Tyrosine kinase inhibitors have revolutionized the treatment of gastrointestinal stromal tumor (GIST), which are mostly driven by mutations in the receptor tyrosine kinase KIT. However, resistance commonly develops, which is associated with second site mutations in KIT. Here, we created the first genetically engineered mouse model of the most common second site mutation, KIT-V654A (mouse KIT-V653A), to study in vivo its oncogenic properties and mechanisms of resistance in order to develop next-generation GIST therapies.

The knock-in strategy consisted of inserting into the endogenous murine Kit locus a targeting vector containing both the KitV558Δ exon 11 mutation and the KitV653A exon 13 mutation under the control of a floxed neomycin-resistance gene-expression cassette which acts similar to a loxP-STOP-loxP cassette. Treatments included imatinib 45 mg/kg i.p., sunitinib 40 mg/kg p.o., and cabozantinib 60 mg/kg p.o. Tumors were assessed by histology, immunohistochemistry, and western blot.

In contrast to our previously published GIST model with a single mutant KitV558Δ allele, the double knock-in KitV558Δ; V653A-neo allele caused perinatal lethality when activated in pre-implantation embryos with a germline EIIa-Cre approach. We therefore sought to restrict the induction of the KitV558Δ;V653A double mutation to the interstitial cells of Cajal (ICC), the cell of origin of GIST, via the essential ICC lineage survival factor ETV1. The KitV558Δ; V653A-neo/+ mouse was crossed with the Etv1Cre-ERT2/+ mouse expressing Cre recombinase under the Etv1-specific promoter. Systemic administration of tamoxifen at birth in KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mice resulted in cecal GIST development with full penetrance. The tumors were histologically similar to human GIST, stained positive for KIT, and displayed active p-KIT signaling. As expected, high-dose imatinib treatment did not inhibit p-KIT Y719 in these GISTs and did not induce a histologic response. Sunitinib and cabozantinib each had significant anti-tumor effects, as assessed by decreased Ki67 index and increased histologic response. Compared to single mutant KitV558Δ-neo/+; Etv1Cre-ERT2/+ mice, double mutant KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mice developed larger cecal GISTs and had decreased survival.

The KitV558Δ; V653A-neo/+; Etv1Cre-ERT2/+ mouse is the first in vivo model of the most common secondary mutation in GIST and the first in vivo demonstration that cell-autonomous expression of mutant KIT in the ICC lineage gives rise to GIST. We have found cabozantinib, an FDA-approved drug, to be an effective therapy in GISTs harboring the V653A mutation. Furthermore, our results suggest the addition of the KIT-V653A second site mutation leads to increased oncogenesis.

Citation Format: Jennifer Q. Zhang, Benedikt Bosbach, Cristina Antonescu, Peter Besmer, Ronald DeMatteo. The KIT-V654A second site mutation confers perinatal lethality and increased oncogenesis in a mouse model of GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1038. doi:10.1158/1538-7445.AM2017-1038