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.

LPS-induced microglial secretion of TNFα increases activity-dependent neuronal apoptosis in the neonatal cerebral cortex.

During the pre- and neonatal period, the cerebral cortex reveals distinct patterns of spontaneous synchronized activity, which is critically involved in the formation of early networks and in the regulation of neuronal survival and programmed cell death (apoptosis). During this period, the cortex is also highly vulnerable to inflammation and in humans prenatal infection may have a profound impact on neurodevelopment causing long-term neurological deficits. Using in vitro and in vivo multi-electrode array recordings and quantification of caspase-3 (casp-3)-dependent apoptosis, we demonstrate that lipopolysaccharide-induced inflammation causes rapid alterations in the pattern of spontaneous burst activities, which subsequently leads to an increase in apoptosis. We show that these inflammatory effects are specifically initiated by the microglia-derived pro-inflammatory cytokine tumor necrosis factor α and the chemokine macrophage inflammatory protein 2. Our data demonstrate that inflammation-induced modifications in spontaneous network activities influence casp-3-dependent cell death in the developing cerebral cortex.

Control of programmed cell death by distinct electrical activity patterns.

Electrical activity and sufficient supply with survival factors play a major role in the control of apoptosis in the developing cortex. Coherent high-frequency neuronal activity, which efficiently releases neurotrophins, is essential for the survival of immature neurons. We studied the influence of neuronal activity on apoptosis in the developing cortex. Dissociated cultures of the newborn mouse cerebral cortex were grown on multielectrode arrays to determine the activity patterns that promote neuronal survival. Cultures were transfected with a plasmid coding for a caspase-3-sensitive fluorescent protein allowing real-time analysis of caspase-3-dependent apoptosis in individual neurons. Elevated extracellular potassium concentrations (5 and 8 mM), application of 4-aminopyridine or the γ-aminobutyric acid-A receptor antagonist Gabazine induced a shift in the frequency distribution of activity toward high-frequency bursts. Under these conditions, a reduction or delay in caspase-3 activation and an overall increase in neuronal survival could be observed. This effect was dependent on the activity of phosphatidylinositol-3 kinase, as blockade of this enzyme abolished the survival-promoting effect of high extracellular potassium concentrations. Our data indicate that increased network activity can prevent apoptosis in developing cortical neurons.

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.

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.

Cross-Species Genomics Identifies TAF12, NFYC, and RAD54L as Choroid Plexus Carcinoma Oncogenes.

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

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.

Glycine receptors influence radial migration in the embryonic mouse neocortex.

To investigate whether glycine receptors influence radial migration in the neocortex, we analyzed the effect of glycine and the glycinergic antagonist strychnine, on the distribution of 5-bromo-2'deoxyuridine-labeled neurons in organotypic slice cultures from embryonic mice cortices. Application of glycine impeded radial migration only in the presence of the glycine-transport blockers, ALX-5407 and ALX-1393. This effect was blocked by the specific glycine receptor antagonist strychnine, whereas application of strychnine in the absence of glycine was without effect. We conclude from these observations that an activation of glycine receptors can impede radial migration, but that the glycinergic system is not directly implicated in the regulation of radial migration in organotypic slice cultures.

Sustained elevation of cyclic guanosine monophosphate induces apoptosis in microglia.

Cyclic nucleotides mediate transient as well as plastic cellular responses. The most ultimate response is cell death. In the present study, we propose that an increase of intracellular cyclic guanosine monophosphate (cGMP) for at least 1h promotes cell death in the murine microglial cell line, BV-2 cells, as well as in primary murine microglia. Cells were exposed to ammonium, the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and to the membrane-permeable cGMP analogue, 8-Bromo-cGMP (8-Br-cGMP), respectively. Cell death was estimated using DAPI labelling and annexin-V labelling of exposed phosphatidylserine, and cGMP level was quantified by an immunoassay. Ammonium not only increased the number of apoptotic cells but also promoted a moderate increase in intracellular cGMP. Addition of ODQ suppressed ammonium-induced apoptosis. Furthermore, we found that 8-Br-cGMP significantly increased the number of BV-2 cells and primary microglia, respectively, containing nuclei with condensed chromatin accumulated at the nuclear periphery. Similarly, cells exposed to 8-Br-cGMP showed significantly more cells with exposed phosphatidylserine compared to control cells. Thus, according to the nuclear structure as well as to changes in the plasma membrane, chronic elevation of cGMP induces apoptosis in microglia.