ABSTRACTE3 ubiquitin ligases are part of various families of proteins and include hundreds of members, which play key roles in all aspects of cell biology. They generally regulate the half-life of other proteins but can also modulate their cellular localization and functions. The MARCH family of ubiquitin ligases is composed of 11 members and two closely related proteins, MARCH1 and MARCH8, share similar targets, while being active in different cell types. Although they appear to target principally immune cell components, such as MHC class II molecules and the co-stimulatory molecule CD86, the repertory of their targets remains to be fully documented. Here, to further define the MARCH1’s interactome, we adapted a proximity-dependent biotin identification (BioID)-based screening approach in live HEK293 cells. We transfected a fusion protein consisting of mouse MARCH1 linked to YFP at its N-terminus and to the biotin ligase ofAquifex aeolicusat its C-terminus. Upon transient overexpression of this construct in the presence of exogenous biotin, we could recover biotinylated proteins that are presumably found within 10nm of MARCH1. To help in the identification ofbona fidedown-regulated specific targets, we compared MARCH1’s interactome with the one obtained using a ubiquitination-deficient MARCH1 mutant (MARCH1W104A). CD98 and CD71, two previously described targets of MARCH1, were identified in this screen. Of 16 other biotinylated proteins identified by semi-quantitative mass spectrometry, 10 were tested directly by flow cytometry to monitor their expression in the presence or absence of transfected MARCH1. The protein levels of five of these endogenous targets, CD29, CD112, NKCC1, CD147 and SNAT2, confirmed their negative regulation by MARCH1 in this system. SNAT2 was particularly sensitive to the presence of MARCH1 and was found to be ubiquitinated on Western blots following immunoprecipitation. Thus, BioID2 is an effective mean of characterizing the interactome of MARCH1 and the identification of SNAT2 suggests a role of this ubiquitin ligase in cellular metabolism.

Background/Objectives: Oncolytic viruses infect and kill tumor cells while leaving normal cells unharmed. They are often attenuated through the reduction in their ability to antagonize antiviral defenses, leading to robust replication in tumor cells, which often possess defects in antiviral pathways, while minimizing replication in normal cells. However, not all tumors have defects in their antiviral defenses, and virus replication in these tumors is minimal, thus limiting therapeutic benefits. Therefore, identifying and modulating host factors that regulate virus replication in oncolytic virus-resistant cancer cells, but not normal cells, could lead to increased replication in these tumors and potentially improved therapeutic outcomes. Methods: To identify host factors that modulate oncolytic virus replication in tumor cells, we conducted an RNA interference screen by using a replication-competent library of Sindbis virus recombinants individually enabled with the capacity to elicit RNA interference in host genes via the expression of artificial microRNAs. Since the expression of artificial microRNAs is coupled to virus replication, this results in the selective enrichment of viral clones which express an artificial microRNA that promotes virus replication. Results: By using this approach, the serial passage of the Sindbis virus-artificial microRNA library in a tumor cell line followed by the deep sequencing of the selected viral populations led to the identification of several artificial microRNA sequences that were enriched. Furthermore, the identified artificial miRNA sequences increased the replication of several oncolytic viruses both in vitro and in vivo, ultimately leading to an enhanced therapeutic effect. Conclusions: Altogether, our study highlights the utility of this screening platform in identifying artificial microRNAs that enhance oncolytic virus efficacy.

Glucose is essential for T cell proliferation and function, yet its specific metabolic roles in vivo remain poorly defined. Here, we identify glycosphingolipid (GSL) biosynthesis as a key pathway fueled by glucose that enables CD8+ T cell expansion and cytotoxic function in vivo. Using 13C-based stable isotope tracing, we demonstrate that CD8+ effector T cells use glucose to synthesize uridine diphosphate-glucose (UDP-Glc), a precursor for glycogen, glycan, and GSL biosynthesis. Inhibiting GSL production by targeting the enzymes UDP-Glc pyrophosphorylase 2 (UGP2), UDP-Gal-4-epimerase (GALE), or UDP-Glc ceramide glucosyltransferase (UGCG) impairs CD8+ T cell expansion upon pathogen challenge. Mechanistically, we show that glucose-dependent GSL biosynthesis is required for plasma membrane lipid raft integrity and optimal T cell receptor (TCR) signaling. Moreover, UGCG-deficient CD8+ T cells display reduced granzyme expression, cytolytic activity, and tumor control in vivo. Together, our data establish GSL biosynthesis as a critical metabolic fate of glucose-beyond energy production-that is required for CD8+ T cell responses in vivo.

Abstract The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKβ independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Background/Objectives: Ovarian cancer is the deadliest gynecologic cancer, and with the majority of patients dying within the first five years of diagnosis, new therapeutic options are required. The small guanosine triphosphatase (GTPase) Ras-related nuclear protein (Ran) has been reported to be highly expressed in high-grade serous ovarian cancers (HGSOCs) and associated with poor outcomes. Blocking Ran function or preventing its expression were shown to be promising treatment strategies, however, there are currently no small molecule inhibitors available to specifically inhibit Ran function. Interestingly, a previous study suggested that the Vesicular stomatitis virus (VSV) could inhibit Ran activity. Given that VSV is an oncolytic virus (OV) and, therefore, has anti-cancer activity, we reasoned that oncolytic VSV (oVSV) might be particularly effective against ovarian cancer via Ran inhibition. Methods: We evaluated the sensitivity of patient-derived ovarian cancer cell lines to oVSV, as well as the impact of oVSV on Ran and vice versa, using overexpression systems, small interfering RNAs (siRNAs), and drug inhibition. Results: In this study, we evaluated the interplay between oVSV and Ran and found that, although oVSV does not consistently block Ran, increased Ran activation allows for better oVSV replication and tumor cell killing. Conclusions: Our study reveals a positive impact of Ran on oVSV sensitivity. Given the high expression of Ran in HGSOCs, which are particularly aggressive ovarian cancers, our data suggest that oVSV could be effective against the deadliest form of the disease.

Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

Infusion of 13 C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13 C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes –infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)—which regulates de novo aspartate synthesis—for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Glycolytic rate assay and ROS staining

Cpt1a, PPARs and FAO-related gene expression

Shows increased IFNgamma mRNA and T-bet expression in GW treated cells

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

The factors that promote T cell expansion are not fully known. Creatine is an abundant circulating metabolite that has recently been implicated in T cell function; however, its cell-autonomous role in immune-cell function is unknown. Here, we show that creatine supports cell-intrinsic CD8(+) T cell homeostasis. We further identify creatine kinase B (CKB) as the creatine kinase isoenzyme that supports these T cell properties. Loss of the creatine transporter (Slc6a8) or Ckb results in compromised CD8(+) T cell expansion in response to infection without influencing adenylate energy charge. Rather, loss of Slc6a8 or Ckb disrupts naive T cell homeostasis and weakens TCR-mediated activation of mechanistic target of rapamycin complex 1 (mTORC1) signaling required for CD8(+) T cell expansion. These data demonstrate a cell-intrinsic role for creatine transport and creatine transphosphorylation, independent of their effects on global cellular energy charge, in supporting CD8(+) T cell homeostasis and effector function.

Advances in immunotherapy have underscored the importance of antitumor immune responses in controlling cancer. However, the tumor microenvironment (TME) imposes several obstacles to the proper function of immune cells, including a metabolically challenging and immunosuppressive microenvironment. The increased metabolic activity of tumor cells can lead to the depletion of key nutrients required by immune cells and the accumulation of byproducts that hamper antitumor immunity. Furthermore, the presence of suppressive immune cells, such as regulatory T cells and myeloid-derived suppressor cells, and the expression of immune inhibitory receptors can negatively impact immune cell metabolism and function. This review summarizes the metabolic reprogramming that is characteristic of various immune cell subsets, discusses how the metabolism and function of immune cells are shaped by the TME, and highlights how therapeutic interventions aimed at improving the metabolic fitness of immune cells and alleviating the metabolic constraints in the TME can boost antitumor immunity.

By conferring systemic protection and durable benefits, cancer immunotherapies are emerging as long-term solutions for cancer treatment. One such approach that is currently undergoing clinical testing is a therapeutic anti-cancer vaccine that uses two different viruses expressing the same tumor antigen to prime and boost anti-tumor immunity. By providing the additional advantage of directly killing cancer cells, oncolytic viruses (OVs) constitute ideal platforms for such treatment strategy. However, given that the targeted tumor antigen is encoded into the viral genomes, its production requires robust infection and therefore, the vaccination efficiency partially depends on the unpredictable and highly variable intrinsic sensitivity of each tumor to OV infection. In this study, we demonstrate that anti-cancer vaccination using OVs (Adenovirus (Ad), Maraba virus (MRB), Vesicular stomatitis virus (VSV) and Vaccinia virus (VV)) co-administered with antigenic peptides is as efficient as antigen-engineered OVs and does not depend on viral replication. Our strategy is particularly attractive for personalized anti-cancer vaccines targeting patient-specific mutations. We suggest that the use of OVs as adjuvant platforms for therapeutic anti-cancer vaccination warrants testing for cancer treatment.

Metabolic reprogramming is an essential part of the T cell activation program. Upon activation, T cells undergo dramatic rewiring of their metabolic pathways to promote ATP production and biosynthesis sufficient to support the rapid exponential growth of antigen-specific T cells. The metabolic profile of T cells is shaped by both cell-intrinsic factors, such as genetics and receptor-mediated signaling, and environmental conditions, such as nutrient availability. However, our understanding of T cell metabolism has largely been shaped by studies in vitro, where oxygen and nutrients are in excess. Here, we combined bioenergetic profiling and 13C-glutamine infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. Similar to in vitro-activated T cells, glutamine is a prominent source of fuel for the TCA cycle during early expansion (3 dpi). However, at later timepoints of infection (6 dpi), the use of glutamine is substantially decreased by Teff cells. Similar to the observation of decreased glucose usage by T cells over the course of infection (Ma et al Immunity 2019). Our data suggests a dynamic flexibly in nutrient usage by T cells in vivo to fuel the different phases of T cell functions, from the early T cell response (3 dpi) to the late (6 dpi). Our work highlights the differences in T cell metabolism in vivo compared to in vitro cultures and a new method to study T cell nutrient utilization in vivo.

Oncolytic viruses (OVs) are a class of biotherapeutics that are currently being explored for the treatment of cancer. While showing promise in several pre-clinical and clinical studies, systemic delivery of these anti-cancer agents is hampered by inefficient tumor targeting and a host immune system that is highly evolved to detect and neutralize pathogens. To shield the virus from immune recognition and destruction, the use of cells as delivery vehicles has been explored for the systemic delivery of OVs. Though several types of cell carriers are able to protect OVs during intravenous delivery, many still lack the ability to specifically home to or accumulate within the tumor microenvironment. Overall, OV-based therapeutics could benefit from tumor targeting strategies to maximize tumor-specific delivery and minimize infection of off-target tissues. In the current study, we examine magnetic targeting as a strategy to improve OV infection of tumor cells in vitro. We found that magnetic targeting of magnetically-labeled VSV particles or VSV-infected cell carriers resulted in increased infection and killing of tumor cells. Furthermore, this enhanced infection of target tumor cells was observed even in the presence of virus-specific neutralizing antibodies. Overall, our findings suggest that magnetic targeting strategies can improve the infection of tumor cells and may be a viable strategy to improve the tumor-targeted delivery of oncolytic VSV-based therapeutics.

Dendritic cells, cells of the innate immune system, are found in a steady state poised to respond to activating stimuli, Once stimulated, they rapidly undergo dynamic changes in gene expression to adopt an activated phenotype capable of stimulating immune responses. We find that the microRNA miR-9 is upregulated in both bone marrow-derived DCs and conventional DC1s but not in conventional DC2s following stimulation. miR-9 expression in BMDCs and conventional DC1s promotes enhanced DC activation and function, including the ability to stimulate T cell activation and control tumor growth. We find that miR-9 regulated the expression of several negative regulators of transcription, including the transcriptional repressor Polycomb group factor 6 (Pcgf6). These findings demonstrate that miR-9 facilitates the transition of DCs from steady state to mature state by regulating the expression of several negative regulators of DC function in a cell-type-specific manner.

One-carbon metabolism fuels the high demand of cancer cells for nucleotides and other building blocks needed for increased proliferation. Although inhibitors of this pathway are widely used to treat many cancers, their global impact on anabolic and catabolic processes remains unclear. Using a combination of real-time bioenergetics assays and metabolomics approaches, we investigated the global effects of methotrexate on cellular metabolism. We show that methotrexate treatment increases the intracellular concentration of the metabolite AICAR, resulting in AMPK activation. Methotrexate-induced AMPK activation leads to decreased one-carbon metabolism gene expression and cellular proliferation as well as increased global bioenergetic capacity. The anti-proliferative and pro-respiratory effects of methotrexate are AMPK-dependent, as cells with reduced AMPK activity are less affected by methotrexate treatment. Conversely, the combination of methotrexate with the AMPK activator, phenformin, potentiates its anti-proliferative activity in cancer cells. These data highlight a reciprocal effect of methotrexate on anabolic and catabolic processes and implicate AMPK activation as a metabolic determinant of methotrexate response.