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Acute myeloid leukemia (AML) is a leading blood cancer subtype that can be caused by 27 gene mutations. Previous studies have explored potential vaccine and drug treatments against AML, but many were proven immunologically insignificant. Here, we targeted this issue and applied various clinical filters to improve immune response. KIT is an oncogenic gene that can cause AML when mutated and is predicted to be a promising vaccine target because of its immunogenic responses when activated. We designed a multi-epitope vaccine targeting mutations in the KIT oncogene using CD8+ and CD4+ epitopes. We selected the most viable vaccine epitopes based on thresholds for percentile rank, immunogenicity, antigenicity, half-life, toxicity, IFNγ release, allergenicity, and stability. The efficacy of data was observed through world and regional population coverage of our vaccine design. Then, we obtained epitopes for optimized population coverage from PCOptim-CD, a modified version of our original Java-based program code PCOptim. Using 24 mutations on the KIT gene, 12 CD8+ epitopes and 21 CD4+ epitopes were obtained. The CD8+ dataset had a 98.55% world population coverage, while the CD4+ dataset had a 65.14% world population coverage. There were five CD4+ epitopes that overlapped with the top CD8+ epitopes. Strong binding to murine MHC molecules was found in four CD8+ and six CD4+ epitopes, demonstrating the feasibility of our results in preclinical murine vaccine trials. We then created three-dimensional (3D) models to visualize epitope–MHC complexes and TCR interactions. The final candidate is a non-toxic and non-allergenic multi-epitope vaccine against KIT mutations that cause AML. Further research would involve murine trials of the vaccine candidates on tumor cells causing AML.

G-quadruplexes (GQs) are highly ordered nucleic acid structures that play fundamental roles in regulating gene expression and maintaining genomic stability. GQs are topologically diverse and enriched in promoter sequences of growth regulatory genes and proto-oncogenes, suggesting they may serve as attractive targets for drug design at the level of transcription rather than inhibiting the activity of the protein products of these genes. The c-kit promoter contains three adjacent GQ-forming sequences that have proposed antagonistic effects on gene expression and thus are promising drug targets for diseases like gastrointestinal stromal tumors, mast cell disease, and leukemia. Since GQ stability is influenced by primary structure, secondary structure, and ion interactions, a greater understanding of GQ structure, dynamics, and ion binding properties is needed to develop novel, GQ-targeting therapeutics. Here, we performed molecular dynamics (MD) simulations to systematically study the c-kit2 and c-kit* GQs, evaluating nonpolarizable and polarizable force fields (FFs) and examining the effects of base substitutions and cation type (K(+), Na(+), and Li(+)) on the dynamics of their isolated and linked structures. We found that the Drude polarizable FF outperformed the additive CHARMM36 FF in two-and three-tetrad GQs and solutions of KCl, NaCl, and LiCl. Drude simulations with different cations agreed with the known GQ stabilization preference (K(+) > Na(+) > Li(+)) and illustrated that tetrad core-ion coordination differs as a function of cation type. Finally, we showed that differences in primary and secondary structure influence loop sampling, ion binding, and core-ion energetics of GQs.

Gastrointestinal stromal tumors (GISTs) are a subtype of soft tissue sarcoma (STS), and have become a concept of oncogenic addiction and targeted therapies.The large majority of these tumors develop after a mutation in KIT or platelet derived growth factor receptor α (PDGFRα), resulting in uncontrolled proliferation. GISTs are highly sensitive to imatinib. GISTs are immune infiltrated tumors with a predominance of tumor-associated macrophages (TAMs) and T-cells, including many CD8+ T-cells, whose numbers are prognostic. The genomic expression profile is that of an inhibited Th1 response and the presence of tertiary lymphoid structures and B cell signatures, which are known as predictive to response to ICI. However, the microtumoral environment has immunosuppressive attributes, with immunosuppressive M2 macrophages, overexpression of indoleamine 2,3-dioxygenase (IDO) or PD-L1, and loss of major histocompatibility complex type 1. In addition to inhibiting the KIT oncogene, imatinib appears to act by promoting cytotoxic T-cell activity, interacting with natural killer cells, and inhibiting the expression of PD-L1. Paradoxically, imatinib also appears to induce M2 polarization of macrophages. There have been few immunotherapy trials with anti-CTLA-4 or anti-PD-L1drugs and available clinical data are not very promising. Based on this comprehensive analysis of TME, we believe three immunotherapeutic strategies must be underlined in GIST. First, patients included in clinical trials must be better selected, based on the identified driver mutation (such as PDGFRα D842V mutation), the presence of tertiary lymphoid structures (TLS) or PD-L1 expression. Moreover, innovative immunotherapeutic agents also provide great interest in GIST, and there is a strong rationale for exploring IDO targeting after disease progression during imatinib therapy. Finally and most importantly, there is a strong rationale to combine of c-kit inhibition with immune checkpoint inhibitors.

Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1–3. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation4,5. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers. Gastrointestinal stromal tumours can be initiated by gain-of-function mutations of the KIT or PDGFRA oncogenes but also by loss of the metabolic complex succinate dehydrogenase (SDH), which leads to DNA hypermethylation; this study shows that in SDH-deficient tumours, displacement of CTCF insulators by DNA methylation activates oncogene expression, illustrating how epigenetic alterations can drive oncogenic signalling in the absence of kinase mutations.

Approximately 15% of acral and mucous melanomas carry activating mutations in KIT oncogene. There is a diversity of spectrum of KIT mutations, with some of them rendering tumors responsive to imatinib, while others being imatinib-resistant or not studied yet. Here we present an acral melanoma patient with KIT р.T632I mutation, who failed to respond to imatinib.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

The formation of G-quadruplex (G4) structures in oncogenic G-rich promoter regions are implicated in their biological functions, especially the inhibition of transcription. The binding of cations is thought to contribute to the stabilization of the G4 formation and competition against the duplex formation in the genomic sequence. Furthermore, it might affect the recognition of DNA-binding proteins. Therefore, measuring the interaction between G4 DNA and cations in a free solution environment is critical for evaluating G4 DNA biological functions. However, how binding to cations (K+ and NH4+) affects the folding equilibrium of the G4 structure remains unclear. In this work, a Taylor dispersion analysis (TDA) method using a capillary electrophoresis (CE) instrument was established for the quantitative characterization of the cation-dependent G4 formation in the human c-KIT oncogene promoter region, as well as diffusivities and hydrodynamic radii of DNA variations before and after folding. Our results showed that both K+ and NH4+ can induce the random-coiled c-KIT DNA to unfold and form a more unstretched intermediate state and then fold into tightly structured G4s with smaller size. The G4 size induced by NH4+ was smaller than that induced by K+ ions, though these two cations induced the c-KIT G4 DNA formation with similar binding constants (order of magnitude around 106 M−1). The TDA method can be widely used for rapid structural analyses of trace amounts of DNA mixtures, which effectively differentiate DNA variations or DNA-ligand complex conformations.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Protein kinase A (PKA) regulatory subunit type 1A (PRKAR1A) defects lead to primary pigmented nodular adrenocortical disease (PPNAD). The KIT protooncogene (c-KIT) is not known to be expressed in the normal adrenal cortex (AC). In this study, we investigated the expression of c-KIT and its ligand, stem cell factor (SCF), in PPNAD and other cortisol-producing tumors of the adrenal cortex. mRNA and protein expression, by qRT-PCR, immunohistochemistry (IHC) and immunoblotting (IB), respectively, were studied. We then tested c-KIT and SCF responses to PRKAR1A introduction and PKA stimulation in adrenocortical cell lines CAR47 and H295R, which were also treated with the KIT inhibitor, imatinib mesylate (IM). Mice xenografted with H295R cells were treated with IM. There was increased c-KIT mRNA expression in PPNAD; IHC showed KIT and SCF immunoreactivity within certain nodular areas in PPNAD. IB data was consistent with IHC and mRNA data. PRKAR1A-deficient CAR47 cells expressed c-KIT; this was enhanced by forskolin and lowered by PRKAR1A reintroduction. Knockdown of PKA’s catalytic subunit (PRKACA) by siRNA reduced c-KIT levels. Treatment of the CAR47 cells with IM resulted in reduced cell viability, growth arrest, and apoptosis. Treatment with IM of mice xenografted with H295 cells inhibited further tumor growth. We conclude that c-KIT is expressed in PPNAD, an expression that appears to be dependent on PRKAR1A and/or PKA activity. In a human adrenocortical cell line and its xenografts in mice, c-KIT inhibition decreased growth, suggesting that c-KIT inhibitors may be a reasonable alternative therapy to be tested in PPNAD, when other treatments are not optimal.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that expand in inflammatory conditions including transplantation. MDSCs may be capable of controlling rejection. The critical mechanisms underlying MDSC mediated alloregulation remain unexplored. G-CSF potently stimulates MDSC expansion. We hypothesized that G-CSF-induced MDSCs use a novel mechanism to suppress T cell responses. G-CSF promoted expansion of MDSCs and enhanced their suppressive function against T cell proliferation. Gene expression analysis revealed MDSCs expanded with G-CSF upregulated immune-related genes, but downregulated proliferation-related genes when compared to naïve control MDSCs. The KIT oncogene, encoding the c-Kit (CD117) transmembrane tyrosine kinase receptor, was the most significantly increased in MDSCs expanded with G-CSF. c-Kit inhibition with both imatinib and monoclonal blocking antibody reduced expression of ARG-1, iNOS, PD-L1, and SAA3. Further, imatinib also reduced MDSC-mediated T cell suppression in vitro. Modulation of c-Kit activity may represent a therapeutic target for alloregulatory MDSCs.

Introduction: MHC class I (MHC I) expression is necessary for the antitumoral lymphocyte response but its relationship to oncogene signaling is not well characterized. Gastrointestinal stromal tumor (GIST) is a common sarcoma caused by an activating Kit mutation and is sensitive to the specific Kit inhibitor imatinib. Using KitV558Δ/+ mice, a murine model of GIST, we found imatinib reduced MHC I expression on tumor cells, which we hypothesized was due to attenuation of type I interferon (IFN) signaling in the setting of oncogene inhibition. Methods: Using flow cytometry and Western blot, we studied tumoral MHC I expression in human GIST cell lines T1 and HG129 under conditions of IFN stimulation and imatinib inhibition. We applied the same methods in vivo in KitV558Δ/+ mice. To show that IFN signaling was responsible for tumoral MHC I expression, we used a blocking anti-IFNAR antibody to abrogate IFN signaling in KitV558Δ/+ mice. Finally, we validated our findings with RNA seq of 35 Kit exon 11 human GIST. Results: In human GIST cell lines, IFNα (1000U/mL) stimulated MHC I expression at 24h as measured on cell surface by mean florescence intensity (MFI) and by Western blot. This was accompanied by increased Stat1 and pStat1 protein. Imatinib (100nM) inhibited Stat1 activity and reduced MHC I expression by 12-34% on MFI, with and without IFNα stimulation. When Stat1 was knocked down with SiRNA, imatinib did not further decrease MHC I. In vivo, KitV558Δ/+ mice treated with imatinib had 50% reduction in tumoral MHC I expression MFI by flow (n=5/group). Concurrently, imatinib diminished mRNA expression of IFNb1 in bulk KitV558Δ/+ tumors by 8-fold. To demonstrate that IFN signaling contributed to GIST MHC I expression, we administered a blocking anti-IFNAR antibody in KitV558Δ/+ mice and found MHC I decreased by 34% on MFI, along with decreased pStat1 on western blot. In human GIST, imatinib significantly decreased MHC I gene expression on RNA seq (p = 0.03) and reduced IFNb1 mRNA by 77% (p = 0.04). Furthermore, there was significant correlation between MHC I genes and Stat1 expression (p< 0.0001). Taken together, this suggests that oncogene-dependent IFN signaling is responsible for MHC I expression in GIST. Conclusion: Kit oncogene inhibition reduced MHC I expression in GIST through decreased type I interferon production and signaling via Stat1. Thus, targeted therapies may reduce the antitumor immune response. Citation Format: Mengyuan Liu, Benjamin D Medina, Geraldo A Vitiello, Mark S Etherington, Nesteene J. Param, Mia E DiLolle, Timothy G. Bowler, Ferdinando Rossi, Ronald P. DeMatteo. Kit inhibition decreases tumoral MHC class I expression in gastrointestinal stromal tumors through reduction of type I interferon signaling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B27.

MicroRNAs (miRNAs) have emerged as crucial players in the development and maintenance of disease. miR-155 is an inflammation-associated, oncogenic miRNA, frequently overexpressed in hematological malignancies and solid tumors. However, the mechanism of oncogenesis by miR-155 is not well characterized, and research has focused primarily on individual, direct targets, which does not recapitulate the complexities of cancer. Using a powerful, inducible transgenic mouse model that overexpresses miR-155 and develops miR-155-addicted hematological malignancy, we describe here a multi-step process of oncogenesis by miR-155, which involves cooperation between miR-155, its direct targets, and other oncogenes. miR-155 is known to target DNA-repair proteins, leading to a mutator phenotype, and we find that over 93% of tumors in our miR-155 overexpressing mice contain activating mutations in a single oncogene, c-Kit. Treating mice with dasatinib or imatinib, which target c-Kit, resulted in complete tumor regression, indicating that c-Kit activity is crucial in the oncogenic process. Interestingly, c-Kit expression is high when miR-155 is overexpressed, indicating further cooperation between miR-155 and c-Kit. Our findings support a multi-step model of oncogenesis by miR-155 in which miR-155 promotes both a mutator phenotype and a cellular environment particularly susceptible to mutations in a given oncogene.

BACKGROUND Patients with mucosal and acral melanomas have limited treatment options and a poor prognosis. Mutations of the KIT oncogene in these melanoma subtypes provide a potential therapeutic target. METHODS A multicenter phase 2 trial of sunitinib was conducted in patients with unresectable stage III or IV melanoma of a mucosal or acral primary origin. Patients were treated in 2 cohorts: cohort A received sunitinib at a dose of 50 mg daily for 4 weeks of a 6-week cycle, and cohort B received sunitinib at a dose of 37.5 mg daily on a continuous basis. Dose reductions were permitted for treatment-related toxicities, and tumor assessments were performed every 2 months. RESULTS Fifty-two patients were enrolled: 21 in cohort A and 31 in cohort B. Four patients had confirmed partial responses, which lasted 5 to 10 months (1 with a KIT mutation). In both cohorts, the proportion of patients alive and progression-free at 2 months was 52% (95% confidence interval, 38%-66%); this was significantly larger than the hypothesized null of 5%. There was no significant difference in response or overall survival between the 25% of patients with a KIT mutation and those without one (response rate, 7.7% vs 9.7%; overall survival, 6.4 vs 8.6 months). The overall disease control rate was 44%, and a high rate of toxicity was associated with the treatment. CONCLUSIONS Sunitinib showed activity in the treatment of mucosal and acral melanoma that was not dependent on the presence of a KIT mutation. However, the medication was poorly tolerated, and there were no prolonged responses. Cancer 2015;121:4007–4015. © 2015 American Cancer Society.

Objective As disruption of epigenetic control is a frequent event in solid tumors and leukemia, we investigated changes in DNA methylation (5mC) and hydroxymethylation (5hmC) in patients with systemic mastocytosis (SM), a rare myeloproliferative disease with a wide spectrum of severity, characterized by the accumulation of mast cells in various organs. Methods We measured overall genomic levels of 5hmC and 5mC in patients with SM by dot blot, as well as by quantitative immunofluorescence in samples of cutaneous mastocytosis. Results Overall 5hmC levels were reduced in all patients with SM, but to a greater extent in the presence of higher D816V mutational load in the KIT oncogene, which affects prognosis and therapeutic options in these patients. Loss of 5hmC was likely due to systemic effects of SM as it did not correlate with overall mast cell burden in these patients, nor it was due to inactivating mutations of TET2 or reduced TET2 expression. Conclusions The correlation between SM diagnosis and significantly low 5hmC levels suggests that reduction of 5hmC represents a systemic effect of SM that may be useful for patient stratification and that measurements of 5hmC levels may serve as a better prognostic marker than TET2 mutations.

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7-day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)-related genes, octamer-4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C-KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.

Background Failure to attain peak lung function by early adulthood is a risk factor for chronic lung diseases. Previously, we reported that C3H/HeJ mice have about twice total lung capacity (TLC) compared to JF1/MsJ mice. We identified seven lung function quantitative trait loci (QTL: Lfnq1-Lfnq7) in backcross/intercross mice derived from these inbred strains. We further demonstrated, superoxide dismutase 3, extracellular (Sod3), Kit oncogene (Kit) and secreted phosphoprotein 1 (Spp1) located on these Lfnqs as lung function determinants. Emanating from the concept of early origin of lung disease, we sought to identify novel candidate genes for pulmonary function by investigating lung transcriptome in C3H/HeJ and JF1/MsJ mice at the completion of embryonic development, bulk alveolar formation and maturity. Methods Design-based stereological analysis was performed to study lung structure in C3H/HeJ and JF1/MsJ mice. Microarray was used for lung transcriptomic analysis [embryonic day 18, postnatal days 28, 70]. Quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunohistochemical analysis were used to confirm selected differences. Results Stereological analysis revealed decreased alveolar number density, elastin to collagen ratio and increased mean alveolar volume in C3H/HeJ mice compared to JF1/MsJ. Gene ontology term “extracellular region” was enriched among the decreased JF1/MsJ transcripts. Candidate genes identified using the expression-QTL strategy include: ATP-binding cassette, sub-family G (WHITE), member 1 (Abcg1), formyl peptide receptor 1 (Fpr1), gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1); histocompatibility 2 genes: class II antigen E beta (H2-Eb1), D region locus 1 (H2-D1), and Q region locus 4 (H2-Q4); leucine rich repeat containing 6 (testis) (Lrrc6), radial spoke head 1 homolog (Rsph1), and surfactant associated 2 (Sfta2). Noteworthy genes selected as candidates for their consistent expression include: Wnt inhibitor factor 1 (Wif1), follistatin (Fst), chitinase-like 1 (Chil1), and Chil3. Conclusions Comparison of late embryonic, adolescent and adult lung transcript profiles between mouse strains with extreme TLCs lead to the identification of candidate genes for pulmonary function that has not been reported earlier. Further mechanistic investigations are warranted to elucidate their mode of action in determining lung function. Electronic supplementary material The online version of this article (doi:10.1186/s12931-017-0629-3) contains supplementary material, which is available to authorized users.

Background: The anorectal location of melanomas is extremely rare (1% - 3% of all melanomas), and the prognosis remains poor because of the aggressiveness and the high metastatic potential of those tumors. The discovery that the KIT oncogene may be aberrantly activated in a subset of patients with anorectal melanoma creates a realm of possibility for the development of targeted molecular therapy. Aim: to show the epidemiologic, clinico-radiological, histological features and treatment management especially in patients with over-expression of C-KIT treated by Imatinib. Methods: It is a retrospective study conducted in the department of medical oncology at Hassan II University Hospital between January 2007 and January 2012, including all patients with histologically proven melanoma of the anorectal area. Results: 11 cases were collected, with slight female predominance. Nine patients were metastatic at the moment of diagnosis, and only two with local stage, but evolution was marked by local and distant recurrence less than 12 months after abdo-minoperineal resection. First line of chemotherapy was based mainly on paclitaxel, carboplatine and dacarbazine. Response was modest with only 3 partial responses, 4 patients with disease stability, and 4 patients with disease progression. Two patients, with over expression of C-KIT, received Imatinib as second line of treatment with significant improvement of symptoms and radiological response reaching 50%. Seven patients died with a median survival of 11 months from diagnosis to the date of death. Conclusion: Primary anorectal melanomas are very rare, with high aggressiveness and poor prognosis. Treatment management is still a big challenge given to the modest efficacy of conventional chemotherapy. Better understanding of carcinogenesis and signaling pathways will allow development of new targeted therapies.

We report here on the screening of a fragment library against a G-quadruplex element in the human c-MYC promoter. The ten fragment hits had significant concordance between a biophysical assay, in silico modelling and c-MYC expression inhibition, highlighting the feasibility of applying a fragment-based approach to the targeting of a quadruplex nucleic acid.

Imatinib reduces tumor cell KIT signaling and causes tumor cell apoptosis, which drives TAMs to shift from M1- to M2-like in mouse and human GIST., Tumor-associated macrophages (TAMs) are a major component of the cancer microenvironment. Modulation of TAMs is under intense investigation because they are thought to be nearly always of the M2 subtype, which supports tumor growth. Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and typically results from an activating mutation in the KIT oncogene. Using a spontaneous mouse model of GIST and 57 freshly procured human GISTs, we discovered that TAMs displayed an M1-like phenotype and function at baseline. In both mice and humans, the KIT oncoprotein inhibitor imatinib polarized TAMs to become M2-like, a process which involved TAM interaction with apoptotic tumor cells leading to the induction of CCAAT/enhancer binding protein (C/EBP) transcription factors. In human GISTs that eventually developed resistance to imatinib, TAMs reverted to an M1-like phenotype and had a similar gene expression profile as TAMs from untreated human GISTs. Therefore, TAM polarization depends on tumor cell oncogene activity and has important implications for immunotherapeutic strategies in human cancers.

Acute myeloid leukemia makes up about 30% of all leukemia cases in adults. Mutations in the genes of the receptor tyrosine kinases KIT and FLT3, along with chromosomal translocations, are frequently found in leukemic cells. In the current work, we show that the transgenic B-cells BAF3/FLT3-ITD are significantly more sensitive to cytotoxic action of the ribonuclease binase than original BAF3 cells. BAF3/FLT3-ITD cells differ from BAF3 in expression of the FLT3-ITD oncogene, which results in the alteration of normal signaling pathways. We observed a similar effect previously when studying binase cytotoxic action in cells Kasumi-1 and FDC-P1-N822K, in which the activated oncogene KIT-N822K was expressed. An elevated cytotoxicity of binase to the cells that express the FLT3-ITD oncogene indicates that, as in case of the FDC-P1 cells transduced by the KIT oncogene, the expression of an activated oncogene determines the cell’s sensitivity to the binase action.

Acute myeloid leukemia is the most common acute leukemia affecting adults, and its incidence increases with age. Along with chromosomal translocations in leukemic cells mutations in the genes of receptor tyrosine kinases KIT and FLT3 were found with a high frequency. Here we show that transgenic progenitor of B-cells BAF3/FLT3-ITD are much more sensitive to the ribonuclease binase cytotoxic effects than the original BAF3 cells. The principal difference between BAF3/FLT3-ITD and the original BAF3 cells is the expression of FLT3-ITD oncogene, which leads to a change in the normal cell signaling pathways. Earlier, we described a similar effect for the cytotoxic action of binase on Kasumi-1 and FDC-P1-N822K cells, which express the activated KIT-N822K oncogene. Increased binase cytotoxicity toward the cells, expressing FLT3-ITD oncogene, suggests that, as in the case of FDC-P1 cells, transduced by KIT oncogene, the expression of an activated oncogene determines the sensitivity of cells to binase.

Introduction: Leiomyosarcomas (LMS) are rare malignant neoplasms with smooth muscle differentiation, which occur more commonly in the uterus, digestive tract or retroperitoneum. Before 2000, the mesenchymal gastric tumors were divided in leiomyomas, leiomyoblastomas and leiomyosarcomas. After discovery of the role of KIT oncogene, a distinct entity in the mesenchymal gastrointestinal tumors line has surfaced: GIST (Gastrointestinal Stomal Tumors). The true gastric leiomyosarcoma has become a rare entity. The presentation with brain metastasis had only been reported in the pre-GIST era. Case report: A female, 57 years old, admitted in October 2013, for investigation of abdominal pain, finding a lesion in the left illiac fossa continuing with the terminal ileum. She was submitted to the resection of the lesion with histopathological result showing leiomyosarcoma (6 mitotic figures/10 high power field; IHQ: vimentin positive, S-100 negative, desmin positive). Started chemotherapy. However, presented progression of the disease for subcutaneous tissue, liver (submitted to radiotherapy) and nervous system (only one lesion in the posterior fossa). The patient was submitted in March 7th 2016 to suboccipital craniotomy and en-block resection of the lesion. The microscopic examination revealed a fasicular spindle cell malignant neoplasm, with necrotic areas, with intense and diffuse desmin and smooth muscle actin-positivity in immunohistochemical study, which confirmed the leiomyosarcoma metastatic lesion hypothesis. The patient evolved well during the postoperative period, it was peformed the postoperative MRI, that showed a satisfactory resection. She remained hospitalized until the fourth postoperative day, with no complications, and was then discharged for outpatient follow-up. Conclusion: We have described a case of gastric leiomyosarcoma, emphasizing that even in front of the rarity of leiomyosarcoma itself as a primary lesion (especially after the discovery of GIST as an independent mesenchymal line tumor), this lesion may progress and present central nervous system metastasis, a fact not before reported in literature.

Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell–cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesisin vivoand duringin vitroculture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus–oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived eitherin vivoor byin vitroculture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the samein vivoandin vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin βA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor β receptor 2 (Tgfbr2)) were downregulated relative toin vivofollicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) andInhba) had increased expression relative toin vivocounterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), andNlrp5(Mater)) were expressedin vitroat the same level and with the same pattern asin vivo-derived follicles. These studies establish the similarities and differences betweenin vivoandin vitrocultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality.

Guanine-rich DNA sequences with the ability to form quadruplex structures are enriched in the promoter regions of protein-coding genes, particularly those of proto-oncogenes. G-quadruplexes are structurally polymorphic and their folding topologies can depend on the sample conditions. We report here on a structural study using solution state NMR spectroscopy of a second G-quadruplex-forming motif (c-kit2) that has been recently identified in the promoter region of the c-kit oncogene. In the presence of potassium ions, c-kit2 exists as an ensemble of structures that share the same parallel-stranded propeller-type conformations. Subtle differences in structural dynamics have been identified using hydrogen–deuterium exchange experiments by NMR spectroscopy, suggesting the coexistence of at least two structurally similar but dynamically distinct substates, which undergo slow interconversion on the NMR timescale.

Gastrointestinal stromal tumors (GISTs) and leiomyosarcomas (LMSs) are common types of mesenchymal tumors that may present a diagnostic challenge. GISTs have frequent overexpression of Kit and often encode for mutation of the KIT oncogene. In addition, patients usually experience a favorable response to targeted therapy with imatinib mesylate (Gleevec®, STI-571), but not cytotoxic chemotherapy. Up to 5% of GISTs can be completely negative for Kit on immunohistochemistry. Conversely, LMSs may rarely express Kit, but are virtually never associated with an activating KIT mutation, and are often effectively treated with cytotoxic chemotherapy but are resistant to imatinib. To aid in resolution of this clincal challenge, we performed a whole-genome gene expression study on 71 well-defined GIST and LMS samples with development of a robust, novel approach to molecular classification discussed herein.

Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real-time PCR (qRT-PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0-22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK inhibitors., FCT(SFRH/BI/15257/2004). This work was partially supported by NOVARTIS Oncology, Portugal, and Breakthrough Breast Cancer, UK.

Background Gastrointestinal stromal tumours (GIST) are rare malignancies characterised by their association with KIT oncogene mutations. Until now, population-based reports of the incidence or survival of kit-confirmed GIST have been rare, and none have originated in Southern Europe. Materials and methods We used the Girona Cancer Registry to identify malignant mesenchymal tumours of the digestive tract between 1994 and 2001, and performed c-kit testing in the tumour samples. Age-adjusted incidence rates and survival rates were calculated, and they were also analysed by sex and NIH risk categories. Results Forty-six cases were categorised as GIST. Fifty percent were localised in the stomach, 43.5% in small intestine, 4.3% in the omentum, and 2.2% in colon. Thirty-seven percent were classified as high risk of an aggressive behaviour, 30.4% as intermediate risk and 32.6% as low or very low risk. Only one patient received treatment with imatinib mesilate. The annual incidence by 100,000 inhabitants in crude rate, European age-standardised rate and world age-standardised rate was, respectively, 1.09, 0.90 and 0.65 cases. The relative 5-year survival rate was 74.7% for the entire cohort, and it was markedly lower in the high-risk cases (20.3%). Conclusions We report the first population-based study of GIST incidence and survival in Southern Europe. The incidence rate is low and comparable with that of cancer registries from Northern Europe. Survival was favourable in our pre-imatinib population although it was low in high risk cases. Prognostic discrimination of the cases with intermediate, low, or very low risk is inadequate, and these categories should be considered jointly in the future. Our results will help researchers in establishing baseline values against which they can compare, in the future, the impact of imatinib and other Kit tyrosine inhibitors on survival.

Mucosal melanoma is an exceedingly rare variant of cutaneous melanoma that, due to its rarity, is poorly described and infrequently studied. Primary sites of origin include the head and neck, anorectum and vulvovaginal regions. It is uniquely different from cutaneous melanoma with respect to epidemiology, etiology, pathogenesis and prognosis. The etiology and pathogenesis remain unclear. Unlike cutaneous melanoma, exposure to UV light is not an apparent risk factor. Furthermore, distinct molecular features including a lower incidence of BRAF oncogene mutations but a higher incidence of KIT oncogene mutations suggest divergent genetic etiologies. Mucosal melanomas generally present at a later stage, are more aggressive and carry a worse prognosis regardless of the stage at diagnosis. Establishing standardized treatment guidelines has been challenging due to the rarity of the disease. Early detection provides the best chance at survival but is often difficult due to anatomic location. Surgery remains the primary therapeutic intervention if complete resection is technically feasible given the anatomic location. Radiotherapy may be used to achieve local control when resection is not feasible, or adjuvantly to enhance locoregional control, but most studies have failed to demonstrate an improvement in overall survival. There are no consensus guidelines on the optimal systemic therapy, and regimens are often extrapolated from data based on therapies used to treat advanced cutaneous melanoma. Clinical trials, particularly utilizing newer targeted therapies and immunotherapies, are investigating novel treatment approaches.

CCDC26 on chromosome 8q24 is considered to encode a long intergenic noncoding RNA because the short open reading frame within the mRNA transcribed from this gene is not conserved in any other species. Genome-wide analysis has revealed association of CCDC26 with certain tumors, for instance low-level glioma. Moreover, 1.5- to 2-fold amplifications of the whole or part of the CCDC26 genetic locus have been observed in pediatric acute myeloid leukemia patients. The CCDC26 gene is amplified in the HL-60 acute myeloid leukemia cell line, in which double minute chromosomes—abnormal tiny chromosomes—harbor the CCDC26 gene. We examined the function of CCDC26 by gene knock-down (KD) using short hairpin RNAs in K562 human myeloid leukemia cells. In four stable KD clones, CCDC26 expression was suppressed to 1% of its normal level by transcriptional gene suppression, not post-transcriptional suppression. The growth rates of these KD clones were reduced compared with those of control cells in media containing high serum concentrations. In contrast, in media containing much lower serum concentrations, the KD clones exhibited significantly higher growth rates than controls, and increased survival after serum withdrawal. Enhanced expression of a receptor tyrosine kinase, KIT , was detected in the KD clones, and treatment with ISCK03, a KIT inhibitor, eliminated their increased survival in the absence of serum. Therefore, CCDC26 seems to control myeloid leukemia cell growth through regulation of KIT expression. These observations suggest that CCDC26 is a tumor-suppressive long noncoding RNA because it suppresses the KIT oncogene that supports survival of cancer cells in the stem cell state.

Background/aims Early germ cell development is deranged in undescended testis, potentially leading to infertility and cancer. Androgens are proposed to regulate gonocyte transformation into stem cells during human ‘minipuberty’ at 3–12 months. We studied genes expressed in germ cells, Sertoli cells, and other somatic cells to determine whether androgen mediates gonocyte transformation. Methods Testes from androgen-receptor knockout (ARKO) and wild-type (WT) littermates were collected at postnatal day P0 (birth), P4, P8 for real-time PCR to measure gene expression of mouse VASA homolog (Mvh), anti-Mullerian hormone (Amh), kit oncogene (c-Kit), matrix metalloproteinase-1 (Mt1-mmp), zinc finger, and BTB domain-containing 16 (Plzf) and octamer-binding protein 4 (Oct4). Data were normalized to ribosomal protein L32 (Rpl32), and reproductive homeobox gene 5 (Rhox5) was a positive control for androgenic response. Changes in gene expression were calculated with GraphPad Prism 5.02. Results There were no statistical differences (p > 0.05) in Mvh, Oct4, c-Kit, Plzf, Amh and Mt1-mmp expression between WT and ARKO testes from P0 to P8. Conclusion These results show that androgen did not influence gene expression in postnatal mouse testis, which coincides with human ‘minipuberty’. The results are consistent with gonocyte transformation being independent of androgens, and that nonandrogenic regulators need to be identified.

The c-kit gene encodes a receptor tyrosine kinase, whose engagement by its ligand triggers signals leading to cell proliferation. c-kit activity is elevated in gastrointestinal stromal tumors (GISTs), and its therapeutic inhibition by small molecules such as imatinib is clinically validated. We identified a putative quadruplex forming 21-nucleotide sequence upstream of the c-kit transcription initiation site (c-kit21), on the G-rich strand, which occupies a site required for core promoter activity. Here, we show by nuclear magnetic resonance (NMR), circular dichroism (CD), and ultraviolet (UV) spectroscopic methods that c-kit21 forms quadruplexes under physiological conditions. Mutational analysis of c-kit21 has provided insights into its structural polymorphism. In particular, one mutated form appears to form a single quadruplex species that adopts a parallel conformation. The quadruplex-forming sequence shows a high level of sequence conservation across human, mouse, rat, and chimpanzee. The small variation in sequence between the quadruplex in human/chimpanzee as compared to the rat/mouse was examined more closely by biophysical methods. Despite a variation in the sequence and length of loop 2, the quadruplexes showed both comparable CD spectra, indicative of parallel quadruplexes, and also similar thermal-stability profiles, suggesting conservation of biophysical characteristics. Collectively, the evidence suggests that this quadruplex is a serious target for a detailed functional investigation at the cell-biology level.

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.

PurposeMembers of a family with hereditary gastrointestinal stromal tumors (GISTs) and a germline KIT oncogene mutation were evaluated for other potential syndrome manifestations. A tumor from the proband was analyzed to compare features with sporadic GISTs.Patients and MethodsMembers of a kindred in which six relatives in four consecutive generations comprised an autosomal dominant pattern of documented GISTs and cutaneous lesions underwent physical examination, imaging studies, and germline KIT analysis. A recurrent GIST from the proband was studied using microarray, karyotypic, immunohistochemical, and immunoblotting techniques.ResultsIn addition to evidence of multiple GISTs, lentigines, malignant melanoma, and an angioleiomyoma were identified in relatives. A previously reported gain-of-function missense mutation in KIT exon 11 (T → C) that results in a V559A substitution within the juxtamembrane domain was identified in three family members. The proband's recurrent gastric GIST had a 44,XY−14,−22 karyotype and immunohistochemical evidence of strong diffuse cytoplasmic KIT expression without expression of actin, desmin, or S-100. Immunoblotting showed strong expression of phosphorylated KIT and downstream signaling intermediates (AKT and MAPK) at levels comparable with those reported in sporadic GISTs. cDNA array profiling demonstrated clustering with sporadic GISTs, and expression of GIST markers comparable to sporadic GISTs.ConclusionThese studies provide the first evidence that gene expression and mechanisms of cytogenetic progression and cell signaling are indistinguishable in familial and sporadic GISTs. Current investigations of molecularly targeted therapies in GIST patients provide opportunities to increase the understanding of features of the hereditary syndrome, and risk factors and molecular pathways of the neoplastic phenotypes.

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

Primary mediastinal B-cell lymphoma (PMBL) is a specific subtype of diffuse large B-cell lymphoma with a distinctive clinical course and a specific genetic profile.1,2 Several prior studies have provided key molecular–genetic aberrations;1,3 however, a targeted next-generation sequencing-based study of relevant oncogenes in all currently available PMBL cell lines has not been performed. Here we report previously unrecognized mutations in known hot-spot regions of the KIT oncogene in ~12.5% of PMBL tumors. Interestingly, activating mutations in the proto-oncogene KIT have already been described in a variety of malignancies, including mast cell leukemia, acute myelogenous leukemia, gastrointestinal stromal tumors, as well as several other solid tumors,4 where they carry, in part, therapeutic relevance due to clinically effective inhibitors.5,6

The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively.

Objective: Multiple sclerosis (MS) is a neurodegenerative disorder of the central nervous system (CNS). Damage of the blood-brain barrier integrity is a key pathogenic event leading to the migration of lymphocytes into the CNS and subsequent demyelination. This process is tightly regulated by chemokines and cytokines which are target of therapeutic strategies in MS, such as anti-inflammatory glucocorticosteroid treatment. Here, we examine the effects of dexamethasone-treatment and MS patient sera on the expression of cytokines and chemokines in brain microvascular cell line, cEND in vitro. Methods: We conducted 96-well Mouse Cytokines and Receptors qPCR arrays to quantitatively compare the cytokine and chemokine expression profiles after treatment with dexamethasone. For selected cytokines, we studied the effects of pre-treatment with MS patient sera from active phase of disease (exacerbation) or in relapse (remission) in combination with dexamethasone. Results: After dexamethasone treatment, colony stimulating factor 3 (Csf3) and interleukin 17F (IL17f) were significantly up-regulated, whereas the chemokine (C-C motif) ligand 12 (CCL12/MCP-5), chemokine (C-X-C motif) receptor 3 (CXCR3) and kit oncogene (Kit) were significantly down-regulated. These results were confirmed in qRT-PCR using gene-specific primers. For Csf3 and CCL12 we analyzed the dexamethasone-mediated changes in protein levels secreted into the cell culture medium. Dexamethasone treatment increased the release of Csf3 into the culture medium and decreased the release of CCL12 by cEND. Additionally, we examined the effects of MS-patient sera on dexamethasone-induced cytokine secretion. Pretreatment with MS-patient serum from exacerbation phase augmented dexamethasone effects on Csf3 and CCL12 release in cEND cells. The expression of Csf3- and CCL12-receptors was demonstrated on protein and mRNA level in cEND cells. Conclusion: We identified Csf3 (G-Csf) and CCL12 as cytokines differentially regulated by dexamethasone on mRNA and protein level. This effect was even more pronounced after pretreatment with MS patient serum, especially from patients with acute relapses.

Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3 0 untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loophelix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation. Med1 ,m icroRNA,Mir146, retinoic acid, spermatogonial differentiation

The term gastrointestinal stromal tumor (GIST) in the description of a specific group of gastrointestinal nonepithelial tumors lacking the microscopic evidence of smooth muscle or characteristics of neural immunoreactivity was first introduced by Mazur and Clark (Mazur & Clark, 1983). The common origin of GIST and interstitial cell of Cajal (ICC), the pacemaker cells in the digestive tract, was proposed due to their immunohistochemical and ultrastructural similarities. The definition of GISTs as tumors originating from ICC was further confirmed according to the findings that both GIST and ICC express KIT and that most GISTs have gain-of-function mutations of KIT, the proto-oncogene that encodes a 145 kDa transmembrane tyrosine kinase KIT receptor. Mutation of different exons of the KIT oncogene results in activation of the tyrosine kinase activity of KIT, leading to ligandindependent kinase activity and uncontrolled cell proliferation as well as resistance to apoptosis (Demetri et al., 2002; Hirota et al., 1998; Kindblom et al., 1998; Savage & Antman, 2002). More than 90% of GIST have constitutive activation of the KIT protein as a result of KIT mutation and in GIST without KIT mutations, gain-of-function of platelet derived growth factor receptor α (PDGFRA) are present in about one-third of cases. It was recently proposed that ETV1, one of the ETS family transcription factor, can be a lineage survival factor that cooperates with KIT in gastrointestinal stromal tumours. GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program (Chi et al., 2010; Roberts & Eisenberg, 2002; Rubin et al., 2001). GIST was estimated to occur in about 14.5 cases per million and was the most common mesenchymal tumor of the gastrointestinal tract. The most common locations of GIST are the stomach (50-60%), small intestine (20-30%), colon and rectum (10%), and esophagus (5%). Patients mostly present with nonspecific symptoms and signs (69%) and initial metastasis was noted in about 15-50% of GISTs (DeMatteo et al., 2000; Fletcher et al., 2002; Nilsson et al., 2005; Roberts & Eisenberg, 2002; Shinomura et al., 2005). The accurate diagnosis of GIST should be based on tumor morphology and immunohistochemistry. GIST tumors grossly appear as well-defined submucosal lesion with prominent vasculature and occasional hemorrhage or ulceration (Fig. 1A and 1B.). Under morphologic examination in the immunohistochemical analysis, GIST tumor cells are

A single institution series of 48 mucosal melanomas (MMs) has been analyzed for the presence of KIT mutations using high-resolution melting and sequencing of abnormally melted DNA fragments. The analysis of exons 9, 11, 13, and 17 has revealed eight of 48 (17%) nonsynonymous alterations, including zero of seven head and neck, six of 24 anorectal, one of 15 genitourinary, one of one gastric, and zero of one mediastinal MMs. Seven of these mutations were potentially associated with the tumor sensitivity to KIT tyrosine kinase inhibitors. One tumor harbored somatically acquired silent nucleotide substitution c.1383A>G (T461T). This study adds to the evidence that a substantial portion of MMs carry a therapeutically relevant mutation in the KIT oncogene.

MicroRNAs (miRNAs) are small ∼22nt single stranded RNAs that negatively regulate protein expression by binding to partially complementary sequences in the 3' untranslated region (3' UTRs) of target gene messenger RNAs (mRNA). Recently, mutations have been identified in both miRNAs and target genes that disrupt regulatory relationships, contribute to oncogenesis and serve as biomarkers for cancer risk. KIT, an established oncogene with a multifaceted role in melanogenesis and melanoma pathogenesis, has recently been shown to be upregulated in some melanomas, and is also a target of the miRNA miR-221. Here, we describe a genetic variant in the 3' UTR of the KIT oncogene that correlates with a greater than fourfold increased risk of acral melanoma. This KIT variant results in a mismatch in the seed region of a miR-221 complementary site and reporter data suggests that this mismatch can result in increased expression of the KIT oncogene. Consistent with the hypothesis that this is a functional variant, KIT mRNA and protein levels are both increased in the majority of samples harboring the KIT variant. This work identifies a novel genetic marker for increased heritable risk of melanoma.

The proto-oncogene c-kit encodes a tyrosine kinase receptor related to the PDGF/CSF-1 receptors. Mutations of this gene result in impairment of hematopoiesis, melanogenesis and gametogenesis. Using monoclonal antibodies to the c-kit gene product, we have analyzed its expression in normal and transformed human tissues. Unexpectedly, the receptor was found to be expressed in normal mammary epithelium. While in benign breast lesions, the c-kit gene product was detected at variable levels in 82% of the instances, in primary tumors, no product could be identified in 87% of the cases. This phenotype is maintained in metastatic foci. These findings were confirmed by paired Northern blot analysis of RNA preparations from normal and tumor tissues. These results demonstrate that the c-kit receptor may also be involved in the growth control of mammary epithelium and that this function may be impaired following malignant transformation and de-differentiation.

Gastrointestinal stromal tumors (GISTs) are rare tumors of mesenchymal origin that develop along the gastrointestinal tract. Over ten years ago, their management dramatically changed following the discovery of an activating mutation of the KIT oncogene, which led to the use of small molecule inhibitors to therapeutically target these mutant kinases. Patients with advanced GIST, who were once a subset of sarcoma with poor prognosis due to their lack of chemosensitivity, may now survive more than 5 years following treatment with tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) and sunitinib malate (SU). After ten years of active clinical and preclinical research, it has become clear that, although relatively homogeneous compared to other solid malignancies, there is still some heterogeneity in GISTs and most notably in regard to response to therapy. Here, we review the current data regarding molecular prediction of response in this disease.