Mouse Model Of Gastrointestinal Stromal Tumor Research Articles

A smooth muscle-derived, Braf-driven mouse model of gastrointestinal stromal tumor (GIST): evidence for an alternative GIST cell-of-origin.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the gut. GISTs are thought to arise solely from interstitial cells of Cajal (ICC), a KIT-positive population that controls gut motility. Activating gain-of-function mutations in KIT and PDGFRA are the most frequent driver events, and most of these tumors are responsive to the tyrosine kinase inhibitor imatinib. Less common drivers include mutant BRAFV600E and these tumors are resistant to imatinib. A mouse model of GIST was recently reported using Etv1, the master transcriptional regulator of ICC-intramuscular (IM) and ICC-myenteric (MY), to induce mutant Braf expression. ICC hyperplasia was observed in Etv1CreERT2 ;BrafLSL-V600E/+ mice but loss of Trp53 was required for development of GIST. We identified previously expression of the pan-ErbB negative regulator, LRIG1, in two distinct subclasses of ICC [ICC-deep muscular plexus (DMP) in small intestine and ICC-submucosal plexus (SMP) in colon] and that LRIG1 regulated their development from smooth muscle cell progenitors. Using Lrig1CreERT2 to induce BrafV600E , we observed ICC hyperplasia beyond the confines of ICC-DMP and ICC-SMP expression, suggesting smooth muscle cells as the cell-of-origin. To examine this possibility, we selectively activated BrafV600E in smooth muscle cells. Myh11CreERT2 ;BrafLSL-V600E/+ mice developed not only ICC hyperplasia but also GIST and in the absence of Trp53 disruption. In addition to providing a simpler model for mutant Braf GIST, these results provide conclusive evidence for smooth muscle cells as an alternative cell-of-origin for GIST. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oncogenic kinase inhibition limits Batf3-dependent dendritic cell development and antitumor immunity.

Gastrointestinal stromal tumor (GIST) is driven by an activating mutation in the KIT proto-oncogene. Using a mouse model of GIST and human specimens, we show that intratumoral murine CD103+CD11b- dendritic cells (DCs) and human CD141+ DCs are associated with CD8+ T cell infiltration and differentiation. In mice, the antitumor effect of the Kit inhibitor imatinib is partially mediated by CD103+CD11b- DCs, and effector CD8+ T cells initially proliferate. However, in both mice and humans, chronic imatinib therapy decreases intratumoral DCs and effector CD8+ T cells. The mechanism in our mouse model depends on Kit inhibition, which reduces intratumoral GM-CSF, leading to the accumulation of Batf3-lineage DC progenitors. GM-CSF is produced by γδ T cells via macrophage IL-1β. Stimulants that expand and mature DCs during imatinib treatment improve antitumor immunity. Our findings identify the importance of tumor cell oncogene activity in modulating the Batf3-dependent DC lineage and reveal therapeutic limitations for combined checkpoint blockade and tyrosine kinase inhibition.

Abstract 1038: The KIT-V654A second site mutation confers perinatal lethality and increased oncogenesis in a mouse model of GIST

Abstract 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

Abnormal elevated PTEN expression in the mouse antrum of a model of GIST KitK641E/K641E

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Approximately 85% of GISTs harbor activating mutations of the KIT or PDGFRA receptor tyrosine kinases. PTEN and SHIP2 are major phosphatases that dephosphorylate PI(3,4,5)P 3, one of the intracellular signal pathways downstream of KIT. PTEN is an important tumor suppressor, whereas the involvement of SHIP2 in cancer has been proposed based essentially on cell line studies. We have used a mouse model of GIST, i.e. Kit K641E knock-in mice, resulting in the substitution of a Lys by Glu at position 641 of Kit. In homozygous Kit K641E mice, PTEN-immunoreactivity (ir) in antrum was found in the hyperplastic Kit-ir layer. The same localization was found for SHIP2. Western blot analysis in antrum showed a large increase in PTEN expression in Kit K641E homozygous mice as compared to wild type. In contrast, SHIP2 expression was not affected between the two genotypes. Erk1, but not PKB, phosphorylation appears to be upregulated in Kit K641E homozygous mice. In the human GIST882 imatinib sensitive cell line, both PTEN and SHIP2 were expressed and showed, in part, a nuclear localization. The upregulation of PTEN in antrum in Kit K641E mice might serve as a feedback mechanism to limit PI 3-kinase activation downstream of Kit in a context of oncogenic mutation.

Nanoneoadjuvant Therapy of Gastrointestinal Stromal Tumor (GIST)

Although it is clear that imatinib mesylate is highly effective in metastatic gastrointestinal stromal tumor (GIST), its optimal perioperative use is undefined. McAuliffe and colleagues now report a provocative study of a brief preoperative imatinib therapy. Seventeen patients were randomized to receive 3, 5, or 7 days of 600 mg imatinib, underwent surgery immediately thereafter, and then received 2 years of adjuvant imatinib therapy. Radiologic, histologic, and surgical outcome measures were analyzed. By the authors’ criteria, the response rate to FDG-PET (F-fluoro-2-deoxy-D-glucose positron emission tomography) and dynamic computed tomography (CT) was approximately 70% after transient preoperative therapy. There was no mention of change in tumor density by CT, which has been the basis of the Choi criteria developed at their institution. Presumably it was unchanged. Pathologic analysis of 10 preoperative biopsy samples and their matched surgical specimens revealed that apoptosis (the primary endpoint of the trial) increased by a mean of 12%. Early apoptosis has been shown previously in a mouse model of GIST after treatment with imatinib. Because early necrosis can be observed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining as well, in the present study, it is uncertain whether apoptosis is the only mechanism responsible for tumor death. Interestingly, histologic cytoreduction was not apparent despite the high radiologic response rate. Whether patients without a radiologic response had evidence of apoptosis was not stated. One item that will be of primary practical interest to surgeons is the fact that there was no difference in intraoperative blood loss, even though blood flow was reduced as measured by dynamic CT. This is surprising and disappointing. It may just reflect patient selection, small sample size, and the constraints of using historical controls. The authors also conclude that compared with historical controls who did not receive neoadjuvant imatinib, there was no change in perioperative morbidity. However, the complication rate was 18% in the present series, versus 44% in controls (P = .1). Similarly, postoperative complications did not appear to be altered in 45 patients with GIST treated with neoadjuvant imatinib on a Radiation Therapy Oncology Group phase II trial. Larger studies with proper controls are needed to identify whether neoadjuvant imatinib alters the rate of surgical complications. Long-term outcome was assessed, although the analysis is limited by the mixture of patients with primary (n = 13) and metastatic (n = 6) disease. Recurrence-free survival exceeded 90% at 1 year, consistent with the preliminary data from the American College of Surgeons Oncology Group intergroup Z9001 adjuvant trial and metastatic GIST trials. Although the current trial has primarily provided biologic and radiologic correlative information regarding neoadjuvant imatinib therapy in GIST, can the data be used in clinical practice? Is there a rationale to treat patients with resectable primary GIST for 1 week with neoadjuvant imatinib if intraoperative blood loss and perioperative morbidity are not affected? In patients with resectable primary GIST, there is a theoretical possibility that intraoperative tumor dissemination may be decreased by diminished tumor blood flow. This unproven benefit must be weighed against the rare occurrence of intraperitoneal tumor rupture induced by imatinib therapy. As more targeted drugs are developed, the other principal reason to use nanoneoadjuvant therapy for resectable GIST could be to select the most effective drug for adjuvant therapy in a given patient. However, until there is a strong clinical rationale, nanoneoadjuvant therapy for GIST may otherwise have a short life span. Society of Surgical Oncology 2009 Published Online: 24 January 2009