Regulated signal transduction in discrete microdomains of the cell surface is an attractive hypothesis for achieving spatial and temporal specificity in signaling. A procedure for purifying caveolae separately from other similarly buoyant microdomains including those rich in glycosylphosphatidylinositol-anchored proteins has been developed (Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J., and Oh, P. (1995) Science 269, 1435-1439) and used here to show that caveolae contain many signaling molecules including select kinases (platelet-derived growth factor (PDGF) receptors, protein kinase C, phosphatidylinositol 3-kinase, and Srclike kinases), phospholipase C, sphingomyelin, and even phosphoinositides. More importantly, two different techniques reveal that caveolae function as signal transducing subcompartments of the plasma membrane. PDGF rapidly induces phosphorylation of endothelial cell plasmalemmal proteins residing in caveolae as detected by membrane subfractionation and confocal immunofluorescence microscopy. This PDGF signaling cascade is halted when the caveolar compartment is disassembled by filipin. Finally, in vitro kinase assays show that caveolae contain most of the intrinsic tyrosine kinase activity of the plasma membrane. As signal transducing organelles, caveolae organize a distinct set of signaling molecules to permit direct regionalized signal transduction within their boundaries.

Background Bromodomain and extra-terminal domain proteins are promising epigenetic anticancer drug targets. This first-in-human study evaluated the safety, recommended phase II dose, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the bromodomain and extra-terminal domain inhibitor molibresib (GSK525762) in patients with nuclear protein in testis (NUT) carcinoma (NC) and other solid tumors. Methods This was a phase I and II, open-label, dose-escalation study. Molibresib was administered orally once daily. Single-patient dose escalation (from 2 mg/d) was conducted until the first instance of grade 2 or higher drug-related toxicity, followed by a 3 + 3 design. Pharmacokinetic parameters were obtained during weeks 1 and 3. Circulating monocyte chemoattractant protein-1 levels were measured as a pharmacodynamic biomarker. Results Sixty-five patients received molibresib. During dose escalation, 11% experienced dose-limiting toxicities, including six instances of grade 4 thrombocytopenia, all with molibresib 60–100 mg. The most frequent treatment-related adverse events of any grade were thrombocytopenia (51%) and gastrointestinal events, including nausea, vomiting, diarrhea, decreased appetite, and dysgeusia (22%–42%), anemia (22%), and fatigue (20%). Molibresib demonstrated an acceptable safety profile up to 100 mg; 80 mg once daily was selected as the recommended phase II dose. Following single and repeat dosing, molibresib showed rapid absorption and elimination (maximum plasma concentration: 2 hours; t1/2: 3–7 hours). Dose-dependent reductions in circulating monocyte chemoattractant protein-1 levels were observed. Among 19 patients with NC, four achieved either confirmed or unconfirmed partial response, eight had stable disease as best response, and four were progression-free for more than 6 months. Conclusions Once-daily molibresib was tolerated at doses demonstrating target engagement. Preliminary data indicate proof-of-concept in NC.

Desensitization plays an important role in the rapid termination of G-protein signaling pathways. This process, which involves phosphorylation by a G-protein-coupled receptor kinase (GRK) followed by arrestin binding, has been studied extensively in the rod photoreceptor cell of the mammalian retina. In contrast, less is known regarding desensitization in cone photoreceptor cells, which occurs more rapidly than in rod cells. Recently, our laboratory has cloned a novel GRK family member, GRK7, from the retina of a cone-dominant mammal, the 13-lined ground squirrel. Here we report the cloning of GRK7 from rod-dominant pig and human retinas, suggesting that this kinase plays a role in human visual signaling. Because GRK1 (rhodopsin kinase), the GRK that mediates rhodopsin desensitization in the rod cell, is reportedly expressed in both rods and cones, a detailed comparison of the localization of the two kinases is a necessary step toward determining their potential roles in cone visual signaling. Immunocytochemical analysis using antibodies selective for these two GRKs unexpectedly demonstrated species-specific differences in GRK7 and GRK1 expression in cones. In pigs and dogs, cones express only GRK7, whereas in mice and rats, we detected only GRK1 in cones. These results suggest that either GRK7 or GRK1 may participate in cone opsin desensitization, depending on the expression pattern of the kinases in different species. In contrast, GRK7 and GRK1 are coexpressed in monkey and human cones, suggesting that coordinate regulation of desensitization by both kinases may occur in primates.

Purpose: Enhancer of zeste homolog 2 (EZH2) activity is dysregulated in many cancers.Patients and Methods: This phase I study determined the safety, maximum-tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of the intravenously administered, highly selective EZH2 inhibitor, GSK2816126, (NCT02082977). Doses of GSK2816126 ranged from 50 to 3,000 mg twice weekly, and GSK2816126 was given 3-weekson/1-week-off in 28-day cycles. Eligible patients had solid tumors or B-cell lymphomas with no available standard treatment regimen.Results: Forty-one patients (21 solid tumors, 20 lymphoma) received treatment. All patients experienced !1 adverse event (AE). Fatigue [22 of 41 (53.7%)] and nausea [20 of 41 (48.8%)] were the most common toxicity. Twelve (32%) patients experienced a serious AE. Dose-limiting elevated liver transaminases occurred in 2 of 7 patients receiving 3,000 mg of GSK2816126; 2,400 mg was therefore established as the MTD. Following intravenous administration of 50 to 3,000 mg twice weekly, plasma GSK2816126 levels decreased biexponentially, with a mean terminal elimination half-life of approximately 27 hours. GSK2816126 exposure (maximum observed plasma concentration and area under the plasmatime curve) increased in a dose-proportional manner. No change from baseline in H3K27me3 was seen in peripheral blood mononuclear cells. Fourteen of 41 (34%) patients had radiological best response of stable disease, 1 patient with lymphoma achieved a partial response, 21 of 41 (51%) patients had progressive disease, and 5 patients were unevaluable for antitumor response.Conclusions: The MTD of GSK2816126 was established at 2,400 mg, but the dosing method and relatively short half-life limited effective exposure, and modest anticancer activity was observed at tolerable doses.

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K m and V max of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser 21 and GRK7 at Ser 23 and Ser 36 in vitro. These sites are also phosphorylated when FLAGtagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca 2؉ /calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases. G protein-coupled receptors (GPCRs)1 belong to the largest family of signal-transducing proteins in eukaryotes. They mediate cellular responses to a variety of environment stimuli, including light, taste, odorants, ions, nucleotides, peptides, and lipids, through the activation of heterotrimeric G proteins (1-4). Phosphorylation of ligand-activated GPCRs by G proteincoupled receptor kinases (GRKs) is the initial step in the rapid termination or desensitization of GPCR signal transduction. For example, in rod cells of the vertebrate retina, desensitization occurs when light-activated rhodopsin is phosphorylated by GRK1, followed by the binding of visual arrestin to the phosphorylated rhodopsin, which blocks its interaction with transducin, the rod cell G protein (5). Although desensitization also occurs in cone cells, less is known about the proteins involved and their regulation. GRK1 and a cone-specific GRK, GRK7, are coexpressed in human and monkey cone cells (6 -8) where both may contribute to the deactivation of cone opsins (7-9). In contrast, only GRK7 is expressed in the cones of pigs and dogs. However, it is absent from the mouse genome altogether and mouse cones express only GRK1 (7). Therefore, differences in cone visual transduction between species may result in part from variations in the expression pattern of GRK1 and GRK7. We recently implicated GRK7 in the phosphorylation of cone o...

Polo-like protein kinase 3 (Plk3) has been proposed to regulate entry into S phase and promote apoptosis in response to oxidative stress. Its mRNA contains three AU-rich elements (AREs) in its 3 untranslated region (3-UTR) that can contribute to the rapid degradation of labile transcripts. We investigated the possibility that tristetraprolin (TTP), a tandem CCCH zinc finger protein, could promote the decay of Plk3 transcripts. TTP is known to stimulate the deadenylation and decay of mRNAs possessing one or more copies of the consensus nonamer motif UUAUUUAUU. In stable mouse fibroblast cell lines derived from wild-type and TTP knockout littermates, the decay of Plk3 transcripts after serum stimulation was slowed in the absence of TTP. The specificity of TTP for promoting the degradation of Plk3 was demonstrated by the unaltered decay of Plk3 mRNA in cell lines deficient in the TTP family members ZFP36L1 and ZFP36L2. We also found that the AREs present in the Plk3 transcript were essential for both the binding of TTP to the 3-UTR and promoting the destruction of target transcripts in cotransfection experiments. The regulation of Plk3 mRNA stability by TTP may influence the control of the cell cycle by this protein kinase.

Background: GSK525762 is a potent, selective pan-BET inhibitor that abrogates binding of BET family proteins (BRD2, BRD3, BRD4 and BRD-T) to acetylated histones. In pre-clinical models this results in suppression of BET target genes that drive oncogenic pathways, resulting in growth inhibition of cancer cell lines (median IC50 of 50-1698 nM for solid tumors and 50 nM for NMC). GSK525762 is being evaluated clinically for treatment of pts with hematologic malignancies and solid tumors including NMC, a rare, aggressive cancer with few treatment options. NMC is characterized by BRD3-/BRD4-NUT fusion oncoproteins that represents a rational therapeutic target for BET inhibitors. Methods: Primary objectives of Part 1 were to determine safety, tolerability, and maximum tolerated dose (MTD) for GSK525762 in solid tumors, using a combined N-CRM and 3+3 dose escalation. Secondary objectives include PK and PD analysis and preliminary evaluation of activity. Oral once daily (qd) and twice daily (bid) schedules have been evaluated. PD activity was assessed with [18F]-deoxy-glucose-PET scans and changes in cytokine release from LPS-stimulated PBMC of treated pts. The dose-limiting toxicity (DLT) observation period was 28 days. Response evaluation was by RECIST 1.1 and pts were followed to disease progression, discontinuation due to adverse events, withdrawal of consent or death. Results: As of 28-Jan 2016, a total of 70 (including 17 NMC) pts have been treated at doses of 2-100 mg qd and 20 and 30 mg bid. The most common adverse events (all grades, ?20%) observed were thrombocytopenia (44%), nausea (40%), vomiting (29%), anemia (26%), fatigue (26%), decreased appetite (24%), diarrhoea (23%) and dysgeusia (20%). DLTs occurred in 5 pts at doses of 60 mg (n = 1, 11%), 80 mg (n = 3, 14%) and 100 mg (n = 1, 11%). The most common AEs leading to dose interruption were thrombocytopenia and hyperbilirubinemia (each n = 7, 10%). Limited data shows similar tolerability at bid doses. PK was dose-proportional after single and repeat dosing with large between patient variability. Dose dependent inhibition of LPS stimulated BRD regulated cytokine (IL-6, TNF-α, MCP-1, IL-8) release suggests target engagement. Of 11 NMC pts treated at 60-100 mg qd, evaluation is awaited on 1 pt. Of 10 pts evaluated, 2 (20%; 95% CI (2.5%, 56%)) had confirmed PR (15 and 23 weeks) and 4 (40%; 95% CI (12%, 74%)) had SD. An 80mg once-daily dose will be evaluated in NMC and other solid tumor expansion cohorts. Conclusion: GSK525762 is an active, orally bio-available BET inhibitor showing dose-proportional PK and good tolerability up to 80 mg qd dosing. Preliminary evidence of clinical activity observed in pts with NMC provides a rational targeted therapy for this aggressive tumor and proof of concept for BET inhibitors in the clinic. Study funded by GSK (NCT01587703). Citation Format: Peter J. O’Dwyer, Sarina A. Piha-Paul, Christopher French, Sara Harward, Gerladine Ferron-Brady, Yuehui Wu, Olena Barbash, Anastasia Wyce, Meg Annan, Thierry Horner, Nigel J. Parr, Rab K. Prinjha, Christopher Carpenter, Geoffrey Shapiro, Arindam Dhar, Christine Hann. GSK525762, a selective bromodomain (BRD) and extra terminal protein (BET) inhibitor: results from part 1 of a phase I/II open-label single-agent study in patients with NUT midline carcinoma (NMC) and other cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT014.

resistant prostate cancer; CTCAE v4.0, Common Terminology Criteria for Adverse Events version 4.0; ECG, electrocardiogram; ECOG, Eastern Cooperative Oncology Group; ER + BC, estrogen receptorpositive breast cancer; FPKM, fragments per kilobase per million reads; FTIH, first time in human; GIST, gastrointestinal stromal tumor; GSEA, gene set enrichment analysis; HIV, human immunodeficiency virus; MSigDB, molecular signatures database; NC, nuclear protein in testis carcinoma; NUT, nuclear protein in testis; ORR, overall response rate; PD, progressive disease; PK, pharmacokinetic; PR, partial response; PSA, prostate-specific antigen; QTcF, QT interval assessment corrected for heart rate by Fridericia's formula; RECIST v1.1, Response Evaluation Criteria In Solid Tumors version 1.1 criteria; RP2D, recommended Phase 2 dose; SAE, serious adverse event; SCLC, small cell lung cancer; SD, stable disease; TNBC, triple-negative breast cancer; ULN, upper limit of normal.Antara Datta was working at GSK at the time of study design and initiation.