TWEAK/FN14 inhibition synergizes with oncogene-directed tyrosine kinase inhibitors to overcome resistance across multiple driver contexts.

Oncogene-directed tyrosine kinase inhibitors (TKIs) have transformed the treatment of molecularly defined cancers; however, durable responses are frequently undermined by therapy-induced adaptive resistance. Beyond secondary kinase mutations, accumulating evidence suggests that stress-responsive, non-genetic survival pathways play a central role in attenuating TKI efficacy across oncogenic contexts. The TWEAK/FN14 signaling axis has been implicated in stress-induced, NF-κB-mediated survival signaling, yet its role as a convergent mediator of adaptive resistance to oncogene-targeted therapies remains incompletely defined. We performed a structure-guided virtual screen of approximately 1.3 million compounds evaluated across multiple FN14 binding interface models, yielding ~3.9 million docking simulations, to identify small molecules capable of disrupting TWEAK/FN14 signaling. Lead candidates were validated using TWEAK/FN14 and TNFα-driven NF-κB reporter assays with cytotoxicity controls. Combination studies were conducted across a broad panel of Ba/F3 models expressing oncogenic drivers-including RET, ALK, ROS1, NTRK, EGFR exon 20 insertion, BRAF V600E, and KRAS G12C-each paired with matched TKIs and resistance mutations. Drug interactions were quantified using Bliss independence and Loewe additivity models. In vivo efficacy was evaluated in a Ba/F3 KIF5B-RET G810R xenograft model. Cabozantinib, zanzalintinib, and selected screening-derived compounds inhibited TWEAK/FN14-induced NF-κB signaling at nanomolar concentrations, with minimal effects on TNFα-mediated signaling and limited intrinsic cytotoxicity. TWEAK/FN14 inhibition consistently enhanced the anti-tumor activity of oncogene-matched TKIs across all seven oncogenic driver classes, including models harboring clinically relevant resistance mutations. Synergistic interactions were observed across multiple TKI combinations, demonstrating greater-than-additive suppression of oncogene-driven cell survival. In vivo, combined selpercatinib and cabozantinib treatment resulted in significantly greater tumor growth inhibition than either monotherapy in a RET G810R resistance model, without evidence of toxicity. These findings are consistent with TWEAK/FN14 signaling functioning as a broadly exploitable adaptive resistance pathway across diverse oncogenic contexts. Pharmacologic disruption of this pathway, including through repurposing of clinically advanced agents such as cabozantinib, represents a rational and testable strategy to enhance the efficacy of oncogene-directed TKIs; genetic validation of the FN14-specific mechanism is warranted for future investigation.