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Summary Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) and Netrin G1 ligand (NGL-1) as promoters of PDAC tumorigenesis. NetG1 + cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1/NGL-1 mediated effect on glutamate/glutamine metabolism. NetG1 + CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These functions were partially mediated by vesicular glutamate transporter 1 and glutamine synthetase. This study uncovered an important link between CAF driven metabolism and its immunosuppressive capacity, suggesting NetG1 and NGL-1 as potential targets in PDAC. Significance PDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered two potential targets, NetG1 in CAFs and its binding partner NGL-1 in tumor cells. NetG1 in CAFs was important for the metabolic support of PDAC cells and for the intrinsic immunosuppressive capacity of CAFs, while NGL-1 in PDAC cells drove tumorigenesis. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Finally, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity. Thus, NetG1/NGL-1 axis mediates cell reciprocal and cell autonomous functions in PDAC, representing new attractive targets for this aggressive disease.

Mutations in the cystathionine beta-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The c.797 G>A (p.R266K) mutation in CBS was originally described in several Norwegian pyridoxine responsive CBS deficient patients, and heterologous gene expression studies have shown that the protein has near wild-type levels of enzyme activity. Here, we characterize a transgenic mouse lacking endogenous Cbs and expressing p.R266K human CBS protein from a zinc inducible metallothionein promoter (Tg-R266K Cbs−/−). Unlike mice expressing other mutant CBS alleles, the Tg-R266K transgene is unable to efficiently rescue neonatal lethality of Cbs−/− on a C57BL/6J background. On a C3H/HeJ background, zinc-induced Tg-R266K Cbs−/− mice express CBS mRNA, but have very low levels of CBS protein and enzyme activity, resulting in extreme elevations in serum total homocysteine (tHcy). Treatment with pyridoxine did not have any appreciable effect on tHcy, indicating this allele is not pyridoxine responsive in mice. However, treatment with the proteasome inhibitor bortezomib resulted in an 97% reduction in tHcy and a 2381% increase in liver CBS activity. These studies show that the p.R266K mutation causes increased proteasomal degradation in vivo, and that treatments that stabilize the protein can be used to reverse its effect.

Predicting the phenotypes of missense mutations uncovered by large-scale sequencing projects is an important goal in computational biology. High-confidence predictions can be an aid in focusing experimental and association studies on those mutations most likely to be associated with causative relationships between mutation and disease. As an aid in developing these methods further, we have derived a set of random mutations of the enzymatic domains of human cystathionine beta synthase. This enzyme is a dimeric protein that catalyzes the condensation of serine and homocysteine to produce cystathionine. Yeast missing this enzyme cannot grow on medium lacking a source of cysteine, while transfection of functional human CBS into yeast strains missing endogenous enzyme can successfully complement for the missing gene. We used PCR mutagenesis with error-prone Taq polymerase to produce 948 colonies, and compared cell growth in the presence or absence of a cysteine source as a measure of CBS function. We were able to infer the phenotypes of 204 single-site mutants, 79 of them deleterious and 125 neutral. This set was used to test the accuracy of six publicly available prediction methods for phenotype prediction of missense mutations: SIFT, PolyPhen, PMut, SNPs3D, PhD-SNP, and nsSNPAnalyzer. The top methods are PolyPhen, SIFT, and nsSNPAnalyzer, which have similar performance. Using kernel discriminant functions, we found that the difference in position-specific scoring matrix values is more predictive than the wild-type PSSM score alone, and that the relative surface area in the biologically relevant complex is more predictive than that of the monomeric proteins.