GIST Cells Research Articles

Fibroblast Growth Factor 2 (FGF2) Activates Vascular Endothelial Growth Factor (VEGF) Signaling in Gastrointestinal Stromal Tumors (GIST): An Autocrine Mechanism Contributing to Imatinib Mesylate (IM) Resistance.

We showed previously that the autocrine activation of the FGFR-mediated pathway in GIST lacking secondary KIT mutations was a result of the inhibition of KIT signaling. We show here that the FGF2/FGFR pathway regulates VEGF-A/VEGFR signaling in IM-resistant GIST cells. Indeed, recombinant FGF2 increased the production of VEGF-A by IM-naive and resistant GIST cells. VEGF-A production was also increased in KIT-inhibited GIST, whereas the neutralization of FGF2 by anti-FGF2 mAb attenuated VEGFR signaling. Of note, BGJ 398, pan FGFR inhibitor, effectively and time-dependently inhibited VEGFR signaling in IM-resistant GIST T-1R cells, thereby revealing the regulatory role of the FGFR pathway in VEGFR signaling for this particular GIST cell line. This also resulted in significant synergy between BGJ 398 and VEGFR inhibitors (i.e., sunitinib and regorafenib) by enhancing their pro-apoptotic and anti-proliferative activities. The high potency of the combined use of VEGFR and FGFR inhibitors in IM-resistant GISTs was revealed by the impressive synergy scores observed for regorafenib or sunitinib and BGJ 398. Moreover, FGFR1/2 and VEGFR1/2 were co-localized in IM-resistant GIST T-1R cells, and the direct interaction between the aforementioned RTKs was confirmed by co-immunoprecipitation. In contrast, IM-resistant GIST 430 cells expressed lower basal levels of FGF2 and VEGF-A. Despite the increased expression VEGFR1 and FGFR1/2 in GIST 430 cells, these RTKs were not co-localized and co-immunoprecipitated. Moreover, no synergy between FGFR and VEGFR inhibitors was observed for the IM-resistant GIST 430 cell line. Collectively, the dual targeting of FGFR and VEGFR pathways in IM-resistant GISTs is not limited to the synergistic anti-angiogenic treatment effects. The dual inhibition of FGFR and VEGFR pathways in IM-resistant GISTs potentiates the proapoptotic and anti-proliferative activities of the corresponding RTKi. Mechanistically, the FGF2-induced activation of the FGFR pathway turns on VEGFR signaling via the overproduction of VEGF-A, induces the interaction between FGFR1/2 and VEGFR1, and thereby renders cancer cells highly sensitive to the dual inhibition of the aforementioned RTKs. Thus, our data uncovers the novel mechanism of the cross-talk between the aforementioned RTKs in IM-resistant GISTs lacking secondary KIT mutations and suggests that the dual blockade of FGFR and VEGFR signaling might be an effective treatment strategy for patients with GIST-acquired IM resistance via KIT-independent mechanisms.

Abstract 956: FBXW7 regulates imatinib chemoresistance through targeting MCL1 for apoptotic regulation in gastrointestinal stromal tumors

Abstract Introduction: Imatinib definitively contribute to improve prognosis for high-risk or unresectable GISTs. Due to therapeutic efficacy is limited by imatinib chemoresistance and toxicity, the exploration of predictive markers of imatinib therapeutic efficacy that early enable patients to utilize more effective therapeutic strategies remains urgent. Here, we evaluate that the ability of FBXW7 acts as a predictor of imatinib therapeutic effect in preclinical models, and the potential of FBXW7-MCL1 axis promote imatinib-induced apoptosis response. Methods: The cellular and in vivo correlation between FBXW7 and imatinib chemoresistance via FBXW7-MCL1 axis were evaluated by detecting IC50 value, apoptosis assay, and GIST xenograft studies. The clinical significance of FBXW7 as a predictor of imatinib treatment efficacy was examined in 140 high-risk patients with GISTs by immunohistochemical staining and multivariate analyses. Results: FBXW7 affects imatinib chemoresistance through regulating cellular apoptosis in GIST-T1 cells. FBXW7 targets MCL1 to regulate cellular apoptosis. MCL1 involves in the regulation of imatinib chemoresistance through inhibiting apoptosis of GIST-T1 cells. FBXW7 regulates imatinib chemoresistance via down-regulating MCL1 for enhancing imatinib-induced apoptosis in vitro. FBXW7 regulates imatinib sensitivity of GIST cells via targeting MCL1 to predict therapeutic efficacy of imatinib treatment in vivo. Through 5-year RFS rates analysis and multivariate analysis, the ability of FBXW7 predicting therapeutic efficacy of high-risk GIST patients with adjuvant imatinib was proved. Conclusions: FBXW7 regulates imatinib chemoresistance through inhibiting MCL1 for enhancing imatinib-induced apoptosis in GIST and acted as a reliable predictor of imatinib treatment efficacy in high-risk GIST patients treated imatinib. Citation Format: Xiyu Wu, Masaaki Iwatsuki, Meiyue Lou, Takatsugu Ishimoto, Yoshifumi Baba, Yuji Miyamoto, Hideo Baba. FBXW7 regulates imatinib chemoresistance through targeting MCL1 for apoptotic regulation in gastrointestinal stromal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 956.

Mesenchymal stromal cells promote the drug resistance of gastrointestinal stromal tumors by activating the PI3K-AKT pathway via TGF-β2

BackgroundGastrointestinal stromal tumors (GISTs) are the prevailing sarcomas of the gastrointestinal tract. Tyrosine kinase inhibitors (TKIs) therapy, exemplified by Imatinib mesylate (IM), constitutes the established adjuvant therapy for GISTs. Nevertheless, post-treatment resistance poses a challenge that all patients must confront. The presence of tumor heterogeneity and secondary mutation mechanisms fail to account for some instances of acquired drug resistance. Certain investigations suggest a strong association between tumor drug resistance and mesenchymal stromal cells (MSC) in the tumor microenvironment, but the underlying mechanism remains obscure. Scarce research has explored the connection between GIST drug resistance and the tumor microenvironment, as well as the corresponding mechanism.MethodsImmunofluorescence and fluorescence-activated cell sorting (FACS) methodologies were employed to detect the presence of MSC in GIST samples. The investigation encompassed the examination of MSC migration towards tumor tissue and the impact of MSC on the survival of GIST cells under IM treatment. Through ELISA, western blotting, and flow cytometry analyses, it was confirmed that Transforming Growth Factor Beta 2 (TGF-β2) triggers the activation of the PI3K-AKT pathway by MSC, thereby facilitating drug resistance in GIST.ResultsOur findings revealed a positive correlation between a high proportion of MSC and both GIST resistance and a poor prognosis. In vitro studies demonstrated the ability of MSC to migrate towards GIST. Additionally, MSC were observed to secrete TGF-β2, consequently activating the PI3K-AKT pathway and augmenting GIST resistance.ConclusionsOur investigation has revealed that MSC within GISTs possess the capacity to augment drug resistance, thereby highlighting their novel mechanism and offering a promising target for intervention in GIST therapy.

Abstract A013: CDK2 and CDK4/6 inhibition in GIST: Mechanisms of response and resistance

Abstract Advanced GIST is characterized by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor (CDK4/6i) therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximizing response to therapeutic CDK4/6 inhibition. Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples (N=18), including 8 metastatic TKI-resistant GISTs. Multiple metastases were analyzed in 3 patients. The impact of CDK2i (CDK2 inhibitor-II), CDK4/6i (palbociclib or abemaciclib), and CDK2/4/6i (PF-06873600) was determined through cell proliferation and protein detection assays in vitro and in vivo. Mechanisms of acquired CDK2i and CDK4/6i resistance were characterized in GIST cell lines after long-term exposure. The results demonstrate recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways. Identical aberrations of p16, RB1, and TP53 were present in all metastases from 3 patients. We show that therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation (P<0.01). Intact RB1 predicted response to treatment, whereas RB1-deficient models were resistant. Moreover, we identify RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) as mechanisms of acquired CDK inhibitor resistance in GIST. The CCND1 rearrangement deleted the cyclin D1 C-terminal Thr286 and Thr288 residues which mediate cyclin D1 proteasomal degradation, resulting in overexpression of an abnormal cyclin D1. CDK inhibitor resistance properties were corroborated by lentiviral transduction of the CCND1 fusion gene into fusion-negative GIST, leiomyosarcoma, and breast cancer cells. These studies establish the biologic rationale for CDK2 and CDK4/6 co-inhibition as therapeutic strategy in patients with advanced GIST, including patients with metastatic GIST progressing on TKIs. In addition, these findings expand the spectrum of potential CDK inhibitor resistance mechanisms with translational potential for improving cell cycle targeted therapies in other cancer types. Citation Format: Inga-Marie Schaefer, Matthew L. Hemming, Meijun Z. Lundberg, Matthew P. Serrata, Isabel Goldaracena, Ninning Liu, Peng Yin, Joao A. Paulo, Steven P. Gygi, Suzanne George, Jeffrey A. Morgan, Monica M. Bertagnolli, Ewa T. Sicinska, Adrian Mariño-Enríquez, Jason L. Hornick, Chandrajit P. Raut, George D. Demetri, Wen-Bin Ou, Sinem K. Saka, Jonathan A. Fletcher. CDK2 and CDK4/6 inhibition in GIST: Mechanisms of response and resistance [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A013.

THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

BackgroundGastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract and are characterized by activating mutations of c-KIT or PDGFRa receptor tyrosine kinases (RTKs). Despite the clinical success of tyrosine kinase inhibitors (TKIs), more than half of GIST patients develop resistance due to a second mutation. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of CDK-activating kinase (CAK), and it plays an important role in the regulation of cell cycle transitions and gene transcription. THZ1, a CDK7 inhibitor, exhibits a dose-dependent inhibitory effect in various cancers.MethodsData from the public GEO database and tissue microarray were used to analyse the gene expression levels of CDKs in GISTs. The impact of CDK7 knockdown and the CDK7 inhibitor THZ1 on GIST progression was investigated in vitro using CCK-8, colony formation, and flow cytometry assays and in vivo using a xenograft mouse model. RNA sequencing was performed to investigate the mechanism of GIST cell viability impairment mediated by THZ1 treatment.ResultsOur study demonstrated that CDK7 is relatively overexpressed in high-risk GISTs and predicts a poor outcome. A low concentration of THZ1 exhibited a pronounced antineoplastic effect in GIST cells in vivo and in vitro. Moreover, THZ1 exerted synergistic anticancer effects with imatinib. THZ1 treatment resulted in transcriptional modulation by inhibiting the phosphorylation of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII). c-KIT, an oncogene driver of GIST, was transcriptionally repressed by THZ1 treatment or CDK7 knockdown. Transcriptome sequencing analysis showed that OSR1 acts as a downstream target of CDK7 and regulates c-KIT expression. Taken together, our results highlight elevated CDK7 expression as a predictor of poor outcome in GIST and present the combination of CDK7 and RTK inhibitors as a potent therapeutic strategy to improve the efficacy of GIST treatment.3dK7zp1xpFMvfXo5mnDRH-Video abstract

Efficacy of SCF drug conjugate targeting c-KIT in gastrointestinal stromal tumor

BackgroundGastrointestinal stromal tumor (GIST) is a rare type of cancer that occurs in the gastrointestinal tract. The majority of GIST cases carry oncogenic forms of KIT, the receptor for stem cell factor (SCF). Small molecule kinase inhibitor imatinib is effective in prolonging the survival of GIST patients by targeting KIT. However, drug resistance often develops during the therapeutic treatment. Here, we produced a SCF-emtansine drug conjugate (SCF-DM1) with favorable drug efficacy towards GIST cells.MethodsRecombinant human SCF (rhSCF) was expressed in E. coli cells and further purified with Ni–NTA Sepharose and Phenyl Sepharose. It was then conjugated with DM1, and the conjugated product SCF-DM1 was evaluated using in vitro cell-based assays and in vivo xenograft mouse model.ResultsSCF-DM1 was effective in inhibiting imatinib-sensitive and -resistant GIST cell lines and primary tumor cells, with IC50 values of < 30 nM. It induced apoptosis and cell cycle arrest in GIST cells. In xenograft mouse model, SCF-DM1 showed favorable efficacy and safety profiles.ConclusionsrhSCF is a convenient and effective vector for drug delivery to KIT positive GIST cells. SCF-DM1 is an effective drug candidate to treat imatinib-sensitive and -resistant GIST.

Abstract 5648: Response and resistance to CDK2 and CDK4/6 inhibition in GIST

Abstract Gastrointestinal stromal tumor (GIST) is the most common GI sarcoma and is generally initiated by KIT or PDGFRA mutations which are compelling therapeutic targets for tyrosine kinase inhibitors (TKI). However, the emergence of secondary mutations results in clinical resistance to available TKIs. GIST progression is driven by genomic events which incrementally target the p16-CDK4/6-RB1 and p14-TP53-RB1 pathways to create CDK4/6 and CDK2 oncogenic co-dependency. Based on limited efficacy of single-agent CDK4/6-inhibitor (CDK4/6i) therapy in GIST, we evaluated strategies of co-targeting CDK2 and CDK4/6. Multiplexed protein imaging (via Immuno-SABER) was validated for the detection of cell cycle regulator aberrations in GIST clinical samples (N=18), 7 of which were TKI-resistant, and including 3 patients in whom multiple metastases were analyzed. The impact of various CDK perturbants using CDK2i (CDK2 inhibitor-II), CDK4/6i (palbociclib or abemaciclib), and CDK2/4/6i (PF-06873600) was determined through cell proliferation and protein detection assays in GIST cell lines and murine xenografts. Mechanisms of acquired CDK2i and CDK4/6i resistance were characterized in GIST cell lines after long-term exposure. Abnormal expression/biallelic inactivation of CDKN2A/p16, RB1, and TP53 were identified in 7 (39%), 2 (11%), and 2 (11%) of 18 GISTs, respectively. Identical aberrations of p16, RB1, and TP53 were present in all metastases from 3 patients. Since 5 of 7 RB1-intact advanced GISTs had co-dysregulation of the CDK2 and CDK4/6 pathways, we evaluated co-inhibition of CDK2 and CDK4/6 in vitro and in vivo which inhibited cell proliferation (P&amp;lt;0.01) and RB1 hyperphosphorylation. Intact RB1 predicted response to treatment, whereas RB1-deficient models were resistant. Two resistant sub-lines emerged after 11 and 14 months of palbociclib exposure: one with biallelic genomic RB1 inactivation and the other with the first known example of a cyclin D1 coding sequence fusion with oncogenic properties (CCND1::chr11.g:70025223). The CCND1 fusion deleted the cyclin D1 C-terminal Thr286 and Thr288 residues which mediate cyclin D1 proteasomal degradation, resulting in overexpression of an abnormal cyclin D1. Palbociclib-resistance properties were corroborated by lentiviral transduction of the CCND1 fusion gene into fusion-negative GIST, leiomyosarcoma, and breast cancer cells. CDK2 and CDK4/6 pathway perturbations with retained RB1 are frequent in advanced GIST and can be conserved across metastases, creating a compelling biologic rationale for therapeutic cell cycle restoration. We show that co-inhibition of CDK2 and CDK4/6 is synergistic in GIST and highlight RB1 inactivation and a novel oncogenic cyclin D1 as mechanisms of acquired CDKi resistance. Hence, combination therapies targeting CDK2 and CDK4/6 with correlative biomarkers predictive of response should be evaluated in patients with metastatic or TKI-resistant GIST. Citation Format: Inga-Marie Schaefer, Meijun Z. Lundberg, Matthew L. Hemming, Sinem K. Saka, Matthew P. Serrata, Isabel Goldaracena, Ninning Liu, Peng Yin, Joao A. Paulo, Steven Gygi, George D. Demetri, Ewa Sicinska, Adrian Mariño-Enríquez, Jason L. Hornick, Chandrajit P. Raut, Wen-Bin Ou, Jonathan A. Fletcher. Response and resistance to CDK2 and CDK4/6 inhibition in GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5648.

The synergistic therapeutic effect of imatinib and protein kinase CK2 Inhibition correlates with PI3K-AKT activation in gastrointestinal stromal tumors

BackgroundGastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Casein kinase 2 (CK2) has been reported to be involved in several cellular processes in multiple cancers. However, the role of CK2 in GIST remains unclear. AimWe aimed to investigate the combinatorial treatment of imatinib (IM) and CK2 inhibition on the progression of GISTs. MethodsGIST biopsies and adjacent normal tissues were collected from patients. GIST882 and GIST48 cell lines were subjected to investigate the effect of IM and CK2 inhibition in GIST cells. CCK-8 assay, Caspase-3 activity assay, western blotting, and flow cytometry analysis were employed in the present investigation. ResultsOur results showed that CK2 was highly expressed in GIST biopsies, and inhibition of CK2 resulted in decrease in cell viability and increase in apoptosis of GIST cells. Moreover, the combination treatment with CX-4945 (CX) and IM resulted in a more significant decrease in cell viability and increase in cell apoptosis compared with mono-treatment. Mechanistically, the combination treatment influenced the activation of the PI3K/AKT pathway. The activation of the PI3K/AKT pathway reversed the synergistic impacts of the combined treatment on cell viability and apoptosis. ConclusionOur results demonstrated that inhibition of CK2 combined with IM exhibited a synergistic anti-cancer effect on GIST cells through inactivation of the PI3K/AKT pathway.

Selected Articles from This Issue

Relapse to MAp. pathway inhibitors (such as BRAF, MEK, and KRAS inhibitors) is common due to the reactivation of ERK and MAPK signaling. In this First Disclosure, Wallis, Munck, and colleagues outline the ERK inhibitor ASTX029. ASTX029 inhibits both the phosphorylation of ERK by MEK as well as its catalytic activity and, therefore, is categorized as a dual-mechanism inhibitor by the authors. The dual-mechanism ERK inhibitor demonstrated anti-tumor activity to MAPK inhibitor-resistant cancers in vitro and in vivo. Their work supports the continued clinical development of ASTX029 in advanced solid tumors.The specific DNA damage O6-methylguanine induced by temozolomide was quantified and set in relation to double-strand breaks, apoptosis and senescence. The findings challenged the model of DNA damage thresholding as O6-methylguanine induced these endpoints in glioblastoma cells without a no-effect threshold. Interestingly, senescence appears to occur at a much lower number of O6-methylguanine molecules than cell death, indicating that low dose temozolomide impedes cytotoxicity by increasing the quiescent population. The data indicate a) need of inhibiting selectively senescence in order to ameliorate the killing effects and b) quantification of O6-methylguanine could serve as a potential biomarker of temozolomide intratumoral dose.Although imatinib can target KIT mutations in gastrointestinal stomach tumor, 40% of localized tumors recur within 5 years. In this study, Sicklick and colleagues report a population of CD34+KITlow GIST cells with a cancer stem cell-like expression profile and were enriched by imatinib treatment. Tumors with low KIT expression had activated Gas6/AXL and NF-κB pathways which could be targeted by bemcentinib (AXL inhibitor) or bardoxolone (NF-κB inhibitor) alone or in combination with imatinib. Their findings present a new population of resistant cells that can be overcome in recurrent GIST disease.Despite advances in experimental therapeutics for refractory acute myeloid leukemia (AML), it remains incurable. The success of CD19 CAR T cells in hematologic malignancies has led to their development in expanding indications. In this manuscript, Rennert and colleagues describe the preclinical development of a CD19-anti-CLEC12A bridge-protein secreting CAR T cell targeting CLEC12A in AML. The CAR19 cells secrete the biparatopic bridging protein to increase the cytotoxicity against CLEC12A-positive AML cells in vitro and in vivo. The efficacy of these cells demonstrates a means to thwart resistance by enhancing CD19-based CAR T cells.

KITlow Cells Mediate Imatinib Resistance in Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by imatinib (IM), a tyrosine kinase inhibitor (TKI). However, IM does not cure GIST, and adjuvant therapy only delays recurrence in high-risk tumors. We hypothesized that GIST contains cells with primary IM resistance that may represent a reservoir for disease persistence. Here, we report a subpopulation of CD34+KITlow human GIST cells that have intrinsic IM resistance. These cells possess cancer stem cell-like expression profiles and behavior, including self-renewal and differentiation into CD34+KIThigh progeny that are sensitive to IM treatment. We also found that TKI treatment of GIST cell lines led to induction of stem cell-associated transcription factors (OCT4 and NANOG) and concomitant enrichment of the CD34+KITlow cell population. Using a data-driven approach, we constructed a transcriptomic-oncogenic map (Onco-GPS) based on the gene expression of 134 GIST samples to define pathway activation during GIST tumorigenesis. Tumors with low KIT expression had overexpression of cancer stem cell gene signatures consistent with our in vitro findings. Additionally, these tumors had activation of the Gas6/AXL pathway and NF-κB signaling gene signatures. We evaluated these targets in vitro and found that primary IM-resistant GIST cells were effectively targeted with either single-agent bemcentinib (AXL inhibitor) or bardoxolone (NF-κB inhibitor), as well as with either agent in combination with IM. Collectively, these findings suggest that CD34+KITlow cells represent a distinct, but targetable, subpopulation in human GIST that may represent a novel mechanism of primary TKI resistance, as well as a target for overcoming disease persistence following TKI therapy.

Abstract 3767: FBXO32 ubiquitin ligase mediates apoptosis evasion and adaptation to KIT inhibition in gastrointestinal stromal tumor - GIST

Abstract Introduction: GIST is the most common malignant mesenchymal neoplasm and features myogenic differentiation. Most GISTs depend upon oncogenic signaling through constitutively activated forms of KIT and exhibit remarkable responses to the first-line KIT inhibitor imatinib. Secondary resistance involves emergence of subclonal populations harboring heterogeneous KIT-secondary mutations that are not collectively inhibited by any approved therapy. We dissected KIT-downstream signaling nodes to identify mediators of the KIT oncogenic program, seeking targets of overarching relevance in polyclonal imatinib-resistant GIST. Methods: using clinically-representative human GIST cell models, we undertook pharmacological and functional screening to identify key targetable nodes within KIT-downstream PI3K/mTOR and RAS/MAPK pathways and to model therapeutic strategies in vitro and in vivo. Transcriptomic analysis (RNAseq) identified genes co-regulated by PI3K/mTOR and RAS/MAPK, and functional evaluations by lentiviral construct overexpression and gene knockdown elucidated roles in GIST biology. Results: PI3K/mTOR and MEK1/2 were the most essential KIT-dependent nodes irrespective of the type of KIT primary or secondary mutation. Single node ablation did not yield sustained antiproliferative and apoptotic effects, but concurrent intermittent inhibition was synergistic in vitro and in vivo, supporting the critical and complementary roles of these two pathways. Transcriptomic analyses underscored FBXO32 (a SCF E3 ubiquitin ligase and the main effector of muscular atrophy) as the most differentially expressed gene co-regulated by the PI3K/mTOR and RAS/MAPK pathways. FBXO32 was also the most differentially expressed gene after KIT inhibition in GIST cell lines, and enrichment in FBXO32 expression was found in post-imatinib GIST samples compared to baseline matched tumor tissue. We demonstrated that FBXO32 is transcriptionally upregulated by FOXO3a upon KIT or concurrent PI3K/mTOR and MEK1/2 blockage, paralleling human muscle regulation. Notoriously, FBXO32 proved to have a pro-survival role in GIST cells, enabling evasion of apoptosis. Microarray and in vitro and in vivo functional assays showed that FBXO32 mediates cell cycle exit and quiescence upon KIT or concurrent KIT-downstream pathways inhibition, thereby minimizing imatinib-induced apoptosis and facilitating GIST cell survival. Conclusions: PI3K/mTOR and MEK1/2 are critical mediators of KIT oncogenic signaling in GIST. FBXO32 emerges as a KIT-dependent E3 ubiquitin ligase transcriptionally repressed through KIT downstream pathways. However, FBXO32 induction upon therapeutic KIT signaling inhibition triggers quiescence and subsequent apoptosis evasion, thus emerging as a novel mechanism of adaptation to therapeutic inhibition of oncogenic KIT. Citation Format: Alfonso García Valverde, Jordi Rosell, Daniel Pilco Janeta, Sergi Sayols, Claudia Valverde, Anna Esteve, Marta Gut, Jordi Barretina, George D. Demetri, Jonathan A. Fletcher, Joan Carles, Joaquín Arribas, César Serrano. FBXO32 ubiquitin ligase mediates apoptosis evasion and adaptation to KIT inhibition in gastrointestinal stromal tumor - GIST [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3767.

Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway.

BackgroundGastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. However, up to 40–50% of GISTs develop secondary resistance after an average of 24 months of imatinib treatment. It has been reported that autophagy can promote the survival of GIST cells and induce drug resistance. Presently, the specific mechanism of autophagy in GISTs with imatinib resistance is not clear.Materials and MethodsThe cell-counting kit (CCK)-8 method and flow cytometry were used for in vitro drug sensitivity testing and autophagy level detection. Detection of the apoptosis level was by flow cytometry with the annexin V Kit. Western blotting was used to analyze the role of autophagy and apoptosis in GIST cells with CQ alone, imatinib alone, or in combination, and to analyze MAPK pathway expression. In vitro results were confirmed by in vivo experiments using the mice model. Hematoxylin and eosin and immunohistochemical staining were used to detect the pathological characteristics and immunophenotype of the transplanted tumor. Detection of KIT and PDGFRA gene mutations in the transplanted imatinib-resistant GIST was done by denaturing high performance liquid chromatography (DHPLC) and direct sequencing. ERK and KIT expression and regulation levels were detected by Western blotting.ResultsIn vitro and vivo experiments, the autophagy level of imatinib-resistant cells was higher than that of normal cells; CQ combined with imatinib can promote apoptosis by blocking autophagy of imatinib-resistant cells. In the meanwhile, we found that the phosphorylation level of ERK may be related to autophagy.ConclusionOur data suggest that autophagy through the MAPK/ERK pathway may play a pivotal role in imatinib-resistant GIST proliferation. Moreover, combining an autophagy inhibitor with imatinib may be a potential valuable strategy in overcoming acquired resistance in GIST patients.

Anti-KIT DNA Aptamer for Targeted Labeling of Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST), the most common sarcoma, is characterized by KIT protein overexpression, and tumors are frequently driven by oncogenic KIT mutations. Targeted inhibition of KIT revolutionized GIST therapy and ushered in the era of precision medicine for the treatment of solid malignancies. Here, we present the first use of a KIT-specific DNA aptamer for targeted labeling of GIST. We found that an anti-KIT DNA aptamer bound cells in a KIT-dependent manner and was highly specific for GIST cell labeling in vitro Functionally, the KIT aptamer bound extracellular KIT in a manner similar to KIT mAb staining, and was trafficked intracellularly in vitro The KIT aptamer bound dissociated primary human GIST cells in a mutation agnostic manner such that tumors with KIT and PDGFRA mutations were labeled. In addition, the KIT aptamer specifically labeled intact human GIST tissue ex vivo, as well as peritoneal xenografts in mice with high sensitivity. These results represent the first use of an aptamer-based method for targeted detection of GIST in vitro and in vivo.

Gastrointestinal stromal tumor after tyrosine kinase inhibition therapy: a review of biopsies of 34 patients with clinically suspected relapse and/or progression of the tumor.

Implementation of combined surgical and targeted therapy strategies using tyrosine kinase inhibitors improved the prognosis of patients with aggressive GISTs. The therapeutic answer may be individually different, some patients do not respond properly, or even progress in spite of the therapy. This together with intratumoral heterogeneity and possible development of secondary phenotypical and genetical changes represents a challenge for pathologists examining a biopsy of relapsed tumors and/or their metastases. For this study biopsy files of the national Slovak GIST registry were reviewed to identify patients examined bioptically both prior the therapy and during the TKI treatment due to suspected tumor relapse and/or progression. All the GIST biopsies were analyzed using a standardized algorithm of histological, immunohistochemical and molecular analyses of exon 7, 9, 11, 13 of c-KIT and exons 12, 14, and 18 of PDGFRA genes, with the aim to identify posttherapeutical changes of these parameters. From 34 patients fulfilling the criteria of selection, all were histologically examined during their clinically suspicious first GIST relaps, eight during the 2nd, three during 3rd and one during 4th and 5th relapse resp. All but one posttherapeutical biopsies showed "viable" GIST tissue and so 44 relapses of 33 patients could be evaluated in comparison with identical parameters of diagnostic biopsies. Distinguishing three major histological types (spindle-, epitheloid-cell and mixed cell type), a change of the GIST type was identified in 1/3 of 1st relapse and ¼ of all relapse biopsies. Evaluation of three phenotypical GIST parameters CD117, CD34 and DOG-1, showed that phenotype alteration was always represented by a single change. The most common was either a gain or loss of CD34 positivity appearing in 1/3 of 1st relapse biopsies, while a loss of CD117 positivity was identified in one patient´s biopsy only. Altogether, the phenotypical changes were in ¼ of all relapses. A changed mutational profile was recognized in 38,2% first relaps biopsies and in 33% of all relapses, the change was mostly isolated (in 10/45 relapses) and less often (in 4/45 relapses) it represented a gain of a new mutation in association with persisting original one. In conclusion, the biopsies of patients showing relapse and/or progression on TKI treatment show predominance of viable GIST cells with limited or even absent signs of scaring, as well as relatively low incidence of morphological, pheno- and genotypical changes.

Abstract A40: MicroRNA-494 inhibits growth of the gastrointesintal stromal tumor by targeting multiple cell cycle regulatory genes

Abstract Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms occurring throughout the entire region of the gastrointestinal tract. Two oncogenes of the receptor tyrosine kinase family, KIT and PDGFRA, have gain-of-function mutations in approximately 70% and 15% of GISTs. Mutations of these 2 genes cause sustained activation, resulting in constant stimulation of the downstream signaling pathways of KIT and platelet-derived growth factor receptor, alpha polypeptide. Although KIT mutations are present in approximately 70% of GISTs,KIT overexpression is observed in more than 90% of GISTs. Dysregulation of microRNAs(miRNA)-494 was demonstrated to be a major cause for the overexppression of mutatnt KIT proteins in GISTs. It was also demonstrated that miR-494 treatment inhibits proliferation of GISTs and promotes apoptosis however, no details mechanisms are not known. The purpose of this study was to provide evidence regarding the roles of miR-494 in GIST suppression. To elucidate the specific pathways affected by miRNA-494 treatment on GIST cell lines, transcriptome analysis of GIST430 and GIST882 cells transfected with nontargeting microRNA or with miR-494 was performed and subsequently validated by qRT-PCR. Based on the transcriptome analysis, we found that multiple cell cycle relevant pathways were down-regulated by miR-494 treatment and importantly, core genes such as KIT, CASP2, CDCA2, AURKB, CDCA8, E2F7, BIRC5 and CKS1B involved in the regulation of cell cycle pathways were identified as novel targets by miR-494. Using reporter assays, we further validated that miR-494 directly binds to the 3'UTR regions of BRAC5, CKS1B and E2F7 and induces biological alterations. To demonstrate the perturbation of cell cycle pathways by miR-494, FACS analysis using five GIST cell lines was performed and we found that all of the five GIST cell lines transfected with miR-494 subjected to cell cycle arrest and apoptosis. Colony forming and proliferation assays also demonstrated that miR-494 treatment induced the growth inhibition of GIST cells which was derived from the alterations of cell cycle pathways. Taken together, our findings suggest that the efficient suppression of GISTs by miR-494 is driven not only by targeting KIT, but also multiple genes involved in cell cycle pathways Citation Format: SeongJu Yun, Won Kyu Kim, Hoguen Kim. MicroRNA-494 inhibits growth of the gastrointesintal stromal tumor by targeting multiple cell cycle regulatory genes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A40.

Targeting Disease Persistence in Gastrointestinal Stromal Tumors.

SummaryGastrointestinal stromal tumors (GISTs) represent 20%-40% of human sarcomas. Although approximately half of GISTs are cured by surgery, prognosis of advanced disease used to be poor due to the high resistance of these tumors to conventional chemo- and radiotherapy. The introduction of molecularly targeted therapy (e.g., with imatinib mesylate) following the discovery of the role of oncogenic mutations in the receptor tyrosine kinases KIT and platelet-derived growth factor α (PDGFRA) significantly increased patient survival. However, GIST cells persist in 95%-97% of imatinib-treated patients who eventually progress and die of the disease because of the emergence of clones with drug-resistant mutations. Because these secondary mutations are highly heterogeneous, even second- and third-line drugs that are effective against certain genotypes have only moderately increased progression-free survival. Consequently, alternative strategies such as targeting molecular mechanisms underlying disease persistence should be considered. We reviewed recently discovered cell-autonomous and microenvironmental mechanisms that could promote the survival of GIST cells in the presence of tyrosine kinase inhibitor therapy. We particularly focused on the potential role of adult precursors for interstitial cells of Cajal (ICCs), the normal counterpart of GISTs. ICC precursors share phenotypic characteristics with cells that emerge in a subset of patients treated with imatinib and in young patients with GIST characterized by loss of succinate dehydrogenase complex proteins and lack of KIT or PDGFRA mutations. Eradication of residual GIST cells and cure of GIST will likely require individualized combinations of several approaches tailored to tumor genotype and phenotype. Gastrointestinal stromal tumors (GISTs) are one of the most common connective tissue cancers. Most GISTs that cannot be cured by surgery respond to molecularly targeted therapy (e.g., with imatinib); however, tumor cells persist in almost all patients and eventually acquire drug-resistant mutations. Several mechanisms contribute to the survival of GIST cells in the presence of imatinib, including the activation of "escape" mechanisms and the selection of stem-like cells that are not dependent on the expression of the drug targets for survival. Eradication of residual GIST cells and cure of GIST will likely require individualized combinations of several approaches tailored to the genetic makeup and other characteristics of the tumors.

Molecularly-guided therapeutic options beyond tyrosine kinase inhibitors (TKIs) for gastrointestinal stromal tumors (GIST).

58 Background: GISTs are characterized by KIT/PDGFRA mutations. A range of multi-targeted tyrosine kinase inhibitors (TKIs) are available for treatment, however, resistance mechanisms inevitably emerge. Recent data (Boichuk, et al 2014) suggests the potential efficacy of various cytotoxic therapies that were identified as being able to effectively kill TKI-responsive and -resistant GIST cells. We sought to investigate the theranostic markers associated with non-TKI therapy options for their potential role in treatment of GIST. Methods: 147 GIST cases were evaluated. A multiplatform approach of biomarker testing was used and included a combination of sequencing (NGS, Sanger), protein expression (IHC) and gene amplification (ISH). Results: Multidrug resistance phenotype was found in 52-68% (PGP, MRP1). Tubulin-binding agents (taxanes, vinca alkaloids) may be of potential use due to the high frequency of low TUBB3 expression (72% or 39/54). Anthracyclines and topoisomerase inhibitors may be of potential benefit in 1/3 of patients based on expression of TOPO2A (32% or 32/99) and TOPO1 (34% or 37/110). Cytotoxic agents used in non-GIST solid tumors, may also be considered, based on high frequency of low expressin of MGMT (47% or 57/122), TS (70% or 76/109) and RRM1 (79% or 88/111). PTEN was intact (positive expression) in the majority of GIST (87%). Nine patients were examined for PD1/PDL1: 56% exhibited positive tumor infiltrating lymphocytes and 33% exhibited PDL1 tumor expression. Only one amplification event was observed: cMET (0/53), HER2 (0/69), EGFR (0/16), PIK3CA (0/1) and TOP2A (1/11). Mutational screening using a hot spot cancer panel (and Sanger sequencing for some genes) resulted in the detection of variants in only 10 genes, excluding KIT (97/132) and PDGFRA (5/55). Variants were detected in the following genes, in decreasing order of frequency: RB1, APC and JAK3 (2/55; 2/55; 2/57), PIK3CA (2/69) and ABL1, cMET, EGFR, KDR, VHL and BRAF (1/55, 1/57, 1/57, 1/57, 1/52, 1/78). Conclusions: A multi-platform approach of theranostic biomarkers identified therapies beyond TKIs for GIST. Various cytotoxics and non-KIT/PDGFRA targeted therapies were identified based on protein expression or gene variations.

Augmentation of multiple protein kinase activities associated with secondary imatinib resistance in gastrointestinal stromal tumors as revealed by quantitative phosphoproteome analysis.

In this study, we performed a quantitative phosphoproteome analysis using a cell culture-based GIST model system. The goal of the study was to investigate the mechanism of acquired resistance in GISTs against imatinib, a molecularly targeted drug that inhibits kinase activity of the KIT protein and that has been approved for the treatment of GISTs. In imatinib-resistant GIST cells, we observed elevated expression of KIT and restoration of its kinase activity, as well as activation of multiple proliferative signaling pathways. Our results indicate that the effects of even so-called 'molecularly targeted' drugs, are broad rather than convergent, and that the mechanisms of action of such drugs during continuous administration are extremely complex.

Abstract 3396: Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates imatinib antitumor effect in GIST oncogenesis

Abstract Gastrointestinal stromal tumour (GIST) is one of the most common types of human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. Despite the initial clinical success of imatinib that specifically target mutant KIT and PDGFRA, imatinib resistance has become the biggest challenge in the management of advanced GIST patient. Novel therapeutics that can improve the efficacy of first line imatinib therapy and/or prevent and overcome imatinib resistance is imperative. The ETS family member, ETV1, has been previously identified as a lineage-specific survival factor that cooperates with mutant KIT by ETV1 protein stabilization by active MAP kinase signaling downstream of KIT signaling in GIST oncogenesis. Here we demonstrate that ETV1 is required for GIST initiation and maintenance in vivo using compound genetically engineered mouse models (GEMM). We further identified that ETV1 forms a feed forward circuit in GIST oncogenesis where the ETV1 protein is stabilized by active MAP kinase signaling downstream of KIT and stabilized ETV1enhances KIT expression through direct binding to the KIT enhancer regions in both GIST mouse models and human GIST cell lines. The dual lineage targeting of KIT by imatinib and ETV1 by MEK162 (an MEK inhibitor) disrupts the ETV1-KIT feed forward circuit and induces more apoptosis than single agent imatinib or MEK162 in human GIST cells. The combination therapy resulted in complete tumor regression whereas single agent imatinib or MEK162 resulted in stabilization of disease in human GIST xenograft studies. Moreover, the combination therapy also induced more tumor fibrosis than single agent imatinib or MEK162 in GIST GEMM. These observations demonstrate the in vivo role of ETV1 in GIST oncogenesis and the feasibility of targeting ETV1 protein stability by inhibiting MAP kinase signaling. They also suggest that the dual lineage targeting of ETV1 and KIT by the combination therapy may provide a more effective therapeutic strategy than imatinib alone in GIST management. Citation Format: Leili Ran, Inna Sirota, Zhen Cao, Devan Murphy, Shipra Shukla, Ferdinando Rossi, John Wongvipat, William D. Tap, Peter Besmer, Cristina R. Antonescu, Yu Chen, Ping Chi. Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates imatinib antitumor effect in GIST oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2014-3396

Effect of secondary KIT mutations on growth of GIST cells in the absence of selective pressure by imatinib in isogenic models of imatinib resistance.

10555 Background: Secondary mutations of KIT are commonly found in progressing lesions of imatinib (IM)-resistant GIST and represent a major factor of drug resistance. Development of salvage treatm...