Fbxo32 Ubiquitin Ligase Mediates Apoptosis Evasion And Adaptation To Kit Inhibition In Gastrointestinal Stromal Tumor - Gist

Introduction: GIST is the most common malignant mesenchymal neoplasm and features myogenic differentiation. Most GISTs depend upon oncogenic signaling through constitutively activated forms of KIT and exhibit remarkable responses to the first-line KIT inhibitor imatinib. Secondary resistance involves emergence of subclonal populations harboring heterogeneous KIT-secondary mutations that are not collectively inhibited by any approved therapy. We dissected KIT-downstream signaling nodes to identify mediators of the KIT oncogenic program, seeking targets of overarching relevance in polyclonal imatinib-resistant GIST. Methods: using clinically-representative human GIST cell models, we undertook pharmacological and functional screening to identify key targetable nodes within KIT-downstream PI3K/mTOR and RAS/MAPK pathways and to model therapeutic strategies in vitro and in vivo. Transcriptomic analysis (RNAseq) identified genes co-regulated by PI3K/mTOR and RAS/MAPK, and functional evaluations by lentiviral construct overexpression and gene knockdown elucidated roles in GIST biology. Results: PI3K/mTOR and MEK1/2 were the most essential KIT-dependent nodes irrespective of the type of KIT primary or secondary mutation. Single node ablation did not yield sustained antiproliferative and apoptotic effects, but concurrent intermittent inhibition was synergistic in vitro and in vivo, supporting the critical and complementary roles of these two pathways. Transcriptomic analyses underscored FBXO32 (a SCF E3 ubiquitin ligase and the main effector of muscular atrophy) as the most differentially expressed gene co-regulated by the PI3K/mTOR and RAS/MAPK pathways. FBXO32 was also the most differentially expressed gene after KIT inhibition in GIST cell lines, and enrichment in FBXO32 expression was found in post-imatinib GIST samples compared to baseline matched tumor tissue. We demonstrated that FBXO32 is transcriptionally upregulated by FOXO3a upon KIT or concurrent PI3K/mTOR and MEK1/2 blockage, paralleling human muscle regulation. Notoriously, FBXO32 proved to have a pro-survival role in GIST cells, enabling evasion of apoptosis. Microarray and in vitro and in vivo functional assays showed that FBXO32 mediates cell cycle exit and quiescence upon KIT or concurrent KIT-downstream pathways inhibition, thereby minimizing imatinib-induced apoptosis and facilitating GIST cell survival. Conclusions: PI3K/mTOR and MEK1/2 are critical mediators of KIT oncogenic signaling in GIST. FBXO32 emerges as a KIT-dependent E3 ubiquitin ligase transcriptionally repressed through KIT downstream pathways. However, FBXO32 induction upon therapeutic KIT signaling inhibition triggers quiescence and subsequent apoptosis evasion, thus emerging as a novel mechanism of adaptation to therapeutic inhibition of oncogenic KIT. Citation Format: Alfonso Garcia Valverde, Jordi Rosell, Daniel Pilco Janeta, Sergi Sayols, Claudia Valverde, Anna Esteve, Marta Gut, Jordi Barretina, George D. Demetri, Jonathan A. Fletcher, Joan Carles, Joaquin Arribas, Cesar Serrano. FBXO32 ubiquitin ligase mediates apoptosis evasion and adaptation to KIT inhibition in gastrointestinal stromal tumor - GIST [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3767.