Abstract Clinical efficacy of small-molecule inhibitors can sometimes be attributed to multiple drug effects acting in concert. Herein, we define an off-target effect in which several clinical BRAFV600 inhibitors, including the widely used dabrafenib and encorafenib, interact directly with GCN2 to activate the Integrated Stress Response and ATF4. Blocking this off-target effect by co-drugging with a GCN2 inhibitor in A375 melanoma cells causes enhancement rather than suppression of cancer cell outgrowth, suggesting that the off-target activation of GCN2 is detrimental to these cells. This result is mirrored in PC9 lung cancer cells treated with erlotinib, an EGFR inhibitor, that shares the same off-target activation of GCN2. Using an in silico kinase inhibitor screen, we identified dozens of FDA-approved drugs that appear to share this off-target activation of GCN2 and ATF4. We further describe the activation of GCN2 signaling in melanoma patient-derived xenograft models treated with dabrafenib and trametinib, and observe a significant decrease in disease-free survival in a cohort of human melanoma patients with altered GCN2 pathway components. Finally, we describe the benefits of combining the next-generation “paradox breaker” RAF inhibitor naporafenib with a GCN2 activator, HC-7366, in A375 melanoma cells. Thus, GCN2 is emerging as a promising cotreatment candidate in melanoma and potentially beyond. Citation Format: C. Ryland Ill, Nasreen C. Marikar, Vu T. Nguyen, Brianna Fernandez, Varuna Nangia, Alicia M. Darnell, Matthew G. Vander Heiden, Philip Reigan, Sabrina L. Spencer. Off-target activation of GCN2 by BRAFV600inhibitors attenuates melanoma outgrowth abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 337.
Ill et al. (Fri,) studied this question.