Pancreatic Ductal Adenocarcinoma Metabolism Research Articles

Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context.

Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate-aspartate shuttle, a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the TME.

A phase I study of first-line L-glutamine (Gln) with gemcitabine (gem) and nab-paclitaxel (nab-p) in advanced pancreatic cancer (GlutaPanc).

TPS636 Background: Cytotoxic chemotherapy remains the preferred first-line treatment for advanced or unresectable pancreatic cancer with combination regimens including 5-fluoruracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX), or gem and nab-p. The use and response of second- and third-line therapies remains dismal, thus the optimization of first-line treatment is critical. Our previous work investigating Gln metabolism in pancreatic ductal adenocarcinoma (PDAC) through glutaminase inhibition indicates that Gln deprivation in PDAC increases cancer cell survival and resistance to chemotherapy. However, when PDAC cell were treated with Gln supplementation, there was an increase in PDAC cell death with increasing concentrations of gem. In this present study we aim to test the feasibility and safety of combining L-Gln with gem and nab-p in treatment-naïve patients with unresectable or metastatic PDAC. Methods: This is a single arm, single center, phase I study. The primary objective is to assess the recommended phase II dose (RP2D) of L-Gln in combination with gem and nab-p. The RP2D will be assessed by escalation of overdose control (EWOC), an adaptive Bayesian design, through determination of the maximum tolerated dose (MTD) across 3 doses of gem, nab-p, and L-Gln. The MTD is defined as the dose such that the probability of dose-limiting toxicities (DLTs) at the MTD is θ = 0.33. The first patient will receive 1000 mg/m2 gem, 125 mg/m2 nab-p, and 0.1 g/kg L-Gln and the subsequent doses will be determined by the EWOC algorithm. We plan to enroll a maximum of 16 patients. L-Gln is administered orally BID throughout the 28-day cycle with a one-week lead-in prior to the beginning of the first cycle where gem and nab-p are administered on days 1, 8, and 15 of each cycle. Key inclusion criteria include: advanced or unresectable, histologically confirmed pancreatic cancer that is either new or recurrent (if recurrent, prior neoadjuvant or adjuvant chemotherapy or chemoradiation is allowed but must have been completed >12 months prior to recurrence), ECOG PS ≤ 2 or KP ≥ 60%, and normal organ and marrow function. Secondary objectives are to describe any preliminary evidence of antitumor activity by assessment of objective response rate, progression-free survival, and overall survival. Since October 2020, 3 patients have been screened and 3 enrolled. Clinical trial information: NCT04634539.

CircMYOF triggers progression and facilitates glycolysis via the VEGFA/PI3K/AKT axis by absorbing miR-4739 in pancreatic ductal adenocarcinoma

Emerging evidence has demonstrated that circular RNAs (circRNAs) take part in the initiation and development of pancreatic ductal adenocarcinoma (PDA), a deadly neoplasm with an extremely low 5-year survival rate. Reprogrammed glucose metabolism is a key feature of tumour development, including PDA. In this research, we evaluated the role of circRNAs in reprogrammed glucose metabolism in PDA. RNA sequencing under various glucose incubation circumstances was performed. A new circMYOF was identified. Sanger sequencing and RNase R treatment confirmed its circular RNA characteristics. Real-time PCR indicated that it was highly expressed in PDA clinical specimens and cell lines. Gain-of- and loss-of-function assays showed that circMYOF induced progression in PDA. Mechanistically, RNA pull-down and luciferase reporter experiments elucidated that circMYOF, as a competing endogenous RNA for miR-4739, facilitated glycolysis via the VEGFA/PI3K/AKT pathway. Taken together, our findings indicate that circMYOF may work as a desirable biomarker and therapeutic target for PDA patients.

Abstract PO-028: Pancreatic ductal adenocarcinoma is dependent on an unconventional pathway for polyamine synthesis

Abstract Targeting altered metabolism in pancreatic ductal adenocarcinoma (PDAC) has been an area of extensive investigation for over a decade now. A major hurdle however, for most anti-tumor metabolic strategies, is the high risk for toxicity, given the essential roles of metabolic pathways in the maintenance of normal tissue homeostasis. Indeed, this has been the case for targeting polyamines in cancers. Polyamines are small, highly positively charged molecules involved in multiple fundamental processes of cell growth and survival, including the synthesis of nucleic acids, modifications of chromatin structure, gene transcription and mRNA translation. Polyamine levels are significantly increased in many cancers, including PDAC. Prior anti-tumor strategies focused on pharmacological inhibition of the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC1) with little success, partially due to risk of harming normal tissues at higher drug doses. In this project, using both in vitro and in vivo mouse models of PDAC, we identified a dependency of PDAC on an unconventional way for the synthesis of the polyamine precursor ornithine, specifically from glutamine via ornithine aminotransferase (OAT); this is in contrast to its synthesis in most adult normal tissues from arginine via arginase (ARG) activity. We also identified potential key players mediating the induction of this metabolic pathway via KRAS, the main oncogenic driver in PDAC and are currently characterizing the downstream effects of polyamines on pancreatic tumor cells. The high dependency of PDAC, compared to normal tissue, on de novo ornithine synthesis via OAT provides an attractive therapeutic window for treating pancreatic cancer patients with minimal toxicity. Citation Format: Min-Sik Lee, Insia Naqvi, Courtney Dennis, Lucas Dailey, Alireza Lorzadeh, Tamara Zaytouni, Ashley Adler, Daniel S. Hitchcock, Lin Lin, Unmesh Jadhav, Clary B. Clish, Nada Y. Kalaany. Pancreatic ductal adenocarcinoma is dependent on an unconventional pathway for polyamine synthesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-028.

Abstract PO-026: CircMYOF acts as a miR-4739 sponge to promote progression and facilitate glycolysis via VEGFA/PI3K/AKT pathway in pancreatic ductal adenocarcinoma

Abstract Emerging evidences has revealed that circular RNAs (circRNAs) participate in the initiation and development of pancreatic ductal adenocarcinoma (PDAC), a deadly malignancy with extremely low 5-year survival rate. Reprogrammed glucose metabolism is a key feature of tumor development, including PDAC. In this study, we aimed to investigate the role of circRNAs in reprogrammed glucose metabolism in PDAC. RNA sequencing under various glucose incubation circumstance was performed. A new circMYOF was identified. Sanger sequencing and RNase R treatment confirmed its circular RNA characteristics. Real time PCR indicated that it was overexpressed in PDAC tissues and cell lines. Gain-of and loss-of function assay implied that circMYOF promoted progression in PDAC. Mechanically, RNA pull down and luciferase reporter assay suggested that circMYOF, exhibiting as a competing endogenous RNA (ceRNA) for miR-4739, facilitated glycolysis via VEGFA/PI3K/AKT pathway. Taken together, our finding indicates that circMYOF may be a potential biomarker and therapeutic target for PDAC patients. Citation Format: Dandan Zheng, Xianxian Huang, Juanfei Peng, Yanyan Zhuang, Yuanhua Li, Junchi Qu, Shineng Zhang, Fengting Huang. CircMYOF acts as a miR-4739 sponge to promote progression and facilitate glycolysis via VEGFA/PI3K/AKT pathway in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-026.

High-Throughput Single-Cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma.

Even populations of clonal cells are heterogeneous, which requires high‐throughput analysis methods with single‐cell sensitivity. Here, we propose a rapid, label‐free single‐cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK‐293T, PANC‐1, CFPAC‐1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.

High‐Throughput Single‐Cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma

AbstractEven populations of clonal cells are heterogeneous, which requires high‐throughput analysis methods with single‐cell sensitivity. Here, we propose a rapid, label‐free single‐cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK‐293T, PANC‐1, CFPAC‐1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.

Abstract 2389: LINC00901 regulates glucose metabolism in pancreatic cancer

Abstract Dysregulation of metabolism is one of the cancer hallmarks. However, little is known whether long coding RNAs (lncRNAs) play a role in the regulation of metabolism in pancreatic ductal adenocarcinoma (PDAC). In the present study, we focused on lncRNA LINC00901 because a high level of LINC00901 is associated with the poor outcome of PDAC patients. Functional studies showed that LINC00901 promotes PDAC cell proliferation and invasion, whereas knockout (KO) of LINC00901 impairs these abilities of PDAC cells. Furthermore, the xenograft animal model suggested that LINC00901 promotes tumorigenesis. To determine the underlying mechanism of LINC00901-mediated tumorigenesis, we performed RNA-seq analysis in LINC00901 overexpression or KO cells. Gene Set Enrichment Analysis (GSEA) analysis showed that gene sets involved in glucose biological processes such as ‘‘MYC targets”, “Glycolysis” and “Gluconeogenesis” are significantly enriched in the LINC00901 overexpression group, whereas these genes are down-regulated in the LINC00901 KO group. The top-scoring genes recurring in the three gene sets include key glycolysis-related genes, lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and enolase 1 (ENO1), all of which were validated by qRT-PCR and western blot. Moreover, we found that LINC00901 upregulates c-Myc by stabilizing c-Myc RNA. Together, these results suggest that LINC00901 is an oncogenic lncRNA capable of regulating glucose metabolism, leading to PDAC development. Experiments are underway to determine how LINC00901 regulates c-Myc RNA stability in pancreatic cancer cells. Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. LINC00901 regulates glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2389.

Overcoming chemoresistance by targeting reprogrammed metabolism: the Achilles' heel of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death due to its late diagnosis that removes the opportunity for surgery and metabolic plasticity that leads to resistance to chemotherapy. Metabolic reprogramming related to glucose, lipid, and amino acid metabolism in PDAC not only enables the cancer to thrive and survive under hypovascular, nutrient-poor and hypoxic microenvironments, but also confers chemoresistance, which contributes to the poor prognosis of PDAC. In this review, we systematically elucidate the mechanism of chemotherapy resistance and the relationship of metabolic programming features with resistance to anticancer drugs in PDAC. Targeting the critical enzymes and/or transporters involved in glucose, lipid, and amino acid metabolism may be a promising approach to overcome chemoresistance in PDAC. Consequently, regulating metabolism could be used as a strategy against PDAC and could improve the prognosis of PDAC.

Autophagy is critical for cysteine metabolism in pancreatic cancer through regulation of SLC7A11

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer. The elevated macroautophagy/autophagy in these tumors supports growth, promotes immune evasion, and increases therapeutic resistance. Therefore, targeting autophagy is a therapeutic strategy that is being pursued to treat PDAC patients. Whereas autophagy inhibition impairs mitochondrial metabolism in PDAC, the specific metabolite(s) that becomes limiting when autophagy is inhibited has not been identified. We report that loss of autophagy specifically results in intracellular cysteine depletion under nutrient-replete conditions. Mechanistically, we show that PDAC cells utilize the autophagy machinery to regulate the activity and localization of the cystine transporter SLC7A11 at the plasma membrane. Upon inhibition of autophagy, SLC7A11 is localized to lysosomes in an MTORC2-dependent manner. Our findings reveal a novel connection between autophagy and cysteine metabolism in pancreatic cancer.

GEO data mining and TCGA analysis reveal altered branched chain amino acid metabolism in pancreatic cancer patients

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor of the digestive system which has a less than 1% 5-year survival rate. The pathogenesis of PDAC development is incompletely understood. Genetic predisposition, disease history of chronic pancreatitis and diabetes elevate the risk of PDAC while environmental and dietary factors including smoking, alcohol abuse, high fat/protein intake as well as air pollution exacerbate PDAC progression. BCAAs, consisting of leucine, isoleucine and valine are essential amino acids that are obtained from food and play versatile roles in carcinogenesis. Recent studies have demonstrated that BCAA metabolism affects PDAC development but the results are controversial. To explore the possible engagement of BCAA metabolism in PDAC, we took advantage of the GEO and TCGA database and discovered that BCAA uptake is closely related to PDAC development while BCAA catabolism is down-regulated in PDAC tissue. Besides, NOTCH and MYC are differentially involved in BCAA metabolism in tumor and muscle, and enhanced lipid synthesis is independent of BCAA catabolism. Altogether, we highlight BCAA uptake as a promising target for PDAC treatment.

Metabolic profiling of a cohort of pancreatic ductal adenocarcinoma (PDAC) patients of african ancestry

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is deadly cancer with dysregulated metabolism being one of its key features, affecting several processes such as immune response. Studying metabolism in PDAC especially in under-studied population groups could lead to the identification of biomarkers and in better understanding of the immune response. We aimed to identify dysregulated metabolites in PDAC progression and their possible role in the immune response. Method: Blood samples were collected from 34 PDAC, 6 CP, and 6 healthy volunteers, all of African ancestry (M190681). Nuclear Magnetic Resonance (NMR) spectroscopy and Reactive Oxygen Species (ROS) analyses were used to identify metabolites and quantify ROS, respectively. The R studio V1.3 was used for statistical analysis and a p-value of <0.05 was considered significant. Results: Preliminary results from the study showed that tyrosine (p-value:0.001), histidine (p-value:0.004), acetate (p-value:0.002) and 3-hydroxybutyrate (p-value:0.018) levels were elevated as the disease progressed. Total Bilirubin exceeded the normal threshold of 5-21 μmol/L for the PDAC patients. Mortality occurred earlier for patients with inorganic phosphates (p-value: 0.229) higher than 1.42 mmol/L and anion gap (p-value: 0.002) higher than the threshold 9-16 mmol/L. Furthermore, patients having C-reactive protein (CRP) < 200mg/L and metabolic value of GlycA < 1.1 survived longer than 150 days. ROS was elevated in resectable (p-value:0.003) and Locally advanced (p-value:0.008) PDAC when compared with HC. Conclusion: Dysregulated metabolism is associated with increased ROS which has been shown to be linked to immunosuppression. The combination of dysregulated metabolites, ROS, and clinical parameters may have potential utility in disease stratification, management, and future therapeutic targeting.

The Replication Stress Response Kinase ATR is an Interferon Signaling Driven Co-Dependency&amp;nbsp;In Pancreas Cancer Cells

An inflammatory, cytokine-rich tumor microenvironment is a defining feature of pancreatic ductal adenocarcinoma (PDAC). Among proinflammatory cytokines, type I interferons (IFNs) are particularly important as they regulate the expression of hundreds of genes. It is however unclear whether IFN signaling in PDAC tumors can be leveraged therapeutically. We determined that a type I IFN response transcriptional signature is enriched in a subset of PDAC and that IFN signaling biomarkers are detectable in patient-derived and cell line xenograft tumors. To systematically identify actionable vulnerabilities induced by IFN, we integrated a mass spectrometry-based phosphoproteomic analysis and a chemical genomics high-throughput synthetic lethality screen which together revealed that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) and sensitizes tumor cells to ATR inhibitors. In vitro, IFNs induce replication stress and cell cycle arrest in S-phase which is accompanied by nucleotide pool insufficiency and enhanced nucleoside efflux into the extracellular environment. In combination with IFN, ATR inhibitors induce lethal DNA damage, downregulate de novo nucleotide biosynthesis mediated by ribonucleotide reductase, and deplete nucleotide pools. In vivo, ATR inhibitors limit the growth of subcutaneous and orthotopic PDAC tumors in which IFN production is driven by the activation of stimulator of interferon genes (STING). These results highlight critical crosstalk between IFN signaling, the DNA replication stress response network, and nucleotide metabolism in PDAC while providing the rationale for exploring targeted therapeutic interventions that leverage the tumor cell autonomous effects of IFN signaling.

A Review of Metabolic Stress and Development of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most notorious malignancies with a 5-year survival rate of less than 8%. Therefore, it is crucial to investigate the molecular mechanism underlining PDAC initiation, promotion, and progression for efficient treatment of PDAC. In order to adapt and survive in an extremely adverse microenvironment of hypoxia and insufficiency of nutrients and energy, PDAC cells undergo extensive metabolic modification triggered by intrinsic signalings which are activated by different genetic events, including mutations occurred at K RAS, TP53, and DPC4/ SMAD4, collaboratively promoting PDAC development. Notably, PDCA cells have extensive crosstalk in the form of reciprocal metabolic flux with its surrounding microenvironment to facilitate tumor advancement and therapy resistance. We herein summarize recent findings of PDAC metabolism and discuss metabolic rewiring-based therapeutic strategies.

Mitochondrial Metabolism in PDAC: From Better Knowledge to New Targeting Strategies.

Cancer cells reprogram their metabolism to meet bioenergetics and biosynthetic demands. The first observation of metabolic reprogramming in cancer cells was made a century ago (“Warburg effect” or aerobic glycolysis), leading to the classical view that cancer metabolism relies on a glycolytic phenotype. There is now accumulating evidence that most cancers also rely on mitochondria to satisfy their metabolic needs. Indeed, the current view of cancer metabolism places mitochondria as key actors in all facets of cancer progression. Importantly, mitochondrial metabolism has become a very promising target in cancer therapy, including for refractory cancers such as Pancreatic Ductal AdenoCarcinoma (PDAC). In particular, mitochondrial oxidative phosphorylation (OXPHOS) is an important target in cancer therapy. Other therapeutic strategies include the targeting of glutamine and fatty acids metabolism, as well as the inhibition of the TriCarboxylic Acid (TCA) cycle intermediates. A better knowledge of how pancreatic cancer cells regulate mitochondrial metabolism will allow the identification of metabolic vulnerabilities and thus novel and more efficient therapeutic options for the benefit of each patient.

Dissecting cell-type-specific metabolism in pancreatic ductal adenocarcinoma

Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between cell types within a mixed population can be challenging due to the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in murine pancreatic cancer organoid-fibroblast co-cultures and tumors. Pancreatic cancer cells exhibited increased pyruvate carboxylation relative to fibroblasts, and this flux depended on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells was necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tissue.

SOAT1 promotes mevalonate pathway dependency in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.

Abstract A41: Nucleotide metabolism heterogeneity in mutant KRAS pancreatic cancer

Abstract Mutant KRAS orchestrates major metabolic adaptations critical for pancreatic ductal adenocarcinoma (PDAC) growth and survival, including changes in glucose, amino acid, lipid and energy metabolism. However, how nucleotide metabolism in PDAC is impacted by mutant KRAS has received much less attention, even though nucleotides play critical roles in major biologic processes including nucleic acid synthesis, phospholipid biosynthesis and protein glycosylation. Pyrimidine nucleotide metabolism is a highly complex and regulated system with multiple pathways contributing to its plasticity and robustness. Pyrimidine nucleotides can be produced via the de novo pathway (DNP) that utilizes glucose and amino acids, and the nucleoside scavenging pathway (NSP) that utilizes extracellular uridine and cytidine. We hypothesize that nucleotide products of RNA turnover can fuel a third route for pyrimidine biosynthesis, which we term the nucleotide recycling pathway (NRP). Among these three major pyrimidine nucleotide biosynthetic pathways, the DNP has been studied most extensively. The biologic and therapeutic significance of the NSP and NRP in PDAC, as well as their regulation and coordination with DNP in the context of mutant KRAS signaling, are poorly understood. We further hypothesize that since PDAC tumor microenvironment is characterized by insufficient supply of nutrients and oxygen, these tumors have limited DNP capacity and increased reliance on alternate pyrimidine biosynthetic pathways. Here, we describe a novel mass spectrometric (MS) method to measure the contributions of pyrimidine nucleotide convergent pathways to RNA and DNA synthesis in PDAC models. Applying this method to a panel of PDAC models, including patient-derived primary lines, revealed previously unappreciated pyrimidine biosynthetic heterogeneity, suggesting the existence of distinct pyrimidine nucleotide metabolic subtypes. Identifying and characterizing these subtypes in the context of oncogenic KRAS signaling may enable stratification of PDAC tumors and guide the development of novel therapeutic approaches. Citation Format: Thuc M. Le, Woosuk Kim, Joseph R. Capri, Anthony E. Cabebe, Wes Armstrong, Evan Abt, Soumya Poddar, Shili Xu, Dave Dawson, Timothy R. Donahue, Caius G. Radu. Nucleotide metabolism heterogeneity in mutant KRAS pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A41.

Loss of heterozygosity for KrasG12D promotes REDD1-dependent, non-canonical glutamine metabolism in pancreatic ductal adenocarcinoma

Pancreatic cancer is associated with high mortality, and pancreatic ductal adenocarcinoma (PDAC) is its most common subtype. The rapid growth of PDAC is dependent on the non-canonical pathway of glutamine (Gln) utilization, and loss of heterozygosity for KrasG12D (KrasG12D-LOH) frequently observed in PDAC is associated with an aggressive and invasive phenotype. However, it remains unclear whether KrasG12D-LOH contributes to non-canonical Gln metabolism in PDAC. Here, we showed that KrasG12D-LOH leads to a substantial increase in non-canonical Gln metabolism in PDAC cells. Importantly, we observed elevated expression of regulated in DNA damage and development 1 (REDD1), which is activated in response to hypoxia and nutrient deprivation, in KrasG12D-LOH PDAC, and that REDD1 knockdown efficiently repressed KrasG12D-LOH-regulated Gln metabolism and suppressed proliferation, migration, and invasion of KrasG12D-LOH PDAC cells. These data provide evidence that REDD1 is a downstream target of KrasG12D-LOH and is involved in promoting non-canonical Gln metabolism in PDAC.

Glycolysis-Based Genes Associated with the Clinical Outcome of Pancreatic Ductal Adenocarcinoma Identified by The Cancer Genome Atlas Data Analysis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadly tumors in digestive tract tumors. Although there has been advancement in PDAC treatment, its prognosis still remains unsatisfactory, mainly because of dismal diagnosis. This article aims to develop new prognostic factors related to energy metabolism in PDAC and to use these genes for novel risk stratification. Hundred fifty messenger RNA (mRNA) expression profiles and clinicopathological data of PDAC were downloaded from The Cancer Genome Atlas dataset. The glycolysis pathway was the significant pathway based on the gene set enrichment analysis. We chose the glycolysis pathway-related 176 genes for further analysis. Multivariate Cox regression analysis and forward stepwise Cox regression model established a novel three-gene glycolytic signature (including MET, B3GNT3, and SPAG4) for PDAC patients' prognosis prediction. All 150 patients were classified into two groups by the median risk score. High-risk group had a worse outcome compared to the low-risk group. The risk score was also significantly correlated with age and radiotherapy. A nomogram, including the glycolytic gene signature, has shown some clinical net benefit for overall survival prediction. We also validated the validity and reliability in the Puleo dataset. This novel gene expression signature may be involved in the pathophysiology and used for risk stratification and prognosis prediction in PDAC.