Inhibit Calcineurin Phosphatase Research Articles

D-2-Hydroxyglutarate Inhibits Calcineurin Phosphatase Activity to Abolish NF-AT Activation and IL-2 Induction in Stimulated Lymphocytes.

Gliomas expressing mutant isocitrate dehydrogenases excessively synthesize d-2-hydroxyglutarate (D2HG), suppressing immune surveillance. A portion of this D2HG is released from these tumor cells, but the way environmental D2HG inhibits lymphocyte function is undefined. We incubated human PBLs or Jurkat T cells with D2HG at concentrations present within and surrounding gliomas or its obverse l-2-hydroxyglutarate (L2HG) stereoisomer. We quantified each 2HG stereoisomer within washed cells by N-(p-toluenesulfonyl)-l-phenylalanyl chloride derivatization with stable isotope-labeled D2HG and L2HG internal standards, HPLC separation, and mass spectrometry. D2HG was present in quiescent cells and was twice as abundant as L2HG. Extracellular 2HG rapidly increased intracellular levels of the provided stereoisomer by a stereoselective, concentration-dependent process. IL-2 expression, even when elicited by A23187 and PMA, was abolished by D2HG in a concentration-dependent manner, with significant reduction at just twice its basal level. In contrast, L2HG was only moderately inhibitory. IL-2 expression is regulated by increased intracellular Ca2+ that stimulates calcineurin to dephosphorylate cytoplasmic phospho-NF-AT, enabling its nuclear translocation. D2HG abolished stimulated expression of a stably integrated NF-AT-driven luciferase reporter that precisely paralleled its concentration-dependent inhibition of IL-2. D2HG did not affect intracellular Ca2+. Rather, surface plasmon resonance showed D2HG, but not L2HG, bound calcineurin, and D2HG, but not L2HG, inhibited Ca2+-dependent calcineurin phosphatase activity in stimulated Jurkat extracts. Thus, D2HG is a stereoselective calcineurin phosphatase inhibitor that prevents NF-AT dephosphorylation and so abolishes IL-2 transcription in stimulated lymphocytes. This occurs at D2HG concentrations found within and adjacent to gliomas independent of its metabolic or epigenetic transcriptional regulation.

Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion.

Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.

Identification of pyrogallol from Awa-tea as an anti-allergic compound that suppresses nasal symptoms and IL-9 gene expression

As the expression level of allergic disease sensitive genes are correlated with the severity of allergic symptoms, suppression of these gene expressions could be promising therapeutics. We demonstrated that protein kinase Cδ / heat shock protein 90-mediated H1R gene expression signaling and nuclear factor of activated T-cells (NFAT)-mediated IL-9 gene expression signaling are responsible for the pathogenesis of pollinosis. Treatment with Awa-tea combined with wild grape hot water extract suppressed these signaling and alleviated nasal symptoms in toluene-2,4-diisocyanate (TDI)-sensitized rats. However, the underlying mechanism of its anti-allergic activity is not elucidated yet. Here, we sought to identify an anti-allergic compound from Awa-tea and pyrogallol was identified as an active compound. Pyrogallol strongly suppressed ionomycin-induced up-regulation of IL-9 gene expression in RBL-2H3 cells. Treatment with pyrogallol in combination with epinastine alleviated nasal symptoms and suppressed up-regulation of IL-9 gene expression in TDI-sensitized rats. Pyrogallol itself did not inhibit calcineurin phosphatase activity. However, pyrogallol suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFAT. These data suggest pyrogallol is an anti-allergic compound in Awa-tea and it suppressed NFAT-mediated IL-9 gene expression through the inhibition of dephosphorylation of NFAT. This might be the underlying mechanism of the therapeutic effects of combined therapy of pyrogallol with antihistamine. J. Med. Invest. 67 : 289-297, August, 2020.

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways.

Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-β (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNβ, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

Structural design, synthesis and substituent effect of hydrazone-N-acylhydrazones reveal potent immunomodulatory agents

4-(Nitrophenyl)hydrazone derivatives of N-acylhydrazone were synthesized and screened for suppress lymphocyte proliferation and nitrite inhibition in macrophages. Compared to an unsubstituted N-acylhydrazone, active compounds were identified within initial series when hydroxyl, chloride and nitro substituents were employed. Structure-activity relationship was further developed by varying the position of these substituents as well as attaching structurally-related substituents. Changing substituent position revealed a more promising compound series of anti-inflammatory agents. In contrast, an N-methyl group appended to the 4-(nitrophenyl)hydrazone moiety reduced activity. Anti-inflammatory activity of compounds is achieved by modulating IL-1β secretion and prostaglandin E2 synthesis in macrophages and by inhibiting calcineurin phosphatase activity in lymphocytes. Compound SintMed65 was advanced into an acute model of peritonitis in mice, where it inhibited the neutrophil infiltration after being orally administered. In summary, we demonstrated in great details the structural requirements and the underlying mechanism for anti-inflammatory activity of a new family of hydrazone-N-acylhydrazone, which may represent a valuable medicinal chemistry direction for the anti-inflammatory drug development in general.

Regulator of Calcineurin (RCAN-1) Regulates Thermotaxis Behavior in Caenorhabditiselegans

Regulator of calcineurin (RCAN) is a calcineurin-interacting protein that inhibits calcineurin phosphatase when overexpressed, often upregulated under neuropathological conditions with impaired learning and memory processes, such as Down syndrome or Alzheimer's disease. Thermotactic behavior in the nematode Caenorhabditis elegans is a form of memory in which calcineurin signaling plays a pivotal role in the thermosensation of AFD neurons. In this study, we found that rcan-1 deletion mutants exhibited cryophilic behavior dependent on tax-6, which was rescued by expressing rcan-1 in AFD neurons. Interaction between RCAN-1 and TAX-6 requires the conserved PxIxIT motif of RCAN-1, without which thermotactic behavior could not be fully rescued. In addition, the loss of crh-1/CREB suppressed the thermotaxis phenotypes of rcan-1 and tax-6 mutants, indicating that crh-1 is crucial in thermotaxis memory in these mutants. Taken together, our results suggest that rcan-1 is an inhibitory regulator of tax-6 and that it acts in the formation of thermosensory behavioral memory in C. elegans.

Calcineurin and Sorting-Related Receptor with A-Type Repeats Interact to Regulate the Renal Na⁺-K⁺-2Cl⁻ Cotransporter.

The furosemide-sensitive Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Aβ isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Aβ in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Aβ. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aβ and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aβ and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.

0151 : Role a calcium/calmodulin-dependent serine protein kinase on Cav1.2 channel regulation

Voltage-dependent calcium channels (Cav) constitute a major pathway for the entry of calcium into excitable cells. The long list of cardiac pathologies highlights the key role played by Cav1.2 channels in cardiac physiology. A calcium/calmodulin-dependent serine protein kinase (CASK) has been recently identified as a potent new protein interacting with Cav1.2 channel. Nevertheless, despite the fact that CASK is expressed in cardiac cells, to this date, no functional experiments have been performed to investigate the role of CASK on Cav1.2 channels regulation. Full length or the C-terminal truncated (2024stop) Cavα1c subunit, both expressed in cardiac cells, was coexpressed with Cavβ2 and Cavα2-δ1 subunits in TsA-201 cells. In addition scramble or silencer of CASK protein was co-transfected with Cav1.2 channels. Barium currents (IBa) and calcium current (ICa) were recorded under voltage-clamp conditions using the whole-cell configuration. After 72 hours post-transfection, the cells silenced for CASK, expressing either the full length or 2024stop Cavα1c subunits, show an increase of ICa. In contrast, in similar condition of transfection, the IBa current is only increased in the condition with the C-terminal truncated Cavα1c subunit suggesting that the C-terminal part of Cavα1c subunit counteracts the CASK silencing effect. Interestingly, the C-terminal part of Cavα1c subunit masks an interacting motif for the calcium-dependent serine-threonine phosphatase: calcineurin. One hour incubation with cyclosporin A, a specific inhibitor of calcineurin phosphatase, blunts the CASK silencing effect on IBa with the C-terminal truncated part of Cavα1c subunit bringing out the key role of calcineurin in the mechanism of Cav1.2 channels regulation by CASK. In the present study, we highlight CASK protein as a novel regulator of voltage-gated calcium channel Cav1.2 via the calcineurin.

Combination therapy for KIT-mutant mast cells: targeting constitutive NFAT and KIT activity.

Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.

Phosphodiesterase type 5 inhibition attenuates cyclosporine A induced nephrotoxicity in mice

We investigated the renal protective effects of phophodiesterase type 5 (PDE5) inhibitors in mice with cyclosporine A (CyA; a calcineurin phosphatase inhibitor) induced nephrotoxicity. Fifty male mice were divided into five groups of 10. Group 1 received no treatment, group 2 received only saline orally, group 3 received 30 mg/kg/day CyA by subcutaneous injection, group 4 received only 30 mg/kg/day vardenafil orally, and group 5 received 30 mg/kg/day CyA by subcutaneous injection and 30 mg/kg/day vardenafil orally. At 28 days, platelet-derived growth factor A (PDGF-A) and C (PDGF-C), transforming growth factor-beta 1 (TGF-β1), cyclo-oxygenase 1 and 2 (COX-1 and COX-2), and P glycoprotein (Pgp) expression levels were measured in the renal tissues. In addition, expressions of COX-1 and COX-2 genes were determined using real-time PCR. PDE5 inhibitor administration ameliorated decreased PDGF-A and C, TGF-β1, COX-1 and -2, and Pgp expression levels by modulation of cyclic guanosine monophosphate (cGMP) activity in kidneys. The relative expressions of COX-1 and COX-2 genes to GAPDH revealed that the maximum increase was obtained in the group treated with CyA and vardenafil for both COX-1 and COX-2 genes. Our study revealed that long term oral treatment with vardenafil protects kidneys from CyA induced nephrotoxicity. We showed that long term oral treatment with PDE5 prevents pathological kidney changes caused by CyA induced nephrotoxicity.

Biomimetic synthesis of the calcineurin phosphatase inhibitor dibefurin.

Dibefurin is a Ci -symmetric natural product that acts as an inhibitor of calcineurin phosphatase. A six-step synthesis of this compound is reported, which features an oxidative dimerization of the aromatic polyketide epicoccine as the key step. Dibefurin is proposed to be related to epicolactone, a complex yet racemic fungal metabolite that has recently been discovered. Attempts to access epicolactone from epicoccine and epicoccone B resulted in an unusual dimer that is formed through a hetero-Diels-Alder reaction of a para-quinone methide with an ortho-quinone.

Biomimetic Synthesis of the Calcineurin Phosphatase Inhibitor Dibefurin

AbstractDibefurin is a Ci‐symmetric natural product that acts as an inhibitor of calcineurin phosphatase. A six‐step synthesis of this compound is reported, which features an oxidative dimerization of the aromatic polyketide epicoccine as the key step. Dibefurin is proposed to be related to epicolactone, a complex yet racemic fungal metabolite that has recently been discovered. Attempts to access epicolactone from epicoccine and epicoccone B resulted in an unusual dimer that is formed through a hetero‐Diels–Alder reaction of a para‐quinone methide with an ortho‐quinone.

Echinomycin, a Potential Binder of FKBP12, Shows Minor Effect on Calcineurin Activity

Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor–1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was –10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR.

Neuroprotective Copper Bis(thiosemicarbazonato) Complexes Promote Neurite Elongation

Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, CuII(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that CuII(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, CuII(atsm), but at a higher concentration. Induction of neurite elongation by CuII(gtsm) was restricted to neurites within the length range of 75–99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM CuII(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that CuII(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM CuII(gtsm), suggesting a potential link between CuII(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes.

Combination Therapy In Mast Cell Neoplasms: Co-Targeting KIT and NFAT Signaling Pathways

Activating KIT mutations are a hallmark of human systemic mast cell disease and have also been identified in gastrointestinal stromal tumors, AML, melanoma, and seminoma. Traditionally, constitutively active KIT mutations have been targeted by tyrosine kinase inhibitors; however, currently available inhibitors have limited activity against resistant mutations and sustainable responses are often not achieved. Our goal has been to identify a synergistic drug combination that increases the effectiveness of KIT inhibitors. A recent study in chronic myelogenous leukemia reported enhanced anti-proliferative/pro-apoptotic effects when combining a BCR-ABL inhibitor with inhibitors targeting a novel Wnt/Ca++/Calcineurin/NFAT pathway. Based on shared downstream signaling mechanisms between BCR-ABL and KIT kinases we hypothesized that a similar combination would work to target KIT mutant kinases.We found that combining a KIT inhibitor with a calcineurin phosphatase inhibitor led to a synergistic decrease (average combination index (CI) values less than 1.0) in cell viability as well as an increase in apoptosis in KIT-mutant cell lines including six mastocytosis cell lines. For example, we found that 1uM of cyclosporine A (CSA) plus 20nM of dasatinib in the p815 cell line consistently reduced cell viability by 50% compared to dasatinib alone. This decrease in cell viability corresponded to a 40 fold increase in capsase 3/7 activity (Figure 1). Similar results were obtained using the other five mastocytosis cell lines. [Display omitted] The activity of NFAT transcription factors is known to be regulated by the phosphatase activity of calcineurin. To confirm our results were calcineurin- and NFAT-dependent, we repeated the previous combination experiments with the NFAT specific inhibitors, rocaglamide and tributylhexadecylphosphonium bromide (THPB). We observed a synergistic decrease (average CI value 0.81 for rocaglamide and 0.43 for THPB) in cell viability and increase in apoptosis when combining these NFAT-specific and KIT kinase inhibitors, indicating NFAT as an important mediator of the observed synergy. Next we characterized NFAT in KIT mutant mast cells and found constitutive NFAT activation in each cell line, as indicated by nuclear localization and dephosphorylation under unstimulated conditions. NFAT-dependent transcriptional activity, as measured by a luciferase reporter system, decreased following treatment with either calcineurin or NFAT inhibitors. Unexpectedly, NFAT-dependent transcriptional activity also decreased following treatment with a number of KIT inhibitors. However, only calcineurin inhibitors and not KIT inhibitors, completely blocked NFAT activation following calcium influx, suggesting a unique mechanism by which KIT inhibition affects NFAT-dependent transcriptional activity. To determine downstream effectors of the observed synergy, we measured changes in RNA expression following mono- and combination therapy. RNA-Seq revealed synergistic decreases in a number of genes including members of the JAK-STAT and MAPK pathways, which are druggable targets downstream of this transcriptional program. Further validation studies of these targets are currently underway and will be reported later.The constitutive activation of NFAT has not been previously reported in KIT mutant cell lines. Further, our data suggest a point of convergence between KIT and NFAT signaling pathways, possibly through the interaction of NFAT with a DNA binding partner. Importantly, our results indicate that combination therapy using an NFAT inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant mast cells. However, existing calcineurin phosphatase inhibitors such as CSA have known chronic long-term toxicities, including immunosuppression. To identify alternative drug targets we used RNA-Seq and found that simultaneous calcineurin and KIT inhibition modulated members of the JAK-STAT and MAPK pathways. Preliminary data indicates that targeting these pathways in combination with dasatinib achieves the same synergistic effect on KIT mutant cancer cells. We are currently testing the potential of these alternative combination therapies for the treatment of advanced mast cell disease. Disclosures:Heinrich: Onyx: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; MolecularMD: Consultancy, Equity Ownership; AROG: Research Funding.

Natural regulatory T cells are resistant to calcium release-activated calcium (CRAC/ORAI) channel inhibition

Organ transplant patients are often treated with immunosuppressants, such as the calcineurin phosphatase inhibitor, cyclosporin A, to block T cell-mediated graft rejection. The calcium release-activated calcium (CRAC/ORAI) channels, which act upstream of calcineurin, are essential for calcium entry and CD4(+) T-cell activation. Although cyclosporine A has also been shown to inhibit FoxP3(+) Tregs both in vitro and in vivo, the role of ORAI channel inhibition in natural Tregs (nTregs) or inducible Tregs (iTregs) has not been investigated. We found that, despite inhibition of calcium influx through the ORAI channels, ORAI channel inhibitors were unable to repress FoxP3 expression in mouse and human nTregs, whereas FoxP3 expression was inhibited in iTregs. In contrast, cyclosporin A inhibited FoxP3 expression in both nTregs and iTregs. We also generated mice with a T cell-specific, conditional knockout of ORAI1 and found that the mice have normal nTreg development and suppressive activity. Moreover, iTregs derived from ORAI1 conditional knockout mice develop normally and are still susceptible to ORAI channel inhibition. Our data indicate that unlike CD4(+) T cells and iTregs, nTregs are resistant to ORAI-mediated inhibition. Targeting ORAI channels potentially offers a novel way to inhibit pathologic T cells, while sparing nTreg-mediated tolerance.

Abstract 3281: Combination therapy to target KIT mutant cells.

Abstract Purpose: Gastrointestinal stromal tumors (GIST), the most common abdominal sarcoma, arise from the interstitial cells of Cajal in the wall of the gut. Eighty percent of GISTs harbor activating mutations in the receptor tyrosine kinase KIT. Despite progress in medical treatment of GIST, advanced disease remains incurable. In addition to GIST, activating KIT mutations are found in cases of mast cell neoplasms, AML, melanoma and seminoma. Therefore, there is a compelling need to identify new targets whose inhibition is synergistic when combined with biochemical inhibition of KIT. Based on shared oncogenic kinase mechanisms with CML, we hypothesized that combined inhibition of KIT and a novel Wnt/Ca++/Calcineurin/NFAT pathway would synergistically inhibit the proliferation of KIT mutant cells. Experimental methods: We sequentially combined four different calcineurin inhibitors with five different KIT inhibitors and measured cell proliferation and caspase 3/7activity in nine unique KIT mutant cell lines. Using the Chou and Talalay method we calculated combination indices to quantify drug-drug interactions. Changes in NFAT phosphorylation and localization in KIT mutant cells were assessed by immunoblotting. Finally, we measured NFAT transcriptional activity using an NFAT-responsive reporter cell line. Results: In contrast to other cellular models, NFAT appeared to be constitutively active and localized in the nucleus in many KIT mutant cell lines. We observed synergy when we combined a calcineurin inhibitor with a KIT inhibitor in all KIT mutant cell lines where NFAT was constitutively active. Combination index (CI) values for the different KIT mutant cell lines ranged from 0.3-0.5. To investigate whether NFAT was required for this synergy, we combined NFAT specific inhibitors with KIT inhibitors and found synergistic inhibition of cell proliferation. We created a KIT-mutant reporter cell line where NFAT binding drives the expression of luciferase. As expected, NFAT transcriptional activity decreased when our promoter-reporter cell line was treated with calcineurin inhibitors and increased with TPA and ionomycin treatment. Unexpectedly, we also found that KIT inhibitors decreased NFAT transcriptional activity in this cell model. Conclusions: These data indicate that combination therapy using a calcineurin phosphatase inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant cells. Additionally, the constitutive activation of NFAT in these cell lines indicate aberrant signaling in the Wnt/Ca2+/calcineurin/NFAT pathway, which has not been previously reported in KIT mutant cell lines. The observed synergy between NFAT inhibitors and KIT inhibitors suggests this effect is mediated by NFAT rather than other calcineurin targets. Finally, the fact that KIT inhibitors modulated NFAT transcriptional activity suggests crosstalk between KIT and NFAT signaling pathways. Citation Format: Alison C. Macleod, Michael C. Heinrich, Diana Griffith, Ajia Town. Combination therapy to target KIT mutant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3281. doi:10.1158/1538-7445.AM2013-3281

Phosphorylation of TRPC4 as a regulatory mechanism of Isoc

Calcium signaling mediates a number of cellular events, and in many non‐excitable cells, store‐operated calcium entry (SOCE) represents the major Ca2+ event for signaling. In SOCE, depletion of ER Ca2+ stores leads to extracellular Ca2+ entry through SOC channels. Pulmonary artery endothelial cells (PAECs) exhibit a Ca2+‐selective SOCE current termed Isoc. Activation of Isoc in PAECs leads to formation of inter‐endothelial cell gaps. TRPC proteins 1 and 4 contribute to Isoc channel architecture. The region encompassing the protein 4.1 binding site and adjacent proline‐rich region on the TRPC4 subunit are essential for Isoc channel activation. Isoc inactivation is phosphorylation‐ and Ca2+‐dependent but the identity of kinases/phosphatases and their targets are still being explored. TRPC4 is a promising target for phosphoregulation of Isoc. Here, we examine the phosphorylation status of TRPC4 following activation of thapsigargin‐induced SOCE in PAECs. Following store depletion, TRPC4 phosphorylation increases as evidenced by western blot. In the presence of a calcineurin phosphatase inhibitor, baseline and thapsigargin‐induced TRPC4 phosphorylation is increased, and thapsigargin‐induced inter‐endothelial gap formation is reduced. These data suggest TRPC4 phosphorylation plays a role in Isoc regulation. Supported by 1F32HL112565–01 and 5R00HL089361

Ablation of Calcineurin Aβ Reveals Hyperlipidemia and Signaling Cross-talks with Phosphodiesterases

Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating β-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aβ (CnAβ(-/-)). We show that mice lacking calcineurin Aβ are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAβ(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by β-adrenergic G-protein-coupled receptor signaling pathways. CnAβ(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAβ(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAβ(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.

The use of ciclosporin A in veterinary dermatology

Ciclosporin A (CsA) has potent immunosuppressive and immunomodulatory activity that has been exploited in human medicine to prevent the rejection of transplanted organs and to manage atopic dermatitis and psoriasis. Over the past decade, CsA has been employed more frequently in veterinary dermatology and its value in the management of several canine and feline dermatoses is now well established. CsA inhibits calcineurin phosphatase, suppressing T cell activation and the synthesis of T cell cytokines consequently impairing the activity of B cells, antigen-presenting cells, mast cells, basophils and eosinophils. The pharmacokinetics of CsA are similar in humans, dogs and cats and the drug has a wide safety margin in dogs, cats and rabbits. Adverse effects, principally transient vomiting and soft faeces/diarrhoea, may be seen shortly after instituting treatment but often resolve despite continuing treatment. Gingival hyperplasia and cutaneous effects such as hirsutism may occur after prolonged treatment.