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39


PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

Ilic, Nina; Birsoy, Kivanc; Aguirre, Andrew J; Kory, Nora; Pacold, Michael E; Singh, Shambhavi; Moody, Susan E; DeAngelo, Joseph D; Spardy, Nicole A; Freinkman, Elizaveta; Weir, Barbara A; Tsherniak, Aviad; Cowley, Glenn S; Root, David E; Asara, John M; Vazquez, Francisca; Widlund, Hans R; Sabatini, David M; Hahn, William C
Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.
PMCID:5410781
PMID: 28396387
ISSN: 1091-6490
CID: 2546302

Extracellular RNAs Are Associated With Insulin Resistance and Metabolic Phenotypes

Shah, Ravi; Murthy, Venkatesh; Pacold, Michael; Danielson, Kirsty; Tanriverdi, Kahraman; Larson, Martin G; Hanspers, Kristina; Pico, Alexander; Mick, Eric; Reis, Jared; de Ferranti, Sarah; Freinkman, Elizaveta; Levy, Daniel; Hoffmann, Udo; Osganian, Stavroula; Das, Saumya; Freedman, Jane E
OBJECTIVE:Insulin resistance (IR) is a hallmark of obesity and metabolic disease. Circulating extracellular RNAs (ex-RNAs), stable RNA molecules in plasma, may play a role in IR, though most studies on ex-RNAs in IR are small. We sought to characterize the relationship between ex-RNAs and metabolic phenotypes in a large community-based human cohort. RESEARCH DESIGN AND METHODS:We measured circulating plasma ex-RNAs in 2,317 participants without diabetes in the Framingham Heart Study (FHS) Offspring Cohort at cycle 8 and defined associations between ex-RNAs and IR (measured by circulating insulin level). We measured association between candidate ex-RNAs and markers of adiposity. Sensitivity analyses included individuals with diabetes. In a separate cohort of 90 overweight/obese youth, we measured selected ex-RNAs and metabolites. Biology of candidate microRNAs was investigated in silico. RESULTS:. In age-, sex-, and BMI-adjusted models across 391 ex-RNAs in FHS, 18 ex-RNAs were associated with IR (of which 16 were microRNAs). miR-122 was associated with IR and regional adiposity in adults and IR in children (independent of metabolites). Pathway analysis revealed metabolic regulatory roles for miR-122, including regulation of IR pathways (AMPK, target of rapamycin signaling, and mitogen-activated protein kinase). CONCLUSIONS:These results provide translational evidence in support of an important role of ex-RNAs as novel circulating factors implicated in IR.
PMCID:5360281
PMID: 28183786
ISSN: 1935-5548
CID: 5688572

Corrigendum: A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate

Pacold, Michael E; Brimacombe, Kyle R; Chan, Sze Ham; Rohde, Jason M; Lewis, Caroline A; Swier, Lotteke J Y M; Possemato, Richard; Chen, Walter W; Sullivan, Lucas B; Fiske, Brian P; Cho, Steve; Freinkman, Elizaveta; Birsoy, Kivanc; Abu, Monther-Remaileh; Shaul, Yoav D; Liu, Chieh Min; Zhou, Minerva; Koh, Min Jung; Chung, Haeyoon; Davidson, Shawn M; Luengo, Alba; Wang, Amy Q; Xu, Xin; Yasgar, Adam; Liu, Li; Rai, Ganesha; Westover, Kenneth D; Heiden, Matthew G Vander; Shen, Min; Gray, Nathanael S; Boxer, Matthew B; Sabatini, David M
PMID: 27434767
ISSN: 1552-4469
CID: 2245272

A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate

Pacold, Michael E; Brimacombe, Kyle R; Chan, Sze Ham; Rohde, Jason M; Lewis, Caroline A; Swier, Lotteke J Y M; Possemato, Richard; Chen, Walter W; Sullivan, Lucas B; Fiske, Brian P; Cho, Steve; Freinkman, Elizaveta; Birsoy, Kivanc; Abu-Remaileh, Monther; Shaul, Yoav D; Liu, Chieh Min; Zhou, Minerva; Koh, Min Jung; Chung, Haeyoon; Davidson, Shawn M; Luengo, Alba; Wang, Amy Q; Xu, Xin; Yasgar, Adam; Liu, Li; Rai, Ganesha; Westover, Kenneth D; Vander Heiden, Matthew G; Shen, Min; Gray, Nathanael S; Boxer, Matthew B; Sabatini, David M
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
PMCID:4871733
PMID: 27110680
ISSN: 1552-4469
CID: 2092382

Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway

Saxton, Robert A; Knockenhauer, Kevin E; Wolfson, Rachel L; Chantranupong, Lynne; Pacold, Michael E; Wang, Tim; Schwartz, Thomas U; Sabatini, David M
Eukaryotic cells coordinate growth with the availability of nutrients through the mechanistic target of rapamycin complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag guanosine triphosphatases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. Here we present the 2.7 angstrom crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway.
PMCID:4698039
PMID: 26586190
ISSN: 1095-9203
CID: 2245262

SHMT2 drives glioma cell survival in ischaemia but imposes a dependence on glycine clearance

Kim, Dohoon; Fiske, Brian P; Birsoy, Kivanc; Freinkman, Elizaveta; Kami, Kenjiro; Possemato, Richard L; Chudnovsky, Yakov; Pacold, Michael E; Chen, Walter W; Cantor, Jason R; Shelton, Laura M; Gui, Dan Y; Kwon, Manjae; Ramkissoon, Shakti H; Ligon, Keith L; Kang, Seong Woo; Snuderl, Matija; Vander Heiden, Matthew G; Sabatini, David M
Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.
PMCID:4533874
PMID: 25855294
ISSN: 1476-4687
CID: 1522042

Dihydropyrimidine accumulation is required for the epithelial-mesenchymal transition

Shaul, Yoav D; Freinkman, Elizaveta; Comb, William C; Cantor, Jason R; Tam, Wai Leong; Thiru, Prathapan; Kim, Dohoon; Kanarek, Naama; Pacold, Michael E; Chen, Walter W; Bierie, Brian; Possemato, Richard; Reinhardt, Ferenc; Weinberg, Robert A; Yaffe, Michael B; Sabatini, David M
It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits.
PMCID:4250222
PMID: 25171410
ISSN: 0092-8674
CID: 1210452

Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites

Schwartz, Schraga; Mumbach, Maxwell R; Jovanovic, Marko; Wang, Tim; Maciag, Karolina; Bushkin, G Guy; Mertins, Philipp; Ter-Ovanesyan, Dmitry; Habib, Naomi; Cacchiarelli, Davide; Sanjana, Neville E; Freinkman, Elizaveta; Pacold, Michael E; Satija, Rahul; Mikkelsen, Tarjei S; Hacohen, Nir; Zhang, Feng; Carr, Steven A; Lander, Eric S; Regev, Aviv
N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing "basal" degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.
PMCID:4142486
PMID: 24981863
ISSN: 2211-1247
CID: 1678992

Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor

Lim, Sang Min; Westover, Kenneth D; Ficarro, Scott B; Harrison, Rane A; Choi, Hwan Geun; Pacold, Michael E; Carrasco, Martin; Hunter, John; Kim, Nam Doo; Xie, Ting; Sim, Taebo; Janne, Pasi A; Meyerson, Matthew; Marto, Jarrod A; Engen, John R; Gray, Nathanael S
We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.
PMCID:3914205
PMID: 24259466
ISSN: 1521-3773
CID: 2245252

Supportive and palliative radiation oncology service: impact of a dedicated service on palliative cancer care

Tseng, Yolanda D; Krishnan, Monica S; Jones, Joshua A; Sullivan, Adam J; Gorman, Daniel; Taylor, Allison; Pacold, Michael; Kalinowski, Barbara; Mamon, Harvey J; Abrahm, Janet; Balboni, Tracy A
PURPOSE/OBJECTIVE:The American Society of Clinical Oncology has recommended tailoring palliative cancer care (PCC) to the distinct and complex needs of advanced cancer patients. The Supportive and Palliative Radiation Oncology (SPRO) service was initiated July 2011 to provide dedicated palliative radiation oncology (RO) care to cancer patients. We used care providers' ratings to assess SPRO's impact on the quality of PCC and compared perceptions of PCC delivery among physicians practicing with and without a dedicated palliative RO service. METHODS AND MATERIALS/METHODS:An online survey was sent to 117 RO care providers working at 4 Boston-area academic centers. Physicians and nurses at the SPRO-affiliated center rated the impact of the SPRO service on 8 PCC quality measures (7-point scale, "very unfavorably" to "very favorably"). Physicians at all sites rated their department's performance on 10 measures of PCC (7-point scale, "very poorly" to "very well"). RESULTS:Among 102 RO care providers who responded (response rate, 89% for physicians; 83% for nurses), large majorities believed that SPRO improved the following PCC quality measures: overall quality of care (physician/nurse, 98%/92%); communication with patients and families (95%/96%); staff experience (93%/84%); time spent on technical aspects of PCC (eg, reviewing imaging) (88%/56%); appropriateness of treatment recommendations (85%/84%); appropriateness of dose/fractionation (78%/60%); and patient follow-up (64%/68%). Compared with physicians practicing in departments without a dedicated palliative RO service, physicians at the SPRO-affiliated department rated the overall quality of their department's PCC more highly (P = .02). CONCLUSIONS:Clinicians indicated that SPRO improved the quality of PCC. Physicians practicing within this dedicated service rated their department's overall PCC quality higher than physicians practicing at academic centers without a dedicated service. These findings point to dedicated palliative RO services as a promising means of improving PCC quality.
PMCID:4617676
PMID: 25012833
ISSN: 1879-8519
CID: 5688562