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Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle

Shin, Jong; Mir, Hannan; Khurram, Maaz A; Fujihara, Kenji M; Dynlacht, Brian D; Cardozo, Timothy J; Possemato, Richard
Metabolism is a fundamental cellular process that is coordinated with cell cycle progression. Despite this association, a mechanistic understanding of cell cycle phase-dependent metabolic pathway regulation remains elusive. Here we report the mechanism by which human de novo pyrimidine biosynthesis is allosterically regulated during the cell cycle. Combining traditional synchronization methods and metabolomics, we characterize metabolites by their accumulation pattern during cell cycle phases and identify cell cycle phase-dependent regulation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase (CAD), the first, rate-limiting enzyme in de novo pyrimidine biosynthesis. Through systematic mutational scanning and structural modelling, we find allostery as a major regulatory mechanism that controls the activity change of CAD during the cell cycle. Specifically, we report evidence of two Animalia-specific loops in the CAD allosteric domain that involve sensing and binding of uridine 5'-triphosphate, a CAD allosteric inhibitor. Based on homology with a mitochondrial carbamoyl-phosphate synthetase homologue, we identify a critical role for a signal transmission loop in regulating the formation of a substrate channel, thereby controlling CAD activity.
PMID: 36747088
ISSN: 2522-5812
CID: 5422782

Iron-sulfur cluster deficiency can be sensed by IRP2 and regulates iron homeostasis and sensitivity to ferroptosis independent of IRP1 and FBXL5

Terzi, Erdem M; Sviderskiy, Vladislav O; Alvarez, Samantha W; Whiten, Gabrielle C; Possemato, Richard
Intracellular iron levels are strictly regulated to support homeostasis and avoid iron-mediated ROS production. Loss of iron-sulfur cluster (ISC) synthesis can increase iron loading and promote cell death by ferroptosis. Iron-responsive element-binding proteins IRP1 and IRP2 posttranscriptionally regulate iron homeostasis. IRP1 binding to target mRNAs is competitively regulated by ISC occupancy. However, IRP2 is principally thought to be regulated at the protein level via E3 ubiquitin ligase FBXL5-mediated degradation. Here, we show that ISC synthesis suppression can activate IRP2 and promote ferroptosis sensitivity via a previously unidentified mechanism. At tissue-level O2 concentrations, ISC deficiency enhances IRP2 binding to target mRNAs independent of IRP1, FBXL5, and changes in IRP2 protein level. Deletion of both IRP1 and IRP2 abolishes the iron-starvation response, preventing its activation by ISC synthesis inhibition. These findings will inform strategies to manipulate ferroptosis sensitivity and help illuminate the mechanism underlying ISC biosynthesis disorders, such as Friedreich's ataxia.
PMID: 34039609
ISSN: 2375-2548
CID: 4888832

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε

Sviderskiy, Vladislav O; Blumenberg, Lili; Gorodetsky, Elizabeth; Karakousi, Triantafyllia R; Hirsh, Nicole; Alvarez, Samantha W; Terzi, Erdem M; Kaparos, Efiyenia; Whiten, Gabrielle C; Ssebyala, Shakirah; Tonzi, Peter; Mir, Hannan; Neel, Benjamin G; Huang, Tony T; Adams, Sylvia; Ruggles, Kelly V; Possemato, Richard
Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.
PMID: 33152268
ISSN: 1097-4164
CID: 4664322

NFS1 undergoes positive selection in lung tumours and protects cells from ferroptosis

Alvarez, Samantha W; Sviderskiy, Vladislav O; Terzi, Erdem M; Papagiannakopoulos, Thales; Moreira, Andre L; Adams, Sylvia; Sabatini, David M; Birsoy, Kivanc; Possemato, Richard
Environmental nutrient levels impact cancer cell metabolism, resulting in context-dependent gene essentiality. Here, using loss-of-function screening based on RNA interference, we show that environmental oxygen levels are a major driver of differential essentiality between in vitro model systems and in vivo tumours. Above the 3-8% oxygen concentration typical of most tissues, we find that cancer cells depend on high levels of the iron-sulfur cluster biosynthetic enzyme NFS1. Mammary or subcutaneous tumours grow despite suppression of NFS1, whereas metastatic or primary lung tumours do not. Consistent with a role in surviving the high oxygen environment of incipient lung tumours, NFS1 lies in a region of genomic amplification present in lung adenocarcinoma and is most highly expressed in well-differentiated adenocarcinomas. NFS1 activity is particularly important for maintaining the iron-sulfur co-factors present in multiple cell-essential proteins upon exposure to oxygen compared to other forms of oxidative damage. Furthermore, insufficient iron-sulfur cluster maintenance robustly activates the iron-starvation response and, in combination with inhibition of glutathione biosynthesis, triggers ferroptosis, a non-apoptotic form of cell death. Suppression of NFS1 cooperates with inhibition of cysteine transport to trigger ferroptosis in vitro and slow tumour growth. Therefore, lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage.
PMCID:5808442
PMID: 29168506
ISSN: 1476-4687
CID: 2792182

Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl-tRNAs

Minton, Denise R; Nam, Minwoo; McLaughlin, Daniel J; Shin, Jong; Bayraktar, Erol C; Alvarez, Samantha W; Sviderskiy, Vladislav O; Papagiannakopoulos, Thales; Sabatini, David M; Birsoy, Kıvanç; Possemato, Richard
Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria.
PMCID:5819360
PMID: 29452640
ISSN: 1097-4164
CID: 2958432

Functional genomics reveal that the serine synthesis pathway is essential in breast cancer

Possemato, Richard; Marks, Kevin M; Shaul, Yoav D; Pacold, Michael E; Kim, Dohoon; Birsoy, Kivanc; Sethumadhavan, Shalini; Woo, Hin-Koon; Jang, Hyun G; Jha, Abhishek K; Chen, Walter W; Barrett, Francesca G; Stransky, Nicolas; Tsun, Zhi-Yang; Cowley, Glenn S; Barretina, Jordi; Kalaany, Nada Y; Hsu, Peggy P; Ottina, Kathleen; Chan, Albert M; Yuan, Bingbing; Garraway, Levi A; Root, David E; Mino-Kenudson, Mari; Brachtel, Elena F; Driggers, Edward M; Sabatini, David M
Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.
PMCID:3353325
PMID: 21760589
ISSN: 0028-0836
CID: 1086622

Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides

Birsoy, Kivanc; Possemato, Richard; Lorbeer, Franziska K; Bayraktar, Erol C; Thiru, Prathapan; Yucel, Burcu; Wang, Tim; Chen, Walter W; Clish, Clary B; Sabatini, David M
As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here we developed a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low-glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNA interference (RNAi) screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the OXPHOS upregulation that is normally caused by glucose limitation as a result of either mitochondrial DNA (mtDNA) mutations in complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, antidiabetic drugs that inhibit OXPHOS, when cancer cells are grown in low glucose or as tumour xenografts. Notably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of complex I function. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.
PMCID:4012432
PMID: 24670634
ISSN: 0028-0836
CID: 1086572

Iron-sulfur cluster metabolism impacts iron homeostasis, ferroptosis sensitivity, and human disease

Chapter by: Sviderskiy, Vladislav O.; Terzi, Erdem M.; Possemato, Richard
in: Ferroptosis in Health and Disease by
[S.l.] : Springer International Publishing, 2019
pp. 215-237
ISBN: 9783030267797
CID: 4508572

The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

Ravn-Boess, Niklas; Roy, Nainita; Hattori, Takamitsu; Bready, Devin; Donaldson, Hayley; Lawson, Christopher; Lapierre, Cathryn; Korman, Aryeh; Rodrick, Tori; Liu, Enze; Frenster, Joshua D; Stephan, Gabriele; Wilcox, Jordan; Corrado, Alexis D; Cai, Julia; Ronnen, Rebecca; Wang, Shuai; Haddock, Sara; Sabio Ortiz, Jonathan; Mishkit, Orin; Khodadadi-Jamayran, Alireza; Tsirigos, Aris; Fenyö, David; Zagzag, David; Drube, Julia; Hoffmann, Carsten; Perna, Fabiana; Jones, Drew R; Possemato, Richard; Koide, Akiko; Koide, Shohei; Park, Christopher Y; Placantonakis, Dimitris G
Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.
PMID: 37938973
ISSN: 2211-1247
CID: 5590372

SHORT CHAIN FATTY ACIDS MITIGATE OSTEOCLAST-MEDIATED ARTHRITIC BONE REMODELLING

Yang, Katharine Lu; Mullins, Briana J; Lejeune, Alannah; Ivanova, Ellie; Shin, Jong; Bajwa, Sofia; Possemato, Richard; Cadwell, Ken; Scher, Jose U; Koralov, Sergei B
OBJECTIVE:To study the effects of Short Chain Fatty Acids (SCFAs) on arthritic bone remodeling. METHODS:CD4Cre mice, with SCFA supplemented water. We also performed in vitro osteoclast differentiation assays in the presence of serum-level SCFAs to evaluate the direct impact of these microbial metabolites on maturation and function of osteoclasts. We further characterized the molecular mechanism of SCFAs by transcriptional analysis. RESULTS:CD4Cre mice. Further interrogation revealed that bone marrow derived OCPs from diseased mice expressed a higher level of SCFA receptors than that of control mice and that the progenitor cells in the bone marrow of SCFA-treated mice presented a modified transcriptomic landscape, suggesting a direct impact of SCFAs on bone marrow progenitors in the context of osteoporosis. CONCLUSION/CONCLUSIONS:We demonstrated how gut microbiota-derived SCFAs can regulate distal pathology, i.e., osteoporosis, and identified a potential therapeutic option for restoring bone density in rheumatic disease, further highlighting the critical role of the gut-bone axis in these disorders.
PMID: 37994265
ISSN: 2326-5205
CID: 5608662