Mesenchymal phenotype predisposes lung cancer cells to impaired proliferation and redox stress in response to glutaminase inhibition.

Recent work has highlighted glutaminase (GLS) as a key player in cancer cell metabolism, providing glutamine-derived carbon and nitrogen to pathways that support proliferation. There is significant interest in targeting GLS for cancer therapy, although the gene is not known to be mutated or amplified in tumors. As a result, identification of tractable markers that predict GLS dependence is needed for translation of GLS inhibitors to the clinic. Herein we validate a small molecule inhibitor of GLS and show that non-small cell lung cancer cells marked by low E-cadherin and high vimentin expression, hallmarks of a mesenchymal phenotype, are particularly sensitive to inhibition of the enzyme. Furthermore, lung cancer cells induced to undergo epithelial to mesenchymal transition (EMT) acquire sensitivity to the GLS inhibitor. Metabolic studies suggest that the mesenchymal cells have a reduced capacity for oxidative phosphorylation and increased susceptibility to oxidative stress, rendering them unable to cope with the perturbations induced by GLS inhibition. These findings elucidate selective metabolic dependencies of mesenchymal lung cancer cells and suggest novel pathways as potential targets in this aggressive cancer type.

Small molecule activation of PKM2 in cancer cells induces serine auxotrophy.

Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.

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. 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 α-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.

Nanostructure initiator mass spectrometry: tissue imaging and direct biofluid analysis.

Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.

Nanostructure-initiator mass spectrometry: a protocol for preparing and applying NIMS surfaces for high-sensitivity mass analysis.

Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid ('initiator') compounds. Analyte materials adsorbed onto this 'clathrate' surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.

Observation of cysteine thiolate and -S...H-O intermolecular hydrogen bond.

The cysteine anion was produced in the gas phase by electrospray ionization and investigated by photoelectron spectroscopy at low temperature (70 K). The cysteine anion was found to exhibit the thiolate form [-SCH2CH(NH2)CO2H], rather than the expected carboxylate form [HSCH2CH(NH2)CO2-]. This observation was confirmed by two control experiments, that is, methyl cysteine [CH3SCH2CH(NH2)CO2-] and cysteine methyl ester [-SCH2CH(NH2)CO2CH3]. The electron binding energy of [-SCH2CH(NH2)CO2H] was measured to be about 0.7 eV blue-shifted relative to [-SCH2CH(NH2)CO2CH3] due to the formation of an intramolecular -S-...HO2C- hydrogen bond in the cysteine thiolate. Theoretical calculations at the CCSD(T)/6-311++G(2df,p) and B3LYP/6-311++G(2df,p) levels were carried out to estimate the strength of this intramolecular -S-...HO2C- hydrogen bond. Combining experimental measurements and theoretical calculations yielded an estimated value of 16.4 +/- 2.0 kcal/mol for the -S-...HO2C- intramolecular hydrogen-bond strength.

Photoelectron spectroscopy of free multiply charged Keggin anions alpha-[PM12O40]3- (M = Mo, W) in the gas phase.

Two polyoxometalate Keggin-type anions, alpha-PM12O40(3-) (M = Mo, W), were transferred to the gas phase by electrospray; their electronic structure and stability were probed by photoelectron spectroscopy. These triply charged anions were found to be highly stable in the gas phase with large adiabatic electron detachment energies of 1.7 and 2.1 eV for M = Mo and W, respectively. The magnitude of the repulsive Coulomb barrier was measured as approximately 3.4 eV for both anions, providing an experimental estimate for the intramolecular Coulomb repulsion present in these highly charged anions. Density functional theory calculations were carried out and compared with the experimental data, providing insight into the electronic structure and valence molecular orbitals of the two Keggin anions. The calculations indicated that the highest occupied molecular orbital and other frontier orbitals for PM12O40(3-) are localized primarily on the mu2-oxo bridging ligands of the polyoxometalate framework, consistent with the reactivity on the mu2-oxo sites observed in solution. It was shown that the HOMO of PW12O40(3-) is stabilized relative to that of PMo12O40(3-) by approximately 0.35 eV. The experimental adiabatic electron detachment energies of PM12O40(3-) (i.e., the electron affinities of PM12O40(2-)) are combined with recent calculations on the proton affinity of PM12O40(3-) to yield O-H bond dissociation energies in PM12O39(OH)2- as approximately 5.1 eV.

Probing the intrinsic electronic structure of the bis(dithiolene) anions [M(mnt)2]2- and [M(mnt)2]1- (M = Ni, Pd, Pt; mnt = 1,2-S2C2(CN)2) in the gas phase by photoelectron spectroscopy.

A detailed understanding of the electronic structure of transition metal bis(dithiolene) complexes is important because of their interesting redox, magnetic, optical, and conducting properties and their relevance to enzymes containing molybdenum and tungsten bis(dithiolene) centers. The electronic structures of the bis(dithiolene) anions [M(mnt)(2)](n-) (M = Ni, Pd, Pt; mnt = 1,2-S(2)C(2)(CN)(2); n = 0-2) were examined by a combination of photodetachment photoelectron spectroscopy (PES) and density functional theory calculations. The combined experimental and theoretical data provide insight into the molecular orbital energy levels of [M(mnt)(2)](2-) and the ground and excited states of [M(mnt)(2)](1-) and [M(mnt)(2)]. Detachment features from ligand-based orbitals of [M(mnt)(2)](2-) occur at similar energies for each species, independent of the metal center, while those arising from metal-based orbitals occur at higher energies for the heavier congeners. Electronic excitation energies inferred for [M(mnt)(2)](1-) from the PES experiments agree well with those obtained in optical absorption experiments in solution, with the PES experiments providing additional insight into the changes in energy of these transitions as a function of metal. The singly charged anions [M(mnt)(2)](1-) were also prepared and studied independently. Electron detachment from the ground states of these doublet anions accessed the lowest singlet and triplet states of neutral [M(mnt)(2)], thereby providing a direct experimental measure of their singlet-triplet splitting.

Probing the low-barrier hydrogen bond in hydrogen maleate in the gas phase: a photoelectron spectroscopy and ab initio study.

The strength of the low-barrier hydrogen bond in hydrogen maleate in the gas phase was investigated by low-temperature photoelectron spectroscopy and ab initio calculations. Photoelectron spectra of maleic and fumaric acid monoanions (cis-/trans-HO(2)CCH=CHCO(2)(-)) were obtained at low temperatures and at 193 nm photon energy. Vibrational structure was observed for trans-HO(2)CCH=CHCO(2)(-) due to the OCO bending modes; however, cis-HO(2)CCH=CHCO(2)(-) yielded a broad and featureless spectrum. The electron binding energy of cis-HO(2)CCH=CHCO(2)(-) is about 1 eV blue-shifted relative to trans-HO(2)CCH=CHCO(2)(-) due to the formation of intramolecular hydrogen bond in the cis-isomer. Theoretical calculations (CCSD(T)/ aug-cc-pVTZ and B3LYP/aug-cc-pVTZ) were carried out to estimate the strength of the intramolecular hydrogen bond in cis-HO(2)CCH=CHCO(2)(-). Combining experimental and theoretical calculations yields an estimate of 21.5 +/- 2.0 kcal/mol for the intramolecular hydrogen bond strength in hydrogen maleate.