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Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.

Understanding mechanisms of immune regulation is key to developing immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes during peripheral T cell regulation in type 1 diabetes and melanoma. MERTK expression and activity in mononuclear phagocytes in the pancreatic islets, promoted islet T cell regulation, resulting in reduced sensitivity of T cell scanning for cognate antigen in pre-diabetic islets. MERTK-dependent regulation led to reduced T cell activation and effector function at the disease site in the islets and prevented rapid progression of type 1 diabetes. In human islets, MERTK-expressing cells were increased in remaining insulin-containing islets of type 1 diabetic patients, suggesting that MERTK protects the islets from autoimmune destruction. MERTK also regulated T cell arrest in melanoma tumors. These data indicate that MERTK signaling in mononuclear phagocytes drives T cell regulation at inflammatory disease sites in peripheral tissues through a mechanism that reduces the sensitivity of scanning for antigen leading to reduced responsiveness to antigen.

Phagocytosis after myocardial infarction (MI) is a prerequisite to cardiac repair. Recruited monocytes clear necrotic cardiomyocytes and differentiate into cardiac macrophages. Some studies have linked apoptotic cell receptors on cardiac macrophages to tissue repair; however, the contribution of precursor monocyte phagocytic receptors, which are the first to interact with the cardiac parenchyma, is unclear. The scavenger receptor cluster of differentiation (CD)36 protein was detected on cardiac Ly6cHI monocytes, and bone marrow–derived Cd36 was essential for both early phagocytosis of dying cardiomyocytes and for smaller infarct sizes in female and male mice after permanent coronary ligation. Cd36 deficiency led to reduced expression of phagocytosis receptor Mertk and nuclear receptor Nr4a1 in cardiac macrophages, the latter previously shown to be required for phagocyte survival. Nr4a1 was required for phagocytosis-induced Mertk expression, and Nr4a1 protein directly bound to Mertk gene regulatory elements. To test the overall contribution of the Cd36-Mertk axis, MI was induced in Cd36−/− Mertk−/− double-knockout mice and led to increases in myocardial rupture. These data implicate monocyte CD36 in the mitigation of early infarct size and transition to Mertk-dependent macrophage function. Increased myocardial rupture in the absence of both Cd36 and Mertk underscore the physiologic significance of phagocytosis during tissue injury.—Dehn, S., Thorp, E. B. Myeloid receptor CD36 is required for early phagocytosis of myocardial infarcts and induction of Nr4a1-dependent mechanisms of cardiac repair.

Background Glioma-associated macrophages and microglia (GAMs) are components of the glioblastoma (GBM) microenvironment that express MerTK, a receptor tyrosine kinase that triggers efferocytosis and can suppress innate immune responses. The aim of the study was to define MerTK as a therapeutic target using an orally bioavailable inhibitor, UNC2025. Methods We examined MerTK expression in tumor cells and macrophages in matched patient GBM samples by double-label immunohistochemistry. UNC2025-induced MerTK inhibition was studied in vitro and in vivo. Results MerTK/CD68+ macrophages increased in recurrent tumors while MerTK/glial fibrillary acidic protein-positive tumor cells did not. Pharmacokinetic studies showed high tumor exposures of UNC2025 in a syngeneic orthotopic allograft mouse GBM model. The same model mice were randomized to receive vehicle, daily UNC2025, fractionated external beam radiotherapy (XRT), or UNC2025/XRT. Although median survival (21, 22, 35, and 35 days, respectively) was equivalent with or without UNC2025, bioluminescence imaging (BLI) showed significant growth delay with XRT/UNC2025 treatment and complete responses in 19%. The responders remained alive for 60 days and showed regression to 1%-10% of pretreatment BLI tumor burden; 5 of 6 were tumor free by histology. In contrast, only 2% of 98 GBM mice of the same model treated with XRT survived 50 days and none survived 60 days. UNC2025 also reduced CD206+ macrophages in mouse tumor samples. Conclusions These results suggest that MerTK inhibition combined with XRT has a therapeutic effect in a subset of GBM. Further mechanistic studies are warranted.

Aim Infection by hepatitis C virus (HCV) can result in the development of liver fibrosis and may eventually progress into cirrhosis and hepatocellular carcinoma but the molecular mechanisms for this process are not fully known. Several genome wide association studies (GWAS) have been performed to pinpoint causative variants in HCV-infected patient cohorts but these variants are usually not the functional ones. The aim of this study was to identify the regulatory SNP associated to the risk of HCV induced liver fibrosis and elucidate its molecular mechanism. Methods We utilized a bioinformatics approach to identify a non-coding regulatory variant, located in an intron of the MERTK gene, based on differential transcription factor binding between the alleles. We validated the results using expression reporter assays and electrophoresis mobility shift assays (EMSA). Results ChIP-sequencing indicates that transcription factor(s) (TF) bind stronger to the A allele of rs6726639. EMSA supports these findings and suggest that the TF is IRF1. Luciferase report assays showed lower enhancer activity from the A allele and that IRF1 may act as a repressor. Conclusions Treatment of hepatitis C with interferon alpha (INF) results in increased IRF1 levels and our data suggest that this leads to an allele-specific down-regulation of MERTK mediated by an allelic effect on the regulatory element containing the functional rs6726639. This variant also shows the hallmarks for being the driver of the GWAS for reduced risk of liver fibrosis and non-alcoholic fatty liver disease (NAFLD) at MERTK.

Background:Despite substantial improvements in long-term clinical outcomes, a significant proportion of rheumatoid arthritis (RA) patients still fail to respond to treatment adequately, and early prognostic biomarkers of response are missing. Single-cell transcriptomic studies on RA synovial tissue (ST) have shown that MerTK is highly expressed in “anti-inflammatory” macrophages [1]. It has also been suggested that synovial macrophages isolated from RA patients in remission are characterised by a CD206+/MerTK+ signature [2]. Finally, monocyte-derived macrophages from RA patients treated with TNF-inhibitors (TNF-i) up-regulate MerTK.Objectives:To assess i) the modulation of synovial tissue MerTK+ macrophages upon treatment with conventional synthetic (cs) disease-modifying anti-rheumatic drugs (DMARDs) and ii) the relationship between baselineMerTKgene expression and response to TNFi.Methods:ST was obtained by US-guided synovial biopsies of an inflamed peripheral joint in patients with early (Results:Before any treatment intervention, the percentage of MerTK+CD206+ macrophages was significantly higher in RA patients with low (DAS285.1) disease activity (24.5±20.1 versus 4.8±4.8, pConclusion:Our whole-tissue protein expression data further support the hypothesis that a selective expansion of the MerTK+ macrophage subset characterise patients achieving remission. Moreover, the pre-treatment up-regulation of the MerTK gene in future responders to TNFi suggest that MerTK is implicated in modulating synovial inflammatory responses and may be exploited as a therapeutic target in RA.References:[1]F. Zhang et al, Nature Immunology, vol. 20, no. 7, pp. 928–942, 2019.[2]S. R. Finlay at al, Annals of the Rheumatic Diseases, vol. 77, Supplement 2, pp. 183–183, 2018.[3]Y. Degboé et al, Frontiers in Immunology, vol. 10, p. 3, 2019.Acknowledgments:Versus ArthritisDisclosure of Interests:None declared

Retinitis pigmentosa (RP) is a group of inherited retinal diseases that result in severe progressive visual impairment.The purpose of this article was to apply targeted next-generation sequencing (NGS) to identify the causative mutation in a Chinese RP family.Blood samples were collected from a Chinese proband diagnosed with RP and her family members. A total of 163 genes that have been previously found to be involved in inherited retinal diseases were selected for NGS. Rigorous NGS data analysis; Sanger sequencing validation; and segregation analysis were applied to evaluate a novel frameshift mutation.Sequence analysis revealed that the proband and her affected sister both carried a novel homozygous frameshift mutation in MERTK (p.I103Nfs*4). Other family members carrying a heterozygous mutation were unaffected. This mutation was found to cosegregate with the disease phenotype in this family. This mutation was not found in 1,000 control individuals.The targeted NGS strategy employed provides an efficient tool for RP pathogenic gene detection. This study identified a new autosomal recessive mutation in the RP-related gene MERTK, which expands the spectrum of RP disease-causing mutations.

Purpose Retinitis pigmentosa (RP) describes a complex group of inherited retinal dystrophies with almost 300 reported genes and loci. We investigated the genetic etiology of autosomal recessive RP (arRP) in a large kindred with 5 affected family members, who reside on the island of Newfoundland, Canada. Methods Genetic linkage analysis was performed on 12 family members (Infinium HumanOmni2.5-8 BeadChip). Whole exome sequencing analysis (Illumina HiSeq) was performed on one affected individual. A custom pipeline was applied to call, annotate, and filter variants. FishingCNV was used to scan the exome for rare copy number variants (CNVs). Candidate CNVs subsequently were visualized from microarray data (CNVPartition v.3.1.6.). MERTK breakpoints were mapped and familial cosegregation was tested using Sanger Sequencing. Results We found strong evidence of linkage to a locus on chromosome 2 (logarithm of the odds [LOD] 4.89 [θ = 0]), at an interval encompassing the MERTK gene. Whole exome sequencing did not uncover candidate point mutations in MERTK, or other known RP genes. Subsequently, CNV analysis of the exome data and breakpoint mapping revealed a 25,218 bp deletion of MERTK, encompassing exons 6 to 8, with breakpoints in introns 5 (chr2:112,725,292) and 8 (chr2:112,750,421). A 48 bp insertion sequence was buried within the breakpoint; 18 bps shared homology to MIR4435-2HG and LINC00152, and 30 bp mapped to MERTK. The deletion cosegregated with arRP in the family. Conclusions This study describes the molecular and clinical characterization of an arRP family segregating a novel 25 kb deletion of MERTK. These findings may assist clinicians in providing a diagnosis for other unsolved RP cases.

Definitive erythropoiesis takes place at erythroblastic islands, where erythroblasts proliferate and differentiate in association with central macrophages. At the final stage of erythropoiesis, pyrenocytes (nuclei surrounded by plasma membranes) are excluded from erythroblasts, expose phosphatidylserine (PtdSer), and are engulfed by the macrophages in a PtdSer-dependent manner. However, the molecular mechanism(s) involved in the engulfment of pyrenocytes are incompletely understood. Here, we constructed an in vitro assay system for the enucleation and engulfment of pyrenocytes using a methylcellulose-based culture. As reported previously, erythroblasts were bound to macrophages via interactions between integrin-α4β1 on erythroblasts and Vcam1 on macrophages. After enucleation, the resulting pyrenocytes exhibited a reduced affinity for Vcam1 that correlated with the presence of inactive integrin-α4β1 complexes. The pyrenocytes were then engulfed by the macrophages via a MerTK-protein S-dependent mechanism. Protein S appeared to function as a bridge between the pyrenocytes and macrophages by binding to PtdSer on the pyrenocytes and MerTK on the macrophages. Normally, NIH3T3 cells do not engulf pyrenocytes, but when they were transformed with MerTK, they efficiently engulfed pyrenocytes in the presence of protein S. These results suggest that macrophages use similar mechanisms to engulf both pyrenocytes and apoptotic cells.

BACKGROUND Glioblastoma-associated macrophages and microglia (GAMs) are the predominant immune cells in the tumor microenvironment. Activation of MerTK, a receptor tyrosine kinase, triggers efferocytosis and polarizes GAMs to an immunosuppressive phenotype, promoting glioma growth. Our previous findings showed that UNC2371, a small-molecule inhibitor of MerTK, induced a less immunosuppressive phenotype of GAMs. Here, we investigate the role of MerTK inhibition on glioblastoma cells in the tumor microenvironment in vitro and in vivo. METHODS Cytotoxicity of UNC2371 in glioblastoma cells was determined by cell viability and colony formation assays. The protein expression of MerTK, AKT, and Erk were quantified by Western blotting in UNC2371-treated glioblastoma cells. A syngeneic GL261 mouse orthotopic glioblastoma model was used to evaluate the survival benefit of UNC2371 treatment. Fluorescent multiplex immunohistochemistry (IHC) was used to evaluate the expression of CD206, an anti-inflammatory marker on GAMs in murine brain tumor tissues. RESULTS UNC2371 inhibited GBM cell growth with an EC50 < 100 nM in both human U251 and mouse GL261 glioma cells, but not in GAMs. UNC2371-induced cell death and decreased cell proliferation were demonstrated by colony formation assays. UNC2371 decreased protein expression of phosphorylated MerTK, AKT, and Erk, which are essential for cell survival signaling, in U251 and GL261 cells. Furthermore, UNC2371 treatment prolonged survival in the mouse orthotopic GL261 glioblastoma model, suggesting that UNC2371 induces glioma cell death. A decreased of CD206+ GAMs was found in mice glioma tissues by fluorescent multiplex IHC, consistent with our previous findings in the in vitro cell-based assays. These data suggest that in addition to alleviate immunosuppression in the glioma microenvironment, UNC2371 directly inhibits GBM cell growth in vitro and in vivo. CONCLUSION Our findings suggest that UNC2371 has a therapeutic benefit via promoting GAM polarization towards proinflammatory status in the glioblastoma microenvironment and unexpectedly, inducing tumor cell death.

The human induced pluripotent stem cell (hiPSC) line RP1-FiPS4F1 generated from the patient with autosomal recessive retinitis pigmentosa (arRP) caused by homozygous Ser331Cysfs*5 mutation in Mer tyrosine kinase receptor (MERTK) was genetically corrected using CRISPR/Cas9 system. Two isogenic hiPSCs lines, with heterozygous and homozygous correction of c.992_993delCA mutation in the MERTK gene were generated. These cell lines demonstrate normal karyotype, maintain a pluripotent state, and can differentiate toward three germ layers in vitro. These genetically corrected hiPSCs represent accurate controls to study the contribution of the specific genetic change to the disease, and potentially therapeutic material for cell-replacement therapy.

Objective— Arch atherosclerosis 4 ( Aath4 ) is a quantitative trait locus for atherosclerotic plaque formation in the inner curve of the aortic arch previously identified in an F2 cross of Apoe −/− mice on DBA/2J and 129S6 backgrounds. C-mer proto-oncogene tyrosine kinase (Mertk ), coding for a ligand-activated transmembrane tyrosine kinase, is a candidate gene within the same chromosomal region. Our objective was to determine whether strain differences in Mertk influence plaque formation. Approach and Results— To dissect the strain effects of Mertk on atherosclerosis, we first established a congenic mouse line ( Aath4a DBA/DBA ) in which a 5′ region of Aath4 of DBA/2J, including Mertk , was backcrossed onto a 129S6- Apoe −/− background. The resulting Aath4a DBA/DBA male mice developed significantly larger plaques compared with control mice ( Aath4a 129/129 ), proving that the DBA/2J allele of Aath4a is proatherogenic. Thioglycollate-elicited peritoneal macrophages from Aath4a DBA/DBA mice express less than 50% of Mertk mRNA and cell-surface MERTK protein compared with those from the control mice. Moreover, both large and small peritoneal Aath4a DBA/DBA macrophages showed reduced phagocytosis of apoptotic cells. When Mertk cDNAs from 129S6 and DBA/2J mice were overexpressed in HEK293T (human embryonic kidney 293T) cells, phagocytosis of apoptotic cells was equally enhanced in direct proportion to Mertk levels, indicating that phagocytosis is modulated by the amount of MERTK, but that it is not affected by MERTK amino acid differences between 129S6 and DBA/2J. Conclusions— Reduced transcription of Mertk , rather than differences in MERTK protein structure, determines the reduced efficiency of apoptotic cell clearance in the Aath4a DBA/DBA mice, which, in turn, contributes to their increased susceptibility to atherosclerosis.

The efferocytic receptor MerTK is expressed on monocytes/macrophages and facilitates tolerogenic T cell suppressive clearance of apoptotic cellular debris. Given the rapid cell turn-over in leukemia, we hypothesized that ongoing efferocytosis is leukemia-permissive and inhibition of MerTK on monocytes/macrophages will reverse T cell suppression leading to enhanced anti-leukemia immunity. To test this hypothesis, we inoculated mice with syngeneic murine BCR-ABL p185 acute lymphoblastic leukemia (ALL) cells, which do not express MerTK. The effects of MerTK inhibition were evaluated in wild-type (WT) mice treated daily with MRX-2843, a MerTK small molecule inhibitor, and in mice with a MerTK knockout mutation (Mertk-/-). Median survival was prolonged in WT mice treated with MRX-2843 (40 days) compared to vehicle treatment (20 days, p To explore the T cell suppressive effects of MerTK inhibition on CD11b+ monocytes/macrophages, we used flow cytometry to study ex vivo mixed cell cultures using Mertk-/- or WT murine splenocytes +/- ALL cells treated +/- MRX-2843. Co-culture with ALL cells for 24 (36.0%), 48 (34.9%) and 72 (36.6%) hours resulted in a significant increase in expression of co-inhibitory ligands PD-L1 and PD-L2 on CD11b+ cells compared to cultures without ALL cells (2.03%, 3.2% and 7.5%, p In these experiments, expression of PD-1, the co-inhibitory receptor which binds PD-L1/PD-L2, on T cells was also assessed. In WT mice inoculated with ALL, expression of PD-1 on T cells from spleens (74.2% CD4+; 49.2% CD8+) and bone marrow (65.9% CD4+; 73.0% CD8+) was significantly increased compared to spleens (10.0% CD4+; 4.1% CD8+) and bone marrow (36.4% CD4+; 8.5% CD8+) from uninoculated control mice (p To determine whether changes in PD-1 expression were indicative of T cell activation or exhaustion, T cells treated with α-CD3 beads were added to splenocyte and ALL cell co-cultures for 24 hours and intracellular IFN-ɣ and TNF-α levels were assessed using flow cytometry. The fraction of T cells producing IFN-ɣ/TNF-α significantly increased in co-cultures treated with MRX-2843 (44.2% CD4+; 51.1% CD8+) compared to vehicle-treated co-cultures (27.4% CD4+, p In conclusion, inhibition of MerTK on CD11b+ monocytes/macrophages reversed T cell suppression in response to leukemia via the PD-1 axis. MerTK inhibitors are in early phase clinical trials and these data demonstrate a potential immunotherapeutic use for MerTK inhibitors in the treatment of ALL. Disclosures Wang: Meryx: Equity Ownership, Patents & Royalties. Frye:Meryx: Equity Ownership, Patents & Royalties. Earp:Meryx: Equity Ownership. DeRyckere:Meryx: Equity Ownership. Graham:Meryx: Equity Ownership.

BACKGROUND: Glioma-associated macrophages and microglia (GAMs) are predominant immune cells in a glioma microenvironment. Receptor tyrosine kinase MerTK is highly expressed in GAMs. Activation of MerTK triggers efferocytosis and polarizes GAMs to an immunosuppressive phenotype, promoting tumor growth. We hypothesize that inhibiting MerTK by UNC2371, a small molecule MerTK inhibitor, may lead to a less immunosuppressive glioma microenvironment. METHODS: Monocytes were isolated from the blood of healthy human donors followed by differentiation and stimulation into proinflammatory (CD80(+)/CD86(+)) and anti-inflammatory (CD163(+)/CD206(+)) macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF)/Interferon-gamma and macrophage colony-stimulating factor (M-CSF)/Interleukin-4 respectively. Phenotyping of the macrophages was done by flow cytometry analysis of the cell surface markers. Cytotoxicity of UNC2371 in GBM cells and macrophages was determined by cell viability. Cytokine profiling from the macrophages and a human microglial cell line was performed using cytokine array analysis. The protein expressions of arginase I, interleukin-8, and MerTK were quantified by Western blotting. RESULTS: We demonstrate that UNC2371 inhibits the growth of GBM cells with EC50 of 95.5 nM in U251 glioma cells, but not macrophages. While cultured in U251 conditioned medium (CM), a decrease in CD163(+)/CD206(+)(anti-inflammatory) macrophages was observed in 43% of healthy donors (n=7) after UNC2371 treatment, suggesting a proinflammatory polarization. UNC2371-treated macrophages or microglia reduces secretion of Interleukin-8, a chemokine known to promote gliomagenesis, was detected by cytokine array analysis. UNC2371 decreases protein expression of MerTK, Interleukin-8, and arginase I in GAMs. These data suggest that UNC2371 inhibits MerTK signaling in GAMs and alleviates immunosuppression in the glioma microenvironment. CONCLUSION: Our findings suggest that UNC2371 is a promising drug to polarize GAMs towards proinflammatory, suppress pro-glioma cytokines and modulate the glioma microenvironment. Preclinical evaluations of UNC2371 as a tumor microenvironment modulator using animal models are currently underway.

Aim: To characterise the ocular phenotype of a family segregating the splice site mutation c.2189+1G>T in the tyrosine kinase receptor gene MERTK . Methods: Five affected children of a consanguineous Moroccan family were investigated by ophthalmic examinations, including fundus photography, autofluorescence (FAF) imaging, optical coherence tomography (OCT), psychophysical and electrophysiological methods. Results: Affected children were between 5 and 19 years of age, allowing an estimation of disease progression. Electroretinography demonstrated loss of scotopic and photopic function in the first decade of life. Younger siblings showed drusen-like deposits with focal relatively increased FAF in the macular area. With increasing age, a yellowish lesion with relatively increased FAF and subsequent macular atrophy developed. Visual acuity deteriorated with age and ranged between 20/50 in the best eye of the youngest affected and 20/400 in the worst eye of the oldest affected sibling. Spectral-domain OCT revealed debris-like material in the subneurosensory space. Conclusion: The splice site mutation c.2189+1G>T in MERTK causes rod–cone dystrophy with a distinct macular phenotype. The debris in the subneurosensory space resembles that in the Royal College of Surgeons (RCS) rat being the mertk animal model. Patients might therefore benefit from advances in gene therapy that were previously achieved in the RCS rat.

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Self-antigens expressed by apoptotic cells (ACs) may become targets for autoimmunity. Tolerance to these antigens is partly established by an ill-defined capacity of ACs to inhibit antigen-presenting cells such as dendritic cells (DCs). We present evidence that the receptor tyrosine kinase Mer (MerTK) has a key role in mediating AC-induced inhibition of DC activation/maturation. Pretreatment of DCs prepared from nonobese diabetic (NOD) mice with AC blocked secretion of proinflammatory cytokines, up-regulation of costimulatory molecule expression, and T cell activation. The effect of ACs on DCs was dependent on Gas6, which is a MerTK ligand. NOD DCs lacking MerTK expression (NOD.MerTK(KD/KD)) were resistant to AC-induced inhibition. Notably, autoimmune diabetes was exacerbated in NOD.MerTK(KD/KD) versus NOD mice expressing the transgenic BDC T cell receptor. In addition, beta cell-specific CD4(+) T cells adoptively transferred into NOD.MerTK(KD/KD) mice in which beta cell apoptosis was induced with streptozotocin exhibited increased expansion and differentiation into type 1 T cell effectors. In both models, the lack of MerTK expression was associated with an increased frequency of activated pancreatic CD11c(+)CD8alpha(+) DCs, which exhibited an enhanced T cell stimulatory capacity. These findings demonstrate that MerTK plays a critical role in regulating self-tolerance mediated between ACs, DCs, and T cells.

Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches. This work was supported by Andalusian Health Council (PI-0324-2013), Instituto de Salud Carlos III (PI13/01331), Spanish Ministry of Economy and Competitiveness-FEDER BFU2012-36845, Instituto de Salud Carlos III RETICS RD12/0034/0010 and Academy of Finland (218050; 272808).

Objective. The MER receptor tyrosine kinase (MERTK) gene is critical for the efficient clearance of apoptotic cells and has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the genetic polymorphisms in MERTK to evaluate it as a potential candidate gene for a host genetic study of SLE and clinical manifestations in patients with SLE. Methods. By resequencing the coding and flanking regions of the MERTK gene in 24 unrelated Koreans, 37 polymorphisms were identified. Based on gene position, minor allele frequency and inter-single-nucleotide polymorphism (SNP) linkage disequilibrium, six of these polymorphisms were selected for subsequent genotyping and association analysis with the risk of SLE and haematological disorders in 350 Korean SLE patients and 330 controls. Results. Although no significant associations with the risk of SLE were found, logistic regression analyses revealed that variants þ465C > G (P ¼ 0.05) and þ130215insdelT (P ¼ 0.0005) were significantly associated with decreased risk of leucopenia in SLE patients. Further, þ465C > G, þ95616G > A, þ123157A > G and the haplotype ht1 also showed significant associations (P ¼ 0.006–0.05) with a decreased risk of lymphopenia in SLE patients. Conclusion. Our findings suggest that polymorphisms in MERTK might be one of the genetic risk factors for presenting leucopenia and lymphopenia in SLE patients.

6 páginas, 8 figuras, 2 tablas.-- Licence Creative Commons, attribution, Non-commercial licence.-- et al., [Background/aim]: MERTK, a tyrosine kinase receptor protein expressed by the retinal pigment epithelium (RPE), is mutated in both rodent models and humans affected by retinal disease. This study reports a survey of families for Mertk mutations and describes the phenotype exhibited by one family. [Methods]: 96 probands with retinal dystrophy, consistent with autosomal recessive segregation, were screened by direct sequencing. A family homozygous for a likely null allele was investigated clinically. [Results]: A novel frame shifting deletion was identified in one of 96 probands. Other polymorphisms were detected. The deletion allele occurred on both chromosomes of four affected family members. Electrophysiology demonstrated early loss of scotopic and macular function with later loss of photopic function. Visual acuities and visual fields were preserved into the second decade. Perception of light vision was present in a patient in the fourth decade. A “bull’s eye” appearance and a hyperautofluorescent lesion at the central macula were consistent clinical findings. [Conclusions]: Mutations in Mertk are a rare cause of ARRP in humans. The study extends the phenotypic characteristics of this retinal dystrophy and shows distinctive clinical signs that may improve its clinical identification. The moderate severity and presence of autofluorescence implies that outer segment phagocytosis is not entirely absent., the special trustees of Moorfields Eye Hospital, the Wellcome Trust, and the Foundation Fighting Blindness for their generous sponsorship.

PURPOSE. Mutations in the MERTK gene are responsible for retinal degeneration in the Royal College of Surgeons (RCS) rat and are a cause of human autosomal recessive retinitis pigmentosa (RP). This study reports the identification and functional analysis of novel MERTK mutations to provide information regarding whether they are causative of severe rod- cone degeneration in a young patient. METHODS. MERTK missense variants identified by single-strand conformational polymorphism (SSCP) and sequence analysis were introduced into expression constructs and used to transfect HEK293T cells. Recombinant protein expression was assayed with anti-MERTK and anti-phosphotyrosine antibodies. Protein turnover was assayed in pulse-chase studies of 35 S-methionine incorporation. Transcript levels were determined by quantitative RT-PCR. RESULTS. Three MERTK sequence variants were identified in a patient with rod-cone dystrophy: R722X in exon 16 and R865W in exon 19 on the paternal allele and R844C in exon 19 on the maternal allele. The R844C sequence change affects an evolutionarily conserved amino acid residue and was not detected in unaffected individuals. In transfected HEK293Tcells, wild-type (wt) and W865 MERTK were expressed at equivalent levels and present in the plasma membrane, stimulated tyrosine phosphorylation, and induced significant rounding of the cell bodies. In contrast, C844 MERTK was expressed at low levels and did not stimulate tyrosine phosphorylation. In addition, the relative stability of C844 MERTK was significantly less than wt in assays of protein turnover. At age 13, the patient had 20/60 and 20/200 acuities, tunnel vision of 5° centrally, and a far temporal peripheral crescent bilaterally, and ERGs were nondetectable. The fundi showed bull's-eye macular atrophy and widespread RPE thinning. CONCLUSIONS. The present study reports the identification of R844C, the first putative pathogenic MERTK missense mutation that results in severe retinal degeneration with childhood onset when in compound heterozygous form with a R722X allele. The loss of function of C844 MERTK is probably due to decreased protein stability.

In the Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) cannot phagocytose the outer segment discs that are continually shed from photoreceptors. The resulting accumulation of debris in the subretinal space leads to a progressive loss of photoreceptors. The defect results from a mutation in the Mertk gene, which is normally expressed in the RPE. Mertk is a receptor tyrosine kinase, involved in the binding of photoreceptor debris. Mutations in MERTK have also been described in patients with retinitis pigmentosa (RP). Here we demonstrate that subretinal injection of recombinant adeno-associated virus (AAV) expressing the murine Mertk gene can significantly prolong photoreceptor cell survival in the RCS rat. Electroretinographic analysis of treated eyes showed that functional photoreceptors were still present at 9 weeks, when there is virtually no activity in untreated control eyes. Histological analysis of treated eyes revealed a decrease in the amount of debris in the subretinal space, suggesting that RPE function was restored. Moreover, 9 weeks after treatment the number of photoreceptors was 2.5-fold higher in treated than in control eyes. This study provides strong support for the development of AAV-mediated gene therapy for RP caused by mutations in the MERTK gene.

International audience; We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3–4 days and in the serum for up to 15–20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases.

The domestic dog segregates a significant number of inherited progressive retinal diseases, several of which mirror human retinal diseases and which are collectively termed progressive retinal atrophy (PRA). In 2014, a novel form of PRA was reported in the Swedish Vallhund breed, and the disease was mapped to canine chromosome 17. The causal mutation was not identified, but expression analyses of the retinas of affected Vallhunds demonstrated a 6-fold increased expression of the MERTK gene compared to unaffected dogs. Using 24 retinopathy cases and 97 controls with no clinical signs of retinopathy, we replicated the chromosome 17 association in Swedish Vallhunds from the UK and aimed to elucidate the causal variant underlying this association using whole genome sequencing (WGS) of an affected dog. This revealed a 6-8 kb insertion in intron 1 of MERTK that was not present in WGS of 49 dogs of other breeds. Sequencing and BLASTN analysis of the inserted segment was consistent with the insertion comprising a full-length intact LINE-1 retroelement. Testing of the LINE-1 insertion for association with retinopathy in the UK set of 24 cases and 97 controls revealed a strong statistical association (P-value 6.0 x 10(-11)) that was subsequently replicated in the original Finnish study set (49 cases and 89 controls (P-value 4.3 x 10(-19)). In a pooled analysis of both studies (73 cases and 186 controls), the LINE-1 insertion was associated with a similar to 20-fold increased risk of retinopathy (odds ratio 23.41, 95% confidence intervals 10.99-49.86, P-value 1.3 x 10(-27)). Our study adds further support for regulatory disruption of MERTK in Swedish Vallhund retinopathy; however, further work is required to establish a functional overexpression model. Future work to characterise the mechanism by which this intronic mutation disrupts gene regulation will further improve the understanding of MERTK biology and its role in retinal function.

Vertebrate photoreceptor cells are the basic sensory apparatus of the retina, capable of converting the energy of absorbed photons into neuronal signals. The proximal portions of mammalian photoreceptor outer segments are synthesized daily by cell bodies, and outer segment tips are shed with a circadian rhythm, resulting in a complete turnover of outer segments about every 9 days. The shed outer segments are phagocytosed by adjacent retinal pigment epithelial (RPE) cells, and metabolites are recycled to photoreceptors. The Royal College of Surgeons (RCS) rat is a widely studied, classic model of recessively inherited retinal degeneration in which the RPE fails to phagocytose shed outer segments, and photoreceptor cells subsequently die. We have used a positional cloning approach to study the rdy (retinal dystrophy) locus of the RCS rat. Within a 0.3 cM genetic inclusion interval, we have discovered a small deletion of RCS DNA that disrupts the gene encoding the receptor tyrosine kinase Mertk. The deletion includes the splice acceptor site upstream of the second coding exon of Mertk and results in a shortened transcript that lacks this exon. The aberrant transcript joins the first and third coding exons, leading to a frameshift and a translation termination signal 20 codons after the AUG. The concordance of these and other data indicate that Mertk is probably the gene for rdy. Our results provide genetic evidence for an essential role of a receptor tyrosine kinase in a specialized form of phagocytosis and suggest a molecular model for ingestion of outer segments by RPE cells.

MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is ectopically expressed in 30-50% of newly diagnosed pediatric acute lymphoblastic leukemia (ALL) patient samples and aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by shRNA or a small molecule inhibitor, MRX-2843, decreased colony-forming potential and induced apoptosis in leukemia cell cultures. Moreover, MERTK inhibition prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with MRX-2843 effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. Additionally, Gas6, a MERTK ligand, is a poor prognostic factor in AML, mediates increased resistance to cytotoxic chemotherapy in leukemia cells, and is expressed in the bone marrow. To determine the role of Gas6 produced by bone marrow stromal cells in mediating resistance to MERTK inhibition by MRX-2843, acute leukemia cell lines were cultured in the presence of a Gas6-producing fibroblast-like cell line (HS27) or bone marrow derived stromal cells (BMDSCs) from wild type or Gas6 knockout mice and induction of apoptosis and cell death was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 300nM MRX-2843 compared to leukemia cells alone (29.4% versus 60.5%, p=0.002). Similar results were observed in Nomo1 AML and 697 pre-B ALL cell cultures. To evaluate whether soluble factors mediated this protective effect, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Interestingly, conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (86.0% vs. 85.5% in unconditioned medium). To more directly assess the role of Gas6, BMDSCs isolated from wild-type and Gas6 knockout C57Bl/6 mice were co-cultured with 697 leukemia cells and sensitivity to MRX-2843 was determined. BMDSCs from wild-type mice protected 697 leukemia cells from MRX-2843 induced cell death much more effectively than BMDSCs from Gas6 knockout mice (4.3% apoptotic and dead cells versus 72.4%, respectively). To investigate biochemical mechanisms of Gas6-mediated protection, Kasumi-1 AML cells were cultured with 200nM MRX-2843 or vehicle in the presence or absence of HS27 cells and expression and activation of MERTK, AXL, TYRO-3, and downstream signaling effectors STAT5, AKT, and ERK1/2 were determined by immunoblot. AXL was not expressed in Kasumi-1 cells with or without co-culture. Treatment with MRX-2843 mediated robust inhibition of MERTK activation indicated by reduced levels of phosphorylated protein in both the presence and absence of stromal cell co-culture. In contrast, activation of TYRO-3 was increased after treatment with MRX-2843 in leukemia cells co-cultured with HS27 stromal cells. Similarly, in the absence of co-culture MRX-2843 inhibited activation of STAT5, AKT, and ERK1/2. However, in the presence of HS27 cells there was robust activation of STAT5 that was sustained even after treatment with MRX-2843. In contrast, MRX-2843 inhibited activation of AKT and ERK1/2 in HS27 co-cultures, although higher doses were required. Together these data support a model whereby Gas6 produced by stromal cells mediates leukemia cell resistance to MERTK inhibition in the bone marrow by inducing activation of TYRO-3, thereby promoting downstream signaling and cell survival despite MERTK inhibition. Thus, combined treatment with MRX-2843 and a TAM ligand sink (eg MERTK-Fc), a TYRO-3 inhibitor, or a bone marrow mobilizing agent may be particularly effective therapeutic strategies. Disclosures Wang: Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Frye:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Earp:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. DeRyckere:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. Graham:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK.

The MerTK receptor tyrosine kinase is aberrantly expressed in 80-100% of acute myeloid leukemia (AML), and inhibition of MerTK prolongs leukemia-free survival in vivo, making it an appealing therapeutic target. MerTK is normally expressed on macrophages (MMs) and dendritic cells (DCs) and facilitates engulfment of apoptotic debris. Through this process, MerTK promotes tolerogenicity and anti-inflammatory cytokine production. We hypothesized that inhibition of MerTK will also mediate anti-leukemia effects through relief of microenvironment immunosuppression and stimulation of innate MM and DC activity leading to enhanced T-cell immunity. To test this hypothesis, we transplanted wild-type C57BL/6 mice with syngeneic murine MLL-ENL fusion AML or primary mouse MLL-AF9 AML. We evaluated the effects of MerTK inhibition using MRX-2843, a MerTK kinase inhibitor. Mice were treated daily and survival was monitored. Treatment with MRX-2843 prolonged median survival (43 days) compared to vehicle treatment (17 days) in mice transplanted with MLL-ENL AML (p To explore the immune effects of MerTK inhibition in AML, we evaluated in vivoMMs and DCs by flow cytometry. Compared to non-leukemic controls, vehicle-treated leukemic mice demonstrated a significant infiltration of MMs and DCs into the primary sites of leukemic burden (spleen, bone marrow). These MMs and DCs expressed high levels of co-inhibitory ligands PD-L1 and PD-L2. Treatment with MRX-2843 significantly decreased PD-L1 and PD-L2 expressing MM and DCs in the spleen (p We next evaluated effects of MerTK inhibition on the leukemia microenvironment. We confirmed that T-cells do not express MerTK in-vivowith or without AML, nor +/- MRX-2843. Compared to non-leukemic mice, leukemic mice significantly upregulated co-inhibitory receptors PD-1 and Tim-3 on CD4 and CD-8 T-cells in the spleen and bone marrow. Treatment with MRX-2843 in leukemic mice significantly decreased CD4 and CD8 T-cell PD-1 and Tim-3 expression in the spleen (p We next evaluated peripheral serum cytokines using Luminex magnetic bead analysis. Treatment with MerTK inhibition significantly increased serum [IL-18] (p In conclusion, inhibition of MerTK decreased infiltration of immunosuppressive co-inhibitory ligand-expressing MMs and DCs into primary sites of leukemic engraftment. Furthermore, inhibition of MerTK altered the leukemia microenvironment to decrease T-cell co-inhibitory receptor expression and improve anti-leukemia effects. These preliminary studies demonstrate the potential of MerTK inhibition as an immunotherapeutic strategy in AML. Disclosures Wang: Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Earp:Meryx: Employment, Patents & Royalties: MRX-2843. Frye:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843.

Retinal pigment epithelium (RPE) is a single layer of epithelial cells lined between the neurosensory retina and choriocapillaris. It is part of the blood-retinal barrier and is a central component of the visual phototransduction pathway. RPE cells regenerate 11-cisretinal by RPE65 isomerase and its related enzymes and chaperones (Moiseyev et al., 2006; Xue et al., 2004). They are professional phagocytes and are responsible for the clearance of daily shed photoreceptor outer segments (POS) (Young, 1967; Young & Bok, 1969). The multi-step process of phagocytosis includes receptor-mediated binding of POS to the RPE (Finnemann et al., 1997), internalization (Feng et al., 2002; Finnemann & Silverstein, 2001), transport to lysosome and degradation. The importance of RPE phagocytosis has been clearly illustrated by the Royal College of Surgeons (RCS) rats, which carry a mutation in Mertk gene (D'Cruz et al., 2000). MERTK is a membrane-associated receptor tyrosine kinase and is activated upon binding of POS to the RPE (Feng et al. 2002). In RCS rats, loss-offunction mutation of Mertk causes defects in phagocytosis and consequently these animals develop inherited retinal dystrophy and photoreceptor apoptosis (Tso et al., 1994). In addition to their roles in the visual cycle, RPE cells provide vital support for the structure and function of the outer retina. They transport ions, water and nutrients between choroidal blood supply and the retina, and synthesize melanin which absorbs light and shields the retina. RPE-produced growth factors, such as vascular endothelial growth factor (VEGF), are indispensable for the choroidal vasculature (Saint-Geniez et al., 2009).

To determine the basis and to characterize the phenotype of a chemically induced mutation in a mouse model of retinal degeneration.Screening by indirect ophthalmoscopy identified a line of N-ethyl-N-nitrosourea (ENU) mutagenized mice demonstrating retinal patches. Longitudinal studies of retinal histologic sections showed photoreceptors in the peripheral retina undergoing slow, progressive degeneration. The mutation was named neuroscience mutagenesis facility 12 (nmf12), and mapping localized the critical region to Chromosome 2.Sequencing of nmf12 DNA revealed a point mutation in the c-mer tyrosine kinase gene, designated Mertk(nmf12). We detected elevated levels of tumor necrosis factor (Tnf, previously Tnfa) in retinas of Mertk(nmf12) homozygotes relative to wild-type controls and investigated whether the increase of TNF, an inflammatory cytokine produced by macrophages/monocytes that signals intracellularly to cause necrosis or apoptosis, could underlie the retinal degeneration observed in Mertk(nmf12) homozygotes. Mertk(nmf12) homozygous mice were mated to mice lacking the entire Tnf gene and partial coding sequences of the Lta (Tnfb) and Ltb (Tnfc) genes.(2) B6.129P2-Ltb/Tnf/Lta(tm1Dvk)/J homozygotes did not exhibit a retinal degeneration phenotype and will, hereafter, be referred to as Tnfabc(-/-) mice. Surprisingly, mice homozygous for both the Mertk(nmf12) and the Ltb/Tnf/Lta(tm1Dvk) allele (Tnfabc(-/-)) demonstrated an increase in the rate of retinal degeneration.These findings illustrate that a mutation in the Mertk gene leads to a significantly slower progressive retinal degeneration compared with other alleles of Mertk. These results demonstrate that TNF family members play a role in protecting photoreceptors of Mertk(nmf12) homozygotes from cell death.

Multiple sclerosis (MS) is a debilitating, chronic demyelinating disease of the central nervous system affecting over 2 million people worldwide. The TAM family of receptor tyrosine kinases (TYRO3, AXL and MERTK) have been implicated as important players during demyelination in both animal models of MS and in the human disease. We therefore conducted an association study to identify single nucleotide polymorphisms (SNPs) within genes encoding the TAM receptors and their ligands associated with MS. Analysis of genotype data from a genome-wide association study which consisted of 1618 MS cases and 3413 healthy controls conducted by the Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene) revealed several SNPs within the MERTK gene (Chromosome 2q14.1, Accession Number NG_011607.1) that showed suggestive association with MS. We therefore interrogated 28 SNPs in MERTK in an independent replication cohort of 1140 MS cases and 1140 healthy controls. We found 12 SNPs that replicated, with 7 SNPs showing p-values of less than 10(-5) when the discovery and replication cohorts were combined. All 12 replicated SNPs were in strong linkage disequilibrium with each other. In combination, these data suggest the MERTK gene is a novel risk gene for MS susceptibility.

Contains fulltext : 87683.pdf (Publisher’s version ) (Closed access) PURPOSE. To identify the genetic defect in a family with variable retinal phenotypes. The proband had a diagnosis of Leber congenital amaurosis (LCA), whereas her two cousins had an early-onset severe retinal dystrophy (EOSRD) with useful vision. A distant family member had retinitis pigmentosa (RP). METHODS. DNA samples of the affected family members were genotyped with 250 K genome-wide SNP microarrays. Genetic defects were localized by linkage analysis and homozygosity mapping, and candidate genes were analyzed by sequencing. Patients underwent a full ophthalmic examination. RESULTS. Compound heterozygous mutations in CEP290 were identified in the proband and her two cousins: the frequent c.2991+1655A>G founder mutation and a novel nonsense mutation in exon 7 (c.451C>T, p.Arg151X). The proband had nystagmus, hyperopia, a flat electroretinogram (ERG), and decreased visual acuity (20/250) from birth. The two cousins had minimal scotopic ERG responses at the age of 2. In one of these patients, visual acuity had reached a level of 20/32 at age 5, which is high for patients with CEP290 mutations. Analysis of the CEP290 mRNA in affected individuals revealed altered splice forms in which either exon 7 or exons 7 and 8 were skipped. In both mutant cDNA products, the open reading frame was not disrupted. Furthermore, homozygosity mapping and mutation analysis in the distant family member affected by RP revealed a homozygous mutation in MERTK, but no CEP290 mutations. This MERTK mutation was heterozygously present in the most severely affected (LCA) patient, but was absent in the two more mildly affected cousins. CONCLUSIONS. A novel nonsense mutation in CEP290 results in nonsense-associated altered splicing. That the remaining open reading frame is intact may explain the less severe phenotype observed in the two affected cousins. The additional heterozygous mutation in MERTK may clarify the more severe phenotype in the proband. This study extends the phenotypic spectrum of CEP290-associated diseases at the mild end. 01 juli 2010

Dendritic cells (DCs) play an instrumental role in regulating tolerance to self-antigens and preventing autoimmunity. One mechanism by which “tolerogenic” DCs are established is through the inhibitory effects of apoptotic cells (ACs). Immature DCs encountering ACs are resistant to stimuli that activate and mature DCs. We have shown that the Mer receptor tyrosine kinase (MerTK) plays a key role in transducing inhibitory signals upon binding of ACs, which in turn involve the phosphatidylinositol 3-kinase (PI3K) pathway. Nevertheless, the molecular basis for AC-induced inhibition of DCs is ill defined. In the current study, the proximal signaling events induced by MerTK after AC binding were studied. AC treatment of bone marrow–derived or splenic DCs established a complex consisting of MerTK, the nonreceptor tyrosine kinase c-Src, the transcription factor STAT3, and PI3K. In contrast, AC treatment of DCs lacking MerTK expression failed to increase c-Src and STAT3 activation. In addition, the inhibitory effects of ACs were blocked by treating DCs with pharmacologic inhibitors or siRNA specific for c-Src and STAT3. These findings demonstrate that AC-induced inhibition of DCs requires MerTK-dependent activation of c-Src and STAT3, and provide evidence for novel roles for c-Src and STAT3 in the immunoregulation of DCs.

Background/aim: Mutations in MERTK, a member of the MER/AXL/TYR03 receptor kinase family, have been associated with disruption of the Retinal Pigment Epithelium (RPE) phagocytosis pathway and settling of autosomal recessive RP (arRP) in humans. This study reports a novel MERTK mutation (IVS16+1G>T) in a Spanish consanguineous family presenting arRP. Methods: 21 genes were screened by high-throughput SNP multiplexing assay. Subsequent direct sequencing was performed in exons and intronic boundaries of the cosegregating gene. The effect of the mutation in mRNA splicing was confirmed by cDNA analysis. Results: Haplotypic data revealed MERTK cosegregation with RP in affected individuals. MERTK sequencing showed a G-to-T substitution at the first nucleotide of intron 16. Finally, cDNA analysis confirmed the lack of exon 16 in the mRNA splicing process. Conclusions: IVS16+1G>T disrupts the splice donor site causing exon 16 skipping. Absence of exon 16 causes a frameshift and, subsequently, the introduction of a premature termination codon into exon 17 creating an altered mRNA transcript with a seriously affected tyrosine kinase domain.

Retinal pigment epithelium (RPE) is essential for the function and survival of photoreceptor cells by playing supporting roles including shedding the outer segments of the photoreceptor cells, removing metabolic wastes, transporting nutrients and maintaining visual cycle. RPE defects have been found in various human retinal disorders, such as age-related macular degeneration (Zarbin, 1998), Best disease (Petrukhin et al., 1998; Marmorstein et al., 2000), Sorsby fundus dystrophy (Weber et al., 1994; Ruiz et al., 1996; Della et al., 1996), and childhood-onset severe retinal dystrophy (Gu et al., 1997). Animal models with RPE defects have been used to study the molecular basis for the function of the RPE cells. The Royal College of Surgeons (RCS) rat, a model for recessive inherited retinal degeneration, is characterized by the dysfunction of RPE due to a null mutation of the receptor tyrosine kinase Mertk gene (D’Cruz et al., 2000). RPE cells fail to shed the outer segments of the photoreceptor cells in the RCS rat (Mullen et al., 1996). Recently, mice with a targeted disruption of the Mertk gene manifest retinal dystrophy similar to RCS rats (Duncan et al., 2003). In addition, mutated Mertk gene has been identified in patients with retinitis pigmentosa (Gal et al., 2002).

s / Digestive and Liver Disease 46S (2014) e136–e141 e137 all 248 patients had reached HBV undetectability

Background: Studies investigating the potential utility of novel small molecule dual MerTK and Flt3 inhibitors (MRX TKIs1) as therapy for AML and ALL will be presented. Methods: MerTK and Flt3 phosphorylation were measured using enzymatic and cell-based assays. Induction of apoptosis was determined using flow cytometry and colony-forming potential was assessed in methylcellulose or soft agar cultures. Orthotopic xenografts were generated in NOD/Scid/IL-2 gamma mice using a luciferase-expressing derivative of the 697 B-ALL cell line, the Molm14 AML cell line, or a Molm14 derivative with the FLT3-ITD D835Y or F691L mutation. Orthotopic patient-derived xenografts (PDX) were generated from primary AML samples. Inhibition of MerTK in bone marrow leukemia cells was assessed by immunoblot. Disease burden was measured using bioluminescence imaging or flow cytometry and median survival was determined. Results: MRX TKI inhibited MerTK and Flt3 with nanomolar potency and had limited off-target activity against 300 other kinases. MRX TKI decreased pro-survival signaling, induced apoptosis, and reduced colony-forming potential in leukemia cells. In B-ALL xenografts, MRX TKI inhibited MerTK phosphorylation in leukemic blasts leading to a dose-dependent reduction in tumor burden and a 2- to 3-fold increase in survival. MRX TKI also increased sensitivity to methotrexate, a chemotherapy currently used for treatment of ALL, resulting in reduced tumor burden and a 50% increase in survival compared to either agent alone. In a MerTK-expressing AML PDX, treatment with MRX TKI resulted in disease regression in blood, spleen, and bone marrow and a 2-fold increase in survival. MRX TKI was similarly effective in xenograft models of FLT3-ITD AML. Importantly, MRX TKI has activity against clinically-relevant FLT3 mutants that mediate resistance to current FLT3 TKIs. Conclusions: The very high potency, relative selectivity, oral bioavailability, target inhibition, and therapeutic efficacy mediated by MRX TKIs in MerTK and FLT3-dependent murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of acute leukemia.

Internal tandem duplication mutations of the FLT3 tyrosine kinase (FLT3-ITDs) are present in 15-30% of cases of pediatric and adult acute myeloid leukemias (AML), and are known to portend a poor prognosis. Over the past decade small molecule inhibitors targeting FLT3 have entered clinical trials. Although initial responses have been observed, patients typically develop resistance to current FLT3 inhibitors during the first year of therapy via acquisition of or selection for point mutations in the FLT3 kinase domain at amino acid F691 or D835. Although FLT3 is the most commonly targeted protein in AML, other candidates for pharmacologic inhibition have been identified. Ectopic expression of MerTK, a receptor tyrosine kinase, has been identified in 80-100% of primary AML patient samples, and previous data demonstrate anti-leukemic effects in response to shRNA-mediated MerTK inhibition. Here we describe MRX2843, a novel small molecule inhibitor of MerTK and FLT3 with activity against FLT3 point mutations and therapeutic efficacy in mouse xenograft models of AC220-resistant AML. MRX2843 potently inhibits MerTK and FLT3 with enzymatic IC50 values of 1.3 and 0.64nM, respectively. Treatment of two AML cell lines that express a FLT3-ITD mutation (MV4;11 has low MerTK expression; MOLM-14 does not express MerTK), and two AML cell lines that express MerTK but do not have activating mutations in FLT3 (U937, Kasumi-1) with 25-300nM MRX2843 abrogated activation of intracellular signaling pathways downstream of FLT3 and MerTK, including AKT and ERK1/2. In addition, 72 hour treatment with MRX2843 led to induction of apoptosis in AML cell lines compared to vehicle-treated controls, as determined by flow cytometic analysis after staining with YO-PRO-1 iodide and propidium iodide. For example, in MOLM-14 cultures, treatment with MRX2843 resulted induction of apoptosis in 84±2% of cells, compared to 5±2% after vehicle treatment (p To determine the effects of MRX2843 treatment in vivo, a patient-derived murine xenograft model was established by intravenous injection of primary AML patient blasts that express both MerTK and FLT3-ITD into NOD-SCID-gamma (NSG) mice. Engraftment was monitored by flow cytometric determination of peripheral blast count. When ~10% peripheral blasts were detected, mice were randomized to treatment with 50mg/kg MRX2843 or vehicle (saline) once daily by oral gavage. Treatment with MRX2843 significantly prolonged survival (median survival of 96 days after MRX2843 treatment versus 16 days in control mice, n=4 per group, p Two derivatives of the human FLT3-ITD AML cell line MOLM-14, which acquired either the D835Y (activation loop) or F691L (gatekeeper) mutation after selection in escalating doses of the FLT3 inhibitor AC220, were used to test the activity of MRX2843 against clinically relevant FLT3 point mutations. Treatment with MRX2843 resulted in a significant reduction in the number of viable cells after 48 hours of culture in MOLM-14, MOLM-14:D834Y, and MOLM-14:F691L with IC50 values of 17nM, 20nM, and 30nM, respectively. In both mutant cell lines, MRX2843 potently inhibited phosphorylation of FLT3 and abrogated activation of downstream intracellular signaling molecules. Treatment with MRX2843 induced cell death in MOLM-14:D835Y (80±7% versus 10±1%, p A murine model of AC220-resistant AML was developed by intravenous injection of MOLM-14:D835Y cells into NSG mice. Daily treatment with saline, 10mg/kg AC220, or 50mg/kg MRX2843 was administered by oral gavage beginning 4 days after transplant. Treatment with MRX2843 significantly prolonged survival when compared to mice treated with saline or AC220 with median survival of 103 days, 33 days, and 48 days, respectively (n=5 per group, p In summary, MRX2843 is a novel MerTK and FLT3 inhibitor that retains activity against clinically relevant, resistance-conferring FLT3 point mutations and prolongs survival in murine models of AML. These data support further development of MRX2843 and advancement to early phase clinical trials. Disclosures DeRyckere: University of Colorado - Denver: targeting of the Mer tyrosine kinase as cancer therapy Patents & Royalties; Meryx, Inc: Equity Ownership. Wang:University of North Carolina Chapel Hill: MRX-2843 Patents & Royalties; Meryx, Inc: Equity Ownership. Frye:University of North Carolina Chapel Hill: MRX-2843 Patents & Royalties; Meryx, Inc: Equity Ownership. Earp:University of North Carolina Chapel Hill: MRX-2843 Patents & Royalties; University of North Carolina Chapel Hill: targeting of the Mer tyrosine kinase as cancer therapy Patents & Royalties; Meryx, Inc: Equity Ownership. Graham:University of Colorado - Denver: MRX-2843 Patents & Royalties; University of Colorado - Denver: targeting of the Mer tyrosine kinase as cancer therapy Patents & Royalties; Meryx, Inc: Equity Ownership.

The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported.An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively.Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months.This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

Mutation of a receptor tyrosine kinase gene, Mertk, in the Royal College of Surgeons (RCS) rat results in defective phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) and retinal degeneration. We screened the human orthologue, MERTK, located at 2q14.1 (ref. 10), in 328 DNA samples from individuals with various retinal dystrophies and found three mutations in three individuals with retinitis pigmentosa (RP). Our findings are the first conclusive evidence implicating the RPE phagocytosis pathway in human retinal disease.

PURPOSE. Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis. METHODS. Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells. RESULTS. Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect. CONCLUSIONS. Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of US.

The TAM receptors are a distinct family of three receptor tyrosine kinases, namely Tyro3, Axl, and MerTK. Since their discovery in the early 1990s, they have been studied for their ability to influence numerous diseases, including cancer, chronic inflammatory and autoimmune disorders, and cardiovascular diseases. The TAM receptors demonstrate an ability to influence multiple aspects of cardiovascular pathology via their diverse effects on cells of both the vasculature and the immune system. In this review, we will explore the various functions of the TAM receptors and how they influence cardiovascular disease through regulation of vascular remodelling, efferocytosis and inflammation. Based on this information, we will suggest areas in which further research is required and identify potential targets for therapeutic intervention.