KIT Protein Expression Research Articles

Tirapazamine combined with photodynamic therapy improves the efficacy of ABZSO nanoparticles on Echinococcosis granulosus via further enhancing "breaking-then-curing".

Photodynamic therapy (PDT) has a promising application prospect in Echinococcus granulosus (Egs), however, the hypoxic environment of Egs and the hypoxia associated with PDT will greatly limit its effects. As a hypoxic-activated pre-chemotherapeutic drug, tirapazamine (TPZ) can be only activated and produce cytotoxicity under hypoxia environment. Albendazole sulfoxide (ABZSO) is the first choice for the treatment of Egs. This study aimed to explore the effects of ABZSO nanoparticles (ABZSO NPs), TPZ combined with PDT on the activity of Egs in vitro and in vivo. The Egs were divided into control, ABZSO NPs, ABZSO NPs+PDT, and ABZSO NPs+TPZ+PDT groups, and the viability of Egs was determined using methylene blue staining. Then, the ROS, LDH and ATP levels were measured using their corresponding assay kit, and H2AX and TopoI protein expression was detected by western blot. The morphology of Egs with different treatments was observed using hematoxylin eosin (HE) staining and scanning electron microscopy (SEM). After that, the in vivo efficacy of ABZSO NPs, TPZ and PDT on Egs was determined in a Egs infected mouse model. In vitro experiments showed that the combined treatment of TPZ, ABZSO NPs and PDT significantly inhibited Egs viability; and significantly increased ROS levels and LDH contents, while decreased ATP contents in Egs; as well as up-regulated H2AX and down-regulated TopoI protein expression. HE staining and SEM results showed that breaking-then-curing treatment seriously damaged the Egs wall. Additionally, in vivo experiments found that the combination of ABZSO NPs, PDT and TPZ had more serious calcification and damage of the wall structure of cysts. ABZSO NPs combined with TPZ and PDT has a better inhibitory effect on the growth of Egs in vitro and in vivo based on the strategy of "breaking-then-curing".

Adeno-associated virus-9 reverses delayed gastric emptying of solids in diabetic mice.

Gastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase-1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin-10 (IL-10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL-10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno-associated viruses (AAVs) in diabetic mice with delayed GE. C57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9-cre, scAAV9-GFP, or scAAV9-HO1 particles. Changes to GE were assessed weekly utilizing our [13 C]-octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression. Delayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9-cre and in 4/5 mice that received the scAAV9-GFP, whereas mice that received scAAV9-HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4-6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9-GFP-treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE. Injection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

Sodium butyrate supresses malignant human mast cell proliferation, downregulates expression of KIT and promotes differentiation.

Sodium butyrate (NaBu) is a class I histone deacetylase inhibitor (HDACi) that can impede the proliferation of transformed cells. Although some HDACi downregulate the expression of the stem cell factor receptor (KIT/CD117), the effect of NaBu on KIT expression and human mast cell proliferation requires further elucidation. In this study, we examined the effects of NaBu on three transformed human mast cell lines, HMC-1.1, HMC-1.2 and LAD2. NaBu (100 µM) inhibited the proliferation and metabolic activity of all three cell lines without significantly affecting their viability, suggesting that although the cells had ceased to divide, they were not yet undergoing apoptosis. Cell cycle analysis using the cell-permeant dye, propidium iodide, indicated that NaBu significantly blocked the cell cycle progression of HMC-1.1 and HMC-1.2 from G1 to G2/M phases. Furthermore, NaBu downregulated the expression of C-KIT mRNA and KIT protein expression in all three cell lines, but this effect was most significant in the HMC-1.1 and HMC-1.2, both of which harbour activating mutations in KIT, which proliferate more rapidly than LAD2. These data support earlier observations showing that human mast cell lines are sensitive to histone deacetylase inhibition. However, our data presents the novel observation that inhibition of cell proliferation by NaBu was not associated with a loss in cell viability but rather an arrest of the cell cycle. Higher concentrations of NaBu led to modest increases in histamine content, tryptase expression, and granularity. In conclusion, NaBu treatment of human mast cell lines led to a modest enhancement of the hallmarks of mature mast cells.

Impact of first chemotherapy exposure on follicle activation and survival in human cryopreserved ovarian tissue.

Does chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) impact the signaling pathways governing follicle activation and survival for prepubertal and postpubertal patients? Chemotherapy exposure prior OTC increases follicle apoptosis rates but not follicular activation, although the PI3K/AKT/mTOR and Hippo signaling pathways were modified in the cortex. OTC is currently the only available fertility preservation procedure for children and for patients who have already started their treatment. While previous studies have not observed harmful impacts of first chemotherapy exposure on OTC outcomes, the consequences of treatment on follicle activation and survival need to be further investigated. To address this question, we evaluated signaling pathway modifications induced by chemotherapy exposure according to pubertal status. Cryopreserved ovarian tissues from postpubertal (12-29 years old, n = 8) and prepubertal (3-10 years old, n = 8) cancer patients donated for research were thawed and cultured for 24 h. Analyses of the survival of the follicles and stroma, and of the PI3K/AKT/mTOR and Hippo signaling pathways, were conducted at thawing and after culture. Ovarian fragments exposed to chemotherapy before collection were compared to non-exposed controls. Histological investigations were performed to assess the distribution of the follicles, stroma fibrosis, vessel integrity, and apoptosis levels. It included follicular counting, collagen staining, immunostaining on CD31 and gH2AX, as well as TUNEL staining. To explore follicle activation in the different groups, the PI3K/AKT/mTOR and Hippo signaling pathways were investigated by gene expression analyses of isolated follicles and protein analyses on whole fragments through western blots and immunostaining. We first assessed the impact of a first exposure to chemotherapy on the collagen density and vessels in ovarian tissues at thawing and after culture. While no differences in collagen density were observed according to age or previous treatment, the vascularization area (CD31+) was significantly lower in tissue from previously exposed patients compared to non-treated ones. Apoptosis analyses (TUNEL) revealed an acute deleterious impact on follicle survival after chemotherapy exposure without affecting the follicular density. Surprisingly, leukemic patients had a significantly higher percentage of gH2AX-positive follicles, indicating a DNA damage response, compared to the other patients. The proportion of activated follicles appeared to decrease following exposure to chemotherapy, suggesting that it at least did not increase activation process. Stable KIT LIGAND gene and protein expression and cKIT protein levels were observed among the groups, confirming the absence of activation. Analysis of the PI3K pathway did not reveal a difference in the AKT phosphorylation level between the groups, but pRPS6 was significantly higher in tissue from patients previously exposed to chemotherapy compared to that from non-exposed patients. Finally, protein and gene analyses on Hippo pathway signaling showed a higher LATS1 protein level in the cortex after chemotherapy exposure. The heterogeneity of the human fragments, and the limited number of patients included in the cohort have to be considered as important study limitations. Moreover, this study did not explore the long-term consequences of chemotherapy on follicular development. Therefore, the results should be interpreted with caution. These results underscore the deleterious effect of previous chemotherapeutic treatment on follicle survival. Although follicular density was not reduced, these data suggested that exposure to chemotherapy impacts follicular apoptosis and the DNA damage response. Chemotherapy-induced activation was not observed despite the impact on mTOR and Hippo signaling pathways in the whole cortex. This work was funded by an Excellence of Science (EOS) Grant (ID: 30443682) and was supported by Fonds Erasme. I.D. and M.-M.D. are associate researchers at Fonds National de la Recherche Scientifique de Belgique (FNRS). There are no competing interests. N/A.

S1846 The GIST of Abdominal Pain With Jaundice

Introduction: Gastrointestinal Stromal Tumors (GISTs) are subepithelial neoplasms characterized by the expression of KIT protein (CD117 antigen). They are the most common non-epithelial neoplasms of the GI tract, often found in the stomach and distal small intestine, with small GISTs often undiagnosed. GISTs have malignant potential, but nodal involvement is rare, explaining why surgical resection is usually first line treatment. Adjuvant treatment following resection or neoadjuvant treatment in the setting of unresectable/metastatic GISTs may produce an improved median survival. We present a patient with painful jaundice found to have GIST. Case Description/Methods: A 70-year-old man with a history of lung cancer, coronary artery disease, and nephrolithiasis presented with waxing and waning diffuse abdominal pain occasionally radiating to the back for three weeks. Associated symptoms included increased eructation, decreased appetite, generalized weakness, steatorrhea, rust-colored urine, and jaundice. The pain worsened with fatty meals and improved with defecation. The patient appeared jaundiced with conjunctival icterus. Abdomen was soft, non-distended with right upper quadrant tenderness, voluntary guarding and hypoactive bowel sounds. Labs revealed a cholestatic pattern of liver enzymes indicating obstruction. Of note, the CA 19-9 level was 142. Biliary imaging was performed (Figure A), which showed peripancreatic mass and severe biliary stricture in the lower third of main bile duct (Figure B). Pathology results of fine needle biopsy indicated low-grade spindle cell neoplasm consistent with GIST, CD117, CD34 and smooth muscle actin positive, S-100 and desmin negative. Given the location of the tumor, elective robotic pancreaticoduodenectomy was performed two months later with nodal resection. Discussion: Historically, the presentation of painful jaundice is associated with extrahepatic cholestasis including intrinsic and extrinsic tumors (cholangiocarcinoma, pancreatic cancer), choledocholithiasis, primary sclerosing cholangitis, acute pancreatitis, sphincter of Oddi dysfunction or biliary tract strictures following invasive procedures. The unusual location of this patient’s GIST led to differential diagnoses that did not include his ultimate diagnosis. This case report highlights the presentation of an often undiagnosed cancer, GIST, especially given its unique location in the GI tract. It is vital that as clinicians we keep a broad differential while performing workups of abdominal pain with jaundice.Figure 1.: Biliary Imaging. A: RUQ abdominal ultrasound demonstrating hydropic gallbladder 11.5 cm in length with sludge. B: ERCP fluoroscopy showing evidence of biliary duct stricture.

Effect of perfluorooctanoic acid on lipid accumulation in rat liver cells BRL-3A and its possible mechanism

To investigate the effect of perfluorooctanoic acid on rat hepatocytes BRL-3 A cell viability and the expression of transcription factor nuclear factor erythroid 2-related factor 2(Nrf2) and arginosuccinate synthase(Ass1). Rat hepatocytes BRL-3 A were cultured and divided into control group(0 μmol/L PFOA), low-dose group(6.25 μmol/L PFOA), and medium-dose group(25 μmol/L PFOA), high-dose group(100 μmol/L PFOA). After 48 hours, cell viability was detected by MTT, ROS content was detected by free radical indicator H_2DCFDA, enzyme activity related to oxidative stress was detected by the kit, Nrf2 and Ass1 protein expression level was detected by Western blot and immunocytochemistry(ICC). Compared with the control group, with the increase of the PFOA concentration, the cell viability of the middle and high dose groups had a downward trend, but there was no statistical significance(P>0.05). The intracellular ROS content increased, among which in the middle and high dose groups significantly increased(P<0.05), and the average fluorescence intensity was(5417.66±161.09) and(5725.50±166.83), respectively. Compared with the control group, the content of intracellular TG, TC and MDA in the low and medium dose groups did not change significantly, and the content of TG, TC and MDA in the high dose group was significantly increased(P<0.05), which was(0.21±0.05) mmol/L, (14.5±6.07) mmol/L and(1.23±0.33) nmol/mL, respectively. According to the ICC and Western blot result, the expression level of Nrf2 protein increased significantly in the high-dose group(P<0.05), and the expression level of Ass1 protein increased significantly in the low-dose group(P<0.05). Exposure to a certain dose of PFOA can lead to the accumulation of ROS in BRL-3 A cells. Nrf2 and Ass1 can play a certain role in eliminating ROS and ammonia detoxification by increasing their expression under the oxidative damage of rat liver cells caused by PFOA.

KIT Expression Is Regulated by DNA Methylation in Uveal Melanoma Tumors.

Uveal melanoma (UM) is an ocular tumor with a dismal prognosis. Despite the availability of precise molecular and cytogenetic techniques, clinicopathologic features with limited accuracy are widely used to predict metastatic potential. In 51 UM tissues, we assessed a correlation between the expression of nine proteins evaluated by immunohistochemistry (IHC) (Melan-A, S100, HMB45, Cyclin D1, Ki-67, p53, KIT, BCL2, and AIFM1) and the presence of UM-specific chromosomal rearrangements measured by multiplex ligation-dependent probe amplification (MLPA), to find IHC markers with increased prognostic information. Furthermore, mRNA expression and DNA methylation values were extracted from the whole-genome data, achieved by analyzing 22 fresh frozen UM tissues. KIT positivity was associated with monosomy 3, increasing the risk of poor prognosis more than 17-fold (95% CI 1.53–198.69, p = 0.021). A strong negative correlation was identified between mRNA expression and DNA methylation values for 12 of 20 analyzed positions, five located in regulatory regions of the KIT gene (r = −0.658, p = 0.001; r = −0.662, p = 0.001; r = −0.816; p < 0.001; r = −0.689, p = 0.001; r = −0.809, p < 0.001, respectively). DNA methylation β values were also inversely associated with KIT protein expression (p = 0.001; p = 0.001; p = 0.015; p = 0.025; p = 0.002). Our findings, showing epigenetic deregulation of KIT expression, may contribute to understanding the past failure to therapeutically target KIT in UM.

Two Hematopoietic Transcription Factors, RUNX1 and FUBP1, Control the Expression of KIT Oncogene in Pre-B Lymphoblasts

Hematopoiesis involves an elaborate network of transcription factors articulated with transcriptional cooperators. RUNX1 geneencodes a transcription factor playing a key role in hematopoiesis. Abnormal functions of RUNX1 protein are implicated in blood cancer, notably in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To unravel the mechanisms behind the modulation of the transcriptional activity of RUNX1, we performed experiments of RUNX1-specific chromatin immunoprecipitation (ChIP) followed by mass spectrometry, and identified Far Upstream Element Binding Protein 1 (FUBP1) as a potential functional cooperator of RUNX1. The helicase and transcriptional regulator FUBP1 has recently been described to be essential for expansion and self-renewal of hematopoietic stem cells and to function as a potential cancer driver gene in lymphoblastic leukemia. Our goals were to validate the protein interaction between RUNX1 and its putative cooperator FUBP1, to identify gene(s) transcriptionally controlled by both RUNX1 and FUBP1, and finally to demonstrate the biological consequence of the cooperation between RUNX1 and its cooperator FUBP1.Co-immunoprecipitation and colocalization experiments demonstrate that RUNX1 colocalizes with FUBP1 in pre-B cell lines, in pre-B lymphoblasts isolated from BCP-ALL patients, and more generally in other hematopoietic progenitors. To characterize some target genes directly regulated by FUBP1 and RUNX1, we performed ChIP combined with sequencing (ChIP-seq) for FUBP1 and RUNX1. Those binding signals were compared to different histone marks and reveal that a subset of FUBP1 and RUNX1 binding signals are enriched within primed enhancer regions characterized by histone H3K4me1 marks. Our data show that RUNX1 signal is also enriched within FUBP1 regions, demonstrating that they are often retrieved on same chromatin regions, bound by FUBP1. Interestingly, FUBP1 is widely distributed in the genome, therefore only a subset of RUNX1 binding signals overlaps with FUBP1 binding signals. To go further, using qPCR, we investigated the expression level of several potential target candidate genes in pre-B cells overexpressed for FUBP1 or RUNX1. We found that both FUBP1 and RUNX1 positively regulate KIT transcript. KIT is a tyrosine kinase receptor expressed on hematopoietic progenitors cell surface and is an oncogene overexpressed notably in different types of leukemia. With the comparison of data from FUBP1 and RUNX1 ChiP-seq, we found that FUBP1 and RUNX1 share two common DNA binding regions on KIT gene, including one overlapping with histone H3K4me1 mark. ChIP-qPCR analyses confirm the binding of FUBP1 and RUNX1 on the KIT enhancer. Moreover, using reporter gene assay, we demonstrated that they can both activate this enhancer. With supervised and unsupervised approaches, we were able to determine FUBP1 and RUNX1 binding motifs that are functionally important for the enhancer activation. Finally, we demonstrated that FUBP1 overexpression in a pre-B cell line increases the expression of KIT protein and exacerbates one of the KIT downstream pathways. FUBP1 overexpression increases also cell proliferation by promoting cell cycle progression in vitro . Concordantly, in vivo, xenografted immunodeficient mice injected with overexpressed FUBP1 cells present a shorter survival time than control mice.To conclude, we demonstrate a regulatory network involving RUNX1 and FUBP1 for the control of a KIT enhancer. Indeed, FUBP1 and RUNX1 can colocalize on chromatin, regulate KIT transcription by binding to a common region in a KIT enhancer, and promote cell cycle progression. These findings suggest a new mechanism for the control of the proliferation of pre-B lymphoblasts. Because FUBP1 and KIT are overexpressed in some types of leukemia, abnormalities in this regulatory network could participate in the onset or the maintenance of RUNX1-related leukemia.This work is supported by Ligue Régionale Contre le Cancer, Région Bretagne, Rennes Métropole, French Research Ministry, Société Française de lutte contre les Cancers et les leucémies de l'Enfant et de l'adolescent, Fédération Enfants et Santé, Société Française de Biochimie et Biologie Moléculaire, Société Française d'Hématologie, Mrs. M-Dominique Blanc-Bert and the Marie Curie Actions of the European Union FP7 under REA grant agreement n°291851. DisclosuresNo relevant conflicts of interest to declare.

Identification of CD44 as a Cell-Surface Marker for Kit Negative Interstitial Cells of Cajal in Adult Mouse Colon

Loss of Kit protein expression is proven to influence the plasticity of interstitial cells of Cajal (ICCs) and may contribute to gastrointestinal (GI) dysfunctions. The role and fate of Kit negative ICCs are unclear, and cell-specific markers for the Kit ICCs are unknown. In this study, we treated adult mice with imatinib (a Kit signaling blocker) for 8 or 16 days and investigated whether CD44 is a specific marker for the Kit negative ICCs in the adult mouse colon. We aimed at examining the protein and mRNA level of CD44 and Kit by using Western blot and real-time RT-PCR, respectively. Our results indicated that Kit expression was downregulated for both protein and mRNA levels after imatinib treatment for 8 or 16 days as compared to the vehicle-treated mice. Interestingly, CD44 expression remained unchanged throughout the treatment. Immunostaining on whole-mount preparations for Kit and CD44 showed that CD44 was exclusively co-localized with Kit in the ICCs of the vehicle-treated mouse colon. After imatinib treatment, a number of CD44⁺/Kit^– cells with elaborated processes were observed with an evident decrease of Kit⁺ cell number within the muscular layers (ICC-IM) and around the myenteric nerve plexus (ICC-MY) as compared to vehicle-treated mice. After discontinuing imatinib for 16 days, Kit⁺ ICC-MY and ICC-IM were completely co-localized with normalization of CD44 and Kit⁺ cell numbers. Overall, our results identify CD44 as a cell-specific surface marker for Kit^–ICCs and may be useful to understand the role and fate of Kit^– ICCs in GI disorders.

Abstract 5181: Therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumor (GIST), with DS-6157a, an antibody-drug conjugate (ADC)

Abstract More than 85% of GISTs are driven by activating mutations in KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Currently, the only approved treatments for GIST are KIT directed tyrosine kinase inhibitors (TKIs). However, treatment with approved TKIs eventually results in disease progression most often due to the development of secondary resistance mutations in KIT. In addition, these agents have limited activity in PDGFRA mutant GIST and KIT/PDGFRA wild type (WT) GIST as primary therapy. Therefore, it is essential to develop novel therapeutic strategies with different modes of action in advanced GIST. G protein-coupled receptor 20 (GPR20) is an orphan GPCR selectively and abundantly expressed in GIST. GPR20 expression is regulated by FOXF1 and ETV1, transcription factors that play critical roles in KIT-driven GIST initiation, proliferation, and survival. We hypothesize that GPR20 is a potential therapeutic target for ADC development for the treatment of GIST. In this study, 1) GPR20 and KIT protein expression was assessed by IHC staining on GIST samples from DFCI (n=144) and NCCHE (n=100) as well as on normal and malignant tissue microarrays obtained commercially, and 2) an anti-GPR20 ADC (DS-6157a) was generated to evaluate antitumor activity in GIST models and to assess safety. GPR20 was expressed in more than 88% of the GIST samples analyzed, with higher expression levels in samples that: (I) received multiple treatment lines compared to naïve/early treated samples, (II) expressed higher KIT levels, (III) were small intestinal GIST, and/or (IV) had no KIT mutation, including succinate dehydrogenase (SDH) deficient GIST and neurofibromatosis type 1 (NF1)-associated GIST. The interstitial cells of Cajal were the only normal cells positive for GPR20. Normal mast cells expressed KIT but not GPR20. DS-6157a is an ADC composed of a humanized anti-GPR20 antibody, a Gly-Gly-Phe-Gly tetra-peptide-based linker, and a DNA topoisomerase I (TOP1) inhibitor Dxd. DS-6157a exhibited GPR20 expression-dependent cell growth-inhibitory activity and induced tumor regression with dosing at 3 to 10 mg/kg in multiple GIST xenograft models. In addition, DS-6157a showed antitumor activity in a GIST patient-derived xenograft model that was resistant to imatinib, sunitinib, and regorafenib. In vitro, DS-6157a induced TOP1 inhibitor-associated markers of DNA damage (phosphorylation of Chk1) and apoptosis (cleaved PARP) in GPR20 expressing cells. In preclinical toxicology studies using rats and cynomolgus monkeys, the pharmacokinetics and safety profile of DS-6157a were favorable at up to 200 mg/kg and 30 mg/kg, respectively. These data support the clinical development of DS-6157a as a potential novel GIST therapy with activity in patients that are resistant, refractory, or intolerant to approved TKIs. Citation Format: Kenji Iida, Amr H. Abdelhamid, Akiko Kawano Nagatsuma, Tomoko Shibutani, Satoru Yasuda, Michiko Kitamura, Chiharu Hattori, Manabu Abe, Jun Hasegawa, Takuma Iguchi, Tsuyoshi Karibe, Takashi Nakada, Koichiro Inaki, Reiko Kamei, Yuki Abe, Jessica L. Andersen, Sandro Santagata, Matthew L. Hemming, Suzanne George, Toshihiko Doi, Atsushi Ochiai, George D. Demetri, Toshinori Agatsuma. Therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumor (GIST), with DS-6157a, an antibody-drug conjugate (ADC) [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 5181.

The Role of miR-375-3p and miR-200b-3p in Gastrointestinal Stromal Tumors.

Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have been observed in virtually all types of human cancer. However, their role in the pathogenesis of rare mesenchymal gastrointestinal stromal tumors (GISTs) is not well defined, yet. In this study, we aimed to investigate the role of two miRNAs strongly downregulated in GIST—miR-375-3p and miR-200b-3p—in the pathogenesis of GIST. To achieve this, miRNA mimics were transfected into GIST-T1 cells and changes in the potential target gene mRNA and protein expression, as well as alterations in cell viability, migration, apoptotic cell counts and direct miRNA–target interaction, were evaluated. Results revealed that overexpression of miR-375-3p downregulated the expression of KIT mRNA and protein by direct binding to KIT 3′UTR, reduced GIST cell viability and migration rates. MiR-200b-3p lowered expression of ETV1 protein, directly targeted and lowered expression of EGFR mRNA and protein, and negatively affected cell migration rates. To conclude, the present study identified that miR-375-3p and miR-200b-3p have a tumor-suppressive role in GIST.

The Relationship between KIT Copy Number Variation, Protein Expression, and Angiogenesis in Sporadic Breast Cancer.

KIT is a protooncogene that encodes for the KIT oncoprotein, which is a transmembrane tyrosine kinase growth factor receptor that holds a critical role in a variety of normal physiological and pathological processes including angiogenesis. KIT has been shown to be involved in tumorigenesis, contributing to the development of gastrointestinal carcinoma and leukemia. A link between KIT overexpression and breast cancer development has previously been reported. In the current study, we explored KIT gene expression and exonic copy number variants (CNV) and the relationship with angiogenesis (CD34) and the clinicopathological features of breast cancer. MLPA technique was used to determine the CNV in 64 breast cancer tumor samples from patients diagnosed with primary sporadic breast cancer. Results were confirmed by quantitative PCR. Expression of KIT and CD34 was determined using immunohistochemistry (IHC). Our results show that 28.1% of the tumor samples from patients with primary sporadic breast cancer had CNV in the KIT gene. Among the breast tumor samples, 54.7% showed positive KIT expression. The expression of the CD34 angiogenesis marker was reported in 43.8% of the tumor samples as low, 42.2% as moderate and 14.1% as high. A significant correlation between increased CNV of KIT exons, a high level of angiogenesis (CD34) and increased tumor grade was observed (p< 0.05). A significant correlation between the KIT CNV and the angiogenesis marker was found. Examining KIT expression and CNV has the potential to function as a biomarker for tyrosine kinase inhibitor drugs in breast cancer.

Silencing of long noncoding RNA TUG1 inhibits viability and promotes apoptosis of acute myeloid leukemia cells by targeting microRNA-221-3p/KIT axis.

Acute myeloid leukemia (AML) is a hematological malignancy. This study was attempted to uncover the effects of long noncoding RNA taurine-upregulated gene1 (TUG1) on the viability and apoptosis of AML cells. QRT-PCR was implemented to examine the expression of TUG1, miR-221-3p and KIT in AML. The correlation between TUG1 and clinicopathological features of AML patients was evaluated. The effect of TUG1 on AML cells were studied by RNA interference approach. AML cells were transfected with miR-221-3p mimic and miR-221-3p inhibitor, respectively. Then the viability and apoptosis of AML cells were examined by MTT and flow cytometry assay, respectively. Additionally, dual-luciferase reporter assay was used to confirm the interactions among TUG1, miR-221-3p and KIT. Western blot was applied to analyze protein expression of KIT. The expression of TUG1 and KIT was up-regulated in AML, but miR-221-3p was down-regulated. TUG1 expression had obviously correlation with World Health Organization (WHO) grade in AML patients. The functional experiment stated that TUG1 silencing suppressed the viability and accelerated the apoptosis of AML cells. Moreover, the mechanical experiment demonstrated that TUG1 and KIT were both targeted by miR-221-3p with the complementary binding sites at 3'UTR. Up-regulation of miR-221-3p inhibited the protein expression of KIT. Furthermore, in the feedback experiment, miR-221-3p inhibition or KIT overexpression reversed the repression of tumor behavior induced by TUG1 silencing. TUG1 silencing retarded viability and promoted apoptosis of AML cells via regulating miR-221-3p/KIT axis, providing a potential therapeutic target for AML.

Prognostic and predictive significance of KIT protein expression and c-kit gene mutation in canine cutaneous mast cell tumours: A consensus of the Oncology-Pathology Working Group.

One of the primary objectives of the Oncology-Pathology Working Group (OPWG), a joint initiative of the Veterinary Cancer Society and the American College of Veterinary Pathologists, is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for oncologic pathology. Consensus is established through critical review of peer-reviewed literature relevant to a subgroup's particular focus. Subsequent acceptance and approval of the document by the OPWG membership at large establishes consensus. The intent of this publication is to help educate practitioners and pathologists on the value of diagnostics related to the KIT receptor tyrosine kinase for canine cutaneous mast cell tumours and to provide a guide for the use of these tests in veterinary medicine. This document represents the opinions of the OPWG and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists or the Veterinary Cancer Society.

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

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

Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19–63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas.Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/μm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

Histocytic-like Atypical Mast Cell Tumours in Horses

This report describes a series of four equine mast cell tumours (MCTs) with atypical morphological features. The tumours were 1-2cm in diameter and mostly localized to the eyes (one eyelid, two conjunctiva). Histologically, they were composed of very large (up to 35μm) round pleomorphic cells with a large central to paracentral nucleus and abundant granular cytoplasm. A large number of viable mature eosinophils were detected intermingled with the large round cells. Histochemical staining (toluidine blue and Perls' Prussian blue) and immunohistochemistry (KIT, mast cell tryptase, lysozyme and proliferating cell nuclear antigen) confirmed the mast cell origin of the atypical cells and identified an aberrant KIT protein expression in three cases. Based on morphological and immunohistochemical features, we propose to call the lesions equine histiocytic-like atypical MCTs.

Gene expression analyses determine two different subpopulations in KIT-negative GIST-like (KNGL) patients.

IntroductionThere are limited findings available on KIT-negative GIST-like (KNGL) population. Also, KIT expression may be post-transcriptionally regulated by miRNA221 and miRNA222. Hence, the aim of this study is to characterize KNGL population, by differential gene expression, and to analyze miRNA221/222 expression and their prognostic value in KNGL patients.MethodsKIT, PDGFRA, DOG1, IGF1R, MIR221 and MIR222 expression levels were determined by qRT-PCR. We also analyzed KIT and PDGFRA mutations, DOG1 expression, by immunohistochemistry, along with clinical and pathological data. Disease-free survival (DFS) and overall survival (OS) differences were calculated using Log-rank test.ResultsHierarchical cluster analyses from gene expression data identified two groups: group I had KIT, DOG1 and PDGFRA overexpression and IGF1R underexpression and group II had overexpression of IGF1R and low expression of KIT, DOG1 and PDGFRA. Group II had a significant worse OS (p = 0.013) in all the series, and showed a tendency for worse OS (p = 0.11), when analyzed only the localized cases. MiRNA222 expression was significantly lower in a control subset of KIT-positive GIST (p < 0.001). OS was significantly worse in KNGL cases with higher expression of MIR221 (p = 0.028) or MIR222 (p = 0.014).ConclusionsWe identified two distinct KNGL subsets, with a different prognostic value. Increased levels of miRNA221/222, which are associated with worse OS, could explain the absence of KIT protein expression of most KNGL tumors.

Two hematopoietic transcription factors, RUNX1 and FUBP1, control the expression of KIT oncogene in pre-B lymphoblasts

Abstract Hematopoiesis involves an elaborate network of transcription factors articulated with transcriptional cooperators. RUNX1 geneencodes a transcription factor playing a key role in hematopoiesis. Abnormal functions of RUNX1 protein are implicated in blood cancer, notably in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To unravel the mechanisms behind the modulation of the transcriptional activity of RUNX1, we performed experiments of RUNX1-specific chromatin immunoprecipitation (ChIP) followed by mass spectrometry, and identified Far Upstream Element Binding Protein 1 (FUBP1) as a potential functional cooperator of RUNX1. The helicase and transcriptional regulator FUBP1 has recently been described to be essential for expansion and self-renewal of hematopoietic stem cells and to function as a potential cancer driver gene in lymphoblastic leukemia. Our goals were to validate the protein interaction between RUNX1 and its putative cooperator FUBP1, to identify gene(s) transcriptionally controlled by both RUNX1 and FUBP1, and finally to demonstrate the biological consequence of the cooperation between RUNX1 and its cooperator FUBP1. Co-immunoprecipitation and colocalization experiments demonstrate that RUNX1 colocalizes with FUBP1 in pre-B cell lines, in pre-B lymphoblasts isolated from BCP-ALL patients, and more generally in other hematopoietic progenitors. To characterize some target genes directly regulated by FUBP1 and RUNX1, we performed ChIP combined with sequencing (ChIP-seq) for FUBP1 and RUNX1. Those binding signals were compared to different histone marks and reveal that a subset of FUBP1 and RUNX1 binding signals are enriched within primed enhancer regions characterized by histone H3K4me1 marks. Our data show that RUNX1 signal is also enriched within FUBP1 regions, demonstrating that they are often retrieved on same chromatin regions, bound by FUBP1. Interestingly, FUBP1 is widely distributed in the genome, therefore only a subset of RUNX1 binding signals overlaps with FUBP1 binding signals. To go further, using qPCR, we investigated the expression level of several potential target candidate genes in pre-B cells overexpressed for FUBP1 or RUNX1. We found that both FUBP1 and RUNX1 positively regulate KIT transcript. KIT is a tyrosine kinase receptor expressed on hematopoietic progenitors cell surface and is an oncogene overexpressed notably in different types of leukemia. With the comparison of data from FUBP1 and RUNX1 ChiP-seq, we found that FUBP1 and RUNX1 share two common DNA binding regions on KIT gene, including one overlapping with histone H3K4me1 mark. ChIP-qPCR analyses confirm the binding of FUBP1 and RUNX1 on the KIT enhancer. Moreover, using reporter gene assay, we demonstrated that they can both activate this enhancer. With supervised and unsupervised approaches, we were able to determine FUBP1 and RUNX1 binding motifs that are functionally important for the enhancer activation. Finally, we demonstrated that FUBP1 overexpression in a pre-B cell line increases the expression of KIT protein and exacerbates one of the KIT downstream pathways. FUBP1 overexpression increases also cell proliferation by promoting cell cycle progression in vitro . Concordantly, in vivo, xenografted immunodeficient mice injected with overexpressed FUBP1 cells present a shorter survival time than control mice. To conclude, we demonstrate a regulatory network involving RUNX1 and FUBP1 for the control of a KIT enhancer. Indeed, FUBP1 and RUNX1 can colocalize on chromatin, regulate KIT transcription by binding to a common region in a KIT enhancer, and promote cell cycle progression. These findings suggest a new mechanism for the control of the proliferation of pre-B lymphoblasts. Because FUBP1 and KIT are overexpressed in some types of leukemia, abnormalities in this regulatory network could participate in the onset or the maintenance of RUNX1-related leukemia. This work is supported by Ligue Regionale Contre le Cancer, Region Bretagne, Rennes Metropole, French Research Ministry, Societe Francaise de lutte contre les Cancers et les leucemies de l'Enfant et de l'adolescent, Federation Enfants et Sante, Societe Francaise de Biochimie et Biologie Moleculaire, Societe Francaise d'Hematologie, Mrs. M-Dominique Blanc-Bert and the Marie Curie Actions of the European Union FP7 under REA grant agreement n°291851. Disclosures No relevant conflicts of interest to declare.

LMTK3 as a novel regulator of oncogenic KIT in KIT-mutant cancers.

11044 Background: Multiple cancers, such as gastrointestinal stromal tumors (GIST) and melanoma, have been shown to be caused by somatic activating mutations in the receptor tyrosine kinase KIT. The major cause of death in patients with advanced KIT -mutant cancers is due to the development of KIT tyrosine kinase inhibitor-resistant (TKI-resistant) metastatic disease. Drug resistance arises almost exclusively from secondary mutations within KIT, highlighting the importance of KIT in the proliferation and survival of these tumors. Methods: We performed a human kinase siRNA screen in multiple KIT -mutant cancer cell lines using viability as a read out. We defined candidate targets as those whose knockdown decreased viability in all cell lines. Validation and mechanistic studies were done using a library of KIT-mutant GIST and melanoma cells. Results: We identified lemur tyrosine kinase 3 (LMTK3) as candidate target in three KIT -mutant cell lines. LMTK3 silencing reduced the viability of all KIT -mutant GIST and melanoma cells tested to date, including cell lines with KIT TKI-resistance mutations. Importantly, LMTK3 silencing decreased the viability of KIT -mutant cells specifically, but not that of KIT-independent GIST and melanoma cells. Further, we found that decreased cell viability was due to induction of apoptosis, as assessed by measuring caspase 3 and 7 activity within 96 hours of LMTK3 silencing. LMTK3 knockdown also reduced tumor growth in vivo in a GIST xenograft model. Because these cells depend so heavily on KIT and the loss of KIT signaling results in cell death, we hypothesized that LMTK3 silencing may affect this pathway. Indeed, LMTK3 silencing decreased levels of autophosphorylated KIT. We also observed a significant decrease in total KIT protein expression. This phenotype and corresponding viability was rescued with exogenous expression of full length LMTK3. Conclusions: LMTK3 is an important regulator of oncogenic KIT expression and activity in KIT-mutant GIST and melanoma and represents a novel, tractable target.