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Inactivation of the M2 form of pyruvate kinase (PKM2) in cancer cells is associated with increased tumorigenicity. To test the hypothesis that tumor growth may be inhibited through the PKM2 pathway, we generated a series of small-molecule PKM2 activators. The compounds exhibited low nanomolar activity in both biochemical and cell-based PKM2 activity assays. These compounds did not affect the growth of cancer cell lines under normal conditions in vitro, but strongly inhibited the proliferation of multiple lung cancer cell lines when serine was absent from the cell culture media. In addition, PKM2 activators inhibited the growth of an aggressive lung adenocarcinoma xenograft. These findings show that PKM2 activation by small molecules influences the growth of cancer cells in vitro and in vivo, and suggest that such compounds may augment cancer therapies. Mol Cancer Ther; 12(8); 1453–60. ©2013 AACR.

Supplementary Figure Legends - PDF file 41K, Legends for supplemental figures 1-4

Supplementary Figures 1-4 - PDF file 294K, Supplementary Figure 1: PKM2 activation by screening hit XC-409; Supplementary Figure 2: Phosphorylation state of PKM2 at Tyr105 in cancer cell lines; supplementary Figure 3: Viability of a selection of lung adenocarcinoma cells in media with and without serine; Supplementary Figure 4: Sensitivity of a panel of adherent cancer cell lines to serine deprivation and treatment with PKM2 activator SGI-10067

Epstein–Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells—part of a phenomenon known as the “Warburg effect”— and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-(13)C(6)–glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.

Epstein-Barr Virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune-suppressed.In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells-part of a phenomenon known as the “Warburg effect,” and is mediated by the monocarboxylate transporters 1 and 4 (MCT1 and MCT4). However, the role of MCT1/4 has yet to be studied in EBV-associated malignancies which display Warburg-like metabolismin vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1 respectively, with MCT1 being induced early after infection and MCT4 late. Remarkably, singular MCT1 inhibition early, and dual MCT1/4 inhibition in LCLs using a novel MCT4-selective inhibitor led to growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significatly reduced oxygen consumption rates (OCR) and NAD+/NADH ratios, contrary to prevous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/MCT4-inhibited cancer cells. Furthermore, U-13C6 glucose labeling of MCT1/4-inhibited LCLs also revealed increased labeling of glutathione in the presence of elevated ROS and depleted glutathione pools, as well as increased labeling ofde novopyrimidine biosynthetic intermediates, suggesting broad effects on LCL metabolism. These vulnerabilities sensitized LCLs as well as EBV+, and the related gammaherpesvirus KSHV+ lymphoma cell lines to killing by metformin and phenformin, pointing at a novel therapeutic approach for viral lymphomas.

The metabolic rewiring of cardiomyocytes is a widely accepted hallmark of heart failure (HF). These metabolic changes include a decrease in mitochondrial pyruvate oxidation and an increased export of lactate. We identify the Mitochondrial Pyruvate Carrier (MPC) and the cellular lactate exporter Monocarboxylate Transporter 4 (MCT4), as pivotal nodes in this metabolic axis. We observed that cardiac assist device-induced myocardial recovery in chronic HF patients was coincident with increased myocardial expression of the MPC. Moreover, the genetic ablation of the MPC in cultured cardiomyocytes and in adult murine hearts was sufficient to induce hypertrophy and HF. Conversely, MPC overexpression attenuated drug-induced hypertrophy in a cell-autonomous manner. We also introduced a novel, highly potent MCT4 inhibitor that mitigated hypertrophy in cultured cardiomyocytes and in mice. Together, we find that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure.

Activators of the pyruvate kinase M2 (PKM2) are currently attracting significant interest as potential anticancer therapies. They may achieve a novel antiproliferation response in cancer cells through modulation of the classic ‘Warburg effect’ characteristic of aberrant metabolism. In this Letter, we describe the optimization of a weakly active screening hit to a structurally novel series of small molecule 3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as potent PKM2 activators.

Epigenetic modification of DNA leads to changes in gene expression. DNA methyltransferases (DNMTs) comprise a family of nuclear enzymes that catalyze the methylation of CpG dinucleotides, resulting in an epigenetic methylome distinguished between normal cells and those in disease states such as cancer. Disrupting gene expression patterns through promoter methylation has been implicated in many malignancies and supports DNMTs as attractive therapeutic targets. This review focuses on the rationale of targeting DNMTs in cancer, the historical approach to DNMT inhibition, and current marketed hypomethylating therapeutics azacytidine and decitabine. In addition, we address novel DNMT inhibitory agents emerging in development, including CP-4200 and SGI-110, analogs of azacytidine and decitabine, respectively; the oligonucleotides MG98 and miR29a; and a number of reversible inhibitors, some of which appear to be selective against particular DNMT isoforms. Finally, we discuss future opportunities and challenges for next-generation therapeutics.

The biogenesis of the large (60S) ribosomal subunit in eukaryotes involves nucleolar, nucleoplasmic, and cytoplasmic steps. The cytoplasmic protein Rei1, found in all eukaryotes, was previously shown to be necessary for the nuclear reimport of 60S subunit export factor Arx1. In this study we investigate the function of Reh1, a protein with high sequence similarity to Rei1. We demonstrate an overlapping function for Reh1 and Rei1 in the cytoplasmic maturation of the 60S subunit that is independent of Arx1 recycling. We observe that strains lacking both Reh1 and Rei1 accumulate salt-labile 60S subunits, suggesting that Reh1/Rei1 is necessary for the cytoplasmic 60S subunit to adopt its mature, stable form.

The peptidyl transferase center of the ribosome is literally the mother of all protein enzymes, yet it was only recently that the content of its active site was found to be RNA and not protein. This review focuses on how this RNA enzyme orients its substrates and promotes the chemical reaction that is center stage in protein synthesis.

The crystal structure of the ribosomal 50S subunit from Haloarcula marismortui in complex with the transition state analog CCdA-phosphate-puromycin (CCdApPmn) led to a mechanistic proposal wherein the universally conversed A2451 in the ribosomal active site acts as an “oxyanion hole” to promote the peptidyl transferase reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. (2000) Science 289, 920–929]. In the model, close proximity (3 Å) between the A2451 N3 and the nonbridging phosphoramidate oxygen of CCdApPmn suggested that the carbonyl oxyanion formed during the tetrahedral transition state is stabilized by hydrogen bonding to the protonated A2451 N3, the pKa of which must be perturbed substantially. We characterize the contribution of the putative hydrogen bond between the N3 of A2451 and the nonbridging phosphoramidate oxygen by using chemical protection and peptidyl transfer inhibition assays. If this putative hydrogen bond makes a significant thermodynamic contribution, then CCdApPmn-binding affinity to the 50S ribosomal subunit should be strongly pH-dependent, with affinity increasing as the pH is lowered. We report that CCdApPmn binds 50S ribosomes with essentially equal affinity at all pH values between 5.0 and 8.5. These data argue against a mechanism for peptidyl transfer in which a residue with near neutral pKa stabilizes the transition-state oxyanion, at least to the extent that CCdApPmn accurately mimics the transition state.

Inactivation of the M2 form of pyruvate kinase (PKM2) in cancer cells is associated with increased tumorigenicity. To test the hypothesis that tumor growth may be inhibited through the PKM2 pathway, we generated a series of small-molecule PKM2 activators. The compounds exhibited low nanomolar activity in both biochemical and cell-based PKM2 activity assays. These compounds did not affect the growth of cancer cell lines under normal conditions in vitro, but strongly inhibited the proliferation of multiple lung cancer cell lines when serine was absent from the cell culture media. In addition, PKM2 activators inhibited the growth of an aggressive lung adenocarcinoma xenograft. These findings show that PKM2 activation by small molecules influences the growth of cancer cells in vitro and in vivo, and suggest that such compounds may augment cancer therapies. Mol Cancer Ther; 12(8); 1453–60. ©2013 AACR.

2-Arylamino-4-aryl-pyrimidines were found to be potent inhibitors of PAK1 kinase. The synthesis and SAR are described. The incorporation of a bromide at the 5-position of the pyrimidine core and in combination with a 1,2-dimethylpiperazine pendant domain yielded a lead compound with potent PAK1 inhibition and anti-proliferative activity in various colon cancer cell lines.

A series of compounds based on a 4-phenyl-2-phenylaminopyridine scaffold that are potent and selective inhibitors of Traf2- and Nck-interacting kinase (TNIK) activity are described. These compounds were used as tools to test the importance of TNIK kinase activity in signaling and proliferation in Wnt-activated colorectal cancer cells. The results indicate that pharmacological inhibition of TNIK kinase activity has minimal effects on either Wnt/TCF4/β-catenin-driven transcription or viability. The findings suggest that the kinase activity of TNIK may be less important to Wnt signaling than other aspects of TNIK function, such as its putative role in stabilizing the TCF4/β-catenin transcriptional complex.

The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.

Among the clinically used nucleoside analogue inhibitors that target human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), there is little detailed mechanistic information on the interactions of 2*,3*-didehydro-2*,3*-dideoxythymidine-5*-triphosphate (d4TTP) with the enzyme z primer-template complex and how these interactions compare with those of the natural substrate, dTTP. Using a pre-steady-state kinetic analysis, we found that d4TTP was incorporated by HIV-1 RT just as efficiently as dTTP during both DNA- and RNA-dependent DNA synthesis. To our knowledge, these results represent the first observation of a 3*-modified nucleoside triphosphate analogue that has an incorporation efficiency comparable to that observed for the natural substrate during DNA synthesis by HIV-1 RT. This information provides a mechanistic basis for understanding the inhibition of HIV-1 RT by d4TTP as well as insight into the clinically observed lack of d4T resistance mutations in HIV-1 RT isolated from AIDS patients. The replication of human immunodeficiency virus (HIV), which causes AIDS, requires the virally encoded enzyme reverse transcriptase (RT). RT converts the single-stranded HIV RNA genome to a double-stranded DNA copy by catalyzing both DNA-dependent and RNA-dependent DNA polymerization as well as RNase H cleavage activity to remove the RNA template once the DNA has been synthesized. Because of its unique catalytic properties, RT has been the target enzyme for many antiviral therapeutic agents used in the treatment of AIDS, including nucleoside and nonnucleoside analogues (2‐4, 8, 26). The nucleoside analogues that are used clinically

Tumor cells primarily utilize aerobic glycolysis, rather than oxidative phosphorylation, to metabolize glucose (the Warburg effect). The M2 splice form of pyruvate kinase (PKM2), the enzyme catalyzing the rate-limiting final step of glycolysis, is highly upregulated in tumors. Unlike the M1 splice form (PKM1), a constitutively active tetramer found predominantly in non-cancerous tissues, PKM2 is an inactive dimer under normal physiological conditions. Tetramerization of PKM2 requires binding of the allosteric activator fructose-1,6-bisphosphate (FBP), an upstream glycolytic intermediate, resulting in a fully active enzyme. Inactivation of PKM2 by cancer cells may allow glycolytic intermediates to be diverted into other biosynthetic pathways necessary for biomass production. The finding that PKM2 rather than PKM1 expression enhances tumorigenicity suggests that activators of PKM2 may have anti-tumor properties. We have identified and developed a series of small molecule PKM2 activators that exhibit low nM activation activity in biochemical and cell-based assays that measure pyruvate and ATP production. The extent of activation of these compounds is equal to or greater than that of FBP in biochemical assays. In addition, preliminary studies show that PKM2 activators inhibit the growth of lung cancer cell lines in vitro. The current lead compound was tested in established subcutaneously implanted A549 lung adenocarcinoma xenografts, where we observed a statistically significant 54% decrease in tumor growth, with no observable toxicity. These data suggest that this class of PKM2 activators is effective as tumor cell metabolic regulators with anti-tumor activity for lung cancer and potentially other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B143.