Gastrointestinal Stromal Tumors Resistance Research Articles

Differential antitumor activity of compounds targeting the ubiquitin-proteasome machinery in gastrointestinal stromal tumor (GIST) cells

The majority of gastrointestinal stromal tumors (GISTs) are driven by oncogenic KIT signaling and can therefore be effectively treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate. However, most GISTs develop imatinib resistance through secondary KIT mutations. The type of resistance mutation determines sensitivity to approved second-/third-line TKIs but shows high inter- and intratumoral heterogeneity. Therefore, therapeutic strategies that target KIT independently of the mutational status are intriguing. Inhibiting the ubiquitin-proteasome machinery with bortezomib is effective in GIST cells through a dual mechanism of KIT transcriptional downregulation and upregulation of the pro-apoptotic histone H2AX but clinically problematic due to the drug’s adverse effects. We therefore tested second-generation inhibitors of the 20S proteasome (delanzomib, carfilzomib and ixazomib) with better pharmacologic profiles as well as compounds targeting regulators of ubiquitination (b-AP15, MLN4924) for their effectiveness and mechanism of action in GIST. All three 20S proteasome inhibitors were highly effective in vitro and in vivo, including in imatinib-resistant models. In contrast, b-AP15 and MLN4924 were only effective at high concentrations or had mostly cytostatic effects, respectively. Our results confirm 20S proteasome inhibitors as promising strategy to overcome TKI resistance in GIST, while highlighting the complexity of the ubiquitin-proteasome machinery as a therapeutic target.

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.

Mechanisms of resistance to a PI3K inhibitor in gastrointestinal stromal tumors: an omic approach to identify novel druggable targets.

Background: Gastrointestinal stromal tumors (GISTs) represent a worldwide paradigm of target therapy. The introduction of tyrosine kinase inhibitors has deeply changed the prognosis of GIST patients, however, the majority of them acquire secondary mutations and progress. Unfortunately, besides tyrosine-kinase inhibitors, no other therapeutic options are available. Therefore, it is mandatory to identify novel molecules and/or strategies to overcome the inevitable resistance. In this context, after promising preclinical data on the novel PI3K inhibitor BYL719, the NCT01735968 trial in GIST patients who had previously failed treatment with imatinib and sunitinib started. BYL719 has attracted our attention, and we comprehensively characterized genomic and transcriptomic changes taking place during resistance.Methods: For this purpose, we generated two in vitro GIST models of acquired resistance to BYL719 and performed an omic-based analysis by integrating RNA-sequencing, miRNA, and methylation profiles in sensitive and resistant cells.Results: We identified novel epigenomic mechanisms of pharmacological resistance in GISTs suggesting the existence of pathways involved in drug resistance and alternatively acquired mutations. Therefore, epigenomics should be taken into account as an alternative adaptive mechanism.Conclusion: Despite the fact that currently we do not have patients in treatment with BYL719 to verify this hypothesis, the most intriguing result is the involvement of H19 and PSTA1 in GIST resistance, which might represent druggable targets.

Molecular Comparison of Imatinib-Naïve and Resistant Gastrointestinal Stromal Tumors: Differentially Expressed microRNAs and mRNAs.

Despite the success of imatinib in advanced gastrointestinal stromal tumor (GIST) patients, 50% of the patients experience resistance within two years of treatment underscoring the need to get better insight into the mechanisms conferring imatinib resistance. Here the microRNA and mRNA expression profiles in primary (imatinib-naïve) and imatinib-resistant GIST were examined. Fifty-three GIST samples harboring primary KIT mutations (exon 9; n = 11/exon 11; n = 41/exon 17; n = 1) and comprising imatinib-naïve (IM-n) (n = 33) and imatinib-resistant (IM-r) (n = 20) tumors, were analyzed. The microRNA expression profiles were determined and from a subset (IM-n, n = 14; IM-r, n = 15) the mRNA expression profile was established. Ingenuity pathway analyses were used to unravel biochemical pathways and gene networks in IM-r GIST. Thirty-five differentially expressed miRNAs between IM-n and IM-r GIST samples were identified. Additionally, miRNAs distinguished IM-r samples with and without secondary KIT mutations. Furthermore 352 aberrantly expressed genes were found in IM-r samples. Pathway and network analyses revealed an association of differentially expressed genes with cell cycle progression and cellular proliferation, thereby implicating genes and pathways involved in imatinib resistance in GIST. Differentially expressed miRNAs and mRNAs between IM-n and IM-r GIST were identified. Bioinformatic analyses provided insight into the genes and biochemical pathways involved in imatinib-resistance and highlighted key genes that may be putative treatment targets.

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.

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.

Downregulation of lncRNA CCDC26 contributes to imatinib resistance in human gastrointestinal stromal tumors through IGF-1R upregulation.

Imatinib is the first line of therapy for patients with metastatic or gastrointestinal stromal tumors (GIST). However, drug resistance limits the long-term effect of imatinib. Long non-coding RNAs (lncRNAs) are emerging as key players in regulating drug resistance in cancer. In this study, we investigated the association between lncRNA CCDC26 and IGF-1R in GIST and their involvement in drug resistance. Considering the key role of lncRNAs in drug resistance in cancer, we hypothesized that IGF-1R is regulated by lncRNAs. The expression of a series of reported drug resistance-related lncRNAs, including CCDC26, ARF, H19, NBR2, NEAT1, and HOTAIR, in GIST cells treated with imatinib H19 was examined at various time-points by qRT-PCR. Based on our results and published literature, CCDC26, a strongly down-regulated lncRNA following imatinib treatment, was chosen as our research target. GIST cells with high expression of CCDC26 were sensitive to imatinib treatment while knockdown of CCDC26 significantly increased the resistance to imatinib. Furthermore, we found that CCDC26 interacted with c-KIT by RNA pull down, and that CCDC26 knockdown up-regulated the expression of IGF-1R. Moreover, IGF-1R inhibition reversed CCDC26 knockdown-mediated imatinib resistance in GIST. These results indicated that treatments targeting CCDC26-IGF-1R axis would be useful in increasing sensitivity to imatinib in GIST.

Targeting of FGF-Signaling Re-Sensitizes Gastrointestinal Stromal Tumors (GIST) to Imatinib In Vitro and In Vivo.

Dysregulation of the fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling pathway is frequently observed in multiple human malignancies, and thus, therapeutic strategies targeting FGFs and FGFRs in human cancer are being extensively explored. We observed the activation of the FGF/FGFR-signaling pathway in imatinib (IM)-resistant gastrointestinal stromal tumor (GIST) cells. Furthermore, we found that the activation of FGFR signaling has a significant impact on IM resistance in GISTs in vitro. Next, we tested the efficacy of BGJ398, a potent and selective FGFR1–3 inhibitor, in xenograft models of GISTs exhibiting secondary IM resistance due to receptor-tyrosine kinase (RTK) switch (loss of c-KIT/gain of FGFR2a). Five to eight-week-old female nu/nu mice were subcutaneously inoculated into the flank areas with GIST T-1R cells. Mice were randomized as control (untreated), IM, BGJ398, or a combination and treated orally for 12 days. IM had a moderate effect on tumor size, thus revealing GIST resistance to IM. Similarly, a minor regression in tumor size was observed in BGJ398-treated mice. Strikingly, a 90% decrease in tumor size was observed in mice treated with a combination of IM and BGJ398. Treatment with BGJ398 and IM also induced major histopathologic changes according to a previously defined histopathologic response score and resulted in massive myxoid degeneration. This was associated with increased intratumoral apoptosis as detected by immunohistochemical staining for cleaved caspase-3 on day 5 of the treatment. Furthermore, treatment with BGJ398 and IM significantly reduced the proliferative activity of tumor cells as measured by positivity for Ki-67 staining. In conclusion, inhibition of FGFR signaling substantially inhibited the growth of IM-resistant GISTs in vitro and showed potent antitumor activity in an IM-resistant GIST model via the inhibition of proliferation, tumor growth, and the induction of apoptosis, thereby suggesting that patients with advanced and metastatic GISTs exhibiting IM resistance might benefit from therapeutic inhibition of FGFR signaling.

MiR-125a-5p regulation increases phosphorylation of FAK that contributes to imatinib resistance in gastrointestinal stromal tumors

The use of imatinib mesylate has greatly improved the clinical outcome for gastrointestinal stromal tumor (GIST) patients. However, imatinib resistance is still a major clinical challenge, and the molecular mechanisms are not fully understood. We have previously shown that miR-125a-5p and its mRNA target PTPN18 modulate imatinib response in GIST cells. Herein, we evaluated phosphorylated FAK (pFAK) as a candidate downstream target of PTPN18 and the possible association of this regulation with imatinib resistance in GIST. FAK and pFAK expressions were evaluated in GIST882 cells transfected with short hairpin RNA or short interfering RNA targeting PTPN18 or miR-125a-5p mimic, imatinib-resistant GIST882R subclones and clinical samples using Western blot analyses. FAK phosphorylation was blocked using the FAK inhibitor 14 (FAKi) and the effects on cell viability and apoptosis were evaluated using WST-1 assay and cleaved PARP expression. Clinical associations of FAK and pFAK expression with imatinib resistance, KIT mutation and patient outcome were assessed by Fisher's exact test or log-rank test. Over-expression of miR-125a-5p and silencing of PTPN18 increased pFAK, but not FAK, expression in GIST cells. Higher pFAK expression was observed in the GIST882R subclones with acquired imatinib resistance compared to their imatinib-sensitive parental cells. Treatment with FAKi in imatinib-resistant GIST882R cells reduced cell viability and increased apoptosis upon imatinib treatment. Additionally, FAKi could rescue the imatinib resistance effect mediated by miR-125a-5p over-expression. In clinical samples, high FAK and pFAK expressions were associated with KIT mutation status, and high FAK expression was also associated with metastasis in GIST. Higher pFAK was found in cases with shorter overall survival. Our findings highlight an important role for miR-125a-5p regulation and its downstream target pFAK for imatinib resistance in GIST. pFAK and FAK may have prognostic values in GIST.

Molecular mechanism of D816X mutation-induced c-Kit activation and -mediated inhibitor resistance in gastrointestinal stromal tumor

The D816X (X = V, H, Y or F) missense mutation constitutively activates c-Kit kinase in gastrointestinal stromal tumor (GIST) and has been observed to cause acquired resistance against first-line and second-line kinase inhibitors. In the present study, the allosteric mechanism of D816X-induced c-Kit conformational change is investigated at molecular level. The Asp816 residue is located at the activation loop (A-loop) of c-Kit and the mutation can eliminate a negative formal charge from the loop region by substituting the acidic asparagic acid residue with neutral valine, histidine, tyrosine or phenylalanine. Here, we classify the c-Kit kinase into four states in terms of its mutation (wild type or mutant) and conformation (DFG-in or DFG-out). The wild-type kinase is electrostatically stabilized in inactive DFG-out conformation, whereas the D816X mutation can promote the conformational conversion to active DFG-in and then activate the kinase. Structural analysis reveals that the Asp816 residue in DFG-out is surrounded by a number of polar and positively charged residues within its first and second shells of protein context, and kinase conformational change to DFG-in brings this residue into a negative electrostatic potential environment. Dynamics simulation characterizes that the c-Kit conformational conversion from DFG-out to DFG-in can cause local unfavorable effect to type-II inhibitor, while the mutation-induced global structural rearrangement would participate in the favorable interaction of c-Kit with type-I inhibitor.

Progress in the study of the mechanism of drug resistance in gastrointestinal stromal tumors

gastrointestinal stromal tumors (GISTs) are one of the most common malignant tumors of digestive tract, and are resistant to cytotoxic radiotherapy and chemotherapy. Tyrosine kinase inhibitors (TKIs) have been used as a miracle drug for clinical treatment. However, the drug resistance has limited its clinical application. With the continuous deepening understanding of the relevant mechanisms, novel therapeutic agents are emerging. Therefore, fundamental studies on the pathogenesis of gastrointestinal stromal tumor are essential for the discovery of novel therapeutic approaches. At present, there are at least three models which are widely applied in the basic research of the pathogenesis of tumor, which are the homologous resistant/sensitive cell line model, the tumor transplanted mouse model, and the human tumor specimen. In this paper, we review the literature on the research methods of gastrointestinal stromal tumor resistance mechanism, and summarize the related research methods and the main mechanisms of drug resistance. Key words: Tumor cell line; Gastrointestinal stromal tumors; Drug resistance; Xenograft model; Human tumor specimen

Epigenetics: an alternative pathway in GISTs tumorigenesis.

Many diseases have different pathological backgrounds responsible for abnormal cell behavior and exhibiting altered function and signal transduction. This is especially true for tumors and although changes affecting DNA sequence, irreversible mutations and chromosomal aberrations in gastrointestinal stromal tumors (GISTs) have been widely studied, the importance of reversible epigenetic changes increasingly recognized in many cancers has received insufficient attention in these tumors. Epigenetic mechanisms are part of normal development and gene expression under normal conditions, but malfunction of these processes leads to malignant transformation by disturbing both intra- and intercellular communication. GISTs are a specific group of gastrointestinal tract tumors resistant to conventional chemotherapy and radiotherapy. Although they account for only 1% to 2% of tumors, they are among the most widespread gastrointestinal mesenchymal tumors. DNA hyper/hypomethylation overexpression/underexpression of miRNAs or abnormal histone modification may provide an alternative to the genetic modifications responsible for GIST pathology, response to treatment, prognosis and overall survival. This review summarizes the known epigenetic mechanisms involved in GIST pathogenesis; including onset, progression, and GISTs resistance. Reversible epigenetic changes are a novel and appropriate approach to halt the spread of metastases and the emergence of resistance in GIST treatment, and these changes depend on the type of epigenetic alternation, including inhibitors of histone acetyltranferase and deacetylase and DNA methyltransferases.

1514P - Serum miRNA abundances discriminate imatinib-naive patients with advanced gastrointestinal stromal tumors (GIST) from those in remission on Imatinib therapy

Background: Deregulation of microRNAs (miRNAs) expression is observed virtually in all major types of neoplasm and miRNAs level in blood circulation are investigated as a potential diagnostics or prognostics biomarkers for neoplastic disorders. Gastrointestinal stromal tumors (GISTs) is the most common sarcoma of the gastrointestinal tract and to date performed studies on GISTs have provided mounting evidence on altered miRNA association with clinical, pathological features and Imatinib resistance in GIST. However, the utility of circulating miRNA as response markers of GIST progression and for Imatinib treatment have not been evaluated Methods: 36 metastatic or unresectable CD-117-positive GIST patients, were enrolled and serum sample was collected prior to Imatinib treatment. All patients responded initially to imatinib therapy. In 12 patients an additional serum sample was collected following targeted treatment at the time of remission. Control group comprised 30 healthy individuals. MiRNAs were isolated from serum with MirVANA miRNA Isolation Kit and then analyzed using deep sequencing on Ion Torrent PGM. Reads were mapped to miRBase miRNA collection with miRDeep2. Differential expression was evaluated with edgeR. Results: Deep sequencing identified 1284 miRNAs. The pair-wise comparison between Imatinib treated and Imatinib-naive GIST samples uncovered 22 miRNAs with differential expression (adjusted p value <0.05) of which 10 (miR-142-5p, let-7d-3p, miR-30e-5p, miR-194-5p, miR-223-5p, miR-223-3p, miR-125a-5p, miR-199b-5p, miR-24-2-5p, miR-641) yielded AUCs (areas under Receiver Operating Characteristic curves) ranging 0.81 and 0.9, thus having a high discriminative properties. A comparison of imatinib-naive GIST and control healthy samples revealed 99 differentially expressed miRNAs (adjusted p value <0.05) of which four (miR-582-5p, miR-150-5p, miR-450b-5p, miR-450a-5p) reached AUCs with high discriminatory power ranging 0.81-0.84. Conclusions: Circulating miRNA abundances can distinguish GIST patients from those in remission following Imatinib therapy as well as from the healthy controls. However, further studies evaluating the potential of designated microRNAs as response markers for treatment or as predictive markers of GIST are warranted. Legal entity responsible for the study: Piotr Rutkowski Funding: the grant from National Science Center [2013/11/B/NZ5/03165 to PR Disclosure: All authors have declared no conflicts of interest.

Abstract 1330: HSP90 inhibitor has a possibility to overcome imatinib resistance in gastrointestinal stromal tumors

Abstract [Background] Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the digestive tract and are reported to harbor gain-of-function mutations in the KIT gene, which contribute to the development of sporadic GISTs. This knowledge has facilitated the development of targeted therapies with tyrosine kinase inhibitors and the revolutionary chemotherapeutic drug imatinib mesylate (IM). In clinical trials, the disease control rate was nearly 85%, and corresponding 2-year overall survival rates ranged from 70-80%, indicating markedly improved patient outcomes compared with anecdotal data in the pre-IM era. Despite its effectiveness, half of GISTs treated with IM develop resistance within 2 years, largely due to the accumulation of additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase, even in the presence of IM. Heat shock protein 90 (HSP90) is one of chaperon molecules required for the proper folding, function, and stability of various client proteins such as KIT. The aim of this study is to clarify the efficacy of HSP90 inhibitor against IM resistant GIST. [Material and Method] We used the established human GIST cell line GIST-T1, and two IM-resistant cell lines (GIST-T1R8, GIST-T1R9), which had additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase same as clinical samples, by exposure to IM. These resistant cell lines exhibited imatinib IC50 values (&amp;gt;10 μM) that were &amp;gt;1000-fold higher than the parental cell line. We investigated the cytotoxicity and signaling inhibition by HSP90 inhibitor using by the WST-8 assay, caspase3/7 apoptosis assay and western blotting. Immunobolts for KIT and for KIT-depending signaling pathways were observed at 12 hours of treatment. Caspase 3/7 apoptosis assay was measured after 24h. [Result] HSP90 inhibitor showed growth inhibition not only for GIST T1 but also GIST-T1R8 and GIST-T1R9. Apoptosis induction of these cell lines were also confirmed by the exposure to HSP90 inhibitor in a dose-dependent manner. To find the mechanism of apoptosis induced by HSP90 inhibitor, we investigated the signaling pathway depending on KIT. HSP90 inhibitor showed inhibition of phosphorylated-KIT, a client for HSP90, in both IM sensitive and resistant GIST cell lines. And, it also inhibited downstream of KIT signaling, e.g. p-ERK and p-AKT. [Conclusions]HSP90 inhibitor showed the anti-tumor efficacy for imatinib resistant GIST in vitro. It might have a possibility to apply for clinical use. Citation Format: Yurina Saito, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Koji Tanaka, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. HSP90 inhibitor has a possibility to overcome imatinib resistance in gastrointestinal stromal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1330. doi:10.1158/1538-7445.AM2017-1330

Imatinib induces autophagy via upregulating XIAP in GIST882 cells

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms originating from the gastrointestinal tract with gain of function mutations in receptor tyrosine kinases KIT or platelet-derived growth factor receptor A (PDGFRA). The main effective treatment for GISTs is tyrosine kinase inhibitors, such as imatinib mesylate. However, GISTs respond to imatinib treatment eventually develop acquired resistance, which is a main obstacle for GISTs therapy. Therefore, it's urgent to have a better understanding of the mechanisms underlying the imatinib resistance in GISTs to develop novel therapeutic strategies. X-linked inhibitor of apoptosis (XIAP) is the most potent apoptosis inhibitor among the inhibitor of apoptosis protein (IAP) family members. Increased cellular expression of XIAP often leads to drug resistance in cancers. Here we report that XIAP is induced upon imatinb treatment in GIST882 cells, leading to imatinib-induced autophagy. Imatinib-induced autophagy was impaired in XIAP-knockout cells generated by CRISPR/Cas9 system demonstrated by the decreasing of LC3 lipidation. XIAP knockout sensitizes GIST882 cells to imatinib-induced apoptotic cell death, suggesting that XIAP protects GIST882 cells from imatinib-induced cell death by inducing autophagy. Thus, the resistance of the GIST882 cells to imatinib appears to be, in part, due to the increasing of XIAP and subsequent induction of autophagy.

Genomic and transcriptomic analysis of imatinib resistance in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors represent the most common mesenchymal tumor of the digestive tract, driven by gain‐of‐function mutations in KIT. Despite its proven benefits, half of the patients treated with imatinib show disease progression within 2 years due to secondary resistance mutations in KIT. It remains unclear how the genomic and transcriptomic features change during the acquisition of imatinib resistance. Here, we performed exome sequencing and microarray transcription analysis for four imatinib‐resistant cell lines and one cell line briefly exposed to imatinib. We also performed exome sequencing of clinical tumor samples. The cell line briefly exposed to imatinib exhibited few single‐nucleotide variants and copy‐number alterations, but showed marked upregulation of genes related to detoxification and downregulation of genes involved in cell cycle progression. Meanwhile, resistant cell lines harbored numerous genomic changes: amplified genes related to detoxification and deleted genes with cyclin‐dependent kinase activity. Some variants in the resistant samples were traced back to the drug‐sensitive samples, indicating the presence of ancestral subpopulations. The subpopulations carried variants associated with cell death. Pre‐existing cancer cells with genetic alterations promoting apoptosis resistance may serve as a basis whereby cancer cells with critical mutations, such as secondary KIT mutations, can establish full imatinib resistance. © 2017 The Authors Genes, Chromosomes and Cancer Published by Wiley Periodicals, Inc.

Assessing tumor vascularization as a potential biomarker of imatinib resistance in gastrointestinal stromal tumors by dynamic contrast-enhanced magnetic resonance imaging

BackgroundMost metastatic gastrointestinal stromal tumors (GISTs) develop resistance to the first-line imatinib treatment. Recently, increased vessel density and angiogenic markers were reported in GISTs with a poor prognosis, suggesting that angiogenesis is implicated in GIST tumor progression and resistance. The purpose of this study was to investigate the relationship between tumor vasculature and imatinib resistance in different GIST mouse models using a noninvasive magnetic resonance imaging (MRI) functional approach.MethodsImmunodeficient mice (n = 8 for each cell line) were grafted with imatinib-sensitive (GIST882 and GIST-T1) and imatinib-resistant (GIST430) human cell lines. Dynamic contrast-enhanced MRI (DCE-MRI) was performed on GIST xenografts to quantify tumor vessel permeability (Ktrans) and vascular volume fraction (vp). Microvessel density (MVD), permeability (mean dextran density, MDD), and angiogenic markers were evaluated by immunofluorescence and western blot assays.ResultsDynamic contrast-enhanced magnetic resonance imaging showed significantly increased vessel density (P < 0.0001) and permeability (P = 0.0002) in imatinib-resistant tumors compared to imatinib-sensitive ones. Strong positive correlations were observed between MRI estimates, Ktrans and vp, and their related ex vivo values, MVD (r = 0.78 for Ktrans and r = 0.82 for vp) and MDD (r = 0.77 for Ktrans and r = 0.94 for vp). In addition, higher expression of vascular endothelial growth factor receptors (VEGFR2 and VEFGR3) was seen in GIST430.ConclusionsDynamic contrast-enhanced magnetic resonance imaging highlighted marked differences in tumor vasculature and microenvironment properties between imatinib-resistant and imatinib-sensitive GISTs, as also confirmed by ex vivo assays. These results provide new insights into the role that DCE-MRI could play in GIST characterization and response to GIST treatment. Validation studies are needed to confirm these findings.

Molecular mechanism of gastrointestinal stromal tumors and progress in drug research

The functional mutation of c-kit and platelet-derived growth factor receptor α (PDGFRA) which encode proto-oncogene receptor tyrosine kinase are the crucial pathogeneses of gastrointestinal stromal tumors(GISTs). 80%-85% c-kit gene mutation including exon 11,exon 9,exon 13,exon 17 and 5%-10% PDGFRA gene mutation such as exon 18, exon 12 are examined in GISTs. Neither of c-kit or PDGFRA gene mutation are called wide type GISTs. The pathogeneses of wild type GISTs are not clear. The deficiency of succinate dehydrogenase B(SDHB)-related insulin-like growth factor 1(IGF-1R) activation, BRAF gene mutation and neurofibromatosis type 1 may be related to progression of wild type GISTs. More than half of metastatic GISTs patients receiving imatinib treatment can develop to c-kit secondary mutations, which are responsible for secondary resistance. However, the reasons of imatinib resistance in GISTs without c-kit secondary mutation need to be explored. At present, many clinical trials are ongoing to evaluate new drugs in GISTs treatment, including nilotinib, masitinib, pazopanib, dovitinib, ponatinib, dasatinib, crenolanib, linsitinib and immunotherapy, which may bring resistance GISTs treatment to new hope. Next generation sequencing (NGS) and liquid biopsy will be very important in GISTs research and clinical practice.

Abstract 290: NF1 loss modulates cellular fitness in KIT-mutant gastrointestinal stromal tumor (GIST)

Abstract Patients with inoperable GIST obtain substantial clinical benefit from imatinib and other KIT/PDGFRA inhibitors. However, secondary resistance develops in 90% of patients within 18-24 months. Reactivation of oncogenic signaling pathways at time of progression is most often due to secondary mutation in KIT or PDGFRA, but downstream RAS or BRAF mutations are occasionally alternative mechanisms of KIT/PDGFRA-inhibitor resistance. We identified deleterious NF1 mutations in 3 KIT-mutant GIST samples from patients progressing on imatinib. In genomic scale loss-of-function shRNA genomic screens, designed to identify genes involved in imatinib resistance, a shRNA targeting NF1 was the most enriched shRNA in imatinib-treated GIST cells out of 54,000 shRNAs (NF1 gene score 29/11,238). To evaluate the contribution of NF1 loss to KIT-mutant GIST resistance, NF1-deficient GIST models were generated from 2 patient-derived, KIT-mutant GIST cell lines, GIST882 and GIST-T1, by sequential cotransfection with Cas9 and 2 independent NF1-targeting CRISPR-sgRNAs. Complete loss of NF1 protein was observed after 5-10 passages due to on-target heterogeneous genomic deletion at the NF1 locus. Orthogonal validations were obtained using stable shRNA-mediated NF1 knockdown with similar results in the same GIST cell lines. NF1-deleted cells retained expression of mutant KIT but were resistant to imatinib (IC50 196 and 500nM, significantly increased from baseline 34 and 110nM, for GIST-T1 and GIST882, respectively). Notably, after interruption of imatinib treatment, NF1-deficient GIST cells showed reduced proliferation (69% and 77% growth reduction at day 7 off imatinib, for GIST-T1 and GIST882, t test p&amp;lt;0.001) despite continuous expression of activated KIT and downstream signaling effectors. In conclusion, acquired NF1 loss induces TKI resistance in KIT-mutant GIST cells but results in reduced cellular fitness in the absence of KIT-inhibitor treatment. These findings suggest that NF1-deficient GIST subclones that expand on imatinib treatment can be destabilized by intermittently discontinuing KIT-inhibitor therapy. Citation Format: Adrian Marino-Enriquez, Bin Li, Nacef Bahri, Alexandra Lauria, Yuexiang Wang, Jonathan A. Fletcher. NF1 loss modulates cellular fitness in KIT-mutant gastrointestinal stromal tumor (GIST). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 290.

RACK1 overexpression is linked to acquired imatinib resistance in gastrointestinal stromal tumor.

Although treatment with imatinib, which inhibits KIT and PDGFR, controls advanced disease in about 80% of gastrointestinal stromal tumor (GIST) patients, resistance to imatinib often develops. RACK1 (Receptor for Activated C Kinase 1) is a ribosomal protein that contributes to tumor progression by affecting proliferation, apoptosis, angiogenesis, and migration. Here, we found that c-KIT binds to RACK1 and increases proteasome-mediated RACK1 degradation. Imatinib treatment inhibits c-KIT activity and prevents RACK1 degradation, and RACK1 is upregulated in imatinib-resistant GIST cells compared to non-resistant parental cells. Moreover, Erk and Akt signaling were reactivated by imatinib in resistant GIST cells. RACK1 functioned as a scaffold protein and mediated Erk and Akt reactivation after imatinib treatment, thereby promoting GIST cell survival even in the presence of imatinib. Combined inhibition of KIT and RACK1 inhibited growth in imatinib-resistant GIST cell lines and reduced tumor relapse in GIST xenografts. These findings provide new insight into the role of RACK1 in imatinib resistance in GIST.