c-Kit Signaling Research Articles

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

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

Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia

BackgroundArteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c-Kit in arteriogenesis. MethodsMutant mice with loss of c-Kit function (KitW/W-v), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre-, post-, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post-ischemia while collaterals remodeling were measured 28 days post-ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c-Kit expression on vascular recovery. ResultsKitW/W-v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. KitW/W-v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow-derived cells, we investigated which source of c-Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. KitW/W-v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted KitW/W-v mice. ConclusionThis study provides evidence that c-Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c-Kit signaling because rescue of systemic c-Kit activity by bone marrow transplantation did not augment the functional recovery of KitW/W-v mouse hindlimbs.

MiR-19b-3p induces cell proliferation and reduces heterochromatin-mediated senescence through PLZF in goat male germline stem cells.

Promyelocytic leukemia zinc finger PLZF, known as ZBTB16 or ZFP145 is a critical zinc finger protein of male germline stem cells (mGSCs), it's an essential transcriptional factor for goat testis development and spermatogenesis. Loss of PLZF results in progressive depletion of SSCs after the first wave of spermatogenesis leading to eventual spermatogenic arrest, apparently the result of a shift in the balance in SSC fate away from self-renewal and toward differentiation. Cumulating evidences have demonstrated that microRNAs are expressed in a cell-specific or stage-specific manner during spermatogenesis and acts as regulators on specific makers such as Stra8, ETV5, and PLZF. However, the post transcriptional function of PLZF still poorly elucidate in mGSCs. Bioinformatic analysis and dual luciferase reporter assay showed that miR-19b-3p binds the 3'UTR of PLZF, suggesting that PLZF is a direct target of miR-19b-3p. The profile of miR-19b-3p and PLZF analyzed in dairy goat testis at different age showed that miR-19b-3p was significantly up-regulated in goat testis at 1, 3, 6 months and downregulated at 12, 18, and 24 months which was inversely correlated with PLZF in the same testis. Focusing on the role of miR-19b-3p, we found that miR-19b-3p changes c-KIT and mTOR signaling through PLZF to promote proliferation in goat nGSCs and infertile mice testes. Over-expression of PLZF significantly reversed miR-19b-3p-mediated proliferation in mice testes. We found also that miR-19b-3p reduced heterochromatin-mediated senescence through PLZF localized on HP1α. Taken together, our findings indicate that miR-19b-3p promotes proliferation and reduces heterochromatin-mediated senescence through PLZF in mGSCs.

Kit W-sh Mutation Prevents Cancellous Bone Loss during Calcium Deprivation.

Calcium is essential for normal bone growth and development. Inadequate calcium intake increases the risk of osteoporosis and fractures. Kit ligand/c-Kit signaling plays an important role in regulating bone homeostasis. Mice with c-Kit mutations are osteopenic. The present study aimed to investigate whether impairment of or reduction in c-Kit signaling affects bone turnover during calcium deprivation. Three-week-old male WBB6F1/J-Kit W /Kit W-v /J (W/W v ) mice with c-Kit point mutation, Kit W-sh /HNihrJaeBsmJ (W sh /W sh ) mice with an inversion mutation in the regulatory elements upstream of the c-Kit promoter region, and their wild-type controls (WT) were fed either a normal (0.6% calcium) or a low calcium diet (0.02% calcium) for 3weeks. μCT analysis indicated that both mutants fed normal calcium diet had significantly decreased cortical thickness and cancellous bone volume compared to WT. The low calcium diet resulted in a comparable reduction in cortical bone volume and cortical thickness in the W/W v and W sh /W sh mice, and their corresponding controls. As expected, the low calcium diet induced cancellous bone loss in the W/W v mice. In contrast, W sh /W sh cancellous bone did not respond to this diet. This c-Kit mutation prevented cancellous bone loss by antagonizing the low calcium diet-induced increase in osteoblast and osteoclast numbers in the W sh /W sh mice. Gene expression profiling showed that calcium deficiency increased Osx, Ocn, Alp, type I collagen, c-Fms, M-CSF, and RANKL/OPG mRNA expression in controls; however, the W sh mutation suppressed these effects. Our findings indicate that although calcium restriction increased bone turnover, leading to osteopenia, the decreased c-Kit expression levels in the W sh /W sh mice prevented the low calcium diet-induced increase in cancellous bone turnover and bone loss but not the cortical bone loss.

Activation of non-canonical cKIT signalling in erythroid progenitor cells from polycythemia vera

Activation of non-canonical cKIT signalling in erythroid progenitor cells from polycythemia vera

Alterations of oncogenes expression in NK cells in patients with cancer.

IntroductionC‐kit/SCF signaling plays a key role in regulating NK cell homeostasis, maturation, proliferation, and cytotoxicity. C‐kit‐deficiency in NK cells results in significant reduction of their number, suggesting an imperative role for c‐kit signaling in NK cell biology. We have recently showed that human NK cells express not only c‐kit‐receptor, but also both membrane‐bound and soluble forms of c‐kit ligand—Stem cell factor. The goal of this study was to characterize the c‐kit/SCF autocrine loop in peripheral blood NK cells obtained from patients with cancer.MethodsUsing Smart Flare and qRT‐PCR, we have characterized expression of c‐kit and two forms of SCF in patients’ NK cells and correlated these results with the expression of c‐myc and STAT3.ResultsOur results demonstrated that the expression of proto‐oncogenes c‐myc and c‐kit was significantly decreased in NK cells from all cancer patients. Expression of membrane‐bound SCF in NK cells correlated with the presence of remote metastases.ConclusionsWe suggest that the abnormal signaling and expression of c‐kit/SCF, c‐myc, and STAT3 in NK cells is responsible for the defect in their cytolytic activity in cancer and these defects at the gene expression level may be the cause rather than the result of tumor progression.

Abstract 463: MicroRNA-7 suppresses RB1 expression leading to chromosomal instability in leukemia cells harboring c-Kit mutation

Abstract C-Kit mutation D816V is a well-known indicator of poor prognosis in acute myeloid leukemia (AML) harboring t(8;21) chromosomal translocations. However, the mechanism the D816V mutation promotes therapeutic resistance is still under investigation. C-Kit V814 mutation is a murine counterpart of the human D816V. We utilized a murine IL-3 dependent cell line Ba/F3 with or without the c-Kit V814 mutation to investigate its downstream signaling pathway and the effect on the expression of bcl-2 family members and critical granulocytic transcription factors. The c-Kit V814 and wild type (WT) c-Kit were retrovirally transduced into Ba/F3 cells. Unexpectedly, Gata1 mRNA was significantly downregulated to one-tenth in V814+Ba/F3 cells (p < 0.01), contrary to the previous reports that WT c-Kit signaling induces Gata1 expression. When V814+Ba/F3 cells were treated with several c-Kit signaling pathway inhibitors, mRNA and protein expression levels of Gata1 recovered to normal levels only with MEK 1/2 inhibitor (PD325901) treatment. We then hypothesized that miRNAs might control Gata1 transcription. We used an array to identify differences in miRNA expression between Ba/F3 cells and V814+Ba/F3 cells, and between V814+Ba/F3 cells treated with or without PD325901. Only miR-7a-5p and miR-706 were significantly upregulated in V814+Ba/F3 cells compared with Ba/F3 without the V814 mutation, and downregulated (> 2-fold) in cells treated with PD325901 compared with controls. We focused our further analysis on miR-7 as it is highly conserved in vertebrates. The differences in miR-7 expression were confirmed using qRT-PCR (10.5-fold change, p < 0.01). In silico analysis implicated Rb1 as a candidate miR-7 target that may regulate Gata1 transcription. Luciferase reporter constructs containing murine and human Rb1 miR-7 target sequence exhibited 0.51 and 0.49 times lower luciferase activity than the controls, respectively (p < 0.01). To further determine the biological significance of the c-Kit mutation that would transform the cells to more aggressive phenotype, we focused on DNA and chromosomal instability caused by RB1 deterioration. We exposed WT c-Kit Ba/F3 and V814+Ba/F3 cells to 4 Gy irradiation and then used immunohistochemistry to analyze the frequency of γH2AX foci in the cells. The percentage of cells with more than 10 γH2AX foci was significantly higher in V814+Ba/F3 cells than WT Ba/F3 cells (27% vs. 14%, p < 0.05), Here, we clearly show that a single mutation of c-Kit is sufficient to regulate miR-7 expression leading to RB1 translational suppression. Since c-Kit mutation requires only a single step to inhibit RB1, this mechanism is more likely to facilitate oncogenesis than bi-allelic RB1 chromosomal and/or gene alterations in leukemia cells. Taken together, we identified miR-7 as a suppressor of RB1 in V814 mutation-positive cells, which might reflect the responsibility of D816V to refractory feature in AML. Citation Format: Keiji Kurata, Shinichiro Kawamoto, Ryota Masutani, Kimikazu Yakushijin, Katsuya Yamamoto, Hiroshi Matsuoka, Takayuki Takubo, Hironobu Minami. MicroRNA-7 suppresses RB1 expression leading to chromosomal instability in leukemia cells harboring c-Kit mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2017-463

The stem cell factor (SCF)/c-KIT signalling in testis and prostate cancer.

The stem cell factor (SCF) is a cytokine that specifically binds the tyrosine kinase receptor c-KIT. The SCF/c-KIT interaction leads to receptor dimerization, activation of kinase activity and initiation of several signal transduction pathways that control cell proliferation, apoptosis, differentiation and migration in several tissues. The activity of SCF/c-KIT system is linked with the phosphatidylinositol 3-kinase (PI3-K), the Src, the Janus kinase/signal transducers and activators of transcription (JAK/STAT), the phospholipase-C (PLC-γ) and the mitogen-activated protein kinase (MAPK) pathways. Moreover, it has been reported that cancer cases display an overactivation of c-KIT due to the presence of gain-of-function mutations or receptor overexpression, which renders c-KIT a tempting target for cancer treatment. In the case of male cancers the most documented activated pathways are the PI3-K and Src, both enhancing abnormal cell proliferation. It is also known that the Src activity in prostate cancer cases depends on the presence of tr-KIT, the cytoplasmic truncated variant of c-KIT that is specifically expressed in tumour tissues and, thus, a very interesting target for drug development. The present review provides an overview of the signalling pathways activated by SCF/c-KIT and discusses the potential application of c-KIT inhibitors for treatment of testicular and prostatic cancers.

MTOR, VEGF, PDGFR, and c-kit signaling pathway activation in Kaposi sarcoma

Kaposi sarcoma (KS) is a locally progressive, intermediate-grade vascular neoplasm with no known cure, high recurrence rates, and potential for wide dissemination. Low efficacy and high toxicity limit current therapeutic options for advanced disease. Activation of mammalian target of rapamycin (mTOR), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and c-kit signaling pathways has been implicated in KS pathogenesis and may suggest a role for targeted inhibitors. KS cases were retrospectively retrieved (N=274), most (90%) associated with human immunodeficiency virus. Tissue microarray slides were stained with human herpes virus-8, Friend leukemia integration 1 transcription factor, CD117 (c-kit), phospho-S6 (pS6), PDGF receptor-β, VEGF, and phospho-mTOR. Both intensity and extent of staining were scored. Multiplying these scores for each core yielded total staining H-scores. Human herpes virus-8 was positive in 87% and Friend leukemia integration 1 transcription factor in 95.7% of cases. Most were also VEGF+ (97.6%), pS6+ (95.7%), CD117+ (92.5%), and PDGFRB+ (87.4%). Approximately half (55.6%) were phospho-mTOR+. There was no significant difference in staining among patients with low (<500 cells/mm3) or preserved CD4 T-cell counts. Immunohistochemistry confirms upregulation of the mTOR, PDGF, VEGF, and c-kit pathways in a large cohort of KS samples. Of proteins tested, pS6, downstream of mTOR, demonstrated the highest proportion of strong positivity (67.1%). These results support the possibility of using targeted inhibitors in KS. Overexpression was independent of CD4 count, suggesting that even patients with low counts may be targeted therapy candidates.

Identifying the niche controlling melanocyte differentiation.

Melanocytes present in hair follicles are responsible for their pigmentation. Melanocyte differentiation and hair pigmentation depend on the stem cell factor (SCF)/c-Kit signaling pathway, but the niche that regulates melanocyte differentiation is not well characterized. In this issue of Genes & Development, Liao and colleagues (pp. 744-756) identify Krox20+-derived cells of the hair shaft as the niche and the essential source of SCF required for melanocyte maturation. This study delineates the niche factors regulating melanocyte differentiation and hair pigmentation and opens up new avenues to further characterize the cross-talk between the hair follicle and melanocytes that controls melanocyte maintenance and differentiation.

A Chemically Inducible Helper Module for Detecting Protein-Protein Interactions with Tunable Sensitivity Based on KIPPIS.

As protein-protein interactions (PPIs) play essential roles in regulating their functional consequences in cells, methods to detect PPIs in living cells are desired for correct understanding of intracellular PPIs and pharmaceutical development therefrom. Here we demonstrate a c-kit-based PPI screening (KIPPIS) system in combination with a chemically inducible helper module for detecting PPIs in living mammalian cells. In this system, a mutant of FK506-binding protein 12 (FKBPF36V) is fused with a protein of interest and the intracellular domain of a receptor tyrosine kinase c-kit. Constitutive expression of two fusion proteins with interacting proteins of interest in interleukin-3 (IL-3)-dependent cells results in dimerization and subsequent activation of the c-kit intracellular domains, which allows cell proliferation in a culture medium devoid of IL-3. A helper ligand, a small synthetic chemical that homodimerizes FKBPF36V, assists the formation of stable complexes of the fusion proteins and serves as a tuner for sensitivity of the system. Using this system, two model PPIs were successfully detected on the basis of cell proliferation, which was featured by the helper-ligand- and PPI-dependent phosphorylation of the Src family kinases, a hallmark of the c-kit signaling. Notably, the inclusion of the helper module enabled PPI detection with tunable sensitivity. The helper-assisted KIPPIS allows us to configure various affinity thresholds by changing the concentration of the helper ligand, which may be applied to select affinity-matured variants using the advantage of cell proliferation.

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling

Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered invivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells invitro and invivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Orai1 mediates tumor-promoting store-operated Ca2+ entry in human gastrointestinal stromal tumors via c-KIT and the extracellular signal-regulated kinase pathway.

Gastrointestinal stromal tumors originate from interstitial cells of Cajal, the pacemaker cells of the gut. Ca2+ regulates the pacemaker activity of interstitial cells of Cajal. Store-operated Ca2+ entry mediates the majority of Ca2+ entry in most cancer cells and may be a factor in regulating intracellular Ca2+ in interstitial cells of Cajal and gastrointestinal stromal tumors. Therefore, a blockade of this mechanism may affect the progression of gastrointestinal stromal tumors. Orai1 is the pore subunit of store-operated Ca2+ channels. Here, we reported that Orai1 was overexpressed in gastrointestinal stromal tumor tissues and was positively correlated with a high-risk grade in gastrointestinal stromal tumor patients. Furthermore, upon Orai1 silencing, the functional store-operated Ca2+ entry in gastrointestinal stromal tumor cells was decreased, indicating that the function of store-operated Ca2+ entry was mediated by Orai1. Inhibition of Orai1-mediated store-operated Ca2+ entry by Orai1 silencing or store-operated Ca2+ entry blockers (SKF-96365 and 2-aminoethyl diphenylborate) induced obvious cell proliferation suppression, cell-cycle distribution, and apoptosis stimulation in GIST-T1 cells. Conversely, Orai1 overexpression increased store-operated Ca2+ entry and cell proliferation in GIST882 cells. In addition, we found that activation of c-KIT and the extracellular signal-regulated kinase pathway participated in the oncogenic functions of Orai1-mediated store-operated Ca2+ entry in gastrointestinal stromal tumor cells. These results revealed that Orai1-mediated store-operated Ca2+ entry is critical for gastrointestinal stromal tumor cell proliferation via c-KIT and ERK signaling pathway activation. Orai1-mediated store-operated Ca2+ entry plays an oncogenic role and may be a novel prognostic factor and therapeutic target for patients with gastrointestinal stromal tumors.

A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis

T helper 9 (Th9) cells contribute to lung inflammation and allergy as sources of interleukin-9 (IL-9). However, the mechanisms by which IL-9/Th9 mediate immunopathology in the lung are unknown. Here we report an IL-9-driven positive feedback loop that reinforces allergic inflammation. We show that IL-9 increases IL-2 production by mast cells, which leads to expansion of CD25+ type 2 innate lymphoid cells (ILC2) and subsequent activation of Th9 cells. Blocking IL-9 or inhibiting CD117 (c-Kit) signalling counteracts the pathogenic effect of the described IL-9-mast cell-IL-2 signalling axis. Overproduction of IL-9 is observed in expectorates from cystic fibrosis (CF) patients, and a sex-specific variant of IL-9 is predictive of allergic reactions in female patients. Our results suggest that blocking IL-9 may be a therapeutic strategy to ameliorate inflammation associated with microbial colonization in the lung, and offers a plausible explanation for gender differences in clinical outcomes of patients with CF.

A Review of the Aetiopathogenesis and Clinical and Histopathological Features of Oral Mucosal Melanoma.

Oral mucosal melanoma is an uncommon, usually heavily melanin-pigmented, but occasionally amelanotic aggressive tumour with a poor prognosis. Despite radical surgery, radiotherapy, or chemotherapy, local recurrence and distant metastasis are frequent. Microscopical examination is essential for diagnosis, and routine histological staining must be supplemented by immunohistochemical studies. The aetiology is unknown, the pathogenesis is poorly understood, and the 5-year survival rate rarely exceeds 30%. In most cases, oral mucosal melanoma arises from epithelial melanocytes in the basal layer of the epithelium and less frequently from immature melanocytes arrested in the lamina propria. In both cases the melanocytes undergo malignant transformation, invade deeper tissues, and metastasize to regional lymph nodes and to distant sites. Very rarely metastasis from skin melanoma may give rise to oral mucosal melanoma that may be mistaken for primary oral mucosal melanoma. The pathogenesis of oral mucosal melanoma is complex involving multiple interactions between cytogenetic factors including dysregulation of the cKit signalling pathways, cell cycle, apoptosis, and cell-to-cell interactions on the one hand and melanin itself, melanin intermediates, and local microenvironmental agents regulating melanogenesis on the other hand. The detailed mechanisms that initiate the malignant transformation of oral melanocytes and thereafter sustain and promote the process of melanomagenesis are unknown.

Phosphoproteomic Landscaping Unveils Constitutive cKIT Activation in Human Erythroblasts from Polycythemia Vera (PV) Patients

PV is characterized by the gain-of-function V617F mutation in JAK2, the gene encoding the first signaling element of the cytokine receptor superfamily. Progenitor cells from PV are more sensitive in vitro to Imatinib, which also inhibits the SCF receptor cKIT, than those from normal sources (Gaikwad, Exp Hemat 2007;35:931) and clinical trials with similar tyrosine kinase inhibitors have been reported to have some efficacy in PV (Nussenzveig, Int J Hematol 2009;90:58; Silver, Leuk Res 2012;36:156). These data led us to examine the mechanism by which this tyrosine kinase inhibitor affects erythropoiesis in PV.We observed that in cultures containing SCF PV progenitor cells generated similar numbers of erythroid cells (Ery) as those from adult (AB) and cord (CB) blood by day 10 [fold increase (FI) ~1-2] but by day 13 PV cells generated significantly greater numbers of Ery than AB and CB [PV FI=11±0.2, p=0.021 vs AB; CB FI=6.2±1.9, p=0.025 vs PV and 0.0055 vs AB; AB FI=2.6±0.5]. Since by day 10 progenitor cells were no longer detected, we hypothesized that increases in Ery at day 13 reflect intrinsically greater Ery proliferation potentials. To test this hypothesis, we compared the phosphoproteomic landscaping of day 10 Ery from 3 PV, 3 AB and 3 CB by Reverse Phase Protein Array (RPPA) using as target >160 signaling events (data are at http://capmm.gmu.edu/data). Overall, 40 proteins were statistically different between PV and AB and 30 proteins were statistically different between CB and AB. Pathway analyses of significant hits identified that PV and CB Ery differ from the AB ones in the activation states of 1-2 proteins involved in stemness and cell cycle control inferring that there is no major change in their cycling or differentiation state. By contrast, the 3 populations showed numerous differences in cKIT signaling. PV Ery differed from AB cells by expressing greater levels of cKITY719 and cKIT703, which were reduced to barely detectable levels by the pan-JAK inhibitor Ruxolitinib, and of elements of PI3K (eNOS/NosIII, PDK1 and PKCd) and MAPK (pMARCKS, MSK1, AMPKα1 and β1 and p38 MAPK) signaling downstream, respectively, to cKITY719 and cKIT703. PV Ery expressed also greater levels of JAK2Y1007/1008 and of its downstream target STAT3Y705. CB Ery showed lower levels of cKIT, cKITY703, cKITY719 and CD63, a member of tetraspanin superfamily that binds cKITY719 switching its intracellular fate from recycling to lysosome degradation, greater phosphorylation of proteins of MAPK (pMARCKS, MSK1, PTEN and Src) and PI3K (PKCd, mTOR, p70S6K and panPKC/βII) signaling than AB Ery. These results were stoichiometrically validated by WB and indicate that PV Ery express greater degrees of cKIT activation than AB Ery suggesting that greater response to SCF might account for their greater amplification in culture. This hypothesis was tested by RPPA analyses (and stoichiometric validation by WB) of Ery from PV, AB and CB growth factor deprived (GFD) for 4h and then stimulated with SCF for 15’ and 2h. GFD altered the activation state of 25 proteins (22 de-activated and 3 activated) in PV, of 12 proteins (10 de-activated and 2 activated) in AB and 8 proteins (4 de-activated and 4 activated) in CB. SCF altered the activation state of 36 proteins in PV (18 activated and 18 de-activated), 23 proteins in CB (all activations) and 6 proteins in AB (all activations). In PV and CB Ery, GFD decreased cKITY719 and cKITY703 and the activation state of their downstream targets JAK2Y1007/1008, MAPKs and mTOR while SCF increased the stoichiometric levels of cKITY719 and cKITY703 and the activation of mTOR. SCF also increased cKITY703 and cKITY719 but did not activate mTOR in Ery from AB. In agreement with the hypothesis that Ery from PV and CB respond more readily to SCF than those from AB, SCF induced greater cell-surface cKIT down-modulation (by flow cytometry) and lower intra-cytoplasmic cKIT/CD63 association (by confocal microscopy and WB) in PV and CB Ery than in AB Ery. Screening of 97 inhibitors against targets analysed by RPPA which are approved for clinical use by FDA revealed that growth of PV Ery was more sensitive than that of AB only to JAK and cKIT inhibitors. In addition, shRNA-CD63 reduced the growth of PV Ery (FI=0.9 vs 1.3 p=0.012) while increased by 2-fold (p=0.02) that of AB Ery. These results provide the first phosphoproteomic landscaping of cKIT signalling in Ery from PV and normal sources and confirm that cKIT is an important therapeutic target for PV. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mast cells promote melanoma colonization of lungs.

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre− R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre− R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre− R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

Suppression of c-Kit signaling induces adult neurogenesis in the mouse intestine after myenteric plexus ablation with benzalkonium chloride.

Adult neurogenesis rarely occurs in the enteric nervous system (ENS). In this study, we demonstrated that, after intestinal myenteric plexus (MP) ablation with benzalkonium chloride (BAC), adult neurogenesis in the ENS was significantly induced in c-kit loss-of-function mutant mice (W/Wv). Almost all neurons and fibers in the MP disappeared after BAC treatment. However, 1 week after ablation, substantial penetration of nerve fibers from the non-damaged area was observed in the MP, longitudinal muscle and subserosal layers in both wildtype and W/Wv mice. Two weeks after BAC treatment, in addition to the penetrating fibers, a substantial number of ectopic neurons appeared in the subserosal and longitudinal muscle layers of W/Wv mice, whereas only a few ectopic neurons appeared in wildtype mice. Such ectopic neurons expressed either excitatory or inhibitory intrinsic motor neuron markers and formed ganglion-like structures, including glial cells, synaptic vesicles and basal lamina. Furthermore, oral administration of imatinib, an inhibitor of c-Kit and an anticancer agent for gastrointestinal stromal tumors, markedly induced appearance of ectopic neurons after BAC treatment, even in wildtype mice. These results suggest that adult neurogenesis in the ENS is negatively regulated by c-Kit signaling in vivo.

Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.

c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-KitW/W-v mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-KitW-sh/W-sh (Wsh/Wsh) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that Wsh/Wsh mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit Wsh mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in Wsh/Wshosteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in Wsh/Wsh osteoclasts. Conditioned medium from Wsh/Wsh osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.

Abstract 3336: ST6Gal-I glycosyltransferase promotes an undifferentiated cell phenotype and enhances c-kit signaling

Abstract Acute myelogenous leukemia (AML) is characterized by an increased number of myeloid cells in the bone marrow. These cells are arrested in their maturation process, frequently resulting in hematopoietic insufficiency. Recent advancements in molecular genetics have identified new markers that may be useful in diagnosing AML, including mutant c-kit (CD117). c-kit is a receptor tyrosine kinase and activating mutations in this receptor have been identified as a functional driver of AML and other myeloproliferative diseases. c-kit also plays a major role in maintaining normal stem cell pools. Prior studies suggest that ST6Gal-I, a unique glycosyltransferase upregulated in many cancers, promotes a stem-like cell phenotype in epithelial tumor cells. ST6Gal-I is a sialyltransferase that adds a negatively charged sialic acid in an α2-6 linkage to membrane glycoproteins. In this study, we show c-kit to be a substrate of ST6Gal-I, with increased sialylation correlating to increased c-kit activation. In order to study the effects of sialylation on signaling, we forced constitutive expression of ST6Gal-I in the U937 monocytic leukemia cell line. c-kit signaling was then induced by treating U937 cells with stem cell factor (SCF), the c-kit ligand. Here we show enhanced SCF-mediated c-kit activation, indicated by increased levels of phospho-c-kit in ST6Gal-I overexpressing cells. This, in turn, is associated with increased activation of downstream effecters of c-kit signaling, phospho-Erk and phospho-Akt. We further hypothesize that ST6Gal-I might play a role in the cell differentiation process. To test the effects of sialylation on differentiation, we used a well-established model to induce monocyte cell differentiation, treatment with the phorbol ester, PMA. PMA is known to induce macrophage differentiation in U937 cells, evidenced by exit from the cell cycle and upregulation of differentiation markers, including CD11b. Macrophage differentiation induces downregulation of endogenous ST6Gal-I, thereby reducing surface sialylation of specific receptors. However, forced overexpression of ST6Gal-I inhibits PMA-induced exit from the cell cycle, suggesting that ST6Gal-I acts to maintain cells in an undifferentiated state. To further understand the effects of receptor sialylation on PMA-induced differentiation, we used flow cytometric analysis to evaluate surface markers on U937 cells with or without ST6Gal-I overexpression. We show that forced expression of ST6Gal-I inhibits PMA-induced upregulation of CD11b, whereas c-kit expression is elevated in ST6Gal-I overexpressing cells relative to parental cells. These results suggest a novel mechanism by which ST6Gal-I contributes to an undifferentiated state in part through the regulation of the c-kit receptor. Citation Format: Kaitlyn A. Dorsett, Susan L. Bellis. ST6Gal-I glycosyltransferase promotes an undifferentiated cell phenotype and enhances c-kit signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3336.