Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy.

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.

Discovery of a Novel and Selective Indoleamine 2,3-Dioxygenase (IDO-1) Inhibitor 3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003) and Its Characterization as a Potential Clinical Candidate.

Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure-activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.

Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis.

Strategies targeting pathological angiogenesis have focused primarily on blocking vascular endothelial growth factor (VEGF), but resistance and insufficient efficacy limit their success, mandating alternative antiangiogenic strategies. We recently provided genetic evidence that the glycolytic activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) promotes vessel formation but did not explore the antiangiogenic therapeutic potential of PFKFB3 blockade. Here, we show that blockade of PFKFB3 by the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) reduced vessel sprouting in endothelial cell (EC) spheroids, zebrafish embryos, and the postnatal mouse retina by inhibiting EC proliferation and migration. 3PO also suppressed vascular hyperbranching induced by inhibition of Notch or VEGF receptor 1 (VEGFR1) and amplified the antiangiogenic effect of VEGF blockade. Although 3PO reduced glycolysis only partially and transiently in vivo, this sufficed to decrease pathological neovascularization in ocular and inflammatory models. These insights may offer therapeutic antiangiogenic opportunities.

Incomplete and transitory decrease of glycolysis: a new paradigm for anti-angiogenic therapy?

During vessel sprouting, a migratory endothelial tip cell guides the sprout, while proliferating stalk cells elongate the branch. Tip and stalk cell phenotypes are not genetically predetermined fates, but are dynamically interchangeable to ensure that the fittest endothelial cell (EC) leads the vessel sprout. ECs increase glycolysis when forming new blood vessels. Genetic deficiency of the glycolytic activator PFKFB3 in ECs reduces vascular sprouting by impairing migration of tip cells and proliferation of stalk cells. PFKFB3-driven glycolysis promotes the tip cell phenotype during vessel sprouting, since PFKFB3 overexpression overrules the pro-stalk activity of Notch signaling. Furthermore, PFKFB3-deficient ECs cannot compete with wild-type neighbors to form new blood vessels in chimeric mosaic mice. In addition, pharmacological PFKFB3 blockade reduces pathological angiogenesis with modest systemic effects, likely because it decreases glycolysis only partially and transiently.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Aging- and activation-induced platelet microparticles suppress apoptosis in monocytic cells and differentially signal to proinflammatory mediator release.

BACKGROUND: Platelet microparticles (PM) are the most abundant cell-derived microparticles in the blood, and accumulate in thrombo-inflammatory diseases. Platelets produce PM upon aging via an apoptosis-like process and by activation with strong agonists. We previously showed that long-term treatment of monocytic cells with apoptosis-induced PM (PMap) promotes their differentiation into resident macrophages. Here we investigated shorter term effects of various types of PM on monocyte signalling and function. METHODS AND RESULTS: Flow cytometry and scanning electron microscopy revealed that PM formed upon platelet aging (PMap) or ultra-sonication (PMsonic) expressed activated αIIbß3 integrins and tended to assemble into aggregates. In contrast, PM formed upon platelet activation with thrombin (PMthr) or Ca(2+) ionophore (PMiono) had mostly non-activated αIIbß3 and little aggregate formation, but had increased CD63 expression. PM from activated and sonicated platelets expressed phosphatidylserine at their surface, while only the latter were enriched in the receptors CD40L and CX3CR1. All PM types expressed P-selectin, interacted with monocytic cells via this receptor, and were internalised into these cells. The various PM types promoted actin cytoskeletal rearrangements and hydrogen peroxide production by monocytic cells. Markedly, both aging- and activation-induced PM types stimulated the phosphoinositide 3-kinase/Akt pathway, suppressing apoptosis induced by several agonists, in a P-selectin-dependent manner. On the other hand, the PM types differentially influenced monocyte signalling in eliciting Ca(2+) fluxes (particularly PMap) and in releasing secondary mediators (complement factor C5a with PMap, and pro-inflammatory tumour necrosis factor-α with PMthr). CONCLUSIONS: In spite of their common anti-apoptotic potential via Akt activation, aging- and activation-induced PM cause different Ca(2+) signalling events and mediator release in monocytic cells. By implication, aging and activated platelets may modulate monocyte function in different way by the shedding of different PM types.

Contribution of platelet CX(3)CR1 to platelet-monocyte complex formation and vascular recruitment during hyperlipidemia.

OBJECTIVE: The chemokine receptor CX(3)CR1 is an inflammatory mediator in vascular diseases. On platelets, its ligation with fractalkine (CX(3)CL1) induces platelet activation followed by leukocyte recruitment to activated endothelium. Here, we evaluated the expression and role of platelet-CX(3)CR1 during hyperlipidemia and vascular injury. METHODS AND RESULTS: The existence of CX(3)CR1 on platelets at mRNA and protein level was analyzed by RT-PCR, quantitative (q)PCR, FACS analysis, and Western blot. Elevated CX(3)CR1 expression was detected on human platelets after activation and, along with increased binding of CX(3)CL1, platelet CX(3)CR1 was also involved in the formation of platelet-monocyte complexes. Interestingly, the expression of CX(3)CR1 was elevated on platelets from hyperlipidemic mice. Accordingly, CX(3)CL1-binding and the number of circulating platelet-monocyte complexes were increased. In addition, CX(3)CR1 supported monocyte arrest on inflamed smooth muscle cells in vitro, whereas CX(3)CR1-deficient platelets showed decreased adhesion to the denuded vessel wall in vivo. CONCLUSIONS: Platelets in hyperlipidemic mice display increased CX(3)CR1-expression and assemble with circulating monocytes. The formation of platelet-monocyte complexes and the detection of platelet-bound CX(3)CL1 on inflamed smooth muscle cells suggest a significant involvement of the CX(3)CL1-CX(3)CR1 axis in platelet accumulation and monocyte recruitment at sites of arterial injury in atherosclerosis.

Loss or inhibition of stromal-derived PlGF prolongs survival of mice with imatinib-resistant Bcr-Abl1(+) leukemia.

Imatinib has revolutionized the treatment of Bcr-Abl1(+) chronic myeloid leukemia (CML), but, in most patients, some leukemia cells persist despite continued therapy, while others become resistant. Here, we report that PlGF levels are elevated in CML and that PlGF produced by bone marrow stromal cells (BMSCs) aggravates disease severity. CML cells foster a soil for their own growth by inducing BMSCs to upregulate PlGF, which not only stimulates BM angiogenesis, but also promotes CML proliferation and metabolism, in part independently of Bcr-Abl1 signaling. Anti-PlGF treatment prolongs survival of imatinib-sensitive and -resistant CML mice and adds to the anti-CML activity of imatinib. These results may warrant further investigation of the therapeutic potential of PlGF inhibition for (imatinib-resistant) CML.

Vessel abnormalization: another hallmark of cancer? Molecular mechanisms and therapeutic implications.

As a result of excessive production of angiogenic molecules, tumor vessels become abnormal in structure and function. By impairing oxygen delivery, abnormal vessels fuel a vicious cycle of non-productive angiogenesis, which creates a hostile microenvironment from where tumor cells escape through leaky vessels and which renders tumors less responsive to chemoradiation. While anti-angiogenic strategies focused on inhibiting new vessel growth and destroying pre-existing vessels, clinical studies showed modest anti-tumor effects. For many solid tumors, anti-VEGF treatment offers greater clinical benefit when combined with chemotherapy. This is partly due to a normalization of the tumor vasculature, which improves cytotoxic drug delivery and efficacy and offers unprecedented opportunities for anti-cancer treatment. Here, we overview key novel molecular players that induce vessel normalization.

Role of membrane cholesterol in platelet calcium signalling in response to VWF and collagen under stasis and flow.

Several studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca(2+) signalling and secretion during the interaction of platelets with VWF and collagen. VWF/ristocetin-induced platelet aggregation was delayed after treatment with methyl beta-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca(2+) response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca(2+) responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A(2) and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca(2+) signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs.

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation.

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661-1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of alpha2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV(-/-) neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV(-/-) neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV(-/-) leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV(-/-) leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of alpha2,3-linked sialic acid-specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

Hemostatic and signaling functions of transfused platelets.

Metabolic studies have revealed a gradual impairment in platelet integrity during storage, a process termed the platelet storage lesion. Recent evidence shows that stored platelets also lose signaling responses to physiological agonists with impaired integrin activation, secretion, and aggregation of the cells. On the other hand, storage leads to a gain in platelet activation properties, such as release of microparticles and appearance of surface epitopes for their clearance by macrophages. New techniques for measuring flow-induced thrombus formation and platelet-dependent coagulation provide evidence that the hemostatic activity of platelets decreases during storage. Besides pharmacological inhibition, novel storage strategies, like metabolic suppression, should be considered to better preserve platelet functionality while limiting the expression of clearance markers. Understanding the changes that occur in association with the platelet storage lesion and the use of updated storage methods will help to generate platelets for transfusion with optimal hemostatic function and a long circulation time after transfusion.

Novel methodology for assessment of prophylactic platelet transfusion therapy by measuring increased thrombus formation and thrombin generation.

Currently, patients developing severe thrombocytopenia during chemotherapy treatment are prophylactically transfused with platelets. We developed two platelet function tests to report the improved haemostasis in the transfused patients, which were capable of detecting aberrant responsiveness of the platelets after transfusion. First, in a whole-blood flow test, platelet adhesion and thrombus formation were determined under high-shear flow conditions. Second, the procoagulant function of platelets was assayed in platelet-rich plasma by measurement of thrombin generation. Experimental conditions were established, where flow-induced adhesion and thrombin generation test parameters increased semi-linearly with the platelet concentration, and informed on the activation properties of platelets. The transfusion effects were evaluated for 38 thrombocytopenic patients, who were transfused with platelets stored in plasma or in synthetic medium (platelet additive solution II). In most but not all patients, transfusion resulted in increased adhesion and thrombus formation, as well as in improved platelet-dependent coagulation. Taken together, the increase in platelet count after transfusion explained 57% of the overall improvement in platelet function. In acute graft-versus-host disease, thrombus formation was normal, while platelet-dependent coagulation was higher than expected. We conclude that assessment of flow-induced adhesion and thrombin generation in acquired thrombocytopenia adequately determines the improved haemostatic activity by transfused platelets.

Shedding of procoagulant microparticles from unstimulated platelets by integrin-mediated destabilization of actin cytoskeleton.

Platelet activation by potent, Ca(2+)-mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from alphaIIbbeta3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin-mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo.

Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation.

BACKGROUND: Platelets (PLTs) contain purinergic receptors for ATP (P2X1) and ADP (P2Y1 and P2Y12) that rapidly desensitize upon stimulation with these nucleotides. In vivo, this is antagonized by ectonucleotidases on the surface of endothelial cells and white blood cells (WBCs). The receptor desensitization of ATP- and ADP-induced responses of PLTs stored in plasma without WBCs was investigated. STUDY DESIGN AND METHODS: ATP- and ADP-induced PLT shape change (shear-induced) aggregation and Ca2+ signaling were measured in the presence or absence of plasma. Degradation of nucleotides in plasma was quantified by high-performance liquid chromatography. RESULTS: Washed PLTs became refractory for ATP and ADP in shape change, aggregation, and Ca2+ responses during a 90-minute incubation at 37 degrees C. The PLT responses mediated by P2X1, P2Y1, and P2Y12 receptors gradually reduced or disappeared. When plasma was present, however, the PLTs persistently showed high responses to ATP and ADP. Heat treatment of plasma abolished this effect. Also under conditions of flow and high shear, PLTs in plasma kept high P2X1 activity, mediating aggregate formation. In isolated plasma, not containing WBCs, nucleotides were degraded in the order of ADP/UDP>ATP/UTP. Degradation of ATP was partly inhibited by blocking the ecto-NTPDase CD39, whereas degradation of both ATP and ADP was inhibited by blocking ectopyrophosphatase/phosphodiesterase activity. Part of the nucleotide-degrading activities appeared to be membrane-bound. CONCLUSION: Ectonucleotidases in plasma preserve the functionality of P2X1 and P2Y receptors. Upon PLT storage, these plasma activities are essential to ensure adequate (shear-dependent) formation of aggregates and thrombi.

Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation.

Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca(2+) signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor VIIa, complexed with tissue factor, evoked a greatly delayed, oscillatory Ca(2+) response, which relied on its catalytic activity and resembled that of SLIGRL, a peptide specifically activating the protease-activated receptor 2 (PAR2). Thrombin even at low concentrations evoked a rapid, mostly non-oscillating Ca(2+) response through activation of PAR1, which reinforced the factor Xa response. The additive Ca(2+) signals persisted, when factor X and prothrombin were activated in situ, or in the presence of plasma that was triggered to coagulate with tissue factor. Further, thrombin reinforced the factor Xa-induced production of IL-8, but not of IL-6. Both interleukins were produced in the presence of coagulating plasma. In conclusion, under coagulant conditions, factor Xa and thrombin appear to contribute in different and additive ways to the Ca(2+)-mobilizing and proinflammatory reactions of endothelial cells. These data provide first evidence that these serine proteases trigger distinct signaling modules in endothelium that is activated by plasma coagulation.

Platelet ADP response deteriorates in synthetic storage media.

BACKGROUND: During storage under blood bank conditions, platelets (PLTs) are known to secrete ADP. PLT stimulation by ADP results in refractoriness to restimulation, making this response one of the most unstable PLT reactions. The goal of this study was to evaluate the ADP-induced responses of PLTs stored in full plasma or in plasma and additive solution (AS). STUDY DESIGN AND METHODS: Surface expression of P-selectin, ADP-induced aggregation, and reconstituted whole-blood thrombus formation were determined on collagen surfaces in a perfusion model with PLTs that were stored for 4 days either in plasma or in the presence of plasma with PAS-II or Composol. RESULTS: After 4 days of storage in PAS-II but not in Composol, the percentage of PLTs that had secreted granule contents (P-selectin) was increased, when compared to PLTs stored in full plasma. Maximal aggregation in response to ADP was reduced for PLTs stored in PAS-II or Composol. Resuspension of these PLTs in plasma at 37 degrees C for 1 hour caused partial recovery of the aggregation response. Addition of apyrase to PLTs in AS preserved the responsiveness toward ADP. Titration experiments indicated that this response gradually decreased with decreasing plasma concentration. The functional significance of these findings was demonstrated by perfusion experiments. Thrombus formation on collagen was significantly higher for PLTs stored in full plasma than for PLTs stored in PAS-II or Composol. CONCLUSIONS: Storage of PLTs in the presence of AS under blood bank conditions induces deterioration of the PLT responsiveness to ADP compared to PLT concentrates in 100 percent plasma. Higher plasma-to-AS ratios result in better preserved responses.

von Willebrand factor but not alpha-thrombin binding to platelet glycoprotein Ibalpha is influenced by the HPA-2 polymorphism.

OBJECTIVE: Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex. The N-terminal domain of the GPIbalpha chain contains binding sites for alpha-thrombin and von Willebrand factor (VWF). The human platelet alloantigen (HPA)-2 polymorphism of the GPIbalpha gene is associated with a C/T transition at nucleotide 1018, resulting in a Thr/Met dimorphism at residue 145 of GPIbalpha. To study the structural and functional effects of this dimorphism, N-terminal fragments (AA1-289) of the HPA-2a and HPA-2b alloform of GPIbalpha expressed in CHO cells were used. METHODS AND RESULTS: Of 74 moAbs directed against human GPIbalpha, 2 antibodies with epitope between AA1-59 could differentiate between both alloforms. In addition, VWF bound with a higher affinity to the recombinant HPA-2a fragment or to homozygous HPA-2a platelets. In contrast, no difference was found in the binding of alpha-thrombin to the recombinant alloform fragments or of antibodies directed against the alpha-thrombin binding anionic sulfated tyrosine sequence (AA269-282). CONCLUSIONS: Whereas the Thr145Met dimorphism does not affect alpha-thrombin binding, it does influence the conformation of the N-terminal flanking region and first leucine-rich repeat of GPIbalpha and by this has an effect on VWF binding.