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KIT or PDGFRA gain-of-function mutations are the primary drivers of gastrointestinal stromal tumor (GIST) growth and progression throughout the disease course. The PI3K/mTOR pathway is critically involved in the transduction of KIT/PDGFRA oncogenic signaling regardless of the type of primary and secondary mutations, and therefore emerges as a relevant targetable node in GIST biology. We evaluated in GIST preclinical models the antitumor activity of copanlisib, a novel pan-class-I PI3K inhibitor with predominant activity against p110α and p110δ isoforms, as single-agent and in combination with first-line KIT inhibitor imatinib. In vitro studies undertaken in one imatinib-sensitive (GIST-T1) and two imatinib-resistant (GIST-T1/670 and GIST430/654) GIST cell models showed that single-agent copanlisib effectively suppressed PI3K pathway activation leading to decreased cell viability and proliferation in both imatinib-sensitive and -resistant cells irrespective of the type of primary or secondary KIT mutations. Simultaneous PI3K and KIT inhibition with copanlisib and imatinib resulted in enhanced impairment of cell viability in both imatinib-sensitive and -resistant GIST cell models, although apoptosis was mostly triggered in GIST-T1. Single-agent copanlisib inhibited GIST growth in vivo, and conjoined inhibition of PI3K and KIT was the most active therapeutic intervention in imatinib-sensitive GIST-T1 xenografts. IHC stain for cleaved-caspase 3 and phospho-S6 support a predominant antiproliferative effect of copanlisib in GIST. In conclusion, copanlisib has single-agent antitumor activity in GIST regardless KIT mutational status or sensitivity to imatinib. Effective KIT inhibition is necessary to achieve synergistic or additive effects with the combination of imatinib and any given PI3K/mTOR pathway inhibition.

Plasma levels of copanlisib of GIST-T1 and GIST-T1/670 xenografts at day 21. Plasma was collected 4 hours after last drug administration.

Tumor volume (mm3) in four treatment arms at different time points in GIST-T1 (A) and GIST-T1/670 (B) in vivo models. Mean {plus minus} SEM.

Drug combination effect of imatinib and copanlisib.

Representative IHC figures across all treatment arms.

Individual IC50s values of copanlisib and imatinib in cell viability and proliferation studies in GIST-T1, GIST-T1/670 and GIST430/654.

Immunohistochemical analysis of the phosphorylation of AKT-serine 473 in A, GIST-T1 and B, GIST-T1/670. Immunohistochemical analysis of the phosphorylation of 4EBP1 in C, GIST-T1 and D, GIST-T1/670. Immunohistochemical analysis of KIT in E, GIST-T1 and F, GIST-T1/670. Graphs are expressed as fold change normalized to vehicle. Differences between treatments was considered to be significant with a p-value *

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 García Valverde, Jordi Rosell, Daniel Pilco Janeta, Sergi Sayols, Claudia Valverde, Anna Esteve, Marta Gut, Jordi Barretina, George D. Demetri, Jonathan A. Fletcher, Joan Carles, Joaquín Arribas, César 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.