Mutant IDH in Gliomas: Role in Cancer and Treatment Options.

Altered metabolism is a common feature of many cancers and, in some cases, is a consequence of mutation in metabolic genes, such as the ones involved in the TCA cycle. Isocitrate dehydrogenase (IDH) is mutated in many gliomas and other cancers. Physiologically, IDH converts isocitrate to α-ketoglutarate (α-KG), but when mutated, IDH reduces α-KG to D2-hydroxyglutarate (D2-HG). D2-HG accumulates at elevated levels in IDH mutant tumours, and in the last decade, a massive effort has been made to develop small inhibitors targeting mutant IDH. In this review, we summarise the current knowledge about the cellular and molecular consequences of IDH mutations and the therapeutic approaches developed to target IDH mutant tumours, focusing on gliomas.

Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors.

Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.

Advances in Research of Adult Gliomas.

Diffuse gliomas are the most frequent brain tumours, representing 75% of all primary malignant brain tumours in adults. Because of their locally aggressive behaviour and the fact that they cannot be cured by current therapies, they represent one of the most devastating cancers. The present review summarises recent advances in our understanding of glioma development and progression by use of various in vitro and in vivo models, as well as more complex techniques including cultures of 3D organoids and organotypic slices. We discuss the progress that has been made in understanding glioma heterogeneity, alteration in gene expression and DNA methylation, as well as advances in various in silico models. Lastly current treatment options and future clinical trials, which aim to improve early diagnosis and disease monitoring, are also discussed.

Anion-exchange chromatography mass spectrometry provides extensive coverage of primary metabolic pathways revealing altered metabolism in IDH1 mutant cells.

Altered central carbon metabolism is a hallmark of many diseases including diabetes, obesity, heart disease and cancer. Identifying metabolic changes will open opportunities for better understanding aetiological processes and identifying new diagnostic, prognostic, and therapeutic targets. Comprehensive and robust analysis of primary metabolic pathways in cells, tissues and bio-fluids, remains technically challenging. We report on the development and validation of a highly reproducible and robust untargeted method using anion-exchange tandem mass spectrometry (IC-MS) that enables analysis of 431 metabolites, providing detailed coverage of central carbon metabolism. We apply the method in an untargeted, discovery-driven workflow to investigate the metabolic effects of isocitrate dehydrogenase 1 (IDH1) mutations in glioblastoma cells. IC-MS provides comprehensive coverage of central metabolic pathways revealing significant elevation of 2-hydroxyglutarate and depletion of 2-oxoglutarate. Further analysis of the data reveals depletion in additional metabolites including previously unrecognised changes in lysine and tryptophan metabolism.

Serum- and Glucocorticoid-induced Kinase Sgk1 Directly Promotes the Differentiation of Colorectal Cancer Cells and Restrains Metastasis.

PURPOSE: The molecular events that determine intestinal cell differentiation are poorly understood and it is unclear whether it is primarily a passive event or an active process. It is clinically important to gain a greater understanding of the process, because in colorectal cancer, the degree of differentiation of a tumor is associated with patient survival. SGK1 has previously been identified as a gene that is principally expressed in differentiated intestinal cells. In colorectal cancer, there is marked downregulation of SGK1 compared with normal tissue.Experimental Design: An inducible SGK1 viral overexpression system was utilized to induce reexpression of SGK1 in colorectal cancer cell lines. Transcriptomic and phenotypic analyses of these colorectal cancer lines was performed and validation in mouse and human cohorts was performed. RESULTS: We demonstrate that SGK1 is upregulated in response to, and an important controller of, intestinal cell differentiation. Reexpression of SGK1 in colorectal cancer cell lines results in features of differentiation, decreased migration rates, and inhibition of metastasis in an orthotopic xenograft model. These effects may be mediated, in part, by SGK1-induced PKP3 expression and increased degradation of MYC. CONCLUSIONS: Our results suggest that SGK1 is an important mediator of differentiation of colorectal cells and may inhibit colorectal cancer metastasis.

The role of inflammation in subventricular zone cancer.

The adult subventricular zone (SVZ) stem cell niche has proven vital for discovering neurodevelopmental mechanisms and holds great potential in medicine for neurodegenerative diseases. Yet the SVZ holds a dark side - it can become tumorigenic. Glioblastomas can arise from the SVZ via cancer stem cells (CSCs). Glioblastoma and other brain cancers often have dismal prognoses since they are resistant to treatment. In this review we argue that the SVZ is susceptible to cancer because it contains stem cells, migratory progenitors and unusual inflammation. Theoretically, SVZ stem cells can convert to CSCs more readily than can postmitotic neural cells. Additionally, the robust long-distance migration of SVZ progenitors can be subverted upon tumorigenesis to an infiltrative phenotype. There is evidence that the SVZ, even in health, exhibits chronic low-grade cellular and molecular inflammation. Its inflammatory response to brain injuries and disease differs from that of other brain regions. We hypothesize that the SVZ inflammatory environment can predispose cells to novel mutations and exacerbate cancer phenotypes. This can be studied in animal models in which human mutations related to cancer are knocked into the SVZ to induce tumorigenesis and the CSC immune interactions that precede full-blown cancer. Importantly inflammation can be pharmacologically modulated providing an avenue to brain cancer management and treatment. The SVZ is accessible by virtue of its location surrounding the lateral ventricles and CSCs in the SVZ can be targeted with a variety of pharmacotherapies. Thus, the SVZ can yield aggressive tumors but can be targeted via several strategies.

Oncogenic IDH1 Mutations Promote Enhanced Proline Synthesis through PYCR1 to Support the Maintenance of Mitochondrial Redox Homeostasis.

Since the discovery of mutations in isocitrate dehydrogenase 1 (IDH1) in gliomas and other tumors, significant efforts have been made to gain a deeper understanding of the consequences of this oncogenic mutation. One aspect of the neomorphic function of the IDH1 R132H enzyme that has received less attention is the perturbation of cellular redox homeostasis. Here, we describe a biosynthetic pathway exhibited by cells expressing mutant IDH1. By virtue of a change in cellular redox homeostasis, IDH1-mutated cells synthesize excess glutamine-derived proline through enhanced activity of pyrroline 5-carboxylate reductase 1 (PYCR1), coupled to NADH oxidation. Enhanced proline biosynthesis partially uncouples the electron transport chain from tricarboxylic acid (TCA) cycle activity through the maintenance of a lower NADH/NAD+ ratio and subsequent reduction in oxygen consumption. Thus, we have uncovered a mechanism by which tumor cell survival may be promoted in conditions associated with perturbed redox homeostasis, as occurs in IDH1-mutated glioma.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

Recurrent chromosomal gains and heterogeneous driver mutations characterise papillary renal cancer evolution.

Papillary renal cell carcinoma (pRCC) is an important subtype of kidney cancer with a problematic pathological classification and highly variable clinical behaviour. Here we sequence the genomes or exomes of 31 pRCCs, and in four tumours, multi-region sequencing is undertaken. We identify BAP1, SETD2, ARID2 and Nrf2 pathway genes (KEAP1, NHE2L2 and CUL3) as probable drivers, together with at least eight other possible drivers. However, only ~10% of tumours harbour detectable pathogenic changes in any one driver gene, and where present, the mutations are often predicted to be present within cancer sub-clones. We specifically detect parallel evolution of multiple SETD2 mutations within different sub-regions of the same tumour. By contrast, large copy number gains of chromosomes 7, 12, 16 and 17 are usually early, monoclonal changes in pRCC evolution. The predominance of large copy number variants as the major drivers for pRCC highlights an unusual mode of tumorigenesis that may challenge precision medicine approaches.

Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche.

Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

HOT mutation screening in human glioblastomas.

AIMS: Somatic mutations in IDH1 and IDH2 are described in glioblastomas (GBMs). Mutant IDH1 and IDH2 reduce α-KG to D-2HG which accumulates, and is proposed to promote tumorigenesis. HOT catalyzes the conversion of γ-hydroxybutyrate to succinic semialdehyde in a reaction that produces D-2HG. Since increased HOT enzyme activity could lead to an accumulation of D-2HG, coupled with the fact that only a minority of GBMs carry IDH1/2 mutations and 2HG accumulation has recently been described in IDH wild-type tumors, we analyzed a set of GBM samples for mutations in the HOT gene. MATERIALS & METHODS: We screened 42 human GBM samples for mutations in HOT. RESULTS: No mutations in HOT were identified in the 42 GBM samples screened. CONCLUSION: Mutations in the coding regions of HOT do not occur at an appreciable frequency in GBM.

IDH mutations in tumorigenesis and their potential role as novel therapeutic targets.

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Somatic mutations in genes encoding IDH1 and IDH2 were first identified in glioma and subsequently in acute myeloid leukemia and other solid tumors. These heterozygous point mutations occur at the arginine residue of the enzyme's active site and cause both loss of normal enzyme function and gain of function, causing reduction of α-KG to D-2-hydroxyglutarate, which accumulates. D-2-hydroxyglutarate may act as an oncometabolite through the inhibition of various α-KG-dependent enzymes, stimulating angiogenesis, histone modifications and aberrant DNA methylation. Possibly, IDH mutations may also cause oncogenic effects through dysregulation of the tricarboxylic acid cycle, or by increasing susceptibility to oxidative stress. Clinically, IDH mutations may be useful diagnostic, prognostic and predictive biomarkers, and it is anticipated that a better understanding of the pathogenesis of IDH mutations will enable IDH-directed therapies to be developed in the future.

Cells lacking the fumarase tumor suppressor are protected from apoptosis through a hypoxia-inducible factor-independent, AMPK-dependent mechanism.

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.

Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1 expression.

Although genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here, we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma, modulates the binding and function of hypoxia-inducible factor (HIF) at a previously unrecognized transcriptional enhancer of CCND1 (encoding cyclin D1) that is specific for renal cancers characterized by inactivation of the von Hippel-Lindau tumor suppressor (pVHL). The protective haplotype impairs binding of HIF-2, resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control.

SDH mutations in cancer.

The SDHA, SDHB, SDHC, SDHD genes encode the four subunits of succinate dehydrogenase (SDH; mitochondrial complex II), a mitochondrial enzyme involved in two essential energy-producing metabolic processes of the cell, the Krebs cycle and the electron transport chain. Germline loss-of-function mutations in any of the SDH genes or assembly factor (SDHAF2) cause hereditary paraganglioma/phaeochromocytoma syndrome (HPGL/PCC) through a mechanism which is largely unknown. Owing to the central function of SDH in cellular energy metabolism it is important to understand its role in tumor suppression. Here is reported an overview of genetics, clinical and molecular progress recently performed in understanding the basis of HPGL/PCC tumorigenesis.

Screen for IDH1, IDH2, IDH3, D2HGDH and L2HGDH mutations in glioblastoma.

Isocitrate dehydrogenases (IDHs) catalyse oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). IDH1 functions in the cytosol and peroxisomes, whereas IDH2 and IDH3 are both localized in the mitochondria. Heterozygous somatic mutations in IDH1 occur at codon 132 in 70% of grade II-III gliomas and secondary glioblastomas (GBMs), and in 5% of primary GBMs. Mutations in IDH2 at codon 172 are present in grade II-III gliomas at a low frequency. IDH1 and IDH2 mutations cause both loss of normal enzyme function and gain-of-function, causing reduction of α-KG to D-2-hydroxyglutarate (D-2HG) which accumulates. Excess hydroxyglutarate (2HG) can also be caused by germline mutations in D- and L-2-hydroxyglutarate dehydrogenases (D2HGDH and L2HGDH). If loss of IDH function is critical for tumourigenesis, we might expect some tumours to acquire somatic IDH3 mutations. Alternatively, if 2HG accumulation is critical, some tumours might acquire somatic D2HGDH or L2HGDH mutations. We therefore screened 47 glioblastoma samples looking for changes in these genes. Although IDH1 R132H was identified in 12% of samples, no mutations were identified in any of the other genes. This suggests that mutations in IDH3, D2HGDH and L2HGDH do not occur at an appreciable frequency in GBM. One explanation is simply that mono-allelic IDH1 and IDH2 mutations occur more frequently by chance than the bi-allelic mutations expected at IDH3, D2HGDH and L2HGDH. Alternatively, both loss of IDH function and 2HG accumulation might be required for tumourigenesis, and only IDH1 and IDH2 mutations have these dual effects.

Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status.

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.

Expression profiling in progressive stages of fumarate-hydratase deficiency: the contribution of metabolic changes to tumorigenesis.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that "pseudohypoxic" stabilization of hypoxia-inducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothesized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as manifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient models represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors.

Fumarase tumor suppressor gene and MET oncogene cooperate in upholding transformation and tumorigenesis.

Loss of the fumarate hydratase (FH) tumor suppressor gene results in the development of benign tumors that rarely, but regrettably, progress to very aggressive cancers. Using mouse embryo fibroblasts (MEFs) to model transformation, we found that fh knockdown results in increased expression of the met oncogene-encoded tyrosine kinase receptor through hypoxia-inducible factor (hif) stabilization. MET-increased expression was alone able to stabilize hif, thus establishing a feed forward loop that might enforce tumor progression. The fh-defective MEFs showed increased motility and protection from apoptosis. Motility, but not survival, relied on hif-1alpha and was greatly enhanced by MET ligand hepatocyte growth factor. Met cooperated with a weakly oncogenic ras in making MEFs transformed and tumorigenic, as shown by in vitro and in vivo assays. Loss of fh was not equally effective by itself but enhanced the transformed and tumorigenic phenotype induced by ras and MET. Consistently, the rescue of fumarase expression abrogated the motogenic and transformed phenotype of fh-defective MEFs. In conclusion, the data suggest that the progression of tumors where FH is lost might be boosted by activation of the MET oncogene, which is able to drive cell-autonomous tumor progression and is a strong candidate for targeted therapy.