C-kit Overexpression Research Articles

Unveiling the potential of bioactive compounds from Linum usitatissimum L. to target Hepatocellular carcinoma: an in silico based approach

Bioactive metabolites from Linum usitatissimum L. have been extensively explored for their anti-cancer and antioxidant potential in several cancer cell lines. The bioactive chemicals that have been reported are being assessed using in silico methods to create a new antagonist that may effectively target hepatocellular carcinoma by inhibiting c-Kit (stem cell factor receptor) and HBXIP (hepatitis B X-interacting protein). Dysregulation in function or overexpression of c-Kit and HBXIP shows the development of hepatocellular carcinoma. The objective of this study is to use computational approaches to choose the most suitable candidate that interacts with our chosen target proteins, c-Kit and HBXIP, derived from flaxseed. From the online resources, namely RCSB and PubChem, the 3D structures of the proteins and bioactive compounds are obtained. The drug likeness study and ADMET profiling of these bioactive compounds were performed, and the selected fourteen compounds were considered for docking analysis. The molecular docking study revealed that secoisolariciresinol (SECO) has a comparable interaction affinity of −5.5 kcal/mol and −7.5 kcal/mol with HBXIP and c-KIT, respectively, when compared to the redock co-crystal complex (-3.5 kcal/mol and −12.4 kcal/mol, respectively). The results of the molecular dynamics simulation study demonstrate the constancy of the SECO_HBXIP and SECO_c-Kit complexes when compared to the redock complex. The average root-mean-square fluctuation (RMSF), root-mean-square deviation (RMSD) values, gyration (Rg) and hydrogen bonding provide additional evidence of the significant binding and stability of the complexes. Moreover, in vitro cell viability assay on HepG2 also showed a significant reduction in the cell proliferation after the treatment with SECO.

Adjuvant therapy with toceranib for hepatocellular carcinoma and cholangiocarcinoma in a Pomeranian

A 10-year-old spayed female Pomeranian dog was referred for hepatic mass evaluation. Blood tests revealed mildly elevated alkaline phosphatase activities. Computed tomography revealed a mass with multiple nodules on the right hepatic medial lobe adjacent to the caudal vena cava; histopathology confirmed mixed hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Because of incomplete resection, adjuvant therapy was recommended. As tumour cells showed PDGFR-α, c-Kit, and FGFR1 overexpression, the anticancer effect of tyrosine kinase inhibitors was evaluated on the cells; toceranib was the most effective and was administered starting with an extra-labelled dose. The dog remained stable for 2.3 years with mild adverse effects. To our knowledge, this is the first successful clinical application of toceranib in a dog with mixed HCC-CC.

Acceleration of melanocyte senescence by the proinflammatory cytokines IFNγ and TNFα impairs the repigmentation response of vitiligo patients to narrowband ultraviolet B (NBUVB) phototherapy

Vitiligo is a chronic autoimmune disease characterized by the T helper 1 (Th1) cytokine-driven immune destruction of melanocytes (MCs). Although narrowband ultraviolet B (NBUVB) phototherapy has been proven to be an effective therapeutic option, the repigmentation response to that phototherapy varies greatly in different vitiligo patients. Here, we demonstrate that there is an increase of NBUVB-induced cellular senescence in vitiligo MCs exposed to Th1 cytokine interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) in lesional vitiligo skin from poor responders who had undergone NBUVB phototherapy. Supplementation with exogenous recombinant human stem cell factor (rhSCF) in the culture medium as well as the lentiviral vector–mediated overexpression of cKIT could prevent the MCs from the IFNγ/TNFα-accelerated cellular senescence. Mechanistic studies indicated that the reduced ratio of membrane-bound KIT (mKIT) to the soluble form of KIT (sKIT) is directly related to the cellular senescence of vitiligo MCs following exposure to IFNγ and TNFα. Furthermore, the matrix metalloprotease 9 (MMP9) inhibitor GM6001 attenuates the production of sKIT via the suppression of cKIT ectodomain shedding. Altogether, our study indicates that the presence of Th1 cytokines IFNγ and/or TNFα in the epidermal milieu might impair the repigmentation response of vitiligo patients to NBUVB phototherapy.

The prognostic role of C-KIT, TET1 and TET2 gene expression in Acute Myeloid Leukemia

Aimwas to assess the role of C-KIT, TET1 and TET2 expression in the diagnosis and prognosis of acute myeloblastic leukemia (AML).MethodsThe expression levels of C-KIT, TET1 and TET2 were assessed in the bone marrow (BM) aspirate of 152 AML patients compared to 20 healthy control using quantitative real-time polymerase chain reaction (qRT-PCR). Data were correlated with the clinico-pathological features of the patients, response to treatment, disease-free survival (DFS), and overall survival (OS) rates.ResultsC-KIT, TET1 and TET2 were significantly upregulated in AML patients [0.25 (0–11.6), 0.0113 (0–3.301), and 0.07 (0–4); respectively], compared to the control group [0.013 (0.005–0.250), P < 0.001, 0.001 (0–0.006), P < 0.001, and 0.02 (0.008–0.055), P = 0.019; respectively]. The sensitivity, specificity, and area under curve of of C-KIT were (48.7%, 100%, 0.855; respectively, P = 0.001), and that of TET1 were (63.4%, 100%, 0.897; respectively, P = 0.001), while that of TET2 were (56.8%, 100%, 0.766; respectively, P = 0.019). When combining the three markers, the sensitivity was 77.5%, however it reached the highest sensitivity (78.6%) and specificity (100%) when combining both c-KIT + TET1 together for the diagnosis of AML. C-KIT overexpression associated with shorter DFS (P = 0.05) and increased incidence of relapse (P = 0.019). Lymph nodes involvement [HR = 2.200, P = 0.005] is an independent risk factor for shorter OS rate of AML patients. Increased BM blast % [HR = 7.768, P = 0.002], and FLT3-ITD mutation [HR = 2.989, P = 0.032] are independent risk factors for shorter DSF rate of the patients.ConclusionC-KIT, TET1, and TET2 could be used as possible useful biomarkers for the diagnosis of AML.

Bufalin Inhibits Tumorigenesis, Stemness, and Epithelial-Mesenchymal Transition in Colorectal Cancer through a C-Kit/Slug Signaling Axis.

Colorectal cancer (CRC) is a major source of morbidity and mortality, characterized by intratumoral heterogeneity and the presence of cancer stem cells (CSCs). Bufalin has potent activity against many tumors, but studies of its effect on CRC stemness are limited. We explored bufalin's function and mechanism using CRC patient-derived organoids (PDOs) and cell lines. In CRC cells, bufalin prevented nuclear translocation of β-catenin and down-regulated CSC markers (CD44, CD133, LGR5), pluripotency factors, and epithelial-mesenchymal transition (EMT) markers (N-Cadherin, Slug, ZEB1). Functionally, bufalin inhibited CRC spheroid formation, aldehyde dehydrogenase activity, migration, and invasion. Network analysis identified a C-Kit/Slug signaling axis accounting for bufalin's anti-stemness activity. Bufalin treatment significantly downregulated C-Kit, as predicted. Furthermore, overexpression of C-Kit induced Slug expression, spheroid formation, and bufalin resistance. Similarly, overexpression of Slug resulted in increased expression of C-Kit and identical functional effects, demonstrating a pro-stemness feedback loop. For further study, we established PDOs from diagnostic colonoscopy. Bufalin differentially inhibited PDO growth and proliferation, induced apoptosis, restored E-cadherin, and downregulated CSC markers CD133 and C-Myc, dependent on C-Kit/Slug. These findings suggest that the C-Kit/Slug axis plays a pivotal role in regulating CRC stemness, and reveal that targeting this axis can inhibit CRC growth and progression.

Abstract 6227: An optimized preclinical antibody-drug conjugate against cancers with cKIT overexpression or activating mutations

Abstract cKIT overexpression and activating mutations have been observed in many types of cancer, including small cell lung cancer (SCLC), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML), and are known to be predictive of poor prognosis. In previously reported work, NN2101-DM1, a fully human, anti-cKIT antibody-drug conjugate (ADC), was developed as a proof-of-concept (PoC) entity by conjugating the site-nonspecific and uncleavable linker SMCC with the microtubule inhibitory payload DM1. This linker-payload system is the same used in ado-trastuzumab emtansine, an approved ADC for HER2-positive breast cancer. NN2101-DM1 showed therapeutic efficacy against SCLC and GIST both in vitro and in xenograft models in vivo, demonstrating the feasibility of ADC development using NN2101. Despite the potent efficacy of NN2101-DM1, there are inherent limitations to SMCC linker and DM1 payload systems. The nonspecific attachment of the linker to the antibody produces highly heterogeneous drug-antibody ratios (DARs) with unpredictable attachment. Additionally, impaired lysosomal proteolytic activity has been shown to contribute to resistance to SMCC-DM1 cleavage. To optimize the ADC configuration for advancement to a clinical candidate, four types of ADC with different linker-payload combinations and DARs were designed, and their efficacy and safety were evaluated in vitro and in vivo. Among them, NN3201, a conjugation of the antimitotic payload MMAE to the cKIT-targeting antibody NN2101 via a site-specific, cleavable peptide linker, with a homogenous (&amp;gt;95%) DAR of 4 was selected as a lead candidate. This linker system utilizes a cysteine re-bridging technology to produce site-specific conjugation with high DAR homogeneity. NN3201 showed consistent sub-nanomolar IC50s in several in vitro cell lines and demonstrated complete remission as monotherapy in SCLC (H526), GIST (GIST-T1), AML (Kasumi-1), and mastocytoma (HMC 1.2) cell-derived xenograft models in doses ranging from 1.0-3.0 mg/kg. There were no significant toxicities detected at doses up to 60 mg/kg in normal mice, suggesting a therapeutic index greater than 20 times. In conclusion, NN3201 showed potent anti-tumor activity and was selected as preclinical candidate. Citation Format: Jin-Ock Kim, Han-Jik Ko, Jae-Won Kim, Sang Gyu Park. An optimized preclinical antibody-drug conjugate against cancers with cKIT overexpression or activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6227.

Antibody-Drug Conjugate Targeting c-Kit for the Treatment of Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15–25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study, we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit and specifically binds to SCLC cells expressing c-Kit with a binding affinity of KD = 5.5 × 10−9 M. Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently induced apoptosis in SCLC with an IC50 ranging from 158 pM to 4 nM. An in vivo assay using a xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59% (5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment.

A novel anti-c-Kit antibody-drug conjugate to treat wild-type and activating-mutant c-Kit-positive tumors.

c‐Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small‐cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti‐c‐Kit antibody–drug conjugate was developed in this study to treat wild‐type and mutant c‐Kit‐positive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N‐succinimidyl‐4‐(N‐maleimidomethyl) cyclohexane‐1‐carboxylate (SMCC) (to give NN2101‐DM1). The antitumor activity of NN2101‐DM1 was evaluated in vitro and in vivo using various cancer cell lines. NN2101‐DM1 exhibited potent growth‐inhibitory activities against c‐Kit‐positive cancer cell lines. In a mouse xenograft model, NN2101‐DM1 exhibited potent growth‐inhibitory activities against imatinib‐resistant GIST and SM cells. In addition, NN2101‐DM1 exhibited a significantly higher anti‐cancer effect than carboplatin/etoposide against SCLC cells where c‐Kit does not mediate cancer pathogenesis. Furthermore, the combination of NN2101‐DM1 with imatinib in imatinib‐sensitive GIST cells induced complete remission compared with treatment with NN2101‐DM1 or imatinib alone in mouse xenograft models. These results suggest that NN2101‐DM1 is a potential therapeutic agent for wild‐type and mutant c‐Kit‐positive cancers.

Evaluation of the Expression of HER2 and c-KIT Proteins as Prognostic Markers in Superficial Bladder Urothelial Carcinoma.

BackgroundThe roles of c-KIT and HER2 protein expression in bladder cancer are still debated, and the prognostic value of these proteins as markers of tumor progression is inconclusive.ObjectiveTo assess the impact of HER2 and c-KIT protein expressions in the progression of non-muscle-invasive bladder cancer.MethodsAll patients undergoing transurethral resection of bladder tumors for non-muscle-invasive urothelial carcinoma, with standard regimen of BCG, between January 2017 and November 2019, were evaluated pathologically and immunohistochemically for HER1 and c-KIT proteins in urothelial carcinoma cells. Follow-up cystoscopy was performed for 100 patients every 3 months for the first 2-years and any recurred tumors were excised and examined pathologically, as well as stained for HER2 and c-KIT protein expression.ResultsHER2 and c-KIT positive expressions were detected in 49% and 38% of cases, respectively. After a mean follow-up of 26.4±7.2 months, the overall recurrence and progression rates were significantly correlated with overexpression of HER2 and c-KIT. In high-grade non-invasive muscle neoplasms, tumor cells showed weak expression for both HER2 and c-KIT proteins, but with progression to muscle-invasion, tumor cells strongly expressed HER2 and lost expression to c-KIT. In the multivariate model, overexpression of HER2 rather than c-KIT protein significantly predicted increased progression.ConclusionRecurrence and progression of non-muscle-invasive bladder cancer correlate with overexpression of HER2 and c-KIT proteins in tumor cells.

C-Kit, a Double-Edged Sword in Liver Regeneration and Diseases.

Previous studies have reported an important role of c-kit in embryogenesis and adulthood. Activation of the SCF/KIT signal transduction pathway is customarily linked to cell proliferation, migration and survival thus influence hematopoiesis, pigmentation, and spermatogenesis. The role of c-kit in the liver is controversial, it is however argued that it is a double-edged sword in liver regeneration and diseases. First, liver c-kit+ cells, including oval cells, bile epithelial cells, and part of hepatocytes, participate in liver tissue repair by regenerating target cells according to the type of liver injury. At the same time, c-kit+ mast cells, act as immature progenitors in circulation, playing a critical role in liver fibrosis. Furthermore, c-kit is also a proto-oncogene. Notably, c-kit overexpression regulates gastrointestinal stromal tumors. Various studies have explored on c-kit and hepatocellular carcinoma, nevertheless, the intricate roles of c-kit in the liver are largely understudied. Herein, we extensively summarize previous studies geared toward providing hints for future clinical and basic research.

P-305 Efficacy and safety of IMATINIB in gastrointestinal stromal tumors: Experience of the medical oncology department of the CHU Hassan II of Fez

Gastrointestinal Stromal Tumors (GIST) are a very rare form of cancers of the digestive tract belonging to the sarcoma family. They are characterized by an overexpression of C-kit in immunohistochemistry and by activating mutations of tyrosine kinase receptors. Targeted therapies against these receptors such as IMATINIB and then SUNITINIB have transformed the management and prognosis of advanced and metastatic forms. The aim of this work was to study the efficacy and tolerance of IMATINIB in gastrointestinal stromal tumors. A retrospective study was carried out in the medical oncology department of the Hassan II University Hospital in Fez, collecting 67 patients with a gastrointestinal stromal tumor during the period from April 2013 to April 2019. The statistical analysis of the results was done by SPSS version 23 software, the survival was calculated by the Kaplan-Meier method. The average age of the patients was 59 years with a clear male predominance (sex ratio of 1.5). The most frequent localization was gastric in 33 cases, followed by the small intestine in 21 cases, then other localizations in 13 cases. The most common symptom was pain, reported in 85% of cases. Surgery was the means of diagnostic confirmation in 76% of the cases. At the end of the radiological and histological assessment, the diagnosis of localized GIST was retained in 38 cases, locally advanced in 11 cases and metastatic in 18 cases. Surgery was performed in 46 patients. All patients received treatment with IMATINIB. The most common side effects were: * Asthenia: observed in 30% of patients. * Hematotoxicity: 15% thrombocytopenia, 20% anemia and 10% leukopenia. * Cutaneous-mucous: 47% of hand-foot syndrome, 25% of mucositis, 15% of skin rash and depigmentation of the hair was observed in 5% of patients. * Digestive: vomiting and nausea occurred in 20% of patients. 35% of patients developed edema of the lower limbs and 25% of periorbital edema. 20% of patients developed dysthyroidism and hypertension was observed in 10% of patients. After a median follow-up of 23 months, all the patients were alive and the median progression-free survival was 7 months. The safety profile of IMATINIB used in our study was comparable to the global tolerance reported in the literature. More studies are needed to investigate the relationship between their toxicity and their efficacy in the Moroccan population.

Development and characterization of a fully human antibody targeting SCF/c-kit signaling

CD117/c-kit, a tyrosine kinase receptor, plays a critical role in hematopoiesis, pigmentation, and fertility. The overexpression and activation of c-kit are thought to promote tumor growth and have been reported in various cancers, including leukemia, glioblastoma and mastocytosis. To disrupt the SCF/c-kit signaling axis in cancer, we generated a c-kit antagonist human antibody (NN2101) that binds to domain 2/3 of c-kit. This completely blocked the SCF-mediated phosphorylation of c-kit and inhibited TF-1 cell proliferation, erythroleukemia. In addition, the examination of binding affinity using surface plasmon resonance (SPR) assay showed that NN2101 can bind to c-kit of monkeys (KD = 2.92 × 10−10 M), rats (KD = 1.68 × 10−6 M), mice (KD = 11.5 × 10−9 M), and humans (KD = 2.83 × 10−12 M). We showed that NN2101 does not cause antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The immunogenicity of NN2101 was similar to that of bevacizumab. Furthermore, the crystal structure of NN2101 Fab was determined and the structure of NN2101 Fab:c-kit complex was modeled. Structural information, as well as mutagenesis results, revealed that NN2101 can bind to the SCF-binding regions of c-kit. Collectively, we generated a c-kit neutralizing human antibody (NN2101) for the treatment of erythroleukemia and characterized its biophysical properties. NN2101 can potentially be used as a therapeutic antibody to treat different cancers.

Tankyrase Inhibitors Target Colorectal Cancer Stem Cells via AXIN-Dependent Downregulation of c-KIT Tyrosine Kinase.

Cancer stem cells (CSC) constitute heterogeneous cell subpopulations of a tumor. Although targeting CSCs is important for cancer eradication, no clinically approved drugs that target CSCs have been established. Tankyrase poly(ADP-ribosyl)ates and destabilizes AXIN, a negative regulator of β-catenin, and promotes β-catenin signaling. Here, we report that tankyrase inhibitors downregulate c-KIT tyrosine kinase and inhibit the growth of CD44-positive colorectal CSCs. c-KIT expression in CD44-positive subpopulations of colorectal cancer COLO-320DM cells is associated with their tumor-initiating potential in vivo Tankyrase inhibitors downregulate c-KIT expression in established cell lines, such as COLO-320DM and DLD-1, and colorectal cancer patient-derived cells. These effects of tankyrase inhibitors are caused by reducing the recruitment of SP1 transcription factor to the c-KIT gene promoter and depend on AXIN2 stabilization but not β-catenin downregulation. Whereas c-KIT knockdown inhibits the growth of CD44-positive COLO-320DM cells, c-KIT overexpression in DLD-1 cells confers resistance to tankyrase inhibitors. Combination of a low-dose tankyrase inhibitor and irinotecan significantly inhibited the growth of COLO-320DM tumors in a mouse xenograft model. These observations suggest that tankyrase inhibitors target c-KIT-positive colorectal CSCs and provide a novel therapeutic strategy for cancer.

Long-term c-Kit overexpression in beta cells compromises their function in ageing mice.

c-Kit signalling regulates intracellular pathways that enhance beta cell proliferation, insulin secretion and islet vascularisation in mice up to 28weeks of age and on short-term high-fat diet. However, long-term c-Kit activation in ageing mouse islets has yet to be examined. This study utilises beta cell-specific c-Kit-overexpressing transgenic (c-KitβTg) ageing mice (~60weeks) to determine the effect of its activation on beta cell dysfunction and insulin secretion. Wild-type and c-KitβTg mice, aged 60weeks, were examined using metabolic tests to determine glucose tolerance and insulin secretion. Pancreas histology and proteins in isolated islets were examined to determine the expression of beta cell transcription factors, proliferation and intracellular signalling. To determine the role of insulin receptor signalling in ageing c-KitβTg mice, we generated beta cell-specific inducible insulin receptor knockout in ageing c-KitβTg mice (c-KitβTg;βIRKO mice) and examined the ageing mice for glucose tolerance and islet histology. Ageing c-KitβTg mice progressively developed glucose intolerance, compared with age-matched wild-type littermates, due to impaired insulin secretion. Increased beta cell mass, proliferation and nuclear forkhead box transcription factor O1 (FOXO1) expression and reduced exocytotic protein levels were detected in ageing c-KitβTg mouse islets. Protein analyses of isolated islets showed increased insulin receptor, phosphorylated IRS-1Ser612 and cleaved poly(ADP-ribose) polymerase levels in ageing c-KitβTg mice. Ageing c-KitβTg mouse islets treated ex vivo with insulin demonstrated reduced Akt phosphorylation, indicating that prolonged c-Kit induced beta cell insulin insensitivity. Ageing c-KitβTg;βIRKO mice displayed improved glucose tolerance and beta cell function compared with ageing c-KitβTg mice. These findings indicate that long-term c-Kit overexpression in beta cells has a negative impact on insulin exocytosis and that temporally dependent regulation of c-Kit-insulin receptor signalling is important for optimal beta cell function.

High-dose intramyocardial HMGB1 induces long-term cardioprotection in sheep with myocardial infarction.

In rodents with acute myocardial infarction (AMI), high mobility group box 1 (HMGB1) injection has produced controversial results. Given the lack of data in large mammals, we searched the dose that would promote angiogenesis and expression of specific regenerative genes in sheep with AMI (protocol 1) and, subsequently, use this dose to study long-term effects on infarct size and left ventricular (LV) function (protocol 2). Protocol 1: Sheep with AMI received 250μg (high-dose, n = 7), 25μg (low-dose, n = 7) HMGB1, or PBS (placebo, n = 7) in 10 intramyocardial injections (0.2ml each) in the peri-infarct area. Seven days later, only the high-HMGB1-dose group exhibited higher microvascular densities, Ki67-positive cardiomyocytes, and overexpression of VEGF, Ckit, Tbx20, Nkx2.5, and Gata4. Protocol 2: Sheep with AMI received HMGB1 250μg (n = 6) or PBS (n = 6). At 60days, HMGB1-treated sheep showed smaller infarcts (8.5 ± 2.11 vs. 12.2 ± 1.97% LV area, P < 0.05, ANOVA-Bonferroni) and higher microvascular density (capillaries, 1798 ± 252 vs. 1266 ± 250/mm2; arterioles, 18.3 ± 3.9 vs. 11.7 ± 2.2/mm2; both P < 0.01). Echocardiographic LV ejection fraction, circumferential shortening, and wall thickening increased from day 3 to 60 with HMGB1 (all P < 0.05). Conclusion: in ovine AMI, high-dose HMGB1 induces angio-arteriogenesis, reduces infarct size, and improves LV function at 2months post-treatment.

The RUNX1-ETO fusion protein trans-activates c-KIT expression by recruiting histone acetyltransferase P300 on its promoter.

The oncoprotein RUNX1-ETO is the fusion product of t(8;21)(q22;q22) and constitutes one of the most common genetic alterations in acute myeloid leukemia (AML). Abnormal c-KIT overexpression is considered an independent negative prognostic factor for relapse and survival in t(8;21) AML patients. However, the molecular mechanism of high c-KIT expression in t(8;21) AML remains unknown. In this study, we detected RUNX1-ETO and c-KIT gene expression in AML-M2 patients and verified the overexpression of c-KIT in t(8;21) AML patients. We also found that c-KIT overexpression was a poor prognostic indicator in RUNX1-ETO positive AML patients, but not in RUNX1-ETO negative AML patients. We used the dual-luciferase and ChIP assays to demonstrate that the RUNX1-ETO protein epigenetically trans-activates c-KIT by binding to the c-KIT promoter and recruiting the histone acetyltransferase P300 to the c-KIT promoter, elucidating the mechanism of the abnormally increased c-KIT expression in t(8;21) AML patients. Moreover, pharmacological studies revealed that C646, a P300 inhibitor, could inhibit proliferation, induce apoptosis and arrest the cell cycle more effectively in RUNX1-ETO positive cells than in negative ones. The levels of c-KIT and RUNX1-ETO proteins were also decreased with C646 treatment in RUNX1-ETO positive cells. These findings suggested that P300 could be a therapeutic target and that C646 could be used as a potential treatment for RUNX1-ETO positive AML patients. Interestingly, using the dual-luciferase assay, we also found that the binding capacity of RUNX1-ETO9a, a truncated RUNX1-ETO isoform, to the c-KIT promoter was stronger than that of RUNX1-ETO, suggesting RUNX1-ETO9a as another valuable therapeutic target in t(8;21) AML.

Nobiletin Down-Regulates c-KIT Gene Expression and Exerts Antileukemic Effects on Human Acute Myeloid Leukemia Cells.

Nobiletin, a dietary citrus flavonoid, has been reported to possess several biological activities such as antioxidant, anti-inflammatory, and anticancer properties. The aim of this study was to investigate the antileukemic effects of nobiletin and its underlying mechanisms on human acute myeloid leukemia (AML) cells. We demonstrated that nobiletin (0-100 μM) significantly reduced cell viability from 100.0 ± 9.6% to 31.1 ± 2.8% in human AML THP-1 cell line. Nobiletin arrested cell cycle progression in G1 phase and induced myeloid cell differentiation in human AML cells. Microarray analysis showed that mRNA expression of the c- KIT gene, a critical proto-oncogene associated with leukemia progression, was dramatically reduced in nobiletin-treated AML cells. Furthermore, we verified that AML cells treated with nobiletin (40 and 80 μM) for 48 h markedly suppressed c-KIT mRNA expression (from 1.00 ± 0.07-fold to 0.62 ± 0.08- and 0.30 ± 0.05-fold) and reduced the level of c-KIT protein expression (from 1.00 ± 0.11-fold to 0.60 ± 0.15- and 0.34 ± 0.05-fold) by inhibition of KIT promoter activity. The knockdown of c-KIT expression by shRNA attenuated cancer cell growth and induced cell differentiation. Moreover, we found that the overexpression of c-KIT abolished nobiletin-mediated cell growth inhibition in leukemia cells. These results indicate that nobiletin exerts antileukemic effects through the down-regulation of c-KIT gene expression in AML cells. Finally, we demonstrated that the combination of a conventional AML chemotherapeutic agent, cytarabine, with nobiletin resulted in more reduction of cell viability in AML cells. Our current findings suggest that nobiletin is a novel c-KIT inhibitor and may serve as a chemo-preventive or -therapeutic agent against human AML.

Preclinical Antitumor Activity of a Novel Anti-c-KIT Antibody-Drug Conjugate against Mutant and Wild-type c-KIT-Positive Solid Tumors.

Purpose: c-KIT overexpression is well recognized in cancers such as gastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC), melanoma, non-small cell lung cancer (NSCLC), and acute myelogenous leukemia (AML). Treatment with the small-molecule inhibitors imatinib, sunitinib, and regorafenib resulted in resistance (c-KIT mutant tumors) or limited activity (c-KIT wild-type tumors). We selected an anti-c-KIT ADC approach to evaluate the anticancer activity in multiple disease models.Experimental Design: A humanized anti-c-KIT antibody LMJ729 was conjugated to the microtubule destabilizing maytansinoid, DM1, via a noncleavable linker (SMCC). The activity of the resulting ADC, LOP628, was evaluated in vitro against GIST, SCLC, and AML models and in vivo against GIST and SCLC models.Results: LOP628 exhibited potent antiproliferative activity on c-KIT-positive cell lines, whereas LMJ729 displayed little to no effect. At exposures predicted to be clinically achievable, LOP628 demonstrated single administration regressions or stasis in GIST and SCLC xenograft models in mice. LOP628 also displayed superior efficacy in an imatinib-resistant GIST model. Further, LOP628 was well tolerated in monkeys with an adequate therapeutic index several fold above efficacious exposures. Safety findings were consistent with the pharmacodynamic effect of neutropenia due to c-KIT-directed targeting. Additional toxicities were considered off-target and were consistent with DM1, such as effects in the liver and hematopoietic/lymphatic system.Conclusions: The preclinical findings suggest that the c-KIT-directed ADC may be a promising therapeutic for the treatment of mutant and wild-type c-KIT-positive cancers and supported the clinical evaluation of LOP628 in GIST, AML, and SCLC patients. Clin Cancer Res; 24(17); 4297-308. ©2018 AACR.

Combination of Luteolin and Solifenacin Improves Urinary Dysfunction Induced by Diabetic Cystopathy in Rats.

BackgroundThe purpose of the present study was to assess the effect of luteolin and solifenacin on diabetic cystopathy (DCP) and to investigate the mechanism of action. A novel link between the overexpression of c-Kit in the bladder and voiding dysfunction was identified in rats with DCP.Material/MethodsA rat model of DCP was successfully established by intraperitoneal injection of streptozotocin and a diet high in glucose and lipids, and animals were treated with luteolin and solifenacin. The effect of luteolin and solifenacin on urinary dysfunction in DCP rats was investigated by assessing bladder pressure and performing a volume test. The protein levels of c-Kit, stem cell factor (SCF), p110, and phosphorylated p110 in the bladder were detected by Western blot analysis and immunohistochemical staining.ResultsIn DCP rats, the protein levels of c-Kit, SCF and phosphorylated p110 in the bladder were significantly increased. However, oral treatment of DCP rats with luteolin combined with solifenacin resulted in effective improvement of overactive bladder and reduced the protein expression of c-Kit, SCF, and phosphorylated p110. Moreover, the effect of luteolin combined with solifenacin on maximum voiding pressure and residual urine volume was improved compared to that of luteolin alone.ConclusionsLuteolin improved overactive bladder in DCP rats, which may be due to SCF/c-kit inhibition, as well as the downregulation of the phosphoinositide-3 kinase signaling pathway. Moreover, solifenacin enhanced the potential pharmacological effect of luteolin in the treatment of DCP.

Expression of c-kit in common benign and malignant breast lesions.

c-kit (CD117) is a transmembrane tyrosine kinase that acts as a type III receptor for mast cell growth factor. In recent years, the role of c-kit in the development of preinvasive and invasive breast carcinomas has been investigated. The aim of our study was to detect c-kit expression in the entire spectrum of common benign and malignant breast lesions in correlation with a well-studied myoepithelial or stem-cell like marker (p63). We evaluated 270 cases of benign and malignant breast lesions including fibrocystic disease, fibroadenoma, sclerosing adenosis, atypical ductal hyperplasia, ductal/lobular carcinoma in situ, and ductal/lobular/mixed type carcinoma. C-kit staining was evaluated in the cytoplasm/cell membrane in epithelial and myoepithelial cells and p63 in the nuclei of myoepithelial cells. c-kit was highly expressed (85.3%) in benign lesions (fibrocystic disease, sclerosing adenosis, fibroadenoma), and p63 expression was 95.5% in the aforementioned lesions. c-kit distribution in preinvasive and invasive lesions was as follows: ductal/lobular carcinoma in-situ, 43%/35%; ductal/lobular carcinoma, 36%/39%; and mixed type carcinoma, 20%. c-kit was highly expressed in myofibroblast/fibroblast cells only in grade III ductal/lobular carcinomas. c-kit was totally absent in stromal cells in benign lesions and in situ carcinomas whereas expression was weak in grade I and II carcinomas. Combined overexpression of c-kit and p63 is indicative of benign breast lesions. In contrast, there is reduced expression of c-kit in in situ and invasive breast carcinomas, with simultaneous overexpression in the stromal cells. This suggests that c-kit may play a role in breast cancer progression.