Restored Protein Phosphatase 2A Activity Research Articles

A unique role for IL-13 in inducing esophageal eosinophilia through MID-1 and STAT6.

Eosinophilic esophagitis (EoE) is associated with allergen-driven inflammation of the esophagus and an upregulated Th2 cytokine signature. Recombinant interleukin (IL)-13 (rIL-13) administration to mice induces some of the hallmark features of EoE, including increased eotaxin expression and eosinophil recruitment. Inflammation in EoE has previously been shown to depend on the expression of TRAIL and MID-1, which reduced protein phosphatase 2A (PP2A) activity. The relationship between IL-13 and TRAIL signalling in esophageal eosinophilia is currently unknown. To investigate the interaction between IL-13-driven eosinophil infiltration and TRAIL or MID-1 in the esophagus. We administered rIL-13 to wild type (WT), TRAIL-deficient (Tnsf10-/-) or STAT6-deficient (STAT6-/-) mice and targeted MID-1 with small interfering RNA. rIL-13 administration to mice increased TRAIL and MID-1 expression in the esophagus while reducing PP2A activity. TRAIL deficient, but not STAT6 deficient mice demonstrated increased MID-1 expression and PP2A reduction upon IL-13 challenge which correlated with eosinophil infiltration into the esophagus. Silencing MID-1 expression with siRNA completely ablated IL-13 induced eosinophil infiltration of the esophagus, restored PP2A activity, and reduced eotaxin-1 expression. IL-13-driven eosinophil infiltration of the esophagus induced eosinophilia and eotaxin-1 expression in a STAT6-dependent and MID-1-dependent manner. This study highlights a novel mechanism employed by IL-13 to perpetuate eosinophil infiltration.

Recombinant Human Proteoglycan 4 Regulates Phagocytic Activation of Monocytes and Reduces IL-1β Secretion by Urate Crystal Stimulated Gout PBMCs.

ObjectivesTo compare phagocytic activities of monocytes in peripheral blood mononuclear cells (PBMCs) from acute gout patients and normal subjects, examine monosodium urate monohydrate (MSU) crystal-induced IL-1β secretion ± recombinant human proteoglycan 4 (rhPRG4) or interleukin-1 receptor antagonist (IL-1RA), and study the anti-inflammatory mechanism of rhPRG4 in MSU stimulated monocytes.MethodsAcute gout PBMCs were collected from patients in the Emergency Department and normal PBMCs were obtained from a commercial source. Monocytes in PBMCs were identified by flow cytometry. PBMCs were primed with Pam3CSK4 (1μg/mL) for 24h and phagocytic activation of monocytes was determined using fluorescently labeled latex beads. MSU (200μg/mL) stimulated IL-1β secretion was determined by ELISA. Reactive oxygen species (ROS) generation in monocytes was determined fluorometrically. PBMCs were incubated with IL-1RA (250ng/mL) or rhPRG4 (200μg/mL) and bead phagocytosis by monocytes was determined. THP-1 monocytes were treated with MSU crystals ± rhPRG4 and cellular levels of NLRP3 protein, pro-IL-1β, secreted IL-1β, and activities of caspase-1 and protein phosphatase-2A (PP2A) were quantified. The peritoneal influx of inflammatory and anti-inflammatory monocytes and neutrophils in Prg4 deficient mice was studied and the impact of rhPRG4 on immune cell trafficking was assessed.ResultsEnhanced phagocytic activation of gout monocytes under basal conditions (p<0.001) was associated with ROS generation and MSU stimulated IL-1β secretion (p<0.05). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1β secretion (p<0.05). rhPRG4 reduced pro-IL-1β content, caspase-1 activity, conversion of pro-IL-1β to mature IL-1β and restored PP2A activity in monocytes (p<0.05). PP2A inhibition reversed rhPRG4’s effects on pro-IL-1β and mature IL-1β in MSU stimulated monocytes. Neutrophils accumulated in peritoneal cavities of Prg4 deficient mice (p<0.01) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (p<0.05).ConclusionsMSU phagocytosis was higher in gout monocytes resulting in higher ROS and IL-1β secretion. rhPRG4 reduced monocyte phagocytic activation to a greater extent than IL-1RA and reduced IL-1β secretion. The anti-inflammatory activity of rhPRG4 in monocytes is partially mediated by PP2A, and in vivo, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare.

Fingolimod Phosphate (FTY720-P) Activates Protein Phosphatase 2A in Human Monocytes and Inhibits Monosodium Urate Crystal-Induced Interleukin-1β Production.

Gout is a chronic inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystal deposits in joints of lower limbs. Phagocytic uptake of MSU crystals by joint-resident macrophages and recruited circulating monocytes results in IL-1β expression and production. Current acute gout treatments have serious toxicities and suffer suboptimal clinical outcomes. Protein phosphatase 2A (PP2A) plays an important role in regulating signaling pathways relevant to inflammation. We hypothesized that innate immune danger signals, e.g., lipopolysaccharide (LPS) and soluble uric acid (sUA), prime human monocytes toward MSU crystal phagocytosis and that increased IL-1β production mediated by a reduction in PP2A activity and restoring PP2A activity exerts an anti-inflammatory effect in this setting. Priming monocytes with LPS + sUA increased cytosolic pro-IL-1β and mature IL-1β and enhanced MSU crystal phagocytosis and its downstream IL-1β expression (P < 0.001). A combination of LPS + sUA priming and MSU crystals reduced PP2A activity in monocytes by 60% (P = 0.013). PP2A catalytic subunit gene knockdown reduced PP2A activity and exacerbated MSU crystal-induced IL-1β expression and secretion (P < 0.0001). Fingolimod (FTY720) and its active metabolite, fingolimod phosphate (FTY720-P), were evaluated for their ability to activate PP2A in human monocytes over 24 hours. FTY720 and FTY720-P activated PP2A to a similar extent, and maximal enzyme activity occurred at 24 hours for FTY720 and at 6 hours for FTY720-P. FTY720-P (2.5 μM) reduced pro-IL-1β production and IL-1β secretion in primed and MSU crystal-stimulated monocytes (P < 0.0001) without changing the magnitude of crystal phagocytosis. We conclude that PP2A is a promising new target in acute gout. SIGNIFICANCE STATEMENT: The activity of protein phosphatase 2A (PP2A) is implicated in the enhanced expression and production of IL-1β by human monocytes in response to priming with soluble uric acid and lipopolysaccharide and phagocytosis of monosodium urate monohydrate (MSU) crystals. Fingolimod phosphate activates PP2A in human monocytes and reduces cytosolic pro-IL-1β content and its conversion to biologically active IL-1β in human monocytes exposed to MSU crystals.

Abstract 425: Protein Kinase Activity of Pi3kγ Regulates Insulin-βArs Cross-talk

β adrenergic receptor (βAR) function is regulated by G-protein coupled receptor (GPCR) kinase (GRK) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization to its classical agonist. Surprisingly, Insulin modulates (βARs) function thus regulating cardiac function. Although insulin is known to modulate βAR function through GRKs, less is known about insulin mediated resensitization mechanisms. PI3Kγ is activated by GPCRs regulates resensitization by inhibiting PP2A activity. Therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Co-immunoprecipitation in cardiac lysates and surface plasmon resonance (SPR) with purified proteins show that PI3Kγ interacts with GRK-2 resulting in recruitment of GRK2-PI3Kγ to the β2AR complex upon insulin. Furthermore, use of PI3K inhibitors significantly reduced Insulin-stimulated β2ARs phosphorylation in HEK293 cells. The role of PI3Kγ was further validated by the loss of insulin stimulated β 2 AR phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs), shRNA and CRIPSR knockdown of PI3Kγ. Data shows that PI3Kγ inhibits PP2A activity at the βAR complex upon insulin while loss of PI3Kγ unravels this inhibition resulting in increased PP2A activity leading to β2AR dephosphorylation and resensitization. Mechanistically, PI3Kγ inhibits PP2A activity at the β2AR complex by phosphorylating an endogenous inhibitor of PP2A (I2PP2A). CRISPR knockout and siRNA knockdown of endogenous I2PP2A in HEK293 cells restored PP2A activity resulting in β2AR dephosphorylation despite PI3Kγ. Furthermore, β blocker (propranolol) pretreatment did not affect β2AR phosphorylation and there was no β-arrestin recruitment to the βAR complex with Insulin. Together these studies show that Insulin mediates β2AR desensitization through β-agonist and β-arrestin independent mechanisms wherein, PI3Kγ-mediated regulation of PP2A activity plays a pivotal role in cardiac βAR function in hyperinsulemic conditions like diabetes and obesity.

Targeting SET to restore PP2A activity disrupts an oncogenic CIP2A-feedforward loop and impairs triple negative breast cancer progression.

BackgroundTriple-negative breast cancer (TNBC) remains difficult to be targeted. SET and cancerous inhibitor of protein phosphatase 2A (CIP2A) are intrinsic protein-interacting inhibitors of protein phosphatase 2A (PP2A) and frequently overexpressed in cancers, whereas reactivating PP2A activity has been postulated as an anti-cancer strategy. Here we explored this strategy in TNBC.MethodsData from The Cancer Genome Atlas (TCGA) database was analyzed. TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. The apoptotic cells were examined by flow cytometry and Western blot. A SET-PP2A protein-protein interaction antagonist TD19 was used to disrupt signal transduction. In vivo efficacy of TD19 was tested in MDA-MB-468-xenografted animal model.FindingsTCGA data revealed upregulation of SET and CIP2A and positive correlation of these two gene expressions in TNBC tumors. Ectopic SET or CIP2A increased cell viability, migration, and invasion of TNBC cells. Notably ERK inhibition increased PP2A activity. ERK activation is known crucial for Elk-1 activity, a transcriptional factor regulating CIP2A expression, we hypothesized an oncogenic feedforward loop consisting of pERK/pElk-1/CIP2A/PP2A. This loop was validated by knockdown of PP2A and ectopic expression of Elk-1, showing reciprocal changes in loop members. In addition, ectopic expression of SET increased pAkt, pERK, pElk-1 and CIP2A expressions, suggesting a positive linkage between SET and CIP2A signaling. Moreover, TD19 disrupted this CIP2A-feedforward loop by restoring PP2A activity, demonstrating in vitro and in vivo anti-cancer activity. Mechanistically, TD19 downregulated CIP2A mRNA via inhibiting pERK-mediated Elk-1 nuclear translocation thereby decreased Elk-1 binding to the CIP2A promoter.InterpretationThese findings suggested that a novel oncogenic CIP2A-feedforward loop contributes to TNBC progression and targeting SET to disrupt this oncogenic CIP2A loop showed therapeutic potential in TNBC.

DR region of Na+-K+-ATPase is a new target to protect heart against oxidative injury

Previous studies have shown that the activity and expression of Na+/K+-ATPase (NKA) are down-regulated in the failing hearts, and that an antibody against the DR-region of NKA (DR-Ab) can stimulate its activity. The present study was designed to investigate the beneficial effects of this antibody against cardiac injury and the underlying mechanisms. We found that DR-Ab improved cardiac function, alleviated cardiac hypertrophy and reduced oxidative stress in isoproterenol-treated mice. In AC16 human cardiomyocytes, DR-Ab increased cell viability and attenuated apoptosis under oxidative stress. Corresponding to the observation of reduced NKA activity, NKA abundance on plasma membrane was lowered during oxidative stress. Suppressed activity of protein phosphatase 2 A (PP2A) was responsible for the loss of membrane NKA due to the increased phosphorylation of key serine residues that trigger endocytosis. Incubation with DR-Ab restored PP2A activity and stabilized NKA expression on the plasma membrane. Inhibitors of PP2A abolished the protective effect of DR-Ab against oxidative stress. In summary, our data indicate that loss of membrane NKA may contribute to cardiac pathologies in heart failure. DR-Ab, by stabilizing membrane NKA, protects cardiomyocytes against oxidative injury and improves cardiac function in the failing hearts, suggesting a novel approach to treat heart failure.

Pharmacological Activation of Protein Phosphatase 2 A (PP2A): A Novel Strategy to Fight Against Human Malignancies?

The serine-threonine protein phosphatase 2A (PP2A) regulates multiple cell signaling cascades and its inactivation by viral oncoproteins, mutation of specific structural subunits or upregulation of the cellular endogenous inhibitors may contribute to malignant transformation by regulating specific phosphorylation events. Pharmacological modulation of PP2A activity is becoming an attractive strategy for cancer treatment. Some compounds targeting PP2A are able to induce PP2A reactivation and subsequent cell death in several types of cancer. We undertook a search of bibliographic databases for peer-reviewed articles focusing on the main item of the review. We selected articles published in indexed journals. The quality of retrieved papers was appraised using the standard bibliometric indicators. One hundred and fourteen papers were included in the review. Twenty-seven papers gave an overview of structure and physiological role of PP2A. Twenty-five papers outlined the role of PP2A in tumor suppression. Forty papers analyzed the mechanism involved in PP2A reactivation by synthetic compounds, and twenty-two papers outlined the capability of natural compounds of restoring PP2A activity and how this could be beneficial. Findings analyzed in this review underline the central role of PP2A as a regulator of cell growth and survival, hence its function as tumor suppressor. The discovery that some compounds, either synthetic or natural, are capable of reactivating PP2A opens up new perspectives for future strategies to fully exploit therapeutic potential in human cancer. Thus, this review could also be of particular interest to pharmaceutical or biotechnology companies for drug design and targeted delivery.

TRAIL deficiency and PP2A activation with salmeterol ameliorates egg allergen-driven eosinophilic esophagitis.

Food antigens are common inflammatory triggers in pediatric eosinophilic esophagitis (EoE). TNF-related apoptosis-inducing ligand (TRAIL) promotes eosinophilic inflammation through the upregulation of the E3 ubiquitin ligase Midline (MID)-1 and subsequent downregulation of protein phosphatase 2A (PP2A), but the role of this pathway in EoE that is experimentally induced by repeated food antigen challenges has not been investigated. Esophageal mucosal biopsies were collected from children with EoE and controls and assessed for TRAIL and MID-1 protein and mRNA transcript levels. Wild-type and TRAIL-deficient (Tnfsf10-/-) mice were administered subcutaneous ovalbumin (OVA) followed by oral OVA challenges. In separate experiments, OVA-challenged mice were intraperitoneally administered salmeterol or dexamethasone. Esophageal biopsies from children with EoE revealed increased levels of TRAIL and MID-1 and reduced PP2A activation compared with controls. Tnfsf10-/- mice were largely protected from esophageal fibrosis, eosinophilic inflammation, and the upregulation of TSLP, IL-5, IL-13, and CCL11 when compared with wild-type mice. Salmeterol administration to wild-type mice with experimental EoE restored PP2A activity and also prevented esophageal eosinophilia, inflammatory cytokine expression, and remodeling, which was comparable to the treatment effect of dexamethasone. TRAIL and PP2A regulate inflammation and fibrosis in experimental EoE, which can be therapeutically modulated by salmeterol.

Silencing [Formula: see text] Rescues Tau Pathologies and Memory Deficits through Rescuing PP2A and Inhibiting GSK-3β Signaling in Human Tau Transgenic Mice.

Increase of inhibitor-2 of protein phosphatase-2A is associated with protein phosphatase-2A (PP2A) inhibition and tau hyperphosphorylation in Alzheimer’s disease (AD). Down-regulating attenuated amyloidogenesis and improved the cognitive functions in transgenic mice expressing amyloid precursor protein (tg2576). Here, we found that silencing by hippocampal infusion of down-regulated (~45%) with reduction of tau phosphorylation/accumulation, improvement of memory deficits, and dendritic plasticity in 12-month-old human tau transgenic mice. Silencing not only restored PP2A activity but also inhibited glycogen synthase kinase-3β (GSK-3β) with a significant activation of protein kinase A (PKA) and Akt. In HEK293/tau and N2a/tau cells, silencing by also significantly reduced tau hyperphosphorylation with restoration of PP2A activity and inhibition of GSK-3β, demonstrated by the decreased GSK-3β total protein and mRNA levels, and the increased inhibitory phosphorylation of GSK-3β at serine-9. Furthermore, activation of PKA but not Akt mediated the inhibition of GSK-3β by silencing. We conclude that targeting can improve tau pathologies and memory deficits in human tau transgenic mice, and activation of PKA contributes to GSK-3β inhibition induced by silencing in vitro, suggesting that is a promising multiple target of AD.

Spermidine promotes adipogenesis of 3T3-L1 cells by preventing interaction of ANP32 with HuR and PP2A

We have shown previously that the polyamine spermidine is indispensable for differentiation of 3T3-L1 preadipocytes. In the present study, we examined the mechanism of spermidine function by using the polyamine biosynthesis inhibitor α-difluoromethylornithine in combination with the metabolically stable polyamine analogues γ-methylspermidine or (R,R)-α,ω-bismethylspermine. At the early phase of differentiation, spermidine-depleted 3T3-L1 cells showed decreased translation of the transcription factor C/EBPβ (CCAAT/enhancer-binding protein β), decreased PP2A (protein phosphatase 2A) activity and increased cytoplasmic localization of the RNA-binding protein HuR (human antigen R). The amount of HuR bound to C/EBPβ mRNA was reduced, whereas the amount of bound CUGBP2, an inhibitor of C/EBPβ translation, was increased. ANP32 (acidic nuclear phosphoprotein 32) proteins, which are known PP2A inhibitors and HuR ligands, bound more PP2A and HuR in spermidine-depleted than in control cells, whereas immunodepletion of ANP32 proteins from the lysate of spermidine-depleted cells restored PP2A activity. Taken together, our data shows that spermidine promotes C/EBPβ translation in differentiating 3T3-L1 cells, and that this process is controlled by the interaction of ANP32 with HuR and PP2A.

Nuclear Export (Karyopherin) Inhibitors: A Novel Therapeutic Strategy for Treating Blast Crisis Chronic Myelogenous Leukemia (CML) and Philadelphia-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Through Interference with hnRNP Nucleocytoplasmic Shuttling and Rescue of Protein Phosphatase 2A (PP2A) Tumor Suppressor Activity,

Abstract 3758Despite the remarkable response in chronic phase CML, tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis CML (CML-BC) and Ph+ ALL, and are unable to kill quiescent Ph+ hematopoietic stem cells. We reported that CML disease progression is characterized by a BCR-ABL1 dose- and kinase-dependent increase in the expression/activity of nucleocytoplasmic-shuttling of heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, which are essential post-transcriptional and translational modulators of critical regulators of cell proliferation, survival and differentiation of CD34+ CML-BC and/or CD34+/CD19+ Ph+ ALL progenitors including SET/PP2A, E2F3, Bcl-xL, C/EBPa, miR-328, and c-Myc. The karyopherin (a class of proteins involved in regulating nuclear import/export) chromosome region maintenance 1 (CRM1) controls the nuclear export of several hnRNPs, and because growth/survival of CML-BC and Ph+ ALL progenitors requires the aberrant cytoplasmic activity of hnRNPs A1, E2 and/or K, we have explored the therapeutic potential of CRM1 inhibitors in p210 and p190 BCR-ABL1+ cell line models of CML-BC and Ph+ ALL, respectively.Thus, the cytokine-dependent myeloid 32Dcl3 and lymphoid BaF3, and the derived cytokine-independent 32D-p210BCR-ABL1 and Baf3-p190BCR-ABL1 mouse progenitors were exposed for 48 hr to different concentrations (0–10 μM) of the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays (n=3) revealed that KPT-185 and KPT-207 decreased viability ∼80% in 32D-p210BCR-ABL1 cells (KPT-185≥207) at concentrations ranging from 150–350 nM. Similar results were obtained in Baf3-p190BCR-ABL1 cells as the EC50 was 300nM for KPT-185 and KPT-207. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70–100% viable cells) at concentrations (150–350 nM) of KPT-185 and KPT-207 that impair survival of BCR-ABL1+ cells.Mechanistically, we found that KPT-SINE CRM1 inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. Indeed, in KPT-207-treated (1 μM; 48h) BCR-ABL1+ cells hnRNP A1, E2 and K accumulated in the cytoplasm. Likewise, KPT-185 treatment (1 μM; 48h) resulted in hnRNP A1 being sequestered in the nucleus whereas hnRNP E2 distribution was not altered and, unexpectedly, levels of hnRNP K expression were markedly decreased in both subcellular compartments.Interestingly, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors.Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization in addition to impairing BCR-ABL1 expression/activity, and suppression of PP2A tumor suppressor activity is essential for both p210 and p190 BCR-ABL1-driven leukemogenesis, it is highly plausible that KPT-207 and KPT-185 negatively regulate the BCR-ABL1-dependent and hnRNP A1-mediated induction of the PP2A inhibitor SET thereby rescuing PP2A phosphatase activity, which in turn decreases BCR-ABL1 expression and kinase activity, and triggers in vitro and in vivo apoptosis of primary CML-BC and Ph+ ALL progenitors. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells.Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Disclosures:Shacham:Karyopharm: Equity Ownership. Kauffman:Karyopharm: Equity Ownership.

FTY720 Restores PP2A Tumor Suppressor Activity in Polycythemia Vera CD34+ Progenitors Through Inhibition of Jak2 V617F- and PI-3Kγ-Dependent SET Serine Phosphorylation and Enhancement of NOS-Dependent PP2A Tyrosine Nitration

Abstract 2494In CD34+ bone marrow (BM) progenitors from Polycythemia Vera (PV) patients and Jak2V617F-expressing cell lines, loss of protein phosphatase 2A (PP2A) tumor suppressor activity is essential for their survival and clonogenic potential, and it depends on signals generated by the mutated Jak2 oncogenic kinase that, in turn, is also negatively regulated by PP2A. Additionally, we showed (Oaks J.J. et al., ASH 2010) that suppression of PP2A requires expression of the Jak2-regulated PP2A inhibitor SET, and it can be reverted by clinically-relevant Jak2 inhibitors or by the sphingosine analog FTY720, which reactivates PP2A and inhibits Jak2V617F expression/activity upon binding/sequestration of SET.Here we report that, unlike the effect of BCR-ABL1 in CML, SET levels do not increase in normal CD34+ BM progenitors engineered to express Jak2V617F; however, SET knock-down restores PP2A activity in both Jak2V617F cell lines and CD34+ PV progenitors, suggesting a post-translational control of SET activity. By using Jak2 and PI-3K kinase inhibitors and SET mutants, we found that PP2A inactivation in PV depends on the Jak2V617F- and PI-3Kγ-induced SET phosphorylation on serine 24. Indeed, a ∼93% restoration of PP2A activity and suppression of SET phosphorylation is achieved upon exposure of Jak2V617F+ cells to the specific PI-3Kγ (AS-604850, 1μM) inhibitor. Likewise, ∼80% loss of SET inhibitory activity is observed upon transduction of S24A but not S9A SET mutant that, albeit essential for PP2A inhibition in other cell types, behaved as wild type SET.Because both Jak2 and PI-3Kγ may regulate nitric oxide synthase (NOS2) that, in turn, inhibits PP2A activity upon induction of peroxynitrate (PN)-mediated nitration of PP2A tyrosine residues with negative regulatory function, we assessed the effect of NOS/PN modulation on the Jak2V617F-dependent inactivation of PP2A. Treatment of Jak2V617F- expressing BaF3 cells with NO donors (1 mM DPTA NONOate and 10μM SIN-1) or PN (500 μM) resulted in 2.7- and 28-fold induction of PP2A activity, respectively. Accordingly, 80% inhibition of PP2A activity was observed in parental BaF3 cells upon inhibition of NOS2 by 3-bromo-7-nitroindazole (5μM). Thus, Jak2V617F inhibits PP2A by inducing SET phosphorylation and inhibiting PP2A tyrosine nitration. Notably, co-existence of both mechanisms of Jak2V617F-dependent PP2A inactivation is also supported by the evidence that NOS2 levels increase upon shRNA-mediated downregulation of SET expression, and by the marked decrease in SET phosphorylation and increase in PP2A tyrosine nitration detected in Jak2V617F cells treated with FTY720 (2.5 mM). Interestingly, similar effects were observed upon exposure of Jak2V617F cells to non-phosphorylatable and non-immunosuppressive FTY720 derivatives (e.g. OSU-2S, QC-FTY720 and SR-FTY720) but not in cells treated with the immunosuppressive phosphorylated FTY720 (FTY720-P).In agreement with our findings indicating that the anti-leukemic activity of FTY720 in Jak2V617F+ primary progenitors and cell lines does not require the sphingosine kinase 2-dependent conversion of FTY720 into FTY720-P and, consequently, its interaction with the S1PR1 receptor (Oaks JJ. et al., ASH 2010), we have now evidence showing that a synthetic FTY720-P, unlike FTY720, impairs (81% inhibition) PP2A activity in normal hematopoietic progenitors, and that the major part of the intracellular FTY720 remains non-phosphorylated. By using S1PR1-selective agonists and S1PR1 receptor knock-down, we also found that inhibition of PP2A activity by synthetic FTY720-P is mediated by the S1PR1-dependent triggering and activation of Jak2 and PI-3Kγ. Accordingly, co-treatment of normal hematopoietic progenitor cells with FTY720-P and either the Jak2 inhibitor AG490 or the PI-3Kγ inhibitor AS-604850 antagonized the inhibitory effects of FTY720-P and restored PP2A activity to levels similar to those detected in untreated cells.Altogether our data indicate that the anti-leukemic effects of FTY720 in CD34+ PV progenitors depends on its ability to antagonize the Jak2V617F- and S1PR1-induced and PI-3Kγ-mediated inactivation of PP2A through inhibition of the PI-3Kγ-dependent SET serine 24 phosphorylation, and via binding/sequestration of phosphorylated SET that, in turn, leads to enhancement of NOS/PN-mediated PP2A activation by tyrosine nitration. Disclosures:No relevant conflicts of interest to declare.

Pharmacologic Restoration of PP2A Activity and Interference with the SET-PP2A Interplay by FTY720 and Its Non-Immunosuppressive Derivative as a Novel and Efficient Therapy for Ph-Negative Myeloproliferative Disorders

AbstractAbstract 775We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2).Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients.To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells.Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min).Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures:Verstovsek:Incyte Corporation: Research Funding.

FTY720 but Not Its Immunosuppressive Phosphorylated Form FTY720-P Exerts Anti-Leukemic Activity towards Ph(+) and Ph(−) Myeloproliferative Disorders through Reactivation of the PP2A Tumor Suppressor.

Abstract 3259Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant.We have shown that the sphingosine analogue FTY720 markedly induces apoptosis of Ph(+) CML (chronic phase and blast crisis) and B-ALL progenitors regardless of their sensitivity to tyrosine kinase inhibitors through the reactivation of the tumor suppressor PP2A (protein phosphatase 2A). Here we report the identification of the molecular mechanism underlying the ability of FTY720 to activate PP2A, and its therapeutic potential towards Ph(+) and Ph(−) myeloproliferative disorders (MPDs).First, we show that the pro-apoptotic and anti-proliferative effect of FTY720 is not limited to BCR/ABL+ leukemias but can be extended to Jak2V617F-driven MPDs that are also characterized by the extensive (80% inhibition) Jak2V617F-mediated suppression of PP2A activity. In fact, shRNA-mediated interference with the hnRNP A1-SET inhibitory pathway in Jak2V617F-expressing erythro-myeloid precursors revealed that Jak2V617F, like BCR/ABL, utilizes these effectors to impair PP2A function in a dose- and kinase-dependent manner. FTY720 treatment (0.5-1mM) fully restored PP2A activity and resulted in decreased BFU-E colony formation of Jak2V617F-transduced Lin- mouse marrow progenitors. Likewise, FTY720 decreased Jak2V617F expression/activity and inhibited clonogenicity of Jak2V617F-expressing 32D-EpoR+, Ba/F3 and TF-1 cells in PP2A/SHP-1-dependent manner.Secondly, we show that PP2A is also inhibited in a SET-dependent manner in CD34+/CD38- stem cells (HSCs) from CML patients. In Ph(+) HSCs, FTY720 but not imatinib or dasatinib decreases the number of long-term culture initiating cells (LTC-IC) and quiescent stem cells (CFSEmax) through activation of BCR/ABL-independent caspase-mediated apoptosis. Importantly, enhanced self-renewal of Ph(+) HSCs seems to depend on constitutive nuclear β-catenin transcriptional activity. In fact, FTY720 or PP2Ac lentiviral transduction, but not imatinib/dasatinib, induces b-catenin inactivation/degradation as measured by in situ immunofluorescence and LET/TCF assays. The effect of FTY720 in Ph(+) HSCs relies on the PP2A ability to override the BCR/ABL-independent inactivation of the β-catenin negative regulator GSK-3β, as treatment with the GSK-3β inhibitors LiCl and SB216763 increased the CFSEmax fraction and hampered the pro-apoptotic effect of FTY720 on quiescent Ph(+) HSCs. Notably, FTY720 did not affect normal stem/progenitor cell viability. Similar results were obtained with the LSK fraction from SCL-tTA/BCR/ABL mice.Reportedly, FTY720 to act as an immunosuppressant requires phosphorylation by sphingosine kinase (SPHK) 2. To determine whether conversion of FTY720 into FTY720-P is important for its anti-leukemic activity in BCR/ABL- and Jak2V617F-expressing cells, we used growth factor-dependent hematopoietic cells transformed by the constitutive activity of these oncogenic tyrosine kinases. In Jak2V617F- and BCR/ABL-transformed cells, PP2A activation by FTY720 (2.5μM, 6h) is not changed when phosphorylation is prevented by treatment with the SPHK inhibitor dimethylsphingosine (2.5μM, 6h). Accordingly, FTY720-P did not activate PP2A and did not affect BCR/ABL- and Jak2V617F-driven colony formation, indicating that the anti-leukemic activity of FTY720 does not require its phosphorylation. Because we have shown that sphingolipid PP2A activator ceramide specifically binds to SET, thus disrupting the SET/PP2A interaction in non hematopoietic cells, we investigated the effect of BCR/ABL or Jak2 V617F and that of FTY720 on ceramide production. LS-MS mass-spectrometry showed that levels of ceramide were similar in untreated and imatinib-treated BCR/ABL+ cells. Similarly, levels of diacylglycerol (DAG) kinase-phosphorylated ceramide were unchanged by treatment with FTY720, suggesting that oncogenic tyrosine kinase-induced suppression of PP2A and FTY720-dependent PP2A reactivation are not ceramide-mediated. Accordingly, FTY720 displaces biotin-labeled C6-ceramide (10μM) from SET, as measured by ceramide affinity chromatography followed by determination of SET levels in the eluted fraction.Thus, FTY720 represents a powerful therapeutic tool as it has the potential to treat and, perhaps, eradicate Ph(+) and Ph(−) MPDs by efficiently inhibiting oncogene-dependent and -;independent signals through the reactivation of the tumor suppressor PP2A via disruption of the SET/PP2A inhibitory complex. Disclosures:Cortes:Novartis: Research Funding. Cancelas:CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein

The oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.