Metabolic Rewiring Is Essential for AML Cell Survival to Overcome Autophagy Inhibition by Loss of ATG3.

Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.

Glutamine Modulates Expression and Function of Glucose 6-Phosphate Dehydrogenase via NRF2 in Colon Cancer Cells.

Nucleotide pools need to be constantly replenished in cancer cells to support cell proliferation. The synthesis of nucleotides requires glutamine and 5-phosphoribosyl-1-pyrophosphate produced from ribose-5-phosphate via the oxidative branch of the pentose phosphate pathway (ox-PPP). Both PPP and glutamine also play a key role in maintaining the redox status of cancer cells. Enhanced glutamine metabolism and increased glucose 6-phosphate dehydrogenase (G6PD) expression have been related to a malignant phenotype in tumors. However, the association between G6PD overexpression and glutamine consumption in cancer cell proliferation is still incompletely understood. In this study, we demonstrated that both inhibition of G6PD and glutamine deprivation decrease the proliferation of colon cancer cells and induce cell cycle arrest and apoptosis. Moreover, we unveiled that glutamine deprivation induce an increase of G6PD expression that is mediated through the activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2). This crosstalk between G6PD and glutamine points out the potential of combined therapies targeting oxidative PPP enzymes and glutamine catabolism to combat colon cancer.

Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.

Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.

Oxidative Pentose Phosphate Pathway Enzyme 6-Phosphogluconate Dehydrogenase Plays a Key Role in Breast Cancer Metabolism.

The pentose phosphate pathway (PPP) plays an essential role in the metabolism of breast cancer cells for the management of oxidative stress and the synthesis of nucleotides. 6-phosphogluconate dehydrogenase (6PGD) is one of the key enzymes of the oxidative branch of PPP and is involved in nucleotide biosynthesis and redox maintenance status. Here, we aimed to analyze the functional importance of 6PGD in a breast cancer cell model. Inhibition of 6PGD in MCF7 reduced cell proliferation and showed a significant decrease in glucose consumption and an increase in glutamine consumption, resulting in an important alteration in the metabolism of these cells. No difference in reactive oxygen species (ROS) production levels was observed after 6PGD inhibition, indicating that 6PGD, in contrast to glucose 6-phosphate dehydrogenase, is not involved in redox balance. We found that 6PGD inhibition also altered the stem cell characteristics and mammosphere formation capabilities of MCF7 cells, opening new avenues to prevent cancer recurrance after surgery or chemotherapy. Moreover, inhibition of 6PGD via chemical inhibitor S3 resulted in an induction of senescence, which, together with the cell cycle arrest and apoptosis induction, might be orchestrated by p53 activation. Therefore, we postulate 6PGD as a novel therapeutic target to treat breast cancer.

Exploratory and confirmatory analysis to investigate the presence of vaginal metabolome expression of microbial invasion of the amniotic cavity in women with preterm labor using high-performance liquid chromatography.

BACKGROUND: Although the influence of microbial invasion of the amniotic cavity on the development of spontaneous preterm delivery is unquestionable, the use of an invasive procedure to diagnose the status of an infection limits its clinical translation. OBJECTIVE: This study aimed to use exploratory and confirmatory analyses to investigate the presence of vaginal metabolome expression of microbial invasion of the amniotic cavity in women diagnosed as having preterm labor using high-performance liquid chromatography. STUDY DESIGN: In 140 women with singleton pregnancies and a diagnosis of preterm labor at <34 weeks' gestation, we analyzed vaginal amino acid concentrations using high-performance liquid chromatography. Vaginal samples were collected shortly after the amniocentesis performed at admission to rule out microbial invasion of the amniotic cavity. Data were normalized for the median of all the amino acid concentrations evaluated. Microbial invasion of the amniotic cavity was defined as a positive aerobic or anaerobic amniotic fluid culture for the presence of bacteria or yeast or Ureaplasma species or Mycoplasma hominis in the mycoplasma culture or a positive polymerase chain reaction result for 16S rRNA gene sequence. Exploratory analysis was performed in half of the sample and confirmatory analysis in the other half. We compared vaginal amino acid concentrations between women with and without microbial invasion of the amniotic cavity in both cohorts. The area under the curve with 95% confidence interval values were calculated for vaginal amino acids with significant differences. RESULTS: In the exploratory cohort (2014-2015), 17 of 76 women (22.3%) had microbial invasion of the amniotic cavity compared with 14 of 72 (19.4%) in the confirmatory cohort (2016-2017). In the exploratory cohort, we found significantly higher amino acid concentrations of vaginal taurine, lysine, and cysteine and significantly lower concentrations of vaginal glutamate, aspartate, and the aspartate to asparagine ratio. These significant differences were confirmed in the confirmatory cohort. The area under the curve of these vaginal amino acids to predict microbial invasion of the amniotic cavity ranged between 0.72 and 0.79, with cysteine being the amino acid with the best performance with an area under the curve of 0.79 (95% confidence interval, 0.71-0.88). CONCLUSION: We found the vaginal metabolome expression of microbial invasion of the amniotic cavity in women with preterm labor and intact membranes. These findings might open the possibility to develop noninvasive diagnostic tools of microbial invasion of the amniotic cavity with the aim of selecting women who would most likely benefit from an amniocentesis for this indication.

The landscape of tiered regulation of breast cancer cell metabolism.

Altered metabolism is a hallmark of cancer, but little is still known about its regulation. In this study, we measure transcriptomic, proteomic, phospho-proteomic and fluxomics data in a breast cancer cell-line (MCF7) across three different growth conditions. Integrating these multiomics data within a genome scale human metabolic model in combination with machine learning, we systematically chart the different layers of metabolic regulation in breast cancer cells, predicting which enzymes and pathways are regulated at which level. We distinguish between two types of reactions, directly and indirectly regulated. Directly-regulated reactions include those whose flux is regulated by transcriptomic alterations (~890) or via proteomic or phospho-proteomics alterations (~140) in the enzymes catalyzing them. We term the reactions that currently lack evidence for direct regulation as (putative) indirectly regulated (~930). Many metabolic pathways are predicted to be regulated at different levels, and those may change at different media conditions. Remarkably, we find that the flux of predicted indirectly regulated reactions is strongly coupled to the flux of the predicted directly regulated ones, uncovering a tiered hierarchical organization of breast cancer cell metabolism. Furthermore, the predicted indirectly regulated reactions are predominantly reversible. Taken together, this architecture may facilitate rapid and efficient metabolic reprogramming in response to the varying environmental conditions incurred by the tumor cells. The approach presented lays a conceptual and computational basis for mapping metabolic regulation in additional cancers.

Synthesis, characterization and biological activity of new cyclometallated platinum(iv) iodido complexes.

The synthesis of six novel cyclometallated platinum(iv) iodido complexes is accomplished by intermolecular oxidative addition of methyl iodide (compounds 2a-2c) or iodine (compounds 3a-3c) upon cyclometallated platinum(ii) compounds [PtX{(CH3)2N(CH2)3NCH(4-ClC6H3)}] (1a-1c: X = Cl, CH3 or I). The X-ray molecular structures of platinum(ii) compound 1c and platinum(iv) compounds 3b and 3a' (an isomer of 3a) are reported. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), DNA interaction, topoisomerase I, IIα, and cathepsin B inhibition, and cell cycle arrest, apoptosis and ROS generation of the investigated complexes are presented. Remarkable antiproliferative activity was observed for most of the synthesized cycloplatinated compounds (series 1-3) in all the selected carcinoma cell lines. The best inhibition was provided for the octahedral platinum(iv) compounds 2a-2c exhibiting a methyl and an iodido axial ligand. Preliminary biological results point to a different mechanism of action for the investigated compounds. Cyclometallated platinum(ii) compounds 1a-1c modify the DNA migration as cisplatin. In contrast, cyclometallated platinum(iv) compounds 2a-2c and 3a-3c did not modify the DNA tertiary structure neither in the absence nor in the presence of ascorbic acid, which made them incapable of reducing platinum(iv) compounds 2b and 2c in a buffered aqueous medium (pH 7.40) according to 1H NMR experiments. Remarkable topoisomerase IIα inhibitory activity is reported for platinum(iv) complexes 2b and 3a and in addition, for the last one, a moderate cathepsin B inhibition is reported. Cell cycle arrest (decrease in G0/G1 and G2 phases and arrest in the S phase), induction of apoptosis and ROS generation are related to the antiproliferative activity of some representative octahedral cyclometallated platinum(iv) compounds (2b and 2c).

Glucose-6-phosphate dehydrogenase and transketolase modulate breast cancer cell metabolic reprogramming and correlate with poor patient outcome.

The pentose phosphate pathway is a fundamental metabolic pathway that provides cells with ribose and NADPH required for anabolic reactions - synthesis of nucleotides and fatty acids - and maintenance of intracellular redox homeostasis. It plays a key role in tumor metabolic reprogramming and has been reported to be deregulated in different types of tumors. Herein, we silenced the most important enzymes of this pathway - glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) - in the human breast cancer cell line MCF7. We demonstrated that inhibition of G6PD, the oxidative branch-controlling enzyme, reduced proliferation, cell survival and increased oxidative stress. At the metabolic level, silencing of both enzymes reduced ribose synthesis. G6PD silencing in particular, augmented the glycolytic flux, reduced lipid synthesis and increased glutamine uptake, whereas silencing of TKT reduced the glycolytic flux. Importantly, we showed using breast cancer patient datasets that expression of both enzymes is positively correlated and that high expression levels of G6PD and TKT are associated with decreased overall and relapse-free survival. Altogether, our results suggest that this metabolic pathway could be subjected to therapeutic intervention to treat breast tumors and warrant further investigation.

On the stability and biological behavior of cyclometallated Pt(IV) complexes with halido and aryl ligands in the axial positions.

A series of cyclometallated platinum(IV) compounds (3a, 3a' and 3b') with a meridional [C,N,N'] terdentate ligand, featuring an halido and an aryl group in the axial positions has been evaluated for electrochemical reduction and preliminary biological behavior against a panel of human adenocarcinoma (A-549 lung, HCT-116 colon, and MCF-7 breast) cell lines and the normal bronquial epithelial BEAS-2B cells. Cathodic reduction potentials (shifting from -1.463 to -1.570V) reveal that the platinum(IV) compounds under study would be highly reluctant to be reduced in a biological environment. Actually ascorbic acid was not able to reduce complex 3a', the most prone to be reduced according its reduction potential, over a period of one week. These results suggest an intrinsic activity for the investigated platinum(IV) complexes (3a, 3a' and 3b'), which exhibit a remarkable cytotoxicity effectiveness (with IC50 values in the low micromolar range), even greater than that of cisplatin. The IC50 for A-549 lung cells and clog P values were found to follow the same trend: 3b'>3a'>3a. However, no correlation was observed between reduction potential and in vitro activity. As a representative example, cyclometallated platinum(IV) compound 3a', exercise its antiproliferative activity directly over non-microcytic A-549 lung cancer cells through a mixture of cell cycle arrest (13% arrest at G1 phase and 46% arrest at G2 phase) and apoptosis induction (increase of early apoptosis by 30 times with regard to control). To gain further insights into the mode of action of the investigated platinum(IV) complexes, drug uptake, cathepsin B inhibition and ROS generation were also evaluated. Interestingly an increased ROS generation could be related with the antiproliferative activity of the cyclometallated platinum(IV) series under study in the cisplatin-resistant A-549 lung and HCT-116 cancer cell lines.

Methylseleninic acid promotes antitumour effects via nuclear FOXO3a translocation through Akt inhibition.

Selenium supplement has been shown in clinical trials to reduce the risk of different cancers including lung carcinoma. Previous studies reported that the antiproliferative and pro-apoptotic activities of methylseleninic acid (MSA) in cancer cells could be mediated by inhibition of the PI3K pathway. A better understanding of the downstream cellular targets of MSA will provide information on its mechanism of action and will help to optimize its use in combination therapies with PI3K inhibitors. For this study, the effects of MSA on viability, cell cycle, metabolism, apoptosis, protein and mRNA expression, and reactive oxygen species production were analysed in A549 cells. FOXO3a subcellular localization was examined in A549 cells and in stably transfected human osteosarcoma U2foxRELOC cells. Our results demonstrate that MSA induces FOXO3a nuclear translocation in A549 cells and in U2OS cells that stably express GFP-FOXO3a. Interestingly, sodium selenite, another selenium compound, did not induce any significant effects on FOXO3a translocation despite inducing apoptosis. Single strand break of DNA, disruption of tumour cell metabolic adaptations, decrease in ROS production, and cell cycle arrest in G1 accompanied by induction of apoptosis are late events occurring after 24h of MSA treatment in A549 cells. Our findings suggest that FOXO3a is a relevant mediator of the antiproliferative effects of MSA. This new evidence on the mechanistic action of MSA can open new avenues in exploiting its antitumour properties and in the optimal design of novel combination therapies. We present MSA as a promising chemotherapeutic agent with synergistic antiproliferative effects with cisplatin.

Cancer cell metabolism as new targets for novel designed therapies.

Metabolic processes are altered in cancer cells, which obtain advantages from this metabolic reprogramming in terms of energy production and synthesis of biomolecules that sustain their uncontrolled proliferation. Due to the conceptual progresses in the last decade, metabolic reprogramming was recently included as one of the new hallmarks of cancer. The advent of high-throughput technologies to amass an abundance of omic data, together with the development of new computational methods that allow the integration and analysis of omic data by using genome-scale reconstructions of human metabolism, have increased and accelerated the discovery and development of anticancer drugs and tumor-specific metabolic biomarkers. Here we review and discuss the latest advances in the context of metabolic reprogramming and the future in cancer research.