Metabolic changes upon GLS inhibition by CB-839 in glioma cell lines

Many tumors use Gln for both energy generation and as a biosynthetic precursor. Glutaminases (GAs) catalyze the first step of glutaminolysis by converting glutamine (Gln) into glutamate and ammonia in the mitochondria. In humans, two genes encode for glutaminases: GLS and GLS2. We examined the metabolic consequences of inhibiting GLS activity in glioma cells by using the clinically relevant inhibitor CB-839. We treated three glioblastoma (GBM) cell lines with CB-839 and performed untargeted metabolomics and isotope tracing experiments using U-13C-labeled Gln and 15N-labeled Gln in the amido group to ascertain the metabolic fates of Gln carbon and nitrogen. Untargeted metabolomics results showed that CB-839 treatment significantly depleted tricarboxylic acid cycle (TCAC) intermediates and related metabolites in the three human glioblastoma cell lines assayed. This result was also confirmed by a lower labeling from U-13C- Gln in these metabolites. U-13C- Gln tracing also revealed reductive carboxylation-related labeling in these cell lines, and this pathways was also suppressed by CB-839. Metabolomics results showed an accumulation of the de novo purine biosynthesis intermediates inosine monophosphate and/or AICAR, and a decrease in uridine monophosphate, while 15N-Gln tracing results showed a decreased labeling from Gln amido group in AMP, GMP, UMP and CTP in T98G cell line when treated with CB-839. Finally, metabolomics showed higher levels of trimethyllysine and, in T98G cells, a 22-fold increase in 5-methyl-cytosine.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Mechanism by which a recently discovered allosteric inhibitor blocks glutamine metabolism in transformed cells

The mitochondrial enzyme glutaminase C (GAC) catalyzes the hydrolysis of glutamine to glutamate plus ammonia, a key step in the metabolism of glutamine by cancer cells. Recently, we discovered a class of allosteric inhibitors of GAC that inhibit cancer cell growth without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we take advantage of mouse embryonic fibroblasts transformed by oncogenic Dbl, which hyperactivates Rho GTPases, together with 13Clabeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis necessary for satisfying the glutamine addiction of cancer cells. We then determine how 968 inhibits the catalytic activity of GAC. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrate how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays with newly developed GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity formonomeric GAC and that the dose-dependent binding of 968 to GAC monomers directlymatches its dose-dependent inhibition of enzyme activity and cellular transformation. Together, these findings highlight the requirement of tetramer formation as the mechanism of GAC activation and shed new light on how a distinct class of allosteric GAC inhibitors impacts the metabolic program of transformed cells