Gastrointestinal Stromal Tumors Cell Lines Research Articles

Synthesis and Cytostatic Activity of (E)-Ethyl-2-Amino-5-(3,3-Dimethyl-4-Oxobutyliden)-4-Oxo-1- (2-Phenylaminobenzamido)-4,5-Dihydro-1Hpyrrol-3-Carboxylate

Interaction of (Z)-N-(5-tert-butyl)-2-oxofuran-3(2H)-yliden-2-phenylaminobenzohydrazide (I) with cyanoacetic ester in the presence of triethylamine was used to synthesize the novel derivative (E)-ethyl-2-amino-5-(3,3-dimethyl-4-oxobutyliden)-4-oxo-1-(2-phenylaminobenzamido)-4,5-dihydro-1H-py rrole-3-carboxylate (II). The acute toxicity of this compound was studied, along with its cytotoxic activity against gastrointestinal stromal tumor (GIST) cell line T-1. Compound II showed a concentration- and time-dependent effect comparable with the antitumor compounds imatinib, paclitaxel, etoposide, and hydroxyurea.

TAS-116 inhibits oncogenic KIT signalling on the Golgi in both imatinib-na\xefve and imatinib-resistant gastrointestinal stromal tumours

BackgroundDespite the effectiveness of imatinib mesylate (IM), most gastrointestinal stromal tumours (GISTs) develop IM resistance, mainly due to the additional kinase-domain mutations accompanied by concomitant reactivation of KIT tyrosine kinase. Heat-shock protein 90 (HSP90) is one of the chaperone molecules required for appropriate folding of proteins such as KIT.MethodsWe used a novel HSP90 inhibitor, TAS-116, which showed specific binding to HSP90α/β with low toxicity in animal models. The efficacy and mechanism of TAS-116 against IM-resistant GIST were evaluated by using IM-naïve and IM-resistant GIST cell lines. We also evaluated the effects of TAS-116 on the other HSP90 client protein, EGFR, by using lung cell lines.ResultsTAS-116 inhibited growth and induced apoptosis in both IM-naïve and IM-resistant GIST cell lines with KIT activation. We found KIT was activated mainly in intracellular compartments, such as trans-Golgi cisternae, and TAS-116 reduced autophosphorylated KIT in the Golgi apparatus. In IM-resistant GISTs in xenograft mouse models, TAS-116 caused tumour growth inhibition. We found that TAS-116 decreased phosphorylated EGFR levels and inhibited the growth of EGFR-mutated lung cancer cell lines.ConclusionTAS-116 may be a novel promising drug to overcome tyrosine kinase inhibitor-resistance in both GIST and EGFR-mutated lung cancer.

Coordinated targeting of CK2 and KIT in gastrointestinal stromal tumours

BackgroundMost gastrointestinal stromal tumours (GIST) are driven by activating oncogenic mutations of KIT/PDGFRA, which provide a compelling therapeutic target. Our previous studies showed that CDC37, regulated by casein kinase 2 (CK2), is a crucial HSP90 cofactor for KIT oncogenic function and a promising and more selective therapeutic target in GIST.MethodsBiologic mechanisms of CK2-mediated CDC37 regulation were assessed in GISTs by immunoblotting, immunoprecipitations, knockdown and inactivation assays. The effects of a combination of KIT and CK2 inhibition were assessed by immunoblotting, cell viability, colony growth, cell cycle analysis, apoptosis, migration and invasiveness.ResultsCK2 overexpression was demonstrated by immunoblotting in GIST cell lines and patient biopsies. Treatment with a specific CK2 inhibitor, CX4945, leads to CDC37 dephosphorylation and inhibits KIT signalling in imatinib-sensitive and in imatinib-resistant GIST cell lines. Immunoprecipitation demonstrated that CK2 inhibition blocks KIT:HSP90:CDC37 interaction in GIST cells. Coordinated inhibition of CK2 and KIT by CX4945 (or CK2 shRNA) and imatinib, respectively, leads to increased apoptosis, anti-proliferative effects and cell cycle arrest and decreased p-AKT and p-S6 expression, migration and invasiveness in all GIST cell lines compared with either intervention alone, indicating additive effects of inhibiting these two important regulators of GIST biology.ConclusionOur findings suggest that combinatorial inhibition of CK2 and KIT warrants evaluation as a novel therapeutic strategy in GIST, especially in imatinib-resistant GIST.

Identification of a Multitargeted Tyrosine Kinase Inhibitor for the Treatment of Gastrointestinal Stromal Tumors and Acute Myeloid Leukemia.

Gastrointestinal stromal tumors (GISTs) are prototypes of stem cell factor receptor (c-KIT)-driven cancer. Two receptor tyrosine kinases, c-KIT and fms-tyrosine kinase (FLT3), are frequently mutated in acute myeloid leukemia (AML) patients, and these mutations are associated with poor prognosis. In this study, we discovered a multitargeted tyrosine kinase inhibitor, compound 15a, with potent inhibition against single or double mutations of c-KIT developed in GISTs. Moreover, crystal structure analysis revealed the unique binding mode of 15a with c-KIT and may elucidate its high potency in inhibiting c-KIT kinase activity. Compound 15a inhibited cell proliferation and induced apoptosis by targeting c-KIT in c-KIT-mutant GIST cell lines. The antitumor effects of 15a were also demonstrated in GIST430 and GIST patient-derived xenograft models. Further studies demonstrated that 15a inhibited the proliferation of c-KIT- and FLT3-driven AML cells in vitro and in vivo. The results of this study suggest that 15a may be a potential anticancer drug for the treatment of GISTs and AML.

KIT-Dependent and KIT-Independent Genomic Heterogeneity of Resistance in Gastrointestinal Stromal Tumors — TORC1/2 Inhibition as Salvage Strategy

Sporadic gastrointestinal stromal tumors (GIST), characterized by activating mutations of KIT or PDGFRA, favorably respond to KIT inhibitory treatment but eventually become resistant. The development of effective salvage treatments is complicated by the heterogeneity of KIT secondary resistance mutations. Recently, additional mutations that independently activate KIT-downstream signaling have been found in pretreated patients-adding further complexity to the scope of resistance. We collected genotyping data for KIT from tumor samples of pretreated GIST, providing a representative overview on the distribution and incidence of secondary KIT mutations (n = 80). Analyzing next-generation sequencing data of 109 GIST, we found that 18% carried mutations in KIT-downstream signaling intermediates (NF1/2, PTEN, RAS, PIK3CA, TSC1/2, AKT, BRAF) potentially mediating resistance to KIT inhibitors. Notably, we found no apparent other driver mutations in refractory cases that were analyzed by whole exome/genome sequencing (13/109). Using CRISPR/Cas9 methods, we generated a panel of GIST cell lines harboring mutations in KIT, PTEN, KRAS, NF1, and TSC2 We utilized this panel to evaluate sapanisertib, a novel mTOR kinase inhibitor, as a salvage strategy. Sapanisertib had potent antiproliferative effects in all cell lines, including those with KIT-downstream mutations. Combinations with KIT or MEK inhibitors completely abrogated GIST-survival signaling and displayed synergistic effects. Our isogenic cell line panel closely approximates the genetic heterogeneity of resistance observed in heavily pretreated patients with GIST. With the clinical development of novel, broad spectrum KIT inhibitors, emergence of non-KIT-related resistance may require combination treatments with inhibitors of KIT-downstream signaling such as mTOR or MEK.

Preclinical development of HQP1351, a multikinase inhibitor targeting a broad spectrum of mutant KIT kinases, for the treatment of imatinib-resistant gastrointestinal stromal tumors

BackgroundImatinib shows limited efficacy in patients with gastrointestinal stromal tumors (GISTs) carrying secondary KIT mutations. HQP1351, an orally bioavailable multikinase BCR-ABL inhibitor, is currently in clinical trials for the treatment of T315I mutant chronic myelogenous leukemia (CML), but the potential application in imatinib-resistant GISTs carrying secondary KIT mutations has not been explored.MethodsThe binding activities of HQP1351 with native or mutant KIT were first analyzed. Imatinib-sensitive GIST T1 and imatinib-resistant GIST 430 cells were employed to test the in vitro antiproliferative activity. Colony formation assay, cell migration assay and cell invasion assay were performed to evaluate the clonogenic, migration and invasion ability respectively. Flow cytometry and western blot analysis were used to detect cell apoptosis, cell cycle and signaling pathway. In vivo antitumor activity was evaluated in mouse xenograft models derived from GIST cell lines.ResultsHQP1351 potently inhibited both wild-type and mutant KIT kinases. In both imatinib-resistant and sensitive GIST cell lines, HQP1351 exhibited more potent or equivalent antiproliferative activity compared with ponatinib, a third generation BCR-ABL and KIT inhibitor. HQP1351 led to more profound inhibition of cell colony formation, cell migration and invasion, cell cycle arrest and cell apoptosis than ponatinib. Furthermore, HQP1351 also inhibited p-KIT, p-AKT, p-ERK1/2, and p-STAT3 to a higher extent than ponatinib. Finally, in xenograft tumor models derived from imatinib-resistant GIST cancer cell lines, HQP1351 exhibited antitumor activity superior to ponatinib.ConclusionsCollectively, our in vitro and in vivo results suggest that the therapeutic application of HQP1351 in imatinib-resistant GIST patients deserves further investigation in clinical trials.

Abstract 1269: P-glycoprotein expression in refractory gastrointestinal stromal tumors and its implication in the efficacy of paclitaxel as a salvage treatment

Abstract BACKGROUND: KIT-targeting tyrosine kinase inhibitors (TKIs) such as imatinib, sunitinib and regorafenib are the standard treatment for patients with gastrointestinal stromal tumor (GIST). However, most patients eventually develop treatment resistance to these standard therapies, and new agents must be introduced upon disease progression. Before TKIs were available, most of the conventional cytotoxic agents did not show sufficient clinical activity in GIST patients. However, a recent preclinical study demonstrated that 37 of the 89 FDA-approved anti-tumor drugs including paclitaxel (PTX) possess antitumor effect in at least one GIST cell line. Therefore, in this study, we aimed to evaluate the efficacy of PTX as a salvage treatment for GIST patients who exhibited treatment failure after standard TKI therapy using in-vitro/vivo models. MATERIALS and METHODS: The effect of PTX in GIST was examined by cell viability assay and rhodamine 123 (Rho123) efflux assay using GIST cells including established patient-derived GIST cell lines, and in animal models with patient-derived xenografts (PDXs) established from patients with GIST tumors refractory to TKIs. Multidrug resistance 1 (MDR1) mRNA expression by reverse transcription-PCR (RT-PCR) and P-glycoprotein (Pgp) expression by Western blotting or immunohistochemistry were evaluated in 20 patients’ tumor tissues, 9 GIST cell lines and 21 PDXs. To investigate the role of Pgp on PTX treatment, a stable MDR1-expressing GIST T1 cell line (GIST-T1-ABCB1#17) was prepared and compared with parent GIST T1 cell line. Verapamil was used as a Pgp inhibitor. RESULTS: Compared to imatinib-sensitive GIST-T1 harboring KIT exon 11 mutation and imatinib-resistant GIST-T1/816 harboring KIT exon 11 and 17 mutations, the patient-derived imatinib- and sunitinib-resistant GIST-R3 cell line harboring KIT exon 11 and 17 mutations was more resistant to PTX. Higher Rho123 efflux was observed in GIST-R3 which had high Pgp expression than in GIST-T1 and GIST-T1/816 which had low Pgp expression. The tumor growth inhibition of PTX was greater in xenografts with low Pgp expression (GIST-T1/816 xenografts and GIST-RX10 PDXs) than in xenografts with high Pgp expression (GIST-R3 xenografts and GIST-RX4 PDX). The GIST-T1-ABCB1#17, a stable MDR1-expressing GIST T1 cell line, exhibited a higher Pgp activity and a less sensitivity to PTX than the parent GIST-T1 cell line. The resistance of GIST-T1-ABCB1#17 to PTX was overcome by verapamil. CONCLUSION: Pgp expression was an important mechanism of resistance to PTX in preclinical GIST models. PTX is worth being tried clinically as a salvage treatment in patients with refractory GISTs with low Pgp expression. Citation Format: Young-Soon Na, Min-Hee Ryu, Young Soo Park, Chae-Won Lee, Ju-Kyung Lee, Jung Min Park, Yangsoon Park, Yoon-Koo Kang. P-glycoprotein expression in refractory gastrointestinal stromal tumors and its implication in the efficacy of paclitaxel as a salvage treatment [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 1269.

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 758: Long non-coding RNAH19 as a competing endogenous RNA of KIT in the progression of GIST

Abstract Background: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor with a wide range of biological behaviors, and activating mutations of KIT are particularly crucial to its development. Long non-coding RNAs (lncRNAs) have been identified as an oncogene in multiple cancer types, but data in GIST are missing. The aim of the study was to evaluate the contribution of lncRNA H19 to the malignant course of GISTs. Materials and Methods: RNA-seq analysis of lncRNA expression in five paired GIST and ANTs were analyzed to screen for differentially expressed lncRNAs. 274 human lncRNAs analyzed, exhibited significantly differential expressions with 186 genes being up-regulated and 88 genes being down-regulated compared to ANT (fold change>5, p<0.05). Unsupervised hierarchical clustering identified 48 lncRNAs and lncRNA H19 was the most overexpressed novel one. To assess the clinical importance of lncRNA H19 upregulation in GISTs, based on the expression levels 92 GIST patients were categorized into two groups: high (n=46) and low (n=46) lncRNA H19 expression. Results: After identifying lncRNA H19 as the most important one, validation in the expanded cohort confirmed the frequent upregulation of lncRNA H19 in GIST. Functional assays revealed that lncRNA H19 promotes GIST cell proliferation and metastasis. We could demonstrate that lncRNA H19 was predicted to sponge 19 miRNAs with six potential binding sites shared with KIT 3’-UTR using bio-informative database analysis. It was confirmed that lncRNA H19 was positively correlated with the expression of KIT, and the knockdown of lncRNA H19 led to the depression of KIT and its downstream pathway. Using luciferase reporter assays, lncRNA H19 was observed to sponge several miRNAs including miR-18a/b-5p, miR-19a/b-3p, miR-193a/b-3p, miR-216b-5p and mIR-3666. The combination of lncRNA H19 and KIT inhibition showed increased growth inhibition in GIST cell lines. Taken together, the data indicate that lncRNA H19 functions as a competing endogenous RNA (ceRNA) of KIT and promotes GIST progression. It could be shown that the expression levels of lncRNA H19 were significantly correlated with the progression markers of GIST, tumor size (P<0.001), mitosis count (P<0.001), and the modified NIH risk criteria (P<0.001) Conclusion: Our results indicate that lncRNA H19 can affect imatinib-induced apoptotic cell death and play a role in facilitating GIST progression by modulating the expression of KIT through the ceRNA network. High lncRNA H19 expression is associated with worse prognosis in patients with GIST and lncRNA H19 may serve as a new predictor of prognosis. Furthermore, targeting lncRNA H19/KIT ceRNA network appears to be a promising treatment for GIST. Citation Format: Peter Hohenberger, Lin Tu, Wenyi Zhao, Ming Wang Wang, Zizheng Zhang, Heike Allgayer, Hui Cao. Long non-coding RNAH19 as a competing endogenous RNA of KIT in the progression of GIST [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 758.

Biochemical Inhibition of DOG1/TMEM16A Achieves Antitumoral Effects in Human Gastrointestinal Stromal Tumor Cells In Vitro.

DOG1 is a calcium-activated chloride channel that has gained attention as a promising drug target due to its involvement in several processes essential for tumor development and progression. DOG1 is overexpressed in >95% of gastrointestinal stromal tumors (GIST). The aim was to determine DOG1 inhibition antitumoral effects on GIST. Human GIST (GIST-T1 and GIST882) cell lines were used to study the effect of DOG1 inhibitors on chloride currents, viability, colony formation, and cell cycle. CaCCinh-A01 decreased chloride currents. CaCCinh-A01 and T16inh-A01 reduced GIST cell viability and CaCCinh-A01 affected cell cycle distribution leading to G1 cell-cycle arrest. CaCCinh-A01 also increased the sub-G1 phase population, indicative of apoptosis, in GIST882. CaCCinh-A01 strongly reduced the colony forming ability of the cells, whereas T16inh-A01 did not. DOG1 inhibition has antitumoral effects in GIST cells in vitro, and could potentially serve as a target for GIST therapy.

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.

Identification of phenothiazine as an ETV1‑targeting agent in gastrointestinal stromal tumors using the Connectivity Map.

Gastrointestinal stromal tumors (GISTs) are gastrointestinal tract sarcomas that commonly contain a mutation in the tyrosine kinases, KIT and platelet‑derived growth factor receptorA(PDGFRA). Imatinib, sunitinib and regorafenib are all effective tyrosine kinase inhibitors; however, acquired resistance is inevitable. The E26 variant1(ETV1) pathway has been found to be a key downstream effector of KIT and is therefore a reasonable therapeutic target for this disease. In this study, we explored the potential agents targeting ETV1 in GISTs by uploading an ETV1 knockout gene signature of GIST cell lines to the pattern‑matching software 'Connectivity Map'. The activity and mechanisms of identified agents were examined using an invitro model. Four drugs were identified: Suberanilohydroxamic acid and trichostatin [two histone deacetylase inhibitors (HDACIs)] and trifluoperazine and thioridazine (two phenothiazine‑class drugs). Western blot analysis demonstrated that all four drugs had ETV1‑downregulating effects. As HDACIs have been previously studied in GISTs, we focused on phenothiazine. Phenothiazine was found to exert cytotoxicity and to induce apoptosis and autophagy in GISTs. Treatment with phenothiazine had little effect on the KIT/AKT/mammalian target of rapamycin (mTOR) pathway, but instead upregulated extracellular‑signal‑regulated kinase(ERK) activity. A combination of phenothiazine and a MEK inhibitor had a synergistic cytotoxic effect on GISTs. Western blot analysis indicated that ELK1 and early growth response1(EGR1) were activated/upregulated following phenothiazine treatment, and the MEK inhibitor/phenothiazine combination downregulated the ERK/ELK1/EGR1 pathway, resulting in diminished autophagy, as well as enhanced apoptosis. On the whole, the findings of this study established phenothiazine as a novel class of therapeutic agents in GIST treatment and demonstrate that a combination of phenothiazine and MEK inhibitor has great potential for use in the treatment of GISTs.

Ethyl-2-amino-pyrrole-3-carboxylates are active against imatinib-resistant gastrointestinal stromal tumors in vitro and in vivo

We showed recently that ethyl-2-amino-pyrrole-3-carboxylates (EAPCs) exhibit potent antiproliferative activities against a broad spectrum of soft tissue sarcoma and gastrointestinal stromal tumor (GIST) cell lines in vitro. The molecular mechanism of action was owing to inhibition of tubulin polymerization and induction of a robust G2/M cell-cycle arrest, leading to the accumulation of tumor cells in the M-phase and induction of apoptosis. Given that more than 50% of the patients with GISTs develop resistance to imatinib (IM) over the 2 years of IM-based therapy, we examined whether EAPCs exhibit activity against IM-resistant GISTs in vitro and in vivo. A real-time antiproliferation assay illustrated the potent antiproliferative activities of EAPCs against IM-sensitive and IM-resistant GISTs. This was in agreement with the colony formation assay, which revealed potent antiproliferative activities of EAPCs against IM-resistant GISTs, being much stronger when compared with IM and doxorubicin, a topoisomerase II inhibitor. Next, we tested the efficacy of EAPCs in the xenograft model of GISTs, exhibiting secondary IM resistance owing to RTK switch (loss of c-KIT/gain of FGFR2α). A total of 30 5- to 8-week-old female nu/nu mice were subcutaneously inoculated into the flank areas with IM-resistant GIST-T1-R cells (100 μl of 1×10 GIST T-1R cells/ml suspension, in Dulbecco's PBS). Mice were randomized as control (untreated), IM (50 mg/kg), EAPC-20 (10 mg/kg) or EAPC-24 (10 mg/kg) and were treated orally for 10 days. IM has a minor inhibitory effect on tumor size, thus revealing GIST resistance to IM. In contrast, both of EAPCs effectively reduced the tumor size. This was associated with an increased intratumoral apoptosis as detected by immunohistochemical staining for cleaved caspase-3 on day 5 of the treatment. Furthermore, both EAPCs significantly reduced the proliferative activity of tumor cells in the central zones of tumors as measured by positivity for Ki-67 staining. More importantly, in EAPC-24-treated GISTs, the histological response was mainly characterized by the induction of necrosis, whereas EAPC-20 induced the signs of intratumoral fibrosis and myxoid degeneration. Collectively, our data suggest that EAPC-20 and EAPC-24 are the perspective antitumor agents that exhibit antiproliferative and cytotoxic activity against GISTs exhibiting secondary resistance to IM.

Creation and characterization of a novel human PDGFRA D842V-mutant GIST cell line.

e22512 Background: Activating PDGFRA mutations are seen in about 10% of gastrointestinal stromal tumors (GIST). Of these, the majority are one specific PDGFRA mutation, D842V, which confers resistance to all clinically approved tyrosine kinase inhibitors (TKI). Much has been learned about the mechanism by which TKIs work in GIST using cell lines derived from human GIST that bear activating KIT mutations. However, despite enormous effort to develop cell lines derived from human tumors over the past 15 years, there are currently no human cell models of PDGFRA D842V GIST. With the development of potent PDGFRA D842V TKI, such as crenolanib, avapritinib, and DCC2618 it is of interest to understand how these drugs affect PDGFRA-mutant GIST biology. Methods: We sought to convert the KIT-driven GIST-T1 cell line to be PDGFRA-driven by transduction with PDGFRA D842V. Endogenous KIT expression was knocked-out using CRISPR Cas9. Knockout was verified at protein and DNA levels. Clonal KIT knockout cell lines were tested for sensitivity to KIT and PDGFRA TKI both in vitro and in vivo models. Kinase dependence was determined by measuring the effect on viability after KIT and/or PDGFRA knockdown by siRNA. Results: GIST-T1 cells transduced with PDGFRA D842V display highly phosphorylated PDGFRA, imatinib resistance, and PDGFRA dependence. Cell lines that express both endogenous mutant KIT and exogenous PDGFRA D842V maintain some dependence on KIT, but less so than the parental KIT-driven GIST-T1 cell line. Complete knockout of KIT protein expression was achieved in GIST-T1 D842V cells using CRISPR-Cas9. GIST-T1 D842V KITko cell lines demonstrate sensitivity to TKI avapritinib, crenolanib, and DCC2618. Xenografts were established using GIST-T1 D842V KITko cell lines and are sensitive to avapritinib in vivo. Conclusions: The GIST-T1 D842V KITko cell line demonstrates ability to switch kinase allegiance resulting in PDGFRA-dependence and KIT-independence. This GIST-T1 D842V KITko cell line represents a novel model of PDGFRA-mutant GIST and can be used to understand the unique biological aspects of these tumors, particularly the response to new potent PDGFRA kinase inhibitors such as avapritinib which will have immense clinical relevance.

Discovery of ( E)- N1-(3-Fluorophenyl)- N3-(3-(2-(pyridin-2-yl)vinyl)-1 H-indazol-6-yl)malonamide (CHMFL-KIT-033) as a Novel c-KIT T670I Mutant Selective Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs).

Gain-of-function mutations of c-KIT kinase play crucial pathological roles for the gastrointestinal stromal tumors (GISTs). Despite the success of imatinib as the first-line treatment of GISTs, dozens of drug-acquired resistant mutations emerge, and c-KIT T670I is one of the most common mutants among them. Although several kinase inhibitors are capable of overcoming the T670I mutant, none of them can achieve the selectivity over the c-KIT wild-type (wt), which also plays important roles in a variety of physiological functions such as hematopoiesis. Starting from axitinib, through fragment hybrid type II kinase inhibitor design approach, we have discovered a novel inhibitor 24, which not only exhibits potent activity to c-KIT T670I mutant but also achieves 12-fold selectivity over c-KIT wt. Compound 24 displays good antiproliferative effects against c-KIT T670I mutant-driven GIST cell lines (GIST-T1/T670I and GIST-5R) and also exhibits suitable in vivo pharmacokinetic profiles as well as dose-dependent antitumor efficacy. This study provides a proof of concept for developing a c-KIT mutant selective inhibitor that theoretically can render a better therapeutic window.

Discovery of Conformational Control Inhibitors Switching off the Activated c-KIT and Targeting a Broad Range of Clinically Relevant c-KIT Mutants.

Drug resistance due to acquired mutations that constitutively activate c-KIT is a significant challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). Herein, we identified 1-(5-ethyl-isoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)pyrimidin-4-ylamino]-thiazol-5-yl}phenyl)urea (10a) as a potent inhibitor against unactivated and activated c-KIT. The binding of 10a induced rearrangements of the DFG motif, αC-helix, juxtamembrane domain, and the activation loop to switch the activated c-KIT back to its structurally inactive state. To the best of our knowledge, it is the first structural evidence demonstrating how a compound can inhibit the activated c-KIT by switching back to its inactive state through a sequence of conformational changes. Moreover, 10a can effectively inhibit various c-KIT mutants and the proliferation of several GIST cell lines. The distinct binding features and superior inhibitory potency of 10a, together with its excellent efficacy in the xenograft model, establish 10a as worthy of further clinical evaluation in the advanced GISTs.

Potentiation of imatinib by cilostazol in sensitive and resistant gastrointestinal stromal tumor cell lines involves YAP inhibition.

Despite the introduction of tyrosine kinase inhibitors, gastrointestinal stromal tumors (GIST) resistance remains a major clinical challenge. We previously identified phosphodiesterase 3A (PDE3A) as a potential therapeutic target expressed in most GIST. The PDE3 inhibitor cilostazol reduced cell viability and synergized with the tyrosine kinase inhibitor imatinib (Gleevec™) in the imatinib-sensitive GIST882 cell line. Here, we found that cilostazol potentiated imatinib also in the imatinib-resistant GIST48 cell line. Cilostazol induced nuclear exclusion, hence inactivation, of the transcriptional co-activator YAP, in a cAMP-independent manner. Verteporfin, a YAP/TEAD interaction inhibitor, reduced by 90% the viability of both GIST882 and GIST48 cells. Our results highlight the potential use of compounds targeting PDE3A or YAP in combined multitherapy to tackle GIST resistance.

Anagrelide for Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is a common type of soft-tissue sarcoma. Imatinib, an inhibitor of KIT, platelet-derived growth factor receptor alpha (PDGFRA), and a few other tyrosine kinases, is highly effective for GIST, but advanced GISTs frequently progress on imatinib and other approved tyrosine kinase inhibitors. We investigated phosphodiesterase 3 (PDE3) as a potential therapeutic target in GIST cell lines and xenograft models. The GIST gene expression profile was interrogated in the MediSapiens IST Online transcriptome database comprising human tissue and cancer samples, and PDE3A and PDE3B expression was studied using IHC on tissue microarrays (TMA) consisting of 630 formalin-fixed human tissue samples. GIST cell lines were screened for sensitivity to 217 anticancer compounds, and the efficacy of PDE inhibitors on GIST was further studied in GIST cell lines and patient-derived mouse xenograft models. GISTs expressed PDE3A and PDE3B frequently compared with other human normal or cancerous tissues both in the in silico database and the TMAs. Anagrelide was identified as the most potent of the PDE3 modulators evaluated. It reduced cell viability, promoted cell death, and influenced cell signaling in GIST cell lines. Anagrelide inhibited tumor growth in GIST xenograft mouse models. Anagrelide was also effective in a GIST xenograft mouse model with KIT exon 9 mutation that may pose a therapeutic challenge, as these GISTs require a high daily dose of imatinib. PDE3A and PDE3B are frequently expressed in GIST. Anagrelide had anticancer efficacy in GIST xenograft models and warrants further testing in clinical trials.

Intracellular concentration and transporters in imatinib resistance of gastrointestinal stromal tumor

Background: We aimed to investigate the role of intracellular imatinib concentration in drug resistance and the expression of candidate drug transporters in gastrointestinal stromal tumor (GIST) cell lines.Method: The imatinib concentrations were measured by the liquid chromatography-tandem mass spectrometry (LC-MS/MS). The expression of candida te drug transporters was detected by qRT-PCR.Results: The tissue imatinib concentrations in imatinib resistant patients were significantly lower than that of sensitive patients (p < .05). Compared with parental cell lines, the intracellular imatinib concentration was notably lower in imatinib resistant GIST cell lines. For candidate transporters, MRP1 and BCRP were overexpressed in resistant GIST cell lines.Conclusion: The intracellular imatinib concentration may play a crucial role in imatinib resistance and the intracellular differences of imatinib concentration may be induced by the upregulation of efflux transporters. Our study highlights the importance of intracellular imatinib concentration and the potential of using imatinib transporters as therapeutic targets for patients with GIST.

Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression

ABSTRACTOncogenic KIT or PDGFRA receptor tyrosine kinase (TK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is the standard of care for patients with metastatic GIST. However, approximately 10% of KIT-positive GIST metastases lose KIT expression at the time of clinical progression during imatinib therapy. In the present report, we performed TK-activation screens, using phosphotyrosine-TK double immunoaffinity purification and mass spectrometry, in GIST in vitro models lacking KIT expression. These studies demonstrated tyrosine-phosphorylated EGFR, AXL, and EPHA2 in four of six KIT-negative GIST lines (GIST62, GIST522, GIST54, GIST226, GIST48B, and GIST430B), and tyrosine-phosphorylated focal adhesion kinase (FAK) in each of the six KIT-negative lines. AXL expression was strong in KIT-negative or -weak clinical GIST samples that were obtained from progressing metastases during imatinib therapy. AXL knockdown inhibited viability in three KIT-negative GIST cell lines (GIST62, GIST54, and GIST522), but not in an AXL-negative, KIT-positive GIST control cell line (GIST430). AXL inhibition by R428, a specific AXL kinase inhibitor, reduced viability in AXL-activated GIST54. AXL knockdown in GIST62, GIST522, and GIST54 was accompanied by an increase in p21, p27, and p53 expression. By contrast, gefitinib-mediated EGFR inhibition, PF562271-mediated FAK inactivation, and shRNA-mediated knockdowns of EPHA2 and FAK had no effect on viability or colony formation of the KIT-negative GISTs. These findings highlight the potential relevance of AXL/p53 signaling as a therapeutic target in a subset of GISTs that have lost KIT oncoprotein expression.