Target KIT Research Articles

Comparative transcriptome and miRNA analysis of skin pigmentation during embryonic development of Chinese soft-shelled turtle (Pelodiscus sinensis)

BackgroundAquatic animals show diverse body coloration, and the formation of animal body colour is a complicated process. Increasing evidence has shown that microRNAs (miRNAs) play important regulatory roles in many life processes. The role of miRNAs in pigmentation has been investigated in some species. However, the regulatory patterns of miRNAs in reptile pigmentation remain to be elucidated. In this study, we performed an integrated analysis of miRNA and mRNA expression profiles to explore corresponding regulatory patterns in embryonic body colour formation in the soft-shelled turtle Pelodiscus sinensis.ResultsWe identified 8 866 novel genes and 9 061 mature miRNAs in the skin of Chinese soft-shelled turtles in three embryonic stages (initial period: IP, middle period: MP, final period: FP). A total of 16 563 target genes of the miRNAs were identified. Furthermore, we identified 2 867, 1 840 and 4 290 different expression genes (DEGs) and 227, 158 and 678 different expression miRNAs (DEMs) in IP vs. MP, MP vs. FP, and IP vs. FP, respectively. Among which 72 genes and 25 miRNAs may be related to turtle pigmentation in embryonic development. Further analysis of the novel miRNA families revealed that some novel miRNAs related to pigmentation belong to the miR-7386, miR-138, miR-19 and miR-129 families. Novel_miR_2622 and novel_miR_2173 belong to the miR-19 family and target Kit and Gpnmb, respectively. The quantification of novel_miR_2622 and Kit revealed negative regulation, indicating that novel_miR_2622 may participate in embryonic pigmentation in P. sinensis by negatively regulating the expression of Kit.ConclusionsmiRNA act as master regulators of biological processes by controlling the expression of mRNAs. Considering their importance, the identified miRNAs and their target genes in Chinese soft-shelled turtle might be useful for investigating the molecular processes involved in pigmentation. All the results of this study may aid in the improvement of P. sinensis breeding traits for aquaculture.

Phenotypes of STAT3 gain-of-function variant related to disruptive regulation of CXCL8/STAT3, KIT/STAT3, and IL-2/CD25/Treg axes.

STAT3 is a cytokine-signaling transcription factor critical for gene regulation. Gain-of-function (GOF) mutations in STAT3 are associated with lymphoproliferation, autoimmune cytopenias, increased susceptibility to infection, early-onset solid-organ autoimmunity, short stature, and eczema. We studied the JAK/STAT signaling pathway gene expression and the cytokine profile in two families carrying STAT3-GOF variants to shed light on the STAT3-GOF-associated variable expressivity, including the identification of disease markers. Considering 92 target genes, KIT and IL2RA were downregulated only in patients with high clinical penetrance, while CXCL8 was markedly downregulated for all of them. Unlike previous studies, SOCS3-a STAT3 inhibitor-was not upregulated in patients. In addition, low levels of IL-2 and a reduced numbers of Tregs cells were strikingly prevalent in patients. This study shows a disruptive role of STAT3-GOF variants in the regulatory axis activities CXCL8/STAT3, KIT/STAT3, IL2/CD25/Treg, which, by slightly different mechanisms, underlie the broad clinical spectrum seen in the studied patients. In addition, we suggest the investigation of CXCL8 as a biomarker for identifying STAT3-GOF mutation.

Targeting KIT by frameshifting mRNA transcripts as a therapeutic strategy for aggressive mast cell neoplasms

Activating mutations in c-KIT are associated with the mast cell (MC) clonal disorders cutaneous mastocytosis and systemic mastocytosis and its variants, including aggressive systemic mastocytosis, MC leukemia, and MC sarcoma. Currently, therapies inhibiting KIT signaling are a leading strategy to treat MC proliferative disorders. However, these approaches may have off-target effects, and in some patients, complete remission or improved survival time cannot be achieved. These limitations led us to develop an approach using chemically stable exon skipping oligonucleotides (ESOs) that induce exon skipping of precursor (pre-)mRNA to alter gene splicing and introduce a frameshift into mature KIT mRNA transcripts. The result of this alternate approach results in marked downregulation of KIT expression, diminished KIT signaling, inhibition ofMC proliferation, and rapid induction of apoptosis in neoplastic HMC-1.2 MCs. We demonstrate that invivo administration of KIT targeting ESOs significantly inhibits tumor growth and systemic organ infiltration using both an allograft mastocytosis model and a humanized xenograft MC tumor model. We propose that our innovative approach, which employs well-tolerated, chemically stable oligonucleotides to target KIT expression through unconventional pathways, has potential as a KIT-targeted therapeutic alone, or in combination with agents that target KIT signaling, in the treatment of KIT-associated malignancies.

Open label phase II trial of cabozantinib (cabo) in patients with metastatic castrate resistant prostate cancer (mCRPC) and known amplifications or activating mutations in gene targets who have received prior anti-androgen therapy.

TPS5095 Background: Despite a variety of therapy classes extending survival in mCRPC – and excepting select population eligible for PARP inhibitors – no molecularly selected drugs are FDA approved in mCRPC. Previously, cabo, an inhibitor of multiple tyrosine kinases ( e.g. MET, VEGFRs 1-3, RET, KIT, TRKB, FLT-3, AXL, TIE-2), was evaluated in phase III trials (COMET-1, COMET-2) in mCRPC. Despite initial promising results, particularly in bone scan responses and rPFS benefit, further application of cabo in mCRPC was halted after improvement in OS was not observed. It is unclear why prolonged rPFS in COMET-1 (vs. prednisone) did not translate into improved OS. Previous failures may reflect inclusion of relatively cabo-insensitive tumors due to an unselected population with regard to presumed cabo activity. Given that mCRPC specimens from our precision medicine cohort have increased expression of target genes MET and KIT, and qualifying genomic alterations (amplifications, activating mutations) are reported in ̃15% of a publicly-available mCRPC cohort, we developed this rationally-designed study. We predict a molecularly-defined mCRPC cohort will identify the population that most benefits from cabo therapy, as reflected by prolonged rPFS and OS, and more frequent PSA declines and CTC conversions. Methods: We have activated a phase II non-randomized, open label trial designed to evaluate treatment response and survival of patients with mCRPC who harbor evidence of increased signaling of the targets of cabo. Study population will have progressed on an ARSI; prior taxane therapy in castration-sensitive PC or CRPC (beyond 12 mos) will be eligible. Molecular eligibility: DNA (tumor or cfDNA) evidence of amplification or activating mutation in selected targets of cabo. Alternatively, IHC confirming high expression (2 or 3+) via CLIA-approved assay is allowed. Overexpression via RNAseq, validated by CLIA-approved IHC, is permitted. All patients will receive 40 mg/d of cabo, with dose-reductions allowed (to 20 mg/d, then 20 mg EOD). Repeat biopsy after 3 weeks on treatment is mandated. Primary endpoint is rPFS. Using median of 5.6 mos (COMET-1) to guide our H0 (50% rPFS rate at 6 mo), the H1 is ≥75% rPFS at 6 mo. Sample size (30) provides 90% power with one-sided alpha of 0.05 via chi-square test. Secondary endpoints include PSA decline by PCWG3, objective radiographic response proportion, OS, and CTC response rate. Exploratory studies will include serial evaluation of cfDNA (via PCF-SELECT); immune tumor microenvironment response via on-treatment biopsy and collection of plasma for circulating immune markers; and exploration of baseline and on-treatment tumor genomic alterations. This trial is multicentered via the Prostate Cancer Clinical Trials Consortium (PCCTC c20-254). Clinical trial information: NCT04631744.

The rs17084733 variant in the KIT 3’ UTR disrupts a miR-221/222 binding site in gastrointestinal stromal tumour: a sponge-like mechanism conferring disease susceptibility

ABSTRACTSeveral miRNAs are dysregulated in gastrointestinal stromal tumours (GIST), and miR-221/222 appear to have a prominent role in GIST biology. Therefore, we investigated the role of DNA variants located in miR-221/222 precursor sequences and their target KIT 3'UTR. Ninety-five polymorphisms were analysed in 115 GIST cases and 88 healthy controls. KIT 3'UTR rs17084733 and pri-miR-222 rs75246947 were found significantly associated with GIST susceptibility. Specifically, KIT rs17084733 A allele was more common in GIST, particularly in KIT wild-type (WT) patients (Padj = 0.017). rs17084733 variant is located within one of the three miR-221/222 binding sites in the KIT 3'UTR, resulting in a mismatch in this seed region. Conversely, KIT mRNA levels were lower in patients carrying the variant allele, except for KIT mutant GIST. Luciferase assay data in GIST cells, generated using a construct containing all the three miR-221/222 binding sites, are consistent with KIT mRNA levels in GIST patients. Reporter assay data, generated using a construct containing only the site encompassing rs17084733, confirmed that this is a functional variant disrupting the miR-221/222 binding site. In conclusion, this is the first study investigating the role of SNPs on miR-221/222 precursor sequences and their binding region on KIT 3'UTR in GIST. We identified the KIT variant rs17084733 as a possible novel genetic biomarker for risk of developing KIT-WT GIST. Moreover, our findings suggest the role of one of the three miR-221/222 binding sites on KIT 3'UTR as endogenous sponge, soaking up and subtracting miR-221/222 to the other two sites characterized by a higher affinity.

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

Combination Therapy In Mast Cell Neoplasms: Co-Targeting KIT and NFAT Signaling Pathways

Activating KIT mutations are a hallmark of human systemic mast cell disease and have also been identified in gastrointestinal stromal tumors, AML, melanoma, and seminoma. Traditionally, constitutively active KIT mutations have been targeted by tyrosine kinase inhibitors; however, currently available inhibitors have limited activity against resistant mutations and sustainable responses are often not achieved. Our goal has been to identify a synergistic drug combination that increases the effectiveness of KIT inhibitors. A recent study in chronic myelogenous leukemia reported enhanced anti-proliferative/pro-apoptotic effects when combining a BCR-ABL inhibitor with inhibitors targeting a novel Wnt/Ca++/Calcineurin/NFAT pathway. Based on shared downstream signaling mechanisms between BCR-ABL and KIT kinases we hypothesized that a similar combination would work to target KIT mutant kinases.We found that combining a KIT inhibitor with a calcineurin phosphatase inhibitor led to a synergistic decrease (average combination index (CI) values less than 1.0) in cell viability as well as an increase in apoptosis in KIT-mutant cell lines including six mastocytosis cell lines. For example, we found that 1uM of cyclosporine A (CSA) plus 20nM of dasatinib in the p815 cell line consistently reduced cell viability by 50% compared to dasatinib alone. This decrease in cell viability corresponded to a 40 fold increase in capsase 3/7 activity (Figure 1). Similar results were obtained using the other five mastocytosis cell lines. [Display omitted] The activity of NFAT transcription factors is known to be regulated by the phosphatase activity of calcineurin. To confirm our results were calcineurin- and NFAT-dependent, we repeated the previous combination experiments with the NFAT specific inhibitors, rocaglamide and tributylhexadecylphosphonium bromide (THPB). We observed a synergistic decrease (average CI value 0.81 for rocaglamide and 0.43 for THPB) in cell viability and increase in apoptosis when combining these NFAT-specific and KIT kinase inhibitors, indicating NFAT as an important mediator of the observed synergy. Next we characterized NFAT in KIT mutant mast cells and found constitutive NFAT activation in each cell line, as indicated by nuclear localization and dephosphorylation under unstimulated conditions. NFAT-dependent transcriptional activity, as measured by a luciferase reporter system, decreased following treatment with either calcineurin or NFAT inhibitors. Unexpectedly, NFAT-dependent transcriptional activity also decreased following treatment with a number of KIT inhibitors. However, only calcineurin inhibitors and not KIT inhibitors, completely blocked NFAT activation following calcium influx, suggesting a unique mechanism by which KIT inhibition affects NFAT-dependent transcriptional activity. To determine downstream effectors of the observed synergy, we measured changes in RNA expression following mono- and combination therapy. RNA-Seq revealed synergistic decreases in a number of genes including members of the JAK-STAT and MAPK pathways, which are druggable targets downstream of this transcriptional program. Further validation studies of these targets are currently underway and will be reported later.The constitutive activation of NFAT has not been previously reported in KIT mutant cell lines. Further, our data suggest a point of convergence between KIT and NFAT signaling pathways, possibly through the interaction of NFAT with a DNA binding partner. Importantly, our results indicate that combination therapy using an NFAT inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant mast cells. However, existing calcineurin phosphatase inhibitors such as CSA have known chronic long-term toxicities, including immunosuppression. To identify alternative drug targets we used RNA-Seq and found that simultaneous calcineurin and KIT inhibition modulated members of the JAK-STAT and MAPK pathways. Preliminary data indicates that targeting these pathways in combination with dasatinib achieves the same synergistic effect on KIT mutant cancer cells. We are currently testing the potential of these alternative combination therapies for the treatment of advanced mast cell disease. Disclosures:Heinrich: Onyx: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; MolecularMD: Consultancy, Equity Ownership; AROG: Research Funding.

MiR-17-92 and miR-221/222 cluster members target KIT and ETV1 in human gastrointestinal stromal tumours

Background:Gastrointestinal stromal tumours (GIST) are characterised by high expression of KIT and ETV1, which cooperate in GIST oncogenesis. Our aim was to identify microRNAs that are deregulated in GIST, have a role in GIST pathogenesis, and could potentially be used as therapeutic tool.Methods:Differentially expressed microRNAs between primary GIST (n=50) and gastrointestinal leiomyosarcomas (GI-LMS, n=10) were determined using microarrays. Selected microRNA mimics were transfected into GIST-882 and GIST-T1 cell lines to study the effects of microRNA overexpression on GIST cells. Luciferase reporter assays were used to establish regulation of target genes by selected microRNAs.Results:MiR-17-92 and miR-221/222 cluster members were significantly (P<0.01) lower expressed in GIST vs GI-LMS and normal gastrointestinal control tissues. MiR-17/20a/222 overexpression in GIST cell lines severely inhibited cell proliferation, affected cell cycle progression, induced apoptosis and strongly downregulated protein and – to a lesser extent – mRNA levels of their predicted target genes KIT and ETV1. Luciferase reporter assays confirmed direct regulation of KIT and ETV1 by miR-222 and miR-17/20a, respectively.Conclusion:MicroRNAs that may have an essential role in GIST pathogenesis were identified, in particular miR-17/20a/222 that target KIT and ETV1. Delivering these microRNAs therapeutically could hold great potential for GIST management, especially in imatinib-resistant disease.

Abstract 3281: Combination therapy to target KIT mutant cells.

Abstract Purpose: Gastrointestinal stromal tumors (GIST), the most common abdominal sarcoma, arise from the interstitial cells of Cajal in the wall of the gut. Eighty percent of GISTs harbor activating mutations in the receptor tyrosine kinase KIT. Despite progress in medical treatment of GIST, advanced disease remains incurable. In addition to GIST, activating KIT mutations are found in cases of mast cell neoplasms, AML, melanoma and seminoma. Therefore, there is a compelling need to identify new targets whose inhibition is synergistic when combined with biochemical inhibition of KIT. Based on shared oncogenic kinase mechanisms with CML, we hypothesized that combined inhibition of KIT and a novel Wnt/Ca++/Calcineurin/NFAT pathway would synergistically inhibit the proliferation of KIT mutant cells. Experimental methods: We sequentially combined four different calcineurin inhibitors with five different KIT inhibitors and measured cell proliferation and caspase 3/7activity in nine unique KIT mutant cell lines. Using the Chou and Talalay method we calculated combination indices to quantify drug-drug interactions. Changes in NFAT phosphorylation and localization in KIT mutant cells were assessed by immunoblotting. Finally, we measured NFAT transcriptional activity using an NFAT-responsive reporter cell line. Results: In contrast to other cellular models, NFAT appeared to be constitutively active and localized in the nucleus in many KIT mutant cell lines. We observed synergy when we combined a calcineurin inhibitor with a KIT inhibitor in all KIT mutant cell lines where NFAT was constitutively active. Combination index (CI) values for the different KIT mutant cell lines ranged from 0.3-0.5. To investigate whether NFAT was required for this synergy, we combined NFAT specific inhibitors with KIT inhibitors and found synergistic inhibition of cell proliferation. We created a KIT-mutant reporter cell line where NFAT binding drives the expression of luciferase. As expected, NFAT transcriptional activity decreased when our promoter-reporter cell line was treated with calcineurin inhibitors and increased with TPA and ionomycin treatment. Unexpectedly, we also found that KIT inhibitors decreased NFAT transcriptional activity in this cell model. Conclusions: These data indicate that combination therapy using a calcineurin phosphatase inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant cells. Additionally, the constitutive activation of NFAT in these cell lines indicate aberrant signaling in the Wnt/Ca2+/calcineurin/NFAT pathway, which has not been previously reported in KIT mutant cell lines. The observed synergy between NFAT inhibitors and KIT inhibitors suggests this effect is mediated by NFAT rather than other calcineurin targets. Finally, the fact that KIT inhibitors modulated NFAT transcriptional activity suggests crosstalk between KIT and NFAT signaling pathways. Citation Format: Alison C. Macleod, Michael C. Heinrich, Diana Griffith, Ajia Town. Combination therapy to target KIT mutant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3281. doi:10.1158/1538-7445.AM2013-3281

Expression profiles of microRNAs and their target genes in papillary thyroid carcinoma

The incidence of thyroid cancer has recently experienced a rapid increase in China, and papillary thyroid carcinoma (PTC) accounts for nearly 80% of human thyroid cancers. In the present study, the differential expression of microRNAs (miRNAs) and their target genes were identified in order to analyze the potential roles of miRNAs as biomarkers and in papillary thyroid carcinogenesis. One hundred and twenty-six PTC samples were collected from patients at the China-Japan Union Hospital, China, and the gene/miRNA expression profiles were examined with Illumina BeadChips and verified by real‑time RT-PCR. Gene Ontology (GO) categories were determined, and pathway analysis was carried out using KEGG. miRNA target genes were predicted by implementing three computational analysis programs: TargetScanS, DIANA-microT and PicTar. Two hundred and forty-eight miRNAs and 3,631 genes were found to be significantly deregulated (gene, P<0.05; miRNA, P<0.01) in PTC tissues when compared with their matching normal thyroid tissues. hsa-miR-206 (target gene, MET), hsa-miR-299-3p (target gene, ITGAV), hsa-miR-101 (target gene, ITGA3), hsa-miR-103 (target gene, ITGA2), hsa-miR‑222 (target genes, KIT and AXIN2), hsa-miR-15a (target genes, AXIN2 and FOXO1) and hsa-mir-221 (target gene, KIT) were identified. Together with the functions of the target genes, we further elucidated the role of miRNAs in papillary thyroid carcinogenesis and suggest the use of miRNAs as biomarkers for early diagnosis. Our findings provide the basis for future studies in the field of miRNA-based cancer therapy.

Imatinib as a Treatment Option for Systemic Non-Langerhans Cell Histiocytoses

Systemic non-Langerhans cell histiocytoses are disorders characterized by the accumulation of histiocytes that do not meet the criteria for Langerhans cells in various organs. So far, no causative treatment is known. Herein, we report the case of a 41-year-old man with Rosai-Dorfman disease, a form of systemic non-Langerhans cell histiocytoses, with histiocytic infiltrations in the skin, bone marrow, liver, and spleen. Histiocytes were positive for the imatinib target proteins platelet-derived growth factor receptor beta and KIT. The disease completely responded to treatment with 400 to 600 mg daily of imatinib for more than 7 months. This case shows that imatinib is a powerful treatment option for patients with non-Langerhans cell histiocytoses.

PDGFRA germline mutation in a family with multiple cases of gastrointestinal stromal tumor

Familial gastrointestinal stromal tumor (GIST) is a rare autosomal dominant genetic disorder associated with KIT germline mutations. In sporadic forms of the disease, somatic mutations target either KIT or PDGFRA genes. In a kindred in which 5 individuals had GIST, no germline mutation in KIT coding sequence has been detected. We hypothesized that the PDGFRA gene could be a predisposing gene in familial GIST. We sequenced PDGFRA exons 12 and 18 because several somatic mutations were identified within this region. We detected a germline PDGFRA missense mutation, 2675G > T, resulting in a tyrosine substitution for the highly conserved aspartic acid at codon 846. This mutation showed perfect cosegregation with the GIST phenotype among the 7 family members tested. Interestingly, PDGFRA Asp846 is homologous to codon 820, which is located in the KIT tyrosine kinase II domain. In a previous study, a KIT germline Asp820Tyr mutation was detected in a Japanese kindred in which 6 individuals had GIST. Transfection of a KIT820Tyr complementary DNA in nude mice was found to be tumorigenic confirming the oncogenic potential of this mutation. The present study shows that PDGFRA is a second familial GIST predisposing gene. These results indicate a further example of involvement of structurally related genes in familial cancer syndromes.

Three highly sensitive "bedside" serum and urine tests for pregnancy compared

We examined three enzyme-linked immunosorbent assay (ELISA) kits for human choriogonadotropin (hCG) (pregnancy tests) for use with urine and serum samples: the Tandem Icon II hCG Urine and Tandem Icon II hCG Serum, the NovoClone Target hCG Test, and the Abbott TestPacks hCG-urine and hCG-serum. Paired comparison of the results from each kit indicated that the NovoClone Target assay showed significantly lower diagnostic sensitivity (P less than 0.05) than did the Tandem Icon II or Abbott TestPack, both for urine and for serum samples. None of the products demonstrated any significant difference (P greater than 0.05) in diagnostic specificity, but the NovoClone Target kit showed several serious false-negative results with both urine and serum. Paired testing of urine kits vs serum kits also showed no significant differences (P greater than 0.05) in diagnostic sensitivity or specificity. We found the Abbott kits to be the most convenient to use and to read.