Preclinical studies of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in pediatric brain tumors.

Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2'-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials

A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo.

Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies

An in vivo screen identifies ependymoma oncogenes and tumor-suppressor genes

Cancers are characterized by non-random chromosome copy number alterations that presumably contain oncogenes and tumor-suppressor genes (TSGs). The affected loci are often large, making it difficult to pinpoint which genes are driving the cancer. Here we report a cross-species in vivo screen of 84 candidate oncogenes and 39 candidate TSGs, located within 28 recurrent chromosomal alterations in ependymoma. Through a series of mouse models, we validate eight new ependymoma oncogenes and ten new ependymoma TSGs that converge on a small number of cell functions, including vesicle trafficking, DNA modification and cholesterol biosynthesis, identifying these as potential new therapeutic targets.We are grateful to F.B. Gertler (Massachusetts Institute of Technology) and S. Gupton (University of North Carolina) for the generous gift of the VAMP7-phlorin construct and the staffs of the Hartwell Center for Bioinformatics and Biotechnology, the Small Animal Imaging Center, the Animal Resources Center, the Cell and Tissue Imaging Center, and the Flow Cytometry and Cell Sorting Shared Resource at St. Jude Children's Research Hospital for technical assistance. This work was supported by grants from the US National Institutes of Health (R01CA129541, P01CA96832 and P30CA021765, R.J.G.), by the Collaborative Ependymoma Research Network (CERN) and by the American Lebanese Syrian Associated Charities (ALSAC)

LPS-induced modifications in spontaneous network activity causes neuronal apoptosis in neonatal cerebral cortex

During the perinatal period the developing brain is most vulnerable to inflammation. Prenatal infection or exposure to inflammatory factors can have a profound impact on fetal neurodevelopment with long-term neurological deficits, such as cognitive impairment, learning deficits, perinatal brain damage and cerebral palsy. Inflammation in the brain is characterized by activation of resident immune cells, especially microglia and astrocytes whose activation is associated with a variety of neurodegenerative disorders like Alzheimer´s disease and Multiple sclerosis. These cell types express, release and respond to pro-inflammatory mediators such as cytokines, which are critically involved in the immune response to infection. It has been demonstrated recently that cytokines also directly influence neuronal function. Glial cells are capable of releaseing the pro-inflammatory cytokines MIP-2, which is involved in cell death, and tumor necrosis factor alpha (TNFalpha), which enhances excitatory synaptic function by increasing the surface expression of AMPA receptors. Thus constitutively released TNFalpha homeostatically regulates the balance between neuronal excitation and inhibition in an activity-dependent manner. Since TNFalpha is also involved in neuronal cell death, the interplay between neuronal activity MIP-2 and TNFalpha may control the process of cell death and cell survival in developing neuronal networks. An increasing body of evidence suggests that neuronal activity is important in the regulation of neuronal survival during early development, e.g. programmed cell death (apoptosis) is augmented when neuronal activity is blocked. In our study we were interested on the impact of inflammation on neuronal activity and cell survival during early cortical development. To address this question, we investigated the impact of inflammation on neuronal activity and cell survival during early cortical development in vivo and in vitro. Inflammation was experimentally induced by application of the endotoxin lipopolysaccharide (LPS), which initiates a rapid and well-characterized immune response. I studied the consequences of inflammation on spontaneous neuronal network activity and cell death by combining electrophysiological recordings with multi-electrode arrays and quantitative analyses of apoptosis. In addition, I used a cytokine array and antibodies directed against specific cytokines allowing the identification of the pro-inflammatory factors, which are critically involved in these processes. In this study I demonstrated a direct link between inflammation-induced modifications in neuronal network activity and the control of cell survival in a developing neuronal network for the first time. Our in vivo and in vitro recordings showed a fast LPS-induced reduction in occurrence of spontaneous oscillatory activity. It is indicated that LPS-induced inflammation causes fast release of proinflammatory factors which modify neuronal network activity. My experiments with specific antibodies demonstrate that TNFalpha and to a lesser extent MIP-2 seem to be the key mediators causing activity-dependent neuronal cell death in developing brain. These data may be of important clinical relevance, since spontaneous synchronized activity is also a hallmark of the developing human brain and inflammation-induced alterations in this early network activity may have a critical impact on the survival of immature neurons.Intrauterine Infektionen und die fötale Immunantwort sind wichtige pathogene Faktoren bei Frühgeburten. Während der frühen Ontogenese können sie Störungen des Gehirnaufbaus und Langzeitschäden induzieren, wie beispielsweise kognitive Störungen, Lerndefizite und zerebrale Lähmungen. Entzündungsreaktionen im Gehirn sind charakterisiert über die Aktivierung von ortsansässigen Immunzellen, wie Astrozyten und Mikroglia. Deren Aktivierung wird mit einigen neurodegenerativen Erkrankungen, wie Alzheimer und Multipler Sklerose in Verbindung gebracht. Die beiden Zelltypen haben die Fähigkeit nach Ihrer Aktivierung pro- inflammatorische Zytokine auszuschütten und ebenso auf diese zu reagieren. Diese Zyotkine spielen eine wichtige Rolle bei der Immunantwort und es wurde erst kürzlich gezeigt, dass sie auch Einfluss auf neuronal Funktionen haben können. Besonderes Augenmerk wurde in meiner Arbeit auf die beiden Zytokine TNFalpha und MIP-2 gelegt. Es wurde bereits gezeigt, dass sowohl MIP-2 als auch TNFalpha im neuronalen Zelltod involviert sind. Im Bezug auf TNFalpha ist ebenso bekannt, dass es zu erhöhter Erregung und zu einer Erhöhung der Anzahl der AMPA Rezeptoren auf der Zelloberfläche von Neuronen führt. Daraus lässt sich schließen, dass kontinuierlich freigesetztes TNFalphawichtig für ein Gleichgewicht zwischen Erregung und Inhibierung in Neuronen ist. Ich vertrete die These, dass ein Zusammenspiel zwischen neuronaler Aktivität, TNFalpha und MIP-2 wichtig für die Regulation des Zelltodes während der frühen Entwicklung ist. In meiner Dissertation wurde der Einfluss von Entzündungsmechanismen auf die neuronale Aktivität und das neuronale Überleben während der frühen Entwicklung in vivo sowie auch in vitro untersucht. Dazu wurden elektrophysiologische Aufnahmen mit Hilfe eines Mulit-Electrode Array Systems und quantitative Analysen des Zelltods verwendet. Die Immunantwort wurde mit Hilfe von Lipopolysaccarid (LPS), einer strukturellen Komponente der Zellwand von gram negativen Bakterien, induzierte. Zusätzlich wurden Zytokin-Arrays und Antikörper gegen spezifische Zytokine zur genaueren Identifizierung der involvierten Faktoren verwendet. In dieser Studie wird zum ersten Mal eine direkte Verbindung zwischen der Veränderung in der neuronalen Netzwerkaktivität nach Entzündung und dem Zellüberleben im sich entwickelnden Kortex gezeigt. In Kooperation mit Jenq-Wei Yang und Shuming An wurden neben in vitro auch in vivo Experimente durchgeführt. Die elektrophysiologischen Aufzeichnungen zeigen eine durch LPS induzierte Reduktion von spontan auftretender oszillatorischer Netzwerkaktivität. Meine Ergebnisse lassen darauf schließen, dass es durch eine LPS induzierte Entzündung in der frühen Entwicklung zur Freisetzung von proinflammatorischen Faktoren kommt, die zu einer Veränderung der spontanen Netzwerkaktivität und darauf folgend zum neuronalen Zelltod führen. Vor allem TNFalpha und MIP-2 spielen eine Schlüsselrolle im aktivitäts-abhängigen neuronalen Zelltod im neonatalen Gehirn. Da spontane Netzwerkaktivität auch im sich entwickelnden menschlichen Gehirn nachweisbar ist, könnten diese Daten zudem von großer klinischer Relevanz sein. Entzündungsreaktionen in diesem frühen Stadium der Entwicklung können zu Veränderungen der Netzwerkaktivität und damit zum Tod von Neuronen führen

Establishing a Preclinical Multidisciplinary Board for Brain Tumors.

Purpose: Curing all children with brain tumors will require an understanding of how each subtype responds to conventional treatments and how best to combine existing and novel therapies. It is extremely challenging to acquire this knowledge in the clinic alone, especially among patients with rare tumors. Therefore, we developed a preclinical brain tumor platform to test combinations of conventional and novel therapies in a manner that closely recapitulates clinic trials.Experimental Design: A multidisciplinary team was established to design and conduct neurosurgical, fractionated radiotherapy and chemotherapy studies, alone or in combination, in accurate mouse models of supratentorial ependymoma (SEP) subtypes and choroid plexus carcinoma (CPC). Extensive drug repurposing screens, pharmacokinetic, pharmacodynamic, and efficacy studies were used to triage active compounds for combination preclinical trials with "standard-of-care" surgery and radiotherapy.Results: Mouse models displayed distinct patterns of response to surgery, irradiation, and chemotherapy that varied with tumor subtype. Repurposing screens identified 3-hour infusions of gemcitabine as a relatively nontoxic and efficacious treatment of SEP and CPC. Combination neurosurgery, fractionated irradiation, and gemcitabine proved significantly more effective than surgery and irradiation alone, curing one half of all animals with aggressive forms of SEP.Conclusions: We report a comprehensive preclinical trial platform to assess the therapeutic activity of conventional and novel treatments among rare brain tumor subtypes. It also enables the development of complex, combination treatment regimens that should deliver optimal trial designs for clinical testing. Postirradiation gemcitabine infusion should be tested as new treatments of SEP and CPC. Clin Cancer Res; 24(7); 1654-66. ©2018 AACR

A novel <i>Atg5</i>-shRNA mouse model enables temporal control of Autophagy <i>in vivo</i>

<p>Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for <i>in vivo</i> studies. Here, we have developed an inducible shRNA mouse model targeting <i>Atg5</i>, allowing us to dynamically inhibit autophagy <i>in vivo</i>, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.</p