Molecular Vulnerability Research Articles

Meta-analysis of synaptic pathology in Alzheimer's disease reveals selective molecular vesicular machinery vulnerability

IntroductionLoss of synapses best correlates to cognitive deficits in Alzheimer's disease (AD) in which oligomeric neurotoxic species of amyloid-β appears to contribute synaptic pathology. Although a number of clinical pathologic studies have been performed with limited sample size, there are no systematic studies encompassing large samples. Therefore, we performed a meta-analysis study. MethodsWe identified 417 publications reporting postmortem synapse and synaptic marker loss from AD patients. Two meta-analyses were performed using a single database of subselected publications and calculating the standard mean differences. ResultsMeta-analysis confirmed synaptic loss in selected brain regions is an early event in AD pathogenesis. The second meta-analysis of 57 synaptic markers revealed that presynaptic makers were affected more than postsynaptic markers. DiscussionThe present meta-analysis study showed a consistent synaptic loss across brain regions and that molecular machinery including endosomal pathways, vesicular assembly mechanisms, glutamate receptors, and axonal transport are often affected.

PTN signaling: Components and mechanistic insights in human ovarian cancer.

Molecular vulnerabilities represent promising candidates for the development of targeted therapies that hold the promise to overcome the challenges encountered with non-targeted chemotherapy for the treatment of ovarian cancer. Through a synthetic lethality screen, we previously identified pleiotrophin (PTN) as a molecular vulnerability in ovarian cancer and showed that siRNA-mediated PTN knockdown induced apoptotic cell death in epithelial ovarian cancer (EOC) cells. Although, it is well known that PTN elicits its pro-tumorigenic effects through its receptor, protein tyrosine phosphatase receptor Z1 (PTPRZ1), little is known about the potential importance of this pathway in the pathogenesis of ovarian cancer. In this study, we show that PTN is expressed, produced, and secreted in a panel of EOC cell lines. PTN levels in serous ovarian tumor tissues are on average 3.5-fold higher relative to normal tissue and PTN is detectable in serum samples of patients with EOC. PTPRZ1 is also expressed and produced by EOC cells and is found to be up-regulated in serous ovarian tumor tissue relative to normal ovarian surface epithelial tissue (P < 0.05). Gene silencing of PTPRZ1 in EOC cell lines using siRNA-mediated knockdown shows that PTPRZ1 is essential for viability and results in significant apoptosis with no effect on the cell cycle phase distribution. In order to determine how PTN mediates survival, we silenced the gene using siRNA mediated knockdown and performed expression profiling of 36 survival-related genes. Through computational mapping of the differentially expressed genes, members of the MAPK (mitogen-activated protein kinase) family were found to be likely effectors of PTN signaling in EOC cells. Our results provide the first experimental evidence that PTN and its signaling components may be of significance in the pathogenesis of epithelial ovarian cancer and provide a rationale for clinical evaluation of MAPK inhibitors in PTN and/or PTPRZ1 expressing ovarian tumors.

Abstract 5118: A functional genetic approach in patient-derived glioblastoma stem cells reveals pre-mRNA splicing components to be cancer-lethal gene targets

Abstract Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults and is among the deadliest cancers with a median survival period of 12-14 months. This poor prognosis despite aggressive therapy underscores the need for novel therapeutic targets specifically required by GBM cells. Many GBM are thought to arise from a neural stem cell (NSC) origin and, consistent with this premise, tumor-initiating GBM stem cells (GSCs) isolated from patients retain the NSC-like phenotype and molecular profile of primary tumors. Importantly, unlike serum-cultured cell lines, GSCs retain the developmental potential and specific genetic mutations acquired as each patient's tumor progressed from its cell of origin. We hypothesized that these genetic alterations driving GBM growth might also give rise to unique molecular vulnerabilities within the cancer cells. To identify such novel gene targets required for GBM cell growth, but which are dispensable to normal cells, we performed genome-scale RNAi screens in multiple patient-derived GSC isolates and simultaneously counter-screened against primary untransformed human NSCs. From these results, we identified and validated the existence of GBM-lethal genes that, when inhibited, render patient GSCs sensitive to cellular stresses arising within these transformed cells. From these targets, we show that GSCs have an increased requirement for the expression and function of multiple specific members of the pre-mRNA splicing machinery. Notably, loss of specific key splicing proteins resulted in cell cycle arrest and subsequent cell death only in GSCs, identifying the spliceosome as a specific molecular vulnerability in GBM. New treatment strategies for this disease are urgently needed. The identification of spliceosomal proteins as essential for the growth and maintenance of GSCs both adds to our understanding of glioblastoma biology and suggests novel targets for therapeutic intervention. 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 5118. doi:1538-7445.AM2012-5118

Bcl-XL represents a druggable molecular vulnerability during aurora B inhibitor-mediated polyploidization

Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This "addiction" can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.

Achilles Heel Molecular Vulnerabilities in Multiple Myeloma Identified From Genome-Scale RNA Interference Screening.

Abstract 2801Poster Board II-777To provide a rationale basis for targeted drug development for multiple myeloma patients, we have conducted high-throughput genome-scale RNA interference (RNAi) synthetic lethality studies in human myeloma cell lines (HMCL) to generate a comprehensive map of critical genes and molecular vulnerabilities in human myeloma tumor cells. KMS11 human myeloma cells were screened with a 13,982-oligo library targeting the ‘druggable' genome (6,991 genes) using optimized conditions that resulted in >95% transfection efficiency. Each gene was screened with 2 or more distinct oligos, in duplicate, using a single-siRNA-per-well format, testing >34,000 wells. Replicate high throughput experiments yielded highly reproducible results (R2=0.82). Viability was measured at 96h by ATP-dependent luminescence. Universally lethal siRNA and non-targeted siRNA were employed as controls. The specificity (FDR) of lethal RNAi was evaluated by custom-developed statistical methods based on RNAi result concordancy. From screening, 5.8% of druggable genome siRNA caused statistically relevant reductions in HMCL viability (greater than three standard deviations from control samples treated with non-silencing siRNA, compared with an anticipated rate due to chance of only 0.135%). Of these, two hundred and nineteen genes, targeted by the most lethal siRNA, were forwarded to validation studies. Validation high-throughput RNAi studies, using 4 oligos per gene, were conducted in order to verify target gene vulnerability. Ultimately, seventy two genes were validated as highly critical for myeloma cell survival (each with multiple concordant siRNA hits plus high reproducibility of R2 =0.94). Among top-ranked lethal molecular vulnerabilities in KMS11 myeloma cells we recurrently identified the proteasome (8 PSM subunits were independently identified as highly vulnerable); and BCL2 family member MCL1; both known therapeutic targets, validating a functional genomics approach to target discovery. In addition, a number of novel, equally lethal, molecular vulnerabilities were identified in KMS11 including WEE1, kinetochore complex component KNTC2, the aurora kinases, polo-like kinase 1, a previously uncharacterized DNA methyl transferase, ribonucleotide reductase, ribosomal protein L38, leptin receptor overlapping transcript LEPROT and SLC25A23 (a carrier responsible for mitochondrial adenine nucleotide flux). Various ubiquitous cellular proteins involved in RNA or protein processing (e.g. SF3a, SNW1, SNRPA1, EIF3s8, amongst others) were also identified as non redundant and critical for cellular viability. Thirty nine top-ranked molecular vulnerabilities in KMS11 were evaluated for comparative vulnerability in a second myeloma cell line, JJN3, and in A549 and 293 epithelial cells. A majority were vulnerable in JJN3. While 25 genes proved equally lethal on silencing in A549 and 293 epithelial cells, several, notably including MCL1 and three proteasome subunits, appear differentially susceptible in myeloma cells versus epithelial cells. From this genome scale study, MCL1 ranks third as a target capable of inducing cell death in myeloma cells, highlighting the potent vulnerability of myeloma cell viability to alterations in this anti-apoptosis regulator. Moreover, from active comparative studies of molecular vulnerability in myeloma versus epithelial cell lines, MCL1 as an RNAi target demonstrates perhaps the greatest cytotoxic selectivity between myeloma and epithelial cell lines of all targets examined. Gene expression studies indicate that MCL1 is highly expressed in myeloma tumor cells but is absent or only weakly expressed in human primary somatic tissues. Overall, therefore, from an unsupervised genome-scale screening approach, our data support MCL1 as an optimal Achilles heel molecular target in multiple myeloma, potentially offering greater specificity and deeper cytotoxic effect than proteasome inhibition. Disclosures:No relevant conflicts of interest to declare.

Management of patients with neurofibromatosis type 1 and studies of pirfenidone

Aim Neurofibromatosis type I (NF 1) is a common tumour predisposing disease in humans. Plexiform neurofibromas are a major cause of morbidity in these patients. Unfortunately, effective medical therapy for these tumours is lacking. The histopathology of neurofibroma is complex, with diverse cellular content and large amount of fibrous tissue. We hypothesised that events connected with fibrogenesis might constitute a point of molecular vulnerability of plexiform neurofibromas. Methods Pirfenidone is a novel, small molecule agent that exhibits potent activity against fibroblasts in vitro and in vivo. We developed an animal model for neurofibroma [human neurofibroma xenotransplants into severe combined immunodeficiency (SCID) mice] and evaluated effects of pirfenidone on neural tumour implants. Results Survival of neurofibroma xenografts in SCID mice treated with oral pirfenidone was significantly lower when compared with survival of xenografts in non-treated animals. Based on encouraging preclinical studies, human studies for patients with plexiform neurofibromas have been initiated. Clinical trial on 24 adults was recently completed at Mayo Clinic and most patients remained stable for 2 years of treatment, not experiencing major toxicity. Paediatric phase I study has been completed and phase II study is still ongoing. Conclusion Pirfenidone is a well-tolerated oral agent. Results from completed trials to date are promising, indicating its potential role in treatment of NF1. Because of the variable and incompletely understood natural history of neurofibromas, interpretation of outcome from clinical trials is difficult and will require longer follow-up of larger population of treated patients and placebo-controlled trials to determine the role of pirfenidone in the therapy of NF1-related tumours.

Disruption of the Y-Box Binding Protein-1 Results in Suppression of the Epidermal Growth Factor Receptor and HER-2

The overexpression of the epidermal growth factor receptor (EGFR) and HER-2 underpin the growth of aggressive breast cancer; still, it is unclear what governs the regulation of these receptors. Our laboratories recently determined that the Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, induced breast tumor cell growth in monolayer and in soft agar. Importantly, mutating YB-1 at Ser(102), which resides in the DNA-binding domain, prevented growth induction. We reasoned that the underlying cause for growth attenuation by YB-1(Ser(102)) is through the regulation of EGFR and/or HER-2. The initial link between YB-1 and these receptors was sought by screening primary tumor tissue microarrays. We determined that YB-1 (n = 389 cases) was positively associated with EGFR (P < 0.001, r = 0.213), HER-2 (P = 0.008, r = 0.157), and Ki67 (P < 0.0002, r = 0.219). It was inversely linked to the estrogen receptor (P < 0.001, r = -0.291). Overexpression of YB-1 in a breast cancer cell line increased HER-2 and EGFR. Alternatively, mutation of YB-1 at Ser(102) > Ala(102) prevented the induction of these receptors and rendered the cells less responsive to EGF. The mutant YB-1 protein was also unable to optimally bind to the EGFR and HER-2 promoters based on chromatin immunoprecipitation. Furthermore, knocking down YB-1 with small interfering RNA suppressed the expression of EGFR and HER-2. This was coupled with a decrease in tumor cell growth. In conclusion, YB-1(Ser(102)) is a point of molecular vulnerability for maintaining the expression of EGFR and HER-2. Targeting YB-1 or more specifically YB-1(Ser(102)) are novel approaches to inhibiting the expression of these receptors to ultimately suppress tumor cell growth.

Etiopatogenia de las alucinaciones auditivas en las psicosis

Auditory hallucinations is one of the core symptoms of psychosis. Two main different approaches have been made in recent years in order to understand this perceptual abnormality. The psycho-social approaches is trying to explain auditory hallucinations as a common human phenomenon that only environmental factors cause them to be pathological. It propose psychotherapeutic techniques based on the cognitive model, paying special attention to the emotional component. The neurobiological approach, specially with neuroimaging techniques, is giving us new information about abnormal activation in specific neural networks, particularly in language areas. It propose biological treatments with antipsychotics and transcranial magnetic stimulation. Neither of these approaches are listening to each other. Neither of them look at the molecular genetic vulnerability to auditory hallucinations in psychosis.From a review of the literature and our own findings, we present a genetic-environmental model of auditory hallucinations in psychoses. We hypothesize two types of genetic vulnerability: vulnerability to hearing voices -that could be related with genes that are expressed in language areas and are related with language abnormalities (FOXP2)-, and vulnerability to abnormal emotional response to voices -that could be related with genes that are involved in the regulation of dopamine in the limbic system (5-HTT, CCK-AR)-. Finally, environmental and cultural factors explain the content and social adjustment to auditory hallucinations.Our model allow the integration of many data from epidemiology and neuroimaging, to psychopharmacology in the understanding of the aetiology of auditory hallucinations.

Dépression et facteurs protéiques de l'adaptation cellulaire : implication des traitements antidépresseurs

Mood disorders remain a puzzling clinical entity in psychiatry, not only at the symptomatic level (maniac versus depressive episodes, unipolar versus bipolar forms…) but also at the therapeutic level. Indeed, despite medications, the treatment responsiveness is rarely above 70%, occurs within the following weeks after initiation and is often not maintained on a half- to 1 year follow-up. The pioneering works of Axelrod’s and Brodie’s groups in the early 60s enlightening the ability of antidepressants to acutely increase central monoaminergic (serotoninergic or 5-HT, and noradrenergic or NAdr) activities have introduced the notion that these therapeutics affect the neuronal physiology. Since then, the affective disorders have been conceptualized as a biochemical disease corresponding to the mirror image of this antidepressant drug effectiveness. However, such understanding of the pathology minimizes or even denies the importance of some pharmacologically-induced events, e.g. the hypersensitization of postsynaptic 5-HT 1 A receptors combined with the desensitization of 5-HT autoreceptors (in particular of the 5-HT 1A subtype) which both occur once symptoms alleviate. This has likely weakened the dogma that treatments affect solely membrane-bound monoaminergic receptors. In addition, the use of drug combinations for fastening the clinical response (association with β-adrenergic drugs, anticonvulsants or thymo-regulators) have pointed out to speculate that the clinical drug effectiveness might be underlined by a sustained suractivation of the intracellular cascades triggered by the central monoaminergic systems (i.e. adenylate cyclase by cyclic adenosine monophosphate [cAMP] for the β-adrenergic, 5-HT 1A and 5-HT 1B/D receptor subtypes and calcium-dependent phospholipase and protein kinases by inositol triphosphate [IP 3] for the 5-HT 1C and 5-HT 2 receptor subtypes via heterotrimeric G protein-coupled receptors; Fig. 1). Accordingly, in the last decade, a new pathophysiological concept of affective disorders has thus emerged which integrates preferentially the molecular and cellular changes that supervene in response to an over-stimulation of these cascades. Most groups have focused their attention to the ability of antidepressants to enhance the expression and activation of a few transcription factors exemplified by CREB (AMPc response element binding protein) and neurotrophic factors such as nervous growth factor (NGF) and brain derived neurotrophic factor (BDNF) in the brain. Here, a review of the literature is synthesized on the topic that underlies the structural adaptations observed in the brain of depressed subjects and animals exposed to antidepressants. In fact, the hypothesis for a molecular vulnerability of mood disorders, a kind of neuronal memory for depression, has emerged in the early 90s when expression (identified by the messenger ribonucleic acid; mRNA) of various biological markers associated with depression [corticotrophin releasing hormone (CRH), tyrosine hydroxylase (TH), gluco-, mineralocorticoid and adrenergic receptors] was found to increase in parallel with the clinical response following a chronic antidepressant medication. Indeed, occurring with a different delay to the well-known mobilization of serotoninergic and noradrenergic receptors located at the plasma membrane level (that occur rapidly after initiation), the clinical response to antidepressant drugs has coincided with the long-term recruitment of various nuclear transcription factors, the mostly documented one being today CREB, and of various neurotrophic factors exemplified by BDNF. This recent hypothesis based on enhanced levels of both transcription and subsequent neurotrophic factors is nowadays getting more attractive due to specific documentation by both animal and human reports. However, whether quantifying expression (assessed by the mean of the mRNA levels) or protein activity of these factors is representative of the biological responsiveness to antidepressants still remains controversial since both hypotheses have now found their own defenders and detractors.

D�pression et facteurs prot�iques de l'adaptation cellulaireB: implication des traitements antid�presseurs

Les adaptations réceptorielles observées au cours du traitement de la dépression ne prédisent pas l'ensemble des ajustements biologiques opérés par la thérapeutique. L'objectif de ce travail est d'apporter les informations les plus récentes dans le domaine qui soient propices à une meilleure compréhension biologique de cette pathologie et de ses thérapeutiques. On sait depuis peu que les antidépresseurs modulent l'expression et l'activation de certains facteurs de transcription comme le CREB (AMPc response element binding protein) et de facteurs neurotrophiques comme le BDNF (brain derived neurotrophic factor) dans le cerveau. L'observation que certaines manifestations dépressives peuvent être induites (ou à l'inverse supprimées) par la réduction (ou à l'inverse l'augmentation) de ces protéines nucléaires suggère ainsi l'existence de véritables « mémoires biologiques » du processus dépressif par épuisement de ces facteurs et ouvre des perspectives nouvelles dans la thérapeutique de la dépression. Mood disorders remain a puzzling clinical entity in psychiatry, not only at the symptomatic level (maniac versus depressive episodes, unipolar versus bipolar forms…) but also at the therapeutic level. Indeed, despite medications, the treatment responsiveness is rarely above 70%, occurs within the following weeks after initiation and is often not maintained on a half- to 1 year follow-up. The pioneering works of Axelrod’s and Brodie’s groups in the early 60s enlightening the ability of antidepressants to acutely increase central monoaminergic (serotoninergic or 5-HT, and noradrenergic or NAdr) activities have introduced the notion that these therapeutics affect the neuronal physiology. Since then, the affective disorders have been conceptualized as a biochemical disease corresponding to the mirror image of this antidepressant drug effectiveness. However, such understanding of the pathology minimizes or even denies the importance of some pharmacologically-induced events, e.g. the hypersensitization of postsynaptic 5-HT1A receptors combined with the desensitization of 5-HT autoreceptors (in particular of the 5-HT1A subtype) which both occur once symptoms alleviate. This has likely weakened the dogma that treatments affect solely membrane-bound monoaminergic receptors. In addition, the use of drug combinations for fastening the clinical response (association with β-adrenergic drugs, anticonvulsants or thymo-regulators) have pointed out to speculate that the clinical drug effectiveness might be underlined by a sustained suractivation of the intracellular cascades triggered by the central monoaminergic systems (i.e. adenylate cyclase by cyclic adenosine monophosphate [cAMP] for the β-adrenergic, 5-HT1A and 5-HT1B/D receptor subtypes and calcium-dependent phospholipase and protein kinases by inositol triphosphate [IP3] for the 5-HT1C and 5-HT2 receptor subtypes via heterotrimeric G protein-coupled receptors; Fig. 1). Accordingly, in the last decade, a new pathophysiological concept of affective disorders has thus emerged which integrates preferentially the molecular and cellular changes that supervene in response to an over-stimulation of these cascades. Most groups have focused their attention to the ability of antidepressants to enhance the expression and activation of a few transcription factors exemplified by CREB (AMPc response element binding protein) and neurotrophic factors such as nervous growth factor (NGF) and brain derived neurotrophic factor (BDNF) in the brain. Here, a review of the literature is synthesized on the topic that underlies the structural adaptations observed in the brain of depressed subjects and animals exposed to antidepressants. In fact, the hypothesis for a molecular vulnerability of mood disorders, a kind of neuronal memory for depression, has emerged in the early 90s when expression (identified by the messenger ribonucleic acid; mRNA) of various biological markers associated with depression [corticotrophin releasing hormone (CRH), tyrosine hydroxylase (TH), gluco-, mineralocorticoid and adrenergic receptors] was found to increase in parallel with the clinical response following a chronic antidepressant medication. Indeed, occurring with a different delay to the well-known mobilization of serotoninergic and noradrenergic receptors located at the plasma membrane level (that occur rapidly after initiation), the clinical response to antidepressant drugs has coincided with the long-term recruitment of various nuclear transcription factors, the mostly documented one being today CREB, and of various neurotrophic factors exemplified by BDNF. This recent hypothesis based on enhanced levels of both transcription and subsequent neurotrophic factors is nowadays getting more attractive due to specific documentation by both animal and human reports. However, whether quantifying expression (assessed by the mean of the mRNA levels) or protein activity of these factors is representative of the biological responsiveness to antidepressants still remains controversial since both hypotheses have now found their own defenders and detractors.

Neuropsychiatric disorders of childhood: Tourette's syndrome as a model.

Neuropsychiatric disorders of childhood include conditions such as autism, pervasive developmental disorder and attention deficit hyperactivity disorder. Tourette's syndrome (TS) of chronic, multiple, motor and vocal tics is a model neuropsychiatric disorder with a childhood onset and long-term, and sometimes life-long, course. Symptoms and signs change during the course of maturation, and include obsessions, compulsions, attentional difficulties and psychological responses to the disability. These clinical features reflect the interaction between genetic and environmental factors. The localization and cloning of the genetic substrate will allow for detailed study of the pathways from molecular vulnerability to clinical syndrome.