Dual targeting of CD19 and CD22 with bicistronic CAR-T cells in patients with relapsed/refractory large B-cell lymphoma.

Relapse after CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy for large B-cell lymphoma (LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multiantigen targeting and programmed cell death protein-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in relapsed/refractory LBCL (NCT03289455). End points include toxicity (primary) and response rates (secondary). Fifty-two patients received AUTO3 and 48/52 received pembrolizumab. Median age was 59 years (range, 27-83), 46/52 had stage III/ IV disease and median follow-up was 21.6 months. AUTO3 was safe; grade 1-2 and grade 3 cytokine release syndrome affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), and hemophagocytic lymphohistiocytosis affected 2 patients. Outpatient administration was tested in 20 patients, saving a median of 14 hospital days per patient. Overall response rates were 66% (48.9%, complete response [CR]; 17%, partial response). Median duration of remission (DOR) for CR patients was not reached and for all responding patients was 8.3 months (95% confidence interval [CI]: 3.0-not evaluable). 54.4% (CI: 32.8-71.7) of CR patients and 42.6% of all responding patients were projected to remain progression-free at ≥12 months. AUTO3 ± pembrolizumab for relapsed/refractory LBCL was safe and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion in vivo, and selection of CAR binders active at low antigen densities.

CAR T cells with dual targeting of CD19 and CD22 in pediatric and young adult patients with relapsed or refractory B cell acute lymphoblastic leukemia: a phase 1 trial.

Chimeric antigen receptor (CAR) T cells targeting CD19 or CD22 have shown remarkable activity in B cell acute lymphoblastic leukemia (B-ALL). The major cause of treatment failure is antigen downregulation or loss. Dual antigen targeting could potentially prevent this, but the clinical safety and efficacy of CAR T cells targeting both CD19 and CD22 remain unclear. We conducted a phase 1 trial in pediatric and young adult patients with relapsed or refractory B-ALL (n = 15) to test AUTO3, autologous transduced T cells expressing both anti-CD19 and anti-CD22 CARs (AMELIA trial, EUDRA CT 2016-004680-39). The primary endpoints were the incidence of grade 3-5 toxicity in the dose-limiting toxicity period and the frequency of dose-limiting toxicities. Secondary endpoints included the rate of morphological remission (complete response or complete response with incomplete bone marrow recovery) with minimal residual disease-negative response, as well as the frequency and severity of adverse events, expansion and persistence of AUTO3, duration of B cell aplasia, and overall and event-free survival. The study endpoints were met. AUTO3 showed a favorable safety profile, with no dose-limiting toxicities or cases of AUTO3-related severe cytokine release syndrome or neurotoxicity reported. At 1 month after treatment the remission rate (that is, complete response or complete response with incomplete bone marrow recovery) was 86% (13 of 15 patients). The 1 year overall and event-free survival rates were 60% and 32%, respectively. Relapses were probably due to limited long-term AUTO3 persistence. Strategies to improve CAR T cell persistence are needed to fully realize the potential of dual targeting CAR T cell therapy in B-ALL.

A preliminary world checklist of fern-mining insects.

Compared to the leaf-miners and stem-miners on flowering plants, the miners on ferns (including both Lycopodiophyta and Polypodiophyta in the broad sense) are less known. Knowledge of miners and their host plants is essential to fully understand plant-insect interactions. Although there are many scattered records on fern miners, a worldwide checklist has not been reported. We provide a preliminary checklist of fern-mining insects and their host plants worldwide. Altogether, we found records for 128 species and 18 families of fern miners, mainly that feed on Dennstaedtiaceae, Equisetaceae, Polypodiaceae and Aspleniaceae. Fern miners belonged to four orders: Diptera (51 species; 39.8%), Coleoptera (33 species; 25.8%), Lepidoptera (28 species; 21.9%) and Hymenoptera (16 species; 12.5%). They are primarily known from the Palaearctic Region, Nearctic Region, Neotropical Region and Oriental Region.

STING Activation and its Application in Immuno-Oncology.

Recent regulatory approval of several immune checkpoint inhibitors has ushered in a new era of cancer immunotherapies with the promise of achieving a durable response. This represents a paradigm shift in cancer treatment from directly targeting tumor cells to harnessing the power of a patient's own immune system to destroy them. The cGAS-STING pathway is the major cytosolic dsDNA sensing pathway that plays a pivotal role in the innate antitumor immune response. With a fundamentally different mode of action (MOA) than immune checkpoint modulators, STING activation can potentially enhance tumor immunogenicity and improve patient responses as a single agent or by synergizing with existing anti-cancer drugs. Therefore, there has been intense interest from the pharmaceutical industry and academic institutions in the search for potent STING agonists as immunotherapies in oncology. In this article, we review briefly the cGAS-STING pathway and STING agonists that are in the clinical and preclinical studies, summarize recently disclosed patent applications and published journal articles in the field and cover both cyclic dinucleotide (CDN) analogs and non-nucleic acid derived STING agonists.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

Discovery and Preclinical Characterization of GSK1278863 (Daprodustat), a Small Molecule Hypoxia Inducible Factor-Prolyl Hydroxylase Inhibitor for Anemia.

Decreased erythropoietin (EPO) production, shortened erythrocyte survival, and other factors reducing the response to EPO contribute to anemia in patients who have a variety of underlying pathologies such as chronic kidney disease. Treatment with recombinant human EPO (rHuEPO) at supraphysiologic concentrations has proven to be efficacious. However, it does not ameliorate the condition in all patients, and it presents its own risks, including cardiovascular complications. The transcription factors hypoxia-inducible factor (HIF) 1α and HIF2α control the physiologic response to hypoxia and invoke a program of increased erythropoiesis. Levels of HIFα are modulated by oxygen tension via the action of a family of HIF-prolyl hydroxylases (PHDs), which tag HIFα for proteasomal degradation. Inhibition of these PHDs simulates conditions of mild hypoxia, leading to a potentially more physiologic erythropoietic response and presenting a potential alternative to high doses of rHuEPO. Here we describe the discovery and characterization of GSK1278863 [2-(1,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido) acetic acid], a pyrimidinetrione-glycinamide low nanomolar inhibitor of PHDs 1-3 that stabilizes HIFα in cell lines, resulting in the production of increased levels of EPO. In normal mice, a single dose of GSK1278863 induced significant increases in circulating plasma EPO but only minimal increases in plasma vascular endothelial growth factor (VEGF-A) concentrations. GSK1278863 significantly increased reticulocytes and red cell mass parameters in preclinical species after once-daily oral administration and has demonstrated an acceptable nonclinical toxicity profile, supporting continued clinical development. GSK1278863 is currently in phase 3 clinical trials for treatment of anemia in patients with chronic kidney disease.

Brain translocator protein occupancy by ONO-2952 in healthy adults: A Phase 1 PET study using [11 C]PBR28.

ONO-2952, a novel antagonist of translocator protein 18 kDa (TSPO), binds with high affinity to TSPO in rat brain and human tumor cell line membrane preparations. This study used the TSPO-specific PET radioligand [11 C]PBR28 to confirm binding of ONO-2952 to brain TSPO in human subjects, and evaluate brain TSPO occupancy and its relationship with ONO-2952 plasma concentration. Sixteen healthy subjects received a single oral dose of 200, 60, 20, or 6 mg ONO-2952 (n = 4 per dose). Two PET scans with [11 C]PBR28 were conducted ≤7 days apart: at baseline and 24 h after ONO-2952 administration. [11 C]PBR28 regional distribution volume (VT ) was derived with kinetic modeling using the arterial input function and a two tissue compartment model. Nonspecific binding (VND ) was obtained on an individual basis for each subject using linear regression as the x-intercept of the Lassen plot. The binding potential relative to VND (BPND ) was derived as the difference between VT in the ROI (VT ROI) and VND , normalized to VND ; BPND = (VT ROI - VND )/VND . TSPO occupancy was calculated as the change in BPND (ΔBPND ) from individual's baseline scan to the on-medication scan to the baseline BPND value. TSPO occupancy by ONO-2952 was dose dependent between 20-200 mg, approaching saturation at 200 mg both in the whole brain and in 15 anatomic regions of interest (ROI). Estimated Ki values ranged from 24.1 to 72.2 nM. This open-label, single-center, single-dose study demonstrated engagement of ONO-2952 to brain TSPO. The relationship between pharmacokinetics and TSPO occupancy observed in this study support the hypothesis that ONO-2952 could potentially modulate neurosteroid production by binding to brain TSPO.

A phase 1 clinical trial of the selective BTK inhibitor ONO/GS-4059 in relapsed and refractory mature B-cell malignancies.

We report the results of a multicenter phase 1 dose-escalation study of the selective Bruton tyrosine kinase (BTK) inhibitor ONO/GS-4059 in 90 patients with relapsed/refractory B-cell malignancies. There were 9 dose-escalation cohorts ranging from 20 mg to 600 mg once daily with twice-daily regimens of 240 mg and 300 mg. Twenty-four of 25 evaluable chronic lymphocytic leukemia (CLL) patients (96%) responded to ONO/GS-4059, with a median treatment duration of 80 weeks; 21 CLL patients remain on treatment. Lymph node responses were rapid and associated with a concurrent lymphocytosis. Eleven of 12 evaluable patients with mantle cell lymphoma (92%) responded (median treatment duration, 40 weeks). Eleven of 31 non-germinal center B-cell diffuse large B-cell lymphoma patients (35%) responded but median treatment duration was 12 weeks due to development of progressive disease. ONO/GS-4059 was very well tolerated with 75% of adverse events (AEs) being Common Toxicity Criteria for Adverse Events version 4.0 grade 1 or grade 2. Grade 3/4 AEs were mainly hematologic and recovered spontaneously during therapy. One CLL patient experienced a grade 3 treatment-related bleeding event (spontaneous muscle hematoma) but no clinically significant diarrhea, cardiac dysrhythmias, or arthralgia were observed. No maximal tolerated dose (MTD) was reached in the CLL cohort. In the non-Hodgkin lymphoma cohort, 4 patients developed a dose-limiting toxicity, yielding an MTD of 480 mg once daily. ONO/GS-4059 has significant activity in relapsed/refractory B-cell malignancies without major drug-related toxicity. The selectivity of ONO/GS-4059 should confer advantages in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT01659255.

Targeting lactate dehydrogenase--a inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor-initiating cells.

The lactate dehydrogenase-A (LDH-A) enzyme catalyzes the interconversion of pyruvate and lactate, is upregulated in human cancers, and is associated with aggressive tumor outcomes. Here we use an inducible murine model and demonstrate that inactivation of LDH-A in mouse models of NSCLC driven by oncogenic K-RAS or EGFR leads to decreased tumorigenesis and disease regression in established tumors. We also show that abrogation of LDH-A results in reprogramming of pyruvate metabolism, with decreased lactic fermentation in vitro, in vivo, and ex vivo. This was accompanied by reactivation of mitochondrial function in vitro, but not in vivo or ex vivo. Finally, using a specific small molecule LDH-A inhibitor, we demonstrated that LDH-A is essential for cancer-initiating cell survival and proliferation. Thus, LDH-A can be a viable therapeutic target for NSCLC, including cancer stem cell-dependent drug-resistant tumors.

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells.

BACKGROUND: Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA. METHODS: High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment. RESULTS: 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells. CONCLUSIONS: Rapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Chronic inhibition of hypoxia-inducible factor prolyl 4-hydroxylase improves ventricular performance, remodeling, and vascularity after myocardial infarction in the rat.

BACKGROUND: Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI). METHODS AND RESULTS: GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group). CONCLUSIONS: Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.

Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist.

Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34(+) bone marrow cells into CD41(+) megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.

Novel ATP-competitive kinesin spindle protein inhibitors.

Kinesin spindle protein (KSP), an ATPase responsible for spindle pole separation during mitosis that is present only in proliferating cells, has become a novel and attractive anticancer target with potential for reduced side effects compared to currently available therapies. We report herein the discovery of the first known ATP-competitive inhibitors of KSP, which display a unique activity profile as compared to the known loop 5 (L5) allosteric KSP inhibitors that are currently under clinical evaluation. Optimization of this series led to the identification of biphenyl sulfamide 20, a potent KSP inhibitor with in vitro antiproliferative activity against human cells with either wild-type KSP (HCT116) or mutant KSP (HCT116 D130V). In a murine xenograft model with HCT116 D130V tumors, 20 showed significant antitumor activity following intraperitoneal dosing, providing in vivo proof-of-principle of the efficacy of an ATP-competitive KSP inhibitor versus tumors that are resistant to the other known KSP inhibitors.

Zebrafish tp53 knockdown extends the survival of irradiated zebrafish embryos more effectively than the p53 inhibitor pifithrin-alpha.

Cancer treatment strategies using radiation therapy have significantly improved outcomes in patients. Although advances in physics and electronics have enabled physicians to plan and deliver radiotherapy regimens precisely and efficiently, studies of the underlying molecular mechanisms of the radiation response could enable more effective and less toxic treatment strategies, as well as protection against radiation damage following unplanned exposure. Using the developing zebrafish embryo as a model system, we hypothesized that knockdown of tp53 mRNA or small molecule inhibition of p53 could alter the efficacy of radiotherapy against developing zebrafish embryos as a model for tumors. We found that interfering with p53 expression and function using hydroxylprolyl-phosphono peptide nucleic acid oligomers or pifithrin-alpha protected developing zebrafish embryos against radiation damage, consistent with our hypothesis.

Cytochrome oxidase and neurofilament reactivity in monocularly deprived human primary visual cortex.

Previous studies of human primary visual cortex (V1) have demonstrated a significant eye-specific decrease in cytochrome oxidase (CO) staining following monocular enucleation. We have extended these results by examining CO staining and neurofilament labeling in V1 from a patient with long-standing monocular blindness. A pattern of reduced neurofilament reactivity was found to align with pale CO-stained ocular dominance columns. Neurons located within deprived ocular dominance columns were significantly smaller compared with those in nondeprived columns. A spatial analysis of the relationship between CO blobs and ocular dominance columns revealed that both deprived and nondeprived blobs tended to align with the centers of ocular dominance columns.

Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos.

PURPOSE: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance the radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. METHODS AND MATERIALS: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. RESULTS: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the "curly up" phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. CONCLUSIONS: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

Coordinate control of cell cycle regulatory genes in zebrafish development tested by cyclin D1 knockdown with morpholino phosphorodiamidates and hydroxyprolyl-phosphono peptide nucleic acids.

During early zebrafish (Danio rerio) development zygotic transcription does not begin until the mid-blastula transition (MBT) 3 h after fertilization. MBT demarcates transition from synchronous short cell cycles of S and M phases exclusively to full cycles encompassing G1 and G2 phases. Transcriptional profiling and RT-PCR analyses during these phases enabled us to determine that this shift corresponds to decreased transcript levels of S/M phase cell cycle control genes (e.g. ccna2, ccnb1, ccnb2 and ccne) and increased transcript levels of ccnd1, encoding cyclin D1, and orthologs of p21 (p21-like) and retinoblastoma (Rb-like 1). To investigate the regulation of this process further, the translation of ccnd1 mRNA, a G1/S checkpoint control element, was impaired by microinjection of ccnd1-specific morpholino phosphorodiamidate (MO) 20mer or hydroxyprolyl-phosphono peptide nucleic acid (HypNA-pPNA) 16mer antisense oligonucleotides. The resulting downregulation of cyclin D1 protein resulted in microophthalmia and microcephaly, but not lethality. The phenotypes were not seen with 3-mismatch MO 20mers or 1-mismatch HypNA-pPNA 16mers, and were rescued by an exogenous ccnd1 mRNA construct with five mismatches. Collectively, these results indicate that transcription of key molecular determinants of asynchronous cell cycle control in zebrafish embryos commences at MBT and that the reduction of cyclin D1 expression compromises zebrafish eye and head development.

Discovery and characterization of a selective, nonpeptidyl thrombopoietin receptor agonist.

OBJECTIVE: Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line. METHODS: STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro. RESULTS: Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey. CONCLUSIONS: SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo.

External imaging of CCND1 cancer gene activity in experimental human breast cancer xenografts with 99mTc-peptide-peptide nucleic acid-peptide chimeras.

UNLABELLED: Detection of a new or recurrent breast cancer lesion relies on physical examination and imaging studies, primarily mammography, followed by histopathologic evaluation of biopsy tissue for morphologic confirmation. Approximately 66%-85% of detected lesions are not malignant. Therefore, biopsies are unnecessary for at least two thirds of patients. Human estrogen receptor-positive breast cancer cells typically display an elevated level of cyclin D1 protein because of the overexpression of CCND1 messenger RNA (mRNA) and an elevated level of insulin-like growth factor 1 (IGF1) receptor (IGF1R) because of the overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of CCND1 peptide nucleic acid (PNA) hybridization probes with a (99m)Tc-chelating peptide on the N terminus and an IGF1 peptide loop on the C terminus could detect CCND1 mRNA in human MCF7 breast cancer xenografts in nude mice from outside the body. METHODS: We prepared the CCND1 probes as well as mismatched controls by solid-phase synthesis. We used fluorescence microscopy to detect the cellular uptake of fluoresceinyl probes and quantitative reverse transcription-polymerase chain reaction to detect the hybridization of probes to mRNA. We imaged (99m)Tc-probes in MCF7 xenografts scintigraphically and measured distribution by scintillation counting of dissected tissues. RESULTS: IGF1R-overexpressing MCF7 cells internalized the fluorescein-chelator-CCND1 PNA-IGF1 peptide but not the mismatched control peptide. The chelator-CCND1 PNA-IGF1 peptide but not the control peptide lowered the level of cyclin D1 protein in IGF1R-overexpressing MCF7 xenografts in nude mice after intratumoral injection. IGF1R-overexpressing MCF7 xenografts in nude mice were visualized at 4, 12, and 24 h after tail vein administration of the (99m)Tc-CCND1 antisense probe but not the control probe. (99m)Tc-chimeras were distributed normally in the kidneys, liver, tumors, and other tissues. CONCLUSION: Cancer gene activity can be detected from outside the body by probing with radionuclide-chelator-PNA-peptide chimeras.