Immunometabolism unveiled: Pioneering breakthroughs in cancer therapeutics.

The field of immunometabolism cannot be considered 'emerging' anymore; it is at the moment one of the most active and rapidly evolving areas of biomedical research. Its hottest zone is cancer immunometabolism. This is partly due to the clinical application of immunotherapy, with either antibodies (checkpoint blockade) or cellular therapies (e.g., CAR-T cells). In addition, the proliferating tumor cells create a nutrient-deprived microenvironment that impairs the metabolic fitness and functionality of infiltrating immune cells such as T cells, NK cells, and macrophages. The key concepts are bidirectional metabolic signaling, plus the conviction that a better understanding of these processes will improve current immunotherapies, and foster new tools and targets for treatment. This collection of reviews will address various exciting aspects from junior and established scientists in the field.

Dependence on mitochondrial respiration of malignant T cells reveals a new therapeutic target for angioimmunoblastic T-cell lymphoma.

Cancer metabolic reprogramming has been recognized as one of the cancer hallmarks that promote cell proliferation, survival, as well as therapeutic resistance. Up-to-date regulation of metabolism in T-cell lymphoma is poorly understood. In particular, for human angioimmunoblastic T-cell lymphoma (AITL) the metabolic profile is not known. Metabolic intervention could help identify new treatment options for this cancer with very poor outcomes and no effective medication. Transcriptomic analysis of AITL tumor cells, identified that these cells use preferentially mitochondrial metabolism. By using our preclinical AITL mouse model, mimicking closely human AITL features, we confirmed that T follicular helper (Tfh) tumor cells exhibit a strong enrichment of mitochondrial metabolic signatures. Consistent with these results, disruption of mitochondrial metabolism using metformin or a mitochondrial complex I inhibitor such as IACS improved the survival of AITL lymphoma-bearing mice. Additionally, we confirmed a selective elimination of the malignant human AITL T cells in patient biopsies upon mitochondrial respiration inhibition. Moreover, we confirmed that diabetic patients suffering from T-cell lymphoma, treated with metformin survived longer as compared to patients receiving alternative treatments. Taking together, our findings suggest that targeting the mitochondrial metabolic pathway could be a clinically efficient approach to inhibit aggressive cancers such as peripheral T-cell lymphoma.

Inhibition of choline metabolism in an angioimmunoblastic T-cell lymphoma preclinical model reveals a new metabolic vulnerability as possible target for treatment.

BACKGROUND: Angioimmunoblastic T-cell lymphoma (AITL) is a malignancy with very poor survival outcome, in urgent need of more specific therapeutic strategies. The drivers of malignancy in this disease are CD4+ follicular helper T cells (Tfh). The metabolism of these malignant Tfh cells was not yet elucidated. Therefore, we decided to identify their metabolic requirements with the objective to propose a novel therapeutic option. METHODS: To reveal the prominent metabolic pathways used by the AITL lymphoma cells, we relied on metabolomic and proteomic analysis of murine AITL (mAITL) T cells isolated from our established mAITL model. We confirmed these results using AITL patient and healthy T cell expression data. RESULTS: Strikingly, the mAITL Tfh cells were highly dependent on the second branch of the Kennedy pathway, the choline lipid pathway, responsible for the production of the major membrane constituent phosphatidylcholine. Moreover, gene expression data from Tfh cells isolated from AITL patient tumors, confirmed the upregulation of the choline lipid pathway. Several enzymes involved in this pathway such as choline kinase, catalyzing the first step in the phosphatidylcholine pathway, are upregulated in multiple tumors other than AITL. Here we showed that treatment of our mAITL preclinical mouse model with a fatty acid oxydation inhibitor, significantly increased their survival and even reverted the exhausted CD8 T cells in the tumor into potent cytotoxic anti-tumor cells. Specific inhibition of Chokα confirmed the importance of the phosphatidylcholine production pathway in neoplastic CD4 + T cells, nearly eradicating mAITL Tfh cells from the tumors. Finally, the same inhibitor induced in human AITL lymphoma biopsies cell death of the majority of the hAITL PD-1high neoplastic cells. CONCLUSION: Our results suggest that interfering with choline metabolism in AITL reveals a specific metabolic vulnerability and might represent a new therapeutic strategy for these patients.

IL-7 germline variant: setting the stage for immune-related adverse events.

Treatment with immune checkpoint inhibitors (ICIs) is frequently associated with immune-related adverse events (irAEs). A new study identified an interleukin 7 (IL-7) allelic variant-rs16906115-as a major risk factor for the development of ICI-associated irAEs. This association is of great significance as it indicates that germline genetic variants influence the occurrence of irAEs, thus opening a new avenue for identifying high-risk patients to enable better management of ICI therapy and associated irAEs.

IRE1α overexpression in malignant cells limits tumor progression by inducing an anti-cancer immune response.

IRE1α is one of the three ER transmembrane transducers of the Unfolded Protein Response (UPR) activated under endoplasmic reticulum (ER) stress. IRE1α activation has a dual role in cancer as it may be either pro- or anti-tumoral depending on the studied models. Here, we describe the discovery that exogenous expression of IRE1α, resulting in IRE1α auto-activation, did not affect cancer cell proliferation in vitro but resulted in a tumor-suppressive phenotype in syngeneic immunocompetent mice. We found that exogenous expression of IRE1α in murine colorectal and Lewis lung carcinoma cells impaired tumor growth when syngeneic tumor cells were subcutaneously implanted in immunocompetent mice but not in immunodeficient mice. Mechanistically, the in vivo tumor-suppressive effect of overexpressing IRE1α in tumor cells was associated with IRE1α RNAse activity driving both XBP1 mRNA splicing and regulated IRE1-dependent decay of RNA (RIDD). We showed that the tumor-suppressive phenotype upon IRE1α overexpression was characterized by the induction of apoptosis in tumor cells along with an enhanced adaptive anti-cancer immunosurveillance. Hence, our work indicates that IRE1α overexpression and/or activation in tumor cells can limit tumor growth in immunocompetent mice. This finding might point toward the need of adjusting the use of IRE1α inhibitors in cancer treatments based on the predominant outcome of the RNAse activity of IRE1α.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

Tumoral microenvironment prevents de novo asparagine biosynthesis in B cell lymphoma, regardless of ASNS expression.

Depletion of circulating asparagine with l-asparaginase (ASNase) is a mainstay of leukemia treatment and is under investigation in many cancers. Expression levels of asparagine synthetase (ASNS), which catalyzes asparagine synthesis, were considered predictive of cancer cell sensitivity to ASNase treatment, a notion recently challenged. Using [U-13C5]-l-glutamine in vitro and in vivo in a mouse model of B cell lymphomas (BCLs), we demonstrated that supraphysiological or physiological concentrations of asparagine prevent de novo asparagine biosynthesis, regardless of ASNS expression levels. Overexpressing ASNS in ASNase-sensitive BCL was insufficient to confer resistance to ASNase treatment in vivo. Moreover, we showed that ASNase's glutaminase activity enables its maximal anticancer effect. Together, our results indicate that baseline ASNS expression (low or high) cannot dictate BCL dependence on de novo asparagine biosynthesis and predict BCL sensitivity to dual ASNase activity. Thus, except for ASNS-deficient cancer cells, ASNase's glutaminase activity should be considered in the clinic.

Novel T Follicular Helper-like T-Cell Lymphoma Therapies: From Preclinical Evaluation to Clinical Reality.

The classification of peripheral T-cell lymphomas (PTCL) is constantly changing and contains multiple subtypes. Here, we focus on Tfh-like PTCL, to which angioimmunoblastic T-cell lymphoma (AITL) belongs, according to the last WHO classification. The first-line treatment of these malignancies still relies on chemotherapy but gives very unsatisfying results for these patients. Enormous progress in the last decade in terms of understanding the implicated genetic mutations leading to signaling and epigenetic pathway deregulation in Tfh PTCL allowed the research community to propose new therapeutic approaches. These findings point towards new biomarkers and new therapies, including hypomethylating agents, such as azacytidine, and inhibitors of the TCR-hyperactivating molecules in Tfh PTCL. Additionally, metabolic interference, inhibitors of the NF-κB and PI3K-mTOR pathways and possibly novel immunotherapies, such as antibodies and chimeric antigen receptors (CAR) directed against Tfh malignant T-cell surface markers, are discussed in this review among other new treatment options.

Meeting report of the 4th biennial Metabolism and Cancer symposium.

The 4th International meeting Metabolism and Cancer initially programed to take place in Bordeaux (France) was held virtually on May 27-29, 2021. The three-day event was followed by around 600 participants daily from 47 countries around the world. The meeting hosted 21 speakers including selected talks and a keynote lecture from the Nobel Prize winner Sir Peter J. Ratcliffe (Oxford, UK). Presentations and discussions were divided in four scientific sessions: (a) Redox and energy metabolism (b) Redox and hypoxia (c) Metabolic profiling and epigenetic control and (d) Signalling, fuelling and metabolism in cancer and a general public session on cancer and nutrition. This report summarises the presentations and outcomes of the 4th annual Metabolism and Cancer symposium. We provide here a summary of the scientific highlights of this exciting meeting.

The prohibitin-binding compound fluorizoline inhibits mitophagy in cancer cells.

Fluorizoline is a prohibitin-binding compound that triggers apoptosis in several cell lines from murine and human origin, as well as in primary cells from hematologic malignancies by inducing the integrated stress response and ER stress. Recently, it was described that PHB (Prohibitin) 1 and 2 are crucial mitophagy receptors involved in mediating the autophagic degradation of mitochondria. We measured mitophagy in HeLa cells expressing Parkin and in A549, a lung cancer cell line that can undergo mitophagy in a Parkin-independent manner, and we demonstrated that both fluorizoline and rocaglamide A, another PHB-binding molecule, inhibit CCCP- and OA-induced mitophagy. Moreover, we demonstrated that PHBs are mediating Parkin-dependent mitophagy. In conclusion, besides being a potent pro-apoptotic compound, we present fluorizoline as a promising new mitophagy modulator that could be used as anticancer agent.

EVT-701 is a novel selective and safe mitochondrial complex 1 inhibitor with potent anti-tumor activity in models of solid cancers.

Targeting the first protein complex of the mitochondrial electron transport chain (MC1) in cancer has become an attractive therapeutic approach in the recent years, given the metabolic vulnerabilities of cancer cells. The anticancer effect exerted by the pleiotropic drug metformin and the associated reduction in hypoxia-inducible factor 1α (HIF-1α) levels putatively mediated by MC1 inhibition led to the development of HIF-1α inhibitors, such as BAY87-2243, with a more specific MC1 targeting. However, the development of BAY87-2243 was stopped early in phase 1 due to dose-independent emesis and thus there is still no clinical proof of concept for the approach. Given the importance of mitochondrial metabolism during cancer progression, there is still a strong therapeutic need to develop specific and safe MC1 inhibitors. We recently reported the synthesis of compounds with a novel chemotype and potent action on HIF-1α degradation and MC1 inhibition. We describe here the selectivity, safety profile and anti-cancer activity in solid tumors of lead compound EVT-701. In addition, using murine models of lung cancer and of Non-Hodgkin's B cell lymphoma we demonstrated that EVT-701 reduced tumor growth and lymph node invasion when used as a single agent therapy. LKB1 deficiency in lung cancer was identified as a potential indicator of accrued sensitivity to EVT-701, allowing stratification and selection of patients in clinical trials. Altogether these results support further evaluation of EVT-701 alone or in combination in preclinical models and eventually in patients.

Physiological impact of in vivo stable isotope tracing on cancer metabolism.

BACKGROUND: There is growing interest in the analysis of tumor metabolism to identify cancer-specific metabolic vulnerabilities and therapeutic targets. Finding of such candidate metabolic pathways mainly relies on the highly sensitive identification and quantitation of numerous metabolites and metabolic fluxes using metabolomics and isotope tracing analyses. However, nutritional requirements and metabolic routes used by cancer cells cultivated in vitro do not always reflect the metabolic demands of malignant cells within the tumor milieu. Therefore, to understand how the metabolism of tumor cells in its physiological environment differs from that of normal cells, these analyses must be performed in vivo. SCOPE OF REVIEW: This review covers the physiological impact of the exogenous administration of a stable isotope tracer into cancer animal models. We discuss specific aspects of in vivo isotope tracing protocols based on discrete bolus injections of a labeled metabolite: the tracer administration per se and the fasting period prior to it. In addition, we illustrate the complex physiological scenarios that arise when studying tumor metabolism - by isotopic labeling in animal models fed with a specific amino acid restricted diet. Finally, we provide strategies to minimize these limitations. MAJOR CONCLUSIONS: There is growing evidence that metabolic dependencies in cancers are influenced by tissue environment, cancer lineage, and genetic events. An increasing number of studies describe discrepancies in tumor metabolic dependencies when studied in in vitro settings or in vivo models, including cancer patients. Therefore, in-depth in vivo profiling of tumor metabolic routes within the appropriate pathophysiological environment will be key to identify relevant alterations that contribute to cancer onset and progression.

Pharmacological preconditioning protects from ischemia/reperfusion-induced apoptosis by modulating Bcl-xL expression through a ROS-dependent mechanism.

Myocardial ischemia/reperfusion (I/R) injury is a frequent perioperative threat, with numerous strategies developed to limit and/or prevent it. One interesting axis of research is the anesthetic preconditioning (APc) agent's hypothesis (such as sevoflurane, SEV). However, APc's mode of action is still poorly understood and volatile anesthetics used as preconditioning agents are often not well suited in clinical practice. Here, in vitro using H9C2 cells lines (in myeloblast state or differentiated toward cardiomyocytes) and in vivo in mice, we identified that SEV-induced APc is mediated by a mild induction of reactive oxygen species (ROS) that activates Akt and induces the expression of the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL), therefore protecting cardiomyocytes from I/R-induced death. Furthermore, we extended these results to human cardiomyocytes (derived from induced pluripotent stem - IPS - cells). Importantly, we demonstrated that this protective signaling pathway induced by SEV could be stimulated using the antidiabetic agent metformin (MET), suggesting the preconditioning properties of MET. Altogether, our study identified a signaling pathway allowing APc of cardiac injuries as well as a rational for the use of MET as a pharmacological preconditioning agent to prevent I/R injuries.

The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway.

Escape from cell death is a key event in cancer establishment/progression. While apoptosis is often considered as the main cell death pathway, upon caspase inhibition, cell death is rather delayed than blocked leading to caspase-independent cell death (CICD). Although described for years, CICD's underlying mechanism remains to be identified. Here, we performed a genome-wide siRNA lethality screening and identified the RING-Type E3 Ubiquitin Transferase (UBR2) as a specific regulator of CICD. Strikingly, UBR2 downregulation sensitized cells towards CICD while its overexpression was protective. We established that UBR2-dependent protection from CICD was mediated by the MAPK/Erk pathway. We then observed that UBR2 is overexpressed in several cancers, especially in breast cancers and contributes to CICD resistance. Therefore, our work defines UBR2 as a novel regulator of CICD, found overexpressed in cancer cells, suggesting that its targeting may represent an innovative way to kill tumor cells.

New preclinical models for angioimmunoblastic T-cell lymphoma: filling the GAP.

Mouse models are essential to study and comprehend normal and malignant hematopoiesis. The ideal preclinical model should mimic closely the human malignancy. This means that these mice should recapitulate the clinical behavior of the human diseases such as cancer and therapeutic responses with high reproducibility. In addition, the genetic mutational status, the cell phenotype, the microenvironment of the tumor and the time until tumor development occurs, should be mimicked in a preclinical model. This has been particularly challenging for human angioimmunoblastic lymphoma (AITL), one of the most prominent forms of peripheral T-cell lymphomas. A complex network of interactions between AITL tumor cells and the various cells of the tumor microenvironment has impeded the study of AITL pathogenesis in vitro. Very recently, new mouse models that recapitulate faithfully the major features of human AITL disease have been developed. Here, we provide a summary of the pathology, the transcriptional profile and genetic and immune-phenotypic features of human AITL. In addition, we give an overview of preclinical models that recapitulate more or less faithfully human AITL characteristics and pathology. These recently engineered mouse models were essential in the evaluation of novel therapeutic agents for possible treatment of AITL, a malignancy in urgent need of new treatment options.

Starvation and antimetabolic therapy promote cytokine release and recruitment of immune cells.

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell death. Cell starvation also triggers adaptive responses, like angiogenesis, that promote tissue reorganization and repair, but other adaptive responses and their mediators are still poorly characterized. To explore this issue, we analyzed secretomes from glucose-deprived cells, which revealed up-regulation of multiple cytokines and chemokines, including IL-6 and IL-8, in response to starvation stress. Starvation-induced cytokines were cell type-dependent, and they were also released from primary epithelial cells. Most cytokines were up-regulated in a manner dependent on NF-κB and the transcription factor of the integrated stress response ATF4, which bound directly to the IL-8 promoter. Furthermore, glutamine deprivation, as well as the antimetabolic drugs 2-deoxyglucose and metformin, also promoted the release of IL-6 and IL-8. Finally, some of the factors released from starved cells induced chemotaxis of B cells, macrophages, and neutrophils, suggesting that nutrient deprivation in the tumor environment can serve as an initiator of tumor inflammation.

GAPDH Overexpression in the T Cell Lineage Promotes Angioimmunoblastic T Cell Lymphoma through an NF-κB-Dependent Mechanism.

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.

GAPDH Expression Predicts the Response to R-CHOP, the Tumor Metabolic Status, and the Response of DLBCL Patients to Metabolic Inhibitors.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease treated with anti-CD20-based immuno-chemotherapy (R-CHOP). We identified that low levels of GAPDH predict a poor response to R-CHOP treatment. Importantly, we demonstrated that GAPDHlow lymphomas use OxPhos metabolism and rely on mTORC1 signaling and glutaminolysis. Consistently, disruptors of OxPhos metabolism (phenformin) or glutaminolysis (L-asparaginase) induce cytotoxic responses in GAPDHlow B cells and improve GAPDHlow B cell-lymphoma-bearing mice survival, while they are low or not efficient on GAPDHhigh B cell lymphomas. Ultimately, we selected four GAPDHlow DLBCL patients, who were refractory to all anti-CD20-based therapies, and targeted DLBCL metabolism using L-asparaginase (K), mTOR inhibitor (T), and metformin (M) (called KTM therapy). Three out of the four patients presented a complete response upon one cycle of KTM. These findings establish that the GAPDH expression level predicts DLBCL patients' response to R-CHOP treatment and their sensitivity to specific metabolic inhibitors.

Mitochondrial defect in muscle precedes neuromuscular junction degeneration and motor neuron death in CHCHD10S59L/+ mouse.

Recently, we provided genetic basis showing that mitochondrial dysfunction can trigger motor neuron degeneration, through identification of CHCHD10 encoding a mitochondrial protein. We reported patients, carrying the p.Ser59Leu heterozygous mutation in CHCHD10, from a large family with a mitochondrial myopathy associated with motor neuron disease (MND). Rapidly, our group and others reported CHCHD10 mutations in amyotrophic lateral sclerosis (ALS), frontotemporal dementia-ALS and other neurodegenerative diseases. Here, we generated knock-in (KI) mice, carrying the p.Ser59Leu mutation, that mimic the mitochondrial myopathy with mtDNA instability displayed by the patients from our original family. Before 14 months of age, all KI mice developed a fatal mitochondrial cardiomyopathy associated with enhanced mitophagy. CHCHD10S59L/+ mice also displayed neuromuscular junction (NMJ) and motor neuron degeneration with hyper-fragmentation of the motor end plate and moderate but significant motor neuron loss in lumbar spinal cord at the end stage of the disease. At this stage, we observed TDP-43 cytoplasmic aggregates in spinal neurons. We also showed that motor neurons differentiated from human iPSC carrying the p.Ser59Leu mutation were much more sensitive to Staurosporine or glutamate-induced caspase activation than control cells. These data confirm that mitochondrial deficiency associated with CHCHD10 mutations can be at the origin of MND. CHCHD10 is highly expressed in the NMJ post-synaptic part. Importantly, the fragmentation of the motor end plate was associated with abnormal CHCHD10 expression that was also observed closed to NMJs which were morphologically normal. Furthermore, we found OXPHOS deficiency in muscle of CHCHD10S59L/+ mice at 3 months of age in the absence of neuron loss in spinal cord. Our data show that the pathological effects of the p.Ser59Leu mutation target muscle prior to NMJ and motor neurons. They likely lead to OXPHOS deficiency, loss of cristae junctions and destabilization of internal membrane structure within mitochondria at motor end plate of NMJ, impairing neurotransmission. These data are in favor with a key role for muscle in MND associated with CHCHD10 mutations.

Regulation of tumor-stroma interactions by the unfolded protein response.

The unfolded protein response (UPR) is a conserved adaptive pathway that helps cells cope with the protein misfolding burden within the endoplasmic reticulum (ER). Imbalance between protein folding demand and capacity in the ER leads to a situation called ER stress that is often observed in highly proliferative and secretory tumor cells. As such, activation of the UPR signaling has emerged as a key adaptive mechanism promoting cancer progression. It is becoming widely acknowledged that, in addition to its intrinsic effect on tumor biology, the UPR can also regulate tumor microenvironment. In this review, we discuss how the UPR coordinates the crosstalk between tumor and stromal cells, such as endothelial cells, normal parenchymal cells, and immune cells. In addition, we further describe the involvement of ER stress signaling in the response to current treatments as well as its impact on antitumor immunity mainly driven by immunogenic cell death. Finally, in this context, we discuss the relevance of targeting ER stress/UPR signaling as a potential anticancer approach.