TRIM24-BRAF fusions are sufficient to induce EGFR TKI resistance. To further evaluate the mechanism, we performed Western blot analysis, which revealed that that osimertinib blocked EGFR phosphorylation in both parental cell lines and cells expressing the TRIM24-BRAF fusion variants. However, osimertinib inhibited MEK phosphorylation only in the parental cell lines, but not in those expressing the TRIM24-BRAF fusions, indicating that cells expressing the TRIM24-BRAF fusion had sustained activation of signal transduction pathways downstream of EGFR after osimertinib treatment. Next, we utilized a panel of compounds targeting the RAS/RAF/MAPK pathway including agents targeting pan-RAS (RMC-6236 and RMC-7977), SHP2 (RMC-4550), BRAF (dabrafenib, encorafenib, tovorafenib and vemurafenib), pan-RAF (TAK-632), MEK (binimetinib, cobimetinib, selumetinib and trametinib), pan-RAF/MEK (NST-628) and assessed whether targeting signal transduction molecule downstream of EGFR could overcome this adaptative resistance mechanism. Our data indicated that trametinib, a MEK inhibitor, and NST-628, a non-degrading pan-RAF/MEK molecular glue, were the most effective against cells expressing TRIM24-BRAF fusions. Moreover, cell viability assays combining osimertinib either with trametinib or with NST-628 showed an addictive effect among all the different TRIM24-BRAF variants and a synergistic effect in some. TRIM24-BRAF expressing cells showed minimal sensitivity to BRAF, SHP2 or RAS targeting single agents. Taken together, our findings indicate that expression of TRIM24-BRAF fusion induce osimertinib resistance in EGFR mutant NSCLC and targeting the MEK pathway using trametinib or NST-628 can overcome osimertinib resistance in this setting. Citation Format: Alvaro Guimaraes Paula, Ximeng Liu, David Molkentine, Monique Nilsson, Junqin He, John V. Heymach. The efficacy of NST-628, a non-degrading pan-RAF/MEK molecular glue, and MEK inhibitors for overcoming acquired osimertinib resistance mediated by TRIM24-BRAF fusions in EGFR mutant NSCLC [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 4440.