Peter Besmer

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit^V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit^V558Δ/+ mice, double-mutant Kit^{V558Δ;Y567F/Y567F} knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit^{V558Δ;Y719F/Y719F} mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit^V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit^{V558Δ;T669I/+} tumors.

The KIT receptor tyrosine kinase has important roles in hematopoiesis. We have recently produced a mouse model for imatinib resistant gastrointestinal stromal tumor (GIST) carrying the KitV558Δ and KitT669I (human KITT670I) mutations found in imatinib-resistant GIST. The KitV558Δ;T669I/+ mice developed microcytic erythrocytosis with an increase in erythroid progenitor numbers, a phenotype previously seen only in mouse models of polycythemia vera with alterations in Epo or Jak2. Significantly, the increased hematocrit observed in KitV558Δ;T669I/+ mice normalized upon splenectomy. In accordance with increased erythroid progenitors, myeloerythroid progenitor numbers were also elevated in the KitV558Δ;T669I/+ mice. Hematopoietic stem cell (HSC) numbers in the bone marrow (BM) of KitV558Δ;T669I/+ mice were unchanged in comparison to wild-type mice. However, increased HSC numbers were observed in fetal livers and the spleen and peripheral blood of adult KitV558Δ;T669I/+ mice. Importantly, HSC from KitV558Δ;T669I/+ BM had a competitive advantage over wild-type HSC. In response to 5-fluorouracil treatment, elevated numbers of dividing Lin−Sca+ cells were found in the KitV558Δ;T669I/+ BM compared to wild type. Our study demonstrates that signaling from the KitV558Δ;T669I/+ receptor has important consequences in hematopoiesis enhancing HSC self-renewal and resulting in increased erythropoiesis.

Background: A subset of KIT/PDGFRA wild-type gastrointestinal stromal tumors (WT GIST) have been associated with alteration of the succinate dehydrogenase (SDH) complex II function. A recent report identified four non-syndromic, KIT/PDGFRA WT GIST harboring compound heterozygous or homozygous mutations in SDHA encoding the main subunit of the SDH complex II. Methods: Next generation sequencing was applied on five pediatric and one young adult WT GIST, by whole exome capture and SOLiD 3-plus system sequencing. The putative mutations were first confirmed by Sanger sequencing and then screened on a larger panel of 11 pediatric and young adult WT GIST, including 5 in the context of Carney triad. Results: A germline p.Arg31X nonsense SDHA mutation was identified in one of the six cases tested by SOLiD platform. An additional p.D38V missense mutation in SDHA exon 2 was identified by Sanger sequencing in the extended KIT/PDGFRA WT GIST patients cohort. Western blotting showed loss of SDHA expression in the two cases harboring SDHA mutations, while expression being retained in the other WT GIST tumors. Results were further confirmed by immunohistochemistry for both SDHA and SDHB, which showed a concurrent loss of expression of both proteins in SDHA-mutant lesions, while the remaining WT tumors showed only loss of SDHB expression. Conclusions: Germline and/or somatic aberrations of SDHA occur in a small subset of KIT/PDGFRA WT GISTs, outside the Carney’s triad and are associated with loss of both SDHA and SDHB protein expression. Mutations of the SDH complex II are more particularly associated with KIT/PDGFRA WT GIST occurring in young adults. Although pediatric GIST consistently display alterations of SDHB protein expression, further molecular studies are needed to identify the crucial genes involved in their tumorigenesis.

Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the Kit ^V558Δ mutation found in a familial case of GIST and the Kit ^T669I (human KIT ^T670I ) “gatekeeper” mutation found in imatinib-resistant GIST patients. Similar to Kit ^V558∆/+ mice, Kit ^{V558∆;T669I/+} mice developed gastric and colonic interstitial cell of Cajal hyperplasia as well as cecal GIST. In contrast to the single-mutant Kit ^V558∆/+ control mice, treatment of the Kit ^{V558∆;T669I/+} mice with either imatinib or dasatinib failed to inhibit oncogenic Kit signaling and GIST growth. However, this resistance could be overcome by treatment of Kit ^{V558∆;T669I/+} mice with sunitinib or sorafenib. Although tumor lesions were smaller in Kit ^{V558∆;T669I/+} mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in Kit ^{V558∆;T669I/+} mice. Strikingly, the Kit ^{V558∆;T669I/+} mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.

Activating mutations in the Kit receptor tyrosine kinase are associated with gastrointestinal stromal tumor (GIST). Imatinib inhibits Kit and is front-line therapy for GIST. However, imatinib most often elicits a partial response or stable disease, and most GIST patients who initially respond to imatinib eventually acquire resistance. Thus, improved treatment strategies for GIST are needed. We investigated the role of Src family kinases (SFK) in tumorigenesis in a mouse model of human GIST. The SFKs Src and Lyn were active in GIST, and surprisingly, imatinib treatment stimulated their phosphorylation/activation. We show that integrin signaling activates focal adhesion kinase and, consequently, SFKs in GIST and that imatinib enhances integrin signaling, implying a role for the extracellular matrix and integrin signaling in tumor maintenance and imatinib resistance. Dasatinib, an inhibitor of SFKs and Kit, inhibited SFK and focal adhesion kinase activation in GIST but also inhibited Kit and Kit-dependent downstream signaling pathways including phosphoinositide 3-kinase and mitogen-activated protein kinase, but not signal transducer and activator of transcription (STAT) signaling. Whereas dasatinib and imatinib alone both produced a minimal histopathologic response, combination therapy improved their efficacy, leading to increased necrosis in GIST. These results highlight the importance of SFK and STAT signaling in GIST and suggest that the clinical efficacy of imatinib may be limited by the stimulation of integrin signaling. Mol Cancer Res; 8(9); 1271–83. ©2010 AACR.

Kit receptor-activating mutations are critical in the pathogenesis of gastrointestinal stromal tumors (GIST). We investigated mechanisms of oncogenic Kit signaling and the consequences of therapeutic intervention in a mouse model of human GIST. Treatment of GIST mice with imatinib decreased cell proliferation and increased apoptosis in the tumor. Analysis of tumor tissue from imatinib-treated mice showed diminished phosphatidylinositol 3-kinase (PI3-kinase) and mammalian target of rapamycin (mTOR) signaling suggesting that oncogenic Kit signaling critically contributes to the translational response in GIST. Treatment with RAD001 (everolimus), an mTOR inhibitor, diminished the translational response and cell proliferation in tumor lesions, pointing to mTOR inhibition as a therapeutic approach for imatinib-resistant GIST. Analysis of RNA expression profiles in GIST lesions with and without imatinib treatment showed changes in expression of IFN-inducible genes and cell cycle regulators. These results convincingly show that Kit ^V558Δ/+ mice represent a unique faithful mouse model of human familial GIST, and they demonstrate the utility of these mice for preclinical investigations and to elucidate oncogenic signaling mechanisms by using genetic approaches and targeted pharmacological intervention.

The Kit receptor tyrosine kinase functions in hematopoiesis, melanogenesis, and gametogenesis and in interstitial cells of Cajal. We previously identified two upstream hypersensitive site (HS) clusters in mast cells and melanocytes. Here we investigated the roles of these 5' HS sequences in Kit expression using transgenic mice carrying Kit-GFP reporter constructs. In these mice there is close correspondence between Kit-GFP reporter and endogenous Kit gene expression in most tissues analyzed. Deletion analysis defined the 5' upstream HS cluster region as critical for Kit expression in mast cells. Furthermore, chromatin immunoprecipitation analysis in mast cells showed that H3 and H4 histone hyperacetylation and RNA polymerase II recruitment within the Kit promoter and in the 5' HS region were associated with Kit expression. Therefore, the 5' upstream hypersensitivity sites appear to be critical components of locus control region-mediated Kit gene activation in mast cells.

The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (Kit^Y719) or Src kinase (Kit^Y567) have been mutated. Whereas steady-state hematopoiesis is normal in Kit^Y719F/Y719F and Kit^Y567F/Y567F mice, lymphopoiesis is affected differentially. The Kit^Y567F mutation, but not the Kit^Y719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec^®) leads to deficits in pro T and pro B cell development, similar to those seen in Kit^Y567F/Y567F and Kit^W/W mice. The two mutations do not affect embryonic gametogenesis but the Kit^Y719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the Kit^Y567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.

Activating mutations of the KIT juxtamembrane region are the most common genetic events in gastrointestinal stromal tumors (GISTs) and have been noted as independent prognostic factors. The impact of KIT mutation in other regions, such as the extracellular or kinase domains, is not well-defined and fewer than 30 cases have been published to date. One hundred twenty GISTs, confirmed by KIT immunoreactivity, were evaluated for the presence of KIT exon 9, 11, 13, and 17 mutations. The relation between the presence/type of KIT mutation and clinicopathological factors was analyzed using Fisher's exact test and log-rank test. Forty-four % of the tumors were located in the stomach, 47% in the small bowel, 6% in the rectum, and 3% in the retroperitoneum. Overall, KIT mutations were detected in 78% of patients as follows: 67% in exon 11, 11% in exon 9, and none in exon 13 or 17. The types of KIT exon 11 mutations were heterogeneous and clustered in the classic "hot spot" at the 5' end of exon 11. Seven % of cases showed internal tandem duplications (ITD) at the 3' end of exon 11, in a region that we designate as a second hot spot for KIT mutations. Interestingly, these cases were associated with: female predominance, stomach location, occurrence in older patients, and favorable outcome. There were significant associations between exon 9 mutations and large tumor size (P < 0.001) and extragastric location (P = 0.02). Ten of these 13 patients with more than 1-year follow-up have developed recurrent disease. Most KIT-expressing GISTs show KIT mutations that are preferentially located within the classic hot spot of exon 11. In addition, we found an association between a second hot spot at the 3'end of exon 11, characterized by ITDs, and a subgroup of clinically indolent gastric GISTs in older females. KIT exon 9 mutations seem to define a distinct subset of GISTs, located predominantly in the small bowel and associated with an unfavorable clinical course.