Uridine-derived ribose fuels glucose-restricted pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy1,2. This is mediated in part by a complex tumour microenvironment3, low vascularity4, and metabolic aberrations5,6. Although altered metabolism drives tumour progression, the spectrum of metabolites used as nutrients by PDA remains largely unknown. Here we identified uridine as a fuel for PDA in glucose-deprived conditions by assessing how more than 175 metabolites impacted metabolic activity in 21 pancreatic cell lines under nutrient restriction. Uridine utilization strongly correlated with the expression of uridine phosphorylase 1 (UPP1), which we demonstrate liberates uridine-derived ribose to fuel central carbon metabolism and thereby support redox balance, survival and proliferation in glucose-restricted PDA cells. In PDA, UPP1 is regulated by KRAS-MAPK signalling and is augmented by nutrient restriction. Consistently, tumours expressed high UPP1 compared with non-tumoural tissues, and UPP1 expression correlated with poor survival in cohorts of patients with PDA. Uridine is available in the tumour microenvironment, and we demonstrated that uridine-derived ribose is actively catabolized in tumours. Finally, UPP1 deletion restricted the ability of PDA cells to use uridine and blunted tumour growth in immunocompetent mouse models. Our data identify uridine utilization as an important compensatory metabolic process in nutrient-deprived PDA cells, suggesting a novel metabolic axis for PDA therapy.

Medium-Chain Acyl-CoA Dehydrogenase Protects Mitochondria from Lipid Peroxidation in Glioblastoma.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.

Cysteine depletion induces pancreatic tumor ferroptosis in mice.

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC - is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC - subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

Tissue of origin dictates GOT1 dependence and confers synthetic lethality to radiotherapy.

BACKGROUND: Metabolic programs in cancer cells are influenced by genotype and the tissue of origin. We have previously shown that central carbon metabolism is rewired in pancreatic ductal adenocarcinoma (PDA) to support proliferation through a glutamate oxaloacetate transaminase 1 (GOT1)-dependent pathway. METHODS: We utilized a doxycycline-inducible shRNA-mediated strategy to knockdown GOT1 in PDA and colorectal cancer (CRC) cell lines and tumor models of similar genotype. These cells were analyzed for the ability to form colonies and tumors to test if tissue type impacted GOT1 dependence. Additionally, the ability of GOT1 to impact the response to chemo- and radiotherapy was assessed. Mechanistically, the associated specimens were examined using a combination of steady-state and stable isotope tracing metabolomics strategies and computational modeling. Statistics were calculated using GraphPad Prism 7. One-way ANOVA was performed for experiments comparing multiple groups with one changing variable. Student's t test (unpaired, two-tailed) was performed when comparing two groups to each other. Metabolomics data comparing three PDA and three CRC cell lines were analyzed by performing Student's t test (unpaired, two-tailed) between all PDA metabolites and CRC metabolites. RESULTS: While PDA exhibits profound growth inhibition upon GOT1 knockdown, we found CRC to be insensitive. In PDA, but not CRC, GOT1 inhibition disrupted glycolysis, nucleotide metabolism, and redox homeostasis. These insights were leveraged in PDA, where we demonstrate that radiotherapy potently enhanced the effect of GOT1 inhibition on tumor growth. CONCLUSIONS: Taken together, these results illustrate the role of tissue type in dictating metabolic dependencies and provide new insights for targeting metabolism to treat PDA.

Abnormal oxidative metabolism in a quiet genomic background underlies clear cell papillary renal cell carcinoma.

While genomic sequencing routinely identifies oncogenic alterations for the majority of cancers, many tumors harbor no discernable driver lesion. Here, we describe the exceptional molecular phenotype of a genomically quiet kidney tumor, clear cell papillary renal cell carcinoma (CCPAP). In spite of a largely wild-type nuclear genome, CCPAP tumors exhibit severe depletion of mitochondrial DNA (mtDNA) and RNA and high levels of oxidative stress, reflecting a shift away from respiratory metabolism. Moreover, CCPAP tumors exhibit a distinct metabolic phenotype uniquely characterized by accumulation of the sugar alcohol sorbitol. Immunohistochemical staining of primary CCPAP tumor specimens recapitulates both the depletion of mtDNA-encoded proteins and a lipid-depleted metabolic phenotype, suggesting that the cytoplasmic clarity in CCPAP is primarily related to the presence of glycogen. These results argue for non-genetic profiling as a tool for the study of cancers of unknown driver.

Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution.

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose- block-2-methylaminoethyl methacrylate) [P(MAG- b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG- b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG- b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG- b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG- b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG- b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG- b-MAEMt) N/P = 5 and P(MAG- b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG- b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG- b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery.

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield pMAT43. The pMAT43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-b-AEMA cationic-block copolymers, pMAT-b-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-b-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-b-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.

Poly(trehalose): sugar-coated nanocomplexes promote stabilization and effective polyplex-mediated siRNA delivery.

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or "poly(trehalose)" and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.