DGKα and DGKζ negatively regulate the DAG/RasGRP1/Ras pathway in T cells. Study of the specific contribution of each isoform to DAG metabolism during immune synapse formation by use of a combination of RNAi and videomicroscopy techniques identifies DGKζ as mainly responsible for DAG consumption at the immunological synapse.

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Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.

c Diacylglycerol kinase ␣ (DGK␣) regulates diacylglycerol levels, catalyzing its conversion into phosphatidic acid. The ␣ isoform is central to immune response regulation; it downmodulates Ras-dependent pathways and is necessary for establishment of the unresponsive state termed anergy. DGK␣ functions are regulated in part at the transcriptional level although the mechanisms involved remain poorly understood. Here, we analyzed the 5= end structure of the mouse DGK␣ gene and detected three binding sites for forkhead box O (FoxO) transcription factors, whose function was confirmed using luciferase reporter constructs. FoxO1 and FoxO3 bound to the 5= regulatory region of DGK␣ in quiescent T cells, as well as after interleukin-2 (IL-2) withdrawal in activated T cells. FoxO binding to this region was lost after complete T cell activation or IL-2 addition, events that correlated with FoxO phosphorylation and a sustained decrease in DGK␣ gene expression. These data strongly support a role for FoxO proteins in promoting high DGK␣ levels and indicate a mechanism by which DGK␣ function is downregulated during productive T cell responses. Our study establishes a basis for a causal relationship between DGK␣ downregulation, IL-2, and anergy avoidance.T he diacylglycerol kinases (DGK) phosphorylate diacylglycerol (DAG) into phosphatidic acid (PA), modulating the levels of these two lipid second messengers, which have several key functions in cells. DAG propagates signals by membrane recruitment of cytosolic proteins containing C1 domains, such as protein kinase C and D (PKC and PKD, respectively), the Ras-guanine nucleotide exchange factor (GEF) RasGRP1, and the Rac-GTPaseactivating protein (GAP) chimaerins (3). DAG deregulation is linked to tumorigenesis, metastasis, diabetes, heart disease, and altered immune responses (9,13,45,53). PA binds and activates proteins involved in cell growth, survival, vesicular trafficking, and cytoskeletal remodeling, and its altered metabolism is also linked to disease onset (7,14,40). Interest in the DGK as key modulators of DAG and PA function has increased in recent years as better understanding of DGK regulatory mechanisms offers opportunities for the development of novel strategies to modulate lipid metabolism for therapeutic purposes (for reviews, see references 32 and 44).DGK function attracted special attention following the characterization of its role in T lymphocyte activation. Productive activation of T lymphocytes requires the integration of the pathways regulated by Ras/mitogen-activated protein kinase (MAPK)/AP1 and Ca 2ϩ /nuclear factor of activated T cells (NFAT). Failure to trigger an adequate balance of these signals, due, for example, to lack of costimulation, drives T cells into a nonresponsive state termed anergy, in which cells survive for long periods in the absence of proliferation (1). DGK␣ is a type I DGK particularly abundant in thymus and mature T lymphocytes (55), and early studies showed its function as a negative modulator of the Ras/ MAPK pathway. DGK␣ limits...

The lack of actionable mutations in patients with non‐small cell lung cancer (NSCLC) presents a significant hurdle in the design of targeted therapies for this disease. Here, we identify somatically mutated ABL1 as a genetic dependency that is required to maintain NSCLC cell survival. We demonstrate that NSCLC cells with ABL1 mutations are sensitive to ABL inhibitors and we verify that the drug‐induced effects on cell viability are specific to pharmacological inhibition of the ABL1 kinase. Furthermore, we confirm that imatinib suppresses lung tumor growth in vivo, specifically in lung cancer cells harboring a gain‐of‐function (GOF) mutation in ABL1. Consistent with structural modeling, we demonstrate that mutations in ABL1 identified in primary NSCLC tumors and a lung cancer cell line increase downstream pathway activation compared to wild‐type ABL1. Finally, we observe that the ABL1 cancer mutants display an increased cytosolic localization, which is associated with the oncogenic properties of the ABL1 kinase. In summary, our results suggest that NSCLC patients with ABL1 mutations could be stratified for treatment with imatinib in combination with other therapies.

Diacylglycerol kinases (DGKs) transform diacylglycerol (DAG) into phosphatidic acid (PA), balancing the levels of these key metabolic and signaling lipids. We previously showed that PA derived from the DGKζ isoform promotes mammalian target of rapamycin complex 1 (mTORC1) activation. This function might be crucial for the growth and survival of cancer cells, especially for those resistant to the allosteric mTOR inhibitor rapamycin. How this positive function of DGKζ coordinates with DAG metabolism and signaling is unknown. In this study, we used a rapamycin-resistant colon cancer cell line as a model to address the role of DGKζ in tumor cells. We found that DGKζ predominated over other PA sources such as DGKα or phospholipase D to activate mTORC1, and that its activity was a component of the rapamycin-induced feedback loops. We show that the DGKζ DAG-consuming function is central to cell homeostasis, as DAG negatively regulates levels of the lipogenic transcription factor SREBP-1. Our findings suggest a model in which simultaneous regulation of DAG and PA levels by DGKζ is integrated with mTOR function to maintain tumor cell homeostasis; we provide new evidence of the crosstalk between mTOR and lipid metabolism that will be advantageous in the design of drug therapies.

RAS is one of the most frequently altered oncogenes, yet RAS-driven tumors are largely refractory to anticancer therapies. Fedele and colleagues demonstrate that SHP2 inhibitors prevent adaptive MEK inhibitor resistance; therefore, combining MEK and SHP2 inhibitors represents an exciting new therapeutic approach for the treatment of RAS-driven cancers. Cancer Discov; 8(10); 1210-2.

Many Gram-negative bacteria produce N-acyl-homoserine lactones (AHLs), quorum sensing (QS) molecules that can be enzymatically inactivated by quorum quenching (QQ) processes; this approach is considered an emerging antimicrobial alternative. In this study, kinetic parameters of several AHLs hydrolyzed by penicillin acylase from Streptomyces lavendulae (SlPA) and aculeacin A acylase from Actinoplanes utahensis (AuAAC) have been determined. Both enzymes catalyze efficiently the amide bond hydrolysis in AHLs with different acyl chain moieties (with or without 3-oxo modification) and exhibit a clear preference for AHLs with long acyl chains (C12-HSL > C14-HSL > C10-HSL > C8-HSL for SlPA, whereas C14-HSL > C12-HSL > C10-HSL > C8-HSL for AuAAC). Involvement of SlPA and AuAAC in QQ processes was demonstrated by Chromobacterium violaceum CV026-based bioassays and inhibition of biofilm formation by Pseudomonas aeruginosa, a process controlled by QS molecules, suggesting the application of these multifunctional enzymes as quorum quenching agents, this being the first time that quorum quenching activity was shown by an aculeacin A acylase. In addition, a phylogenetic study suggests that SlPA and AuAAC could be part of a new family of actinomycete acylases, with a preference for substrates with long aliphatic acyl chains, and likely involved in QQ processes.