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Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling and metabolically leverages iron are poorly understood. We demonstrate that the liver-derived, endocrine regulator of systemic iron balance, hepcidin, is activated ectopically in CRC. Hepcidin binds to the only known mammalian iron exporter ferroportin, resulting in degradation of ferroportin and intracellular iron trapping. Mice deficient for the hepcidin gene specifically in colon tumor epithelium exhibited significant decreases in tumor number, burden, and size compared to wild-type littermates in a sporadic model of CRC, whereas ferroportin deletion exacerbated these tumor parameters. To further understand the biochemical and metabolic utilization of iron in CRC, we subjected a three-dimensional patient-derived CRC tumor enteroid model to metabolomics and found that iron is prioritized in CRC for the production of nucleotides. These metabolomics findings were recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that a decrease in mitochondrial function alters nucleotide synthesis following iron chelation. Restoration of nucleotide metabolism with exogenous supplementation of nucleosides led to a partial rescue of growth in patient-derived tumor enteroids and CRC cell lines in the presence of an iron chelator. Moreover, aspartate, a critical metabolite which links mitochondrial respiration and nucleotide synthesis, also partially rescued growth of iron deficient CRC cells. Collectively, these data suggest that ectopic hepcidin in the tumor epithelium establishes an axis to degrade ferroportin and sequester iron in colorectal tumors in order to maintain the nucleotide pool and sustain proliferation.

Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis(1-3). We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis(4). To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation(5), as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.

Summary Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.

Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC.

Adults diagnosed with acute myeloid leukemia have a poor prognosis, with only 30% surviving at five years. Despite advances in our understanding of the molecular underpinnings of AML, the chemotherapy backbone used to treat most patients (combination cytarabine and anthracycline) remains unchanged since 1973. With the goal of identifying a new differentiation therapy, we previously performed a small molecule phenotypic screen to find compounds that triggered myeloid maturation. Unexpectedly, our most active hits were inhibitors of the enzyme dihydroorotate dehydrogenase (DHODH). The DHODH-inhibitor brequinar demonstrated dramatic activity in vitro and in vivo (Figure 1A), leading to differentiation (Figure 1B) and prolonged survival across multiple AML models when compared to standard induction chemotherapy (iCT) (Figure 1C). Brequinar treatment also depleted leukemia initiating cells in vivo as measured by secondary transplant analysis (Figure 1D). Cancer therapy requires a 'therapeutic window' such that the treatment must target and kill malignant cells before killing normal cells and causing unacceptable side effects. An essential and ubiquitously expressed enzyme, DHODH is not typical among most cancer drug targets and is not known to be mutated or overexpressed in malignant cells. Yet empirically, a DHODH-inhibitor therapeutic window exists. The competitive transplantation of hematopoietic stem cells following 36-days of brequinar treatment (at the same dose that is anti-leukemic and pro-differentiation) does not lead to a competitive disadvantage or deleterious differentiation of normal stem cells (Figure 1E). The basis of this therapeutic window in the case of DHODH inhibitors is not known. Here we address the question: how are malignant cells metabolically programmed such that they are preferentially sensitive to pyrimidine starvation following inhibition of DHODH? Uridine is involved in myriad cellular processes beyond DNA and RNA synthesis, implicating many metabolic pathways (Figure 1F). We are taking an approach of (1) measuring metabolite levels and (2) metabolic pathway flux, as well as (3) using small molecule and genetic perturbations (shRNA) to unravel the differences in pyrimidine metabolism between normal and malignant cells. The mechanism through which treatment with brequinar and inhibition of pyrimidine synthesis results in myeloid differentiation is also not known. Our preliminary data suggest that brequinar treatment not only leads to a rapid depletion of pyrimidines and downstream metabolites, but also to changes in TCA cycle metabolites including succinate and fumarate. Given the importance of these metabolites in the regulation of the TET-family of proteins, we measured changes in 5-hydroxy-methylcytosine as a surrogate for TET-enzyme activity following treatment with brequinar (Figure 1G). An increase in global 5hmC, suggesting an increase in TET-enzyme activity, paralleled the cellular differentiation over the first 72-hours. This is an intriguing association given the importance of TET2 in myeloid differentiation and the observation that inhibition of TET2 contributes to myeloid differentiation arrest. As of summer 2018, two small molecule DHODH inhibitors are in early stage human clinical trials with two more inhibitors poised for 2019. A careful understanding of these metabolic pathways is important to inform the clinical experience of this class of anti-metabolite therapeutics. Understanding the basis of this therapeutic window will be critical to the safe and effective use of DHODH inhibitors in patients with advanced hematologic malignancies. Figure 1. Figure 1. Disclosures Vander Heiden: Agios: Membership on an entity's Board of Directors or advisory committees; Aeglea: Membership on an entity's Board of Directors or advisory committees. Sykes:Clear Creek Bio: Equity Ownership, Other: Co-founder.

Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells.

Chromosomal rearrangements leading to fusion transcripts represent a class of oncogenic aberrations that are of high interest for understanding cancer biology, treating cancer patients, and as targets for the development of new therapies. Transcriptome sequencing via RNA-Seq coupled with downstream bioinformatics software applications offers an effective method for identifying candidate fusion transcripts, and is more targeted and cost-effective than whole genome sequencing. Although many such fusion detection software tools have recently been made available, they often differ greatly in prediction accuracy, execution times, computational requirements, installation complexity, and in not being readily accessible to non-bioinformatician cancer researchers. Here we present the Trinity Cancer Transcriptome Analysis Toolkit (CTAT), a newly developed suite of RNA-Seq targeted fusion detection tools leveraging a combination of reference genome read-mapping and de novo transcriptome assembly, coupled to in silico fusion transcript validation, annotation, and visualization. We show that components of our Trinity CTAT fusion detection toolkit, including STAR-Fusion and FusionInspector, yield improved accuracy and run times as compared to alternative leading tools. As a demonstration of the utility of Trinity CTAT, we explore fusion transcript discovery in a large cohort of ∼300 chronic lymphocytic leukemia (CLL) patient samples. We identify several novel recurrent fusions that may represent drivers of CLL etiology and provide new avenues to therapies. In addition to fusion transcript identification, Trinity CTAT aims to provide cancer researchers with easy access to methods for analyzing cancer RNA-Seq, including transcript reconstruction, identification of mutations, expression analysis, and tumor heterogeneity. Trinity CTAT is freely available open source software and readily accessible to all cancer researchers via our public Galaxy web portal: https://galaxy.ncgas-trinity.indiana.edu/. Citation Format: Brian Haas, Timothy Tickle, Nathalie Pochet, Jing Sun, Peggy Hsu, Chip Stewart, Carrie Ganote, Ben Fulton, Catherine Wu, Aviv Regev. Fast and accurate fusion transcript detection using the Trinity Cancer Transcriptome Analysis Toolkit. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5266.

September 21 Author Manuscript, The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology., National Institutes of Health (U.S.) (Grant CA103866), National Institutes of Health (U.S.) (Grant AI47389), United States. Department of Defense (W81XWH-07-0448), W. M. Keck Foundation, LAM Foundation, American Cancer Society, Howard Hughes Medical Institute (Investigator)

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation[superscript 1, 2]. 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[superscript 3]. 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., Susan G. Komen Breast Cancer Foundation (Fellowship), Life Sciences Research Foundation (Fellowship), W. M. Keck Foundation, David H. Koch Cancer Research Fund, Alexander and Margaret Stewart Trust, National Institutes of Health (U.S.) (Grant CA103866)

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p–14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.