Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the ...IDH1 active site, resulting in loss of the enzyme's ability to catalyse conversion of isocitrate to alpha-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of alpha-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert alpha-ketoglutarate to 2HG. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.
Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozygous, and affect a ...single arginine residue. Recently, IDH1 mutations were identified in 8% of acute myelogenous leukemia (AML) patients. A glioma study revealed that IDH1 mutations cause a gain-of-function, resulting in the production and accumulation of 2-hydroxyglutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant samples. Liquid chromatography-mass spectrometry metabolite screening revealed increased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1 R132C and IDH2 R172K proteins catalyze the novel nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of alpha-ketoglutarate (alpha-KG) to 2-HG. The IDH1 R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases the affinity for NADPH and alpha-KG. This prevents the oxidative decarboxylation of isocitrate to alpha-KG, and facilitates the conversion of alpha-KG to 2-HG. IDH1/2 mutations confer an enzymatic gain of function that dramatically increases 2-HG in AML. This provides an explanation for the heterozygous acquisition of these mutations during tumorigenesis. 2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.
Mutations of the isocitrate dehydrogenase 1 and 2 genes (
IDH1
and
IDH2
) are commonly found in primary brain cancers. We previously reported that a novel enzymatic activity of these mutations ...results in the production of the putative oncometabolite, R(−)-2-hydroxyglutarate (2-HG). Here we investigated the ability of magnetic resonance spectroscopy (MRS) to detect 2-HG production in order to non-invasively identify patients with
IDH1
mutant brain tumors. Patients with intrinsic glial brain tumors (
n
= 27) underwent structural and spectroscopic magnetic resonance imaging prior to surgery. 2-HG levels from MRS data were quantified using LC-Model software, based upon a simulated spectrum obtained from a GAMMA library added to the existing prior knowledge database. The resected tumors were then analyzed for
IDH1
mutational status by genomic DNA sequencing, Ki-67 proliferation index by immunohistochemistry, and concentrations of 2-HG and other metabolites by liquid chromatography–mass spectrometry (LC–MS). MRS detected elevated 2-HG levels in gliomas with
IDH1
mutations compared to those with wild-type
IDH1
(
P
= 0.003). The 2-HG levels measured in vivo with MRS were significantly correlated with those measured ex vivo from the corresponding tumor samples using LC–MS (
r
2
= 0.56;
P
= 0.0001). Compared with wild-type tumors, those with
IDH1
mutations had elevated choline (
P
= 0.01) and decreased glutathione (
P
= 0.03) on MRS. Among the
IDH1
mutated gliomas, quantitative 2-HG values were correlated with the Ki-67 proliferation index of the tumors (
r
2
= 0.59;
P
= 0.026). In conclusion, water-suppressed proton (
1
H) MRS provides a non-invasive measure of 2-HG in gliomas, and may serve as a potential biomarker for patients with
IDH1
mutant brain tumors. In addition to 2-HG, alterations in several other metabolites measured by MRS correlate with
IDH1
mutation status.
The CREB family of proteins are critical mediators of gene expression in response to extracellular signals and are essential regulators of adaptive behavior and long-term memory formation 1–4. The ...TORC proteins were recently described as potent CREB coactivators, but their role in regulation of CREB activity remained unknown 5, 6. TORC proteins were found to be exported from the nucleus in a CRM1-dependent fashion. A high-throughput microscopy-based screen was developed to identify genes and pathways capable of inducing nuclear TORC accumulation. Expression of the catalytic subunit of PKA and the calcium channel TRPV6 relocalized TORC1 to the nucleus. Nuclear accumulation of the three human TORC proteins was induced by increasing intracellular cAMP or calcium levels. TORC1 and TORC2 translocation in response to calcium, but not cAMP, was mediated by calcineurin, and TORC1 was shown to be directly dephosphorylated by calcineurin. TORC function was shown to be essential for CRE-mediated gene expression induced by cAMP, calcium, or GPCR activation, and nuclear transport of TORC1 was sufficient to activate CRE-dependent transcription. Drosophila TORC was also shown to translocate in response to calcineurin activation in vivo. Thus, TORC nuclear translocation is an essential, conserved step in activation of cAMP-responsive genes.
Immune checkpoint blockade improves survival in a subset of patients with non-small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. ...Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited.
We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC).
Cytometric profiling identified an immunologically "hot" cluster with abundant CD8
T cells expressing high levels of PD-1 and TIM-3 and an immunologically "cold" cluster with lower relative abundance of CD8
T cells and expression of inhibitory markers. The "hot" cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the "hot" cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10.
Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer.
The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation.
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. ...Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.
Antibiotics are administered near-universally to very low birth weight (VLBW) infants after birth for suspected early-onset sepsis (EOS). We previously identified a phenotypic group of VLBW infants, ...referred to as low-risk for EOS (LRE), whose risk of EOS is low enough to avoid routine antibiotic initiation. In this cohort study, we compared 18 such infants with 30 infants categorized as non-LRE to determine if the lower risk of pathogen transmission at birth is accompanied by differences in microbiome acquisition and development. We did shotgun metagenomic sequencing of 361 fecal samples obtained serially. LRE infants had a higher human-to-bacterial DNA ratio than non-LRE infants in fecal samples on days 1-3 after birth, confirming lower bacterial acquisition among LRE infants. The microbial diversity and composition in samples from days 4-7 differed between the groups with a predominance of Staphylococcus epidermidis in LRE infants and Enterobacteriaceae sp. in non-LRE infants. Compositional differences were congruent with the distribution of virulence factors and antibiotic resistant genes. After the first week, the overall composition was similar, but changes in relative abundance for several taxa with increasing age differed between groups. Of the nine late-onset bacteremia episodes, eight occurred in non-LRE infants. Species isolated from the blood culture was detected in the pre-antibiotic fecal samples of the infant for all episodes, though these species were also found in infants without bacteremia. In conclusion, LRE infants present a distinct pattern of microbiome development that is aligned with their low risk for EOS. Further investigation to determine the impact of these differences on later outcomes such as late-onset bacteremia is warranted.
Highlights • We identified a group of R/M SCCHN tumors with an inflamed immunophenotype. • We identified a non-inflamed tumor immunophenotype in the same population. • Patients with the inflamed ...tumor phenotype had improved survival. • Patients with the inflamed tumor phenotype may benefit from checkpoint blockade.
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