Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination.

Imatinib (IM) has significantly improved the prognosis of gastrointestinal stromal tumor (GIST) patients, but some patients still have primary resistance to IM, and approximately half of patients develop acquired drug resistance within 2 years of treatment, necessitating exploration of new treatment strategies. Targeting ferroptosis as a novel approach to tumor treatment has gained attention. Yet, there is limited research on ferroptosis in GIST, and the underlying mechanism remains unclear. In this study, we revealed that IM increased lipid reactive oxygen species and intracellular Fe2+ levels, and decreased glutathione levels in GIST. This effect could be partially inhibited by Ferrostatin-1. Additionally, knocking down STUB1 and overexpressing GPX4 reversed the IM-induced ferroptosis effect. Moreover, STUB1 was identified as a novel E3 ubiquitin ligase of GPX4, promoting the ubiquitination at site K191 of GPX4. The combination of the GPX4 inhibitor RSL3 and IM synergistically induces ferroptosis, inhibiting GIST proliferation both in vivo and in vitro. Furthermore, STUB1 and GPX4 expression serve as independent prognostic factors for GIST. In conclusion, This study is the first to demonstrate that IM induces ferroptosis by promoting STUB1-mediated GPX4 ubiquitination in GIST, and the combination of RSL3 and IM emerges as a promising therapeutic strategy for GIST.

CLINICOPATHOLOGICAL AND IMMUNO-HISTOCHEMICAL CHARACTERIZATION OF GASTROINTESTINAL STROMAL TUMOUR AT FOUR TERTIARY HEALTH CENTERS IN NIGERIA USING CD117, DOG1, AND HER-2 BIOMARKERS.

Introduction: Gastrointestinal stromal tumours (GISTs) are neoplastic lesions that primarily affect the digestive tract and develop from interstitial cells of Cajal. Due to their malignant potential and personalized treatment, these lesions require histopathologic and immunohistochemical characterization. In this investigation, the sex, age, lesional sites of origin, histopathologic types, the prevalence of HER-2 expression, prognostic indices (based on tumour size and mitotic figures), expression of CD117 and DOG1, and characteristics of patients with GIST were all characterized. Methodology: This is a retrospective cross-sectional analysis of GIST cases seen at four tertiary healthcare centers in Nigeria over ten years (2008 to 2017) and investigated utilizing histopathological and immunohistochemical (CD117, DOG1, and HER-2) methods. Result: In this study, there were twenty GIST cases. Notably, the majority (40%) of the cases had tumours with sizes between 7.0 and 8.0, the stomach was the most frequent site (70%) and the spindle cell type of GIST was the most prevalent (80%) histopathological type. Additionally, the stomach was significantly associated with GIST as an origin site (with a P value of 0.001), and 100% and 50% of these tumours were immunoreactive with CD117 and DOG1 respectively. Finally, HER-2 immunoreactivity was negatively stained with GIST tumour. Conclusion: In our study, GISTs most frequently develop in the stomach, and CD117& DOG1 are essential for correctly diagnosing these tumours. However, HER-2 immunoreactivity is a predictive marker of survival for personalized care.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIß activation in gastrointestinal stromal tumor cells.

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIß (PKD2-PI4KIIIß) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

Targeting BCL6 in Gastrointestinal Stromal Tumor Promotes p53-Mediated Apoptosis to Enhance the Antitumor Activity of Imatinib.

Imatinib mesylate (IM) has revolutionized the treatment of gastrointestinal stromal tumor (GIST). However, most patients inevitably acquire IM resistance. Second- and third-line treatments exhibit modest clinical benefits with a median time to disease progression of 4 to 6 months, highlighting the urgency for novel therapeutic approaches. Here, we report that the expression of BCL6, a known oncogenic driver and transcriptional repressor, was significantly induced in GIST cells following IM treatment. Elevated BCL6 levels suppressed apoptosis and contributed to IM resistance. Mechanistically, BCL6 recruited SIRT1 to the TP53 promoter to modulate histone acetylation and transcriptionally repress TP53 expression. The reduction in p53 subsequently attenuated cell apoptosis and promoted tolerance of GIST cells to IM. Concordantly, treatment of GIST cells showing high BCL6 expression with a BCL6 inhibitor, BI-3802, conferred IM sensitivity. Furthermore, BI-3802 showed striking synergy with IM in IM-responsive and IM-resistant GIST cells in vitro and in vivo. Thus, these findings reveal a role for BCL6 in IM resistance and suggest that a combination of BCL6 inhibitors and IM could be a potentially effective treatment for GIST. SIGNIFICANCE: BCL6 drives resistance to imatinib by inhibiting p53-mediated apoptosis and can be targeted in combination with imatinib to synergistically suppress tumor growth, providing a therapeutic strategy for treating gastrointestinal stromal tumor.

Inhibition of human UDP-glucuronosyltransferase enzyme by ripretinib: Implications for drug-drug interactions.

Ripretinib, a tyrosine kinase inhibitor (TKI), is the first FDA approved fourth-line therapy for adults with advanced gastrointestinal stromal tumor (GIST). Studies have shown that several TKIs for treating GIST were potent inhibitors of human UDP-glucosyltransferase (UGTs) enzymes. However, whether ripretinib affects the activity of UGTs remains unclear. The aim of this study was to investigate the effects of ripretinib on major UGT isoforms, as well as to evaluate its potential drug-drug interactions (DDIs) risk caused by the inhibition of UGTs activities. The inhibitory effects and inhibition modes of ripretinib on UGTs were systematically evaluated using high-performance liquid chromatography (HPLC) and enzyme kinetic studies, respectively. Our data showed that ripretinib exhibited potent inhibition against UGT1A1, UGT1A3, UGT1A4, UGT1A7 and UGT1A8. Enzyme kinetic studies indicated that ripretinib was not only a competitive inhibitor of UGT1A1, UGT1A4 and UGT1A7, but also a noncompetitive inhibitor of UGT1A3, as well as a mixed inhibitor of UGT1A8. The prediction results of in vitro-in vivo extrapolation (IVIVE) demonstrated that ripretinib might bring the potential risk of DDIs when combined with substrates of UGT1A1, UGT1A3, UGT1A4, UGT1A7 or UGT1A8. Therefore, special attention should be paid when ripretinib is used in conjunction with other drugs metabolized by UGTs to avoid risk of DDIs in clinic.

MITF Downregulation Induces Death in Human Mast Cell Leukemia Cells and Impairs IgE-Dependent Degranulation.

Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.

Diagnostic role of DOG-1, GFAP and B-catenin in Basal cell Adenoma and Cellular Pleomorphic Adenoma of the Salivary Gland.

BACKGROUND: Pleomorphic Adenoma (PA) and Basal cell adenoma (BCA) are benign salivary gland tumors that may pose a diagnostic challenge if typical features are not present. Due to the increased relapse and malignant transformation rate of the former, a correct diagnosis carries relevant prognostic information. Even though immunohistochemistry (IHC) plays a limited role in the diagnosis of these tumors, the use of IHC panels could increase diagnostic accuracy. In the present work, we aimed to demonstrate that the use of an IHC panel consisting of Glial Fibrillary Acid Protein (GFAP), B-Catenin and Discovered On GIST 1 (DOG-1) can aid in the differential diagnosis between PA and BCA. METHODS: We analyzed 18 cases of benign salivary gland tumors (Pleomorphic adenomas and Basal cell adenomas) with overlapping histologic features. First, a head and neck pathologist diagnosed the cases relying on morphology alone. Afterwards, cases were re-evaluated considering the IHC panel results. Inter-observer IHC scoring concordance was evaluated with pre-defined marker cut-off points using Cohen's Kappa scores. RESULTS: Based on morphology alone, 9 cases were classified as PA while the remaining tumors were considered to be BCA. Five out of nine BCA cases showed GFAP staining and absent nuclear B-catenin and DOG-1 positivity. Conversely, 2 PA cases showed absent GFAP and positive nuclear B-catenin with concurrent DOG-1 expression. Therefore, after IHC evaluation, up to 40% of morphologic diagnoses were reconsidered. Overall, the inter-observer concordance for IHC evaluation was good (resulting Kappa Scores between 0.78 and 1). CONCLUSION: Our work supports the use of a concise IHC panel to improve the diagnostic accuracy of benign salivary gland tumors with overlapping histologic features.

Tumor-associated macrophages mediate gastrointestinal stromal tumor cell metastasis through CXCL2/CXCR2.

BACKGROUND: Tumor-associated macrophages (TAMs) are linked with the progression and poor prognosis of multifarious solid tumors, but the regulatory mechanisms involved in gastrointestinal stromal tumors (GIST) remain indistinct. This study intended to delve into the job of TAM-derived chemokines in promoting metastasis in GIST microenvironment. METHODS: Expression levels of M2-TAM markers and CXCL2 in primary and metastatic tissues of GIST were analyzed by bioinformatics methods, and we analyzed the correlation between CXCL2 and M2-TAM markers. Immunofluorescence was applied to assay CXCL2 and M2-TAM marker protein (CD68 and CD206) expression in tumor tissues. Serum CXCL2 concentration in metastatic and non-metastatic patients was assayed by ELISA. The differentiation of THP-1 cells was tested by flow cytometry. Cell function test was utilized to analyze the viability, invasion and migration of GIST cells. Western blot was used to examine the expression of epithelial-mesenchymal transition (EMT)-related proteins. The mouse liver metastasis model was established, and the effects of CXCL2 and EMT-related genes on metastasis were confirmed by hematoxylin-eosin staining and immunohistochemistry experiments. RESULTS: Bioinformatics analysis ascertained that M2-TAM marker proteins and chemokine CXCL2 were highly expressed in GIST metastatic tissues, and CXCL2 and TAM were co-located in tumor tissues. Results of in vitro cell function experiments displayed that CXCL2 secreted by M2-TAM promoted the invasion, migration and EMT of GIST tumor cells, and the anti-CXCL2 antibody could block the metastasis promoting effect of CXCL2. Additionally, the silencing of CXCR2 in GIST cells inhibited the metastasis promoting effect of CXCL2. Animal studies further confirmed that CXCL2 promoted liver metastasis of GIST in vivo. CONCLUSION: This study preliminarily revealed the mechanism of M2-TAM promoting tumor metastasis by secreting CXCL2 in GIST tumor microenvironment, and proffered theoretical reference for the development of immunotherapy strategies targeting M2-TAM.

Clinicopathological and immunohistochemical characterization of gastrointestinal stromal tumour at four tertiary health centers in Nigeria using CD117, DOG1, and human epidermal growth factor receptor-2 biomarkers.

Aims: Gastrointestinal stromal tumors (GISTs) are neoplastic lesions that primarily affect the digestive tract and develop from interstitial cells of Cajal. These lesions require histopathological and immunohistochemical characterization due to their malignant potential and personalized treatment. In this investigation, the sex, age, lesional sites of origin, histopathological types, the prevalence of human epidermal growth factor receptors (HER-2) expression, prognostic indices (based on tumor size and mitotic figures), expression of CD117 and DOG1, and characteristics of patients with GIST were all characterized. Materials and Methods: This was a retrospective cross-sectional analysis of GIST cases seen at four tertiary health-care centers in Nigeria over a 10-year period (2008-2017) and investigated utilizing histopathological and immunohistochemical (CD117, DOG1, and HER-2) methods. Results: In this investigation, there were twenty GIST cases. Notably, the majority (40%) of the cases had tumors with sizes between 7.0 and 8.0 cm; the stomach was the most frequent site (70%) and the spindle cell type of GIST was the most prevalent (80%) histopathological type. In addition, the stomach was significantly associated with GIST as an origin site (with a P = 0.001), and 100% and 50% of these tumors were immunoreactive with CD117 and DOG1, respectively. Conclusions: In our study, GISTs most frequently develop in the stomach, and CD117 and DOG1 are essential for correctly diagnosing these tumors. However, HER-2 immunoreactivity is a predictive marker of survival for personalized care.

Résumé Objectifs: Les tumeurs stromales gastro-intestinales (GIST) sont des lésions néoplasiques qui affectent principalement le tube digestif et se développent à partir des cellules interstitielles de Cajal. Ces lésions nécessitent une caractérisation histopathologique et immunohistochimique en raison de leur potentiel malin et d'un traitement personnalisé. Dans cette enquête, le sexe, l'âge, les sites d'origine des lésions, les types histopathologiques, la prévalence de l'expression des récepteurs du facteur de croissance épidermique humain (HER-2), les indices pronostiques (basés sur la taille de la tumeur et les chiffres mitotiques), l'expression de CD117 et DOG1, et les caractéristiques des patients atteints de GIST ont toutes été caractérisées. Matériels et méthodes: Il s'agissait d'une analyse transversale rétrospective de cas de GIST observés dans quatre centres de soins de santé tertiaires au Nigeria sur une période de 10 ans (2008-2017) et étudiée à l'aide d'analyses histopathologiques et immunohistochimiques (CD117, DOG1 et HER). 2) méthodes. Résultats: Dans cette enquête, il y a eu vingt cas de GIST. Notamment, la majorité (40 %) des cas présentaient des tumeurs mesurant entre 7,0 et 8,0 cm ; l'estomac était le site le plus fréquent (70 %) et le type de GIST à cellules fusiformes était le type histopathologique le plus répandu (80 %). De plus, l'estomac était significativement associé au GIST comme site d'origine (avec un P = 0,001) et 100 % et 50 % de ces tumeurs étaient immunoréactives avec CD117 et DOG1, respectivement. Conclusions: Dans notre étude, les GIST se développent le plus souvent dans l'estomac, et CD117 et DOG1 sont essentiels pour diagnostiquer correctement ces tumeurs. Cependant, l'immunoréactivité HER-2 est un marqueur prédictif de survie pour une prise en charge personnalisée. Mots-clés: Biomarqueurs, tumeurs stromales gastro-intestinales, histopathologie, immunohistochimie.

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

BACKGROUND: Gastrointestinal 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. METHODS: Data 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. RESULTS: Our 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. Video abstract.

Pimitespib: First Approval.

Pimitespib (Jeselhy®) is an oral small molecule inhibitor of the α and ß isoforms of heat shock protein 90 (HSP90). HSP90α and HSP90ß regulate the stability and activity of a number of proteins that are crucial for tumour development. Pimitespib is being developed by Taiho Pharmaceutical for the treatment of solid tumours, including gastrointestinal stromal tumour (GIST), and in June 2022 it received its first approval in Japan for GIST that has progressed after chemotherapy. Pimitespib is undergoing phase I development for the treatment of solid tumours in the EU and the USA. This article summarizes the milestones in the development of pimitespib leading to this first approval for GIST that has progressed after chemotherapy.

Characteristics and clinical significance of lipid metabolism in patients with gastrointestinal stromal tumor.

BACKGROUND: To investigate the characteristics and clinical significance of serum lipids in patients with gastrointestinal stromal tumors (GISTs). METHODS: The clinical and pathological data of 694 GIST patients in Liyuan hospital and Union hospital from 2012 to 2016 were retrospectively analyzed. Blood lipid levels in patients with varying degrees of risk were compared. RESULTS: The findings showed that LDL-C, HDL-C, and CHOL increased significantly in women, and CD34 positive. In patients with tumors size less than 5 cm in diameter, TG, HDL-C, and CHOL were significantly higher. TG levels were significantly higher in DOG-1 (a marker and has a high specificity and sensitivity in the diagnosis of GIST) positive patients than in DOG-1 negative patients (P < 0.05). S-100 positive patients had lower HDL-C levels than S-100 negative patients (P < 0.05). Lipids indexes were found to be correlated with GIST risk stratification and tumor site (P < 0.05). TG/HDL-C was were significantly different among patients with GIST in different locations (P < 0.05). CONCLUSION: The clinical and pathological characteristics of the patients with GIST are closely related to the level of blood lipids. To a certain extent, information about level of blood lipids can be helpful for distinguishing benign and malignant GIST.

Caveolin-1 expression predicts favourable outcome and correlates with PDGFRA mutations in gastrointestinal stromal tumours (GISTs).

AIMS: Novel prognostic markers are warranted for gastrointestinal stromal tumours. Caveolin-1 is a multifunctional protein that proved to be associated with outcome in multiple tumour types. Aim of this study was to investigate Caveolin-1 expression and prognostic efficacy in a series of gastrointestinal stromal tumours. METHODS: Caveolin-1 expression was assessed by immunohistochemistry in a retrospective series of 66 gastrointestinal stromal tumours representative of the different molecular subtypes. Correlations with clinical, histopathological and molecular features were investigated. Statistical analyses were performed as appropriate. RESULTS: Thirty-five cases out of 66 (53.0%) expressed Caveolin-1. Presence of Caveolin-1 expression correlated with favourable histopathologic and clinical traits, including a lower mitotic count (p=0.003) and lower relapse rate (p=0.005). Caveolin-1 expression also resulted associated with the presence of PDGFRA mutations (p=0.010). Outcome analyses showed a favourable prognostic significance of Caveolin-1 expression in terms of relapse-free survival (HR=0.14; 95% CI=0.03 to 0.63) and overall survival (HR=0.29; 95% CI=0.11 to 0.74), even after adjusting for the mutational subgroup (relapse-free survival: HR=0.14, 95% CI=0.04 to 0.44; overall survival: HR=0.29, 95% CI=0.11 to 0.51) and imatinib treatment (relapse-free survival: HR=0.14, 95% CI=0.02 to 0.81; overall survival: HR=0.29, 95% CI=0.17 to 0.48). CONCLUSION: Caveolin-1 represents a novel prognostic marker in gastrointestinal stromal tumours. Further studies are warranted to validate these results and to explore the mechanisms linking Caveolin-1 expression with the PDGFRA oncogenic pathway.

The Relationship of Clinicopathological Findings and PDGFR-ß Expression With Tumor Recurrence in Gastrointestinal Stromal Tumors.

BACKGROUND: Considering the difficulty in predicting the biological behavior of gastrointestinal stromal tumors (GISTs) based on histological findings alone, genetic abnormalities have recently become an area of focus. Platelet-derived growth factor receptor (PDGFR), with 2 isoforms (α and ß) is one of the mutations that play a role in the development of GIST. There are very little data determining the relationship of GIST with PDGFRß which is associated with poor prognosis in other mesenchymal and epithelial tumors. In this study, we aimed to show the relationship between clinicopathological criteria and recurrence. We also wanted to evaluate the effect of PDGFRß expression on recurrence and clinicopathological findings. METHODS: We evaluated 40 GIST patients retrospectively for detailed clinicopathological findings, postoperative immunohistochemical tumor markers (CD117, Ki67), and also for tumor recurrence. Immunohistochemical examination for PDGFRß was performed for the all GIST cases. RESULTS: Tumor recurrence was related to male gender (P = .003), serosal localization (P = .004), surgical margins positivity (P = .001), risk group (P = .011), mitotic activity (P = .000), and Ki67 proliferation index (P = .000). PDGFRß was not significantly associated with tumor recurrence (P = .277). CONCLUSION: We can say that the most important parameters related with recurrence of GISTs are mitotic activity and the Ki67 proliferation index. The determination of the cut-off value of the Ki67 proliferation index as 13% instead of 10% would be much more specific and sensitive. Although PDGFRß may be used for the diagnosis of GIST as an alternative for PDGFRα in cases with cKIT negativity, it is not an indicator of tumor recurrence as in other tumors.

Imatinib Regulates miR-483-3p and Mitochondrial Respiratory Complexes in Gastrointestinal Stromal Tumors.

Metabolic adaptation to increased oxidative phosphorylation (OXPHOS) has been found in gastrointestinal stromal tumor (GIST) upon imatinib treatment. However, the underlying mechanism of imatinib-induced OXPHOS is unknown. Discovering molecules that mediate imatinib-induced OXPHOS may lead to the development of therapeutic strategies synergizing the efficacy of imatinib. In this study, we explored the role of microRNAs in regulating OXPHOS in GIST upon imatinib treatment. Using a microarray approach, we found that miR-483-3p was one of the most downregulated miRNAs in imatinib-treated tumors compared to untreated tumors. Using an extended series of GIST samples, we further validated the downregulation of miR-483-3p in imatinib-treated GIST samples by RT-qPCR. Using both gain- and loss-of-function experiments, we showed that miR-483-3p could regulate mitochondrial respiratory Complex II expression, suggesting its role in OXPHOS regulation. Functionally, miR-483-3p overexpression could rescue imatinib-induced cell death. These findings provide the molecular link for imatinib-induced OXPHOS expression and the biological role of miR-483-3p in regulating cell viability upon imatinib treatment.

E3 ubiquitin ligase Atrogin-1 mediates adaptive resistance to KIT-targeted inhibition in gastrointestinal stromal tumor.

KIT/PDGFRA oncogenic tyrosine kinase signaling is the central oncogenic event in most gastrointestinal stromal tumors (GIST), which are human malignant mesenchymal neoplasms that often feature myogenic differentiation. Although targeted inhibition of KIT/PDGFRA provides substantial clinical benefit, GIST cells adapt to KIT/PDGFRA driver suppression and eventually develop resistance. The specific molecular events leading to adaptive resistance in GIST remain unclear. By using clinically representative in vitro and in vivo GIST models and GIST patients' samples, we found that the E3 ubiquitin ligase Atrogin-1 (FBXO32)-the main effector of muscular atrophy in cachexia-resulted in the most critical gene derepressed in response to KIT inhibition, regardless the type of KIT primary or secondary mutation. Atrogin-1 in GISTs is transcriptionally controlled by the KIT-FOXO3a axis, thus indicating overlap with Atrogin-1 regulation mechanisms in nonneoplastic muscle cells. Further, Atrogin-1 overexpression was a GIST-cell-specific pro-survival mechanism that enabled the adaptation to KIT-targeted inhibition by apoptosis evasion through cell quiescence. Buttressed on these findings, we established in vitro and in vivo the preclinical proof-of-concept for co-targeting KIT and the ubiquitin pathway to maximize the therapeutic response to first-line imatinib treatment.

Malignant gastrointestinal neuroectodermal tumor: Cytologic, histologic, immunohistochemical, and molecular pitfalls.

Malignant gastrointestinal neuroectodermal tumor (GNET) is a rare malignant primary gastrointestinal mesenchymal tumor which can be diagnosed via fine-needle aspiration (FNA) cytology. In the context of FNA, the diagnosis requires a cell block and the use of significant resources including immunohistochemical stains and molecular testing. The differential diagnosis of GNET includes clear cell sarcoma (CCS), gastrointestinal stromal tumor (GIST), gastric schwannoma, metastatic melanoma, malignant perivascular epithelioid cell tumor (PEComa) and granular cell tumor, among others. Here we describe a case which was initially diagnosed as malignant granular cell tumor by FNA which was later revised to GNET following the finding of an EWSR1-ATF1 fusion gene rearrangement.

Beyond the Driver Mutation: Immunotherapies in Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are a subtype of soft tissue sarcoma (STS), and have become a concept of oncogenic addiction and targeted therapies.The large majority of these tumors develop after a mutation in KIT or platelet derived growth factor receptor α (PDGFRα), resulting in uncontrolled proliferation. GISTs are highly sensitive to imatinib. GISTs are immune infiltrated tumors with a predominance of tumor-associated macrophages (TAMs) and T-cells, including many CD8+ T-cells, whose numbers are prognostic. The genomic expression profile is that of an inhibited Th1 response and the presence of tertiary lymphoid structures and B cell signatures, which are known as predictive to response to ICI. However, the microtumoral environment has immunosuppressive attributes, with immunosuppressive M2 macrophages, overexpression of indoleamine 2,3-dioxygenase (IDO) or PD-L1, and loss of major histocompatibility complex type 1. In addition to inhibiting the KIT oncogene, imatinib appears to act by promoting cytotoxic T-cell activity, interacting with natural killer cells, and inhibiting the expression of PD-L1. Paradoxically, imatinib also appears to induce M2 polarization of macrophages. There have been few immunotherapy trials with anti-CTLA-4 or anti-PD-L1drugs and available clinical data are not very promising. Based on this comprehensive analysis of TME, we believe three immunotherapeutic strategies must be underlined in GIST. First, patients included in clinical trials must be better selected, based on the identified driver mutation (such as PDGFRα D842V mutation), the presence of tertiary lymphoid structures (TLS) or PD-L1 expression. Moreover, innovative immunotherapeutic agents also provide great interest in GIST, and there is a strong rationale for exploring IDO targeting after disease progression during imatinib therapy. Finally and most importantly, there is a strong rationale to combine of c-kit inhibition with immune checkpoint inhibitors.

Expression of fatty acid-binding protein-4 in gastrointestinal stromal tumors and its significance for prognosis.

BACKGROUND: Fatty acid-binding proteins (FABPs) have been found to be involved in tumorigenesis and development. However, the role of FABP4, a member of the FABPs, in GISTs (Gastrointestinal stromal tumors) remains unclear. This study aimed to investigate the expression of FABP4 and its prognostic value in GISTs. METHODS: FABP4 expression in 125 patients with GISTs was evaluated by immunohistochemical analysis of tissue microarrays. The relationship between FABP4 expression and clinicopathological features and prognosis of GISTs was analyzed. RESULTS: Multiple logistic regression analysis showed that expression of FABP4 correlated with tumor size and mitotic index. Furthermore, FABP4 level, tumor size, mitotic index, and high AFIP-Miettinen risk were independent prognostic factors in GISTs. The Kaplan-Meier survival curve showed that the 5-year survival rate of patients with high-FABP4 expression GISTs was lower. CONCLUSIONS: These results suggested that high-FABP4 expression might be a marker of malignant phenotype of GISTs and poor prognosis.

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.