A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance.

A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care for GBMs include maximally safe tumor resection, followed by concurrent adjuvant radiation treatment and chemotherapy with the DNA alkylating agent temozolomide (TMZ), which was approved by the FDA in 2005 based on a marginal increase (~2 months) in overall survival (OS) levels. This treatment approach, while initially successful in containing and treating GBM, almost invariably fails to prevent tumor recurrence. In addition to the limited therapeutic benefit, TMZ also causes debilitating adverse events (AEs) that significantly impact the quality of life of GBM patients. Some of the most common AEs include hematologic (e.g., thrombocytopenia, neutropenia, anemia) and non-hematologic (e.g., nausea, vomiting, constipation, dizziness) toxicities. Recurrent GBMs are often resistant to TMZ and other DNA-damaging agents. Thus, there is an urgent need to devise strategies to potentiate TMZ activity, to overcome drug resistance, and to reduce dose-dependent AEs. Here, we analyze major mechanisms of the TMZ resistance-mediated intracellular signaling activation of DNA repair pathways and the overexpression of drug transporters. We review some of the approaches investigated to counteract these mechanisms of resistance to TMZ, including the use of chemosensitizers and drug delivery strategies to enhance tumoral drug exposure.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma.

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5'-monophosphate-activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Brain pharmacokinetics and metabolism of the AMP-activated protein kinase selective inhibitor SBI-0206965, an investigational agent for the treatment of glioblastoma.

PURPOSE: Emerging evidence suggests that 5' Adenosine Monophosphate-Activated Protein Kinase (AMPK), a key regulator of cellular bioenergetics, is a novel target for the treatment of glioblastoma (GBM), a lethal brain tumor. SBI-0206965, an aminopyrimidine derivative, is a potent AMPK inhibitor being investigated for the treatment of GBM. Here we characterized the systemic and brain pharmacokinetics (PK) and hepatic metabolism of SBI-0206965. METHODS: We performed intracerebral microdialysis to determine brain partitioning of SBI-0206965 in jugular vein cannulated rats. We assessed systemic PK of SBI-0206965 in rats and C57BL/6 mice following oral administration. Employing human, mouse, and rat liver microsomes we characterized the metabolism of SBI-0206965. RESULTS: SBI-0206965 is quickly absorbed, achieving plasma and brain extracellular fluid (ECF) peak levels within 0.25 - 0.65 h. Based on the ratio of Cmax and AUC in brain ECF to plasma (corrected for protein binding), brain partitioning is ~ 0.6-0.9 in rats. However, the compound has a short elimination half-life (1-2 h) and low relative oral bioavailability (~ 0.15). The estimated in-vitro hepatic intrinsic clearance of SBI-0206965 in mouse, rat and human was 325, 76 and 68 mL/min/kg, respectively. SBI-0206965 metabolites included desmethylated products, and the metabolism was strongly inhibited by ketoconazole, a CYP3A inhibitor. CONCLUSION: SBI-0206965 has adequate brain permeability but low relative oral bioavailability which may be due to rapid hepatic metabolism, likely catalyzed by CYP3A enzymes. Our observations will facilitate further development of SBI-0206965, and/or other structurally related molecules, for the treatment of GBM and other brain tumors.

Copb2 is essential for embryogenesis and hypomorphic mutations cause human microcephaly.

Primary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome-editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2R254C/Zfn) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2R254C/Zfnbrain revealed a reduction in layer V (CTIP2+) neurons, while the overall cell density of the cortex is unchanged. Moreover, neurospheres derived from animals with Copb2 variants grew less than control. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest.

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK.

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis--two crucial arms of cancer growth--AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

The protein tyrosine phosphatase Shp2 is required for the generation of oligodendrocyte progenitor cells and myelination in the mouse telencephalon.

The protein tyrosine phosphatase Shp2 (PTPN11) is crucial for normal brain development and has been implicated in dorsal telencephalic neuronal and astroglia cell fate decisions. However, its roles in the ventral telencephalon and during oligodendrogenesis in the telencephalon remain largely unknown. Shp2 gain-of-function (GOF) mutations are observed in Noonan syndrome, a type of RASopathy associated with multiple phenotypes, including cardiovascular, craniofacial, and neurocognitive abnormalities. To gain insight into requirements for Shp2 (LOF) and the impact of abnormal Shp2 GOF mutations, we used a Shp2 conditional mutant allele (LOF) and a cre inducible Shp2-Q79R GOF transgenic mouse in combination with Olig2(cre/+) mice to target embryonic ventral telencephalic progenitors and the oligodendrocyte lineage. In the absence of Shp2 (LOF), neuronal cell types originating from progenitors in the ventral telencephalon were generated, but oligodendrocyte progenitor cell (OPC) generation was severely impaired. Late embryonic and postnatal Shp2 cKOs showed defects in the generation of OPCs throughout the telencephalon and subsequent reductions in white matter myelination. Conversely, transgenic expression of the Shp2 GOF Noonan syndrome mutation resulted in elevated OPC numbers in the embryo and postnatal brain. Interestingly, expression of this mutation negatively influenced myelination as mice displayed abnormal myelination and fewer myelinated axons in the white matter despite elevated OPC numbers. Increased proliferating OPCs and elevated MAPK activity were also observed during oligodendrogenesis after expression of Shp2 GOF mutation. These results support the notion that appropriate Shp2 activity levels control the number as well as the differentiation of oligodendrocytes during development.

Coordinated Targeting of S6K1/2 and AXL Disrupts Pyrimidine Biosynthesis in PTEN-Deficient Glioblastoma.

Intrinsic resistance to targeted therapeutics in PTEN-deficient glioblastoma (GBM) is mediated by redundant signaling networks that sustain critical metabolic functions. Here, we demonstrate that coordinated inhibition of the ribosomal protein S6 kinase 1 (S6K1) and the receptor tyrosine kinase AXL using LY-2584702 and BMS-777607 can overcome network redundancy to reduce GBM tumor growth. This combination of S6K1 and AXL inhibition suppressed glucose flux to pyrimidine biosynthesis. Genetic inactivation studies to map the signaling network indicated that both S6K1 and S6K2 transmit growth signals in PTEN-deficient GBM. Kinome-wide ATP binding analysis in inhibitor-treated cells revealed that LY-2584702 directly inhibited S6K1, and substrate phosphorylation studies showed that BMS-777607 inactivation of upstream AXL collaborated to reduce S6K2-mediated signal transduction. Thus, combination targeting of S6K1 and AXL provides a kinase-directed therapeutic approach that circumvents signal transduction redundancy to interrupt metabolic function and reduce growth of PTEN-deficient GBM. SIGNIFICANCE: Therapy for glioblastoma would be advanced by incorporating molecularly targeted kinase-directed agents, similar to standard of care strategies in other tumor types. Here, we identify a kinase targeting approach to inhibit the metabolism and growth of glioblastoma.

A Phase 0/I Pharmacokinetic and Pharmacodynamics and Safety and Tolerability Study of Letrozole in Combination with Standard Therapy in Recurrent High-Grade Gliomas.

PURPOSE: High-grade gliomas (HGG) carry a poor prognosis, with glioblastoma accounting for almost 50% of primary brain malignancies in the elderly. Unfortunately, despite the use of multiple treatment modalities, the prognosis remains poor in this population. Our preclinical studies suggest that the presence of aromatase expression, encoded by CYP19A1, is significantly upregulated in HGGs. Remarkably, we find that letrozole (LTZ), an FDA-approved aromatase inhibitor, has marked activity against HGGs. PATIENTS AND METHODS: We conducted a phase 0/I single-center clinical trial (NCT03122197) to assess the tumoral availability, pharmacokinetics (PK), safety, and tolerability of LTZ in recurrent patients with HGG. Planned dose cohorts included 2.5, 5, 10, 12.5, 15, 17.5, and 20 mg of LTZ administered daily pre- and postsurgery or biopsy. Tumor samples were assayed for LTZ content and relevant biomarkers. The recommended phase 2 dose (R2PD) was determined as the dose that resulted in predicted steady-state tumoral extracellular fluid (ECF; Css,ecf) >2 µmol/L and did not result in ≥33% dose-limiting adverse events (AE) assessed using CTCAE v5.0. RESULTS: Twenty-one patients were enrolled. Common LTZ-related AEs included fatigue, nausea, musculoskeletal, anxiety, and dysphoric mood. No DLTs were observed. The 15 mg dose achieved a Css,ecf of 3.6 ± 0.59 µmol/L. LTZ caused dose-dependent inhibition of estradiol synthesis and modulated DNA damage pathways in tumor tissues as evident using RNA-sequencing analysis. CONCLUSIONS: On the basis of safety, brain tumoral PK, and mechanistic data, 15 mg daily is identified as the RP2D for future trials.

A Multimodal Drug-Diet-Immunotherapy Combination Restrains Melanoma Progression and Metastasis.

The genetic landscape of cancer cells can lead to specific metabolic dependencies for tumor growth. Dietary interventions represent an attractive strategy to restrict the availability of key nutrients to tumors. In this study, we identified that growth of a subset of melanoma was severely restricted by a rationally designed combination therapy of a stearoyl-CoA desaturase (SCD) inhibitor with an isocaloric low-oleic acid diet. Despite its importance in oncogenesis, SCD underwent monoallelic codeletion along with PTEN on chromosome 10q in approximately 47.5% of melanoma, and the other SCD allele was methylated, resulting in very low-SCD expression. Although this SCD-deficient subset was refractory to SCD inhibitors, the subset of PTEN wild-type melanoma that retained SCD was sensitive. As dietary oleic acid could potentially blunt the effect of SCD inhibitors, a low oleic acid custom diet was combined with an SCD inhibitor. The combination reduced monounsaturated fatty acids and increased saturated fatty acids, inducing robust apoptosis and growth suppression and inhibiting lung metastasis with minimal toxicity in preclinical mouse models of PTEN wild-type melanoma. When combined with anti-PD1 immunotherapy, the SCD inhibitor improved T-cell functionality and further constrained melanoma growth in mice. Collectively, these results suggest that optimizing SCD inhibitors with diets low in oleic acid may offer a viable and efficacious therapeutic approach for improving melanoma treatment. Significance: Blockade of endogenous production of fatty acids essential for melanoma combined with restriction of dietary intake blocks tumor growth and enhances response to immunotherapy, providing a rational drug-diet treatment regimen for melanoma.

Chronic AMPK inactivation slows SHH medulloblastoma progression by inhibiting mTORC1 signaling and depleting tumor stem cells.

We show that inactivating AMPK in a genetic medulloblastoma model depletes tumor stem cells and slows progression. In medulloblastoma, the most common malignant pediatric brain tumor, drug-resistant stem cells co-exist with transit-amplifying cells and terminally differentiated neuronal progeny. Prior studies show that Hk2-dependent glycolysis promotes medulloblastoma progression by suppressing neural differentiation. To determine how the metabolic regulator AMPK affects medulloblastoma growth and differentiation, we inactivated AMPK genetically in medulloblastomas. We bred conditional Prkaa1 and Prkaa2 deletions into medulloblastoma-prone SmoM2 mice and compared SmoM2-driven medulloblastomas with intact or inactivated AMPK. AMPK-inactivation increased event-free survival (EFS) and altered cellular heterogeneity, increasing differentiation and decreasing tumor stem cell populations. Surprisingly, AMPK-inactivation decreased mTORC1 activity and decreased Hk2 expression. Hk2 deletion similarly depleted medulloblastoma stem cells, implicating reduced glycolysis in the AMPK-inactivated phenotype. Our results show that AMPK inactivation disproportionately impairs medulloblastoma stem cell populations typically refractory to conventional therapies.

Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme.

Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.

AMPK in the brain: its roles in glucose and neural metabolism.

The adenosine monophosphate-activated protein kinase (AMPK) is an integrative metabolic sensor that maintains energy balance at the cellular level and plays an important role in orchestrating intertissue metabolic signaling. AMPK regulates cell survival, metabolism, and cellular homeostasis basally as well as in response to various metabolic stresses. Studies so far show that the AMPK pathway is associated with neurodegeneration and CNS pathology, but the mechanisms involved remain unclear. AMPK dysregulation has been reported in neurodegenerative diseases such as amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, and other neuropathies. AMPK activation appears to be both neuroprotective and pro-apoptotic, possibly dependent upon neural cell types, the nature of insults, and the intensity and duration of AMPK activation. While embryonic brain development in AMPK null mice appears to proceed normally without any overt structural abnormalities, our recent study confirmed the full impact of AMPK loss in the postnatal and aging brain. Our studies revealed that Ampk deletion in neurons increased basal neuronal excitability and reduced latency to seizure upon stimulation. Three major pathways, glycolysis, pentose phosphate shunt, and glycogen turnover, contribute to utilization of glucose in the brain. AMPK's regulation of aerobic glycolysis in astrocytic metabolism warrants further deliberation, particularly glycogen turnover and shuttling of glucose- and glycogen-derived lactate from astrocytes to neurons during activation. In this minireview, we focus on recent advances in AMPK and energy-sensing in the brain.

Potentiation of temozolomide activity against glioblastoma cells by aromatase inhibitor letrozole.

PURPOSE: The DNA alkylating agent temozolomide (TMZ), is the first-line therapeutic for the treatment of glioblastoma (GBM). However, its use is confounded by the occurrence of drug resistance and debilitating adverse effects. Previously, we observed that letrozole (LTZ), an aromatase inhibitor, has potent activity against GBM in pre-clinical models. Here, we evaluated the effect of LTZ on TMZ activity against patient-derived GBM cells. METHODS: Employing patient-derived G76 (TMZ-sensitive), BT142 (TMZ-intermediately sensitive) and G43 and G75 (TMZ-resistant) GBM lines we assessed the influence of LTZ and TMZ on cell viability and neurosphere growth. Combination Index (CI) analysis was performed to gain quantitative insights of this interaction. We then assessed DNA damaging effects by conducting flow-cytometric analysis of Ë H2A.X formation and induction of apoptotic signaling pathways (caspase3/7 activity). The effects of adding estradiol on LTZ-induced cytotoxicity and DNA damage were also evaluated. RESULTS: Co-treatment with LTZ at a non-cytotoxic concentration (40 nM) reduced TMZ IC50 by 8, 37, 240 and 640 folds in G76, BT-142, G43 and G75 cells, respectively. The interaction was deemed to be synergistic based on CI analysis. LTZ co-treatment also significantly increased DNA damaging effects of TMZ. Addition of estradiol abrogated these LTZ effects. CONCLUSIONS: LTZ increases DNA damage and synergistically enhances TMZ activity in TMZ sensitive and TMZ-resistant GBM lines. These effects are abrogated by the addition of exogenous estradiol underscoring that the observed effects of LTZ may be mediated by estrogen deprivation. Our study provides a strong rationale for investigating the clinical potential of combining LTZ and TMZ for GBM therapy.

Analysis of reactive astrogliosis in mouse brain using in situ hybridization combined with immunohistochemistry.

Reactive astrogliosis is characterized by a profound change in astrocyte phenotype in response to all CNS injuries. Here, we present a revised in situ hybridization and immunohistochemistry (IHC) protocol to label the reactive astrocytes in the mouse brain. Several approaches for quantifying astrocyte reactivity lacked sensitivity to discriminate across the spectrum. We optimized in situ hybridization followed by IHC. We provide a staining protocol for quantitative measures of astrocyte reactivity as an independent confirmation of the magnitude of reactive gliosis. For complete details on the use and execution of this protocol, please refer to Muraleedharan et al. (2020).

Mechanisms of stearoyl CoA desaturase inhibitor sensitivity and acquired resistance in cancer.

The lipogenic enzyme stearoyl CoA desaturase (SCD) plays a key role in tumor lipid metabolism and membrane architecture. SCD is often up-regulated and a therapeutic target in cancer. Here, we report the unexpected finding that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and unintentional monoallelic co-deletion with phosphatase and tensin homolog (PTEN) in a subset of patients. Cell lines from this subset expressed undetectable SCD, yet retained residual SCD enzymatic activity. Unexpectedly, these lines evolved to survive independent of SCD through unknown mechanisms. Cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Last, we identify that SCD-dependent lines acquire resistance through a previously unknown FBJ murine osteosarcoma viral oncogene homolog B (FOSB)-mediated mechanism. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor-bearing mice treated with SCD inhibitor.

Insight on Transcriptional Regulation of the Energy Sensing AMPK and Biosynthetic mTOR Pathway Genes.

The Adenosine Monophosphate-activated Protein Kinase (AMPK) and the Mechanistic Target of Rapamycin (mTOR) are two evolutionarily conserved kinases that together regulate nearly every aspect of cellular and systemic metabolism. These two kinases sense cellular energy and nutrient levels that in turn are determined by environmental nutrient availability. Because AMPK and mTOR are kinases, the large majority of studies remained focused on downstream substrate phosphorylation by these two proteins, and how AMPK and mTOR regulate signaling and metabolism in normal and disease physiology through phosphorylation of their substrates. Compared to the wealth of information known about the signaling and metabolic pathways modulated by these two kinases, much less is known about how the transcription of AMPK and mTOR pathway genes themselves are regulated, and the extent to which AMPK and mTOR regulate gene expression to cause durable changes in phenotype. Acute modification of cellular systems can be achieved through phosphorylation, however, induction of chronic changes requires modulation of gene expression. In this review we will assemble evidence from published studies on transcriptional regulation by AMPK and mTOR and discuss about the putative transcription factors that regulate expression of AMPK and mTOR complex genes.

AMPK-Regulated Astrocytic Lactate Shuttle Plays a Non-Cell-Autonomous Role in Neuronal Survival.

Lactate is used as an energy source by producer cells or shuttled to neighboring cells and tissues. Both glucose and lactate fulfill the bioenergetic demand of neurons, the latter imported from astrocytes. The contribution of astrocytic lactate to neuronal bioenergetics and the mechanisms of astrocytic lactate production are incompletely understood. Through in vivo1H magnetic resonance spectroscopy, 13C glucose mass spectroscopy, and electroencephalographic and molecular studies, here we show that the energy sensor AMP activated protein kinase (AMPK) regulates neuronal survival in a non-cell-autonomous manner. Ampk-null mice are deficient in brain lactate and are seizure prone. Ampk deletion in astroglia, but not neurons, causes neuronal loss in both mammalian and fly brains. Mechanistically, astrocytic AMPK phosphorylated and destabilized thioredoxin-interacting protein (TXNIP), enabling expression and surface translocation of the glucose transporter GLUT1, glucose uptake, and lactate production. Ampk loss in astrocytes causes TXNIP hyperstability, GLUT1 misregulation, inadequate glucose metabolism, and neuronal loss.

Spatiotemporal differences in CXCL12 expression and cyclic AMP underlie the unique pattern of optic glioma growth in neurofibromatosis type 1.

Astrocytoma (glioma) formation in neurofibromatosis type 1 (NF1) occurs preferentially along the optic pathway during the first decade of life. The molecular basis for this unique pattern of gliomagenesis is unknown. Previous studies in mouse Nf1 optic glioma models suggest that this patterning results from cooperative effects of Nf1 loss in glial cells and the action of factors derived from the surrounding Nf1+/- brain. Because CXCL12 is a stroma-derived growth factor for malignant brain tumors, we tested the hypothesis that CXCL12 functions in concert with Nf1 loss to facilitate NF1-associated glioma growth. Whereas CXCL12 promoted cell death in wild-type astrocytes, it increased Nf1-/- astrocyte survival. This increase in Nf1-/- astrocyte survival in response to CXCL12 was due to sustained suppression of intracellular cyclic AMP (cAMP) levels. Moreover, the ability of CXCL12 to suppress cAMP and increase Nf1-/- astrocyte survival was a consequence of mitogen-activated protein/extracellular signal-regulated kinase kinase-dependent inhibition of CXCL12 receptor (CXCR4) desensitization. In support of an instructive role for CXCL12 in facilitating optic glioma growth, we also show that CXCL12 expression along the optic pathway is higher in infant children and young mice and is associated with low levels of cAMP. CXCL12 expression declines in multiple brain regions with increasing age, correlating with the age-dependent decline in glioma growth in children with NF1. Collectively, these studies provide a mechanism for the unique pattern of NF1-associated glioma growth.

Plasma and brain pharmacokinetics of letrozole and drug interaction studies with temozolomide in NOD-scid gamma mice and sprague dawley rats.

PURPOSE: The aromatase inhibitor, letrozole, is being investigated in experimental animal models as a novel treatment for high-grade gliomas (HGGs). To facilitate optimal dosing for such studies, we evaluated the plasma and brain pharmacokinetics (PK) of letrozole in NOD-scid gamma (NSG) mice, which are frequently employed for assessing efficacy against patient-derived tumor cells. Furthermore, we evaluated the potential PK interactions between letrozole and temozolomide (TMZ) in Sprague-Dawley rats. METHODS: NSG mice were administered letrozole (8 mg/kg; i.p) as a single or multiple dose (b.i.d, 10 days). Brain tissue and blood samples were collected over 24 h. Letrozole and TMZ interaction study employed jugular vein-cannulated rats (three groups; TMZ alone, letrozole alone and TMZ + letrozole). Intracerebral microdialysis was performed for brain extracellular fluid (ECF) collection simultaneously with venous blood sampling. Drug levels were measured employing HPLC and PK analysis was conducted using Phoenix WinNonlin®. RESULTS: In NSG mice, peak plasma and brain tissue letrozole concentrations (Cmax) were 3-4 and 0.8-0.9 µg/ml, respectively. The elimination half-life was 2.6 h with minimal accumulation following multiple dosing. In the drug interaction study, no PK changes were evident when TMZ and letrozole were given in combination. For instance, peak plasma and brain ECF TMZ levels when given alone were 14.7 ± 1.1 and 4.6 ± 0.6 µg/ml, respectively, and 12.6 ± 2.4 and 3.4 ± 0.8 µg/ml, respectively, when given with letrozole. CONCLUSIONS: These results will guide the optimization of dosing regimen for further development of letrozole for HGG treatment.

Neurofibromatosis 1: closing the GAP between mice and men.

Neurofibromatosis 1 (NF1) is a common genetic condition in which affected individuals are prone to the development of benign and malignant tumors. The NF1 tumor suppressor encodes a protein product, neurofibromin, which functions in part as a negative regulator of RAS. Loss of neurofibromin expression in NF1-associated tumors or Nf1-deficient mouse cells is associated with elevated RAS activity and increased cell proliferation. Despite this straightforward pathophysiologic association between neurofibromin, RAS, and tumorigenesis, recent insights from mouse and Drosophila modeling studies have suggested additional functions for neurofibromin and have implicated Nf1 heterozygosity in tumor formation. Lastly, Nf1 knockout mouse studies have also demonstrated important roles for cooperating genetic changes that accelerate tumorigenesis as well as modifier genes that impact on cancer susceptibility.