C-kit Inhibitor Research Articles

Rational Design, Synthesis and Biological Evaluation of Pyrimidine-4,6-diamine derivatives as Type-II inhibitors of FLT3 Selective Against c-KIT

FMS-like Tyrosine Kinase 3 (FLT3) is a clinically validated target for acute myeloid leukemia (AML). Inhibitors targeting FLT3 have been evaluated in clinical studies and have exhibited potential to treat FLT3-driven AML. A frequent, clinical limitation is FLT3 selectivity, as concomitant inhibition of FLT3 and c-KIT is thought to cause dose-limiting myelosuppression. Through a rational design approach, novel FLT3 inhibitors were synthesized employing a pyridine/pyrimidine warhead. The most potent compound identified from the studies is compound 13a, which exhibited an IC50 value of 13.9 ± 6.5 nM against the FLT3 kinase with high selectivity over c-KIT. Mechanism of action studies suggested that 13a is a Type-II kinase inhibitor, which was also supported through computer aided drug discovery (CADD) efforts. Cell-based assays identified that 13a was potent on a variety of FLT3-driven cell lines with clinical relevance. We report herein the discovery and therapeutic evaluation of 4,6-diamino pyrimidine-based Type-II FLT3 inhibitors, which can serve as a FLT3-selective scaffold for further clinical development.

Final toxicity results from a phase II study of 5-fluorouracil, oxaliplatin, and dasatinib (FOLFOX-D) in previously untreated metastatic pancreatic adenocarcinoma.

504 Background: Systemic chemotherapy for pancreatic adenocarcinoma (PCa) improves survival but most targeted agents have consistently failed to demonstrate clinical benefits. Src is overexpressed in PCa and promotes an aggressive cancer phenotype. Dasatinib (D) is an oral multi-tyrosine kinase inhibitor (TKI) affecting tumor proliferation through inhibition of Src, Bcr-Abl, CKit and other pathways. Inhibition of Src is associated with biologic modifications favorably modifying the PCa phenotype and has synergy with restoring inherent chemosensitivity. Src inhibition can also increase oxaliplatin activity and modulate Tcell responses. The addition of D to the well-established backbone of FOLFOX represents a multifaceted line of scientific investigation. Methods: This single arm phase II trial is to determine activity and toxicity of FOLFOX + D in previously untreated metastatic PCa. Pts must have at least 1 RECIST measurable target lesion, ECOG PS 0-2, normal QTc and adequate organ function. Treatment is standard q14d cycles of mFOLFOX6 with continuous D (150mg PO daily). Tumor assessments occur q8w. Endpoints are PFS (primary), objective and biochemical response rates, clinical benefit rate, freedom from metastases, overall survival (OS), toxicity, and quality of life. Exploratory tissue, circulating tumor cell and serum analyses to identify predictors of response are planned, particularly in those with durable disease control or exceptional response. Sample size is based on a 50% improved median PFS from 4 (historical) to 6 months. Results: Enrollment is now closed on this prospective phase II study with 42 of 42 evaluable pts. Baseline demographics are in Table 1. Toxicity and outcomes continue to be assessed. Conclusions: Final toxicity data will be presented at the meeting for this novel combination treatment in advanced PCa. Clinical trial information: NCT01652976. [Table: see text]

Abstract IA24: Identification of therapies for plexiform neurofibromas, a precursor of malignant peripheral nerve sheath tumors

Abstract Plexiform neurofibromas (PNs) are complex nerve and soft tissue tumors that affect 25-50% of individuals with neurofibromatosis type 1 (NF1), the most common genetic disease in man with a predisposition to cancer. PNs cause lifelong morbidity ranging from deformity to paralysis and mortality due to organ compression, and progress to MPNST in ~8-13% of patients. The generation of genetically engineered mouse models that closely recapitulate the development of human plexiform neurofibromas has allowed our laboratory and others to pursue genetic and pharmacologic studies to identify novel therapies. Genetic and adoptive transfer studies conducted by our lab in collaboration with Dr. Luis Parada identified a key role for c-kit/kit-ligand in tumor initiation and progression. These data led to a clinical trial administering imatinib mesylate to target aberrant stem cell factor (SCF)/c-kit signaling in the tumor microenvironment, which induced the only objective clinical responses ever obtained in a therapeutic clinical trial in pNF. Based on the observation that young children with head, neck, and airway tumors were most likely to have partial tumor regression, a registration trial in this patient population is currently being pursued. Further, additional phase 2 trials using other receptor tyrosine kinases that include c-kit are under way as well. More recently, studies in GEM models informed clinical trials of MEK inhibitors led by Dr. Brigitte Widemann (Pediatric Branch of NCI) to directly inhibit aberrant Ras signaling in PN cells. Studies are ongoing to extend these promising initial clinical findings to investigate the developmental, molecular, and pharmacokinetic effects of c-kit and MEK inhibition alone and in combination in preclinical models. Specifically, studies to explore adaptive responses to MEK inhibition monotherapy in mice and patients treated with selumetinib via integrated exome, RNAseq, and kinome studies in pre- and post-treatment tumor tissue are under way. Citation Format: D. Wade Clapp. Identification of therapies for plexiform neurofibromas, a precursor of malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA24.

(+)-Usnic Acid Inhibits Migration of c-KIT Positive Cells in Human Colorectal Cancer.

Inhibition of tumor cell migration is a treatment strategy for patients with colorectal cancer (CRC). SCF-dependent activation of c-KIT is responsible for migration of c-KIT positive [c-KIT(+)] cells of CRC. Drug resistance to Imatinib Mesylate (c-KIT inhibitor) has emerged. Inhibition of mTOR can induce autophagic degradation of c-KIT. (+)-usnic acid [(+)-UA], isolated from lichens, has two major functions including induction of proton shuttle and targeting inhibition of mTOR. To reduce hepatotoxicity, the treatment concentration of (+)-UA should be lower than 10 μM. HCT116 cells and LS174 cells were employed to investigate the inhibiting effect of (+)-UA (<10 μM) on SCF-mediated migration of c-KIT(+) CRC cells. HCT116 cells were employed to investigate the molecular mechanisms. The results indicated that firstly, 8 μM (+)-UA decreased ATP content via uncoupling; secondly, 8 μM (+)-UA induced mTOR inhibition, thereby mediated activation suppression of PKC-A, and induced the autophagy of the completed autophagic flux that resulted in the autophagic degradation and transcriptional inhibition of c-KIT and the increase in LDH release; ultimately, 8 μM (+)-UA inhibited SCF-mediated migration of CRC c-KIT(+) cells. Taken together, 8 μM could be determined as the effective concentration for (+)-UA to inhibit SCF-mediated migration of CRC c-KIT(+) cells.

Case: Secondary polycythemia due to pazopanib in patients with metastatic renal cell carcinoma.

Clear-cell renal cell carcinoma (RCC) is the most common primary malignant renal neoplasm. Up to 30% of patients have metastatic disease at the time of initial diagnosis.1 Small molecule inhibitors of vascular endothelial growth factor receptor tyrosine kinase (VEGFR TKIs) are used as first-line treatment for most patients with incurable RCC. Pazopanib is a TKI inhibiting VEGFR,1-3 as well as plateletderived growth factor receptors (PDGFRs) α and β and stem cell receptor c-kit. The most common side effects of pazopanib are diarrhea, hypertension, changes in hair colour, anorexia, nausea, and vomiting. Severe hepatic toxicity is the adverse effect of greatest concern,but is uncommon. Myelosuppressive effects are also noted, such as anemia, thrombocytopenia, and leukopenia, and are presumably mediated through c-kit inhibition. Erythrocytosis is a wellrecognized paraneoplastic syndrome associated with RCC; however, erythrocytosis due to pazopanib therapy has only recently been described. We report two cases and review of the literature related to this phenomenon.

Estrogen induces c-Kit and an aggressive phenotype in a model of invasive lobular breast cancer

Among the molecular subtypes of breast cancer are luminal (A or B) estrogen receptor positive (ER+), HER2+, and triple negative (basal-like). In addition to the molecular subtypes, there are 18 histologic breast cancer subtypes classified on appearance, including invasive lobular breast carcinoma (ILC), which are 8–15% of all breast cancers and are largely ER+ tumors. We used a new model of ER+ ILC, called BCK4. To determine the estrogen regulated genes in our ILC model, we examined BCK4 xenograft tumors from mice supplemented with or without estrogen using gene expression arrays. Approximately 3000 genes were regulated by estrogen in vivo. Hierarchical cluster analyses of the BCK4 derived tumors compared with ER+ and ER- breast cancer cell lines show the estrogen treated BCK4 tumors group with ER- breast cancers most likely due to a high proliferation score, while tumors from cellulose supplemented mice were more related to ER+ breast tumor cells. To elucidate genes regulated in vitro by estrogen in BCK4 cells, we performed expression profiling using Illumina arrays of the BCK4 cell line, treated with or without estrogen in vitro. A set of ~200 overlapping genes were regulated by estrogen in the BCK4 cell line and xenograft tumors, and pathway analysis revealed that the c-Kit pathway might be a target to reduce estrogen-induced proliferation. Subsequent studies found that inhibition of c-Kit activity using imatinib mesylate (Gleevec®) blocked estrogen mediated stimulation of BCK4 tumors and BCK4 cells in vitro as effectively as the anti-estrogen fulvestrant (Faslodex®). Decreased expression of c-Kit using shRNA also decreased baseline and estrogen induced proliferation in vitro and in vivo. These studies are the first to indicate that c-Kit inhibition is an effective approach to target c-Kit+ ILC.

Dual targeting of C-kit and Integrin Linked Kinase abrogates the phosphorylation of S6 ribosomal protein and reduces melanoma cell growth

Background: Activating mutations in c-kit have been observed in distinct subtypes of melanoma and are associated with disease progression and the development of resistance to treatment. Mutant c-kit uses mainly the phosphatidylinositol 3 kinase (PI3k)/Akt pathway and to a lesser extent the mitogen activated protein kinase (MAPK) pathway in order to promote characteristics of the cancer phenotype, such as excessive proliferation, angiogenesis and invasiveness. However, clinical studies on the inhibition of c-kit in melanoma patients have yielded disappointing results. The reactivation of these pathways have been reported in treatment resistant melanoma cells. Previously we showed that downregulation of integrin linked kinase (ILK), an intracellular adapter molecule that promotes tumor progression and angiogenesis when dysregulated, suppressed Akt phosphorylation and inhibited tumor growth. Therefore we investigated the effects of dual inhbition of c-kit and ILK on tumor cell growth and angiogenesis. Methods: The effects of dual blocking of c-kit and ILK-down regulation using siRNA on tumor cell growth were evaluated in an in vitro co-culture system that contained human vein endothelial cells (HUVECs) and melanoma cells. To investigate the effects of blocking c-kit and ILK on angiogenesis, the migration and invasion of HUVECs in response to conditioned medium from melanoma cells was studied using the xCELLigence system, while endothelial cell tube formation was studied using a collagen-based assay. Protein array studies were employed to investigate the expression patterns of angiogenic proteins in mono- and co-cultured cells. Further marker expression was determined using immunocytochemistry. Results: Significant inhibition of melanoma cell growth was observed in treated co-cultures, and the inhibition correlated with reduced expression of S6 ribosomal protein and S6 kinase. ILK-knockdown inhibited endothelial cell migration and invasion, as well as endothelial cell morphogenesis induced by melanoma conditioned medium. Results also revealed an increase in the expression of Platelet Factor 4 (PF4), and a suppression of proangiogenic factors, including leptin, vascular endothelial growth factor and basic fibroblast growth factor. Conclusions: Findings from this study suggest that the cross-talk between melanoma and endothelial cells which supports tumor cell growth, can be disrupted by dual targeting of c-kit and ILK. Furthermore, dual targeting of c-kit and ILK which also resulted in the altered expression profile of angiogenesis markers to favor antiangiogenesis, may imply that such dual targeting could contribute to enhancing complimentary approaches for melanoma therapy. Legal entity responsible for the study: Angiogenesis Laboratory, University of Pretoria Funding: National Research Foundation, University of Pretoria Disclosure: All authors have declared no conflicts of interest.

Possibilities of Radiosensitization and Reduced Invasion After Targeting the SCF/c-Kit Pathway in Human Cancer Cells

The SCF/c-Kit pathway is often overexpressed in human tumors leading to an enhanced proliferation and invasion. Resulting therapy resistance and metastasis often are limiting factors of a successfull radiotherapy. It was now tested for NSCLC and prostate cancer cells growing in 2D and 3D whether the inhibition of this pathway can be used to achieve a reduced cancer cell invasion and whether a significant increased radiosensitivity could be achieved. Experiments were performed using different NSCLC (H23, H226, H460, H520, H1299, H1975, A549) and prostate cancer cell lines (DU 145, PC3, LnCAP) growing either under 2D or 3D conditions. SCF was knocked down by siRNA, c-Kit was inhibited by ISCK03 inhibitor and cells were irradiated with 2 Gy or 6 Gy Photons. Cell survival was determined by colony formation assay. Invasion was measured using BioCoat™ Matrigel™ invasion chambers. When grown in 3D a 2-3-fold increase in invasion capability was examined in all NSCLC and prostate cancer cell lines except H520. This could be explained by EMT-like changes (enhanced expression of vimentin and N-cadherin and reduction of E-cadherin) when cells were grown in 3D. Just the opposite trends were found for H520 cells. Knock down of SCF was generally found to result in significantly reduced invasion and in only a minor increase of cellular radiosensitivity (2D and 3D). Only in cells characterized by a massive overexpression of SCF (H520) a significant increase of cellular radiosensitivity could be obtained after SCF knock down. The inhibition of c-Kit by a specific inhibitor was also found to result only in minor radiosensitization. Photon irradiation did not modulate cancer cell invasion (2D and 3D). For the regulation of tumor cell motility and invasion the SCF/c-Kit pathway appears to take a key position. Inhibition of this pathway significantly reduces the cell invasion of human NSCLC and prostate cancer cells under 2D and 3D conditions. In addition our data indicate that generally cell survival and radioresponse are not strongly determined by the SCF/c-Kit pathway. As a consequence targeting SCF or c-Kit only results in increased radiosensitivity when SCF is strongly over expressed as found for H520 cells.

Estrogen-induced transcription factor EGR1 regulates c-Kit transcription in the mouse uterus to maintain uterine receptivity for embryo implantation.

Early growth response 1 (Egr1) is a key transcription factor that mediates the action of estrogen (E2) to establish uterine receptivity for embryo implantation. However, few direct target genes of EGR1 have been identified in the uterus. Here, we demonstrated that E2 induced EGR1-regulated transcription of c-Kit, which plays a crucial role in cell fate decisions. Spatiotemporal expression of c-Kit followed that of EGR1 in uteri of ovariectomized mice at various time points after E2 treatment. E2 activated ERK1/2 and p38 to induce EGR1, which then activated c-Kit expression in the uterus. EGR1 transfection produced rapid and transient induction of c-KIT in a time- and dose-dependent manner. Furthermore, luciferase assays to measure c-Kit promoter activity confirmed that a functional EGR1 binding site(s) (EBS) was located within-1kb of the c-Kit promoter. Site-directed mutagenesis and chromatin immunoprecipitation-PCR for three putative EBS within-1kb demonstrated that the EBS at -818/-805 was critical for EGR1-dependent c-Kit transcription. c-Kit expression was significantly increased in the uterus on day 4 and administration of Masitinib, a c-Kit inhibitor, effectively interfered with embryo implantation. Collectively, our results showed that estrogen induces transcription factor EGR1 to regulate c-Kit transcription for uterine receptivity for embryo implantation in the mouse uterus.

Platelet Derived Growth Factor Alpha (PDGFRα) Induces the Activation of Cardiac Fibroblasts by Activating c-Kit.

BackgroundEnhanced platelet-derived growth factor receptor α (PDGFRα) signaling pathway activity leads to cardiac fibrosis. However, because of the pleiotropic effects of PDGFR signaling, its role in mediating the cardiac fibrotic response remains poorly understood. This study aimed to investigate the regulatory effect of c-Kit in cardiac fibroblasts activated by PDGFRα signaling.Material/MethodsA cardiac fibrosis mice model was induced using isoproterenol, and the heart tissues of mice were tested through western blotting and real-time quantitative PCR (RT-qPCR). The cardiac fibroblasts of neonatal mice were treated with PDGF-AA or transfected with small interfering RNAs (siRNAs) specific for the mouse c-Kit gene. The levels of collagen I, collagen III, and alpha-smooth muscle actin (α-SMA) were analyzed using western blotting and RT-qPCR.ResultsIn the heart of the cardiac fibrosis mice model, the activity of c-Kit was enhanced. PDGF-AA treatment accelerated the activity of c-Kit in cardiac fibroblasts. In addition, imatinib inhibited the activity of c-Kit in vivo and in vitro. Moreover, inhibition of c-Kit by siRNAs reduced the expression of α-SMA and collagens in the activated cardiac fibroblasts. Furthermore, PDGFRα directly bound c-Kit in cardiac fibroblasts and stimulated the expression of stem cell factor (SCF).ConclusionsOur data demonstrated that PDGF/PDGFRα induced the activation of cardiac fibroblasts by activating c-Kit. This study indicated that c-Kit could be used as a potential therapeutic target for treatment of cardiac fibrosis.

The clinical significance of c-Kit mutations in metastatic oral mucosal melanoma in China.

c-Kit mutations are frequently detected in mucosal melanomas, but their clinical significance in metastatic oral mucosal melanomas (OMM) remains unclear. The main purpose of this study was to investigate the clinical and pathological features of metastatic OMMs with c-Kit mutations and the efficiency of the tyrosine kinase inhibitor imatinib in treating metastatic OMMs. We found thatresidual primary lesion and neck lymph nodes could act as independent prognostic factors in metastatic OMM patients. c-Kit mutations were detected in 22 out of 139 (15.8%) metastatic OMM patients. Under chemotherapy, the overall survival (OS) of c-Kit mutant patients was significantly shorter than that of wild-type patients. The Ki67 expression was significantly higher in c-Kit mutant patients than in wild-type patients. In distant metastatic OMM patients with c-Kit mutations, the treatment with c-Kit inhibitor resulted in a better OS. In conclusion, residual primary lesion, cervical lymph nodes and c-Kit mutations act as adverse prognostic factors of metastatic OMMs. The Kit inhibitor imatinib could benefit metastatic OMM patients with c-Kit mutations.

Abstract TMEM-016: DENDRITIC CELL VACCINATION AND INHIBITION OF TUMOR–ASSOCIATED IMMUNE SUPPRESSION

Abstract PURPOSE: Th17 cell infiltration correlates with markedly prolonged overall survival among ovarian cancer patients, prompting the question of whether Th17 cells could be induced or expanded to therapeutic advantage. To address this question, a phase I clinical trial to test the safety and immunogenicity of Th17-inducing dendritic cell (DC) vaccination for stage IIIC–IV ovarian cancer patients (Clinicaltrials.gov NCT02111941) is nearing its accrual goal (n = 22). Interim analyses show that 8 of 9 patients completing the induction phase of five DC vaccinations mounted Th17 and antibody responses to FRα target antigen. These results are encouraging, but we found that ovarian tumor-associated CD14+ macrophages strongly suppress Th17-inducing DC-stimulated tumor antigen-specific Th1/Th17 effector CD4+ T cells responses. The purpose of this project was to elucidate the mechanisms by which ovarian tumor ascites CD14+ cells suppress DC-stimulated T cell responses, and to explore innovative strategies for alleviation of CD14+ cell-mediated immune suppression, or redirection of CD14+ cells from a suppressive phenotype to a pro-inflammatory phenotype. EXPERIMENTAL PLAN: Primary ovarian ascites CD14+ myeloid cells were tested for their ability suppress DC-stimulated tumor antigen-specific CD4+ T cell responses in contact or transwell coculture assays. Ascites fluid and ascites CD14+ cells were screened for expression of key chemokines and cytokines associated with T cell migration and immune suppression. Ascites CD14+ cells were also screened for PD-L1 expression, indoleamine 2,3-dioxygenase (IDO) activity, and key signaling pathways that determine the balance between M1 and M2 function. We also tested the ability of small molecule inhibitors to block key functions and alleviate immune suppression. SUMMARY OF RESULTS: Ovarian ascites CD14+ cells suppress DC-stimulated CD4+ T cell responses at least in part through expression of IL-10 and IDO. PD-L1 and PD-1 are consistently expressed by ascites CD14+ cells. Flow cytometric phosphoprotein assays with ascites CD14+ cells (n = 9) revealed consistent activation of AktS473, ERK1/2T202/Y204, CREBS133 and STAT3S727. Ovarian tumor-associated CD14+ macrophages consistently show high activity of a non-canonical STAT3 signaling pathway revealed by phosphorylation of S727 rather than the canonical Y705 phosphorylation seen in the classical JAK-STAT3 pathway. This innovative finding is consistent for every patient sample tested to date - canonical STAT3Y705 is absent. Inhibition of p38T180/Y182 MAPK signaling reduced expression of IL-10, RANTES, MIP-1β and IP-10. CCL22 (MDC) expression was also limited by p38 inhibition, but markedly elevated by blockade of JAK1/2 signaling. Inhibition of c-Kit and PI3K/Akt signaling showed a trend towards reduced K/T ratios, whereas inhibition of STAT3 reduced kynurenine production, suggesting that IDO activity is regulated at least in part by non-canonical STAT3S727 signaling. CONCLUSIONS: Multiple pathways regulate mechanisms of immune suppression by ovarian tumor ascites CD14+ myeloid cells. The p38 MAPK pathway regulates IL-10 expression, consistent with our published work on DC function. The non-canonical STAT3S727 pathway may also play a central role in regulating immune function by myeloid cells in the tumor microenvironment. These studies are of fundamental importance for development of adjuvant treatments to enhance the clinical efficacy of Th-17-inducing DC vaccination for ovarian cancer. Citation Format: Martin J Cannon, PhD. DENDRITIC CELL VACCINATION AND INHIBITION OF TUMOR–ASSOCIATED IMMUNE SUPPRESSION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-016.

In cancer cell lines inhibition of SCF/c-Kit pathway leads to radiosensitization only when SCF is strongly over-expressed

Background and purposeThe SCF/c-Kit pathway is often overexpressed in human tumors leading to an enhanced tumorigenesis, proliferation and migration. It was now tested for NSCLC and prostate cancer cells growing in 2D and 3D whether the inhibition of this pathway can be used to achieve a significant radiosensitization and whether a respective biomarker may be identified. Material and methodsExperiments were performed with different cancer cell lines (NSCLC: H23, H520, H226, H1975 and PrCa: DU145) growing either under 2D or 3D conditions. Expression of SCF and c-Kit was determined by RT-PCR and Western blot, SCF was knocked down by siRNA, c-Kit was inhibited by ISCK03 inhibitor and cell survival was determined by colony formation assay. ResultsThere is a profound variation in the expression of both c-Kit and SCF with no association between each other. Neither levels did correlate with the respective cellular radiosensitivity determined for 2D or 3D with only a trend seen for SCF. Knock-down of SCF was generally found to result in no or only minor reduction of plating efficiency or cellular radioresistance. A significant reduction was only obtained for H520 cells characterized by an extreme over-expression of SCF. The inhibition of c-Kit by a specific inhibitor was also found to result only in minor radiosensitization. ConclusionGenerally, the SCF/c-Kit pathway does not have a dominant effect on both, cell survival and radioresponse and, as a consequence, knockdown of this pathway does not result in a strong effect on radioresistance, except when SCF is strongly over-expressed.

5-fluorouracil, oxaliplatin and dasatinib (FOLFOX-D) for previously untreated metastatic pancreatic adenocarcinoma.

TPS517 Background: Systemic chemotherapy for pancreatic adenocarcinoma improves survival and cancer related symptoms. Most targeted agents combined with gemcitabine have consistently failed to demonstrate clinical benefits. Src is overexpressed in pancreatic cancer and promotes an aggressive cancer phenotype. Dasatinib (D) is an oral multi-tyrosine kinase inhibitor (TKI) affecting tumor proliferation through inhibition of Src, Bcr-Abl, CKit and other pathways. Inhibition of Src is associated with biologic modifications favorably modifying the pancreatic cancer phenotype and has synergy with restoring inherent chemosensitivity. Src inhibition can also increase oxaliplatin activity and modulate Tcell responses. The addition of D to the well-established backbone of FOLFOX represents a multifaceted line of scientific investigation. Methods: This is a multicenter phase II trial to determine activity and toxicity of FOLFOX + D in previously untreated metastatic pancreatic adenocarcinoma. Eligible pts must have at least 1 measurable target lesion by RECIST, ECOG PS 0-2, normal QTc and adequate organ function. Pts received standard cycles of mFOLFOX6 repeated q14d with continuous D (150mg PO daily). Tumor assessments occur q8w. Endpoints included progression-free survival (primary; PFS), objective and biochemical response rates, clinical benefit rate, freedom from metastases, overall survival (OS), toxicity, and quality of life. Exploratory tissue, circulating tumor cell and serum analyses to identify predictors of response are planned, particularly in those demonstrating durable disease control or exceptional response. Sample size was based on a 50% improved median PFS from 4 (historical) to 6 months. NCT01652976. Results: Enrollment continues on this prospective phase II study with 38 of 42 evaluable patients. There are no unexpected toxicities noted thus far. Clinical trial information: NCT01652976. [Table: see text]

Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells.

c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence.

Stem cell factor promotes in vitro ovarian follicle development in the domestic cat by upregulating c-kit mRNA expression and stimulating the phosphatidylinositol 3-kinase/AKT pathway.

In the present study we examined the effects of stem cell factor (SCF; 50 vs 100ngmL-1) alone or in combination with epidermal growth factor (EGF; 100ngmL-1) on: (1) the in vitro viability and growth of cat follicles within ovarian cortices; (2) phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) phosphorylation; and (3) c-kit and FSH receptor (FSHr) mRNA expression. At 100ngmL-1, SCF increased (P≤0.05) the percentage and size of secondary follicles after 14 days of in vitro culture and sustained AKT phosphorylation after 3 days incubation. EGF suppressed this beneficial effect and reduced (P≤0.05) the percentage of structurally normal follicles and FSHr expression when combined with 100ngmL-1 SCF. Expression of c-kit mRNA was higher (P≤0.05) in the presence of 100ngmL-1 SCF compared with fresh follicles and cohorts cultured under other conditions. A c-kit inhibitor suppressed follicle growth and reduced AKT phosphorylation. Collectively, the results demonstrate that SCF promotes cat follicle development by upregulating c-kit mRNA expression and AKT phosphorylation. EGF suppresses the stimulating effect of SCF, leading to downregulation of FSHr expression.

C-Kit inhibitors prevent respiratory viral infection-induced acute asthma via the blockade of innate lymphoid cell activation

Abstract Asthma is a major public health problem that affects nearly 10% of the general population and is thought to be mediated by allergen-specific Th2 cells, adaptive immunity and allergic inflammation. Current treatments for asthma generally focus on minimizing the Th2-driven eosinophilic inflammation with the use of anti-inflammatory therapies such as corticosteroids. However, this approach, targeting the adaptive Th2 cell immunity with corticosteroids, has not reduced the high rates of hospitalizations for acute asthma, most often caused by acute viral infection, including influenza. Recently, type 2 innate lymphoid cells (ILC2) expressing c-Kit have been shown to expand during influenza infection and directly caused airway hyper reactivity (AHR), a cardinal feature of asthma. We now show that the treatment of influenza-infected mice with c-Kit kinase inhibitors blocked the development of AHR and airway inflammation and greatly reduced the number and function of ILC2 cells, whereas the treatment with corticosteroids, commonly used to treat acute asthma, failed to prevent the influenza-induced AHR. We also demonstrated that c-Kit function is required for normal ILC2 cell function and for influenza-induced AHR, and that c-Kit kinase inhibitor inhibits murine lung ILC2 cell proliferation and cytokine productions in vitro and in vivo. Furthermore, the treatment of c-Kit inhibitor did not affect in vivo viral clearance. Our study demonstrated the crucial role of c-Kit in modulating the ILC2 activation in the lungs, particularly in respiratory viral infection induced acute asthma.

Discovery of N-(3-((1-Isonicotinoylpiperidin-4-yl)oxy)-4-methylphenyl)-3-(trifluoromethyl)benzamide (CHMFL-KIT-110) as a Selective, Potent, and Orally Available Type II c-KIT Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs).

c-KIT kinase is a validated drug discovery target for gastrointestinal stromal tumors (GISTs). Clinically used c-KIT kinase inhibitors, i.e., Imatinib and Sunitinib, bear other important targets such as ABL or FLT3 kinases. Here we report our discovery of a more selective c-KIT inhibitor, compound 13 (CHMFL-KIT-110), which completely abolished ABL and FLT3 kinase activity. KinomeScan selectivity profiling (468 kinases) of 13 exhibited a high selectivity (S score (1) = 0.01). 13 displayed great antiproliferative efficacy against GISTs cell lines GIST-T1 and GIST-882 (GI50: 0.021 and 0.043 μM, respectively). In the cellular context, it effectively affected c-KIT-mediated signaling pathways and induced apoptosis as well as cell cycle arrest. In addition, 13 possessed acceptable bioavailability (36%) and effectively suppressed the tumor growth in GIST-T1 cell inoculated xenograft model without apparent toxicity. 13 currently is undergoing extensive preclinical evaluation and might be a potential drug candidate for GISTs.

Targeted activation of primitive neural stem cells in the mouse brain.

Primitive neural stem cells (pNSCs) are the earliest NSCs to appear in the developing forebrain. They persist into the adult forebrain where they can generate all cells in the neural lineage and therefore hold great potential for brain regeneration. Thus, pNSCs are an ideal population to target to promote endogenous NSC activation. pNSCs can be isolated from the periventricular region as leukaemia inhibitory factor-responsive cells, and comprise a rare population in the adult mouse brain. We hypothesized that the pup periventricular region gives rise to more clonal pNSC-derived neurospheres but that pup-derived pNSCs are otherwise comparable to adult-derived pNSCs, and can be used to identify selective markers and activators of endogenous pNSCs. We tested the self-renewal ability, differentiation capacity and gene expression profile of pup-derived pNSCs and found them each to be comparable to adult-derived pNSCs, including being GFAP(-) , nestin(mid) , Oct4(+) . Next, we used pup pNSCs to test pharmacological compounds to activate pNSCs to promote endogenous brain repair. We hypothesized that pNSCs could be activated by targeting the cell surface proteins C-Kit and ErbB2, which were enriched in pNSCs relative to definitive NSCs (dNSCs) in an invitro screen. C-Kit and ErbB2 signalling inhibition had distinct effects on pNSCs and dNSCs invitro, and when infused directly into the adult brain invivo. Targeted activation of pNSCs with C-Kit and ErbB2 modulation is a valuable strategy to activate the earliest cell in the neural lineage to contribute to endogenous brain regeneration.

The cKit Inhibitor, Masitinib, Prevents Diabetes-Induced Retinal Vascular Leakage.

Stem cell factor (SCF) has recently demonstrated activity as a novel endothelial permeability factor that contributes to the development of diabetes-induced hyperpermeable retinal vasculature. This study investigated the therapeutic potential of masitinib, a pharmacologic inhibitor of the SCF receptor cKit, for prevention of diabetes-induced breakdown of blood retinal barrier (BRB). Permeability assays were performed with human retinal microvascular endothelial cells (HRMECs) and murine retinal vasculature. Localization of vascular endothelial (VE)-cadherin and activation of SCF signaling pathway was determined by immunofluorescence and Western blotting assays. Mice and rats with streptozotocin (STZ)-induced diabetes were used to investigate the role of cKit and masitinib in diabetes-induced retinal vascular hyperpermeability. Masitinib substantially blocked SCF-induced phosphorylation of cKit in HRMECs. In vitro and in vivo vascular permeability assays showed that masitinib significantly inhibited SCF-induced endothelial hyperpermeability and junctional loss of VE-cadherin. Streptozotocin-induced diabetes was induced in cKit-mutant mice with low cKit expression in their endothelial cells. Although diabetic wild-type mice exhibited enhanced retinal vascular leakage, diabetic cKit-mutant mice showed no increase in retinal vascular leakage or alteration in the distribution of VE-cadherin; this indicates the crucial role of cKit in diabetes-induced breakdown of BRB. Moreover, in vivo prevention experiments showed that an intravitreal injection of masitinib substantially inhibited the development of hyperpermeable retinal vasculature. These results provide the first demonstration that cKit inhibitors, such as masitinib, might be promising therapeutics for prevention of diabetes-induced breakdown of the BRB.