CK1δ and CK1ε Signaling Sustains Mitochondrial Metabolism and Cell Survival in Multiple Myeloma.

Multiple myeloma remains an incurable malignancy due to acquisition of intrinsic programs that drive therapy resistance. Here we report that casein kinase-1δ (CK1δ) and CK1ε are therapeutic targets in multiple myeloma that are necessary to sustain mitochondrial metabolism. Specifically, the dual CK1δ/CK1ε inhibitor SR-3029 had potent in vivo and ex vivo anti-multiple myeloma activity, including against primary multiple myeloma patient specimens. RNA sequencing (RNA-seq) and metabolic analyses revealed inhibiting CK1δ/CK1ε disables multiple myeloma metabolism by suppressing genes involved in oxidative phosphorylation (OxPhos), reducing citric acid cycle intermediates, and suppressing complexes I and IV of the electron transport chain. Finally, sensitivity of multiple myeloma patient specimens to SR-3029 correlated with elevated expression of mitochondrial genes, and RNA-seq from 687 multiple myeloma patient samples revealed that increased CSNK1D, CSNK1E, and OxPhos genes correlate with disease progression and inferior outcomes. Thus, increases in mitochondrial metabolism are a hallmark of multiple myeloma progression that can be disabled by targeting CK1δ/CK1ε. SIGNIFICANCE: CK1δ and CK1ε are attractive therapeutic targets in multiple myeloma whose expression increases with disease progression and connote poor outcomes, and that are necessary to sustain expression of genes directing OxPhos.

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.

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon.

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

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.

Metabolic remodeling contributes towards an immune-suppressive phenotype in glioblastoma.

Glioblastoma (GBM) is one of the most aggressive tumors. Numerous studies in the field of immunotherapy have focused their efforts on identifying various pathways linked with tumor-induced immunosuppression. Recent research has demonstrated that metabolic reprogramming in a tumor can contribute towards immune tolerance. To begin to understand the interface between metabolic remodeling and the immune-suppressive state in GBM, we performed a focused, integrative analysis coupling metabolomics with gene-expression profiling in patient-derived GBM (n = 80) and compared them to low-grade astrocytoma (LGA; n = 28). Metabolic intermediates of tryptophan, arginine, prostaglandin, and adenosine emerged as immuno-metabolic nodes in GBM specific to the mesenchymal and classical molecular subtypes of GBM. Integrative analyses emphasized the importance of downstream metabolism of several of these metabolic pathways in GBM. Using CIBERSORT to analyze immune components from the transcriptional profiles of individual tumors, we demonstrated that tryptophan and adenosine metabolism resulted in an accumulation of Tregs and M2 macrophages, respectively, and was recapitulated in mouse models. Furthermore, we extended these findings to preclinical models to determine their potential utility in defining the biologic and/or immunologic consequences of the identified metabolic programs. Collectively, through integrative analysis, we uncovered multifaceted ways by which metabolic reprogramming may contribute towards immune tolerance in GBM, providing the framework for further investigations designed to determine the specific immunologic consequence of these metabolic programs and their therapeutic potential.

Integrative cross-platform analyses identify enhanced heterotrophy as a metabolic hallmark in glioblastoma.

BACKGROUND: Although considerable progress has been made in understanding molecular alterations driving gliomagenesis, the diverse metabolic programs contributing to the aggressive phenotype of glioblastoma remain unclear. The aim of this study was to define and provide molecular context to metabolic reprogramming driving gliomagenesis. METHODS: Integrative cross-platform analyses coupling global metabolomic profiling with genomics in patient-derived glioma (low-grade astrocytoma [LGA; n = 28] and glioblastoma [n = 80]) were performed. Identified programs were then metabolomically, genomically, and functionally evaluated in preclinical models. RESULTS: Clear metabolic programs were identified differentiating LGA from glioblastoma, with aberrant lipid, peptide, and amino acid metabolism representing dominant metabolic nodes associated with malignant transformation. Although the metabolomic profiles of glioblastoma and LGA appeared mutually exclusive, considerable metabolic heterogeneity was observed in glioblastoma. Surprisingly, integrative analyses demonstrated that O6-methylguanine-DNA methyltransferase methylation and isocitrate dehydrogenase mutation status were equally distributed among glioblastoma metabolic profiles. Transcriptional subtypes, on the other hand, tightly clustered by their metabolomic signature, with proneural and mesenchymal tumor profiles being mutually exclusive. Integrating these metabolic phenotypes with gene expression analyses uncovered tightly orchestrated and highly redundant transcriptional programs designed to support the observed metabolic programs by actively importing these biochemical substrates from the microenvironment, contributing to a state of enhanced metabolic heterotrophy. These findings were metabolomically, genomically, and functionally recapitulated in preclinical models. CONCLUSION: Despite disparate molecular pathways driving the progression of glioblastoma, metabolic programs designed to maintain its aggressive phenotype remain conserved. This contributes to a state of enhanced metabolic heterotrophy supporting survival in diverse microenvironments implicit in this malignancy.

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.

Histologically defined intratumoral sequencing uncovers evolutionary cues into conserved molecular events driving gliomagenesis.

BACKGROUND: Glioblastoma represents an archetypal example of a heterogeneous malignancy. To understand the diverse molecular consequences of this complex tumor ecology, we analyzed RNA-seq data generated from commonly identified intratumoral structures in glioblastoma enriched using laser capture microdissection. METHODS: Raw gene-level values of fragments per kilobase of transcript per million reads mapped and the associated clinical data were acquired from the publicly available Ivy Glioblastoma Atlas Project database and analyzed using MetaboAnalyst (v3.0). The database includes gene expression data generated from multiple structural features commonly identified in glioblastoma enriched by laser capture microdissection. RESULTS: We uncovered a relationship between subtype heterogeneity in glioblastoma and its unique tumor microenvironment, with infiltrating cells harboring a proneural signature while the mesenchymal subtype was enriched in perinecrotic regions. When evaluating the tumors' transcriptional profiles in the context of their derived structural regions, there was a relatively small amount of intertumoral heterogeneity in glioblastoma, with individual regions from different tumors clustering tightly together. Analyzing the transcriptional profiles in the context of evolutionary progression identified unique cellular programs associated with specific phases of gliomagenesis. Mediators of cell signaling and cell cycle progression appear to be critical events driving proliferation in the tumor core, while in addition to a multiplex strategy for promoting angiogenesis and/or an immune-tolerant environment, transformation to perinecrotic zones involved global metabolic alterations. CONCLUSION: These findings suggest that intratumoral heterogeneity in glioblastoma is a conserved, predictable consequence to its complex microenvironment, and combinatorial approaches designed to target these unequivocally present tumor biomes may lead to therapeutic gains.

The interplay between metabolic remodeling and immune regulation in glioblastoma.

The fields of tumor metabolism and immune oncology have both independently received considerable attention over the last several years. The majority of research in tumor metabolism has largely focused on the Warburg effect and its resulting biologic consequences, including energy and macromolecule production. However, recent investigations have identified elegant, multifaceted strategies by which alterations in tumor metabolism can also contribute to a potent tolerogenic immune environment. One of the most notable is increased tryptophan metabolism through activation of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO). However, this pathway represents one of numerous metabolic pathways that may modulate the immune system. For example, metabolites associated with aerobic glycolysis, adenosine, arginine, and prostaglandin metabolism have all been implicated in cancer-mediated immune tolerance and represent attractive therapeutic targets. In this review, we will provide an overview of the emerging interface between these 2 timely areas of cancer research and provide an overview of strategies currently being tested to target these next-generation metabolic immune checkpoints.

Ras-mediated modulation of pyruvate dehydrogenase activity regulates mitochondrial reserve capacity and contributes to glioblastoma tumorigenesis.

BACKGROUND: Even though altered metabolism representing a hallmark of cancer was proposed nearly a century ago, recent technological advances have allowed investigators to continue uncovering a previously unrecognized complexity of metabolic programs that drive tumorigenesis beyond that of aerobic glycolysis. METHODS: The bioenergetic state of a diverse panel of glioblastoma models, including isogenic lines derived from a genetically engineered adult astrocytic mouse model and patient-derived glioblastoma stem cells, was determined at baseline and in stressed conditions. Mechanisms contributing to the discovered metabolic phenotypes were determined through molecular and chemical perturbation, and their biological consequences were evaluated in vivo and in patient samples. RESULTS: Attenuated mitochondrial reserve capacity was identified as a common metabolic phenotype in glioblastoma lines. This phenotype was linked mechanistically with the capacity of Ras-mediated signaling to inhibit pyruvate dehydrogenase (PDH) activity through downregulation of PDH phosphatase (PDP) expression. PDP1 repression was validated clinically in patient-derived samples, suggesting that aberrant cellular signaling typical of glioblastoma actively modulates PDH activity. This phenotype was reversed through both chemical and molecular perturbation. Restoration of PDH activity through stable expression of PDP1-impaired tumorigenic potential. CONCLUSIONS: These findings support the central role that PDH regulation plays as a downstream consequence of aberrant signaling associated with gliomagenesis and the scientific rationale to continue to develop and test clinical strategies designed to activate PDH as a form of anticancer therapy in glioblastoma.

Cysteine catabolism: a novel metabolic pathway contributing to glioblastoma growth.

The relevance of cysteine metabolism in cancer has gained considerable interest in recent years, largely focusing on its role in generating the antioxidant glutathione. Through metabolomic profiling using a combination of high-throughput liquid and gas chromatography-based mass spectrometry on a total of 69 patient-derived glioma specimens, this report documents the discovery of a parallel pathway involving cysteine catabolism that results in the accumulation of cysteine sulfinic acid (CSA) in glioblastoma. These studies identified CSA to rank as one of the top metabolites differentiating glioblastoma from low-grade glioma. There was strong intratumoral concordance of CSA levels with expression of its biosynthetic enzyme cysteine dioxygenase 1 (CDO1). Studies designed to determine the biologic consequence of this metabolic pathway identified its capacity to inhibit oxidative phosphorylation in glioblastoma cells, which was determined by decreased cellular respiration, decreased ATP production, and increased mitochondrial membrane potential following pathway activation. CSA-induced attenuation of oxidative phosphorylation was attributed to inhibition of the regulatory enzyme pyruvate dehydrogenase. Studies performed in vivo abrogating the CDO1/CSA axis using a lentiviral-mediated short hairpin RNA approach resulted in significant tumor growth inhibition in a glioblastoma mouse model, supporting the potential for this metabolic pathway to serve as a therapeutic target. Collectively, we identified a novel, targetable metabolic pathway involving cysteine catabolism contributing to the growth of aggressive high-grade gliomas. These findings serve as a framework for future investigations designed to more comprehensively determine the clinical application of this metabolic pathway and its contributory role in tumorigenesis.

The metabolomic signature of malignant glioma reflects accelerated anabolic metabolism.

Although considerable progress has been made toward understanding glioblastoma biology through large-scale genetic and protein expression analyses, little is known about the underlying metabolic alterations promoting their aggressive phenotype. We conducted global metabolomic profiling on patient-derived glioma specimens and identified specific metabolic programs differentiating low- and high-grade tumors, with the metabolic signature of glioblastoma reflecting accelerated anabolic metabolism. When coupled with transcriptional profiles, we identified the metabolic phenotype of the mesenchymal subtype to consist of accumulation of the glycolytic intermediate phosphoenolpyruvate and decreased pyruvate kinase activity. Unbiased hierarchical clustering of metabolomic profiles identified three subclasses, which we term energetic, anabolic, and phospholipid catabolism with prognostic relevance. These studies represent the first global metabolomic profiling of glioma, offering a previously undescribed window into their metabolic heterogeneity, and provide the requisite framework for strategies designed to target metabolism in this rapidly fatal malignancy.

Class I histone deacetylases localize to the endoplasmic reticulum and modulate the unfolded protein response.

Post-translational modification through protein acetylation is emerging as an important mode of cellular regulation. We have previously demonstrated the role that glucose-regulated protein 78 kDa (GRP78) acetylation and subsequent activation of the unfolded protein response (UPR) play in the antitumor activity of class I histone deacetylase (HDAC) inhibitors, which primarily target class I HDACs. In this study, we explored the contributory role these class I HDACs may play in UPR regulation. Binding studies were performed using immunoprecipitation/immunoblotting following dual-transfection with HA-tagged GRP78 and FLAG-tagged HDACs. Subcellular localization was performed using Western blot of fractionated cell lysates and confocal microscopy. Individual HDACs were inhibited using RNA interference. We identified the potential of HDACs 1, 2, and 3 to bind to GRP78. These HDACs colocalized with GRP78 in the endoplasmic reticulum (ER). Inhibition of individual HDACs resulted in GRP78 acetylation and selective activation of the UPR. Although traditionally viewed as nuclear enzymes, we demonstrate that Class I HDACs localize to the ER, bind to GRP78, and selectively activate the UPR, representing a novel mode of UPR regulation and mechanism of action of HDAC inhibitors.

Targeting the unfolded protein response in glioblastoma cells with the fusion protein EGF-SubA.

Rapidly growing tumors require efficient means to allow them to adapt to fluctuating microenvironments consisting of hypoxia, nutrient deprivation, and acidosis. The unfolded protein response (UPR) represents a defense mechanism allowing cells to respond to these adverse conditions. The chaperone protein GRP78 serves as a master UPR regulator that is aberrantly expressed in a variety of cancers, including glioma. Therefore, cancer cells may be particularly reliant upon the adaptive mechanisms offered by the UPR and targeting GRP78 may represent a unique therapeutic strategy. Here we report that diffuse expression of GRP78 protein is present in Grade III-IV, but not Grade I-II glioma. To determine the role GRP78 plays in glioblastoma tumorigenesis, we explored the anti-tumor activity of the novel fusion protein EGF-SubA, which combines EGF with the cytotoxin SubA that has been recently shown to selectively cleave GRP78. EGF-SubA demonstrated potent tumor-specific proteolytic activity and cytotoxicity in glioblastoma lines and potentiated the anti-tumor activity of both temozolomide and ionizing radiation. To determine if the tumor microenvironment influences EGF-SubA activity, we maintained cells in acidic conditions that led to both UPR activation and increased EGF-SubA induced cytotoxicity. EGF-SubA was well tolerated in mice and led to a significant tumor growth delay in a glioma xenograft mouse model. The UPR is emerging as an important adaptive pathway contributing to glioma tumorigenesis. Targeting its primary mediator, the chaperone protein GRP78, through specific, proteolytic cleavage with the immunotoxin EGF-SubA represents a novel and promising multi-targeted approach to cancer therapy.

Phase I trial of vorinostat combined with bevacizumab and CPT-11 in recurrent glioblastoma.

A phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of vorinostat with bevacizumab and CPT-11 in recurrent glioblastoma. Vorinostat was combined with bevacizumab and CPT-11 and was escalated using a standard 3 + 3 design. Vorinostat was escalated up to 2 actively investigated doses of this compound or until the MTD was identified on the basis of DLTs. Correlative science involving proteomic profiling of serial patient plasma samples was performed. Nineteen patients were treated. The MTD of vorinostat was established at 400 mg on days 1-7 and 15-21 every 28 days when combined with bevacizumab and CPT-11. Common toxicities were fatigue and diarrhea. DLTs included fatigue, hypertension/hypotension, and central nervous system ischemia. Although the MTD was established, CPT-11 dose reductions were common early in therapy. High-dose vorinostat had an improved progression-free survival and overall survival when compared with low-dose vorinostat. Serum proteomic profiling identified IGFBP-5 and PDGF-AA as markers for improved PFS and recurrence, respectively. A MTD for the combination of vorinostat with bevacizumab and CPT-11 has been established, although it has poor long-term tolerability. With the increased toxicities associated with CPT-11 coupled with its unclear clinical significance, investigating the efficacy of vorinostat combined with bevacizumab alone may represent a more promising strategy to evaluate in the context of a phase II clinical trial.

Targeting radiation-induced G(2) checkpoint activation with the Wee-1 inhibitor MK-1775 in glioblastoma cell lines.

The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G(2) checkpoint arrest and modulate radiosensitivity in glioblastoma cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G(2) checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G(2) checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells.