CRISPR/Cas9-Mediated Modification of PTP Expression.

Alteration of protein tyrosine phosphatase (PTP) gene expression is a commonly used approach to experimentally analyze their function in the cell physiology of mammalian cells. Here, exemplified for receptor-type PTPRJ (Dep-1, CD148) and PPTRC (CD45), we provide the CRISPR/Cas9-mediated approaches for their inactivation and transcriptional activation using genome editing. These methods are generally applicable to any other protein of interest.

Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD.

The receptor protein tyrosine phosphatase (RPTP) PTPRJ (also known as DEP-1) has been identified as a negative regulator of the receptor tyrosine kinase FLT3 signalling in vitro. The inactivation of the PTPRJ gene in mice expressing the constitutively active, oncogenic receptor tyrosine kinase FLT3 ITD aggravated known features of leukaemogenesis, revealing PTPRJ's antagonistic role. FLT3 ITD mutations resulting in constitutively kinase activity and cell transformation frequently occur in patients with acute myeloid leukaemia (AML). Thus, in situ activation of PTPRJ could be used to abrogate oncogenic FLT3 signalling. The activity of PTPRJ is suppressed by homodimerization, which is mediated by transmembrane domain (TMD) interactions. Specific Glycine-to-Leucine mutations in the TMD disrupt oligomerization and inhibit the Epidermal Growth Factor Receptor (EGFR) and EGFR-driven cancer cell phenotypes. To study the effects of PTPRJ TMD mutant proteins on FLT3 ITD activity in cell lines, endogenous PTPRJ was inactivated and replaced by stable expression of PTPRJ TMD mutants. Autophosphorylation of wild-type and ITD-mutated FLT3 was diminished in AML cell lines expressing the PTPRJ TMD mutants compared to wild-type-expressing cells. This was accompanied by reduced FLT3-mediated global protein tyrosine phosphorylation and downstream signalling. Further, PTPRJ TMD mutant proteins impaired the proliferation and in vitro transformation of leukemic cells. Although PTPRJ's TMD mutant proteins showed impaired self-association, the specific phosphatase activity of immunoprecipitated proteins remained unchanged. In conclusion, this study demonstrates that the destabilization of PTPRJ TMD-mediated self-association increases the activity of PTPRJ in situ and impairs FLT3 activity and FLT3-driven cell phenotypes of AML cells. Thus, disrupting the oligomerization of PTPRJ in situ could prove a valuable therapeutic strategy to restrict oncogenic FLT3 activity in leukemic cells.

PI3Kγ is a novel regulator of TNFα signaling in the human colon cell line HT29/B6.

Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes phosphorylating phospholipids in the membrane, thereby, promoting the PI3K/AKT signaling cascade. PI3Ks are involved in a variety of fundamental cellular functions, including tumor necrosis factor α (TNFα)-induced tight junction (TJ) impairment-a hallmark of inflammatory bowel diseases. Most of the studies analyzing the role of class I PI3K signaling in epithelial barrier maintenance did not decipher which of the isoforms are responsible for the observed effects. By using wild-type and PI3Kγ-deficient HT-29/B6 cells, we characterized the functional role of PI3Kγ in these cells under inflammatory conditions. Measurement of the transepithelial electrical resistance and the paracellular flux of macromolecules revealed that monolayers of PI3Kγ-deficient cells, compared with wild-type cells, were protected against TNFα-induced barrier dysfunction. This effect was independent of any PI3K activity because treatment with a pan-PI3K inhibitor did not alter this observation. By immunostaining, we found correlative changes in the distribution of the TJ marker ZO-1. Furthermore, the absence of PI3Kγ reduced the basal level of the pore-forming TJ protein claudin-2. Our study suggests a novel noncanonical, kinase-independent scaffolding function of PI3Kγ in TNFα-induced barrier dysfunction.

PI3Kγ Mediates Microglial Proliferation and Cell Viability via ROS.

(1) Background: Rapid microglial proliferation contributes to the complex responses of the innate immune system in the brain to various neuroinflammatory stimuli. Here, we investigated the regulatory function of phosphoinositide 3-kinase γ (PI3Kγ) and reactive oxygen species (ROS) for rapid proliferation of murine microglia induced by LPS and ATP. (2) Methods: PI3Kγ knockout mice (PI3Kγ KO), mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) and wild-type mice were assessed for microglial proliferation using an in vivo wound healing assay. Additionally, primary microglia derived from newborn wild-type, PI3Kγ KO and PI3Kγ KD mice were used to analyze PI3Kγ effects on proliferation and cell viability, senescence and cellular and mitochondrial ROS production; the consequences of ROS production for proliferation and cell viability after LPS or ATP stimulation were studied using genetic and pharmacologic approaches. (3) Results: Mice with a loss of lipid kinase activity showed impaired proliferation of microglia. The prerequisite of induced microglial proliferation and cell viability appeared to be PI3Kγ-mediated induction of ROS production. (4) Conclusions: The lipid kinase activity of PI3Kγ plays a crucial role for microglial proliferation and cell viability after acute inflammatory activation.

Biocompatibility, uptake and subcellular localization of bacterial magnetosomes in mammalian cells.

Magnetosomes represent biogenic, magnetic nanoparticles biosynthesized by magnetotactic bacteria. Subtle biological control on each step of biomineralization generates core-shell nanoparticles of high crystallinity, strong magnetization and uniform shape and size. These features make magnetosomes a promising alternative to chemically synthesized nanoparticles for many applications in the biotechnological and biomedical field, such as their usage as biosensors in medical diagnostics, as drug-delivery agents, or as contrast agents for magnetic imaging techniques. Thereby, the particles are directly applied to mammalian cells or even injected into the body. In the present work, we provide a comprehensive characterization of isolated magnetosomes as potential cytotoxic effects and particle uptake have not been well studied so far. Different cell lines including cancer cells and primary cells are incubated with increasing particle amounts, and effects on cell viability are investigated. Obtained data suggest a concentration-dependent biocompatibility of isolated magnetosomes for all tested cell lines. Furthermore, magnetosome accumulation in endolysosomal structures around the nuclei is observed. Proliferation rates are affected in the presence of increasing particle amounts; however, viability is not affected and doubling times can be restored by reducing the magnetosome concentration. In addition, we evidence magnetosome-cell interactions that are strong enough to allow for magnetic cell sorting. Overall, our study not only assesses the biocompatibility of isolated magnetosomes, but also evaluates effects on cell proliferation and the fate of internalized magnetosomes, thereby providing prerequisites for their future in vivo application as biomedical agents.

Aberrant Bone Homeostasis in AML Is Associated with Activated Oncogenic FLT3-Dependent Cytokine Networks.

Acute myeloid leukaemia (AML) is a haematopoietic malignancy caused by a combination of genetic and epigenetic lesions. Activation of the oncoprotein FLT3 ITD (Fms-like tyrosine kinase with internal tandem duplications) represents a key driver mutation in 25-30% of AML patients. FLT3 is a class III receptor tyrosine kinase, which plays a role in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Mutant FLT3 ITD results in an altered signalling quality, which causes cell transformation. Recent evidence indicates an effect of FLT3 ITD on bone homeostasis in addition to haematological aberrations. Using gene expression data repositories of FLT3 ITD-positive AML patients, we identified activated cytokine networks that affect the formation of the haematopoietic niche by controlling osteoclastogenesis and osteoblast functions. In addition, aberrant oncogenic FLT3 signalling of osteogenesis-specific cytokines affects survival of AML patients and may be used for prognosis. Thus, these data highlight the intimate crosstalk between leukaemic and osteogenic cells within the osteohaematopoietic niche.

Novel Approaches to Target Mutant FLT3 Leukaemia.

Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinases (RTK) and is involved in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Oncogenic mutations in the FLT3 gene resulting in constitutively active FLT3 variants are frequently found in acute myeloid leukaemia (AML) patients and correlate with patient's poor survival. Targeting FLT3 mutant leukaemic stem cells (LSC) is a key to efficient treatment of patients with relapsed/refractory AML. It is therefore essential to understand how LSC escape current therapies in order to develop novel therapeutic strategies. Here, we summarize the current knowledge on mechanisms of FLT3 activity regulation and its cellular consequences. Furthermore, we discuss how aberrant FLT3 signalling cooperates with other oncogenic lesions and the microenvironment to drive haematopoietic malignancies and how this can be harnessed for therapeutical purposes.

SIRT7: an influence factor in healthy aging and the development of age-dependent myeloid stem-cell disorders.

Molecular alterations within the hematopoietic system influence cellular longevity and development of age-related myeloid stem-cell disorders like acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). A reduced SIRT7-expression in aged murine hematopoietic stem cells (HSC) resulted in reduced longevity and increased proliferation. In this study we investigated age-related changes of SIRT7-expression in healthy humans and relevant pathomechanisms in AML and CML. SIRT7-expression in leukocytes of healthy people decreased in an age-dependent manner. Low SIRT7 mRNA levels were also detected in AML and CML patients. With positive treatment response, SIRT7-expression increased, but showed reduction when patients progressed or relapsed. Pharmacologic inhibition of driver mutations in AML (FLT3-ITD) or CML (BCR-ABL) also restored SIRT7 levels in cell lines and patient samples. Furthermore, SIRT7-expression increased with time during PMA-mediated monocyte differentiation of THP-1 cells. SIRT7-overexpression in THP-1 cells resulted in increased expression of differentiation markers. BCR-ABL, FLT3-ITD, and differentiation-associated SIRT7-expression in general were positively regulated by C/EBPα, -ß, and -ε binding to two different C/EBP-binding sites within the SIRT7 promoter. SIRT7 is important in human hematopoietic cell aging and longevity. It might act as tumor suppressor and could potentially serve as general biomarker for monitoring treatment response in myeloid stem-cell disorders.

Wild-type FLT3 and FLT3 ITD exhibit similar ligand-induced internalization characteristics.

Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)-dependent internalization of surface-bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4-11 and MV4-11 cells with endogenous wild-type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild-type, or FLT3 ITD-GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL-stimulated surface-exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL-induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin-dependent endocytosis. Internalization of kinase-inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes.

Knockout of LASP1 in CXCR4 expressing CML cells promotes cell persistence, proliferation and TKI resistance.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR-ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR-ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1-mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell-mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.

Lack of CD45 in FLT3-ITD mice results in a myeloproliferative phenotype, cortical porosity, and ectopic bone formation.

The receptor tyrosine kinase FLT3 is expressed in myeloid and lymphoid progenitor cells. Activating mutations in FLT3 occur in 25-30% of acute myeloid leukaemia (AML) patients. Most common are internal tandem duplications of sequence (ITD) leading to constitutive FLT3-ITD kinase activity with an altered signalling quality promoting leukaemic cell transformation. Here, we observed the attenuating role of the receptor-like protein tyrosine phosphatase (RPTP) CD45/Ptprc in FLT3 signalling in vivo. Low level expression of this abundant RPTP correlates with a poor prognosis of FLT3-ITD-positive AML patients. To get a further insight into the regulatory role of Ptprc in FLT3-ITD activity in vivo, Ptprc knock-out mice were bred with FLT3-ITD knock-in mice. Inactivation of the Ptprc gene in FLT3-ITD mice resulted in a drastically shortened life span and development of severe monocytosis, a block in B-cell development and anaemia. The myeloproliferative phenotype was associated with extramedullary haematopoiesis, splenohepatomegaly and severe alterations of organ structures. The phenotypic alterations were associated with increased transforming signalling of FLT3-ITD, including activation of its downstream target STAT5. These data reveal the capacity of Ptprc for the regulation of FLT3-ITD signalling activity in vivo. In addition, histopathology and computed tomography (CT) revealed an unexpected bone phenotype; the FLT3-ITD Ptprc-/- mice, but none of the controls, showed pronounced alterations in bone morphology and, in part, apparent features of osteoporosis. In the spleen, ectopic bone formation was observed. The observed bone phenotypes suggest a previously unappreciated capacity of FLT3-ITD (and presumably FLT3) to regulate bone development/remodelling, which is under negative control of CD45/Ptprc.

Hyperglycaemia-induced methylglyoxal accumulation potentiates VEGF resistance of diabetic monocytes through the aberrant activation of tyrosine phosphatase SHP-2/SRC kinase signalling axis.

Diabetes mellitus (DM) is a major cardiovascular risk factor contributing to cardiovascular complications by inducing vascular cell dysfunction. Monocyte dysfunction could contribute to impaired arteriogenesis response in DM patients. DM monocytes show blunted chemotactic responses to arteriogenic stimuli, a condition termed as vascular endothelial growth factor (VEGF) resistance. We hypothesize that methylglyoxal (MG), a glucose metabolite, induces monocyte dysfunction and aimed to elucidate the underlying molecular mechanisms. Human monocytes exposed to MG or monocytes from DM patients or mice (db/db) showed VEGF-resistance secondary to a pro-migratory phenotype. Mechanistically, DM conditions or MG exposure resulted in the upregulation of the expression of SHP-2 phosphatase. This led to the enhanced activity of SHP-2 and aided an interaction with SRC kinase. SHP-2 dephosphorylated the inhibitory phosphorylation site of SRC leading to its abnormal activation and phosphorylation of cytoskeletal protein, paxillin. We demonstrated that MG-induced molecular changes could be reversed by pharmacological inhibitors of SHP-2 and SRC and by genetic depletion of SHP-2. Finally, a SHP-2 inhibitor completely reversed the dysfunction of monocytes isolated from DM patients and db/db mice. In conclusion, we identified SHP-2 as a hitherto unknown target for improving monocyte function in diabetes. This opens novel perspectives for treating diabetic complications associated with impaired monocyte function.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis.

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

Synergistic killing of FLT3ITD-positive AML cells by combined inhibition of tyrosine-kinase activity and N-glycosylation.

Fms-like tyrosine kinase 3 (FLT3) with internal tandem duplications (ITD) is a major oncoprotein in acute myeloid leukemia (AML), and confers an unfavorable prognosis. Interference with FLT3ITD signaling is therefore pursued as a promising therapeutic strategy. In this study we show that abrogation of FLT3ITD glycoprotein maturation using low doses of the N-glycosylation inhibitor tunicamycin has anti-proliferative and pro-apoptotic effects on FLT3ITD-expressing human and murine cell lines. This effect is mediated in part by arresting FLT3ITD in an underglycosylated state and thereby attenuating FLT3ITD-driven AKT and ERK signaling. In addition, tunicamycin caused pronounced endoplasmatic reticulum stress and apoptosis through activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of the gene encoding CCAAT-enhancer-binding protein homologous protein (CHOP). PERK inhibition with a small molecule attenuated CHOP induction and partially rescued cells from apoptosis. Combination of tunicamycin with potent FLT3ITD kinase inhibitors caused synergistic cell killing, which was highly selective for cell lines and primary AML cells expressing FLT3ITD. Although tunicamycin is currently not a clinically applicable drug, we propose that mild inhibition of N-glycosylation may have therapeutic potential in combination with FLT3 kinase inhibitors for FLT3ITD-positive AML.

Isoform localization of Dectin-1 regulates the signaling quality of anti-fungal immunity.

Dectin-1 is recognized as a major receptor for fungal ß-glucans and contributes to anti-fungal immunity. Human monocyte populations express Dectin-1 isoforms A and B, which differ by the presence of a stalk region and its N-linked glycosylation site. Here, we analyzed the expression of both isoforms in human monocyte-derived cells. The cellular localization on cell lines stably expressing either Dectin-1 isoform A or B was studied by flow cytometry and confocal laser scanning microscopy. Intracellular protein signaling and cytokine production were analyzed by immunoblotting and cytometric bead array, respectively. Monocyte-derived cells showed cell type-specific expression of the two isoforms. Glycosylated Dectin-1 isoform A was predominantly localized at the cell surface, non-glycosylated isoform B was retained intracellularly. Inhibition of glycosylation resulted in efficient abrogation of cell surface expression of isoform A. Signaling quality following Dectin-1 stimulation was reduced in isoform B cells. Differential isoform specific cytokine secretion was observed by cytometric bead array. We show here that n-glycosylation of Dectin-1 is crucial for its cell surface expression and consequently signal transduction. Taken together, unique cytokine secretion and varying expression levels of human Dectin-1 isoforms on monocyte-derived cells may indicate distinct isoform usage as a cell type-specific mechanism of regulating anti-fungal immunity.

Phosphoinositide 3-kinase γ ties chemoattractant- and adrenergic control of microglial motility.

Microglial motility is tightly controlled by multitude of agonistic and antagonistic factors. Chemoattractants, released after infection or damage of the brain, provoke directed migration of microglia to the pathogenic incident. In contrast, noradrenaline and other stress hormones have been shown to suppress microglial movement. Here we asked for the signaling reactions involved in the positive and negative control of microglial motility. Using pharmacological and genetic approaches we identified the lipid kinase activity of phosphoinositide 3-kinase species γ (PI3Kγ) as an essential mediator of microglial migration provoked by the complement component C5a and other chemoattractants. Inhibition of PI3Kγ lipid kinase activity by protein kinase A was disclosed as mechanism causing suppression of microglial migration by noradrenaline. Together these data characterize PI3Kγ as a nodal point in the control of microglial motility.

The protein-tyrosine phosphatase DEP-1 promotes migration and phagocytic activity of microglial cells in part through negative regulation of fyn tyrosine kinase.

Microglia cells are brain macrophages whose proper functioning is essential for maintenance and repair processes of the central nervous system (CNS). Migration and phagocytosis are critical aspects of microglial activity. By using genetically modified cell lines and knockout mice we demonstrate here that the receptor protein-tyrosine phosphatase (PTP) DEP-1 (also known as PTPRJ or CD148) acts as a positive regulator of both processes in vitro and in vivo. Notably, reduced microglial migration was detectable in brains of Ptprj-/- mice using a wounding assay. Mechanistically, density-enhanced phosphatase-1 (DEP-1) may in part function by inhibiting the activity of the Src family kinase Fyn. In the microglial cell line BV2 DEP-1 depletion by shRNA-mediated knockdown resulted in enhanced phosphorylation of the Fyn activating tyrosine (Tyr420 ) and elevated specific Fyn-kinase activity in immunoprecipitates. Moreover, Fyn mRNA and protein levels were reduced in DEP-1 deficient microglia cells. Consistent with a negative regulatory role of Fyn for microglial functions, which is inhibited by DEP-1, microglial cells from Fyn-/- mice exhibited elevated migration and phagocytosis. Enhanced microglia migration to a site of injury was also observed in Fyn-/- mice in vivo. Taken together our data revealed a previously unrecognized role of DEP-1 and suggest the existence of a potential DEP-1-Fyn axis in the regulation of microglial functions. GLIA 2017;65:416-428.