TAMI-65. FASN-MEDIATED LIPID SYNTHESIS HAMPERS ANTI-CANCER IMMUNITY OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumor in adults. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-cancer immunity by at least generating type I interferon (IFN-I) responses. However, RT also exacerbates potent immune inhibitory mechanisms that facilitate immune evasion. Notably, increased lipid synthesis by the fatty acid synthase (FASN) is an emerging mechanism that can account for the deceiving treatment efficacy and immune escape of GBM. Therefore, we hypothesize that FASN-mediated lipid synthesis represents an innate immune evasion mechanism of irradiated GBM. Supporting this hypothesis, we observed that 10 gray (Gy) irradiation of murine GBM cell lines, GL261 and CT2A, upregulates FASN protein expression and increase cellular lipid content determined by BODIPY staining and electronic microscopy. Interestingly, this effect was abrogated when GBM cells were incubated with an inhibitor of FASN. Next, to ask whether FASN was impairing RT-induced IFN-I, GL261 and CT2A cells were engineered to express an inducible shRNA silencing FASN (GBMshFASN) or its non-silencing control (GBMshNS). Irradiation of GBMshNS cells enhanced the secretion of IFN-beta and CXCL10, but this effect was more pronounced when FASN was blocked. Finally, GBMshNS and GBMshFASN cells were orthotopically implanted in mice on day 0. On day 10, selective irradiation (10Gy) was performed to the tumor. Tumor growth and immune contexture were evaluated on day 17. Magnetic resonance imaging revealed that FASN knockdown reduces tumor growth independently from RT. However, in situ immunofluorescence of GBMshFASN tumors showed increased infiltration of CD8+ T cells and CD11c+ cells only in irradiated mice bearing GBMshFASN tumors. Altogether, our data suggest that RT rewires the energy supply of GBM by promoting FASN-mediated lipid synthesis to foster immune evasion. Targeting FASN is a promising strategy to promote anti-cancer immunity and sensitize irradiated GBM to immunotherapies.

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905 FASN prevents immunogenicity of irradiated glioblastoma by inhibiting ER stress

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.905 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A950-A950

Author(s):

Mara De Martino ◽

Camille Daviaud ◽

Claire Vanpouille-Box

Keyword(s):

Er Stress ◽

Lipid Accumulation ◽

Fatty Acid Synthase ◽

Immune Escape ◽

De Novo ◽

Lipid Synthesis ◽

Adult Brain ◽

Immune Recognition

BackgroundGlioblastoma (GBM) is the most aggressive and incurable adult brain tumor. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-tumor immunity by inducing immunogenic cell death (ICD) subsequent to endoplasmic reticulum (ER) stress. However, RT also exacerbates potent immunosuppressive mechanisms that facilitate immune evasion. Notably, increased de novo lipid synthesis by the fatty acid synthase (FASN) is emerging as a mechanism of therapy resistance and immune escape. Here, we hypothesize that RT induces FASN to promote GBM survival and evade immune recognition by inhibiting ER stress and ICD.MethodsTo determine if lipid synthesis is altered in response to RT, we first assessed FASN expression by western blot (WB) and lipid accumulation by BODIPY staining in murine (CT2A and GL261) and human (U118) GBM cell lines. Next, FASN expression was blocked in CT2A cells using CRISPR-Cas9 or an inducible shRNA directed against Fasn to evaluate ICD and ER stress markers by ELISA, WB, and electron microscopy. Finally, CT2AshFASN cells or its non-silencing control (CT2AshNS) were orthotopically implanted and FASN knockdown was induced by feeding the mice with doxycycline. The immune contexture was determined by in situ immunofluorescence (n=3/group). Remaining mice were followed for survival (n=7/group).ResultsWe found that in vitro irradiation of GBM cells induces lipid accumulation in a dose-dependent fashion; an effect that is magnified over time lasting at least 6/7 days. Consistent with these findings, FASN expression was upregulated in irradiated GBM cells. Confirming the role of FASN, RT-induced accumulation of lipids was reverted when GBM cells were incubated with a FASN inhibitor. Next, we found that FASN ablation in CT2A cells induces mitochondria disruption and was sufficient to increase the expression of the ER stress makers BIP and CHOP. Along similar lines, shFASN enhances the secretion of the ICD markers HMGB1, IFN-beta and CXCL10 in irradiated CT2A cells. In vivo, CT2AshFASN tumors presented increased infiltration of CD11c+ cells and CD8+ T cells, consistent with prolonged mice survival (56 days vs. 28 days for CT2AshNS). Importantly, 43% of CT2AshFASN-bearing mice remained tumor-free for more than 70 days, while none of the CT2AshNS-bearing mice survived.ConclusionsAltogether, our data suggest that FASN-mediated lipid synthesis is an important mechanism to prevent ER stress, ICD, and anti-tumor immune responses in GBM. While much work remains to be done, our data propose FASN as a novel therapeutic target to overcome immunosuppression and sensitize GBM to immunotherapies.

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Human Cytomegalovirus miR-US33as-5p Targets IFNAR1 to Achieve Immune Evasion During Both Lytic and Latent Infection

Frontiers in Immunology ◽

10.3389/fimmu.2021.628364 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qian Zhang ◽

Xin Song ◽

Ping Ma ◽

Liping Lv ◽

Yangyang Zhang ◽

...

Keyword(s):

Viral Load ◽

Human Cytomegalovirus ◽

Immune Evasion ◽

Latent Infection ◽

Signal Transduction Pathway ◽

Luciferase Reporter ◽

Type I ◽

Immune Evasion Mechanism ◽

Reporter Assays

As the first line of antiviral defense, type I interferon (IFN) binds IFN receptor 1 (IFNAR1) and IFNAR2 to activate the Jak-STAT signal transduction pathway, producing IFN-stimulated genes (ISGs) to control viral infection. The mechanisms by which human cytomegalovirus (HCMV) counteracts the IFN pathway are only partially defined. We show that miR-US33as-5p encoded by HCMV is expressed in both lytic and latent infection. By analysis with RNA hybrid and screening with luciferase reporter assays, we identified IFNAR1 as a target of hcmv-miR-US33as-5p, which was further verified by examining the expression of two IFNAR1 mutants and the binding of IFNAR1 to miR-US33as-5p/miR-US33as-5p-M1/miR-US33as-5p-M2. We found that after the transfection of miR-US33as-5p mimics into different cell lines, the phosphorylation of downstream proteins and ISG expression were downregulated. Immunofluorescence showed that the miR-US33as-5p mimics also inhibited STAT1 translocation into the nucleus. Furthermore, we constructed HCMV with mutant miR-US33as-5p and determined that the mutation did not affect HCMV replication. We found that MRC-5/human foreskin fibroblast (HFF) cells infected with ΔmiRNA HCMV exhibited higher IFNAR1 and ISG expression and a reduced viral load in the presence of exogenous IFN than cells infected with WT HCMV did, confirming that the knockout of miR-US33as-5p impaired viral resistance to IFN. Finally, we tested the effect of ΔmiRNA HCMV on THP-1 and d-THP-1 cells, common in vitro models of latent infection and reactivation, respectively. Again, we found that cells infected with ΔmiRNA HCMV showed a reduced viral load in the presence of IFN than the control cells did, confirming that miR-US33as-5p also affects IFN resistance during both latency and reactivation. These results indicate a new microRNA (miRNA)-based immune evasion mechanism employed by HCMV to achieve lifelong infection.

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The US3 kinase of Herpes Simplex virus phosphorylates the RNA sensor RIG-I to suppress innate immunity

Journal of Virology ◽

10.1128/jvi.01510-21 ◽

2021 ◽

Author(s):

Michiel van Gent ◽

Jessica J. Chiang ◽

Santoshi Muppala ◽

Cindy Chiang ◽

Walid Azab ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Immune Escape ◽

Type I ◽

Dependent Manner ◽

Herpes Simplex Virus 1 ◽

E3 Ligases ◽

Immune Evasion Mechanism ◽

Simplex Virus ◽

Hsv 1

Recent studies demonstrated that the signaling activity of the cytosolic pathogen sensor retinoic acid-inducible gene-I (RIG-I) is modulated by a variety of post-translational modifications (PTMs) to fine-tune the antiviral type I interferon (IFN) response. Whereas K63-linked ubiquitination of the RIG-I caspase activation and recruitment domains (CARDs) catalyzed by TRIM25 or other E3 ligases activates RIG-I, phosphorylation of RIG-I at S8 and T170 represses RIG-I signal transduction by preventing the TRIM25-RIG-I interaction and subsequent RIG-I ubiquitination. While strategies to suppress RIG-I signaling by interfering with its K63-polyubiquitin-dependent activation have been identified for several viruses, evasion mechanisms that directly promote RIG-I phosphorylation to escape antiviral immunity are unknown. Here, we show that the serine/threonine (Ser/Thr) kinase US3 of herpes simplex virus 1 (HSV-1) binds to RIG-I and phosphorylates RIG-I specifically at S8. US3-mediated phosphorylation suppressed TRIM25-mediated RIG-I ubiquitination, RIG-I-MAVS binding, and type I IFN induction. We constructed a mutant HSV-1 encoding a catalytically-inactive US3 protein (K220A) and found that, in contrast to the parental virus, the US3 mutant HSV-1 is unable to phosphorylate RIG-I at S8 and elicited higher levels of type I IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in a RIG-I-dependent manner. Finally, we show that this RIG-I evasion mechanism is conserved among the alphaherpesvirus US3 kinase family. Collectively, our study reveals a novel immune evasion mechanism of herpesviruses in which their US3 kinases phosphorylate the sensor RIG-I to keep it in the signaling-repressed state. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes life-long latency in the majority of the human population worldwide. HSV-1 occasionally reactivates to produce infectious virus and to facilitate dissemination. While often remaining subclinical, both primary infection and reactivation occasionally cause debilitating eye diseases, which can lead to blindness, as well as life-threatening encephalitis and newborn infections. To identify new therapeutic targets for HSV-1-induced diseases, it is important to understand the HSV-1-host interactions that may influence infection outcome and disease. Our work uncovered direct phosphorylation of the pathogen sensor RIG-I by alphaherpesvirus-encoded kinases as a novel viral immune escape strategy and also underscores the importance of RNA sensors in surveilling DNA virus infection.

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Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0709819105 ◽

2008 ◽

Vol 105 (3) ◽

pp. 871-876 ◽

Cited By ~ 73

Author(s):

A. Miyanaga ◽

N. Funa ◽

T. Awakawa ◽

S. Horinouchi

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Lipid Synthesis ◽

Polyketide Synthases ◽

Type I ◽

Direct Transfer ◽

Type Iii

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Curcumin as an Adjuvant to Cancer Immunotherapy

Frontiers in Oncology ◽

10.3389/fonc.2021.675923 ◽

2021 ◽

Vol 11 ◽

Author(s):

Silpita Paul ◽

Gaurisankar Sa

Keyword(s):

Immune Evasion ◽

Immune Escape ◽

Growth Inhibitors ◽

Invasion And Metastasis ◽

Hallmarks Of Cancer ◽

Tumor Immune Escape ◽

Tissue Invasion ◽

Anti Cancer ◽

Immune Adjuvant ◽

Over Time

The components of the immune system play a very sincere and crucial role in combating tumors. However, despite their firm efforts of elimination, tumor cells cleverly escape the surveillance process by adopting several immune evasion mechanisms. The conversion of immunogenicity of tumor microenvironment into tolerogenic is considered as a prime reason for tumor immune escape. Therapeutically, different immunotherapies have been adopted to block such immune escaping routes along with better clinical outcomes. Still, the therapies are haunted by several drawbacks. Over time, curcumin has been considered as a potential anti-cancer molecule. Its potentialities have been recorded against the standard hallmarks of cancer such as continuous proliferation, escaping apoptosis, continuous angiogenesis, insensitivity to growth inhibitors, tissue invasion, and metastasis. Hence, the diversity of curcumin functioning has already been established and exploration of its application with immunotherapies might open up a new avenue for scientists and clinicians. In this review, we briefly discuss the tumor’s way of immune escaping, followed by various modern immunotherapies that have been used to encounter the escaping paths and their minute flaws. Finally, the conclusion has been drawn with the application of curcumin as a potential immune-adjuvant, which fearlessly could be used with immunotherapies for best outcomes.

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Keratin 17 Is Required for Lipid Metabolism in Keratinocytes and Benefits Epidermal Permeability Barrier Homeostasis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.779257 ◽

2022 ◽

Vol 9 ◽

Author(s):

Bingyu Pang ◽

Zhenlai Zhu ◽

Chunying Xiao ◽

Yixin Luo ◽

Hui Fang ◽

...

Keyword(s):

Lipid Metabolism ◽

Fatty Acid Synthase ◽

Lipid Synthesis ◽

Skin Inflammation ◽

Permeability Barrier ◽

Type I ◽

Epidermal Permeability Barrier ◽

Keratin 17

The epidermal barrier refers to the stratum corneum, the uppermost layer of the skin, and constitutes the first line of defense against invasion by potentially harmful pathogens, diminishes trans-epidermal water loss, and plays a crucial role in the maintenance of skin homeostasis. Keratin 17 (K17) is a type I epithelial keratin with multiple functions, including in skin inflammation, epithelial cell growth, protein synthesis, and tumorigenesis. However, the relationship between K17 and the skin barrier has yet to be systematically investigated. In this study, we found that acute disruption of the epidermal permeability barrier led to a rapid increase in epidermal K17 expression in vivo. Krt17 gene deficiency in mice resulted in decreased expression of lipid metabolism-related enzymes and antimicrobial peptides, while also delaying epidermal permeability barrier recovery after acute disruption. Adenovirus-mediated overexpression of K17 enhanced, whereas siRNA-mediated knockdown of Krt17 inhibited, the expression of fatty acid synthase (FASN) and that of the transcription factors SREBP-1 and PPARγ in vitro. We further confirmed that K17 can facilitate the nuclear transportation of SREBP-1 and PPARγ and promote lipid synthesis in keratinocytes. This study demonstrated that K17 contributes to the restoration of the epidermal permeability barrier via stabilizing lipid metabolism in keratinocytes.

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