Hong Yin

Metabolic Signatures Uncover Distinct Targets in Molecular Subsets of Diffuse Large B Cell Lymphoma

Molecular signatures have identified several subsets of diffuse large B cell lymphoma (DLBCL) and rational targets within the B cell receptor (BCR) signaling axis. The OxPhos-DLBCL subset, which harbors the signature of genes involved in mitochondrial metabolism, is insensitive to inhibition of BCR survival signaling but is functionally undefined. We show that, compared with BCR-DLBCLs, OxPhos-DLBCLs display enhanced mitochondrial energy transduction, greater incorporation of nutrient-derived carbons into the tricarboxylic acid cycle, and increased glutathione levels. Moreover, perturbation of the fatty acid oxidation program and glutathione synthesis proved selectively toxic to this tumor subset. Our analysis provides evidence for distinct metabolic fingerprints and associated survival mechanisms in DLBCL and may have therapeutic implications.

Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5.

Tumors put in a vulnerable position Cancer cells often display alterations in metabolism that help fuel their growth. Such metabolic “rewiring” may also work against the cancer cells, however, by creating new vulnerabilities that can be exploited therapeutically. A variety of human tumors show changes in methionine metabolism caused by loss of the gene coding for 5-methylthioadenosine phosphorylase (MTAP). Mavrakis et al. and Kryukov et al. found that the loss of MTAP renders cancer cell lines sensitive to growth inhibition by compounds that suppress the activity of a specific arginine methyltransferase called PRMT5. Conceivably, drugs that inhibit PRMT5 activity could be developed into a tailored therapy for MTAP-deficient tumors. Science, this issue pp. 1208 and 1214 Tumors cope with a genomic change by rewiring their metabolism, but this makes them more susceptible to certain drugs. 5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA–mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP–deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.

M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth

Many cancer cells have increased rates of aerobic glycolysis, a phenomenon termed the Warburg effect. In addition, in tumors there is a predominance of expression of the M2 isoform of pyruvate kinase (PKM2). M2 expression was previously shown to be necessary for aerobic glycolysis and to provide a growth advantage to tumors. We report that knockdown of pyruvate kinase in tumor cells leads to a decrease in the levels of pyruvate kinase activity and an increase in the pyruvate kinase substrate phosphoenolpyruvate. However, lactate production from glucose, although reduced, was not fully inhibited. Furthermore, we are unique in reporting increased serine and glycine biosynthesis from both glucose and glutamine following pyruvate kinase knockdown. Although pyruvate kinase knockdown results in modest impairment of proliferation in vitro, in vivo growth of established xenograft tumors is unaffected by PKM2 absence. Our findings indicate that PKM2 is dispensable for tumor maintenance and growth in vivo, suggesting that other metabolic pathways bypass its function.

IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae—Erg26p, Homo sapiens—NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound.

Lack of Involvement of CEP Adducts in TLR Activation and in Angiogenesis

Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an in vivo TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. In vivo, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, in vivo inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others.

Abstract LB-139: IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH alters central carbon metabolism, though, remains unclear. To address this question, we performed 13C metabolic flux analysis (MFA) on an isogenic cell panel containing heterozygous IDH1/2 mutations. We observe a dramatic and consistent decrease in the ability of IDH1, but not IDH2, mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate to isocitrate. Additionally we find that cells with IDH1 mutations exhibit increased oxidative tricarboxylic acid (TCA) metabolism. Similar metabolic trends were observed in vivo as well, and also in endogenous, non-engineered IDH1/2 mutant cell lines. Interestingly, IDH1-mutant specific inhibitors were unable to reverse the decrease in reductive metabolism, suggesting that this metabolic phenotype is independent of 2-HG. Furthermore, this metabolic reprogramming increases the sensitivity of IDH1 mutant cells to hypoxia or electron transport chain (ETC) inhibition in vitro . IDH1 mutant cells also grow poorly as subcutaneous xenografts within hypoxic in vivo microenvironments. These results suggest that exploiting metabolic defects specific to IDH1 mutant cells could be an interesting avenue to explore therapeutically. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Ajit Divakaruni, Courtney Green, Xiamei Zhang, Kelly Slocum, Minying Pu, Fallon Lin, Chad Vickers, Carol Joud-Caldwell, Franklin Chung, Hong Yin, Erika Handly, Christopher Straub, Joseph D. Growney, Matt Vander Heiden, Anne Murphy, Raymond Pagliarini, Christian Metallo. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2014-LB-139

Abstract LB-017: Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to marked dependence on PRMT5

Metabolic genes are increasingly recognized as targets of somatic genetic alteration in human cancer often leading to profound changes in intracellular metabolite concentrations. 5-Methylthioadenosine Phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that metabolizes methylthioadenosine (MTA) to adenine and methionine. Its chromosomal position proximal to CDKN2A results in frequent collateral homozygous deletion in a wide range of human cancers. By interrogating data from a large scale deep-coverage pooled shRNA screen across 390 cancer cell line models we found that the viability of MTAP null cancer cells is strongly impaired upon shRNA-mediated depletion of the protein arginine methyltransferase PRMT5. In MTAP deleted cells there is marked accumulation of the substrate MTA and surprisingly, we find that MTA is a specific inhibitor of the catalytic activity of PRMT5. In keeping with these data, knockout of MTAP in an MTAP-proficient cell line led to increased MTA levels and rendered them sensitive to PRMT5 depletion. Moreover, reconstitution of MTAP in an MTAP-deficient cell line fully rescued PRMT5 dependence. Collectively, these findings indicate that the collateral loss of MTAP in CDNK2A deleted cancers leads to accumulation of MTA that thereby creates a hypomorphic PRMT5 state that is selectively sensitized towards further PRMT5 inhibition. Citation Format: Konstantinos Mavrakis, E Robert McDonald III, Michael R. Schlabach, Eric Billy, Gregory R. Hoffman, Antoine deWeck, David A. Ruddy, Kavitha Venkatesan, Greg McAllister, Rosalie deBeaumont, Samuel Ho, Yue Liu, Yan Yan-Neale, Guizhi Yang, Fallon Lin, Hong Yin, Hui Gao, David Randal Kipp, Songping Zhao, Joshua T. McNamara, Elizabeth R. Sprague, Young Shin Cho, Justin Gu, Ken Crawford, Vladimir Capka, Kristen Hurov, Jeffrey A. Porter, John Tallarico, Craig Mickanin, Emma Lees, Raymond Pagliarini, Nicholas Keen, Tobias Schmelzle, Francesco Hofmann, Frank Stegmeier, William R. Sellers. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to marked dependence on PRMT5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-017.

Abstract B159: Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types. Although these mutations are loss-of-function for conversion of isocitrate to α-ketoglutarate, the mutant enzymes greatly increase the production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). However the full metabolic consequences of IDH1/2 mutation in their heterozygous cellular context have yet to be fully explored. To address this question, we utilized a panel of isogenic cell lines with wild-type IDH1/2 or clinically relevant IDH1/2 mutations and examined the metabolic consequences of IDH mutation using (13)C metabolic flux analysis (MFA). We observe a dramatic and consistent decrease in the ability of IDH1 mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate back to isocitrate. This was not seen either in IDH2 mutant cell lines or in wild-type cell lines treated with exogenous 2-HG. Consistent with these changes, the IDH1 mutant cell lines, but not IDH2 mutant or 2-HG treated cells, were deficient in the utilization of glutamine for de novo lipogenesis. Similar trends were observed in endogenous, non-engineered IDH1/2 mutant cell lines. The decrease in reductive carboxylation in the IDH1 mutant cell lines raises the hypothesis that these cells may be more reliant on mitochondrial metabolism. Indeed, IDH1 mutant cells were more sensitive to either treatment with an electron transport chain inhibitor or growth in hypoxia (which also inhibits mitochondrial metabolism). These results show heterozygous IDH1 mutation robustly impacts wild-type cellular metabolism in a different manner than IDH2 mutation. Furthermore, these results suggest that IDH1 and IDH2 mutant tumors may be differentially sensitive to inhibitors of specific metabolic pathways. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B159. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Courtney Green, Fallon Lin, Carol Joud-Caldwell, Hong Yin, Franklin Chung, Christopher Straub, Matthew Vander Heiden, Raymond Pagliarini, Christian Metallo. Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B159.