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Background Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line–derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood‐brain‐barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. Methods We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin‐induced cell death, to activate intracellular signaling pathways both in vitro and in vivo , and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. Results BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild‐type but not from RET‐knockout mice. BT13 protects cultured dopamine neurons from 6‐Hydroxydopamine (6‐OHDA) and 1‐methyl‐4‐phenylpyridinium (MPP+)–induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. Conclusion The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease‐modifying treatments against PD. © 2019 The Authors. Movement Disorders published by Wiley Periodicals LLC. on behalf of International Parkinson and Movement Disorder Society.

CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming., CRISPRa is an attractive tool for cellular reprogramming due to its multiplexing capacity and direct targeting of genomic loci. Here the authors demonstrate the reprogramming of human fibroblasts into iPSCs, which is enhanced by targeting a conserved Alu-motif.

In this study, the feasibility of direct infusion electrospray ionization microchip mass spectrometry (chip-MS) was compared to the commonly used liquid chromatography-mass spectrometry (LC-MS) in non-targeted metabolomics analysis of human foreskin fibroblasts (HFF) and human induced pluripotent stem cells (hiPSC) reprogrammed from HFF. The total number of the detected features with chip-MS and LC-MS were 619 and 1959, respectively. Approximately 25% of detected features showed statistically significant changes between the cell lines with both analytical methods. The results show that chip-MS is a rapid and simple method that allows high sample throughput from small sample volumes and can detect the main metabolites and classify cells based on their metabolic profiles. However, the selectivity of chip-MS is limited compared to LC-MS and chip-MS may suffer from ion suppression.

Motor symptoms of Parkinson’s disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently no cure for this disease is available. Existing drugs alleviate PD symptoms, but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neuronsin vitroand in animal models of PD, but its clinical use is complicated by pharmacokinetic properties. In the present study we demonstrate the ability of a small molecule agonist of GDNF receptor RET to support the survival of cultured dopamine neurons only when they express GDNF receptors. In addition, BT13 activates intracellular signaling cascadesin vivo, stimulates release of dopamine and protect the function of dopaminergic neurons in a 6-hydroxydopamine (6-OHDA) rat model of PD. In contrast to GDNF, BT13 is able to penetrate through the blood-brain-barrier. Thus, BT13 serves as an excellent tool compound for the development of novel disease-modifying treatments against PD.

We present an individualized systems medicine (ISM) approach to optimize cancer drug therapies one patient at a time. ISM is based on (i) molecular profiling and ex vivo drug sensitivity and resistance testing (DSRT) of patients' cancer cells to 187 oncology drugs, (ii) clinical implementation of therapies predicted to be effective, and (iii) studying consecutive samples from the treated patients to understand the basis of resistance. Here, application of ISM to 28 samples from patients with acute myeloid leukemia (AML) uncovered five major taxonomic drug-response subtypes based on DSRT profiles, some with distinct genomic features (e.g., MLL gene fusions in subgroup IV and FLT3-ITD mutations in subgroup V). Therapy based on DSRT resulted in several clinical responses. After progression under DSRT-guided therapies, AML cells displayed significant clonal evolution and novel genomic changes potentially explaining resistance, whereas ex vivo DSRT data showed resistance to the clinically applied drugs and new vulnerabilities to previously ineffective drugs. Significance: Here, we demonstrate an ISM strategy to optimize safe and effective personalized cancer therapies for individual patients as well as to understand and predict disease evolution and the next line of therapy. This approach could facilitate systematic drug repositioning of approved targeted drugs as well as help to prioritize and de-risk emerging drugs for clinical testing. Cancer Discov; 3(12); 1416–29. ©2013 AACR. See related commentary by Hourigan and Karp, p. 1336 This article is highlighted in the In This Issue feature, p. 1317

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-β signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-β signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.

An epidemic of Zika virus (ZIKV) infection associated with congenital abnormalities such as microcephaly, is ongoing in the Americas and the Pacific. Currently there are no approved therapies to treat this emerging viral disease. Here, we tested three cell-directed broad-spectrum antiviral compounds against ZIKV replication using human retinal pigment epithelial (RPE) cells and a low-passage ZIKV strain isolated from fetal brain. We found that obatoclax, SaliPhe, and gemcitabine inhibited ZIKV infections at noncytotoxic concentrations. Moreover, all three compounds prevented production of viral RNA and proteins as well as activation of cellular caspase 8, 3 and 7. However, these compounds differentially affected ZIKV-mediated transcription, translation and posttranslational modifications of cellular factors as well as metabolic pathways indicating that these agents possess different mechanisms of action. Interestingly, combination of obatoclax and SaliPhe at nanomolar concentrations had a synergistic effect against ZIKV infection. Thus, our results provided the foundation for development of broad-spectrum cell-directed antivirals or their combinations for treatment of ZIKV and other emerging viral diseases.

Neuropathic pain is a chronic pain condition caused by lesion or disease affecting the somatosensory system. The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) alleviate symptoms of NP and stimulate regeneration of sensory neurons in vivo. Here we report the development of the compound BT18 that selectively activates GFLreceptors, alleviates pain and restores damaged dorsal root ganglion (DRG) neurons in rat models of NP.Significance statementNeuropathic pain (NP) is a chronic syndrome caused by different diseases and lesions affecting nervous system. Earlier studies demonstrated that neurotrophic factors - the glial cell line-derived neurotrophic factor (GDNF) and artemin - could reverse the damage done by lesions in animal models of NP. We demonstrate for the first time that a small molecule can activate receptor of GDNF and artemin, it alleviates pain symptoms in vivo in two animal models of NP and restores to normal the molecular markers expressed in sensory neurons. This compound, termed BT18, can pave way for creating novel disease modifying therapies for NP.

In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue. (C) 2016 Elsevier Inc. All rights reserved.

Syndecan-3 may act alone or as a coreceptor with RET to promote cell spreading, neurite outgrowth, and migration of cortical neurons by GNDF, NRTN, and ARTN., Glial cell line–derived neurotrophic factor (GDNF) family ligands (GFLs) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson’s disease. Soluble GFLs bind to a ligand-specific glycosylphosphatidylinositol-anchored coreceptor (GDNF family receptor α) and signal through the receptor tyrosine kinase RET. In this paper, we show that all immobilized matrix-bound GFLs, except persephin, use a fundamentally different receptor. They interact with syndecan-3, a transmembrane heparan sulfate (HS) proteoglycan, by binding to its HS chains with high affinity. GFL–syndecan-3 interaction mediates both cell spreading and neurite outgrowth with the involvement of Src kinase activation. GDNF promotes migration of cortical neurons in a syndecan-3–dependent manner, and in agreement, mice lacking syndecan-3 or GDNF have a reduced number of cortical γ-aminobutyric acid–releasing neurons, suggesting a central role for the two molecules in cortical development. Collectively, syndecan-3 may directly transduce GFL signals or serve as a coreceptor, presenting GFLs to the signaling receptor RET.

Glial cell line-derived neurotrophic factor (GDNF) protects and repairs dopamine neurons. It binds to GDNF family receptor alpha1 (GFRalpha1) and activates receptor tyrosine kinase. Heparan sulphate proteoglycans (HSPGs) also participate in the signalling of GDNF, though binding to HS may hinder the diffusion of infused GDNF. We assessed the importance of heparin-binding determinants in the neuroprotective effects of GDNF in the 6-OHDA rat model of Parkinson's disease. We utilized a truncated, non-heparin-binding Delta38N-GDNF or combined wtGDNF with heparin-binding growth-associated molecule (HB-GAM, pleiotrophin). Tissue diffusion of wtGDNF+/-HB-GAM and Delta38N-GDNF was also compared. A protective effect against ipsilateral d-amphetamine-induced turning was seen with 10 microg wtGDNF, 17 microg HB-GAM+10 microg wtGDNF or 10 microg Delta38N-GDNF at 8 weeks post lesion. This effect was most pronounced with wtGDNF alone. HB-GAM (17 or 50 microg) also reduced rotational behaviour, but did not protect dopaminergic cells. Otherwise, the survival of TH-positive cells in the substantia nigra correlated with the behavioural data. Although Delta38N-GDNF was more widely distributed than wtGDNF (irrespective of its origin), stable in a brain extract, and potent in mitogen-activated kinase assay, it was inferior in vivo. The results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect.

HSEPI (glucuronyl C5-epimerase) catalyzes the conversion of d-glucuronic acid to l-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mice yielded a lethal phenotype with selective organ defects but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi(-)(/)(-) mouse. The HS produced by these cells is devoid of l-iduronic acid residues but shows up-regulated N- and 6-O-sulfation compared with wild type (WT) MEF HS. In medium fortified with 10% fetal calf serum, the Hsepi(-)(/)(-) MEFs proliferated and migrated similarly to WT cells. Under starvation conditions, both cell types showed attenuated proliferation and migration that could be restored by the addition of FGF2 to WT cells, whereas Hsepi(-)(/)(-) cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi(-)(/)(-) compared with WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed a strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor to Hsepi(-)(/)(-) but not to WT HS. glia-derived neurotropic factor has a key role in kidney development, defective in Hsepi(-)(/)(-) mice. By contrast, Hsepi(-)(/)(-) and WT HS interacted similarly and in conventional mode with FGF10. These findings correlate defective function of growth factors with their mode of HS interaction and may help explain the partly modest organ phenotypes observed after genetic ablation of selected enzymes in HS biosynthesis.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor α1 (GFRα1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF2·GFRα12 complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF2·GFRα12 complex and the previously published artemin2·GFRα32 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFRα interface between the two structures, in particular ones that buttress the key conserved ArgGFRα-Gluligand-ArgGFRα interaction. Second, the flexible GDNF ligand “finger” loops fit differently into the GFRαs, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand2-co-receptor2-RET2 heterohexamer complexes. Consistent with this, we showed that GDNF2·GFRα12 and artemin2·GFRα32 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFRα1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg190-Lys202 region in GFRα1 domain 2. This may explain how GDNF·GFRα1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), which consist of GDNF, neurturin, artemin and persephin, regulate the development and maintenance of the nervous system. GDNF protects and repairs dopamine-containing neurons, which degenerate in Parkinson's disease, and motoneurons, which die in amyotrophic lateral sclerosis. GDNF and neurturin have shown promise in clinical trials of Parkinson's disease, and artemin is currently undergoing clinical trials for chronic pain treatment. However, the delivery of GFLs into the brain through invasive approaches such as neurosurgery, viral vectors or by the use of encapsulated cells is associated with multiple obstacles. The development of small molecules that specifically activate GFL receptors and that can be applied systemically would overcome most of these problems. The unique nature of the GFL receptors, recent progress in elucidation of the 3D structures of GFLs and GFL-receptor complexes and the use of high-throughput screening have resulted in the development of the first small molecules that mimic the effects of the different GFLs.

*Institute of Microbiology, Russian Academy of Sciences, pr. 60-1etiya Oktyabrya 7, k. 2, Moscow, 117811 Russia **Kazan State University, ul. Kremlevskaya 18, Kazan, 420008 Russia ***Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia Received July 12, 1999 Abstract--Alkyl-substituted hydroxybenzenes (AHBs), autoinducers of microbial dormancy (or d I factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with the Bacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renatur- ation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made that d I factors may play the role of natural chemical chaper- ons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable com- plexes with enzymes. Key words: dormancy, dormancy autoinducers, metabolic block, thermostability of enzymes, dl factors of microorganisms, chemical chaperons, aikyl-substituted hydroxybenzenes It has recently been shown that autoregulatory d~ factors, accumulating in the medium to a threshold level, induce the transition of vegetative microbial cells to a dormant state [1, 2]. In their chemical nature, the known dl factors represent mixtures of the isomers and homologues of alkyl-substituted hydroxybenzenes (AHBs) 13, 4]. The mechanism of action of dl factors during the development of a metabolically quiescent state is twofold. First, they interact with membrane lipid, causing the crystallization of the membrane lipid stroma, inhibition of the functional activity of mem- branes (including membrane-associated energy-produc- ing processes), an increase in membrane permeability to monovalent ions, and dehydration of the cell protoplast [5, 6]. Second, AHBs function as low-molecular-weight protein modifiers inhibiting the catalytic activity of enzymes (including nucleic acid depolymerases) and providing for the survival of dormant cells in the absence of functionally active energy-producing pro- cesses. The inactivation of enzymes by AHBs has been demonstrated in experiments with chymotrypsin, trypsin, RNase, invertase, and glucose oxidase [7]. The inhibition of metabolism by AHBs is associated with IE-mail for correspondence: andlm@mail.ru an enhanced resistance of resting cells to various stress factors; therefore, it can be anticipated that AHBs may enhance the stability of protein molecules. In view of the foregoing, the aim of the present work was to study the stabilizing effect of d I factors on enzymes as one of the possible mechanisms of resis- tance of dormant cells to stresses. MATERIALS AND METHODS Experiments on the structural modification of enzymes were carried out using C6-AHB, an amphiphilic compound with pK, = 9. C6-AHB (herein- after, also referred to as the dl factor) was added to reaction mixtures in the form of an ethanol solution to give the final ethanol concentration of no more than 5 vol % (in control experiments, equivalent amounts of ethanol were added). Taking into account that RNases play an important role in the functioning of the hereditary apparatus of cells, are involved in trophic chains, and serve as the extracellular autoregulators of physiological activity, experiments were primarily performed with the RNase (EC 3.1.27.5) of Bacillus intermedius purified to an apparent homogeneity (Mr = 12 300 Da) [8]. Some 0026-2617/00/6902-0180525.00 9 2000 MA1K "Nauka/Interperiodica"

Neurotrophic factors promote survival, proliferation and differentiation of neurons inducing intracellular signaling via specific receptors. The conventional biochemical methods often fail to reveal full repertoire of neurotrophic factor-receptor interactions because of their limited sensitivity. We evaluated several approaches to study signaling of Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands and found that reporter-gene systems possess exceptionally high sensitivity and a heuristic power to identify novel biologically relevant growth factor-receptor interactions. We identified persephin, a GDNF family member, as a novel ligand for GFRalpha1/RET receptor complex. We confirmed this finding by several independent methods, including neurite outgrowth assay from the explants of sympathetic ganglia expressing Gfralpha1 and Ret mRNA but not persephin's conventional receptor GFRalpha4. As the activation of GFRalpha1/RET was shown to rescue dopaminergic neurons, our results suggest the potential of persephin for the treatment of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor alpha1 (GFR alpha 1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF(2).GFR alpha 1(2) complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF(2).GFR alpha 1(2) complex and the previously published artemin(2).GFR alpha 3(2) complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR alpha interface between the two structures, in particular ones that buttress the key conserved Arg(GFR alpha)-Glu(ligand)-Arg(GFR alpha) interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR alphas, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand(2)-co-receptor(2)-RET(2) heterohexamer complexes. Consistent with this, we showed that GDNF(2).GFR alpha1(2) and artemin(2).GFR alpha 3(2) signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR alpha 1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg(190)-Lys(202) region in GFR alpha 1 domain 2. This may explain how GDNF.GFR alpha 1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.

Dopamine is a crucial neurotransmitter responsible for functioning and maintenance of the nervous system. Dopamine has also been implicated in a number of diseases including schizophrenia, Parkinson's disease and drug addiction. Dopamine agonists are used in early Parkinson's disease treatment. Dopamine antagonists suppress schizophrenia. Therefore, molecules modulating dopamine receptors activity are vastly important for understanding the nervous system functioning and for the treatment of neurological diseases. In this study we describe novel computational models that efficiently predict binding affinity of the existing small molecule dopamine analogs to dopamine receptor. The model provides the set of molecular descriptors that can be used for the development of new small molecule dopamine agonists.

The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRalpha1 (GDNF family receptor alpha1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRalpha1 and RET. Moreover, soluble full-length GFRalpha1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRalpha1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRalpha1 by stabilizing the interaction between GFRalpha1 and GDNF.

Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.

Introduction Adult acute myeloid leukemia (AML) exemplifies the challenges of modern cancer drug discovery and development in that molecularly targeted therapies are yet to be translated into clinical use. No effective second-line therapy exists once standard chemotherapy fails. While many genetic events have been linked with the onset and progression of AML, the fundamental disease mechanisms remain poorly understood. There is significant genomic and molecular heterogeneity among patients. Several targeted therapies have been investigated for improved second-line AML therapy but none has been approved for clinical use to date. It would be critically important to identify patient subgroups that would benefit from such therapies and to identify combinations of drugs that are likely to be effective. Methods To identify and optimize novel therapies for AML, we studied 28 samples from 18 AML patients with an individualized systems medicine (ISM) approach. The ISM platform includes functional profiling of AML patient cells ex vivo with drug sensitivity and resistance testing (DSRT), comprehensive molecular profiling as well as clinical background information. Data integration was done to identify disease- and patient-specific molecular vulnerabilities for translation in the clinic. The DSRT platform comprises 306 anti-cancer agents, each tested in a dose response series. We calculated differential drug sensitivity scores by comparing AML responses to those of control cells in order to distinguish cancer-specific drug effects. Next generation RNA- and exome-sequencing was used to identify fusion transcripts and mutations that link to drug sensitivities. Results Individual AML patient samples had a distinct drug sensitivity pattern, but unsupervised hierarchical clustering of the drug sensitivity profiles of the 28 AML patient samples identified 5 functional AML drug response subtypes. Each subtype was characterized by distinct combinations of sensitivities: Bcl-2 inhibitors (e.g. navitoclax; Group 1), JAK inhibitors (e.g. ruxolitinib) (Group 2) and MEK inhibitors (e.g. trametinib) (Groups 2 and 4), PI3K/mTOR inhibitors (e.g. temsirolimus; Groups 4 and 5), broad spectrum receptor tyrosine kinase inhibitors (e.g. dasatinib) (Groups 3, 4 and 5) and FLT3 inhibitors (e.g. quizartinib, sunitinib) (Group 5). Correlation of overall drug responses with genomic profiles revealed that RAS and FLT3 mutations were significantly linked with the drug response subgroups 4 and 5, respectively. Activating FLT3 mutations contributed to sensitivity to FLT3 inhibitors, as expected, but also to tyrosine kinase inhibitors not targeting FLT3, such as dasatinib. Hence, these data point to the potential synergistic combinatorial effects of FLT3 inhibitors with dasatinib for improved therapy outcome (Figure). Early clinical translational results based on compassionate use support this hypothesis. Therefore, by combinations of drugs we expect to see synergistic drug responses that can be translated into efficacious and safe therapies for relapsed AML cases in the clinic. Clinical application of DSRT results in the treatment of eight recurrent chemorefractory patients led to objective responses in three cases according to ELN criteria, whereas four of the remaining five patients had meaningful responses not meeting ELN criteria. After disease progression, AML patient cells showed ex vivo resistance to the drugs administered to the patients, as well as significant changes in clonal architecture during treatment response. Furthermore, we saw genomic alterations potentially explaining drug resistance, such as appearance of novel fusion genes. Summary The ISM approach represents an opportunity for improving therapies for cancer patients, one patient at the time. We show that the platform can be used to identify functional groups of AML linking to vulnerabilities to single targeted drugs and, importantly, unexpected drug combinations. This information can in turn be used for personalized medicine strategies and for creating hypotheses to be explored in systematic clinical trials, both for approved and investigational drugs. Disclosures: Off Label Use: Many of the compounds included in our DSRT platform are not indicated for AML therapy. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Honoraria. Porkka:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Kallioniemi:Medisapiens: Membership on an entity’s Board of Directors or advisory committees; Roche: Research Funding.

Identification of signaling pathways that are required for the growth and differentiation block of cells from adult acute myeloid leukemia (AML) is urgently required to facilitate development of novel therapies. Here, we describe an approach to functionally determine molecular drivers of AML by quantitative drug sensitivity and resistance testing (DSRT) of AML blast cells in primary culture ex vivo. The selection of drugs covered the entire cancer pharmacopeia and much of the pipeline of drugs under development in the industry: 120 FDA approved small molecular cancer drugs and 120 emerging drugs, investigational compounds and signal transduction inhibitors. All compounds were tested over a 10,000-fold concentration range to generate quantitative and reliable dose-response data. In addition, whole exome and transcriptome sequencing and phophoproteomic profiling were also performed to derive a comprehensive understanding of the molecular AML-related aberrations on an individual basis. Comparison of 17 AML patient samples and 3 healthy bone marrow control samples based on ex vivo drug responses identified several classes of approved and investigational drugs that showed selective anti-AML activities: mTOR inhibitors (e.g. temsirolomus, everolimus, sirolimus), MEK inhibitors (e.g. AS703026, GSK1120212, RDEA119, selumetinib), tyrosine kinase inhibitors (e.g. dasatinib, ponatinib, sunitinib), Bcl-2 inhibitors (navitoclax) and HSP90 inhibitors (e.g. BIIB021, NVP-AUY922, tanespimycin). In particular, the rapamycin class of mTOR inhibitors and allosteric MEK inhibitors stood out as effective and selective inhibitors in 8/17 (47%) and 9/17 (52%) of the patients, respectively. Simultaneous data from other targeted inhibitors made it possible to dissect the critical steps in signaling and therapeutic efficacy. For example, PI3K and Akt inhibitors were not effective in these patients, suggesting that the mTOR dependency is mediated through a PI3K-Akt-independent pathway. Similarly, the dependency of MEK signaling appears to be through a Ras-Raf-independent pathway since Raf inhibitors were not effective. In conclusion, the DSRT platform allows us to derive quantitative data on the ex vivo drug response profiles of AML cells from individual patients. This information could be used as a diagnostic tool to optimize personalized therapies in the future. Our data demonstrate that mTOR and MEK signaling and the associated inhibitors are the most promising leads for improved AML therapeutics. This analysis also demonstrates gaps in our current understanding of the redundancy of key cancer cell signaling pathways and proves the significant value of data from experimental drug response testing ex vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 895. doi:1538-7445.AM2012-895

In order to discover unexpected anti-cancer efficacies of approved and emerging drugs, we established a diagnostic ex vivo drug sensitivity and resistance testing (DSRT) platform covering the entire cancer pharmacopeia as well as emerging anti-cancer compounds. Here, the platform was applied to analyze bone marrow (BM) mononuclear cells from 17 adult acute myeloid leukemia (AML) patients, 3 healthy donors as well as 7 AML cell lines. The DSRT panel covered FDA-approved small molecule oncology drugs (n=120), as well as emerging, investigational and pre-clinical oncology compounds (n=120), such as kinase (e.g. RTKs, checkpoint and mitotic kinases, Raf, MEK, JAKs, mTOR, PI3K), and non-kinase inhibitors (e.g. HSP, Bcl, activin, HDAC, PARP, Hh). To generate dose-response curves, each of the drugs was applied over a 10,000-fold concentration range. In addition, the samples underwent deep molecular profiling including exome- and transcriptome sequencing, as well as phosphoproteomic analysis. DSRT provided consistent and reliable data from ex vivo samples with a high correlation between data from individual healthy BM samples (r=0.93). Bioinformatic processing of the data from AML resulted in several key observations. First, overall drug response profiles of AML blast cells were distinctly different from healthy BM controls suggesting several potential leukemia-selective effects, such as multi-kinase (dasatinib), MEK, and mTOR inhibitors. Second, the overall drug responses from AML cell lines and the patient ex vivo samples showed differences, suggesting that ex vivo testing may reveal cancer-selective effects not previously seen in established cancer cell line panels. Third, the response data from patient samples clustered many drugs consistently into the expected functional classes (such as topoisomerase II inhibitors, MEK inhibitors and rapalogs), whereas other drug classes were more dispersed (such as FLT3 inhibitors with quizartinib clustering away from all other tyrosine kinase inhibitors), suggesting secondary targets playing a key role in drug efficacy. Fourth, analysis of serial samples from patients developing clinical resistance to targeted agents showed striking agreement between the ex-vivo DSRT profiles and clinical responses. In conclusion, comprehensive DSRT platform generated powerful novel insights on AML drug response and may enable individual optimization of therapies, particularly for recurrent leukemias. DSRT will also serve as a powerful hypothesis-generator for clinical trials, particularly for emerging drugs. The ability to correlate ex vivo response profiles for hundreds of drugs in clinical samples with deep molecular profiling data will yield exciting new translational and pharmacogenomic opportunities for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3188. doi:1538-7445.AM2012-3188

Abstract 2487 Introduction: The molecular drivers of adult AML as well as the determinants of drug response are poorly understood. While AML genomes have recently been sequenced, many cases do not harbor druggable mutations. Treatment options are particularly limited for relapsed and refractory AML. Due to the molecular heterogeneity of the disease, optimal therapy would likely consist of individualized combinations of targeted and non-targeted drugs, which poses significant challenges for the conventional paradigm of clinical drug testing. In order to better understand the molecular driver signals, identify individual variability of drug response, and to discover clinically actionable therapeutic combinations and future opportunities with emerging drugs, we established a diagnostic ex-vivo drug sensitivity and resistance testing (DSRT) platform for adult AML covering the entire cancer pharmacopeia as well as many emerging anti-cancer compounds. Methods: DSRT was implemented for primary cells from adult AML patients, focusing on relapsed and refractory cases. Fresh mononuclear cells from bone marrow aspirates (>50% blast count) were screened in a robotic high-throughput screening system using 384-well plates. The primary screening panel consisted of a comprehensive collection of FDA/EMA-approved small molecule and conventional cytotoxic drugs (n=120), as well as emerging, investigational and pre-clinical oncology compounds (currently n=90), such as major kinase (e.g. RTKs, checkpoint and mitotic kinases, Raf, MEK, JAKs, mTOR, PI3K), and non-kinase inhibitors (e.g. HSP, Bcl, activin, HDAC, PARP, Hh). The drugs are tested over a 10,000-fold concentration range resulting in a dose-response curve for each compound and with combinations of effective drugs explored in follow-up screens. The same samples also undergo deep molecular profiling including exome- and transcriptome sequencing, as well as phosphoproteomic analysis. Results: DSRT data from 11 clinical AML samples and 2 normal bone marrow controls were bioinformatically processed and resulted in several exciting observations. First, overall drug response profiles of the AML samples and the controls were distinctly different suggesting multiple leukemia-selective inhibitory effects. Second, the MEK and mTOR signaling pathways emerged as potential key molecular drivers of AML cells when analyzing targets of leukemia-specific active drugs. Third, potent new ex-vivo combinations of approved targeted drugs were uncovered, such as mTOR pathway inhibitors with dasatinib. Fourth, data from ex-vivo DSRT profiles showed excellent agreement with clinical response when serial samples were analyzed from leukemia patients developing clinical resistance to targeted agents. Summary: The rapid and comprehensive DSRT platform covering the entire cancer pharmacopeia and many emerging agents has already generated powerful insights into the molecular events underlying adult AML, with significant potential to facilitate individually optimized combinatorial therapies, particularly for recurrent leukemias. DSRT will also serve as a powerful hypothesis-generator for clinical trials, particularly for emerging drugs and drug combinations. The ability to correlate response profiles of hundreds of drugs in clinical ex vivo samples with deep molecular profiling data will yield exciting new translational and pharmacogenomic opportunities for clinical hematology. Disclosures: Mustjoki: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Porkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Kallioniemi:Abbot/Vysis: Patents & Royalties; Medisapiens: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer Schering Pharma: Research Funding; Roche: Research Funding.

Alkyl-substituted hydroxybenzenes (AHBs), which are autoinducers of microbial dormancy (d 1 factors), were found to stabilize the structure of protein macromolecules and modify the catalytic activity of enzymes. In vitro experiments showed that C6-AHB at concentrations from 10−4 to 10−2 M, at which it occurs in the medium as a true solution and a micellar colloid, respectively, nonspecifically inhibited the activity of chymotrypsin, RNase, invertase, and glucose oxidase. C6-AHB-induced conformational alterations in protein macromolecules were due to the formation of complexes, as evidenced by differences in the fluorescence spectra of individual RNase and C6-AHB and their mixtures and in the surface tension isotherms of C6-AHB and trypsin solutions. Data on the involvement of dormancy autoinducers in the posttranslational modification of enzymes and their inhibition will provide further insight into the mechanisms of development and maintenance of dormant microbial forms.