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Background: Fluorine-18 fluorodeoxyglucose (FDG) with positron emission tomography (PET) is the standard for detecting myocardial inflammation in cardiac sarcoidosis, requiring preparation with the ketogenic diet (KD) to achieve myocardial glucose suppression. Despite this, incomplete myocardial glucose suppression remains a significant issue, and strategies to reduce myocardial glucose uptake (MGU) and identify incomplete myocardial glucose suppression are required. This study sought to understand the relationship between point-of-care beta-hydroxybutyrate (BHB) and different patterns of MGU and between KD and fasting duration with MGU in patients undergoing evaluation for cardiac sarcoidosis., Methods: We prospectively included 471 outpatients who underwent FDG-PET for cardiac sarcoidosis evaluation, followed the KD for 1 (n=100), 2 (n=29), and ≥3 days (n=342), fasted for at least 12 hours, and had BHB levels measured immediately before FDG injection. Images were classified as (1) no MGU (negative), (2) focal/multifocal (positive), (3) diffuse (nondiagnostic), or (4) nonspecific uptake (NS-MGU)., Results: Cardiac FDG-PET scans were interpreted as the following: 376 (79.83%) negative; 61 (12.95%) positive; 14 (2.97%) diffuse; and 20 (4.25%) NS-MGU. There was a strong negative relationship between BHB levels and MGU ( P <0.0001). BHB levels increased significantly with KD duration ( P <0.0001) and fasting time ( P =0.0067). The combined rate of diffuse, NS-MGU, and positive scans (34%, 28%, 16%) decreased inversely with KD duration (1, 2, and ≥3 days, respectively). However, MGU was not different across different fasting times ( P =0.6). Blood glucose levels were not associated with MGU ( P =0.17) and only weakly associated with BHB levels (R 2 =0.03; P <0.001)., Conclusions: We observed a strong inverse relationship between ketosis and patterns of MGU. Longer KD and fasting durations are associated with higher ketosis. However, only KD duration was associated with lower rates of MGU. Measurement of BHB levels before FDG-PET using point-of-care testing is feasible and may facilitate the management of patients referred for myocardial inflammation., Competing Interests: None.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K) δ inhibitors in B-cell malignancies is hampered by hepatotoxicity, requiring dose reduction, treatment interruption, and/or discontinuation of therapy. In addition, potential molecular mechanisms by which resistance to this class of drugs occurs have not been investigated. Parsaclisib (INCB050465) is a potent and selective next-generation PI3K δ inhibitor that differs in structure from first-generation PI3K δ inhibitors and has shown encouraging anti-B-cell tumor activity and reduced hepatotoxicity in phase 1/2 clinical studies. Here, we present preclinical data demonstrating parsaclisib as a potent inhibitor of PI3K δ with over 1000-fold selectivity against other class 1 PI3K isozymes. Parsaclisib directly blocks PI3K signaling-mediated cell proliferation in B-cell lines in vitro and in vivo and indirectly controls tumor growth by lessening immunosuppression through regulatory T-cell inhibition in a syngeneic lymphoma model. Diffuse large B-cell lymphoma cell lines overexpressing MYC were insensitive to proliferation blockade via PI3K δ signaling inhibition by parsaclisib, but their proliferative activities were reduced by suppression of MYC gene transcription. Molecular structure analysis of the first- and next-generation PI3K δ inhibitors combined with clinical observation suggests that hepatotoxicity seen with the first-generation inhibitors could result from a structure-related off-target effect. Parsaclisib is currently being evaluated in multiple phase 2 clinical trials as a therapy against various hematologic malignancies of B-cell origin (NCT03126019, NCT02998476, NCT03235544, NCT03144674, and NCT02018861). SIGNIFICANCE STATEMENT: The preclinical properties described here provide the mechanism of action and support clinical investigations of parsaclisib as a therapy for B-cell malignancies. MYC overexpression was identified as a resistance mechanism to parsaclisib in DLBCL cells, which may be useful in guiding further translational studies for the selection of patients with DLBCL who might benefit from PI3Kδ inhibitor treatment in future trials. Hepatotoxicity associated with first-generation PI3Kδ inhibitors may be an off-target effect of that class of compounds., (Copyright © 2020 by The Author(s).)

Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombinant human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations., Competing Interests: Holly Koblish, Liangxing Wu, Kevin Bowman, Sharon Diamond, Darlise DiMatteo, Yue Zhang, Michael Hansbury, Mark Rupar, Xiaoming Wen, Paul Collier, Patricia Feldman, Ronald Klabe, Krista A. Burke, Maxim Soloviev, Christine Gardiner, Xin He, Alla Volgina, Maryanne Covington, Richard Wynn, Timothy C. Burn, Swamy Yeleswaram, Wenqing Yao are employees of Incyte Corporationand own Incyte stock. Phillip C. C. Liu, Bruce Ruggeri, Peggy Scherle, Reid Huber, and Gregory Hollis were employees of Incyte employees Corporation at the time of this work and own Incyte stock.Our commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

In an effort to understand the effect of N-alkylation of triarylimidazoles on Tie2 inhibition, ortho-substituted C-2 aryl analogs were synthesized to investigate the effect of different torsion angles on potency. This exercise resulted in the identification of a potent and selective tetrasubstituted imidazole that was efficacious in an animal model of angiogenesis.

This communication details the evolution of the screening lead SB-203580, a known CSBP/p38 kinase inhibitor, into a potent and selective Tie2 tyrosine kinase inhibitor. The optimized compound 5 showed efficacy in an in vivo model of angiogenesis and a MOPC-315 plasmacytoma xenograft model.

Purpose: ErbB receptor signaling pathways are important regulators of cell fate, and their dysregulation, through (epi)genetic alterations, plays an etiologic role in multiple cancers. ErbB ligands are synthesized as membrane-bound precursors that are cleaved by members of the ADAM family of zinc-dependent metalloproteases. This processing, termed ectodomain shedding, is essential for the functional activation of ErbB ligands. Recent studies suggest that elevated levels of ErbB ligands may circumvent the effectiveness of ErbB-targeted therapeutics. Here, we describe the discovery and preclinical development of potent, selective inhibitors of ErbB ligand shedding., Experimental Design: A series of biochemical and cell-based assays were established to identify selective inhibitors of ErbB ligand shedding. The therapeutic potential of these compounds was assessed in multiple in vivo models of cancer and matrix metalloprotease-related toxicity., Results: INCB3619 was identified as a representative selective, potent, orally bioavailable small-molecule inhibitor of a subset of ADAM proteases that block shedding of ErbB ligands. Administration of INCB3619 to tumor-bearing mice reduced ErbB ligand shedding in vivo and inhibited ErbB pathway signaling (e.g., phosphorylation of Akt), tumor cell proliferation, and survival. Further, INCB3619 synergized with clinically relevant cancer therapeutics and showed no overt or compounding toxicities, including fibroplasia, the dose-limiting toxicity associated with broad-spectrum matrix metalloprotease inhibitors., Conclusions: Inhibition of ErbB ligand shedding offers a potentially novel and well-tolerated therapeutic strategy for the treatment of human cancers and is currently being evaluated in the clinic.

Angiopoietin-1 (Ang-1) is one of a family of ligands for the Tie-2 receptor which has been demonstrated to be involved in angiogenesis. Little is known about the regulation of Ang-1 gene expression. We have previously demonstrated that TNF-alpha is able to up-regulate the expression of Ang-1 mRNA in synovial fibroblasts. This present study investigated the signal transduction pathways involved in the TNF-alpha induced expression of Ang-1. TNF-alpha signals primarily through the p38, JNK, MAP kinase, and IKK pathways resulting in the activation of the transcription factors AP-1 and NF-kappa B. Experiments with inhibitors and siRNA for these various signal transduction pathways revealed that TNF-alpha stimulation of Ang-1 expression occurs via the NF-kappa B signal transduction pathway.

There are at least four distinct families of enzymes that recognize and remove uracil from DNA. Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro. Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system. These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair. When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs. The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair. When uracil-DNA glycosylase is deleted (deltaung1), cells continue through S-phase and arrest at G(2)/M, presumably due to the effects of stable uracil misincorporation in DNA. Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair. As a result, these cells experience early protection from antifolate-induced cytotoxicity. When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest. The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE. Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.

The Tie2 receptor and its known ligands, the angiopoietins, play a critical role in endothelial cell differentiation during the process of angiogenesis. Recent experimental observations indicate that the agonistic ligand, angiopoietin-1, can stimulate endothelial cell sprouting and act as a chemo-attractant in vitro and induce increased and enhanced angiogenesis both alone and in conjunction with vascular endothelial growth factor (VEGF) in vivo. Here, we present a monoclonal antibody (MAb), which binds to the extracellular portion of the Tie2 receptor and elicits similar agonist effects. Upon MAb binding to the native Tie2 receptor of cultured human umblical vein endothelial cells (HUVEC), there is a rapid increase in receptor autophosphorylation with a concomitant enhancement in the recruitment and association of the signalling intermediates Grb2 and SH-PTP2. The antibody further demonstrates functional activity in vascular tissues. In vitro, the antibody promotes the survival of cultured HUVEC and elicits a dose dependent outgrowth and branching of microvessels from cultured explants of rat aorta. When administered in vivo, the antibody enhances the vascularization of subcutaneous Matrigel implants in mice. Together these data suggest that the antibody is capable of acting as a surrogate ligand for Tie2 and further confirms the role of Tie2 in the differentiation of endothelial cells during angiogenesis.

CD23, the low affinity IgE receptor, is up-regulated on the surface of IL-4-treated B cells and monocytes and is immediately proteolytically processed, releasing soluble fragments of CD23. Here, we report that inhibitors of the p38 mitogen-activated kinase (p38 MAPK), SK&F 86002 or the more selective inhibitor, SB 203580, reduce the levels of soluble CD23 formed by IL-4-stimulated human monocytes or the human monocytic cell line, U937. In contrast to compounds such as the metalloprotease inhibitor batimastat ([4-(N-hydroxyamino)-2-(R)-isobutyl-3-(S)-(2-thiophenethiomethyl)s uccinyl]-(S)-phenylalanine-N-methylamide, sodium salt), p38 MAPK inhibitors do not directly inhibit proteolytic processing of CD23. Further, evaluation of surface intact CD23 (iCD23) by flow cytometry demonstrated that SK&F 86002 and SB 203580 reduced the surface expression of iCD23 in a concentration-dependent fashion, while batimastat increased the surface expression of iCD23. The decrease in surface iCD23 was accompanied by a decrease in total cell-associated CD23 protein levels but not CD23 mRNA. IL-4 induced a late (>4-h) increase in p38 MAPK activity and corresponding activation of its substrate MAPKAPK-2. This activation was blocked by addition of SB 203580 before IL-4 induction, in parallel with the inhibition of CD23 expression. Modulation of CD23 by antibodies has been shown to alleviate the symptoms of murine collagen-induced arthritis, implicating CD23 as an important proinflammatory agent. These data show that in addition to the known cytokine inhibitory actions of SK&F 86002 and SB 203580, they also confer an additional potential anti-inflammatory activity through modulation of CD23 expression.

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells. We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells. This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by the Bacillus subtilis bacteriophage, PBS2. Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS-PAGE and silver staining. In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations. Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell. Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1. With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment. Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes. Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently post-translationally processed into a lower molecular weight form. This is substantiated by mitochondrial import studies which reveal that in vitro expressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria. Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria. Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000. SDS-PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell. Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus. Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA. These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme.

Molecular analysis of the induction of the lutropin/choriogonadotropin receptor during the process of luteinization of the rat ovary was performed. The appearance of receptor binding activity and an immunological analysis of the receptor using Triton solubilized membrane proteins show little receptor present in luteal tissue through day 3 subsequent to hCG treatment, with some in day 4, and a marked increase by day 5. A similar pattern was found in the analysis of RNA hybridizing to several probes derived from the published cDNA sequence suggesting that receptor induction occurs primarily at the level of transcription.