Quinolinate promotes macrophage-induced immune tolerance in glioblastoma through the NMDAR/PPARγ signaling axis.

There has been considerable scientific effort dedicated to understanding the biologic consequence and therapeutic implications of aberrant tryptophan metabolism in brain tumors and neurodegenerative diseases. A majority of this work has focused on the upstream metabolism of tryptophan; however, this has resulted in limited clinical application. Using global metabolomic profiling of patient-derived brain tumors, we identify the downstream metabolism of tryptophan and accumulation of quinolinate (QA) as a metabolic node in glioblastoma and demonstrate its critical role in promoting immune tolerance. QA acts as a metabolic checkpoint in glioblastoma by inducing NMDA receptor activation and Foxo1/PPARγ signaling in macrophages, resulting in a tumor supportive phenotype. Using a genetically-engineered mouse model designed to inhibit production of QA, we identify kynureninase as a promising therapeutic target to revert the potent immune suppressive microenvironment in glioblastoma. These findings offer an opportunity to revisit the biologic consequence of this pathway as it relates to oncogenesis and neurodegenerative disease and a framework for developing immune modulatory agents to further clinical gains in these otherwise incurable diseases.

Intratumoural haematopoietic stem and progenitor cell differentiation into M2 macrophages facilitates the regrowth of solid tumours after radiation therapy.

BACKGROUND: Bone-marrow-derived haematopoietic stem and progenitor cells (HSPCs) are a prominent part of the highly complex tumour microenvironment (TME) where they localise within tumours and maintain haematopoietic potency. Understanding the role HSPCs play in tumour growth and response to radiation therapy (RT) may lead to improved patient treatments and outcomes. METHODS: We used a mouse model of non-small cell lung carcinoma where tumours were exposed to RT regimens alone or in combination with GW2580, a pharmacological inhibitor of colony stimulating factor (CSF)-1 receptor. RT-PCR, western blotting and immunohistochemistry were used to quantify expression levels of factors that affect HSPC differentiation. DsRed+ HSPC intratumoural activity was tracked using flow cytometry and confocal microscopy. RESULTS: We demonstrated that CSF-1 is enhanced in the TME following exposure to RT. CSF-1 signaling induced intratumoural HSPC differentiation into M2 polarised tumour-associated macrophages (TAMs), aiding in post-RT tumour survival and regrowth. In contrast, hyperfractionated/pulsed radiation therapy (PRT) and GW2580 ablated this process resulting in improved tumour killing and mouse survival. CONCLUSIONS: Tumours coopt intratumoural HSPC fate determination via CSF-1 signaling to overcome the effects of RT. Thus, limiting intratumoural HSPC activity represents an attractive strategy for improving the clinical treatment of solid tumours.

Dacomitinib and gedatolisib in combination with fractionated radiation in head and neck cancer.

BACKGROUND AND PURPOSE: There has been little success targeting individual genes in combination with radiation in head and neck cancer. In this study we investigated whether targeting two key pathways simultaneously might be more effective. MATERIALS AND METHODS: We studied the effect of combining dacomitinib (pan-HER, irreversible inhibitor) and gedatolisib (dual PI3K/MTOR inhibitor) with radiation in well characterized, low passage xenograft models of HNSCC in vitro and in vivo. RESULTS: Dacomitinib showed differential growth inhibition in vitro that correlated to EGFR expression whilst gedatolisib was effective in both cell lines. Neither agent radiosensitized the cell lines in vitro. In vivo studies demonstrated that dacomitinib was an effective agent alone and in combination with radiation whilst the addition of gedatolisib did not enhance the effect of these two modalities despite inhibiting phosphorylation of key genes in the PI3K/MTOR pathway. CONCLUSIONS: Our results showed that combining two drugs with radiation provided no added benefit compared to the single most active drug. Dacomitinib deserves more investigation as a radiation sensitizing agent in HNSCC.

Perhexiline Demonstrates FYN-mediated Antitumor Activity in Glioblastoma.

Glioblastoma is the most common primary malignant brain tumor in adults. Despite aggressive treatment, outcomes remain poor with few long-term survivors. Therefore, considerable effort is being made to identify novel therapies for this malignancy. Targeting tumor metabolism represents a promising therapeutic strategy and activation of fatty acid oxidation (FAO) has been identified as a central metabolic node contributing toward gliomagenesis. Perhexiline is a compound with a long clinical track record in angina treatment and commonly described as an FAO inhibitor. We therefore sought to determine whether this compound might be repurposed to serve as a novel therapy in glioblastoma. Perhexiline demonstrated potent in vitro cytotoxicity, induction of redox stress and apoptosis in a panel of glioblastoma cell lines. However, the antitumor activity of perhexiline was distinct when compared with the established FAO inhibitor etomoxir. By evaluating mitochondrial respiration and lipid dynamics in glioblastoma cells following treatment with perhexiline, we confirmed this compound did not inhibit FAO in our models. Using in silico approaches, we identified FYN as a probable target of perhexiline and validated the role of this protein in perhexiline sensitivity. We extended studies to patient samples, validating the potential of FYN to serve as therapeutic target in glioma. When evaluated in vivo, perhexiline demonstrated the capacity to cross the blood-brain barrier and antitumor activity in both flank and orthotopic glioblastoma models. Collectively, we identified potent FYN-dependent antitumor activity of perhexiline in glioblastoma, thereby, representing a promising agent to be repurposed for the treatment of this devastating malignancy.

Enhanced fatty acid oxidation provides glioblastoma cells metabolic plasticity to accommodate to its dynamic nutrient microenvironment.

Despite advances in molecularly characterizing glioblastoma (GBM), metabolic alterations driving its aggressive phenotype are only beginning to be recognized. Integrative cross-platform analysis coupling global metabolomic and gene expression profiling on patient-derived glioma identified fatty acid ß-oxidation (FAO) as a metabolic node in GBM. We determined that the biologic consequence of enhanced FAO is directly dependent upon tumor microenvironment. FAO serves as a metabolic cue to drive proliferation in a ß-HB/GPR109A dependent autocrine manner in nutrient favorable conditions, while providing an efficient, alternate source of ATP only in nutrient unfavorable conditions. Rational combinatorial strategies designed to target these dynamic roles FAO plays in gliomagenesis resulted in necroptosis-mediated metabolic synthetic lethality in GBM. In summary, we identified FAO as a dominant metabolic node in GBM that provides metabolic plasticity, allowing these cells to adapt to their dynamic microenvironment. Combinatorial strategies designed to target these diverse roles FAO plays in gliomagenesis offers therapeutic potential in GBM.

Low Dose Brain Irradiation Reduces Amyloid-ß and Tau in 3xTg-AD Mice.

We have previously reported that low doses of external beam ionizing irradiation reduced amyloid-ß (Aß) plaques and improved cognition in APP/PS1 mice. In this study we investigated the effects of radiation in an age-matched series of 3xTg-AD mice. Mice were hemibrain-irradiated with 5 fractions of 2 Gy and sacrificed 8 weeks after the end of treatment. Aß and tau were assessed using immunohistochemistry and quantified using image analysis with Definiens Tissue Studio. We observed a significant reduction in Aß plaque burden and tau staining; these two parameters were significantly correlated. This preliminary data is further support that low doses of radiation may be beneficial in Alzheimer's disease.

Amelioration of Mucositis in Proton Therapy of Fanconi Anemia Fanca-/- Mice by JP4-039.

BACKGROUND/AIM: We tested JP4-039, a GS-nitroxide radiation damage mitigator in proton therapy of Fanconi anemia (FA) mice. MATERIALS AND METHODS: Fanca-/- and Fanca+/+ bone marrow stromal cells were pre-treated with JP4-039 and irradiated with either protons or photons (0-10 GyRBE) followed by clonogenic survival and ß-Galactosidase senescence analysis. Fanca-/- and Fanca+/+ mice were pretreated with JP4-039 for 10 min prior to oropharyngeal irradiation with either protons or photons (0 or 30 GyRBE) followed by sacrifice and measurement of oral cavity ulceration, distant hematopoietic suppression, and real-time polymerase chain reaction analysis. RESULTS: JP4-039 reduced oral cavity ulceration in Fanca-/- mice, transcripts Nfkb, Ap1, Sp1, and Nrf2, and proton therapy induced distant marrow suppression. CONCLUSION: JP4-039 protected Fanca-/- and Fanca+/+ cells and mouse oral cavity from both proton and photon radiation.

Tryptophan Metabolism Contributes to Radiation-Induced Immune Checkpoint Reactivation in Glioblastoma.

Purpose: Immune checkpoint inhibitors designed to revert tumor-induced immunosuppression have emerged as potent anticancer therapies. Tryptophan metabolism represents an immune checkpoint, and targeting this pathway's rate-limiting enzyme IDO1 is actively being investigated clinically. Here, we studied the intermediary metabolism of tryptophan metabolism in glioblastoma and evaluated the activity of the IDO1 inhibitor GDC-0919, both alone and in combination with radiation (RT).Experimental Design: LC/GC-MS and expression profiling was performed for metabolomic and genomic analyses of patient-derived glioma. Immunocompetent mice were injected orthotopically with genetically engineered murine glioma cells and treated with GDC-0919 alone or combined with RT. Flow cytometry was performed on isolated tumors to determine immune consequences of individual treatments.Results: Integrated cross-platform analyses coupling global metabolomic and gene expression profiling identified aberrant tryptophan metabolism as a metabolic node specific to the mesenchymal and classical subtypes of glioblastoma. GDC-0919 demonstrated potent inhibition of this node and effectively crossed the blood-brain barrier. Although GDC-0919 as a single agent did not demonstrate antitumor activity, it had a strong potential for enhancing RT response in glioblastoma, which was further augmented with a hypofractionated regimen. RT response in glioblastoma involves immune stimulation, reflected by increases in activated and cytotoxic T cells, which was balanced by immune checkpoint reactivation, reflected by an increase in IDO1 expression and regulatory T cells (Treg). GDC-0919 mitigated RT-induced Tregs and enhanced T-cell activation.Conclusions: Tryptophan metabolism represents a metabolic node in glioblastoma, and combining RT with IDO1 inhibition enhances therapeutic response by mitigating RT-induced immunosuppression. Clin Cancer Res; 24(15); 3632-43. ©2018 AACR.

Recruitment of histone methyltransferase G9a mediates transcriptional repression of Fgf21 gene by E4BP4 protein.

The liver responds to fasting-refeeding cycles by reprogramming expression of metabolic genes. Fasting potently induces one of the key hepatic hormones, fibroblast growth factor 21 (FGF21), to promote lipolysis, fatty acid oxidation, and ketogenesis, whereas refeeding suppresses its expression. We previously reported that the basic leucine zipper transcription factor E4BP4 (E4 binding protein 4) represses Fgf21 expression and disrupts its circadian oscillations in cultured hepatocytes. However, the epigenetic mechanism for E4BP4-dependent suppression of Fgf21 has not yet been addressed. Here we present evidence that histone methyltransferase G9a mediates E4BP4-dependent repression of Fgf21 during refeeding by promoting repressive histone modification. We find that Fgf21 expression is up-regulated in E4bp4 knock-out mouse liver. We demonstrate that the G9a-specific inhibitor BIX01294 abolishes suppression of the Fgf21 promoter activity by E4BP4, whereas overexpression of E4bp4 leads to increased levels of dimethylation of histone 3 lysine 9 (H3K9me2) around the Fgf21 promoter region. Furthermore, we also show that E4BP4 interacts with G9a, and knockdown of G9a blocks repression of Fgf21 promoter activity and expression in cells overexpressing E4bp4. A G9a mutant lacking catalytic activity, due to deletion of the SET domain, fails to inhibit the Fgf21 promoter activity. Importantly, acute hepatic knockdown by adenoviral shRNA targeting G9a abolishes Fgf21 repression by refeeding, concomitant with decreased levels of H3K9me2 around the Fgf21 promoter region. In summary, we show that G9a mediates E4BP4-dependent suppression of hepatic Fgf21 by enhancing histone methylation (H3K9me2) of the Fgf21 promoter.

Peroxisomal localization and circadian regulation of ubiquitin-specific protease 2.

Temporal regulation of nutrient and energy metabolism is emerging as an important aspect of metabolic homeostasis. The regulatory network that integrates the timing cues and nutritional signals to drive diurnal metabolic rhythms remains poorly defined. The 45-kDa isoform of ubiquitin-specific protease 2 (USP2-45) is a deubiquitinase that regulates hepatic gluconeogenesis and glucose metabolism. In this study, we found that USP2-45 is localized to peroxisomes in hepatocytes through a canonical peroxisome-targeting motif at its C-terminus. Clustering analysis indicates that the expression of a subset of peroxisomal genes exhibits robust diurnal rhythm in the liver. Despite this, nuclear hormone receptor PPARα, a known regulator of peroxisome gene expression, does not induce USP2-45 in hepatocytes and is dispensible for its expression during starvation. In contrast, a functional liver clock is required for the proper nutritional and circadian regulation of USP2-45 expression. At the molecular level, transcriptional coactivators PGC-1α and PGC-1ß and repressor E4BP4 exert opposing effects on USP2-45 promoter activity. These studies provide insights into the subcellular localization and transcriptional regulation of a clock-controlled deubiquitinase that regulates glucose metabolism.

USP2a protein deubiquitinates and stabilizes the circadian protein CRY1 in response to inflammatory signals.

The mammalian circadian clock coordinates various physiological activities with environmental cues to achieve optimal adaptation. The clock manifests oscillations of key clock proteins, which are under dynamic control at multiple post-translational levels. As a major post-translational regulator, the ubiquitination-dependent proteasome degradation system is counterbalanced by a large group of deubiquitin proteases with distinct substrate preference. Until now, whether deubiquitination by ubiquitin-specific proteases can regulate the clock protein stability and circadian pathways remains largely unclear. The mammalian clock protein, cryptochrome 1 (CRY1), is degraded via the FBXL3-mediated ubiquitination pathway, suggesting that it is also likely to be targeted by the deubiquitination pathway. Here, we identified that USP2a, a circadian-controlled deubiquitinating enzyme, interacts with CRY1 and enhances its protein stability via deubiquitination upon serum shock. Depletion of Usp2a by shRNA greatly enhances the ubiquitination of CRY1 and dampens the oscillation amplitude of the CRY1 protein during a circadian cycle. By stabilizing the CRY1 protein, USP2a represses the Per2 promoter activity as well as the endogenous Per2 gene expression. We also demonstrated that USP2a-dependent deubiquitination and stabilization of the CRY1 protein occur in the mouse liver. Interestingly, the pro-inflammatory cytokine, TNF-α, increases the CRY1 protein level and inhibits circadian gene expression in a USP2a-dependent fashion. Therefore, USP2a potentially mediates circadian disruption by suppressing the CRY1 degradation during inflammation.