A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation1,2. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model3 and human biopsies4 of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus5 exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

SUMMARY Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.

BUR1, which was previously identified by a selection for mutations that have general effects on transcription in Saccharomyces cerevisiae, encodes a cyclin-dependent kinase that is essential for viability, but none of its substrates have been identified to date. Using an unbiased biochemical approach, we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, as a Bur1 substrate. Phosphorylation of Rpb1 by Bur1 is likely to be physiologically relevant, since bur1 mutations interact genetically with rpb1 CTD truncations and with mutations in other genes involved in CTD function. Several genetic interactions are presented, implying a role for Bur1 during transcriptional elongation. These results identify Bur1 as a fourth S. cerevisiae CTD kinase and provide striking functional similarities between Bur1 and metazoan P-TEFb.The largest subunit of RNA polymerase II (Pol II), Rpb1, contains a highly conserved carboxy-terminal domain (CTD) that has a central role in transcriptional regulation in vivo (3, 11). The Rpb1 CTD consists of multiple repeats of the consensus heptapeptide sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, which is repeated 26 times in Saccharomyces cerevisiae, 42 times in Drosophila melanogaster, and 52 times in humans and mice (9). Although the CTD is not required for RNA polymerase activity in promoter-independent assays, it is essential in vivo; deletion of the entire CTD in Drosophila and S. cerevisiae results in lethality, while truncation to 11 repeats in yeast confers conditional growth and promoter-specific transcriptional defects (36).Phosphorylation of the CTD is important for regulation of Pol II activity during the transcription cycle: unphosphorylated Pol II is preferentially recruited into the preinitiation complex (PIC) (33) and then becomes phosphorylated during the transition from initiation to elongation (28). CTD phosphorylation thus has both stimulatory and inhibitory roles; phosphorylation prior to PIC assembly inhibits initiation, while phosphorylation after PIC assembly stimulates promoter escape and elongation. Phosphorylation occurs primarily on serine 2 and serine 5 of the consensus CTD repeat, with serine 2-phosphorylated Rpb1 being enriched distally from the promoter and serine 5-phosphorylated Rpb1 being enriched at promoter-proximal regions (26). Hyperphosphorylation of the CTD is also linked to other essential events during mRNA synthesis, including recruitment of mRNA modification enzymes and pre-mRNA splicing factors (reviewed in reference 50).The importance of CTD phosphorylation for Pol II regulation has prompted efforts to identify the kinases and phosphatases that determine the CTD phosphorylation state. Several kinases capable of phosphorylating the CTD in vitro have been identified in Drosophila, human, and rodent cell extracts (reviewed in reference 11), but it is not clear whether they all function as CTD kinases in vivo. In S. cerevisiae, where sophisticated genetic analysis can be readily combined with biochemistry to de...

Key Points• IDH2 R140Q expression in TF-1 cells can induce DNA and histone hypermethylation that mirrors human IDH2 mutant acute myeloid leukemia.• The hypermethylation can be reversed on treatment with AGI-6780, an IDH2 mutantspecific small-molecule inhibitor.Mutations of IDH1 and IDH2, which produce the oncometabolite 2-hydroxyglutarate (2HG), have been identified in several tumors, including acute myeloid leukemia. Recent studies have shown that expression of the IDH mutant enzymes results in high levels of 2HG and a block in cellular differentiation that can be reversed with IDH mutant-specific smallmolecule inhibitors. To further understand the role of IDH mutations in cancer, we conducted mechanistic studies in the TF-1 IDH2 R140Q erythroleukemia model system and found that IDH2 mutant expression caused both histone and genomic DNA methylation changes that can be reversed when IDH2 mutant activity is inhibited. Specifically, histone hypermethylation is rapidly reversed within days, whereas reversal of DNA hypermethylation proceeds in a progressive manner over the course of weeks. We identified several gene signatures implicated in tumorigenesis of leukemia and lymphoma, indicating a selective modulation of relevant cancer genes by IDH mutations. As methylation of DNA and histones is closely linked to mRNA expression and differentiation, these results indicate that IDH2 mutant inhibition may function as a cancer therapy via histone and DNA demethylation at genes involved in differentiation and tumorigenesis. (Blood. 2015;125(2):296-303) IntroductionActive site mutations in IDH1 (R132) and IDH2 (R172 and R140) that produce high levels of 2-hydroxyglutarate (2HG) have been identified in several human cancers.1-3 IDH mutations have been shown to cause DNA hypermethylation in both gliomas and leukemias via inhibition of methylcytosine dioxygenase TET2.4,5 Mutant IDH can also promote histone hypermethylation through competitive inhibition of a-ketoglutarate (aKG)-dependent Jumonji-C histone demethylases, thereby activating or deactivating expression of associated genes. 4,6,7 We have shown that mutant IDH1 and IDH2 can affect cell differentiation in solid and liquid tumors. [8][9][10] An IDH1 R132H inhibitor, AGI-5198, delayed growth and promoted differentiation of glioma cells while reducing histone H3K9 trimethylation. 8 Leukemic cell differentiation was also induced in primary human patient samples harboring an IDH2 R140Q mutation when they were treated ex vivo with AGI-6780, an IDH2 R140Q allosteric inhibitor.9 However, the mechanism by which IDH2 mutant activity and 2HG levels contribute to cellular differentiation and tumorigenesis is not fully understood. High levels of 2HG have been shown to competitively inhibit aKGdependent dioxygenases, leading to broad epigenetic changes. Therefore, we sought to investigate the global and gene-specific effects of mutant IDH2 inhibition in TF-1 cells expressing IDH2 R140Q. Probing the effects of IDH2 R140Q expression on histone and DNA methylation and gene expres...

PU.1 and GATA-1 are two hematopoietic specific transcription factors that play key roles in development of the myeloid and erythroid lineages, respectively. The two proteins bind to one another and inhibit each other's function in transcriptional activation and promotion of their respective differentiation programs. This mutual antagonism may be an important aspect of lineage commitment decisions. PU.1 can also act as an oncoprotein since deregulated expression of PU.1 in erythroid precursors causes erythroleukemias in mice. Studies of cultured mouse erythroleukemia cell lines indicate that one aspect of PU.1 function in erythroleukemogenesis is its ability to block erythroid differentiation by repressing GATA-1 (N. Rekhtman, F. Radparvar, T. Evans, and A. I. Skoultchi, Genes Dev. 13:1398-1411, 1999). We have investigated the mechanism of PU.1-mediated repression of GATA-1. We report here that PU.1 binds to GATA-1 on DNA. We localized the repression activity of PU.1 to a small acidic N-terminal domain that interacts with the C pocket of pRB, a well-known transcriptional corepressor. Repression of GATA-1 by PU.1 requires pRB, and pRB colocalizes with PU.1 and GATA-1 at repressed GATA-1 target genes. PU.1 and pRB also cooperate to block erythroid differentiation. Our results suggest that one of the mechanisms by which PU.1 antagonizes GATA-1 is by binding to it at GATA-1 target genes and tethering to these sites a corepressor that blocks transcriptional activity and thereby erythroid differentiation.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-hydroxyglutaric aciduria (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2-hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the accumulation of D-2HG, we generated transgenic mice with conditionally activated IDH2 R140Q and IDH2 R172K alleles. Global induction of mutant IDH2 expression in adults resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2 resulted in more pronounced phenotypes, including runting, hydrocephalus, and shortened life span, recapitulating the abnormalities observed in D2HGA patients. The diseased hearts exhibited mitochondrial damage and glycogen accumulation with a concordant up-regulation of genes involved in glycogen biosynthesis. Notably, mild cardiac hypertrophy was also observed in nude mice implanted with IDH2 R140Q -expressing xenografts, suggesting that 2HG may potentially act in a paracrine fashion. Finally, we show that silencing of IDH2 R140Q in mice with an inducible transgene restores heart function by lowering 2HG levels. Together, these findings indicate that inhibitors of mutant IDH2 may be beneficial in the treatment of D2HGA and suggest that 2HG produced by IDH mutant tumors has the potential to provoke a paraneoplastic condition.

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.