Merry Nishimura

Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons

Homologous recombination was utilized to generate mice with a deletion in the coding sequence of the nerve growth factor (NGF) gene. Animals homozygous for NGF disruption failed to respond to noxious mechanical stimuli, and histological analysis revealed profound cell loss in both sensory and sympathetic ganglia. Within dorsal root ganglia, effects of the mutation appeared to be restricted to small and medium peptidergic neurons. These observations confirm the critical dependence of sensory and sympathetic neurons on NGF and demonstrate that other neurotrophins are not able to compensate for the loss of NGF action on these cells. Examination of the central nervous system revealed that, in marked contrast with neurons of sensory and sympathetic ganglia, basal forebrain cholinergic neurons differentiate and continue to express phenotypic markers for the life span of the null mutant mice. Thus, differentiation and initial survival of central NGF-responsive neurons can occur in the absence of NGF.

Persephin, a Novel Neurotrophic Factor Related to GDNF and Neurturin

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

A Hierarchy of Self-Renewing Tumor-Initiating Cell Types in Glioblastoma

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent

Significance Therapeutic antibodies have revolutionized the treatment of human disease. Despite these advances, antibody bivalency limits their utility against some targets. Here, we describe the development of a one-armed (monovalent) antibody, onartuzumab, targeting the receptor tyrosine kinase MET. While initial screening of bivalent antibodies produced agonists of MET, engineering them into monovalent antibodies produced antagonists instead. We explain the structural basis of the mechanism of action with the crystal structure of onartuzumab antigen-binding fragment in complex with MET and HGF-β. These discoveries have led to an additional antibody-based therapeutic option and shed light on the underpinnings of HGF/MET signaling. Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

3D micro-CT imaging of the Postmortem Brain

Magnetic resonance microscopy (microMRI) is becoming an important tool for non-destructive analysis of fixed brain tissue. However, unlike MRI, X-ray computed tomography (CT) scans show little native soft tissue contrast. In this paper, we explored the use of contrast enhanced (brains immersion stained in iodinated CT contrast media) micro-CT (microCT) for high resolution 3D imaging of fixed normal and pathological brains, compared to microMRI and standard histopathology. An optimum iodine concentration of 0.27 M resulted in excellent contrast between gray and white matter in normal brain and a wide range of anatomical structures were identified. In glioma bearing mouse brains, there was clear deliniation of tumor margin which closely matched that seen on histopathology sections. microCT tumor volume was strongly correlated with histopathology volume. Our data suggests that microCT image contrast in the immersion-stained brains is related to axonal density and myelin content. Compared to traditional histopathology, our microCT approach is relatively rapid and less labor intensive. In addition, compared to microMRI, microCT is robust and requires much lower equipment and maintenance costs. For simple measurements, such as tumor volume and non-destructive postmortem brain screening, microCT may prove to be a valuable alternative to standard histopathology or microMRI.

Targeting the PI3K Pathway in the Brain - Efficacy of a PI3K Inhibitor Optimized to Cross the Blood-Brain Barrier

Purpose: Glioblastoma (GBM), the most common primary brain tumor in adults, presents a high frequency of alteration in the PI3K pathway. Our objectives were to identify a dual PI3K/mTOR inhibitor optimized to cross the blood–brain barrier (BBB) and characterize its brain penetration, pathway modulation in the brain and efficacy in orthotopic xenograft models of GBM. Experimental Design: Physicochemical properties of PI3K inhibitors were optimized using in silico tools, leading to the identification of GNE-317. This compound was tested in cells overexpressing P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP). Following administration to mice, GNE-317 plasma and brain concentrations were determined, and phosphorylated biomarkers (pAkt, p4EBP1, and pS6) were measured to assess PI3K pathway suppression in the brain. GNE-317 efficacy was evaluated in the U87, GS2, and GBM10 orthotopic models of GBM. Results: GNE-317 was identified as having physicochemical properties predictive of low efflux by P-gp and BCRP. Studies in transfected MDCK cells showed that GNE-317 was not a substrate of either transporter. GNE-317 markedly inhibited the PI3K pathway in mouse brain, causing 40% to 90% suppression of the pAkt and pS6 signals up to 6-hour postdose. GNE-317 was efficacious in the U87, GS2, and GBM10 orthotopic models, achieving tumor growth inhibition of 90% and 50%, and survival benefit, respectively. Conclusions: These results indicated that specific optimization of PI3K inhibitors to cross the BBB led to potent suppression of the PI3K pathway in healthy brain. The efficacy of GNE-317 in 3 intracranial models of GBM suggested that this compound could be effective in the treatment of GBM. Clin Cancer Res; 18(22); 6239–48. ©2012 AACR.

Hominoid-specific enzyme GLUD2 promotes growth of IDH1R132H glioma

Significance Mutation of isocitrate dehydrogenase 1 (IDH1) is believed to be the initiating event for the majority of secondary glioblastomas and lower-grade diffuse gliomas; however, the basis for tissue specificity of oncogenesis initiated by IDH1 mutation has not been apparent. We report evidence to suggest that specialization of human neocortex for glutaminergic neurotransmission provides a metabolic niche particularly suited for growth of IDH1R132H glioma. Our findings reveal that IDH1-mutant enzyme challenges growth of murine glioma progenitor cells but that these cells thrive if they are engineered to express the hominoid-specific brain enzyme GLUD2, a mitochondrial enzyme that converts glutamate to alpha-ketoglutarate in human cortex. The current findings raise the possibility that evolutionary changes contributing to human cognitive abilities may have conferred vulnerability to brain tumors driven by IDH1 mutation. Somatic mutation of isocitrate dehydrogenase 1 (IDH1) is now recognized as the most common initiating event for secondary glioblastoma, a brain tumor type arising with high frequency in the frontal lobe. A puzzling feature of IDH1 mutation is the selective manifestation of glioma as the only neoplasm frequently associated with early postzygotic occurrence of this genomic alteration. We report here that IDH1R132H exhibits a growth-inhibitory effect that is abrogated in the presence of glutamate dehydrogenase 2 (GLUD2), a hominoid-specific enzyme purportedly optimized to facilitate glutamate turnover in human forebrain. Using murine glioma progenitor cells, we demonstrate that IDH1R132H exerts a growth-inhibitory effect that is paralleled by deficiency in metabolic flux from glucose and glutamine to lipids. Examining human gliomas, we find that glutamate dehydrogenase 1 (GLUD1) and GLUD2 are overexpressed in IDH1-mutant tumors and that orthotopic growth of an IDH1-mutant glioma line is inhibited by knockdown of GLUD1/2. Strikingly, introduction of GLUD2 into murine glioma progenitor cells reverses deleterious effects of IDH1 mutation on metabolic flux and tumor growth. Further, we report that glutamate, a substrate of GLUD2 and a neurotransmitter abundant in mammalian neocortex, can support growth of glioma progenitor cells irrespective of IDH1 mutation status. These findings suggest that specialization of human neocortex for high glutamate neurotransmitter flux creates a metabolic niche conducive to growth of IDH1 mutant tumors.

PET of Glial Metabolism Using 2-18F-Fluoroacetate

Imaging of the glial activation that occurs in response to central nervous system trauma and inflammation could become a powerful technique for the assessment of several neuropathologies. The selective uptake and metabolism of 2-18F-fluoroacetate (18F-FAC) in glia may represent an attractive strategy for imaging glial metabolism. Methods: We have evaluated the use of 18F-FAC as a specific PET tracer of glial cell metabolism in rodent models of glioblastoma, stroke, and ischemia–hypoxia. Results: Enhanced uptake of 18F-FAC was observed (6.98 ± 0.43 percentage injected dose per gram [%ID/g]; tumor-to-normal ratio, 1.40) in orthotopic U87 xenografts, compared with healthy brain tissue. The lesion extent determined by 18F-FAC PET correlated with that determined by MRI (R2 = 0.934, P = 0.007). After transient middle cerebral artery occlusion in the rat brain, elevated uptake of 18F-FAC (1.00 ± 0.03 %ID/g; lesion-to-normal ratio, 1.90) depicted the ischemic territory and correlated with infarct volumes as determined by 2,3,5-triphenyltetrazolium chloride staining (R2 = 0.692, P = 0.010) and with the presence of activated astrocytes detected by anti–glial fibrillary acidic protein. Ischemia–hypoxia, induced by permanent ligation of the common carotid artery with transient hypoxia, resulted in persistent elevation of 18F-FAC uptake within 30 min of the induction of hypoxia. Conclusion: Our data support the further evaluation of 18F-FAC PET for the assessment of glial cell metabolism associated with neuroinflammation.

Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib (GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastoma models-assessment by matrix-assisted laser desorption ionization imaging.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM.

Brain Distribution and Efficacy of the Brain Penetrant PI3K Inhibitor GDC-0084 in Orthotopic Mouse Models of Human Glioblastoma

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.