Branched-chain amino acid transaminase 1 regulates glioblastoma cell plasticity and contributes to immunosuppression.

BACKGROUND: Glioblastoma is the most common malignant brain tumor in adults. Cellular plasticity and the poorly differentiated features result in a fast relapse of the tumors following treatment. Moreover, the immunosuppressive microenvironment proved to be a major obstacle to immunotherapeutic approaches. Branched-chain amino acid transaminase 1 (BCAT1) was shown to drive the growth of glioblastoma and other cancers;however, its oncogenic mechanism remains poorly understood. METHODS: Using human tumor data, cell line models and orthotopic immuno-competent and -deficient mouse models, we investigated the phenotypic and mechanistic effects of BCAT1 on glioblastoma cell state and immunomodulation. RESULTS: Here, we show that BCAT1 is crucial for maintaining the poorly differentiated state of glioblastoma cells and that its low expression correlates with a more differentiated glioblastoma phenotype. Furthermore, orthotopic tumor injection into immunocompetent mice demonstrated that the brain microenvironment is sufficient to induce differentiation of Bcat1-KO tumors in vivo. We link the transition to a differentiated cell state to the increased activity of ten-eleven translocation demethylases and the hypomethylation and activation of neuronal differentiation genes. In addition, the knockout of Bcat1 attenuated immunosuppression, allowing for an extensive infiltration of CD8+ cytotoxic T-cells and complete abrogation of tumor growth. Further analysis in immunodeficient mice revealed that both tumor cell differentiation and immunomodulation following BCAT1-KO contribute to the long-term suppression of tumor growth. CONCLUSIONS: Our study unveils BCAT1's pivotal role in promoting glioblastoma growth by inhibiting tumor cell differentiation and sustaining an immunosuppressive milieu. These findings offer a novel therapeutic avenue for targeting glioblastoma through the inhibition of BCAT1.

Methylation-directed regulatory networks determine enhancing and silencing of mutation disease driver genes and explain inter-patient expression variation.

BACKGROUND: Common diseases manifest differentially between patients, but the genetic origin of this variation remains unclear. To explore possible involvement of gene transcriptional-variation, we produce a DNA methylation-oriented, driver-gene-wide dataset of regulatory elements in human glioblastomas and study their effect on inter-patient gene expression variation. RESULTS: In 175 of 177 analyzed gene regulatory domains, transcriptional enhancers and silencers are intermixed. Under experimental conditions, DNA methylation induces enhancers to alter their enhancing effects or convert into silencers, while silencers are affected inversely. High-resolution mapping of the association between DNA methylation and gene expression in intact genomes reveals methylation-related regulatory units (average size = 915.1 base-pairs). Upon increased methylation of these units, their target-genes either increased or decreased in expression. Gene-enhancing and silencing units constitute cis-regulatory networks of genes. Mathematical modeling of the networks highlights indicative methylation sites, which signified the effect of key regulatory units, and add up to make the overall transcriptional effect of the network. Methylation variation in these sites effectively describe inter-patient expression variation and, compared with DNA sequence-alterations, appears as a major contributor of gene-expression variation among glioblastoma patients. CONCLUSIONS: We describe complex cis-regulatory networks, which determine gene expression by summing the effects of positive and negative transcriptional inputs. In these networks, DNA methylation induces both enhancing and silencing effects, depending on the context. The revealed mechanism sheds light on the regulatory role of DNA methylation, explains inter-individual gene-expression variation, and opens the way for monitoring the driving forces behind deferential courses of cancer and other diseases.

Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B-cell lymphomas.

Histone methylation-modifiers, such as EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%), also identified by prior exome or RNA-sequencing studies, we here found recurrent alterations to KDM4C in chromosome 9p24, encoding a histone demethylase. Focal structural variation was the main mechanism of KDM4C alterations, and was independent from 9p24 amplification. We also identified KDM4C alterations in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNA-sequencing and genome sequencing data we predict that KDM4C structural variants result in loss-offunction. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as a tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as a putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas.

BCAT1 redox function maintains mitotic fidelity.

The metabolic enzyme branched-chain amino acid transaminase 1 (BCAT1) drives cell proliferation in aggressive cancers such as glioblastoma. Here, we show that BCAT1 localizes to mitotic structures and has a non-metabolic function as a mitotic regulator. Furthermore, BCAT1 is required for chromosome segregation in cancer and induced pluripotent stem cells and tumor growth in human cerebral organoid and mouse syngraft models. Applying gene knockout and rescue strategies, we show that the BCAT1 CXXC redox motif is crucial for controlling cysteine sulfenylation specifically in mitotic cells, promoting Aurora kinase B localization to centromeres, and securing accurate chromosome segregation. These findings offer an explanation for the well-established role of BCAT1 in promoting cancer cell proliferation. In summary, our data establish BCAT1 as a component of the mitotic apparatus that safeguards mitotic fidelity through a moonlighting redox functionality.

Rapid, adaptable and sensitive Cas13-based COVID-19 diagnostics using ADESSO.

During the ongoing COVID-19 pandemic, PCR testing and antigen tests have proven critical for helping to stem the spread of its causative agent, SARS-CoV-2. However, these methods suffer from either general applicability and/or sensitivity. Moreover, the emergence of variant strains creates the need for flexibility to correctly and efficiently diagnose the presence of substrains. To address these needs we developed the diagnostic test ADESSO (Accurate Detection of Evolving SARS-CoV-2 through SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) Optimization) which employs Cas13 to diagnose patients in 1 h without sophisticated equipment. Using an extensive panel of clinical samples, we demonstrate that ADESSO correctly identifies infected individuals at a sensitivity and specificity comparable to RT-qPCR on extracted RNA and higher than antigen tests for unextracted samples. Altogether, ADESSO is a fast, sensitive and cheap method that can be applied in a point of care setting to diagnose COVID-19 and can be quickly adjusted to detect new variants.

Mutational mechanisms shaping the coding and noncoding genome of germinal center derived B-cell lymphomas.

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis.

A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors.

Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific Pten/p53 double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.

Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype.

Glioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural-mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.

Leucine and branched-chain amino acid metabolism contribute to the growth of bone sarcomas by regulating AMPK and mTORC1 signaling.

Osteosarcoma and chondrosarcoma are sarcomas of the bone and the cartilage that are primarily treated by surgical intervention combined with high toxicity chemotherapy. In search of alternative metabolic approaches to address the challenges in treating bone sarcomas, we assessed the growth dependence of these cancers on leucine, one of the branched-chain amino acids (BCAAs), and BCAA metabolism. Tumor biopsies from bone sarcoma patients revealed differential expression of BCAA metabolic enzymes. The cytosolic branched-chain aminotransferase (BCATc) that is commonly overexpressed in cancer cells, was down-regulated in chondrosarcoma (SW1353) in contrast with osteosarcoma (143B) cells that expressed both BCATc and its mitochondrial isoform BCATm. Treating SW1353 cells with gabapentin, a selective inhibitor of BCATc, further revealed that these cells failed to respond to gabapentin. Application of the structural analog of leucine, N-acetyl-leucine amide (NALA) to disrupt leucine uptake, indicated that all bone sarcoma cells used leucine to support their energy metabolism and biosynthetic demands. This was evident from the increased activity of the energy sensor AMP-activated protein kinase (AMPK), down-regulation of complex 1 of the mammalian target of rapamycin (mTORC1), and reduced cell viability in response to NALA. The observed changes were most profound in the 143B cells, which appeared highly dependent on cytosolic and mitochondrial BCAA metabolism. This study thus demonstrates that bone sarcomas rely on leucine and BCAA metabolism for energy and growth; however, the differential expression of BCAA enzymes and the presence of other carbon sources may dictate how efficiently these cancer cells take advantage of BCAA metabolism.

Pilocytic astrocytoma demethylation and transcriptional landscapes link bZIP transcription factors to immune response.

BACKGROUND: Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. While genome and transcriptome landscapes are well studied, data of the complete methylome, tumor cell composition, and immune infiltration are scarce. METHODS: We generated whole genome bisulfite sequence (WGBS) data of 9 PAs and 16 control samples and integrated available 154 PA and 57 control methylation array data. RNA sequence data of 49 PAs and 11 control samples as well as gene expression arrays of 248 PAs and 28 controls were used to assess transcriptional activity. RESULTS: DNA-methylation patterns of partially methylated domains suggested high stability of the methylomes during tumorigenesis. Comparing tumor and control tissues of infra- and supratentorial location using methylation arrays revealed a site specific pattern. Analysis of WGBS data revealed 9381 significantly differentially methylated regions (DMRs) in PA versus control tissue. Enhancers and transcription factor (TF) motifs of five distinct TF families were found to be enriched in DMRs. Methylation together with gene expression data-based in silico tissue deconvolution analysis indicated a striking variation in the immune cell infiltration in PA. A TF network analysis showed a regulatory relation between basic leucine zipper (bZIP) transcription factors and genes involved in immune-related processes. CONCLUSION: We provide evidence for a link of focal methylation differences and differential gene expression to immune infiltration.

GPD1 Specifically Marks Dormant Glioma Stem Cells with a Distinct Metabolic Profile.

Brain tumor stem cells (BTSCs) are a chemoresistant population that can drive tumor growth and relapse, but the lack of BTSC-specific markers prevents selective targeting that spares resident stem cells. Through a ribosome-profiling analysis of mouse neural stem cells (NSCs) and BTSCs, we find glycerol-3-phosphate dehydrogenase 1 (GPD1) expression specifically in BTSCs and not in NSCs. GPD1 expression is present in the dormant BTSC population, which is enriched at tumor borders and drives tumor relapse after chemotherapy. GPD1 inhibition prolongs survival in mouse models of glioblastoma in part through altering cellular metabolism and protein translation, compromising BTSC maintenance. Metabolomic and lipidomic analyses confirm that GPD1+ BTSCs have a profile distinct from that of NSCs, which is dependent on GPD1 expression. Similar GPD1 expression patterns and prognostic associations are observed in human gliomas. This study provides an attractive therapeutic target for treating brain tumors and new insights into mechanisms regulating BTSC dormancy.

Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic Burkitt lymphoma.

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.

Evolutionary Trajectories of IDHWT Glioblastomas Reveal a Common Path of Early Tumorigenesis Instigated Years ahead of Initial Diagnosis.

We studied how intratumoral genetic heterogeneity shapes tumor growth and therapy response for isocitrate dehydrogenase (IDH)-wild-type glioblastoma, a rapidly regrowing tumor. We inferred the evolutionary trajectories of matched pairs of primary and relapsed tumors based on deep whole-genome-sequencing data. This analysis suggests both a distant origin of de novo glioblastoma, up to 7 years before diagnosis, and a common path of early tumorigenesis, with one or more of chromosome 7 gain, 9p loss, or 10 loss, at tumor initiation. TERT promoter mutations often occurred later as a prerequisite for rapid growth. In contrast to this common early path, relapsed tumors acquired no stereotypical pattern of mutations and typically regrew from oligoclonal origins, suggesting sparse selective pressure by therapeutic measures.

Author Correction: BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online.

BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

Branched-chain ketoacids secreted by glioblastoma cells via MCT1 modulate macrophage phenotype.

Elevated amino acid catabolism is common to many cancers. Here, we show that glioblastoma are excreting large amounts of branched-chain ketoacids (BCKAs), metabolites of branched-chain amino acid (BCAA) catabolism. We show that efflux of BCKAs, as well as pyruvate, is mediated by the monocarboxylate transporter 1 (MCT1) in glioblastoma. MCT1 locates in close proximity to BCKA-generating branched-chain amino acid transaminase 1, suggesting possible functional interaction of the proteins. Using in vitro models, we demonstrate that tumor-excreted BCKAs can be taken up and re-aminated to BCAAs by tumor-associated macrophages. Furthermore, exposure to BCKAs reduced the phagocytic activity of macrophages. This study provides further evidence for the eminent role of BCAA catabolism in glioblastoma by demonstrating that tumor-excreted BCKAs might have a direct role in tumor immune suppression. Our data further suggest that the anti-proliferative effects of MCT1 knockdown observed by others might be related to the blocked excretion of BCKAs.

PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate.

The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can't be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors' functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.

Atypical Teratoid/Rhabdoid Tumors Are Comprised of Three Epigenetic Subgroups with Distinct Enhancer Landscapes.

Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in infants. Although the prognosis of ATRT patients is poor, some patients respond favorably to current treatments, suggesting molecular inter-tumor heterogeneity. To investigate this further, we genetically and epigenetically analyzed 192 ATRTs. Three distinct molecular subgroups of ATRTs, associated with differences in demographics, tumor location, and type of SMARCB1 alterations, were identified. Whole-genome DNA and RNA sequencing found no recurrent mutations in addition to SMARCB1 that would explain the differences between subgroups. Whole-genome bisulfite sequencing and H3K27Ac chromatin-immunoprecipitation sequencing of primary tumors, however, revealed clear differences, leading to the identification of subgroup-specific regulatory networks and potential therapeutic targets.