Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.metabolite profiling | metabolic subtypes in PDAC | glycolysis | lipid synthesis | biomarkers for metabolic inhibitors M etabolic reprogramming during tumorigenesis is an essential process in nearly all cancer cells. Tumors share a common phenotype of uncontrolled cell proliferation and must efficiently generate the energy and macromolecules required for cellular growth. The first example of metabolic reprogramming was discovered more than 80 y ago by Otto Warburg: tumor cells can shift from oxidative to fermentative metabolism in the course of oncogenesis (1). More recently, there has been a resurgence of interest in targeting cancer metabolism (2-4) because it may not only be effective in inhibiting tumor growth, but may also provide a therapeutic window (5, 6). For example, inactivation of lactate dehydrogenase-A (LDHA), an enzyme that catalyzes the final step of aerobic glycolysis, thereby reducing pyruvate to lactate, decreases tumorigenesis and induces regression of established tumors in mouse models of lung cancer driven by oncogenic KRAS or epidermal growth factor receptor (EGFR) while minimally affecting normal cell function (7). The finding that cancers have altered metabolism has prompted substantial investigation, both preclinically and in clinical trials, of several metabolically targeted agents, including those that elevate reactive oxygen species (ROS) or block glycolysis, lipid synthesis, mitochondrial function, and glutamine synthesis pathways (8).The identification of distinct metabolic reprogramming events or metabolic subtypes in cancer may inform patient selection for investigational...

Th17 cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 differentiation is relatively unknown. We demonstrate that CD4+ T cells deficient in Itk, a tyrosine kinase required for full TCR-induced activation of PLC-γ, exhibit decreased IL-17A expression, yet relatively normal expression of RORγT, RORα and IL-17F. IL-17A expression was rescued by pharmacologically-induced Ca2+ influx or expression of activated NFATc1. Conversely, decreased TCR stimulation or FK506 treatment preferentially reduced expression of IL-17A. The promoter of IL-17A but not IL-17F has conserved NFAT binding sites that bind NFATc1 in WT, but not Itk-deficient cells, even though both promoters exhibit epigenetic modifications consistent with open chromatin. Finally, defective IL-17A expression and differential regulation of IL-17A and IL-17F were observed in vivo in Itk−/− mice in an allergic asthma model. Our results suggest that Itk specifically couples TCR signaling strength to IL-17A expression through NFATc1.

Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-defi cient C57BL/6 ⌬ dblGATA mice (eosinophil-null mice via the ⌬ DblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway infl ammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophildefi cient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infi ltration and airway infl ammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.

The role of essential amino acids in metabolic reprogramming of cancer cells is now well established, whereas the role of non-essential amino acids (NEAAs) in malignancy remains less clear. Here, we have identified an important role for the NEAA proline in the tumorigenic potential of a subset of cancer cells. By profiling a large panel of cancer cell lines, we observed that proline consumption and expression of proline biosynthesis enzymes were well correlated with clonogenic and tumorigenic potential. Moreover, proline starvation or inhibition of proline biosynthesis enzymes impaired clonogenic/tumorigenic potential. Cancer cells exhibiting dependency on exogenous proline displayed hyperactivation of the mTORC1-mediated 4EBP1 signaling axis, as well as unresolved ER stress. Exogenous proline alleviated ER stress and promoted cellular homeostasis and clonogenicity. Increased dependence on proline may therefore define a specific vulnerability in some cancers that can be exploited by proline depletion.

T cells with a memory-like phenotype and possessing innate immune function have been previously identified as CD8(+)CD44(hi) cells. These cells rapidly secrete IFN-gamma upon stimulation with IL-12/IL-18 and are involved in innate responses to infection with Listeria monocytogenes. The signals regulating these cells are unclear. The Tec kinase Itk regulates T cell activation and we report here that a majority of the CD8(+) T cells in Itk null mice have a phenotype of CD44(hi) similar to memory-like innate T cells. These cells are observed in mice carrying an Itk mutant lacking the kinase domain, indicating that active Tec kinase signaling suppresses their presence. These cells carry preformed message for and are able to rapidly produce IFN-gamma upon stimulation in vitro with IL-12/IL-18, and endow Itk null mice the ability to effectively respond to infection with L. monocytogenes or exposure to lipopolysaccharides by secretion of IFN-gamma. Transfer of these cells rescues the ability of IFN-gamma null mice to reduce bacterial burden following L. monocytogenes infection, indicating that these cells are functional CD8(+)CD44(hi) T cells previously detected in vivo. These results indicate that active signals from Tec kinases regulate the development of memory-like CD8(+) T cells with innate function.

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The Tec family of tyrosine kinases transduces signals from antigen and other receptors in cells of the hematopoietic system. In particular, interleukin-2 inducible T cell kinase (Itk) plays an important role in modulating T cell development and activation. Itk is activated by receptors via a phosphatidylinositol 3-kinase-mediated pathway, which results in recruitment of Itk to the plasma membrane via its pleckstrin homology domain. We show here that membrane localization of Itk results in the formation of clusters of at least two molecules within 80 Å of each other, which is dependent on the integrity of its pleckstrin homology domain. By contrast, the proline-rich region within the Tec homology domain, SH3 or SH2 domains, or kinase activity were not required for this event. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane as only receptors that recruit phosphatidylinositol 3-kinase reside in the same membrane vicinity as the recruited Itk. Our results indicate that Itk forms dimers in the membrane and that receptors that recruit Itk do so to specific membrane regions.The Tec family of non-receptor tyrosine kinases is the second largest family of non-receptor tyrosine kinases (1). This family, which includes Itk, 2 is involved in transducing signals from a number of receptors, including the T cell receptor, B cell receptor, Fc⑀R, c-Kit, CD28, CD2, CD32, and the erythropoietin receptor (1-6). These kinases play important roles in regulating cytokine and immune receptor signals that regulate the immune response (7,8). Itk in particular is involved in early T cell receptor signaling and regulates increases in intracellular calcium and activation of the nuclear factor of activated T cells family of transcription factors (9, 10). In addition, Itk regulates the development of Th2 cells and their subsequent cytokine secretion, thereby modulating the immune response (10 -13). A common feature of these kinases is that they require the activation of PI3 kinase, as well as Src kinase activation for their activity (14 -17). These kinases, including Itk, have a PH domain that allows them to be recruited to the plasma membrane by an activated PI3 kinase (1). Thus, membrane recruitment is a critical component of their activation, and Itk can be found at the plasma membrane of cells, although other events are required for its full activity (17)(18)(19). It is, however, not clear whether this is general membrane localization of the protein in anticipation of activation or a specific localization in certain regions of the plasma membrane.The structure of Itk in cells is not known, although it has been suggested to form dimers in its inactive state, with a resultant monomerization upon activation (20,21). We have examined these issues using a split YFP system (22) and find that Itk forms dimers or higher order clusters; however, it does so only at the plasma membrane and only in the vicinity of a receptor that can recruit PI3 kinase. Our data shed new light on the regulation, struct...