Background <p>Imatinib (IM), a tyrosine kinase inhibitor (TKI), is the first-line treatment for patients with gastrointestinal stromal tumors (GISTs). However, its efficacy is limited due to acquired resistance induced by secondary KIT mutations in most patients with GIST. Furthermore, new challenges have emerged following the clarification that KIT-independent GISTs exhibit strong resistance to small molecule inhibitors targeting KIT/ platelet-derived growth factor receptor alpha (PDGFRA). Therefore, investigating the underlying therapeutic targets for imatinib-resistant GISTs is urgently necessitated.</p> Patients and Methods <p>Through both in vitro and in vivo experiments, along with the analysis of alterations in the FBXW7–MCL1 axis and the YAP/TAZ–CCND1 pathway in patients with GISTs, before and after IM treatment.</p> Results <p>MCL1 overexpression and activation of the YAP/TAZ–CCND1 pathway are induced in IM-resistant GIST cells and post-IM GIST samples. AT101, a BCL-2 inhibitor, exerts a pro-apoptotic effect on GIST cells by suppressing MCL1 overexpression, and the combination therapy of AT101 and IM exerts a stronger pro-apoptotic effect through modulation of IM activity regulated by the FBXW7–MCL1 axis. Furthermore, the suppression of AT101 on GIST growth and metastasis, by targeting the YAP/TAZ–CCND1 pathway, was confirmed through xenograft and metastasis mouse models. Notably, the antitumor activity of AT101 is maintained regardless of the IM sensitivity of GIST cells, whereas AT101 enhances and restores IM activities in both GIST-T1 and IM-resistant GIST cells.</p> Conclusions <p>AT101 exerts a strong antitumor activity by targeting both the FBXW7–MCL1 axis and the YAP/TAZ–CCND1 pathway, suggesting that AT101 monotherapy, and its combination with IM, are worth further investigating in clinical trials.</p>