Beyond BCR::ABL1-The Role of Genomic Analyses in the Management of CML.

Chronic myeloid leukemia (CML) is a model of genomically based diagnosis and management where BCR::ABL1 is successfully targeted by tyrosine kinase inhibitor (TKI) therapy in most patients. The dynamics of BCR::ABL1 transcript decline during therapy is a dependable biomarker of response, relapse, and drug resistance. Missense mutations acquired within the BCR::ABL1 kinase domain that disrupt TKI binding can evolve during therapy and are frequently detected in patients for whom TKI treatment fails. Importantly, specific BCR::ABL1 missense mutations are targetable alterations and direct therapeutic decisions based on the individual mutant TKI sensitivity profile. Nevertheless, BCR::ABL1 mutations are only implicated in approximately half of the cases of acquired resistance. Furthermore, not all patients with a single BCR::ABL1 mutation that is predicted to be sensitive to a specific TKI will experience a response when switched to that TKI. Progression to blast phase heralds independence from BCR::ABL1, and this phase of the disease is notoriously difficult to treat. The independent drivers of resistance and disease progression have long been investigated to both predict progression and to find targets for therapeutic intervention. Recent data reaffirm that drug resistance and disease progression is a mutation-driven process in CML, and somatic variants in genes that are known to drive acute myeloid and lymphoid leukemia have been detected in patients in the advanced phases of CML. Genomic testing over the last few decades for patients with blood cancer has revealed of variety of genomic aberrations that drive disease. Consequently, incorporation of genomic factors into patient management for a range of blood cancers has led to the implementation of high-throughput gene testing to detect clinically actionable variants. Is it time to integrate broader genomic screening into clinical management strategies for patients with CML?

Impact of additional genetic abnormalities at diagnosis of chronic myeloid leukemia for first-line imatinib-treated patients receiving proactive treatment intervention.

The BCR::ABL1 gene fusion initiates chronic myeloid leukemia (CML); however, evidence has accumulated from studies of highly selected cohorts that variants in other cancer-related genes are associated with treatment failure. Nevertheless, the true incidence and impact of additional genetic abnormalities (AGA) at diagnosis of chronic phase (CP)-CML is unknown. We sought to determine whether AGA at diagnosis in a consecutive imatinib-treated cohort of 210 patients enrolled in the TIDEL-II trial influenced outcome despite a highly proactive treatment intervention strategy. Survival outcomes including overall survival, progression-free survival, failure-free survival, and BCR::ABL1 kinase domain mutation acquisition were evaluated. Molecular outcomes were measured at a central laboratory and included major molecular response (MMR, BCR::ABL1 ≤0.1%IS), MR4 (BCR::ABL1 ≤0.01%IS), and MR4.5 (BCR::ABL1 ≤0.0032%IS). AGA included variants in known cancer genes and novel rearrangements involving the formation of the Philadelphia chromosome. Clinical outcomes and molecular response were assessed based on the patient's genetic profile and other baseline factors. AGA were identified in 31% of patients. Potentially pathogenic variants in cancer-related genes were detected in 16% of patients at diagnosis (including gene fusions and deletions) and structural rearrangements involving the Philadelphia chromosome (Ph-associated rearrangements) were detected in 18%. Multivariable analysis demonstrated that the combined genetic abnormalities plus the EUTOS long-term survival clinical risk score were independent predictors of lower molecular response rates and higher treatment failure. Despite a highly proactive treatment intervention strategy, first-line imatinib-treated patients with AGA had poorer response rates. These data provide evidence for the incorporation of genomically-based risk assessment for CML.

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2.

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

Potent antileukemic activity of curaxin CBL0137 against MLL-rearranged leukemia.

Around 10% of acute leukemias harbor a rearrangement of the MLL/KMT2A gene, and the presence of this translocation results in a highly aggressive, therapy-resistant leukemia subtype with survival rates below 50%. There is a high unmet need to identify safer and more potent therapies for MLL-rearranged (MLL-r) leukemia that can be combined with established chemotherapeutics to decrease treatment-related toxicities. The curaxin, CBL0137, has demonstrated nongenotoxic anticancer and chemopotentiating effects in a number of preclinical cancer models and is currently in adult Phase I clinical trials for solid tumors and hematological malignancies. The aim of our study was to investigate whether CBL0137 has potential as a therapeutic and chemopotentiating compound in MLL-r leukemia through a comprehensive analysis of its efficacy in preclinical models of the disease. CBL0137 decreased the viability of a panel of MLL-r leukemia cell lines (n = 12) and xenograft cells derived from patients with MLL-r acute lymphoblastic leukemia (ALL, n = 3) in vitro with submicromolar IC50s. The small molecule drug was well-tolerated in vivo and significantly reduced leukemia burden in a subcutaneous MV4;11 MLL-r acute myeloid leukemia model and in patient-derived xenograft models of MLL-r ALL (n = 5). The in vivo efficacy of standard of care drugs used in remission induction for pediatric ALL was also potentiated by CBL0137. CBL0137 exerted its anticancer effect by trapping Facilitator of Chromatin Transcription (FACT) into chromatin, activating the p53 pathway and inducing an Interferon response. Our findings support further preclinical evaluation of CBL0137 as a new approach for the treatment of MLL-r leukemia.

CD30 and ALK combination therapy has high therapeutic potency in RANBP2-ALK-rearranged epithelioid inflammatory myofibroblastic sarcoma.

BACKGROUND: Epithelioid inflammatory myofibroblastic sarcoma (eIMS) is characterised by perinuclear ALK localisation, CD30 expression and early relapse despite crizotinib treatment. We aimed to identify therapies to prevent and/or treat ALK inhibitor resistance. METHODS: Malignant ascites, from an eIMS patient at diagnosis and following multiple relapses, were used to generate matched diagnosis and relapse xenografts. RESULTS: Xenografts were validated by confirmation of RANBP2-ALK rearrangement, perinuclear ALK localisation and CD30 expression. Although brentuximab-vedotin (BV) demonstrated single-agent activity, tumours regrew during BV therapy. BV resistance was associated with reduced CD30 expression and induction of ABCB1. BV resistance was reversed in vitro by tariquidar, but combination BV and tariquidar treatment only briefly slowed xenograft growth compared with BV alone. Combining BV with either crizotinib or ceritinib resulted in marked tumour shrinkage in both xenograft models, and resulted in prolonged tumour-free survival in the diagnosis compared with the relapse xenograft. CONCLUSIONS: CD30 is a therapeutic target in eIMS. BV efficacy is limited by the rapid emergence of resistance. Prolonged survival with combination ALK and CD30-targeted-therapy in the diagnosis model provides the rationale to trial this combination in eIMS patients at diagnosis. This combination could also be considered for other CD30-positive, ALK-rearranged malignancies.

Integrative genomic analysis reveals cancer-associated mutations at diagnosis of CML in patients with high-risk disease.

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P = .007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1 The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P = .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach.

Dipeptidyl peptidase 9 subcellular localization and a role in cell adhesion involving focal adhesion kinase and paxillin.

Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed member of the DPP4 gene and protease family. Deciphering the biological functions of DPP9 and its roles in pathogenesis has implicated DPP9 in tumor biology, the immune response, apoptosis, intracellular epidermal growth factor-dependent signaling and cell adhesion and migration. We investigated the intracellular distribution of DPP9 chimeric fluorescent proteins and consequent functions of DPP9. We showed that while some DPP9 is associated with mitochondria, the strongest co-localization was with microtubules. Under steady state conditions, DPP9 was not seen at the plasma membrane, but upon stimulation with either phorbol 12-myristate 13-acetate or epidermal growth factor, some DPP9 re-distributed towards the ruffling membrane. DPP9 was seen at the leading edge of the migrating cell and co-localized with the focal adhesion proteins, integrin-ß1 and talin. DPP9 gene silencing and treatment with a DPP8/DPP9 specific inhibitor both reduced cell adhesion and migration. Expression of integrin-ß1 and talin was decreased in DPP9-deficient and DPP9-enzyme-inactive cells. There was a concomitant decrease in the phosphorylation of focal adhesion kinase and paxillin, indicating that DPP9 knockdown or enzyme inhibition suppressed the associated adhesion signaling pathway, causing impaired cell movement. These novel findings provide mechanistic insights into the regulatory role of DPP9 in cell movement, and may thus implicate DPP9 in tissue and tumor growth and metastasis.

The protein tyrosine phosphatase Pez regulates TGFbeta, epithelial-mesenchymal transition, and organ development.

Epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.

Defining Higher-Risk Chronic Myeloid Leukemia: Risk Scores, Genomic Landscape, and Prognostication.

PURPOSE OF REVIEW: The chronic myeloid leukemia (CML) treatment success story is incomplete as some patients still fail therapy, leading to end-stage disease and death. Here we discuss recent research into CML incidence, the role of comorbidities on survival and detecting patients at risk of failing therapy. RECENT FINDINGS: The incidence of CML has fallen markedly in high social-demographic index (SDI) regions of the world but there is disturbing evidence that this is not the case in low and low-middle SDI countries. Now that CML patients more frequently die from their co-morbid conditions than from CML the Adult Comorbidity Evaluation-27 score can assist in risk assessment at diagnosis. Non-adherence to therapy contributes greatly to treatment failure. A good doctor-patient relationship and social support promote good adherence, but patient age, gender, and financial burden have negative effects, suggesting avenues for intervention. Mutations in cancer-associated genes adversely affect outcome and their detection at diagnosis may guide therapeutic choice and offer non-BCR::ABL1 targeted therapies. A differential gene expression signature to assist risk detection is a highly sought-after diagnostic tool being actively researched on several fronts. Detecting patients at risk of failing therapy is being assisted by recent technological advances enabling highly sensitive genomic and expression analysis of insensitive cells. However, patient lifestyle, adherence to therapy, and comorbidities are critical risk factors that need to be addressed by interventions such as social and financial support.

RNA-Based Targeted Gene Sequencing Improves the Diagnostic Yield of Mutant Detection in Chronic Myeloid Leukemia.

Mutation detection is increasingly used for the management of hematological malignancies. Prior whole transcriptome and whole exome sequencing studies using total RNA and DNA identified diverse mutation types in cancer-related genes associated with treatment failure in patients with chronic myeloid leukemia. Variants included single-nucleotide variants and small insertions/deletions, plus fusion transcripts and partial or whole gene deletions. The hypothesis that all of these mutation types could be detected by a single cost-effective hybridization capture next-generation sequencing method using total RNA was assessed. A method was developed that targeted 130 genes relevant for myeloid and lymphoid leukemia. Retrospective samples with 121 precharacterized variants were tested using total RNA and/or DNA. Concordance of detection of precharacterized variants using RNA or DNA was 96%, whereas the enhanced sensitivity identified additional variants. Comparison between 24 matched DNA and RNA samples demonstrated 95.3% of 170 variants detectable using DNA were detected using RNA, including all but one variant predicted to activate nonsense-mediated decay. RNA identified an additional 10 variants, including fusion transcripts. Furthermore, the true effect of splice variants on RNA splicing was only evident using RNA. In conclusion, capture sequencing using total RNA alone is suitable for detecting a range of variants relevant in chronic myeloid leukemia and may be more broadly applied to other hematological malignancies where diverse variant types define risk groups.

In vitro and in vivo drug screens of tumor cells identify novel therapies for high-risk child cancer.

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid high-throughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1.

The transactivation of enhanced growth factor receptor (EGFR) by G protein-coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase sphingosine kinase-1 (SphK1). We show that E2 stimulates SphK1 activation and the release of sphingosine 1-phosphate (S1P), by which E2 is capable of activating the S1P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease-dependent manner. Thus, these findings reveal a key role for SphK1 in the coupling of the signals between three membrane-spanning events induced by E2, S1P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., "criss-cross" transactivation.

Aberrant RAG-mediated recombination contributes to multiple structural rearrangements in lymphoid blast crisis of chronic myeloid leukemia.

Blast crisis of chronic myeloid leukemia is associated with poor survival and the accumulation of genomic lesions. Using whole-exome and/or RNA sequencing of patients at chronic phase (CP, n = 49), myeloid blast crisis (MBC, n = 19), and lymphoid blast crisis (LBC, n = 20), we found 25 focal gene deletions and 14 fusions in 24 patients in BC. Deletions predominated in LBC (83% of structural variants). Transcriptional analysis identified the upregulation of genes involved in V(D)J recombination, including RAG1/2 and DNTT in LBC. RAG recombination is a reported mediator of IKZF1 deletion. We investigated the extent of RAG-mediated genomic lesions in BC. Molecular hallmarks of RAG activity; DNTT-mediated nucleotide insertions and a RAG-binding motif at structural variants were exclusively found in patients with high RAG expression. Structural variants in 65% of patients in LBC displayed these hallmarks compared with only 5% in MBC. RAG-mediated events included focal deletion and novel fusion of genes associated with hematologic cancer: IKZF1, RUNX1, CDKN2A/B, and RB1. Importantly, 8/8 patients with elevated DNTT at CP diagnosis progressed to LBC by 12 months, potentially enabling early prediction of LBC. This work confirms the central mutagenic role of RAG in LBC and describes potential clinical utility in CML management.

Integration of genomics, high throughput drug screening, and personalized xenograft models as a novel precision medicine paradigm for high risk pediatric cancer.

Pediatric high grade gliomas (HGG) are primary brain malignancies that result in significant morbidity and mortality. One of the challenges in their treatment is inter- and intra-tumoral heterogeneity. Precision medicine approaches have the potential to enhance diagnostic, prognostic and/or therapeutic information. In this case study we describe the molecular characterization of a pediatric HGG and the use of an integrated approach based on genomic, in vitro and in vivo testing to identify actionable targets and treatment options. Molecular analysis based on WGS performed on initial and recurrent tumor biopsies revealed mutations in TP53, TSC1 and CIC genes, focal amplification of MYCN, and copy number gains in SMO and c-MET. Transcriptomic analysis identified increased expression of MYCN, and genes involved in sonic hedgehog signaling proteins (SHH, SMO, GLI1, GLI2) and receptor tyrosine kinase pathways (PLK, AURKA, c-MET). HTS revealed no cytotoxic efficacy of SHH pathway inhibitors while sensitivity was observed to the mTOR inhibitor temsirolimus, the ALK inhibitor ceritinib, and the PLK1 inhibitor BI2536. Based on the integrated approach, temsirolimus, ceritinib, BI2536 and standard therapy temozolomide were selected for further in vivo evaluation. Using the PDX animal model (median survival 28 days) we showed significant in vivo activity for mTOR inhibition by temsirolimus and BI2536 (median survival 109 and 115.5 days respectively) while ceritinib and temozolomide had only a moderate effect (43 and 75.5 days median survival respectively). This case study demonstrates that an integrated approach based on genomic, in vitro and in vivo drug efficacy testing in a PDX model may be useful to guide the management of high risk pediatric brain tumor in a clinically meaningful timeframe.

Activation of the sphingosine kinase-signaling pathway by high glucose mediates the proinflammatory phenotype of endothelial cells.

Vascular endothelial cells are key targets for hyperglycemic damage that facilitates vascular inflammation and the vasculopathy associated with diabetes mellitus. However, the mechanisms underlying this damage remain undefined. We now demonstrate that hyperglycemia induces activation of sphingosine kinase (SphK), which represents a novel signaling pathway that mediates endothelial damage under ambient high glucose conditions. SphK activity was significantly increased in aorta and heart of streptozotocin-induced diabetic rats. Interestingly, this increase in SphK activity was prevented by insulin treatment, which achieved euglycemia in the diabetic animals. Hyperglycemia-induced increase in SphK activity was also evident in endothelial cells that received long-term exposure to high glucose (22 mmol/L). Studies using a small interfering RNA strategy demonstrated that endogenous SphK1, but not SphK2, is the major isoenzyme that was activated by high glucose. In addition, an increase in SphK1 phosphorylation was detected in a protein kinase C- and extracellular signal-regulated kinase 1/2-dependent manner, which accounts for the high glucose-induced increases in SphK activity. Importantly, inhibition of SphK1 by either a chemical inhibitor (N',N'-dimethylsphingosine) or expression of a dominant-negative mutant of SphK1 (SphK(G82D)), or SphK1-specific small interfering RNA, strongly protected endothelial cells against high glucose-induced damage, as characterized by an attenuation in the expression of proinflammatory adhesion molecules, adhesion of leukocytes to endothelial cells, and nuclear factor kappaB activation. Thus, interventions that target the SphK-signaling pathway may have the potential to prevent vascular lesions under hyperglycemic conditions.

The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin.

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.

The role of sphingolipid signalling in diabetes­associated pathologies (Review).

Sphingosine kinase (SphK) is an important signalling enzyme that catalyses the phosphorylation of sphingosine (Sph) to form sphingosine­1­phosphate (S1P). The multifunctional lipid, S1P binds to a family of five G protein-coupled receptors (GPCRs). As an intracellular second messenger, S1P activates key signalling cascades responsible for the maintenance of sphingolipid metabolism, and has been implicated in the progression of cancer, and the development of other inflammatory and metabolic diseases. SphK and S1P are critical molecules involved in the regulation of various cellular metabolic processes, such as cell proliferation, survival, apoptosis, adhesion and migration. There is strong evidence supporting the critical roles of SphK and S1P in the progression of diabetes mellitus, including insulin sensitivity and insulin secretion, pancreatic ß­cell apoptosis, and the development of diabetic inflammatory state. In this review, we summarise the current state of knowledge for SphK/S1P signalling effects, associated with the development of insulin resistance, pancreatic ß­cell death and the vascular complications of diabetes mellitus.

PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase.

Subject- Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are emerging as potential protectors against inflammatory cardiovascular diseases including atherosclerosis and diabetic complications. However, their molecular mechanism of action within vasculature remains unclear. We report here that PPARgamma agonists, thiazolidinedione class drugs (TZDs), or 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) were capable of activating diacylglycerol (DAG) kinase (DGK), resulting in attenuation of DAG levels and inhibition of protein kinase C (PKC) activation. The PPARgamma agonist-induced DGK was completely blocked by a dominant-negative mutant of PPARgamma, indicating an essential receptor-dependent action. Importantly, the suppression of DAG-PKC signaling pathway was functional linkage to the anti-inflammatory properties of PPARgamma agonists in endothelial cells (EC), characterized by the inhibition of proinflammatory adhesion molecule expression and adherence of monocytes to the activated EC induced by high glucose. These findings thus demonstrate a novel molecular action of PPARgamma agonists to suppress the DAG-PKC signaling pathway via upregulation of an endogenous attenuator, DGK.

High-density lipoproteins neutralize C-reactive protein proinflammatory activity.

BACKGROUND: C-reactive protein (CRP), a well-recognized marker of atherosclerosis, has recently been suggested to have a direct proinflammatory effect. The constitutive expression of low levels of CRP in normal plasma suggests the likelihood that a natural factor exists to neutralize the effect of CRP. This factor(s) has not yet been identified. Method and Results- The proinflammatory effect of CRP was measured by the induction of inflammatory adhesion molecules in human umbilical vein endothelial cells (HUVECs). We show that CRP significantly induced upregulation of adhesion molecules in both protein and mRNA levels. The CRP-induced expression of these inflammatory adhesion molecules was completely suppressed when the cells were preincubated with a physiological concentration (1 mg/mL apolipoprotein A-I) of HDLs derived from human plasma (native HDL) or reconstituted HDL (rHDL) at a very low concentration (0.01 mg/mL apolipoprotein A-I). A novel mechanism of HDL inhibition is likely to operate, because (1) rHDL was 100 times more potent than native HDL, (2) preincubation with HDL and its sustained presence were obligatory, and (3) oxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine was the fundamental active component. CONCLUSIONS: The CRP-induced upregulation of inflammatory adhesion molecules in HUVECs was completely prevented by HDL via their oxidized phospholipid components.

Sphingosine-1-phosphate receptor 1 transmits estrogens' effects in endothelial cells.

We have previously reported that the steroid hormone estrogens stimulate activation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) receptors in breast cancer cells. Both estrogens and S1P are potent biological modulators of endothelial function in vasculature able to activate multiple effectors, including endothelial nitric oxide synthase (eNOS). In this study we report that treatment of endothelial cells (ECs) with 17ß-estradiol (E2) resulted in a rapid, transient, and dose-dependent increase in SphK activity and increased S1P production. The effect was not reproduced by the inactive E2 analogue 17α-E2. Expression of the dominant-negative mutant SphK1(G82D) or transfection of SphK1-targeted siRNA in ECs caused not only a defect in SphK activation by E2, but also a significant inhibition of E2-induced activation of Akt/eNOS. Furthermore, E2 treatment induced internalization of plasma membrane S1P1 receptor, accompanied with an increase in the amount of cytosolic S1P1. By down-regulating S1P1 receptor expression, the S1P1-specific antisense oligonucleotides significantly inhibited E2-induced activation of Akt/eNOS in ECs. E2-induced EC migration and tube formation were also inhibited by S1P1 down-regulation. Thus, the findings indicate an important role of the SphK1/S1P1 pathway in mediating estrogen signaling and its actions in vasculature.