Clinical Findings and Outcome in 30 Dogs with Presumptive or Confirmed Nerve Sheath Tumors.

Nerve sheath tumors (NSTs) are well-recognized primary nervous system tumors, but there is relatively limited information in dogs including comparison of NSTs in different anatomical locations. This retrospective study describes the clinical features and outcomes in a group of dogs with NSTs affecting the cranial nerves or spinal nerves. Thirty dogs were included, 25 with a presumptive diagnosis and five confirmed by histopathologic analysis. Seven dogs also had cytology of tumor samples, which were supportive of the NST diagnosis in four. Eight dogs had cranial nerve-associated NSTs, with six involving the trigeminal nerve. Twenty-two dogs had spinal nerve-associated NSTs including 13 invading the spinal canal and nine peripheral to the spinal canal, with the majority affecting nerves or nerve roots of the brachial plexus. The prognosis was poor, with dogs being euthanized eventually because of disease progression. Among dogs alive 1 week after diagnosis, the median survival time was 4 months but ranged from 2 weeks to >2 years. While there was a broad overlap between NST locations, survival was generally longer for dogs without spinal canal or intracranial involvement. The results expand available information on NSTs in dogs but should be interpreted with caution given the small number of dogs with a definitive diagnosis. Further investigation is warranted to determine how tumor location, invasiveness, and treatments pursued impact outcome.

Oncogenic Control of Metabolism.

A cell committed to proliferation must reshape its metabolism to enable robust yet balanced production of building blocks for the assembly of proteins, lipids, nucleic acids, and other macromolecules, from which two functional daughter cells can be produced. The metabolic remodeling associated with proliferation is orchestrated by a number of pro-proliferative signaling nodes, which include phosphatidylinositol-3 kinase (PI3K), the RAS family of small GTPases, and transcription factor c-myc In metazoan cells, these signals are activated in a paracrine manner via growth factor-mediated activation of receptor (or receptor-associated) tyrosine kinases. Such stimuli are limited in duration and therefore allow the metabolism of target cells to return to the resting state once the proliferation demands have been satisfied. Cancer cells acquire activating genetic alterations within common pro-proliferative signaling nodes. These alterations lock cellular nutrient uptake and utilization into a perpetual progrowth state, leading to the aberrant accumulation and spread of cancer cells.

Lactate activates the mitochondrial electron transport chain independently of its metabolism.

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.

Lactate activates the mitochondrial electron transport chain independent of its metabolism.

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.

Oncogenic IDH mutations increase heterochromatin-related replication stress without impacting homologous recombination.

Oncogenic mutations in isocitrate dehydrogenases 1 and 2 (IDH1/2) produce 2-hydroxyglutarate (2HG), which inhibits dioxygenases that modulate chromatin dynamics. The effects of 2HG have been reported to sensitize IDH tumors to poly-(ADP-ribose) polymerase (PARP) inhibitors. However, unlike PARP-inhibitor-sensitive BRCA1/2 tumors, which exhibit impaired homologous recombination, IDH-mutant tumors have a silent mutational profile and lack signatures associated with impaired homologous recombination. Instead, 2HG-producing IDH mutations lead to a heterochromatin-dependent slowing of DNA replication accompanied by increased replication stress and DNA double-strand breaks. This replicative stress manifests as replication fork slowing, but the breaks are repaired without a significant increase in mutation burden. Faithful resolution of replicative stress in IDH-mutant cells is dependent on poly-(ADP-ribosylation). Treatment with PARP inhibitors increases DNA replication but results in incomplete DNA repair. These findings demonstrate a role for PARP in the replication of heterochromatin and further validate PARP as a therapeutic target in IDH-mutant tumors.

Hypoxia Potentiates the Inflammatory Fibroblast Phenotype Promoted by Pancreatic Cancer Cell-Derived Cytokines.

Cancer-associated fibroblasts (CAF) are a major cell type in the stroma of solid tumors and can exert both tumor-promoting and tumor-restraining functions. CAF heterogeneity is frequently observed in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by a dense and hypoxic stroma that features myofibroblastic CAFs (myCAF) and inflammatory CAFs (iCAF) that are thought to have opposing roles in tumor progression. While CAF heterogeneity can be driven in part by tumor cell-produced cytokines, other determinants shaping CAF identity and function are largely unknown. In vivo, we found that iCAFs displayed a hypoxic gene expression and biochemical profile and were enriched in hypoxic regions of PDAC tumors, while myCAFs were excluded from these regions. Hypoxia led fibroblasts to acquire an inflammatory gene expression signature and synergized with cancer cell-derived cytokines to promote an iCAF phenotype in a HIF1α-dependent fashion. Furthermore, HIF1α stabilization was sufficient to induce an iCAF phenotype in stromal cells introduced into PDAC organoid cocultures and to promote PDAC tumor growth. These findings indicate hypoxia-induced HIF1α as a regulator of CAF heterogeneity and promoter of tumor progression in PDAC. SIGNIFICANCE: Hypoxia in the tumor microenvironment of pancreatic cancer potentiates the cytokine-induced inflammatory CAF phenotype and promotes tumor growth. See related commentary by Fuentes and Taniguchi, p. 1560.

Activating mTOR Mutations Are Detrimental in Nutrient-Poor Conditions.

The mTOR is a key regulator of cell growth that integrates growth factor signaling and nutrient availability and is a downstream effector of oncogenic receptor tyrosine kinases (RTK) and PI3K/Akt signaling. Thus, activating mTOR mutations would be expected to enhance growth in many tumor types. However, tumor sequencing data have shown that mTOR mutations are enriched only in renal clear cell carcinoma, a clinically hypervascular tumor unlikely to be constrained by nutrient availability. To further define this cancer-type-specific restriction, we studied activating mutations in mTOR. All mTOR mutants tested enhanced growth in a cell-type agnostic manner under nutrient-replete conditions but were detrimental to cell survival in nutrient-poor conditions. Consistently, analysis of tumor data demonstrated that oncogenic mutations in the nutrient-sensing arm of the mTOR pathway display a similar phenotype and were exceedingly rare in human cancers of all types. Together, these data suggest that maintaining the ability to turn off mTOR signaling in response to changing nutrient availability is retained in most naturally occurring tumors. SIGNIFICANCE: This study suggests that cells need to inactivate mTOR to survive nutrient stress, which could explain the rarity of mTOR mutations and the limited clinical activity of mTOR inhibitors in cancer.

Hyperpolarized [5-13C,4,4-2H2,5-15N]-L-glutamine provides a means of annotating in vivo metabolic utilization of glutamine.

Glutamine is consumed by rapidly proliferating cells and can provide the carbon and nitrogen required for growth through various metabolic pathways. However, delineating the metabolic fate of glutamine is challenging to interrogate in vivo. Hyperpolarized magnetic resonance, by providing high transient nuclear magnetic resonance signals, provides an approach to measure fast biochemical processes in vivo. Aminohydrolysis of glutamine at carbon-5 plays an important role in providing nitrogen and carbon for multiple pathways. Here, we provide a synthetic strategy for isotope-enriched forms of glutamine that prolongs glutamine-C5 relaxation times and thereby reveals in vivo reactions involving carbon-5. We investigate multiple enrichment states, finding [5-13C,4,4-2H2,5-15N]-L-glutamine to be optimal for hyperpolarized measurement of glutamine conversion to glutamate in vivo. Leveraging this compound, we explore pancreatic cancer glutamine metabolism in vivo. Taken together, this work provides a means for studying glutamine metabolic flux in vivo and demonstrates on-target effects of metabolic enzyme inhibitors.

Ketohexokinase-mediated fructose metabolism is lost in hepatocellular carcinoma and can be leveraged for metabolic imaging.

The ability to break down fructose is dependent on ketohexokinase (KHK) that phosphorylates fructose to fructose-1-phosphate (F1P). We show that KHK expression is tightly controlled and limited to a small number of organs and is down-regulated in liver and intestinal cancer cells. Loss of fructose metabolism is also apparent in hepatocellular adenoma and carcinoma (HCC) patient samples. KHK overexpression in liver cancer cells results in decreased fructose flux through glycolysis. We then developed a strategy to detect this metabolic switch in vivo using hyperpolarized magnetic resonance spectroscopy. Uniformly deuterating [2-13C]-fructose and dissolving in D2O increased its spin-lattice relaxation time (T1) fivefold, enabling detection of F1P and its loss in models of HCC. In summary, we posit that in the liver, fructolysis to F1P is lost in the development of cancer and can be used as a biomarker of tissue function in the clinic using metabolic imaging.

The hallmarks of cancer metabolism: Still emerging.

Metabolism of cancer cells is geared toward biomass production and proliferation. Since the metabolic resources within the local tissue are finite, this can lead to nutrient depletion and accumulation of metabolic waste. To maintain growth in these conditions, cancer cells employ a variety of metabolic adaptations, the nature of which is collectively determined by the physiology of their cell of origin, the identity of transforming lesions, and the tissue in which cancer cells reside. Furthermore, select metabolites not only serve as substrates for energy and biomass generation, but can also regulate gene and protein expression and influence the behavior of non-transformed cells in the tumor vicinity. As they grow and metastasize, tumors can also affect and be affected by the nutrient distribution within the body. In this hallmark update, recent advances are incorporated into a conceptual framework that may help guide further research efforts in exploring cancer cell metabolism.

Safety and efficacy of dedicated guidewire, microcatheter, and guide catheter extension technologies for chronic total coronary occlusion revascularization: Primary results of the Teleflex Chronic Total Occlusion Study.

BACKGROUND: Description of procedural outcomes using contemporary techniques that apply specialized coronary guidewires, microcatheters, and guide catheter extensions designed for chronic total occlusion (CTO) percutaneous revascularization is limited. METHODS: A prospective, multicenter, single-arm study was conducted to evaluate procedural and in-hospital outcomes among 150 patients undergoing attempted CTO revascularization utilizing specialized guidewires, microcatheters and guide extensions. The primary endpoint was defined as successful guidewire recanalization and absence of in-hospital cardiac death, myocardial infarction (MI), or repeat target lesion revascularization (major adverse cardiac events, MACE). RESULTS: The prevalence of diabetes was 32.7%; prior MI, 48.0%; and previous bypass surgery, 32.7%. Average (mean ± standard deviation) CTO length was 46.9 ± 20.5 mm, and mean J-CTO score was 1.9 ± 0.9. Combined radial and femoral arterial access was performed in 50.0% of cases. Device utilization included: support microcatheter, 100%; guide catheter extension, 64.0%; and mean number of study guidewires/procedure was 4.8 ± 2.6. Overall, procedural success was achieved in 75.3% of patients. The rate of successful guidewire recanalization was 94.7%, and in-hospital MACE was 19.3%. Achievement of TIMI grade 2 or 3 flow was observed in 93.3% of patients. Crossing strategies included antegrade (54.0%), retrograde (1.3%) and combined antegrade/retrograde techniques (44.7%). Clinically significant perforation resulting in hemodynamic instability and/or requiring intervention occurred in 16 (10.7%) patients. CONCLUSIONS: In a multicenter, prospective registration study, favorable procedural success was achieved despite high lesion complexity using antegrade and retrograde guidewire maneuvers and with acceptable safety, yet with comparably higher risk than conventional non-CTO PCI.

Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment.

The aberrant production of collagen by fibroblasts is a hallmark of many solid tumours and can influence cancer progression. How the mesenchymal cells in the tumour microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumour-associated fibroblasts contributes to tumour fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain tricarboxylic acid (TCA) cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumour microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as a potential target to inhibit tumour desmoplasia.

Case Report: Suspected Solitary Osseous Plasmacytoma in a Cat: Use of Magnetic Resonance Imaging to Diagnose and Confirm Resolution of Disease Following Chemotherapy.

A 9-year-old female spayed Domestic Shorthair cat presented for pain, reluctance to jump, and hyporexia of 14 days duration. Neurologic examination was consistent with C6-T2 myelopathy. Magnetic resonance imaging (MRI) revealed a solitary, contrast-enhancing lesion within the T2 vertebral body. Solitary osseous plasmacytoma was diagnosed based on neurologic examination, advanced imaging, and clinicopathologic findings. Melphalan and prednisolone therapy were initiated. Complete resolution of clinical signs and the vertebral lesion were documented at a 2-year follow up examination with neurologic examination and repeat spinal MRI, respectively. Solitary osseous plasmacytoma are rare neoplasms in humans and domestic animals. As such, there is a paucity of published information regarding diagnostic criteria, MRI findings, treatment modalities, progression, and remission of disease in the feline patient. Most data are extrapolated from human medicine. The purpose of this report is to document neurologic exam and MR findings at the time of diagnosis and complete resolution of a solitary osseous vertebral plasmacytoma following melphalan and prednisolone therapy.

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo.

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.

Mitochondrial NADP(H) generation is essential for proline biosynthesis.

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but rather led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses.

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.

Translation in amino-acid-poor environments is limited by tRNAGln charging.

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.