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Pancreatic ductal adenocarcinoma (PDAC) is predicted to soon become the second killer cancer in the USA. One of the hallmarks of PDAC is desmoplasia, which consists of an expansion in activated stellate cells, known as cancer associated fibroblasts (CAFs), and remodeling of their extracellular matrix. Desmoplasia is a major culprit for the unique PDAC microenvironment, which both collapses blood vessels restricting nutrients and fosters immunosuppression. Crosstalk between CAFs, PDAC, and immune cells, within this microenvironment, is not fully understood. To study the functions of CAFs on PDAC development, we first performed a microarray comparing patient matched normal pancreatic stellate cells and CAFs cultured using our stromal 3D system. We identified proteins upregulated in CAFs that have not been previously implicated in desmoplasia. To understand the functional role of these proteins, CRISPR/Cas9 mediated deletion of target proteins was undertaken and cells were subjected to a number of co-culture assays, with PDAC cells and NK cells. Assays consisted of material transfer, cell engagement, proliferation, survival, NK cell activation and PDAC killing. Syngeneic/orthotopic in vivo models, RNAseq analyzes and quantitative multi-plex microscopy, on pathological samples, were also used. We uncovered the ectopic expressions of NetrinG1 (NG1), a neural pre-synaptic protein, in CAFs, and of NG1’s post-synaptic binding partner, NGL1, in PDAC cells. Using our 3D system, we observed that heterotypic CAF-to-PDAC interactions, via NG1/NGL1 engagement, are critical for providing survival advantages to nutrient deprived PDAC cells and for CAFs to protect PDAC from NK cell-driven elimination. Mechanistically, we uncovered that PDAC starvation is overcome by NG1 expressing CAFs that provide nutrition via material transfer. Further, knockout of NG1, in CAFs, significantly reduced the production of immunosuppressive cytokines, such as IL-6, IL-8, and TGF-β. Moreover, loss of NG1 from CAFs resulted in anti-tumor NK cell activation. Unexpectedly, we observed a link between glutamate metabolism in CAFs and the inactivation of anti-tumor NK cells, and this was dependent on CAFs’ NG1 expression. These results were confirmed in an orthotopic PDAC mouse model in which ablation of NGL1, in syngeneic mouse PDAC cells, significantly halted tumor growth. RNAseq comparing CON or NG1+ CAFs vs. knockout NG1- CAFs rendered changes in neural, metabolic and fibrous signature pathways. Translationally, we saw clear expression of stromal NG1 only in PDAC affected pancreata. Overall, this study identifies two novel targets for PDAC; a cancer that lacks effective therapies. Inhibition of the NG1/NGL1 axis, constitutes a potential future therapeutic approach, as it alters CAF mediated metabolism and reverts immunosuppression, stunting PDAC growth in vitro and in vivo. Citation Format: Ralph Francescone, Débora Barbosa Vendramini-Costa, Janusz Franco-Barraza, Jessica Wagner, Alex Muir, Linara Gabitova, Tatiana Pazina, Tiffany Luong, Neelima Shah, Dustin Rollins, Ruchi Malik, Sapna Gupta, Roshan Thapa, Diana Restifo, Allison Lau, Yan Zhou, Kathy Q. Cai, Harvey Harvey Hensley, Emmanuelle Nicolas, Warren D. Kruger, Karthik Devarajan, Siddharth Balachandran, Wafik S. El-Deiry, Matthew Vander Heiden, Kerry Campbell, Igor Astsaturov, Edna Cukierman. NG1/NGL1 engagement supports PDAC development via CAF to PDAC nutrition and CAF-regulated immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2038.

Pancreatic Ductal Adenocarcinoma (PDAC) is the 5th leading cause of cancer death in the U.S. and Europe. It is a very aggressive form of cancer in which patients have a 5-year survival rate lower than 5%. PDAC arises from alterations in the KRAS and TP53 genes. The cell lines used were derived from 2 different mouse models where KRASG12D was combined with a deletion or point mutation of TP53 (TP53-/-, TP53R172H respectively) to drive oncogenic activation of PDAC. The TP53-/- model exhibits rapid pancreatic tumor growth, thus not allowing metastasis to occur, in contrast to the TP53R172H model, where tumors metastasize to the lungs or liver. TP53R172H cells also show a noticeable dependency on pyruvate availability for proliferation in contrast to the opposing model. Other NAD+ replenishing molecules such as duroquinone can have similar effects on proliferation. Cell lines from the primary tumors of both models as well as from the metastasized liver tumors were used for in vitro experimentation. Epithelial-Mesenchymal Transition (EMT) is important for metastasis and cell state can be qualified by measuring E-cadherin expression, which has also been shown to be influenced by pyruvate availability. This project had 2 aims: 1) determine the effect of pyruvate availability on EMT by measuring E-cadherin levels and TWIST1 relative expression and 2) measure the effect that availability of both pyruvate and duroquinone have on cell proliferation. Cells were cultured in 6-well plates. After cell adhesion, they were treated with media containing pyruvate and media lacking pyruvate for comparison. Cells were grown for 24-96 hour periods after which they would be recollected for our designated analyses; western blot, total cell count and qPCR. Measured E-cadherin levels showed that there is higher protein expression in the TP53R172H model. Furthermore, it is shown that E-cadherin levels seem to be regulated by pyruvate in this model. Increase in protein quantity with time also suggests that these cells may be shifting towards an epithelial phenotype. Duroquinone did not rescue proliferation, contrary to the hypothesis. Surprisingly, there was a decrease in proliferation in the TP53-/- model. This could be attributed to experimental errors or loss of pyruvate sensitivity in the cell line. Expression levels were measured for Twist (TWIST1), a transcription factor that serves as a negative regulator for E-cadherin. RNA was isolated from cell lysates and reversed transcribed to measure relative expression of Twist by qPCR. A pronounced effect of pyruvate dependency on Twist expression was observed in both models. This may be meaningful since it could offer a possible explanation on how pyruvate could be influencing EMT in these cells. Citation Format: Andrés E. Martínez-Muñiz, Sharanya Sivanand, Matthew Vander Heiden. Characterization of primary and metastatic pancreatic tumors in pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4358.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types. Although these mutations are loss-of-function for conversion of isocitrate to α-ketoglutarate, the mutant enzymes greatly increase the production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). However the full metabolic consequences of IDH1/2 mutation in their heterozygous cellular context have yet to be fully explored. To address this question, we utilized a panel of isogenic cell lines with wild-type IDH1/2 or clinically relevant IDH1/2 mutations and examined the metabolic consequences of IDH mutation using (13)C metabolic flux analysis (MFA). We observe a dramatic and consistent decrease in the ability of IDH1 mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate back to isocitrate. This was not seen either in IDH2 mutant cell lines or in wild-type cell lines treated with exogenous 2-HG. Consistent with these changes, the IDH1 mutant cell lines, but not IDH2 mutant or 2-HG treated cells, were deficient in the utilization of glutamine for de novo lipogenesis. Similar trends were observed in endogenous, non-engineered IDH1/2 mutant cell lines. The decrease in reductive carboxylation in the IDH1 mutant cell lines raises the hypothesis that these cells may be more reliant on mitochondrial metabolism. Indeed, IDH1 mutant cells were more sensitive to either treatment with an electron transport chain inhibitor or growth in hypoxia (which also inhibits mitochondrial metabolism). These results show heterozygous IDH1 mutation robustly impacts wild-type cellular metabolism in a different manner than IDH2 mutation. Furthermore, these results suggest that IDH1 and IDH2 mutant tumors may be differentially sensitive to inhibitors of specific metabolic pathways. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B159. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Courtney Green, Fallon Lin, Carol Joud-Caldwell, Hong Yin, Franklin Chung, Christopher Straub, Matthew Vander Heiden, Raymond Pagliarini, Christian Metallo. Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B159.