Michael E. Pacold

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

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation (1,2). RNAi-based loss of function screening has proven powerful for the identification of novel and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumor suppressor genes (3). Here, we developed a method for identifying novel cancer targets via negative selection RNAi screening in solid tumours. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumourigenesis. 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 ER-negative breast cancers. PHGDH catalyzes the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have elevations in serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not 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 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 over-expression and demonstrate the utility of in vivo negative selection RNAi screens for finding potential anticancer targets.

mTORC1 Phosphorylation Sites Encode Their Sensitivity to Starvation and Rapamycin

Introduction The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes cell growth by controlling major anabolic and catabolic processes in response to a variety of environmental and intracellular stimuli, and is deregulated in aging and human diseases such as cancer and diabetes. Rapamycin, an allosteric inhibitor of mTORC1, is used clinically in organ transplantation and the treatment of certain cancers. Exactly how rapamycin perturbs mTORC1 signaling is poorly understood and it remains unknown why certain mTORC1 phosphorylation sites are sensitive to the drug whereas others are not. Here, we test the hypothesis that the inherent capacity of a phosphorylation site to serve as an mTORC1 substrate (a property we call substrate quality) is a key determinant of its sensitivity to rapamycin as well as nutrient and growth factor starvation. mTORC1 Phosphorylation sites encode their sensitivity to physiological and pharmacological modulators of mTORC1. Substrate quality is an important determinant of how effectively mTORC1 phosphorylates its substrates in the response to both pharmacological and natural regulators ofthe kinase. Methods We measured the in vitro kinase activity of mTORC1 towards short synthetic peptides encompassing single mTORC1 phosphorylation sites and refined the established mTORC1 phosphorylation motif. We introduced subtle mutations into bona fide mTORC1 phosphorylation sites that we found to enhance or reduce their phosphorylation by mTORC1 in vitro and monitored the corresponding changes in the sensitivity of these sites to rapamycin treatment within cells. Finally, we assessed whether the modifications of the mTORC1 phosphorylation sites also altered their sensitivities to nutrient and growth factor starvation. Results The response of an mTORC1 phosphorylation site to rapamycin treatment should depend on the balance between the activity of mTORC1 and of the protein phosphatase(s) that dephosphorylates it. We found that the in vitro kinase activity of mTORC1 toward peptides containing established phosphorylation sites strongly correlates with the resistance of the sites to rapamycin within cells. Moreover, the relative affinities of the mTOR kinase domain for the peptides also correlated with its capacity to phosphorylate them. In addition to a preference for either proline or a nonproline hydrophobic residue in the +1 position, our refinement of the mTORC1 phosphorylation motif revealed preferences for noncharged residues surrounding the phosphoacceptor site and for serine over threonine as the phosphoacceptor. Utilizing this improved understanding of the sequence motif specificity of mTORC1, we were able to manipulate mTORC1 activity toward its phosphorylation sites in vitro and alter their sensitivities to rapamycin treatment within cells. Interestingly, mTORC1 phosphorylation sites also varied in their sensitivities to nutrient and growth factor levels and manipulations in substrate quality were sufficient to alter their responses to nutrient and growth factor starvation. Discussion Our findings suggest that the sequence composition of an mTORC1 phosphorylation site, including the presence of serine or threonine as the phosphoacceptor, is one of the key determinants of whether the site is a good or poor mTORC1 substrate within cells. Even though the phosphorylation of mTORC1 sites is subject to varied regulatory mechanisms, we propose that differences in substrate quality are one mechanism for allowing downstream effectors of mTORC1 to respond differentially to temporal and intensity changes in the levels of nutrients and growth factors as well as pharmacological inhibitors such as rapamycin. Such differential responses are likely important for mTORC1 to coordinate and appropriately time the myriad processes that make up the vast starvation program it controls. Lastly, it is likely that the form of hierarchical regulation we describe for mTORC1 substrates also exists in other kinase-driven signaling pathways. Not mTORCing Inhibition of the protein kinase complex mTORC1 has potentially beneficial therapeutic affects that include inhibition of cancer and extension of life span. However, effects of its inhibition in vivo have sometimes been disappointing. One reason may be that the well-studied inhibitor of mTORC1, rapamycin, inhibits some effects of mTORC1 but not others. In line with this idea, Kang et al. (1236566) show that the effect of rapamycin depends on the substrate. Characteristics of the phosphorylation sites on various substrates caused them to be phosphorylated with different efficiency by mTORC1. The substrates that were most efficiently phosphorylated were resistant to inhibition of mTORC1. The results explain how various sites, sometimes within the same protein, can differ in their sensitivity to rapamycin. Inhibition of a protein kinase differentially affects its targets, depending on phosphorylation site characteristics. The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.

The bromodomain protein Brd4 insulates chromatin from DNA damage signalling

DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.

Therapeutic Targeting of Oncogenic K‐Ras by a Covalent Catalytic Site Inhibitor

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.

Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway.

From sensing leucine to metabolic control The mTORC1 protein kinase complex plays central roles in regulating cell growth and metabolism and is implicated in common human diseases such as diabetes and cancer. The level of the amino acid leucine tells an organism a lot about its physiological state, including how much food is available, how much insulin is going to be needed, and whether new muscle mass can be made (see the Perspective by Buel and Blenis). Wolfson et al. identified a biochemical sensor of leucine, Sestrin2, which connects the concentration of leucine to the control of organismal metabolism and growth. When leucine bound to Sestrin2, it was released from a complex with the mTORC1 regulatory factor GATOR2, activating the mTORC1 complex. Saxton et al. describe the crystal structure of Sestrin2 and show how it specifically detects leucine. Aylett et al. determined the structure of human mTORC1 by cryoelectron microscopy and the crystal structure of a regulatory subunit, Raptor. The results reveal the structural basis for the function and intricate regulation of this important enzyme, which is also a strategic drug target. Science, this issue p. 43, p. 48, p. 53; see also p. 25 A crystal structure reveals how cells sense leucine for metabolic regulation. [Also see Perspective by Buel and Blenis] 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.

Revival of the abandoned therapeutic wortmannin by nanoparticle drug delivery

One of the promises of nanoparticle (NP) carriers is the reformulation of promising therapeutics that have failed clinical development due to pharmacologic challenges. However, current nanomedicine research has been focused on the delivery of established and novel therapeutics. Here we demonstrate proof of the principle of using NPs to revive the clinical potential of abandoned compounds using wortmannin (Wtmn) as a model drug. Wtmn is a potent inhibitor of phosphatidylinositol 3′ kinase-related kinases but failed clinical translation due to drug-delivery challenges. We engineered a NP formulation of Wtmn and demonstrated that NP Wtmn has higher solubility and lower toxicity compared with Wtmn. To establish the clinical translation potential of NP Wtmn, we evaluated the therapeutic as a radiosensitizer in vitro and in vivo. NP Wtmn was found to be a potent radiosensitizer and was significantly more effective than the commonly used radiosensitizer cisplatin in vitro in three cancer cell lines. The mechanism of action of NP Wtmn radiosensitization was found to be through the inhibition of DNA-dependent protein kinase phosphorylation. Finally, NP Wtmn was shown to be an effective radiosensitizer in vivo using two murine xenograft models of cancer. Our results demonstrate that NP drug-delivery systems can promote the readoption of abandoned drugs such as Wtmn by overcoming drug-delivery challenges.

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

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use 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 suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Yeast N-glycanase distinguishes between native and non-native glycoproteins

N‐glycanase from Saccharomyces cerevisiae (Png1) preferentially removes N‐glycans from misfolded proteins. The ability of Png1 to distinguish between folded and misfolded glycoproteins is reminiscent of substrate recognition by UDP‐glucose glycoprotein glucosyl transferase, an enzyme that possesses this trait. The only known in vivo substrates of Png1 are aberrant glycoproteins that originate in the endoplasmic reticulum, and arrive in the cytoplasm for proteasomal degradation. The substrate specificity of Png1 is admirably suited for this task.

Extracellular RNAs Are Associated With Insulin Resistance and Metabolic Phenotypes

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 The mean age of FHS participants was 65.8 years (56% female), with average BMI 27.7 kg/m2; participants in the youth cohort had a mean age of 15.5 years (60% female), with mean BMI 33.8 kg/m2. 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.

PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

Significance Oncogenic lesions give rise to genotype-specific dependencies in tumors by altering cell physiology. Understanding how oncogenes drive cell transformation will therefore help identify strategies to target tumors harboring these mutations. Although targeting certain oncogenes has led to clinical responses in some cases, PIK3CA inhibition has been disappointing to date. Here, we show that cell proliferation and tumor growth of PIK3CA mutant cancers is inhibited by suppression 2-oxoglutarate dehydrogenase, which leads to increased metabolite 2-oxoglutarate (2OG) levels. Elevated 2OG affects the function of the malate–aspartate shuttle, which is important because of the glycolytic nature of these cancers. This work provides novel insights into how mutant PIK3CA drives tumor proliferation and identifies a metabolic dependency that can be exploited in these cancers. 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.