Metabolic programs of T cell tissue residency empower tumour immunity.

Tissue resident memory CD8+ T (TRM) cells offer rapid and long-term protection at sites of reinfection1. Tumour-infiltrating lymphocytes with characteristics of TRM cells maintain enhanced effector functions, predict responses to immunotherapy and accompany better prognoses2,3. Thus, an improved understanding of the metabolic strategies that enable tissue residency by T cells could inform new approaches to empower immune responses in tissues and solid tumours. Here, to systematically define the basis for the metabolic reprogramming supporting TRM cell differentiation, survival and function, we leveraged in vivo functional genomics, untargeted metabolomics and transcriptomics of virus-specific memory CD8+ T cell populations. We found that memory CD8+ T cells deployed a range of adaptations to tissue residency, including reliance on non-steroidal products of the mevalonate-cholesterol pathway, such as coenzyme Q, driven by increased activity of the transcription factor SREBP2. This metabolic adaptation was most pronounced in the small intestine, where TRM cells interface with dietary cholesterol and maintain a heightened state of activation4, and was shared by functional tumour-infiltrating lymphocytes in diverse tumour types in mice and humans. Enforcing synthesis of coenzyme Q through deletion of Fdft1 or overexpression of PDSS2 promoted mitochondrial respiration, memory T cell formation following viral infection and enhanced antitumour immunity. In sum, through a systematic exploration of TRM cell metabolism, we reveal how these programs can be leveraged to fuel memory CD8+ T cell formation in the context of acute infections and enhance antitumour immunity.

ReIGNITE Radiation Therapy Boost: A Prospective, International Study of Radiation Oncologists' Accuracy in Contouring Prostate Tumors for Focal Radiation Therapy Boost on Conventional Magnetic Resonance Imaging Alone or With Assistance of Restriction Spectrum Imaging.

PURPOSE: In a phase III randomized trial, adding a radiation boost to tumor(s) visible on MRI improved prostate cancer (PCa) disease-free and metastasis-free survival without additional toxicity. Radiation oncologists' ability to identify prostate tumors is critical to widely adopting intraprostatic tumor radiotherapy boost for patients. A diffusion MRI biomarker, called the Restriction Spectrum Imaging restriction score (RSIrs), has been shown to improve radiologists' identification of clinically significant PCa. We hypothesized that (1) radiation oncologists would find accurately delineating PCa tumors on conventional MRI challenging and (2) using RSIrs maps would improve radiation oncologists' accuracy for PCa tumor delineation. METHODS AND MATERIALS: In this multi-institutional, international, prospective study, 44 radiation oncologists (participants) and 2 expert radiologists (experts) contoured prostate tumors on 39 total patient cases using conventional MRI with or without RSIrs maps. Participant volumes were compared to the consensus expert volumes. Contouring accuracy metrics included percent overlap with expert volume, Dice coefficient, conformal number, and maximum distance beyond expert volume. RESULTS: 1604 participant volumes were produced. 40 of 44 participants (91%) completely missed ≥1 expert-defined target lesion without RSIrs, compared to 13 of 44 (30%) with RSIrs maps. On conventional MRI alone, 134 of 762 contour attempts (18%) completely missed the target, compared to 18 of 842 (2%) with RSIrs maps. Use of RSIrs maps improved all contour accuracy metrics by approximately 50% or more. Mixed effects modeling confirmed that RSIrs maps were the main variable driving improvement in all metrics. System Usability Scores indicated RSIrs maps significantly improved the contouring experience (72 vs. 58, p < 0.001). CONCLUSIONS: Radiation oncologists struggle with accurately delineating visible PCa tumors on conventional MRI. RSIrs maps improve radiation oncologists' ability to target MRI-visible tumors for prostate tumor boost.

Cellular sensing of extracellular purine nucleosides triggers an innate IFN-ß response.

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-ß (IFN-ß) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-ß. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-ß. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-ß production.

HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity.

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells.

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

SGEF Is Regulated via TWEAK/Fn14/NF-κB Signaling and Promotes Survival by Modulation of the DNA Repair Response to Temozolomide.

UNLABELLED: Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide, GB tumors develop treatment resistance and ultimately recur. Impaired response to treatment occurs rapidly, conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance to develop targeted therapies to combat this refractory disease. Previous observations indicated that SGEF (ARHGEF26), a RhoG-specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and plays a role in promoting TWEAK-Fn14-mediated glioma invasion. Here, further investigation revealed an important role for SGEF in glioma cell survival. SGEF expression is upregulated by TWEAK-Fn14 signaling via NF-κB activity while shRNA-mediated reduction of SGEF expression sensitizes glioma cells to temozolomide-induced apoptosis and suppresses colony formation following temozolomide treatment. Nuclear SGEF is activated following temozolomide exposure and complexes with the DNA damage repair (DDR) protein BRCA1. Moreover, BRCA1 phosphorylation in response to temozolomide treatment is hindered by SGEF knockdown. The role of SGEF in promoting chemotherapeutic resistance highlights a heretofore unappreciated driver, and suggests its candidacy for development of novel targeted therapeutics for temozolomide-refractory, invasive GB cells. IMPLICATION: SGEF, as a dual process modulator of cell survival and invasion, represents a novel target for treatment refractory glioblastoma.

Mcl-1 mediates TWEAK/Fn14-induced non-small cell lung cancer survival and therapeutic response.

UNLABELLED: Insensitivity to standard clinical interventions, including chemotherapy, radiotherapy, and tyrosine kinase inhibitor (TKI) treatment, remains a substantial hindrance towards improving the prognosis of patients with non-small cell lung cancer (NSCLC). The molecular mechanism of therapeutic resistance remains poorly understood. The TNF-like weak inducer of apoptosis (TWEAK)-FGF-inducible 14 (TNFRSF12A/Fn14) signaling axis is known to promote cancer cell survival via NF-κB activation and the upregulation of prosurvival Bcl-2 family members. Here, a role was determined for TWEAK-Fn14 prosurvival signaling in NSCLC through the upregulation of myeloid cell leukemia sequence 1 (MCL1/Mcl-1). Mcl-1 expression significantly correlated with Fn14 expression, advanced NSCLC tumor stage, and poor patient prognosis in human primary NSCLC tumors. TWEAK stimulation of NSCLC cells induced NF-κB-dependent Mcl-1 protein expression and conferred Mcl-1-dependent chemo- and radioresistance. Depletion of Mcl-1 via siRNA or pharmacologic inhibition of Mcl-1, using EU-5148, sensitized TWEAK-treated NSCLC cells to cisplatin- or radiation-mediated inhibition of cell survival. Moreover, EU-5148 inhibited cell survival across a panel of NSCLC cell lines. In contrast, inhibition of Bcl-2/Bcl-xL function had minimal effect on suppressing TWEAK-induced cell survival. Collectively, these results position TWEAK-Fn14 signaling through Mcl-1 as a significant mechanism for NSCLC tumor cell survival and open new therapeutic avenues to abrogate the high mortality rate seen in NSCLC. IMPLICATIONS: The TWEAK-Fn14 signaling axis enhances lung cancer cell survival and therapeutic resistance through Mcl-1, positioning both TWEAK-Fn14 and Mcl-1 as therapeutic opportunities in lung cancer.

Implications of Rho GTPase Signaling in Glioma Cell Invasion and Tumor Progression.

Glioblastoma (GB) is the most malignant of primary adult brain tumors, characterized by a highly locally invasive cell population, as well as abundant proliferative cells, neoangiogenesis, and necrosis. Clinical intervention with chemotherapy or radiation may either promote or establish an environment for manifestation of invasive behavior. Understanding the molecular drivers of invasion in the context of glioma progression may be insightful in directing new treatments for patients with GB. Here, we review current knowledge on Rho family GTPases, their aberrant regulation in GB, and their effect on GB cell invasion and tumor progression. Rho GTPases are modulators of cell migration through effects on actin cytoskeleton rearrangement; in non-neoplastic tissue, expression and activation of Rho GTPases are normally under tight regulation. In GB, Rho GTPases are deregulated, often via hyperactivity or overexpression of their activators, Rho GEFs. Downstream effectors of Rho GTPases have been shown to promote invasiveness and, importantly, glioma cell survival. The study of aberrant Rho GTPase signaling in GB is thus an important investigation of cell invasion as well as treatment resistance and disease progression.

The Src homology 3 domain-containing guanine nucleotide exchange factor is overexpressed in high-grade gliomas and promotes tumor necrosis factor-like weak inducer of apoptosis-fibroblast growth factor-inducible 14-induced cell migration and invasion via tumor necrosis factor receptor-associated factor 2.

Glioblastoma (GB) is the highest grade of primary adult brain tumors, characterized by a poorly defined and highly invasive cell population. Importantly, these invading cells are attributed with having a decreased sensitivity to radiation and chemotherapy. TNF-like weak inducer of apoptosis (TWEAK)-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival and is dependent upon the activity of multiple Rho GTPases, including Rac1. Here we report that Src homology 3 domain-containing guanine nucleotide exchange factor (SGEF), a RhoG-specific guanine nucleotide exchange factor, is overexpressed in GB tumors and promotes TWEAK-Fn14-mediated glioma invasion. Importantly, levels of SGEF expression in GB tumors inversely correlate with patient survival. SGEF mRNA expression is increased in GB cells at the invasive rim relative to those in the tumor core, and knockdown of SGEF expression by shRNA decreases glioma cell migration in vitro and invasion ex vivo. Furthermore, we showed that, upon TWEAK stimulation, SGEF is recruited to the Fn14 cytoplasmic tail via TRAF2. Mutation of the Fn14-TRAF domain site or depletion of TNF receptor-associated factor 2 (TRAF2) expression by siRNA oligonucleotides blocked SGEF recruitment to Fn14 and inhibited SGEF activity and subsequent GB cell migration. We also showed that knockdown of either SGEF or RhoG diminished TWEAK activation of Rac1 and subsequent lamellipodia formation. Together, these results indicate that SGEF-RhoG is an important downstream regulator of TWEAK-Fn14-driven GB cell migration and invasion.