Interferon-gamma contributes to disease progression in the Ndufs4(-/-) model of Leigh syndrome.

AIM: Leigh syndrome (LS), the most common paediatric presentation of genetic mitochondrial dysfunction, is a multi-system disorder characterised by severe neurologic and metabolic abnormalities. Symmetric, bilateral, progressive necrotizing lesions in the brainstem are defining features of the disease. Patients are often symptom free in early life but typically develop symptoms by about 2 years of age. The mechanisms underlying disease onset and progression in LS remain obscure. Recent studies have shown that the immune system causally drives disease in the Ndufs4(-/-) mouse model of LS: treatment of Ndufs4(-/-) mice with the macrophage-depleting Csf1r inhibitor pexidartinib prevents disease. While the precise mechanisms leading to immune activation and immune factors involved in disease progression have not yet been determined, interferon-gamma (IFNγ) and interferon gamma-induced protein 10 (IP10) were found to be significantly elevated in Ndufs4(-/-) brainstem, implicating these factors in disease. Here, we aimed to explore the role of IFNγ and IP10 in LS. METHODS: To establish the role of IFNγ and IP10 in LS, we generated IFNγ and IP10 deficient Ndufs4(-/-)/Ifng(-/-) and Ndufs4(-/-)/IP10(-/-) double knockout animals, as well as IFNγ and IP10 heterozygous, Ndufs4(-/-)/Ifng(+/-) and Ndufs4(-/-)/IP10(+/-), animals. We monitored disease onset and progression to define the impact of heterozygous or homozygous loss of IFNγ and IP10 in LS. RESULTS: Loss of IP10 does not significantly impact the onset or progression of disease in the Ndufs4(-/-) model. IFNγ loss significantly extends survival and delays disease progression in a gene dosage-dependent manner, though the benefits are modest compared to Csf1r inhibition. CONCLUSIONS: IFNγ contributes to disease onset and progression in LS. Our findings suggest that IFNγ targeting therapies may provide some benefits in genetic mitochondrial disease, but targeting IFNγ alone would likely yield only modest benefits in LS.

Volatile anaesthetic toxicity in the genetic mitochondrial disease Leigh syndrome.

BACKGROUND: Volatile anaesthetics are widely used in human medicine. Although generally safe, hypersensitivity and toxicity can occur in rare cases, such as in certain genetic disorders. Anaesthesia hypersensitivity is well-documented in a subset of mitochondrial diseases, but whether volatile anaesthetics are toxic in this setting has not been explored. METHODS: We exposed Ndufs4(-/-) mice, a model of Leigh syndrome, to isoflurane (0.2-0.6%), oxygen 100%, or air. Cardiorespiratory function, weight, blood metabolites, and survival were assessed. We exposed post-symptom onset and pre-symptom onset animals and animals treated with the macrophage depleting drug PLX3397/pexidartinib to define the role of overt neuroinflammation in volatile anaesthetic toxicities. RESULTS: Isoflurane induced hyperlactataemia, weight loss, and mortality in a concentration- and duration-dependent manner from 0.2% to 0.6% compared with carrier gas (O2 100%) or mock (air) exposures (lifespan after 30-min exposures ∗P<0.05 for isoflurane 0.4% vs air or vs O2, ∗∗P<0.005 for isoflurane 0.6% vs air or O2; 60-min exposures ∗∗P<0.005 for isoflurane 0.2% vs air, ∗P<0.05 for isoflurane 0.2% vs O2). Isoflurane toxicity was significantly reduced in Ndufs4(-/-) exposed before CNS disease onset, and the macrophage depleting drug pexidartinib attenuated sequelae of isoflurane toxicity (survival ∗∗∗P=0.0008 isoflurane 0.4% vs pexidartinib plus isoflurane 0.4%). Finally, the laboratory animal standard of care of 100% O2 as a carrier gas contributed significantly to weight loss and reduced survival, but not to metabolic changes, and increased acute mortality. CONCLUSIONS: Isoflurane is toxic in the Ndufs4(-/-) model of Leigh syndrome. Toxic effects are dependent on the status of underlying neurologic disease, largely prevented by the CSF1R inhibitor pexidartinib, and influenced by oxygen concentration in the carrier gas.

Peripheral macrophages drive CNS disease in the Ndufs4(-/-) model of Leigh syndrome.

Subacute necrotizing encephalopathy, or Leigh syndrome (LS), is the most common pediatric presentation of genetic mitochondrial disease. LS is a multi-system disorder with severe neurologic, metabolic, and musculoskeletal symptoms. The presence of progressive, symmetric, and necrotizing lesions in the brainstem are a defining feature of the disease, and the major cause of morbidity and mortality, but the mechanisms underlying their pathogenesis have been elusive. Recently, we demonstrated that high-dose pexidartinib, a CSF1R inhibitor, prevents LS CNS lesions and systemic disease in the Ndufs4(-/-) mouse model of LS. While the dose-response in this study implicated peripheral immune cells, the immune populations involved have not yet been elucidated. Here, we used a targeted genetic tool, deletion of the colony-stimulating Factor 1 receptor (CSF1R) macrophage super-enhancer FIRE (Csf1rΔFIRE), to specifically deplete microglia and define the role of microglia in the pathogenesis of LS. Homozygosity for the Csf1rΔFIRE allele ablates microglia in both control and Ndufs4(-/-) animals, but onset of CNS lesions and sequalae in the Ndufs4(-/-), including mortality, are only marginally impacted by microglia depletion. The overall development of necrotizing CNS lesions is not altered, though microglia remain absent. Finally, histologic analysis of brainstem lesions provides direct evidence of a causal role for peripheral macrophages in the characteristic CNS lesions. These data demonstrate that peripheral macrophages play a key role in the pathogenesis of disease in the Ndufs4(-/-) model.

AAV-vector based gene therapy for mitochondrial disease: progress and future perspectives.

Mitochondrial diseases are a group of rare, heterogeneous diseases caused by gene mutations in both nuclear and mitochondrial genomes that result in defects in mitochondrial function. They are responsible for significant morbidity and mortality as they affect multiple organ systems and particularly those with high energy-utilizing tissues, such as the nervous system, skeletal muscle, and cardiac muscle. Virtually no effective treatments exist for these patients, despite the urgent need. As the majority of these conditions are monogenic and caused by mutations in nuclear genes, gene replacement is a highly attractive therapeutic strategy. Adeno-associated virus (AAV) is a well-characterized gene replacement vector, and its safety profile and ability to transduce quiescent cells nominates it as a potential gene therapy vehicle for several mitochondrial diseases. Indeed, AAV vector-based gene replacement is currently being explored in clinical trials for one mitochondrial disease (Leber hereditary optic neuropathy) and preclinical studies have been published investigating this strategy in other mitochondrial diseases. This review summarizes the preclinical findings of AAV vector-based gene replacement therapy for mitochondrial diseases including Leigh syndrome, Barth syndrome, ethylmalonic encephalopathy, and others.

Comprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine Pathways.

Reprograming of cellular metabolism is a hallmark of cancer. Altering metabolism allows cancer cells to overcome unfavorable microenvironment conditions and to proliferate and invade. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC defines a subset of poor-prognosis medulloblastoma. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in three different conditions-in vitro, in flank xenografts and in orthotopic xenografts in the cerebellum. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and in vitro MYC-amplified cells. Compared to normal brain, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of the TCA cycle as well as the synthesis of nucleotides, hexosamines, amino acids and glutathione. There was significantly higher glucose uptake and usage in orthotopic xenograft tumors compared to flank xenograft tumors and cells in culture. In orthotopic tumors, glucose was the main carbon source for the de novo synthesis of glutamate, glutamine and glutathione through the TCA cycle. In vivo, the glutaminase II pathway was the main pathway utilizing glutamine. Glutathione was the most abundant upregulated metabolite in orthotopic tumors compared to normal brain. Glutamine-derived glutathione was synthesized through the glutamine transaminase K (GTK) enzyme in vivo. In conclusion, high MYC medulloblastoma cells have different metabolic profiles in vitro compared to in vivo, and key vulnerabilities may be missed by not performing in vivo metabolic analyses.

TORC1/2 kinase inhibition depletes glutathione and synergizes with carboplatin to suppress the growth of MYC-driven medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. An in silico analysis of these cells identified mTOR inhibitors as potential therapeutic agents. We hypothesized that the orally bioavailable TORC1/2 kinase inhibitor TAK228 would have activity against MYC-driven medulloblastoma. TAK228 inhibited mTORC1/2, decreased cell growth and caused apoptosis in high-MYC medulloblastoma cell lines. Comprehensive metabolic profiling of medulloblastoma orthotopic xenografts showed upregulation of glutathione compared to matched normal brain. TAK228 suppressed glutathione production. Because glutathione is required to detoxify platinum-containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin in medulloblastoma. TAK228 synergized with carboplatin to inhibit cell growth and induce apoptosis and extended survival in orthotopic xenografts of high-MYC medulloblastoma. Brain-penetrant TORC1/2 inhibitors and carboplatin may be an effective combination therapy for high-risk medulloblastoma.

Orally bioavailable glutamine antagonist prodrug JHU-083 penetrates mouse brain and suppresses the growth of MYC-driven medulloblastoma.

A subset of poor-prognosis medulloblastoma has genomic amplification of MYC. MYC regulates glutamine metabolism in multiple cellular contexts. We modified the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) to mask its carboxylate and amine functionalities, creating a prodrug termed JHU-083 with increased oral bioavailability. We hypothesized that this prodrug would kill MYC-expressing medulloblastoma. JHU-083 treatment caused decreased growth and increased apoptosis in human MYC-expressing medulloblastoma cell lines. We generated a mouse MYC-driven medulloblastoma model by transforming C57BL/6 mouse cerebellar stem and progenitor cells. When implanted into the brains of C57BL/6 mice, these cells formed large cell/anaplastic tumors that resembled aggressive medulloblastoma. A cell line derived from this model was sensitive to JHU-083 in vitro. Oral administration of JHU-038 led to the accumulation of micromolar concentrations of DON in the mouse brain. JHU-083 treatment significantly increased the survival of immune-competent animals bearing orthotopic tumors formed by the mouse cerebellar stem cell model as well as immune-deficient animals bearing orthotopic tumors formed by a human MYC-amplified medulloblastoma cell line. These data provide pre-clinical justification for the ongoing development and testing of orally bioavailable DON prodrugs for use in medulloblastoma patients.

Unbiased Metabolic Profiling Predicts Sensitivity of High MYC-Expressing Atypical Teratoid/Rhabdoid Tumors to Glutamine Inhibition with 6-Diazo-5-Oxo-L-Norleucine.

PURPOSE: Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the MYC proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT. EXPERIMENTAL DESIGN: Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT. RESULTS: Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT. CONCLUSIONS: Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.

DiSCoVERing Innovative Therapies for Rare Tumors: Combining Genetically Accurate Disease Models with In Silico Analysis to Identify Novel Therapeutic Targets.

PURPOSE: We used human stem and progenitor cells to develop a genetically accurate novel model of MYC-driven Group 3 medulloblastoma. We also developed a new informatics method, Disease-model Signature versus Compound-Variety Enriched Response ("DiSCoVER"), to identify novel therapeutics that target this specific disease subtype. EXPERIMENTAL DESIGN: Human neural stem and progenitor cells derived from the cerebellar anlage were transduced with oncogenic elements associated with aggressive medulloblastoma. An in silico analysis method for screening drug sensitivity databases (DiSCoVER) was used in multiple drug sensitivity datasets. We validated the top hits from this analysis in vitro and in vivo RESULTS: Human neural stem and progenitor cells transformed with c-MYC, dominant-negative p53, constitutively active AKT and hTERT formed tumors in mice that recapitulated Group 3 medulloblastoma in terms of pathology and expression profile. DiSCoVER analysis predicted that aggressive MYC-driven Group 3 medulloblastoma would be sensitive to cyclin-dependent kinase (CDK) inhibitors. The CDK 4/6 inhibitor palbociclib decreased proliferation, increased apoptosis, and significantly extended the survival of mice with orthotopic medulloblastoma xenografts. CONCLUSIONS: We present a new method to generate genetically accurate models of rare tumors, and a companion computational methodology to find therapeutic interventions that target them. We validated our human neural stem cell model of MYC-driven Group 3 medulloblastoma and showed that CDK 4/6 inhibitors are active against this subgroup. Our results suggest that palbociclib is a potential effective treatment for poor prognosis MYC-driven Group 3 medulloblastoma tumors in carefully selected patients. Clin Cancer Res; 22(15); 3903-14. ©2016 AACR.