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Pancreatic ductal adenocarcinoma is a notoriously difficult-to-treat cancer and patients are in need of novel therapies. We have shown previously that these tumours have altered metabolic requirements, making them highly reliant on a number of adaptations including a non-canonical glutamine (Gln) metabolic pathway and that inhibition of downstream components of Gln metabolism leads to a decrease in tumour growth. Here we test whether recently developed inhibitors of glutaminase (GLS), which mediates an early step in Gln metabolism, represent a viable therapeutic strategy. We show that despite marked early effects on in vitro proliferation caused by GLS inhibition, pancreatic cancer cells have adaptive metabolic networks that sustain proliferation in vitro and in vivo . We use an integrated metabolomic and proteomic platform to understand this adaptive response and thereby design rational combinatorial approaches. We demonstrate that pancreatic cancer metabolism is adaptive and that targeting Gln metabolism in combination with these adaptive responses may yield clinical benefits for patients. Glutaminase inhibition (GLSi) has promising activity against certain cancers. Here, the authors show that GLSi has no effect on multiple mouse models of pancreatic cancer and characterize the metabolic pathways activated in response to GLSi whose concomitant inhibition may have therapeutic utility.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.

NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting. The cells of nearly all organisms need iron as this metal plays an important role in a wide range of biological processes. However, iron can also trigger the formation of harmful molecules that can damage cells. It is therefore crucial that the amount of iron in cells is tightly controlled and that any extra iron is safely stored away. Most of the iron in the body is stored within a protein called ferritin, which is then broken down to release iron as it is needed, in a process known as ferritinophagy. Cells use several systems to break down proteins, one of which, called autophagy, has been linked to ferritinophagy. During autophagy, a bubble-like structure called an autophagosome engulfs proteins that need to be removed and delivers them to a compartment in the cell where they can be broken down. In 2014, researchers showed that a protein called NCOA4 on the surface of autophagosomes targets ferritin for destruction. When iron levels are high in the cell, the amount of NCOA4 on the autophagosomes is low. This leads to fewer ferritin molecules being broken down. In contrast, low iron levels lead to an increase of NCOA4 on autophagosomes, which promotes ferritinophagy and increases the amount of iron in the cell. Now, Mancias, Vaites et al—including several of the researchers involved in the 2014 work—investigate the role of NCOA4 in ferritinophagy in more detail. Biochemical experiments revealed that a region of NCOA4 directly interacts with a particular subunit of ferritin and this interaction is necessary to deliver ferritin to autophagosomes. Mancias, Vaites et al. then used laboratory grown-cells to investigate why the amount of NCOA4 changes in response to the amount of iron in the cell. The experiments show the amount of NCOA4 varies depending on whether it interacts with another protein called HERC2, which targets proteins for destruction by a structure called the proteasome. HERC2 only binds to NCOA4 when iron levels are high, which leads to NCOA4 being broken down by the proteasome. When iron levels are low, HERC2 does not interact with NCOA4. The presence of more NCOA4 then leads to more ferritinophagy, and so increases the amount of iron in the cell. Mancias, Vaites et al. also found that red blood cells, which depend highly on iron, do not develop properly in zebrafish that have lower amounts of the NCOA4 protein. Further work is needed to see whether NCOA4 is also important for the development of other cells and tissues.

Pancreatic adenocarcinoma cells drive autophagy in tumour microenvironment-associated stellate cells, which release alanine that is used by the cancer cells as a carbon source for a variety of metabolic processes in an otherwise nutrient-poor environment. A cancer cell support network dissected Cancer cells generally have metabolic needs that differ from those of neighbouring normal cells, and hence display rewired metabolic networks. Cristovão Sousa et al . show that, in pancreatic cancers, stellate cells in the tumour environment supply cancer cells with the amino acid alanine as the carbon needed for anabolic processes when other sources are scarce. Tumour cells in turn stimulate autophagy in stellate cells, which is needed for alanine secretion. This cross-talk allows pancreatic cancer cells to fulfil their metabolic requirements in an environment lacking in other essential nutrients. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism 1 , 2 , 3 , 4 . The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context 5 , 6 , 7 , 8 , 9 , 10 . The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment 4 , 11 , 12 . As such, these tumours must alter how they capture and use nutrients to support their metabolic needs 11 , 13 . Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour’s dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment 4 , 11 . Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment.

During tumorigenesis, the high metabolic demand of cancer cells results in increased production of reactive oxygen species. To maintain oxidative homeostasis, tumor cells increase their antioxidant production through hyperactivation of the NRF2 pathway, which promotes tumor cell growth. Despite the extensive characterization of NRF2-driven metabolic rewiring, little is known about the metabolic liabilities generated by this reprogramming. Here, we show that activation of NRF2, in either mouse or human cancer cells, leads to increased dependency on exogenous glutamine through increased consumption of glutamate for glutathione synthesis and glutamate secretion by xc- antiporter system. Together, this limits glutamate availability for the tricarboxylic acid cycle and other biosynthetic reactions creating a metabolic bottleneck. Cancers with genetic or pharmacological activation of the NRF2 antioxidant pathway have a metabolic imbalance between supporting increased antioxidant capacity over central carbon metabolism, which can be therapeutically exploited.

We have previously demonstrated that endothelial targeting of gold nanoparticles followed by external beam irradiation can cause specific tumor vascular disruption in mouse models of cancer. The induced vascular damage may lead to changes in tumor physiology, including tumor hypoxia, thereby compromising future therapeutic interventions. In this study, we investigate the dynamic changes in tumor hypoxia mediated by targeted gold nanoparticles and clinical radiation therapy (RT). By using noninvasive whole-body fluorescence imaging, tumor hypoxia was measured at baseline, on day 2 and day 13, post-tumor vascular disruption. A 2.5-fold increase (P<0.05) in tumor hypoxia was measured two days after combined therapy, resolving by day 13. In addition, the combination of vascular-targeted gold nanoparticles and radiation therapy resulted in a significant (P<0.05) suppression of tumor growth. This is the first study to demonstrate the tumor hypoxic physiological response and recovery after delivery of vascular-targeted gold nanoparticles followed by clinical radiation therapy in a human non-small cell lung cancer athymic Foxn1nu mouse model.

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18 O 2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18 O 2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18 O under different physiological O 2 tensions. The most highly 18 O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies 1 – 4 and cancers with HPDL overexpression 5 . 18 O 2 labelling is used to identify metabolites in human cells that incorporate gaseous oxygen, including 4-hydroxymandelate, an intermediate in the synthesis of the coenzyme Q10 head group.

As nanoparticle solutions move towards human clinical trials in radiation therapy, the influence of key clinical beam parameters on therapeutic efficacy must be considered. In this study, we have investigated the clinical radiation therapy delivery variables that may significantly affect nanoparticle-mediated radiation dose amplification. We found a benefit for situations which increased the proportion of low energy photons in the incident beam. Most notably, “unflattened” photon beams from a clinical linear accelerator results in improved outcomes relative to conventional “flat” beams. This is measured by significant DNA damage, tumor growth suppression, and overall improvement in survival in a pancreatic tumor model. These results, obtained in a clinical setting, clearly demonstrate the influence and importance of radiation therapy parameters that will impact clinical radiation dose amplification with nanoparticles.

Pancreatic ductal adenocarcinoma (PDAC) cells use glutamine (Gln) to support proliferation and redox balance. Early attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. Here, we demonstrated that treating PDAC cells with a Gln antagonist, 6-diazo-5-oxo- l -norleucine (DON), led to a metabolic crisis in vitro. In addition, we observed a profound decrease in tumor growth in several in vivo models using sirpiglenastat (DRP-104), a pro-drug version of DON that was designed to circumvent DON-associated toxicity. We found that extracellular signal-regulated kinase (ERK) signaling is increased as a compensatory mechanism. Combinatorial treatment with DRP-104 and trametinib led to a significant increase in survival in a syngeneic model of PDAC. These proof-of-concept studies suggested that broadly targeting Gln metabolism could provide a therapeutic avenue for PDAC. The combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome.

Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy 1 – 3 . However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB 4 , mutations that cause loss of MHC-I are rarely found 5 despite the frequent downregulation of MHC-I expression 6 – 8 . Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8 + T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC. Inhibition of the autophagy–lysosome system upregulates surface expression of MHC class I proteins and enhances antigen presentation, and evokes a potent anti-tumour immune response that is mediated by CD8 + T cells.