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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.

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.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy 1 , 2 . This is mediated in part by a complex tumour microenvironment 3 , low vascularity 4 , and metabolic aberrations 5 , 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. A metabolite screen of pancreatic cells shows that pancreatic cancer cells metabolize uridine-derived ribose via UPP1, supporting redox balance, survival and proliferation.

Glioblastoma (GBM) is among the deadliest of human cancers. Despite extensive efforts, it has proven to be highly resistant to chemo- and immune-based therapeutic strategies, and little headway has been made with targeted inhibitors. Like many cancers, metabolism is dysregulated in GBM. Thus, to identify new vulnerabilities and drug targets in GBM, we conducted genetic screens using pooled RNAi libraries targeting metabolic enzymes. We screened multiple glioma stem cell-derived (GSC) xenograft models, which revealed that several enzymes involved in the mitochondrial metabolism of fatty acids were required for tumor cell proliferation. From among these, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids, due to its consistently high score across all of our screens, as well as its high expression level in multiple GSC models and its upregulation in GBM compared to normal brain. In this manuscript, we describe the dependence of GBM on sustained fatty acid metabolism to actively catabolize lipid species that would otherwise damage the mitochondrial structure. The uptake of mediumchain fatty acids lacks negative feedback regulation; therefore, in the absence of MCAD, medium-chain fatty acids accumulate to toxic levels, inducing reactive oxygen species (ROS), mitochondrial damage and failure, and apoptosis. Taken together, our findings uncover a previously unappreciated protective role exerted by MCAD in GBM cells, making it a unique and therapeutically exploitable vulnerability.

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. Here we tested if tissue type impacted GOT1 dependence by comparing PDA and colorectal cancer (CRC) cell lines and tumor models of similar genotype. We found CRC to be insensitive to GOT1 inhibition, contrasting markedly with PDA, which exhibit profound growth inhibition upon GOT1 knockdown. Utilizing a combination of metabolomics strategies and computational modeling, we found that GOT1 inhibition disrupted glycolysis, nucleotide metabolism, and redox homeostasis in PDA but not CRC. These insights were leveraged in PDA, where we demonstrate that radiotherapy potently enhanced the effect of GOT1 inhibition on tumor growth. Taken together, these results illustrate the role of tissue type in dictating metabolic dependencies and provide new insights for targeting metabolism to treat PDA.

Pancreatic ductal adenocarcinoma (PDA) is the third-leading cause of cancer mortality in the US and is highly resistant to classical, targeted, and immune therapies. We show that human PDA cells are dependent on the provision of exogenous cystine to avert a catastrophic accumulation of lipid reactive oxygen species (ROS) that, left unchecked, leads to ferroptotic cell death, both in vitro and in vivo. Using a dual-recombinase genetically engineered model, we found that acute deletion of Slc7a11 led to tumor-selective ferroptosis, tumor stabilizations/regressions, and extended overall survival. The mechanism of ferroptosis induction in PDA cells required the concerted depletion of both glutathione and coenzyme A, highlighting a novel branch of ferroptosis-relevant metabolism. Finally, we found that cystine depletion in vivo using the pre-IND agent cyst(e)inase phenocopied Slc7a11 deletion, inducing tumor-selective ferroptosis and disease stabilizations/regressions in the well-validated KPC model of PDA.

Bioconjugates consist of a synthetic polymer covalently attached to a biomolecule. Protein-polymer conjugates are becoming increasingly important materials in the fields of biotechnology, nanotechnology, and medicine. Chapter 1 focuses on conjugates of polymers produced via the controlled radical polymerization (CRP) techniques atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization with proteins. ATRP is a powerful technique to synthesize well-defined functional polymers. Chapters 2 and 3 describe the synthesis of protected aminooxy- and protected maleimide-functionalized ATRP initiators for the polymerization and and subsequent dimerization of styrene. The resulting polymers contained aminooxy or maleimide end-groups for conjugation to ketones/aldehydes or thiols, respectively following deprotection. Conjugation of aminooxy polystyrene to 4- bromobenzaldehyde via oxime bond formation verified reactivity of the polymer end groups. Maleimide-functionalized polystyrene reactivity was verified via Michael addition of benzyl mercaptan and then bioconjugation to the amino acid N-acetyl-L-cysteine methyl ester. Conjugations were confirmed by 1H NMR. Chapter 4 describes the synthesis of Nitrilotriacetic acid (NTA) functionalized ATRP initiators. NTAs are strong chelators and can bind oligohistidine(His)-tagged proteins via nickel mediated complexation (kD ∼ 10 μM). The NTA functionalized intitators were utilized for the polymerization of poly(ethylene glycol) methacrylate (PEGMA) and 2-hydroxyethyl methacrylate (HEMA). Conjugation to His6-tagged proteins was attempted but unsuccessful Thus, a tri-NTA (KD ∼ 20 nM) functionalized intiator was designed and is currently being synthesized. Chapter 5 describes the synthesis of a thiol-modified neurotransmitter. Neurotransmitters are chemicals that transmit signals from a neuron to a target cell across a synapse. They are a key part of the central nervous system. Starting from ethyl (S)-pyroglutamate the thiol glutamate was synthesized in 4 steps and 8.2% overall yield. The structure of the functionalized neurotransmitter was verified by 2D 1H NMR spectroscopy. Conjugation of this neurotransmitter to various thiol-funtionalized biomolecules is currently underway. Chapter 6 describes the fabrication of a micron-scale dual click surface via microcontact printing. The patterning of proteins individually on reactive alkanethiols using both oxime chemistry and the copper catalyzed Huisgen 1,3-dipolar cycloaddition is described - horse heart myoglobin modified with an α-oxoamide on an aminooxy functionalized monolayer and a red-fluorescent protein (RFP) containing an N-terminal alkyne on an azide functionalized monolayer. The methodologies were combined on a single surface by patterning the aminooxy alkanethiol while backfilling with the azide alkanethiol. The patterns were stained using Click-iT® Alexa Fluor® 594 DIBO alkyne followed by FluoSpheres aldehyde–sulfate spheres (20 nm, yellow-green) with immobilization of each only occurring where the appropriate click coupling partner had been patterned.