BACKGROUND: Zinc finger SWIM-type containing 4 (ZSWIM4) is a zinc finger protein with its function largely uncharacterized. In this study, we aimed to investigate the role of ZSWIM4 in gastrointestinal stromal tumors (GISTs).
RESULTS: We found that ZSWIM4 expression is inhibited by the predominantly mutated protein KIT in GISTs, while conversely, ZSWIM4 inhibits KIT expression and downstream signaling. Consistent with the observation, ZSWIM4 inhibited GIST cell survival and proliferation in vitro. RNA sequencing of GISTs from KITV558A/WT mice and KITV558A/WT/ZSWIM4-/- mice showed that loss of ZSWIM4 expression increases the expression of circadian clock pathway member BMAL1 which contributes to GIST cell survival and proliferation. In addition, we found that KIT signaling increases the distribution of ZSWIM4 in the nucleus of GIST cells, and which is important for its inhibition of KIT and BMAL1. In agreement with the results in vitro, the in vivo studies showed that ZSWIM4 deficiency increases the tumorigenesis of GISTs in KITV558A/WT mice.
CONCLUSIONS: Taken together, our results revealed that the entry of ZSWIM4 to the nucleus is important for its inhibition of KIT and BMAL1, ultimately attenuating GIST tumorigenesis. The results provide a novel insight in the understanding of signal transduction in GISTs and lay strong theoretical basis for the advancement of GIST treatment.

Gastrointestinal stromal tumors (GISTs) are predominately induced by KIT mutants. In this study, we found that four and a half LIM domains 2 (FHL2) was highly expressed in GISTs and KIT signaling dramatically increased FHL2 transcription while FHL2 inhibited KIT transcription. In addition, our results showed that FHL2 associated with KIT and increased the ubiquitination of both wild-type KIT and primary KIT mutants in GISTs, leading to decreased expression and activation of KIT although primary KIT mutants were less inhibited by FHL2 than wild-type KIT. In the animal experiments, loss of FHL2 expression in mice carrying germline KIT/V558A mutation which can develop GISTs resulted in increased tumor growth, but increased sensitivity of GISTs to imatinib treatment which is used as the first-line targeted therapy of GISTs, suggesting that FHL2 plays a role in the response of GISTs to KIT inhibitor. Unlike wild-type KIT and primary KIT mutants, we further found that FHL2 didn\'t alter the expression and activation of drug-resistant secondary KIT mutants. Taken together, our results indicated that FHL2 acts as the negative feedback of KIT signaling in GISTs while primary KIT mutants are less sensitive and secondary KIT mutants are resistant to the inhibition of FHL2.

Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases.
A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated.
A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than - 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%.
In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.

Gastrointestinal mesenchymal tumors, as the most common mesenchymal tumors in the gastrointestinal tract, are adjuvantly treated with multi-targeted tyrosine kinase inhibitors, such as imatinib and sunitinib, but there are problems of drug resistance and complex methods of monitoring therapeutic agents. The pathogenesis of this disease is related to mutations in tyrosine kinase (KIT) or platelet-derived growth factor receptor α, an important target for drug therapy. In recent years, the screening of relevant tyrosine kinase inhibitors from traditional Chinese medicine has become a hotspot in antitumor drug research. In the current study, the KIT-SNAP-tag cell membrane chromatography (KIT-SNAP-tag/CMC) column was prepared with satisfying specificity, selectivity, and reproducibility by chemically bonding high KIT expression cell membranes to the silica gel surface using the SNAP-tag technology. The KIT-SNAP-tag/CMC-HPLC-MS two-dimensional coupling system was investigated using the positive drug imatinib, and the results showed that the system was a reliable model for screening potential antitumor compounds from complex systems. This system screened and identified three potential active compounds of evodiamine (EVO), rutaecarpin (RUT), and dehydroevodiamine (DEVO), which possibly target the KIT receptor, from the alcoholic extract of the traditional Chinese medicine Evodia rutaecarpa. Then, the KD values of the interaction of EVO, RUT, and DEVO with KIT receptors measured using nonlinear chromatography were 7.75 (±4.93) × 10-6, 1.42 (±0.71) × 10-6, and 2.34 (±1.86) × 10-6 mol/L, respectively. In addition, the methyl thiazolyl tetrazolium assay validated the active effects of EVO and RUT in inhibiting the proliferation of high KIT-expressing cells in the ranges of 0.1-10 µmol/L and 0.1-50 µmol/L, respectively. In conclusion, the KIT-SNAP-tag/CMC could be a reliable model for screening antitumor components from complex systems.

BACKGROUND: Mutations in the receptor tyrosine kinase KIT are the main cause of gastrointestinal stromal tumor (GIST), and the KIT mutants mediated PI3 kinase activation plays a key role in the tumorigenesis of GIST. In this study, we aimed to block PI3 kinase activation by cell-permeable peptide and investigate its possible application in the treatment of GIST.
RESULTS: We designed cell-permeable peptides based on the binding domain of PI3 kinase subunit p85 to KIT or PI3 kinase subunit p110, respectively, in order to compete for the binding between p85 and KIT or p110 and therefore inhibit the activation of PI3 kinases mediated by KIT. The results showed that the peptide can penetrate the cells, and inhibit the activation of PI3 kinases, leading to reduced cell survival and cell proliferation mediated by KIT mutants in vitro. Treatment of mice carrying germline KIT/V558A mutation, which can develop GIST, with the peptide that can compete for the binding between p85 and p110, led to reduced tumorigenesis of GIST. The peptide can further enhance the inhibition of the tumor growth by imatinib which is used as the first line targeted therapy of GIST.
CONCLUSIONS: Our results showed that cell-permeable PI3 kinase competitive peptide can inhibit KIT-mediated PI3 kinase activation and tumorigenesis of GIST, providing a rationale to further test the peptide in the treatment of GIST and even other tumors with over-activation of PI3 kinases.

Understanding the molecular pathogenesis of acute myeloid leukemia (AML) with well-defined genomic abnormalities has facilitated the development of targeted therapeutics. Patients with t(8;21) AML frequently harbor a fusion gene RUNX1-RUNX1T1 and KIT mutations as \"secondary hit\", making the disease one of the ideal models for exploring targeted treatment options in AML. In this study we investigated the combination therapy of agents targeting RUNX1-RUNX1T1 and KIT in the treatment of t(8;21) AML with KIT mutations. We showed that the combination of eriocalyxin B (EriB) and homoharringtonine (HHT) exerted synergistic therapeutic effects by dual inhibition of RUNX1-RUNX1T1 and KIT proteins in Kasumi-1 and SKNO-1 cells in vitro. In Kasumi-1 cells, the combination of EriB and HHT could perturb the RUNX1-RUNX1T1-responsible transcriptional network by destabilizing RUNX1-RUNX1T1 transcription factor complex (AETFC), forcing RUNX1-RUNX1T1 leaving from the chromatin, triggering cell cycle arrest and apoptosis. Meanwhile, EriB combined with HHT activated JNK signaling, resulting in the eventual degradation of RUNX1-RUNX1T1 by caspase-3. In addition, HHT and EriB inhibited NF-κB pathway through blocking p65 nuclear translocation in two different manners, to synergistically interfere with the transcription of KIT. In mice co-expressing RUNX1-RUNX1T1 and KITN822K, co-administration of EriB and HHT significantly prolonged survival of the mice by targeting CD34+CD38- leukemic cells. The synergistic effects of the two drugs were also observed in bone marrow mononuclear cells (BMMCs) of t(8;21) AML patients. Collectively, this study reveals the synergistic mechanism of the combination regimen of EriB and HHT in t(8;21) AML, providing new insight into optimizing targeted treatment of AML.

Gastrointestinal stromal tumors (GISTs) are predominantly initiated by KIT mutations. In this study, we observed that discoidin domain receptors 1 and 2 (DDR1 and DDR2) exhibited high expression in GISTs, were associated with KIT, and enhanced the activation of both wild-type KIT and primary KIT mutants. Inhibition of DDR1/2 led to a reduction in the activation of KIT and its downstream signaling molecules, ultimately impairing GIST cell survival and proliferation in vitro. Consequently, treatment of mice carrying germline KIT/V558A mutation with DDR1/2 inhibitor significantly impeded tumor growth, and the combined use of DDR1/2 inhibitor and imatinib, the first-line targeted therapeutic agent for GISTs, markedly enhanced tumor growth suppression. In addition, DDR1/2 inhibition resulted in decreased KIT expression, while KIT inhibition led to upregulation of DDR1/2 expression in GISTs. The presence of DDR1/2 also decreased the sensitivity of wild-type KIT or primary KIT mutants to imatinib, indicating a possible role for DDR1/2 in promoting GIST survival during KIT-targeted therapy. The development of drug-resistant secondary KIT mutations is a primary factor contributing to GIST recurrence following targeted therapy. Similar to primary KIT mutants, DDR1/2 can associate with and enhance the activation of secondary KIT mutants, further diminishing their sensitivity to imatinib. In summary, our data demonstrate that DDR1/2 contribute to KIT activation in GISTs and strengthen resistance to imatinib for both primary and secondary KIT mutants, providing a rationale for further exploration of DDR1/2 targeting in GIST treatment.

UNASSIGNED: Mutations in the receptor tyrosine kinase KIT are the major cause of gastrointestinal stromal tumors. KIT-mediated activation of the RAS/RAF/MEK/ERK and PI3 kinase/AKT pathways plays an important role in KIT mutant-mediated cell transformation.
UNASSIGNED: The frequently seen primary KIT mutations W557K558del and V560D, and the secondary KIT mutations V654A and N822K, in gastrointestinal stromal tumors were stably transfected into Ba/F3 cells. Cell proliferation was examined with a CCK kit, and cell survival and cell cycle were examined by flow cytometry. Cell signaling was examined by western blot.
UNASSIGNED: We found that farnesyltransferase inhibitors tipifarnib and lonafarnib, which inhibit RAS activity, inhibited ERK activation mediated by both wild-type and KIT mutants, which often occur in gastrointestinal stromal tumors. Correspondingly, both wild-type and KIT mutant-mediated cell survival and proliferation were inhibited by both inhibitors. Imatinib is used as the first-line targeted therapy for gastrointestinal stromal tumors in the clinic. In our study, both inhibitors increased imatinib-mediated inhibition of cell survival and proliferation induced by both wild-type and KIT mutants. Similar to the primary KIT mutations, secondary mutations of KIT-induced ERK activation and cell response were inhibited by both inhibitors.
UNASSIGNED: Our results suggested the potential benefit of farnesyltransferase inhibitors either alone or combined with imatinib in the treatment of gastrointestinal stromal tumors carrying KIT mutations.

Thyroid cancer (TC) is one of the fastest-growing malignant tumors. This study sought to explore the mechanism of immune escape mediated by receptor tyrosine kinase (KIT) in TC.
The expression microarray of TC was acquired through the GEO database, and the difference analysis and Kyoto encyclopedia of genes and genomes pathway enrichment analysis were carried out. KIT levels in TC cell lines (K1/SW579/BCPAP) and human normal thyroid cells were detected using reverse transcription quantitative polymerase chain reaction and western blot analysis. TC cells were transfected with overexpressed (oe)-KIT and CD8+ T cells were cocultured with SW579 cells. Subsequently, cell proliferation, migration, and invasion abilities, CD8+ T cell proliferation, cytokine levels (interferon-γ [IFN-γ]/tumor necrosis factor-α [TNF-α]) were determined using colony formation assay, Transwell assays, flow cytometry, and enzyme-linked immunosorbent assay. The phosphorylation of MAPK pathway-related protein (ERK) was measured by western blot analysis. After transfection with oe-KIT, cells were treated with anisomycin (an activator of the MAPK pathway), and the protein levels of p-ERK/ERK and programmed death-ligand 1 (PD-L1) were detected.
Differentially expressed genes (N = 2472) were obtained from the GEO database. KIT was reduced in TC samples and lower in tumor cells than those in normal cells. Overexpression of KIT inhibited immune escape of TC cells. Specifically, the proliferation, migration, and invasion abilities of TC cells were lowered, the proliferation level of CD8+ T cells was elevated, and IFN-γ and TNF-α levels were increased. KIT inhibited the activation of the MAPK pathway in TC cells and downregulated PD-L1.
KIT suppressed immune escape of TC by blocking the activation of the MAPK pathway and downregulating PD-L1.

Gastrointestinal stromal tumors are the most common mesenchymal tumors of the digestive tract, most of which are sporadic, and familial GISTs with germline mutations are rarely seen. Here, we report a 26-year-old female with a germline p. W557R mutation in exon 11 of the KIT gene. The proband and her father and sister presented with multifocal GIST and pigmented nevi. All 3 patients underwent surgery and imatinib therapy. To date, only 49 kindreds with germline KIT mutations and 6 kindreds with germline PDGFRA mutations have been reported. Summarizing the reported kindreds, the majority of familial GISTs manifest as multiple primary GISTs complicated with special clinical manifestations, including cutaneous hyperpigmentation, dysphagia, mastocytosis, inflammatory fibrous polyps, and large hands. Familial GISTs are generally thought to exhibit TKI sensitivity similar to that of sporadic GISTs with the same mutation.