c-Kit Signaling Research Articles

Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown.To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors.To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. DisclosuresNo relevant conflicts of interest to declare.

Mast Cells Modulate the Immune Response and Redox Status of the Gastrointestinal Tract in Induced Venom Pathogenesis

The pathogenesis of Androctonus autralis hector (Aah) scorpion venom involved cellular and molecular mechanisms resulting in multi-organ dysfunction. However, little is reported about the effects of venom on the gastrointestinal axis. Mast cells (MCs) are known to play a crucial role in modulating immune response of the gut. This study aims to investigate the involvement of this cell type in venom-induced gastric and intestinal disorders in a time course (3 and 24h). The obtained results revealed that Aah scorpion venom induced inflammatory cell infiltration as shown by the increase of the myeloperoxidase and eosinophil peroxidase activities. Overexpression of the c-kit receptor (CD117) severely imbalanced the redox status with depletion of antioxidant systemic accompanied by gastrointestinal tissue damage. Moreover, an increased level of lactate dehydrogenase in the serum was correlated with tissue injuries. Pharmacological inhibition of MCs targeting tyrosine kinase (TK) reduces the generation of reactive oxygen species and normalizes catalase, and gluthation S-transferase activities to their physiological levels. In addition, histopathological alterations were restored after pretreatment with c-kit receptor inhibitor associated with a considerable reduction of MC density. Interestingly, obtained results indicate that MCs might be involved in gastric modulation and intestinal inflammation through c-kit signaling following sub-cutaneous Aah venom injection.

The Effect of Shaoyao Gancao Decoction on Sphincter of Oddi Dysfunction in Hypercholesterolemic Rabbits via Protecting the Enteric Nervous System-Interstitial Cells of Cajal-Smooth Muscle Cells Network.

ObjectiveThis study observes the morphological changes in the enteric nervous system (ENS) – interstitial cells of Cajal (ICC) – smooth muscle cells (SMC) network in sphincter of Oddi dysfunction (SOD) in hypercholesterolemic rabbits following treatment with Shaoyao Gancao decoction (SGD), as well as the apoptosis of the ICC.MethodsIn this study, 48 healthy adult New Zealand rabbits are randomly divided into three groups (n = 16 in each group): the control, the model, and the SGD treatment groups. The hypercholesterolemic rabbit model is established. Hematoxylin and eosin staining, transmission electron microscopy, immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, immunohistochemistry, Western blot analysis, and reverse transcription-polymerase chain reaction are used to detect the morphological changes in the ENS–ICC–SMC network, the expression of apoptosis-related proteins in the ICC, and to observe the curative effect of SGD after treatment.ResultsCompared with the control group, the morphology and the ultrastructure of the SO are destroyed in the model group. In addition, the protein gene product 9.5 (PGP9.5), nitric oxide (NO), the SMCs, and the ICC all significantly decreased while substance P (SP) significantly increased. Compared with the model group, the SO morphology and ultrastructure are repaired in the SGD group. In addition, the PGP9.5, NO, the SMCs, and the ICC significantly increased while SP decreased. In addition, SGD may activate the stem cell factor (SCF)/c-Kit signaling pathway to treat SO dysfunction by up-regulating the expression of c-Kit and SCF. Similarly, this pathway restores SO by up-regulating the expression of Bcl2 and inhibiting cleaved caspase-3, Bax, and the tumor necrosis factor.ConclusionShaoyao Gancao decoction can promote the recovery of sphincter of Oddi dysfunction in hypercholesterolemic rabbits by protecting the ENS–ICC–SMC network.

Therapeutic effect of Cistanche deserticola on defecation in senile constipation rat model through stem cell factor/C-kit signaling pathway.

BACKGROUNDConstipation is one of the chronic gastrointestinal functional diseases. It seriously affects the quality of life. Cistanche deserticola (C. deserticola) can treat constipation obviously, but its mechanism has not been clarified. We supposed that mechanism of it improved the intestinal motility by stimulating interstitial Cajal cells (ICC). Activation of the C-kit receptor on the surface of ICC is closely related to ICC function, and the stem cell factor (SCF)/C-kit signaling pathways plays an important role on it. To investigate the mechanism of how C. deserticola treats constipation, this study aimed to establish a constipation model in rats and explore the role of SCF/C-kit signaling pathway in the treatment. AIMTo explore the SCF/C-kit signaling pathways in the role of C. deserticola for treatment of constipation by a constipation rat model.METHODSForty-eight 8-mo-old Sprague–Dawley rats were divided into 4 groups by random weight method: Normal group (n = 12), model group (n = 12), C. deserticola group (n = 12) and blocker group (n = 12). The normal group received normal saline by gavage; the model group received loperamide by gavage; the blocker group received loperamide and C. deserticola by gavage, and STI571 was injected by intraperitoneally. During treatment, the weight, fecal granules and fecal quality were recorded every 10 d. On day 20 after model induction, the colon tissues of each group were removed. Hematoxylin and eosin staining was used to observe pathological changes. Expression levels of SCF, C-kit and Aquaporin genes were detected by immunohistochemistry, western blotting, and real-time-quantitative polymerase chain reaction. The colonic epithelial mitochondria and goblet cells were observed by transmission electron microscopy.RESULTSCompared with the normal group, as treatment progressed, the weight of rats in the model and blocker groups decreased significantly, the stool weight decreased, and the stool quality was dry (P < 0.05). C. deserticola reversed the decrease in body weight and stool weight and improved stool quality. Histopathological analysis indicated that the colonic mucosal epithelium in the model group was incomplete, and the arrangement of the glands was irregular or damaged. Treatment with C. deserticola improved the integrity and continuity of the epithelial cells and regular arrangement of the glands. The blocking agents inhibited the effects of C. deserticola Immunohistochemistry and real-time-quantitative polymerase chain reaction showed that expression of SCF and C-kit protein or genes in the colonic tissue of the model group was decreased (P < 0.05), while treatment with C. deserticola increased protein or gene expression (P < 0.05). Western blotting showed that expression of aquaporin APQ3 was increased, while the expression of Cx43 decreased in the model group. Treatment with C. deserticola inhibited expression of APQ3 and promoted expression of Cx43. Transmission electron microscopy showed that the mitochondria of the colonic epithelium in the model group were swollen and arranged disorderly, and microvilli were sparse. The condition was better in the C. deserticola group. Mice treated with STI571 blocker confirmed that blocking the SCF/C-kit pathway inhibited the improvement of constipation by C. deserticola.CONCLUSIONC. deserticola improved defecation in rats with constipation, and the SCF/C-kit signaling pathway, which is a key link of ICC function, played an important role of the treatment.

A Fully Human Monoclonal Antibody Targeting cKIT Is a Potent Inhibitor of Pathological Choroidal Neovascularization in Mice.

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

Mangiferin mitigates di-(2-ethylhexyl) phthalate-induced testicular injury in rats by modulating oxidative stress-mediated signals, inflammatory cascades, apoptotic pathways, and steroidogenesis

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor that causes reproductive defects in male animal models. This study was conducted to explore the plausible modulatory effects of mangiferin (MF) against DEHP-induced testicular injury in rats. Thirty-two adult male albino rats were allocated into four groups. Two groups were given DEHP (2 g/kg/day, p.o) for 14 days. One of these groups was treated with MF (20 mg/kg/day, i.p) for 7 days before and 14 days after DEHP administration. A vehicle-treated control was included, and another group of rats was given MF only. Results revealed that MF treatment suppressed oxidative testicular injury by amplifying the mRNA expression of nuclear factor-erythroid 2 related factor-2 (Nrf2) and increasing hemoxygenase-1 (HO-1), glutathione, and total antioxidant capacity (TAC) levels. This treatment also enhanced superoxide dismutase activity, but it decreased malondialdehyde and nitric oxide levels. MF had an anti-inflammatory characteristic, as demonstrated by the downregulation of the mRNA of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The content of tumor necrosis factor-alpha also decreased. MF modulated the apoptotic pathway by suppressing the mRNA of cytochrome c (Cyt c), Fas ligand content, Bax IHC expression, caspase-3 activity and cleaved caspase-3 IHC expression. It also upregulated the expression levels of heat-shock protein 70 (HSP70) and B-cell lymphoma 2. Moreover, MF upregulated the mRNA expression levels of HSP70 and c-kit and enriched the content of steroidogenic acute regulatory (StAR) protein, which were reflected in serum testosterone levels. This result indicated that MF played crucial roles in steroidogenesis and spermatogenesis. Besides, the activities of testicular marker enzymes, namely, acid and alkaline phosphatases, and lactate dehydrogenase, significantly increased. Histopathological observations provided evidence supporting the biochemical and molecular measurements. In conclusion, MF provided protective mechanisms against the DEHP-mediated deterioration of testicular functions partially through its antioxidant, anti-inflammatory, and anti-apoptotic properties. It also involved the restoration of steroidogenesis and spermatogenesis through the modulation of Nrf2/HO-1, NF-κB/Cyt c/HSP70, and c-Kit signaling cascades.

Molecular Determinants for Polycomb Group Protein YY1 Control of Hematopoietic Stem Cell Quiescence

Hematopoietic stem cells (HSCs) are undifferentiated, long-lived cells that give rise to lineage-specific progenitors and retain their stem cell identity by undergoing self-renewal. Adult HSCs can remain in a quiescent state for a prolonged time, and quiescence is a fundamental characteristic of adult bone marrow-resident HSCs. A precisely regulated cell cycle is required for HSC-mediated generation of mature hematopoietic cells, while preventing stem cell exhaustion. Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group Protein (PcG) with important functions to regulate embryonic development, lineage differentiation and cell proliferation. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that catalyze histone modifications. Many questions remain unanswered regarding how cell- and tissue-specific regulation is achieved by PcG proteins. Herein, we conditionally deleted Yy1 in mouse hematopoietic cells (Yy1f/f Mx1-Cre) and demonstrated that Yy1 deficiency decreases hematopoietic stem cells (HSCs) long-term repopulating activity. Moreever, ectopic YY1 expression expands HSCs. Yy1-/- mice were pancytopenic with a significantly shorter median survival time compared with wild type mice. YY1 deficiency deregulated the genetic network governing HSC proliferation and impaired Stem Cell Factor (SCF) /c-Kit signaling, thus disrupting mechanisms conferring HSC quiescence. As our prior study demonstrated that YY1 PcG function is required for Igκ chain rearrangement in early B cell development, we evaluated the role of YY1 PcG function in HSC self-renewal and addressed whether the requirement for YY1 PcG function is context-dependent in adult hematopoiesis. Ectopic expression of a YY1 mutant lacking the PcG function (YY1ΔREPO) in Yy1-/- mice rescued SCF/c-Kit signaling, HSC quiescence and maintained the capacity to stimulate HSC self-renewal. Thus, YY1 regulates adult hematopoiesis in both PcG-dependent and independent manners. These results reveal a unique mechanism for how a ubiquitously expressed transcriptional repressor mediates lineage-specific functions to control adult hematopoiesis. Currently, we are testing how YY1 differentially controls HSCs and B cells via PcG-independent and -dependent mechanisms respectively. DisclosuresNo relevant conflicts of interest to declare.

Germline and Somatic Genetic Variants in the p53 Pathway Interact to Affect Cancer Risk, Progression, and Drug Response.

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

SCF Improves In Vitro Differentiation of SSCs Through Transcriptionally Up-regulating PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 Genes.

Several lines of evidence strongly suggest that retinoic acid (RA) and stem cell factor (SCF)/c-Kit signal transduction pathways are involved in the differentiation of spermatogonial stem cells (SSCs). This study was aimed to investigate the effect of RA and SCF on in vitro differentiation of SSCs via evaluation of the mRNA expression of meiosis-specific genes in cultured testicular tissues. Testicular tissue samples were obtained from bilaterally vasectomized rats and also healthy adult rats and then were cultured for 25, 30, and 35days on different conditions. The cultured testicular pieces were sectioned and stained with PAS to histological analysis. The total RNA was extracted from cultured testicular samples, and the expression of ACR, PRTM1, SYCP3, STRA8, c-KIT, PIWIL2, and OCT4 genes at mRNA level was quantified using real-time polymerase chain reaction (qPCR) procedure. After 1-month surgery, bilateral testicular weight showed a significant decrease in vasectomized adult rats compared with healthy adult rats (P < 0.05). Reduction in the diameter of the seminiferous tubules and depletion of advanced germinal elements in vasectomized rats compared with healthy adult rats were also observed. Our findings also demonstrated that the mRNA expression level of PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes in cultured testicular tissues significantly up-regulated in experimental group II compared with the control group (P < 0.001). Our findings lead us to conclude that SCF improves in vitro differentiation of SSCs in the OA rats, at least partially, by transcriptionally upregulating PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes.

LY3214996 relieves acquired resistance to sorafenib in hepatocellular carcinoma cells.

Background: Sorafenib, an oral multi-kinase inhibitor of rapidly accelerated fibrosarcoma; vascular endothelial growth factor receptor-2/3, platelet-derived growth factor receptor, c-Kit, and Flt-3 signaling, is approved for treatment of advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib is often diminished because of acquired resistance through the reactivation of ERK signaling in sorafenib-resistant HCC cells. In this work, we investigated whether adding LY3214996, a selective ERK1/2 inhibitor, to sorafenib would increase the anti-tumor effectiveness of sorafenib to HCC cells.Methods: The Huh7 cell line was used as a cell model for treatment with sorafenib, LY3214996, and their combination. Phosphorylation of the key kinases in the Ras/Raf/MAPK and PI3K/Akt pathways, protein expression of the cell cycle, and apoptosis migration were assessed with western blot. MTT and colony-formation assays were used to evaluate cell proliferation. Wound-healing assay was used to assess cell migration. Cell cycle and apoptosis analyses were conducted with flow cytometry.Results: LY3214996 decreased phosphorylation of the Ras/Raf/MAPK and PI3K/Akt pathways, including p-c-Raf, p-P90RSK, p-S6K and p-eIF4EBP1 activated by sorafenib, despite increased p-ERK1/2 levels. LY3214996 increased the anti-proliferation, anti-migration, cell-cycle progression, and pro-apoptotic effects of sorafenib on Huh7R cells.Conclusions: Reactivation of ERK1/2 appears to be a molecular mechanism of acquired resistance of HCC to sorafenib. LY3214996 combined with sorafenib enhanced the anti-tumor effects of sorafenib in HCC. These findings form a theoretical basis for trial of LY3214996 combined with sorafenib as second-line treatment of sorafenib-resistant in advanced HCC.

C-KIT-ERK1/2 signaling activated ELK1 and upregulated carcinoembryonic antigen expression to promote colorectal cancer progression.

Carcinoembryonic antigen (CEA) is highly expressed in embryo and colorectal cancer (CRC) and has been widely used as a marker for CRC. Emerging evidence has demonstrated that elevated CEA levels promote CRC progression. However, the mechanism of the increased CEA expression in patients with primary and recurrent CRC is still an open question. In this study, we showed that c‐KIT, ELK1, and CEA were hyperexpressed in patients with CRC, especially patients with recurrent disease. From bioinformatics analysis, we picked ELK1 as a candidate transcription factor (TF) for CEA; the binding site of ELK1 within the CEA promoter was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays. Overexpression of ELK1 increased CEA expression in vitro, while knockdown of ELK1 decreased CEA. Upregulated ELK1 promoted the adhesion, migration, and invasion of CRC cells, however knockdown of CEA blocked the activities of ELK1‐overexpressed CRC cells. Furthermore, we explored the role of c‐KIT‐ERK1/2 signaling in activation of ELK1. Blocking c‐KIT signaling using Imatinib or ISCK03 reduced p‐ELK1 expression and consequently decreased CEA levels in CRC cells, as did blocking the ERK1/2 pathway by U0126. Compared with wild type littermates, the c‐kit loss‐of‐functional Wadsm/m mice showed lowered c‐KIT, ELK1, and CEA expression. In conclusion, our study revealed that ELK1, which was activated by c‐KIT‐ERK1/2 signaling, was a key TF for CEA expression. Blocking ELK1 or its upstream signaling could be an alternative way to decelerate CRC progression. Besides being a biomarker for CRC, CEA could be used for guiding targeted therapy.

The Samd14-Capping Protein Complex Controls Cell Signaling in the Erythropoietic Stress Response

In anemia, restoring homeostatic levels of erythrocytes requires an erythropoietic regenerative response to accelerate red blood cell (RBC) production. Elucidating mechanisms that drive the process of erythropoiesis in the context of regeneration or "stress erythropoiesis" can reveal new strategies for targeting ineffective erythropoiesis. An important component of stress erythropoiesis involves stress-dependent activation of genes/proteins through transcriptional enhancers. We discovered an enhancer in intron 1 of the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene elevates Samd14 expression, facilitates SCF/c-Kit signaling, and is needed for survival in a hemolytic anemia model. However, it is dispensable for erythropoietic development. Our prior work demonstrated that the SAM domain of Samd14 promotes c-Kit-mediated cellular signaling to regulate progenitor function, and this SAM domain has functional attributes unique from those of structurally related SAM domains. Using immunoprecipitation-mass spectrometry, we determined that Samd14 interacts with the Capzβ protein. Capzβ is a component of the actin capping protein (CP) complex, which interact as α- and β heterodimers during actin filament assembly/disassembly. CP exerts diverse functions in cell motility, vesicular transport, cell signaling and cytokinesis. Using a series of Samd14 deletion constructs, we tested whether the Samd14-Capzβ interaction is important for Samd14 promotion of c-Kit signaling in stress erythroid progenitors. Our findings determined that the region of Samd14 required for binding to Capzβ (amino acids 38-54) were required to restore c-Kit signaling. Our ongoing studies are examining whether Samd14-Capzβ is similarly required for colony formation and cell survival of stress erythroid progenitors, and whether additional SAM domain-containing proteins have a similar role in regulating stress erythropoiesis. Understanding the fundamental drivers of regenerative erythropoiesis can lead to new therapeutic strategies and prognostic/diagnostic markers. Disclosures No relevant conflicts of interest to declare.

Expression differences of miR-142-5p between treatment-na\xefve chronic myeloid leukemia patients responding and non-responding to imatinib therapy suggest a link to oncogenic ABL2, SRI, cKIT and MCL1 signaling pathways critical for development of therapy resistance

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Although the introduction of tyrosine kinase inhibitors (TKIs) has substantially improved patients’ prognosis, drug resistance remains one of the major challenges in CML therapy. MicroRNAs (miRNAs), a class of short non-coding RNAs acting as post-transcriptional regulators, are implicated in CML progression and drug resistance. The aim of the present study was to analyze the miRNA expression profiles of 45 treatment-naïve CML patients in chronic phase (28 peripheral blood and 17 bone marrow samples) with respect to future response to imatinib therapy.MethodsTaqMan low density arrays were used to analyze the miRNA expression pattern of the patient samples. For selected microRNAs, reporter gene assays were performed to study their ability to regulate CML associated target genes.ResultsSignificant lower expression levels of miR-142-5p were identified in both, peripheral blood and bone marrow samples of future non-responders suggesting a potential tumor suppressor role of this miRNA. This was supported by reporter gene assays that identified the survival, proliferation and invasion promoting CML related genes ABL2, cKIT, MCL1 and SRI as targets of miR-142-5p and miR-365a-3p, the latter identified as potential biomarker in peripheral blood samples.ConclusionMiR-142-5p and to a certain extend also miR-365a-3p were able to discriminate treatment-naïve CML patients not responding to imatinib in the course of their treatment from patients, who responded to therapy. However, further large-scale studies should clarify if the identified miRNAs have the potential as predictive biomarkers for TKI resistance.

Pressure overload by suprarenal aortic constriction in mice leads to left ventricular hypertrophy without c-Kit expression in cardiomyocytes

Animal models of pressure overload are valuable for understanding hypertensive heart disease. We characterised a surgical model of pressure overload-induced hypertrophy in C57BL/6J mice produced by suprarenal aortic constriction (SAC). Compared to sham controls, at one week post-SAC systolic blood pressure was significantly elevated and left ventricular (LV) hypertrophy was evident by a 50% increase in the LV weight-to-tibia length ratio due to cardiomyocyte hypertrophy. As a result, LV end-diastolic wall thickness-to-chamber radius (h/R) ratio increased, consistent with the development of concentric hypertrophy. LV wall thickening was not sufficient to normalise LV wall stress, which also increased, resulting in LV systolic dysfunction with reductions in ejection fraction and fractional shortening, but no evidence of heart failure. Pathological LV remodelling was evident by the re-expression of fetal genes and coronary artery perivascular fibrosis, with ischaemia indicated by enhanced cardiomyocyte Hif1a expression. The expression of stem cell factor receptor, c-Kit, was low basally in cardiomyocytes and did not change following the development of robust hypertrophy, suggesting there is no role for cardiomyocyte c-Kit signalling in pathological LV remodelling following pressure overload.

Abstract 3765: c-Kit mediated phosphorylation of Spred1 regulates Neurofibromin-Spred1 interaction

Abstract Spred1 negatively regulates Ras/MAPK signaling following growth factor stimulation. Spred1 inhibits Ras by binding and localizing Neurofibromin, a RasGAP, to the plasma membrane to accelerate Ras GTPase activity. c-Kit, a receptor tyrosine kinase (RTK), is known to interact with Spred1 but the consequence of this interaction is unknown. Here we demonstrate that c-Kit signaling regulates Neurofibromin-Spred1 interaction. Stimulation with c-Kit ligand, SCF, results in a transient disruption in Neurofibromin-Spred1 binding which corresponds to increased Ras signaling, followed by restoration of Neurofibromin-Spred binding which corresponds to nearly basal levels of Ras signaling. Mass spectrometry analysis identified potential phosphorylation sites on Spred1 that correspond to the initial disruption and later restoration of Neurofibromin-Spred1 binding. Phosphomimetic and phosphodeficient mutants affect the interaction. Our findings provide a potential mechanism by which RTK signaling regulates negative feedback to allow transient activation and subsequent termination of Ras signaling. Citation Format: Claire Lorenzo, Lucy C. Young, Alexandra Tannka, Anatoly Urisman, Frank McCormick. c-Kit mediated phosphorylation of Spred1 regulates Neurofibromin-Spred1 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3765.

SCF/c-kit signaling pathway participates in ICC damage in neurogenic bladder

ABSTRACT Neurogenic bladder (NB) is a type of double renal dysfunction caused by nerve lesions. The interstitial cells of Cajal (ICC) damage are involved in bladder dysfunction. The aim of this study is to investigate the effect of stem cell factor (SCF)/c-kit signaling pathway on ICC damage in NB model rats. Maximum cystometric capacity (MCC), bladder leak point pressures (BLPP), and bladder compliance (BC) were measured in sham-operated and NB model rats. Immunofluorescent staining for c-kit was performed to determine ICC count in rat bladder trigone. The morphology and ultrastructure changes of ICCs were observed under an electron microscope. The mRNA levels of c-kit and SCF in bladder tissues were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein levels of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in bladder tissues were determined by western blot. ICC proliferation was detected by CCK-8 assay. NB resulted in changes in ultrastructure changes of ICCs and a decrease in the number of ICCs and in expression of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in NB tissues. Inhibition of SCF/c-kit signaling pathway suppressed ICC proliferation by inhibiting JAK/STAT3 pathway. Moreover, inhibition of SCF/c-kit signaling pathway impaired the SCF-induced attenuation of ICC damage in NB model rats. Collectively, our data indicate that SCF/c-kit signaling pathway participates in ICC damage in NB.

Sterile α-motif domain requirement for cellular signaling and survival

Hundreds of sterile α-motif (SAM) domains have predicted structural similarities and are reported to bind proteins, lipids, or RNAs. However, the majority of these domains have not been analyzed functionally. Previously, we demonstrated that a SAM domain-containing protein, SAMD14, promotes SCF/proto-oncogene c-Kit (c-Kit) signaling, erythroid progenitor function, and erythrocyte regeneration. Deletion of a Samd14 enhancer (Samd14–Enh), occupied by GATA2 and SCL/TAL1 transcription factors, reduces SAMD14 expression in bone marrow and spleen and is lethal in a hemolytic anemia mouse model. To rigorously establish whether Samd14–Enh deletion reduces anemia-dependent c-Kit signaling by lowering SAMD14 levels, we developed a genetic rescue assay in murine Samd14–Enh−/− primary erythroid precursor cells. SAMD14 expression at endogenous levels rescued c-Kit signaling. The conserved SAM domain was required for SAMD14 to increase colony-forming activity, c-Kit signaling, and progenitor survival. To elucidate the molecular determinants of SAM domain function in SAMD14, we substituted its SAM domain with distinct SAM domains predicted to be structurally similar. The chimeras were less effective than SAMD14 itself in rescuing signaling, survival, and colony-forming activities. Thus, the SAMD14 SAM domain has attributes that are distinct from other SAM domains and underlie SAMD14 function as a regulator of cellular signaling and erythrocyte regeneration.

Tyrosine kinase inhibitors modulate dendritic cell activity via confining c-Kit signaling and tryptophan metabolism

Dendritic cell (DC)-based vaccine has been established in tumor immunotherapy. Importantly, the efficiency of anti-tumor T-cells in draining lymph nodes is dependent on the status of DCs surrounding in tumors. It has been shown that Indoleamine 2,3-dioxygenase (IDO) plays a key role to induce tolerogenic DCs in tumor microenvironment, and tyrosine kinase inhibitors (TKIs) can suppress the function of IDO in DCs. However, the stimulatory effect of TKI-modified DCs on T cells remains unclear. In this report, we found that one type of TKI-dasatinib can modify DCs to increasing the activation of allogenic T cells. These TKI-modified DCs delayed the onset of B16 melanoma progression in mice. In mechanistic studies, TKIs did not increase the maturation but reduce the expression and phosphorylation levels of IDO and IDO mediated tryptophan metabolism in DCs. In addition, the suppressive effect of TKIs on tryptophan metabolism may be caused by blocking c-Kit pathway in DCs. Furthermore, the increased phosphorylation of general control nonderepressible (GCN2) and decreased expression of aryl hydrocarbon receptor (AhR)/aryl hydrocarbon receptor nuclear translocator (ARNT) were observed in the T cells activated by TKI-modified DCs, suggesting the enhancement of effector function of T cells. These results indicate that TKI could be used to modulate DC immunogenic activity and may potentially be applied in DC-based cancer immunotherapy.

Xiaozhang Tie Improves Intestinal Motility in Rats With Cirrhotic Ascites by Regulating the Stem Cell Factor/c-kit Pathway in Interstitial Cells of Cajal.

We previously discovered that Xiaozhang Tie (XZT) was helpful for cirrhotic ascites, with obvious abdominal distention relief, suggesting that it may improve gastrointestinal (GI) motility. However, the underlying mechanisms of GI motility in cirrhotic ascites are unclear. Here, we aimed to discover explored the effect of XZT on GI motility in animal cirrhotic ascites and probed the action mechanism affecting GI motility by regulating the stem cell factor (SCF)/c-kit pathway in interstitial cells of Cajal (ICCs) and GI hormones. First, rat models of cirrhotic ascites were developed and then divided randomly into the following three subgroups: model control, XZT group, and mosapride group. The efficacy of XZT on treating cirrhotic ascites was evaluated on the basis of ascites weight and volume, 24 h urine volume, and feces water content. GI motility of the cirrhotic model, intestine propulsion, and gastric residue were detected using the migration distance of ink in vivo, and the frequency of contraction and tension of isolated gastric and jejunal muscle strips were measured after incubation with XZT extracts. Serum GI hormone content, including motilin (MTL), substance P (SP), somatostatin (SS), and vasoactive intestinal polypeptide were assayed. Subsequently, ICCs were isolated from jejunum, and primarily cultured ICCs were incubated with and without XZT and SCF. The cell vitality of the ICCs was measured. A whole-cell patch recording technique was used to record the current of K+ and Na+ channels in the ICC membrane. Expressions of c-kit/p-c-kit, p-Akt, p-STAT3, and p-Erk1/2 were detected in vivo and in vitro. The results revealed that XZT significantly reduced ascites weight and increased urine volume and fecal water content in model rats. XZT promoted intestinal motility and increased MTL level but reduced SP and SS levels. It enhanced the current of Na+ and K+ in ICCs and improved c-kit expression and signaling mediator phosphorylation in SCF/c-kit, which was inhibited by imatinib in vitro and downregulated in model rats in vivo. Our study concluded that XZT reduced the amount of ascites and improved intestinal motility in cirrhotic rats, which may be associated with its effect on ascites and was involved in the mechanisms regulating the SCF/c-kit signaling pathway in ICCs and improving gastrointestinal hormone secretion.

Interstitial cells of Cajal in Wsh/Wsh c-kit mutant mice.

The c-Kit receptor tyrosine kinase regulates the development and differentiation of several progenitor cells. In the gastrointestinal (GI) tract, the c-Kit regulates the development of the interstitial cells of Cajal (ICC) that are responsible for motility regulation of the GI musculature. W-sash (Wsh) is an inversion mutation upstream of the c-kit promoter region that affects a key regulatory element, resulting in cell-type-specific altered gene expression, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the GI tract of Wsh/Wsh mice using immunohistochemistry and electron microscopy. Although the musculature of the Wsh/Wsh mice did not show any c-Kit immunoreactivity, we detected intensive immunoreactivity for transmembrane member 16A (TMEM16A, anoctamin-1), another ICC marker. TMEM16A immunopositive cells were observed as ICC-MY in the gastric corpus-antrum and the large intestine, ICC-DMP in the small intestine, and ICC-SM in the colon. Electron microscopic analysis revealed these cells as ICC from their ultrastructural features, such as numerous mitochondria and caveolae, and their close contact with nerve terminals. In the developmental period, we examined 14.5 and 18.5 day embryos but did not observe c-Kit immunoreactivity in the Wsh/Wsh small intestine. From this study, ICC subtypes developed and maturated structurally without c-Kit expression. Wsh/Wsh mice are a new model to investigate the effects of c-Kit and unknown signaling on ICC development and function.