Human Gastrointestinal Stromal Tumors Cell Lines Research Articles

Multiple intratumoral sources of kit ligand promote gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit or PDGFRA receptor. While highly effective, tyrosine kinase inhibitors (TKIs) are not curative. The natural ligand for the Kit receptor is Kit ligand (KitL), which exists in both soluble and membrane-bound forms. While KitL is known to stimulate human GIST cell lines in vitro, we used a genetically engineered mouse model of GIST containing a common human KIT mutation to investigate the intratumoral sources of KitL, importance of KitL during GIST oncogenesis, and contribution of soluble KitL to tumor growth in vivo. We discovered that in addition to tumor cells, endothelia and smooth muscle cells produced KitL in KitV558Δ/+ tumors, even after imatinib therapy. Genetic reduction of total KitL in tumor cells of KitV558Δ/+ mice impaired tumor growth in vivo. Similarly, genetic reduction of tumor cell soluble KitL in KitV558Δ/+ mice decreased tumor size. By RNA sequencing, quantitative PCR, and immunohistochemistry, KitL expression was heterogeneous in human GIST specimens. In particular, PDGFRA-mutant tumors had much higher KitL expression than Kit-mutant tumors, suggesting the benefit of Kit activation in the absence of mutant KIT. Serum KitL was higher in GIST patients with tumors resistant to imatinib and in those with tumors expressing more KitL RNA. Overall, KitL supports the growth of GIST at baseline and after imatinib therapy and remains a potential biomarker and therapeutic target.

Oncogenic KIT Modulates Type I IFN-Mediated Antitumor Immunity in GIST.

Type I IFNs are implicated in tumor immunogenicity and response to systemic therapy, but their interaction with oncogene signaling is not well understood. Here, we studied oncogenic KIT, which drives gastrointestinal stromal tumor (GIST), the most common sarcoma. Using mouse models of GIST, we found that KIT inhibition reduced type I IFN production and signaling, which downregulated tumor MHC class I expression. Absence of type I IFN signaling increased tumor size, in part due to CD8+ T-cell impairment. Oncogenic KIT was required for GIST type I IFN signal transduction via STAT1. In human GIST cell lines and surgical specimens, type I IFN signaling contributed to human lymphocyte antigen class I expression and correlated with tumor immunogenicity. Augmenting the type I IFN response partially compensated for the immunosuppressive effects of KIT inhibition. Thus, KIT signaling contributes to type I IFN signaling, whereas KIT inhibition attenuates tumor immunogenicity and is partly rescued by innate immune stimulation.See related Spotlight on p. 489.

Enhancer of zeste homolog 2-mediated paired box 8 methylation promotes gastrointestinal stromal tumor progression through Wnt4 downregulation.

Gastrointestinal stromal tumor (GIST) is a refractory malignant tumor without satisfactory therapy. In recent years, aberrant gene methylation has been highlighted as an inducer for tumor progression. In this study, we explored whether enhancer of zeste homolog 2 (EZH2)-mediated paired box 8 (PAX8) methylation affects GIST development through regulation of Wnt4. A total of 50 cases of GIST tissues were collected and the human GIST cell lines were cultured. PAX8 methylation was examined using MS-PCR. Following loss- and gain-function approaches, GIST cell proliferation, migration, invasion, and apoptosis were examined by CCK-8 assay, Transwell assay and flow cytometry. The expression of proliferation related factors and apoptosis related factors was determined. Finally, xenograft tumors in nude mice were observed to examine in vivo tumorigenicity of GIST cells. Downregulated PAX8 and upregulated EZH2 expression was found in GIST tissues. Overexpression of PAX8 or suppression of PAX8 methylation using DNA methyltransferase inhibitor 5-Aza-dC inhibited the proliferation, migration, and invasion of GIST cells while promoting their apoptosis (diminished PCNA, Ki67 and Bcl-2, elevated Bax, and cleaved caspase-3). EZH2 promoted PAX8 methylation to inhibit its expression. Downregulated PAX8 decreased Wnt4 expression to accelerate GIST progression both in vitro and in vivo. Collectively, EZH2 inhibits PAX8 expression by promoting its methylation, which thus downregulates Wnt4 expression, thereby promoting the development of GIST.

Indocyanine green-labeled dasatinib as a new fluorescent probe for molecular imaging of gastrointestinal stromal tumors.

It is difficult to differentiate gastrointestinal stromal tumors (GISTs) from other subepithelial lesions under gastrointestinal endoscopy. Because most GISTs express tyrosine kinase receptor c-KIT, fluorescence-labeled c-KIT-specific tyrosine kinase inhibitors seem to be useful agents for molecular imaging of GIST. We aimed to develop a near-infrared fluorescent imaging technology for GIST targeting c-KIT using the novel fluorescent probe indocyanine green-labeled dasatinib (ICG-dasatinib) and to investigate the antitumor effect of ICG-dasatinib on GIST cells. Indocyanine green-labeled dasatinib was synthesized by labeling linker-induced dasatinib with ICG derivative 3-indocyanine-green-acyl-1,3-thiazolidine-2-thione. Human GIST cell lines GIST-T1 and GIST-882M were incubated with ICG-dasatinib and observed by fluorescent microscopy. GIST cells were incubated with ICG-dasatinib, unlabeled dasatinib, or imatinib, and cell viabilities were evaluated. Subcutaneous GIST model mice or orthotopic GIST model rats were intravenously injected with ICG-dasatinib and observed using an IVIS Spectrum. Strong fluorescent signals of ICG-dasatinib were observed in both GIST cell lines in vitro. IC50 values for ICG-dasatinib, unlabeled dasatinib, and imatinib were 13.9, 1.17, and 16.2nM in GIST-T1 and 26.6, 3.63, and 47.6nM in GIST-882M cells, respectively. ICG-dasatinib accumulated in subcutaneous xenografts in mice. Fluorescent signals were also observed in liver and gallbladder, indicating biliary excretion; however, fluorescence intensity of tumors was significantly higher than that of intestine after washing. Strong fluorescent signals were observed in orthotopic xenografts through the covering normal mucosa in rats. Indocyanine green-labeled dasatinib could visualize GIST cells and xenografted tumors. The antitumor effect of ICG-dasatinib was preserved to the same degree as imatinib.

The role of novel fusion genes in human GIST cell lines derived from imatinib-resistant GIST patients: A therapeutic potential of fusion gene

Gastrointestinal stromal tumor (GIST) is the most common sarcoma in the gastrointestinal (GI) tract. Approximately 85% of the GIST is associated with a c-KIT mutation. A few GISTs show mutations in the gene encoding platelet-derived growth factor receptor alpha (PDGFR α or PDGFRA) without c-KIT gene mutation. GIST without c-KIT or PDGFRA mutations, which called wild type GIST, is about 5–10% of the total GIST. Fusion genes were also reported as one of the factors associated with carcinogenesis and drug resistance. With five cell lines derived from imatinib-resistant patients, novel fusion genes were identified from RNA sequencing and both physiological role and therapeutic potential were elucidated. Next-generation sequencing (NGS) analysis and lentiviral transduction were used to effect of fusion gene on GISTs. All the GIST cell lines carried c-KIT-positivity. Three different fusion gene analysis methods were used to find candidate fusion genes, including EIF3K-ACTN4, SYNCRIP-SNX14 and EXOC2-AK7. A novel interchromosomal fusion gene of the candidates, especially EXOC2-AK7, was confirmed in both tissue and cell line. The transduction of fusion gene increased the proliferation compared with the control group. Additionally, the fusion gene increased wound coverage capability. The fusion gene-transduced cell lines were more sensitive than the control group in the treatment of imatinib. In conclusion, five different imatinib-resistant GIST cell lines including the EXOC2-AK7 fusion gene derived from GIST-R5 represent important research tools for the investigation of cancer cell mechanisms underlying drug resistance and genetic variation. Furthermore, our study may facilitate pre-clinical studies of new therapeutic strategies.

Abstract B27: Kit inhibition decreases tumoral MHC class I expression in gastrointestinal stromal tumors through reduction of type I interferon signaling

Abstract Introduction: MHC class I (MHC I) expression is necessary for the antitumoral lymphocyte response but its relationship to oncogene signaling is not well characterized. Gastrointestinal stromal tumor (GIST) is a common sarcoma caused by an activating Kit mutation and is sensitive to the specific Kit inhibitor imatinib. Using KitV558Δ/+ mice, a murine model of GIST, we found imatinib reduced MHC I expression on tumor cells, which we hypothesized was due to attenuation of type I interferon (IFN) signaling in the setting of oncogene inhibition. Methods: Using flow cytometry and Western blot, we studied tumoral MHC I expression in human GIST cell lines T1 and HG129 under conditions of IFN stimulation and imatinib inhibition. We applied the same methods in vivo in KitV558Δ/+ mice. To show that IFN signaling was responsible for tumoral MHC I expression, we used a blocking anti-IFNAR antibody to abrogate IFN signaling in KitV558Δ/+ mice. Finally, we validated our findings with RNA seq of 35 Kit exon 11 human GIST. Results: In human GIST cell lines, IFNα (1000U/mL) stimulated MHC I expression at 24h as measured on cell surface by mean florescence intensity (MFI) and by Western blot. This was accompanied by increased Stat1 and pStat1 protein. Imatinib (100nM) inhibited Stat1 activity and reduced MHC I expression by 12-34% on MFI, with and without IFNα stimulation. When Stat1 was knocked down with SiRNA, imatinib did not further decrease MHC I. In vivo, KitV558Δ/+ mice treated with imatinib had 50% reduction in tumoral MHC I expression MFI by flow (n=5/group). Concurrently, imatinib diminished mRNA expression of IFNb1 in bulk KitV558Δ/+ tumors by 8-fold. To demonstrate that IFN signaling contributed to GIST MHC I expression, we administered a blocking anti-IFNAR antibody in KitV558Δ/+ mice and found MHC I decreased by 34% on MFI, along with decreased pStat1 on western blot. In human GIST, imatinib significantly decreased MHC I gene expression on RNA seq (p = 0.03) and reduced IFNb1 mRNA by 77% (p = 0.04). Furthermore, there was significant correlation between MHC I genes and Stat1 expression (p< 0.0001). Taken together, this suggests that oncogene-dependent IFN signaling is responsible for MHC I expression in GIST. Conclusion: Kit oncogene inhibition reduced MHC I expression in GIST through decreased type I interferon production and signaling via Stat1. Thus, targeted therapies may reduce the antitumor immune response. Citation Format: Mengyuan Liu, Benjamin D Medina, Geraldo A Vitiello, Mark S Etherington, Nesteene J. Param, Mia E DiLolle, Timothy G. Bowler, Ferdinando Rossi, Ronald P. DeMatteo. Kit inhibition decreases tumoral MHC class I expression in gastrointestinal stromal tumors through reduction of type I interferon signaling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B27.

Abstract 263: KITlow cells mediate Imatinib resistance and disease persistence in gastrointestinal stromal tumor

Abstract INTRODUCTION: Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by Imatinib (IM). However, IM does not cure GIST; adjuvant therapy only delays recurrence in high-risk tumors. Therefore, we hypothesized that GIST contains cells with primary IM resistance, representing a critical target for overcoming disease persistence. METHODS: Gene Set Enrichment Analysis (GSEA) was performed with publicly available data (GSE1596636). Human GIST cell lines (GIST-T1 and 882) and primary KIT-mutant GIST (IRB #090401) were analyzed by fluorescence-activated cell sorting (FACS) to sort KITloCD34+ (KITlo) vs KIThiCD34+ (KIThi) subpopulations using anti-human KIT and CD34 antibodies. RNA was extracted from cell lysates for analysis by quantitative RT-PCR. Cell viability was assessed by CellTiter-Glo or MTT assays following drug treatments. RESULTS: We performed GSEA on 27 matched GISTs comparing pre- and post-neoadjuvant IM treatment (RTOG S0132). Post-IM samples had 50% lower KIT expression (P=0.002) and were enriched in “cancer stem cell” and Axl/Gas6/NF-κB signaling gene signatures. Similarly, in vitro IM treatment of GIST cell lines resulted in 2-fold reduction of KIT expression and 1.9 to 4.4-fold increased expression of stem-associated transcription factors (SATFs: OCT4, SOX2, KLF4, NANOG). Parallel FACS analysis of IM-treated GIST cell lines (125 nM, 72-h) demonstrated the presence of a KITlo subpopulation (GIST-T1: 0.75%; GIST882: 4%) while untreated cells had 3- to 11-fold fewer KITlo cells. Primary human GISTs also had KITlo cells by FACS (4-8%; N=4), suggesting that the population is not an artifact of in vitro culture. KITlo GIST882 cells were IM-resistant (IC50: KITlo 3641 nM vs KIThi 180 nM) and significantly overexpressed all SATFs by qRT-PCR (2.0 to 4.1-fold; P<0.001) consistent with RTOG S0132 analysis. Moreover, RNAseq confirmed that KITlo cells (relative to KIThi) are enriched in factors present in the “cancer stem cell” gene signature identified by GSEA (KLF4, CCL5, ATF3, JUN, IFIT1, PMP22). Lastly, we tested candidate drugs against targets overexpressed in IM-treated tumors. Unsorted cells were pre-treated with IM (185 nM, 48-h) to enrich for KITlo cells. Subsequent treatment with R428 (AXL inhibitor) or bardoxolone (NF-κB inhibitor) resulted in 70% (R428, 5 μM) and 80% (bardoxolone, 5 μM) cell death. Finally, FACS-sorted KITlo cells were sensitive to both drugs (50% and 88% killing, respectively). CONCLUSIONS: KITlo cells are a distinct subpopulation in human GIST with intrinsic IM-resistance and may represent a novel mechanism of GIST persistence. These cells overexpress stem cell transcriptional programs, including the Axl/Gas6/NF-κB pathway, which represent novel therapeutic targets in vitro. Further studies are needed to explore the in vivo efficacy of combination or sequential targeting of KITlo cells in GIST. Citation Format: Sudeep Banerjee, Chih-Min Tang, Mayra Yebra, Kwat Medetgul, Adam M. Burgoyne, Pablo Tamayo, Robert Wechsler-Reya, Jason K. Sicklick. KITlow cells mediate Imatinib resistance and disease persistence in gastrointestinal stromal tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 263.

Abstract 1260: Inhibition of DOG1, or TMEM16A, results in antitumoral effects in gastrointestinal stromal tumors (GIST) in vitro

Abstract DOG1/TMEM16A, over-expressed in >95% of all GISTs, is a calcium-activated chloride channel that has gained attention as a promising drug target due to its involvement in several tumor-physiological processes. Two well-established human GIST cell lines (GIST-T1 and GIST882) were used to study DOG1 inhibitors effect on viability, colony formation, and cell cycle. In this study, we show that specific DOG1/TMEM16A-inhibitors (T16inh-A01 and CaCCinh-A01) decrease chloride currents, and reduces cell viability in GIST cells. However, only CaCCinh-A01 changes the distribution of the cell cycle in GIST cells, putting them into G1 cell cycle arrest, and increases sub-G1 phase population in GIST882 cells. CaCCinh-A01 strongly reduces the colony forming ability of the cells, whereas limited effect by T16inh-A01. Conclusions: DOG1/TMEM16A inhibition has antitumoral effects in GIST cells in vitro, and could potentially serve as a target for GIST therapy. CaCCinh-A01 seems to be the more potent inhibitor, presumably due to its effect not only on chloride currents but also on DOG1/TMEM16A expression. Citation Format: Robin Fröbom, Erik Berglund, Robert Bränström. Inhibition of DOG1, or TMEM16A, results in antitumoral effects in gastrointestinal stromal tumors (GIST) in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1260.

Mitochondrial Inhibition Augments the Efficacy of Imatinib by Resetting the Metabolic Phenotype of Gastrointestinal Stromal Tumor.

Purpose: Imatinib dramatically reduces gastrointestinal stromal tumor (GIST) 18F-FDG uptake, providing an early indicator of treatment response. Despite decreased glucose internalization, many GIST cells persist, suggesting that alternative metabolic pathways are used for survival. The role of mitochondria in imatinib-treated GIST is largely unknown.Experimental Design: We quantified the metabolic activity of several human GIST cell lines. We treated human GIST xenografts and genetically engineered KitV558del/+ mice with the mitochondrial oxidative phosphorylation inhibitor VLX600 in combination with imatinib and analyzed tumor volume, weight, histology, molecular signaling, and cell cycle activity. In vitro assays on human GIST cell lines were also performed.Results: Imatinib therapy decreased glucose uptake and downstream glycolytic activity in GIST-T1 and HG129 cells by approximately half and upregulated mitochondrial enzymes and improved mitochondrial respiratory capacity. Mitochondrial inhibition with VLX600 had a direct antitumor effect in vitro while appearing to promote glycolysis through increased AKT signaling and glucose transporter expression. When combined with imatinib, VLX600 prevented imatinib-induced cell cycle escape and reduced p27 expression, leading to increased apoptosis when compared to imatinib alone. In KitV558del/+ mice, VLX600 alone did not induce tumor cell death, but had a profound antitumor effect when combined with imatinib.Conclusions: Our findings show that imatinib alters the metabolic phenotype of GIST, and this may contribute to imatinib resistance. Our work offers preclinical proof of concept of metabolic targeting as an effective strategy for the treatment of GIST. Clin Cancer Res; 24(4); 972-84. ©2017 AACR.

Abstract A34: Inhibition of autophagy sensitizes gastrointestinal stromal tumor cells to TKI/Bcl-2 inhibitors-induced apoptosis

Abstract Background: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the GI tract. Most GISTs are driven by mutations in KIT or platelet-derived growth factor receptor-α (PDGFRA), which responds well to imatinib, a tyrosine kinase inhibitor (TKI) that blocks KIT and PDGFR-α signaling. Bcl-2 family plays a critical role in the regulation of cell apoptosis in GISTs. ABT-737 as an inhibitor of Bcl-2/Bcl-xL can result in a time- and dose-dependent activation of apoptosis. Autophagy is a key mechanism to promote tumor cells survival, inhibition of which can induce the cell death in GISTs. Chloroquine, an antimalarial drug, has been also identified as an autophagy inhibitor. In this study, we assessed the combinational effects of imatinib, ABT-737, and chloroquine in GIST cells. Methods: Human GIST cell lines, GIST-T1 and GIST-882, were employed in our study. Cells were treated with imatinib, ABT-737, and chloroquine either separately or in different combinations. Cell viability was tested by means of MTS and synergistic effects were analyzed by isobologram software. The levels of related proteins of apoptosis (PARP, Caspase-3) and autophagy (LC3-II, beclin-1) were measured by Western blot. Cell apoptosis and cell cycle were tested by flow cytometry. Results: Cell viability assay indicated that the cell survival percentage of double- or triple-drug combinations dramatically decreased (less than 5%) compared to that of single-drug treatment (42%, 36%, or 12%) (P<0.05). Isobologram analysis revealed a strongly synergistic drug interaction, with combination indices (CI) < 0.5 in most of double and triple combinations. Importantly, triple-drugs combination showed even more strongly synergistic effects compared to double-drugs combinations (CI =0.204 vs. 0.309 or 0.356, P<0.05). Cell apoptosis assay showed that the cell apoptosis percentage of double- (32.9% or 36.6%) or triple-drugs combinations (66.5%) also significantly increased over that of single-drug treatments (6.1%, 6.1%, or 13.1%) (P<0.05). Finally, Western blot discovered that double- or triple-drugs combinations increased the cleavage of PARP as well as expression of Caspase-3, but inhibited autophagy in comparison to single-drug treatments. Conclusions: The combination of imatinib, ABT-737, and chloroquine has collaborative effects on the treatment of GISTs in vitro. The combined strategy may enhance the clinical efficacy, which provides a rationale for the clinical evaluation of these drug combinations in GISTs treatment. Citation Format: Shuchao Zhang, Guozhi Hu, Ana cristina Paz-mejia, Luyuan Li, Jonathan C. Trent. Inhibition of autophagy sensitizes gastrointestinal stromal tumor cells to TKI/Bcl-2 inhibitors-induced apoptosis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A34.

A Novel Theranostic Combination of Near-infrared Fluorescence Imaging and Laser Irradiation Targeting c-KIT for Gastrointestinal Stromal Tumors.

It is difficult to distinguish gastrointestinal stromal tumors (GISTs) from other types of submucosal tumors under conventional gastrointestinal endoscopy. We aimed to detect GISTs by molecular fluorescence imaging using a near-infrared (NIR) photosensitizer (IR700)-conjugated anti-c-KIT antibody and to treat GISTs by photoimmunotherapy with NIR irradiation as a non-invasive theranostic procedure. We also investigated the therapeutic mechanisms.Methods: Human GIST cell lines GIST-T1 and GIST-882M were incubated with IR700-conjugated anti-c-KIT antibody, IR700-12A8, and observed by confocal laser microscopy. Mice with GIST-T1 xenografts or rats with orthotopic xenografts were injected with IR700-12A8 or AF488-conjugated antibody, and observed under IVIS or autofluorescence imaging (AFI) endoscopy. GIST cells were treated with IR700-12A8 and NIR light in vitro and vivo, and cell viability, histology and apoptosis were evaluated.Results: Strong red fluorescence of IR700-12A8 was observed on the cell membrane of GIST cells and was gradually internalized into the cytoplasm. Tumor-specific accumulation of IR700-12A8 was observed in GIST-T1 xenografts in mice. Under AFI endoscopy, a strong fluorescence signal was observed in orthotopic GIST xenografts in rats through the normal mucosa covering the tumor. The percentage of dead cells significantly increased in a light-dose-dependent manner and both acute necrotic and late apoptotic cell death was observed with annexin/PI staining. Cleaved PARP expression was significantly increased after IR700-12A8-mediated NIR irradiation, which was almost completely reversed by NaN3. All xenograft tumors (7/7) immediately regressed and 4/7 tumors completely disappeared after IR700-12A8-mediated NIR irradiation. Histologic assessment and TUNEL staining revealed apoptosis in the tumors.Conclusion: NIR fluorescence imaging using IR700-12A8 and subsequent NIR irradiation could be a very effective theranostic technology for GIST, the underlying mechanism of which appears to involve acute necrosis and supposedly late apoptosis induced by singlet oxygen.

ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma, often resulting from a KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutation. The lineage transcription factor ETV1 is expressed similarly in GISTs regardless of malignant potential. Although the related transcription factor ETV4 has been associated with metastasis and tumor progression in other cancers, its role in GIST is unknown. In this study, we found that ETV4 levels were high in a subset of human GISTs and correlated with high mitotic rate. Through Gene Set Enrichment Analysis in selected human GISTs, we identified a relationship between ETV4 levels and β-catenin signaling, especially in advanced GISTs. GIST specimens with high ETV4 levels overexpressed cell cycle regulating genes and had aberrant activation of the canonical Wnt pathway. In human GIST cell lines, ETV4 RNA interference suppressed cell cycle genes and Wnt/β-catenin signaling. ETV4 knockdown also reduced tumor cell proliferation, invasion, and tumor growth in vivo. Conversely, ETV4 overexpression increased cyclin D1 expression and Wnt/β-catenin signaling. Moreover, we determined that ETV4 knockdown destabilized nuclear β-catenin and increased its degradation via COP1, an E3 ligase involved in both ETV4 and β-catenin turnover. Aberrant accumulation of ETV4 and nuclear β-catenin was found in patient derived xenografts created from metastatic GISTs that became resistant to tyrosine kinase inhibitors. Collectively, our findings highlight the significance of ETV4 expression in GIST and identify ETV4 as a biomarker in human GISTs.

Inhibition of autophagy sensitizes gastrointestinal stromal tumor cells to TKI/Bcl-2 inhibitors-induced apoptosis.

11036 Background: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the GI tract. Most GISTs are driven by mutations in KIT or platelet-derived growth factor receptor-α (PDGFRA), which responds well to imatinib, a tyrosine kinase inhibitor (TKI) that blocks KIT and PDGFR-α signaling. Bcl-2 family plays a critical role in the regulation of cell apoptosis in GISTs. ABT-737 as an inhibitor of Bcl-2/Bcl-xL can result in a time and dose-dependent activation of apoptosis. Autophagy is a key mechanism to promote tumor cells survival, inhibition of which can induce the cell death in GISTs. Chloroquine, an antimalarial drug, has been also identified as an autophagy inhibitor. In this study, we assessed the combinational effects of imatinib, ABT-737 and chloroquine in GIST cells. Methods: Human GIST cell lines, GIST-T1 and GIST-882, were employed in our study. Cells were treated with imatinib, ABT-737 and chloroquine either separately or in different combinations. Cell viability was tested by means of MTS and synergistic effects were analyzed by isobologram software. The levels of related proteins of apoptosis (PARP, Caspase-3) and autophagy (LC3-II, beclin-1) were measured by western blot. Cell apoptosis and cell cycle were tested by flow cytometry. Results: Cell viability assay indicated cell survival percentage of double or triple drug combinations ( < 5%) dramatically decreased compared to single drug treatments (42%, 36% or 12%) ( P< 0.05). Isobologram analysis revealed triple drugs combination had stronger synergistic effects than double drugs combinations (CI = 0.204 vs 0.309 or 0.356, P< 0.05). Cell apoptosis percentage of double (32.9% or 36.6%) or triple drugs combinations (66.5%) significantly increased compared to single treatments (6.1%, 6.1% or 13.1%) ( P< 0.05). Western blot showed drugs combinations increased cleavage of PARP and Caspase-3 levels, but inhibited autophagy. Conclusions: The combination of imatinib, ABT-737 and chloroquine has collaborative effects on the treatment of GISTs in vitro. The combined strategy may enhance the clinical efficacy, which provides a rationale for the clinical evaluation of these drug combinations in GISTs treatment.

PD-1/PD-L1 Blockade Enhances T-cell Activity and Antitumor Efficacy of Imatinib in Gastrointestinal Stromal Tumors.

Tyrosine kinase inhibitors are effective in gastrointestinal stromal tumors (GISTs) but often are of transient benefit as resistance commonly develops. Immunotherapy, particularly blockade of the inhibitory receptor programmed death 1 (PD-1) or the ligand programmed death ligand 1 (PD-L1), has shown effectiveness in a variety of cancers. The functional effects of PD-1/PD-L1 blockade are unknown in GISTs. We analyzed tumor and matched blood samples from 85 patients with GISTs and determined the expression of immune checkpoint molecules using flow cytometry. We investigated the combination of imatinib with PD-1/PD-L1 blockade in KitV558Δ/+ mice that develop GISTs. The inhibitory receptors PD-1, lymphocyte activation gene 3, and T-cell immunoglobulin mucin-3 were upregulated on tumor-infiltrating T cells compared with T cells from matched blood. PD-1 expression on T cells was highest in imatinib-treated human GISTs. Meanwhile, intratumoral PD-L1 expression was variable. In human GIST cell lines, treatment with imatinib abrogated the IFNγ-induced upregulation of PD-L1 via STAT1 inhibition. In KitV558Δ/+ mice, imatinib downregulated IFNγ-related genes and reduced PD-L1 expression on tumor cells. PD-1 and PD-L1 blockade in vivo each had no efficacy alone but enhanced the antitumor effects of imatinib by increasing T-cell effector function in the presence of KIT and IDO inhibition. PD-1/PD-L1 blockade is a promising strategy to improve the effects of targeted therapy in GISTs. Collectively, our results provide the rationale to combine these agents in human GISTs. Clin Cancer Res; 23(2); 454-65. ©2016 AACR.

Opposing roles of KIT and ABL1 in the therapeutic response of gastrointestinal stromal tumor (GIST) cells to imatinib mesylate.

Most gastrointestinal stromal tumors (GISTs) are caused by activating mutations of the KIT receptor tyrosine kinase. The small molecule inhibitor imatinib mesylate was initially developed to target the ABL1 kinase, which is constitutively activated through chromosomal translocation in BCR-ABL1-positive chronic myeloid leukemia. Because of cross-reactivity of imatinib against the KIT kinase, the drug is also successfully used for the treatment of GIST. Although inhibition of KIT clearly has a major role in the therapeutic response of GIST to imatinib, the contribution of concomitant inhibition of ABL in this context has never been explored. We show here that ABL1 is expressed in the majority of GISTs, including human GIST cell lines. Using siRNA-mediated knockdown, we demonstrate that depletion of KIT in conjunction with ABL1 – hence mimicking imatinib treatment – leads to reduced apoptosis induction and attenuated inhibition of cellular proliferation when compared to depletion of KIT alone. These results are explained by an increased activity of the AKT survival kinase, which is mediated by the cyclin-dependent kinase CDK2, likely through direct phosphorylation. Our results highlight that distinct inhibitory properties of targeted agents can impede antitumor effects and hence provide insights for rational drug development. Novel KIT-targeted agents to treat GIST should therefore comprise an increased specificity for KIT while at the same time displaying a reduced ability to inhibit ABL1.

Abstract 2690: DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft models

Abstract Introduction: KIT kinase mutations are causative of a number of human cancers, including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), mast cell leukemia (MCL), and subtypes of melanoma and acute myeloid leukemia (AML). DCC-2618 is a robust Type II switch pocket control inhibitor which potently inhibits exon 17 KIT mutations that are resistant to conventional TKIs. Experimental procedures: DCC-2618 was tested for inhibition of KIT isoforms using a standard PK/LDH coupled spectrophotometric assay. CHO cells were transiently transfected to express mutant KIT or PDGFRα constructs. Transfected cells were treated with a range of DCC-2618 and levels of phosphorylated KIT or PDGFRα in cell lysates were determined by ELISA or western blot. Cell proliferation of several cell lines was measured using the fluorescent dye resazurin. Experiments were performed in triplicate. In vivo xenograft models were performed at Molecular Imaging, Inc. (Ann Arbor, MI) or Molecular Response, LLC (San Diego, CA). Summary of results: DCC-2618 inhibited various forms of KIT with nanomolar potency: WT (IC50 4 nM), V654A (8 nM), T670I (18 nM), D816H (5 nM), D816V (14 nM). In CHO cells transiently transfected with both single and double (primary/secondary) KIT mutants, DCC-2618 robustly inhibited exon 17, exon 9/13, exon 9/14, and exon 9/17 KIT mutants, as well as exon 11/17 KIT mutants, including exon 17 D816V, D816G, D820A, D820E, D820Y, N822K, N822Y, N822H, and Y823D primary or secondary mutations. DCC-2618 inhibited wild type KIT phosphorylation in the MO7e cell line (IC50 36 nM). DCC-2618 potently inhibited KIT activation in human GIST cell lines, including GIST T1 (exon 11 deletion, IC50 2 nM), GIST 430 (exon 11 deletion/exon 13 V654A, IC50 7 nM), and GIST 48 (exon 11 V560D/exon 17 D820A, IC50 53 nM). In the murine mastocytosis P815 cell line expressing the exon 17 D816Y mutation, DCC-2618 potently inhibited cell proliferation (IC50 2 nM). In vivo, DCC-2618 administration at 50 mg/kg afforded an ED90 for inhibition of KIT phosphorylation in the GIST T1 xenograft model, corresponding to an EC90 concentration of ∼ 470 ng/mL. When give twice daily, this oral dose resulted in almost complete tumor stasis. This dose of DCC-2618 produced tumor regressions in a patient derived xenograft (PDX) GIST expressing KIT exon 11 delW557K558/exon 17 Y823D, and also in a KIT exon 17 N822K AML xenograft model. Conclusion: DCC-2618 is a potent inhibitor of singly and doubly mutated KIT characterized by primary exon 9 or exon 11 mutations paired with secondary mutations in exons 13, 14 or 17. DCC-2618 inhibits exon 17 mutations, including the D816V mutation refractory to currently marketed KIT inhibitors. DCC-2618 has the potential to treat KIT mutant-driven cancers including GIST, systemic mastocytosis, AML, or melanoma. DCC-2618 has been selected for formal IND-enabling clinical development. Citation Format: Bryan D. Smith, Molly M. Hood, Scott C. Wise, Michael D. Kaufman, Wei-Ping Lu, Thomas Rutkoski, Daniel L. Flynn, Michael C. Heinrich. DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2690. doi:10.1158/1538-7445.AM2015-2690

Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1

In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1. We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation. KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration= 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA. In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.

Increased KIT Inhibition Enhances Therapeutic Efficacy in Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and a model of targeted molecular therapy. GIST depends on oncogenic KIT signaling and responds to the tyrosine kinase inhibitor imatinib. However, imatinib is rarely curative. We hypothesized that PLX3397, which inhibits KIT and colony-stimulating-factor-1 receptor (CSF1R), would be more efficacious than imatinib in GIST by also depleting tumor-associated macrophages, which are generally thought to support tumor growth. We treated Kit(V558del/+) mice that develop GIST or mice with subcutaneous human GIST xenografts with imatinib or PLX3397 and analyzed tumor weight, cellular composition, histology, molecular signaling, and fibrosis. In vitro assays on human GIST cell lines were also performed. PLX3397 was more effective than imatinib in reducing tumor weight and cellularity in both Kit(V558del)(/+) murine GIST and human GIST xenografts. The superiority of PLX3397 did not depend on depletion of tumor-associated macrophages, because adding CSF1R inhibition did not improve the effects of imatinib. Instead, PLX3397 was a more potent KIT inhibitor than imatinib in vitro. PLX3397 therapy also induced substantial intratumoral fibrosis, which impaired the subsequent delivery of small molecules. PLX3397 therapy has greater efficacy than imatinib in preclinical GIST models and warrants study in patients with GIST. The resultant intratumoral fibrosis may represent one of the barriers to achieving complete tumor eradication.

MicroRNA-218 inhibits gastrointestinal stromal tumor cell and invasion by targeting KIT

The objectives of this study were to detect the expressions of microRNA-218 (miR-218) in human gastrointestinal stromal tumor (GIST) tissues and cells and explore its effects on the biological features of GIST-T1 cells and the expression of its target gene KIT, so as to provide new insights for GIST treatment. Using quantitative real-time polymerase chain reaction (qRT-PCR), we detected the expressions of miR-218 in the tissues and adjacent tissues of GIST and in the GIST cell lines including GIST882, GIST430, GIST48, and GIST-T1. Forty-eight hours after the miR-218 mimic was transfected into the GIST-T1 cells, the expression of miR-218 in the GIST-T1 cells was detected by qRT-PCR. The effect of miR-218 on the GIST-T1 cell viability was detected using MTT. The effect of miR-218 on the proliferation and apoptosis of GIST-T1 cell was analyzed using flow cytometry. Transwell invasion chamber was applied to detect the effect of miR-218 on the invasion of GIST-T1 cells. KIT was identified to be a target gene of miR-218 by the luciferase reporter enzyme system, and the effect of miR-218 on the expression of KIT protein in cells was determined using Western blotting. As shown by qRT-PCR, compared with that in the GIST adjacent tissue, the expressions of miR-218 in the tumor tissue and GIST cell lines were significantly decreased (P < 0.0001). Compared with the control group, the expression of miR-218 increased significantly in GIST-T1 cells transfected with miR-218 mimic for 48h (P < 0.01). MTT showed that the cell viability decreased significantly after the overexpression of miR-218 in the GIST-T1 cells (P < 0.01). Flow cytometry showed that the cell proliferation index significantly declined after the overexpression of miR-218 (P < 0.01); meanwhile, the apoptosis of cells also significantly increased (P < 0.01). Detection using the Transwell invasion chamber showed that the number of cells passing through the Transwell chamber significantly dropped after the enhanced expression of miR-218 (P < 0.01). Luciferase reporter gene assay showed that, compared with the control group, the relative luciferase activity significantly declined in the miR-218 mimic transfection group (P < 0.01). Compared with the control group, the expression of KIT protein in the GIST-T1 cells transfected with miR-218 mimic for 48h significantly decreased (P < 0.01). In conclusion, the expression of miR-218 decreases in human GIST tissue and cell lines. miR-218 can negatively regulate the expression of KIT protein and inhibit the proliferation and invasion of GIST cells. Treatment based on the enhanced expression of miR-218 may be a promising strategy for GIST.

Establishment and characterization of novel cell lines and xenografts from patients with gastrointestinal stromal tumors

At present, no suitable GIST model exists for the analysis of drug resistance or metastasis using established human gastrointestinal stromal tumor (GIST) cell lines or xenografts even though the molecular mechanisms of drug resistance, progression and metastasis require clarification. The aim of this study was to establish and characterize human GIST cell lines and xenografts that can be used for evaluating drug resistance or various new molecularly targeted therapies. GIST tissues from patients were cultured and implanted under the skin of NOG (NOD/Shi-scid, IL-2Rrnu) mice. Two new cell lines (GK1C and GK3C) and three xenografts (GK1X, GK2X and GK3X) were generated from these clinical samples. The established GIST cell lines and xenografts were investigated for tumorigenesis and imatinib sensitivity. These cell lines and xenografts showed characteristic GIST morphology and exhibited KIT expression profiles similar to those of the patient samples. In addition, these GIST cell lines and xenografts were sensitive to imatinib. In conclusion, new human GIST cell lines and xenografts were established and maintained through repeated passages. These models will enable further study of combination therapies and the mechanisms of resistance, and allow testing of novel targeted monotherapies and combination therapies.