Distinct Metabolic Programs Research Articles

134P Unraveling functionally distinct metabolic programs to predict immunotherapy response in non-small cell lung cancer (NSCLC)

134P Unraveling functionally distinct metabolic programs to predict immunotherapy response in non-small cell lung cancer (NSCLC)

Abstract 1592: Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer related death in the US. This mortality is strongly linked to the complex PDA tumor microenvironment that drives resistance to chemotherapy and immune suppression. Most patients succumb to metastatic disease, with liver being the most frequently colonized organ. Despite this, most pre-clinical studies have focused on primary PDA models to study the disease. Accordingly, the extent that the liver metastatic niche promotes immune evasion and resistance to treatment remains unclear.To directly compare primary vs. metastatic tumors in PDA tumors, we have optimized a syngeneic experimental model to contrast primary vs. metastatic lesions. To define the similarities and differences between cell types present in these tumors, we employed a single-cell transcriptomics approach. Our analysis has identified shared populations of malignant epithelial, stromal, and immune cells between lesions, allowing for a direct comparison of programming. Here, we have observed that the dominant populations of PDA cells in pancreas vs. liver tumors engage in distinct metabolic programs that suggest different actionable vulnerabilities.Beyond these, we also observed fibroblasts and myeloid sub-populations that are exclusive to liver tumors. These data further support our hypothesis that alternative mechanisms of metabolic, immune, and stromal interactions take place in the metastatic tumor microenvironment. To define these crosstalk networks, we employed the interactome tool CellChat, which has uncovered unique signaling interactions between the CAF and cancer cell populations in pancreatic or liver tumors. Overall, these data have the potential to lead to new avenues to directly target cancer cells or engage an effective immune response that can be designed to treat metastatic pancreatic cancer- an urgent clinical need. Citation Format: Rima Singh, Christopher Halbrook. Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1592.

Abstract C024: Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer related deaths in the US. This mortality is strongly linked to the complex PDA tumor microenvironment that drives resistance to chemotherapy and immune suppression. Most patients succumb to metastatic disease, with liver being the most frequently colonized organ. Despite this, most pre-clinical studies have focused on primary PDA models to study the disease. Accordingly, the extent that the liver metastatic niche promotes immune evasion and resistance to treatment remains unclear. To directly compare primary vs. metastatic tumors in PDA tumors, we have optimized a syngeneic experimental model to contrast primary vs. metastatic lesions. To define the similarities and differences between cell types present in these tumors, we employed a single-cell transcriptomics approach. Our analysis has identified shared populations of malignant epithelial, stromal, and immune cells between lesions, allowing for a direct comparison of programming. Here, we have observed that the dominant populations of PDA cells in pancreas vs. liver tumors engage in distinct metabolic programs that suggest different actionable vulnerabilities. Beyond these, we also observed fibroblasts and myeloid sub-populations that are exclusive to liver tumors. These data further support our hypothesis that alternative mechanisms of metabolic, immune, and stromal interactions take place in the metastatic tumor microenvironment. To define these crosstalk networks, we employed the interactome tool CellChat, which has uncovered unique signaling interactions between the CAF and cancer cell populations in pancreatic and liver tumors. Overall, these data have the potential to lead to new avenues to directly target cancer cells or engage an effective immune response that can be designed to treat metastatic pancreatic cancer- an urgent clinical need. Citation Format: Rima Singh, Natalie Yousefian, Nina Steele, Christopher Halbrook. Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C024.

Endocrine-disrupting compounds and metabolomic reprogramming in breast cancer.

Endocrine-disrupting chemicals pose a growing threat to human health through their increasing presence in the environment and their potential interactions with the mammalian endocrine systems. Due to their structural similarity to hormones like estrogen, these chemicals can interfere with endocrine signaling, leading to many deleterious effects. Exposure to estrogenic endocrine-disrupting compounds (EDC) is a suggested risk factor for the development of breast cancer, one of the most frequently diagnosed cancers in women. However, the mechanisms through which EDCs contribute to breast cancer development remain elusive. To rapidly proliferate, cancer cells undertake distinct metabolic programs to utilize existing nutrients in the tumor microenvironment and synthesize macromolecules de novo. EDCs are known to dysregulate cell signaling pathways related to cellular metabolism, which may be an important mechanism through which they exert their cancer-promoting effects. These altered pathways can be studied via metabolomic analysis, a new advancement in -omics technologies that can interrogate molecular pathways that favor cancer development and progression. This review will summarize recent discoveries regarding EDCs and the metabolic reprogramming that they may induce to facilitate the development of breast cancer.

DIPG-56. PEDIATRIC GLIOMAS EXHIBIT A UNIQUE LIPID METABOLIC PHENOTYPE DISTINCT FROM ADULT GBMS

Abstract Pediatric and adult IDH-wildtype gliomas (GBM) differ in both their clinical and molecular characteristics. However, whether distinct metabolic vulnerabilities exist between these two types of malignant brain tumors is unknown. Here, we performed integrated molecular and lipidomic profiling of both adult IDHwt and pediatric gliomas to identify distinguishable lipid phenotypes and consequent vulnerabilities. This analysis revealed IDHwt gliomas have heightened de novo lipid biosynthetic programs with an elevated abundance of triacylglycerides (TAGs). By contrast, pediatric gliomas exhibited lower lipid biosynthetic capacity and contained a higher enrichment for phospholipids. As a consequence of this unique metabolic program, pediatric gliomas relied of uptake of environmental complex fatty acids derived from normal resident brain cells for growth. Accordingly, therapeutic abrogation of lipid scavenging pathways selectively inhibited proliferation of pediatric gliomas compared to adult IDHwt counterparts. Together, these studies identify distinct metabolic programs among adult and pediatric gliomas, revealing new therapeutically actionable targets to slow pediatric glioma growth.

Rewiring of the Host Cell Metabolome and Lipidome during Lytic Gammaherpesvirus Infection Is Essential for Infectious-Virus Production.

Oncogenic virus infections are estimated to cause ~15% of all cancers. Two prevalent human oncogenic viruses are members of the gammaherpesvirus family: Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV). We use murine herpesvirus 68 (MHV-68), which shares significant homology with KSHV and EBV, as a model system to study gammaherpesvirus lytic replication. Viruses implement distinct metabolic programs to support their life cycle, such as increasing the supply of lipids, amino acids, and nucleotide materials necessary to replicate. Our data define the global changes in the host cell metabolome and lipidome during gammaherpesvirus lytic replication. Our metabolomics analysis found that MHV-68 lytic infection induces glycolysis, glutaminolysis, lipid metabolism, and nucleotide metabolism. We additionally observed an increase in glutamine consumption and glutamine dehydrogenase protein expression. While both glucose and glutamine starvation of host cells decreased viral titers, glutamine starvation led to a greater loss in virion production. Our lipidomics analysis revealed a peak in triacylglycerides early during infection and an increase in free fatty acids and diacylglyceride later in the viral life cycle. Furthermore, we observed an increase in the protein expression of multiple lipogenic enzymes during infection. Interestingly, pharmacological inhibitors of glycolysis or lipogenesis resulted in decreased infectious virus production. Taken together, these results illustrate the global alterations in host cell metabolism during lytic gammaherpesvirus infection, establish essential pathways for viral production, and recommend targeted mechanisms to block viral spread and treat viral induced tumors. IMPORTANCE Viruses are intracellular parasites which lack their own metabolism, so they must hijack host cell metabolic machinery in order to increase the production of energy, proteins, fats, and genetic material necessary to replicate. Using murine herpesvirus 68 (MHV-68) as a model system to understand how similar human gammaherpesviruses cause cancer, we profiled the metabolic changes that occur during lytic MHV-68 infection and replication. We found that MHV-68 infection of host cells increases glucose, glutamine, lipid, and nucleotide metabolic pathways. We also showed inhibition or starvation of glucose, glutamine, or lipid metabolic pathways results in an inhibition of virus production. Ultimately, targeting changes in host cell metabolism due to viral infection can be used to treat gammaherpesvirus-induced cancers and infections in humans.

Role of UPRmt and mitochondrial dynamics in host immunity: it takes two to tango.

The immune system of a host contains a group of heterogeneous cells with the prime aim of restraining pathogenic infection and maintaining homeostasis. Recent reports have proved that the various subtypes of immune cells exploit distinct metabolic programs for their functioning. Mitochondria are central signaling organelles regulating a range of cellular activities including metabolic reprogramming and immune homeostasis which eventually decree the immunological fate of the host under pathogenic stress. Emerging evidence suggests that following bacterial infection, innate immune cells undergo profound metabolic switching to restrain and countervail the bacterial pathogens, promote inflammation and restore tissue homeostasis. On the other hand, bacterial pathogens affect mitochondrial structure and functions to evade host immunity and influence their intracellular survival. Mitochondria employ several mechanisms to overcome bacterial stress of which mitochondrial UPR (UPRmt) and mitochondrial dynamics are critical. This review discusses the latest advances in our understanding of the immune functions of mitochondria against bacterial infection, particularly the mechanisms of mitochondrial UPRmt and mitochondrial dynamics and their involvement in host immunity.

L-arginine homeostasis governs adult neural stem cell activation by modulating energy metabolism invivo.

Neurogenesis in the developing and adult brain is intimately linked to remodeling of cellular metabolism. However, it is still unclear how distinct metabolic programs and energy sources govern neural stem cell (NSC) behavior and subsequent neuronal differentiation. Here, we found that adult mice lacking the mitochondrial urea metabolism enzyme, Arginase-II (Arg-II), exhibited NSC overactivation, thereby leading to accelerated NSC pool depletion and decreased hippocampal neurogenesis over time. Mechanistically, Arg-II deficiency resulted in elevated L-arginine levels and induction of a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) caused by impaired attachment of hexokinase-I to mitochondria. Notably, selective inhibition of OXPHOS ameliorated NSC overactivation and restored abnormal neurogenesis in Arg-II deficient mice. Therefore, Arg-II-mediated intracellular L-arginine homeostasis directly influences the metabolic fitness of neural stem cells that is essential to maintain neurogenesis with age.

317 Healthy repair of injured skin requires mitochondrial repurposing in wound macrophages

Cells of the monocyte/macrophage lineage (MFs) are an essential component of the body’s innate ability to restore tissue function after injury. Yet, the molecular signals required to coordinate the transition from pro-inflammatory clearance of injury-associated MFs to pro-resolution signals that rebuild tissue architecture and restore homeostasis remain unresolved. Here we decipher whether metabolic reprogramming drives MF activation and function in healing tissues. By combining multiple methods including generation of genetically modified mouse models, bulk and single cell transcriptomic analysis of wound MFs at different stages and functional metabolic analysis, we demonstrate that skin injury induces distinct metabolic programs in wound MFs, which drive stage specific and critical repair functions over the time course of healing.

Single-Cell Proteomic Interrogation of FLT3-ITD Leukemia Compartment Revealsdistinct Leukemia Cell Populations Resistant to Quizartinib, Decitabine and Venetoclax

Triplet therapy (TT) combining Quizartinib(Quiz), Decitabine(Dec) and Venetoclax(Ven) is highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates (Yilmaz M, ASCO 2022).Signaling mutations, particularly those involving RAS/MAPK pathway, have emerged as key contributors to primary and secondary resistance to TT. Lack or loss of response to TT is not limited to presence or emergence of clones with signaling mutations and disease relapse occurs in FLT3-ITD AML patients with a variety of other mutations. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by therapy pressure, and aid in identifying proteomic correlates of response and therapeutic vulnerabilities. To this end, we performed comprehensive single-cell proteomic analysis of serially collected samples (n:162) from patients treated with TT (n:14) and doublet (n:10, DEC+Quiz) therapy, using CyTOF and interrogated leukemia phenotypic profiles, apoptotic networks, signaling pathways, differentiation states and trajectories. First, we performed unsupervised analyses to identify leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner, interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features that may correlate with clinical response and assessed whether distinct mutation profiles are associated with specific proteomic profiles that may confer resistance to therapy. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators across leukemia landscapes and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC), generally situated in close proximity to leukemia cells in UMAPs, lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells (Figure 1). Due to their contrasting apoptosis regulatory networks, we observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Of note, leukemia cell compartments harbor distinct differentiation hierarchies, the frequencies of which varied across patients. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered and reshaped the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutation diversified the leukemia landscape into a diverse continuum of differentiation states. Remarkably, we observed that RAS/MAPK mutations in FLT3-ITD AML overcame differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells(Figure1). Importantly, we observed that transitional and CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs preferentially persisted at D28, had active signaling pathways and expressed CD36, which indicates distinct metabolic programs could ensure survival under therapy pressure and also had lower levels of BCL2.The presence of CD34+ leukemia cells at D28, adapted under therapy pressure with persistent signaling at D28, both in CD34+ leukemia or MCs, was indicative of poor clinical outcomes. Conclusion: Single-cell proteomic assessment of FLT3-ITD AML treated with Quiz/Dec/Ven (TT) identifies a unique mode of resistance and proteomic features of surviving cells at D28, and elucidates how RAS/MAPK mutations diversify the leukemia proteomic landscape and modulate the architectural organization which eventually gives rise to emerging clones resistant to therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A functional analysis of 180 cancer cell lines reveals conserved intrinsic metabolic programs

Cancer cells reprogram their metabolism to support growth and invasion. While previous work has highlighted how single altered reactions and pathways can drive tumorigenesis, it remains unclear how individual changes propagate at the network level and eventually determine global metabolic activity. To characterize the metabolic lifestyle of cancer cells across pathways and genotypes, we profiled the intracellular metabolome of 180 pan‐cancer cell lines grown in identical conditions. For each cell line, we estimated activity for 49 pathways spanning the entirety of the metabolic network. Upon clustering, we discovered a convergence into only two major metabolic types. These were functionally confirmed by 13C‐flux analysis, lipidomics, and analysis of sensitivity to perturbations. They revealed that the major differences in cancers are associated with lipid, TCA cycle, and carbohydrate metabolism. Thorough integration of these types with multiomics highlighted little association with genetic alterations but a strong association with markers of epithelial–mesenchymal transition. Our analysis indicates that in absence of variations imposed by the microenvironment, cancer cells adopt distinct metabolic programs which serve as vulnerabilities for therapy.

Regulation of immune cell metabolism in health and disease: Special focus on T and B cell subsets.

Metabolism is a dynamic process and keeps changing from time to time according to the demand of a particular cell to meet its bio-energetic requirement. Different immune cells rely on distinct metabolic programs which allow the cell to balance its requirements for energy, molecular biosynthesis, and effector activity. In the aspect of infection and cancer immunology, effector T and B cells get exhausted and help tumor cells to evade immunosurveillance. On the other hand, T cells become hyperresponsive in the scenario of autoimmune diseases. In this article, we have explored the uniqueness and distinct metabolic features of key CD4+ T and B helper cell subsets, CD4+ T, B regulatory cell subsets and CD8+ T cells regarding health and disease. Th1 cells rely on glycolysis and glutaminolysis; inhibition of these metabolic pathways promotes Th1 cells in Treg population. However, Th2 cells are also dependent on glycolysis but an abundance of lactate within TME shifts their metabolic dependency to fatty acid metabolism. Th17 cells depend on HIF-1α mediated glycolysis, ablation of HIF-1α reduces Th17 cells but enhance Treg population. In contrast to effector T cells which are largely dependent on glycolysis for their differentiation and function, Treg cells mainly rely on FAO for their function. Therefore, it is of utmost importance to understand the metabolic fates of immune cells and how it facilitates their differentiation and function for different disease models. Targeting metabolic pathways to restore the functionality of immune cells in diseased conditions can lead to potent therapeutic measures.

CD4+ T cell metabolism, gut microbiota, and autoimmune diseases: implication in precision medicine of autoimmune diseases.

CD4+ T cells are critical to the development of autoimmune disorders. Glucose, fatty acids, and glutamine metabolisms are the primary metabolic pathways in immune cells, including CD4+ T cells. The distinct metabolic programs in CD4+ T cell subsets are recognized to reflect the bioenergetic requirements, which are compatible with their functional demands. Gut microbiota affects T cell responses by providing a series of antigens and metabolites. Accumulating data indicate that CD4+ T cell metabolic pathways underlie aberrant T cell functions, thereby regulating the pathogenesis of autoimmune disorders, including inflammatory bowel diseases, systemic lupus erythematosus, and rheumatoid arthritis. Here, we summarize the current progress of CD4+ T cell metabolic programs, gut microbiota regulation of T cell metabolism, and T cell metabolic adaptions to autoimmune disorders to shed light on potential metabolic therapeutics for autoimmune diseases.

Interplay between intestinal microbiota and PRDM16‐mediated metabolic regulation of intestinal stem and progenitor cells

Distinct metabolic programs regulate intestinal stem cell renewal and differentiation. However, the mechanisms that rewire metabolism during differentiation are poorly defined. Previously, we have shown that the transcription factor PRDM16 is a critical regulator of intestinal metabolism and differentiation. Acute deletion of Prdm16 in adult mice causes intestinal wasting, apoptosis, and an accumulation of poorly differentiated cells in the small intestinal crypt, primarily in the duodenum and proximal jejunum. PRDM16 has emerged as a regulator of metabolic signaling in multiple tissues including thermogenic adipose tissue and the small intestine. RNA‐seq and tracer metabolomic studies show that PRDM16 regulates fatty acid oxidation (FAO) in the intestinal crypt, leading to impaired differentiation of stem and transit amplifying cells into mature cells.Our current studies have focused on the effects of the intestinal microbiota, which produces significant quantities of metabolites that could affect cells in the digestive tract. We postulated that altering the microbiome of mice in the context of Prdm16 deletion could modify the phenotype. Strikingly, pre‐treatment of animals with an antibiotic cocktail including ampicillin, neomycin, vancomycin and metronidazole almost completely rescues the Prdm16 deletion phenotype. While deletion of Prdm16 typically leads to death within 10 days, antibiotic treated mice survive at least 12 weeks after deletion. An early hallmark of the Prdm16‐deletion phenotype is the induction of the p53 pathway in the crypt. Apoptotic cells are present in high numbers, particularly in transit amplifying progenitors. Gene expression profiling of isolated small intestinal crypts indicates that the early p53 signature that is typical of Prdm16 deletion does not develop in antibiotic treated mice. Furthermore, pretreatment of mice with antibiotics prevents the severe defects normally observed when Prdm16 is deleted ex vivo in organoid cultures. However, treatment of organoids with the same antibiotics only in culture does not lead to a phenotypic rescue. We are currently characterizing how deletion of Prdm16 alters the intestinal microbiota and the effects that those changes could have on metabolite levels in the tissue. Overall, these studies aim to understand how changes in metabolic signaling can affect stem and progenitor cell behaviors and how the microbiome and, potentially, antibiotics themselves can affect the function of the intestine.

A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy

Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer Tcells toward a desired function has broad implications for the armamentarium of Tcell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ Tcells and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knockin or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and invivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic, and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together, these findings provide a system for identification of GOF immune boosters and demonstrate PRODH2 as a target to enhance CAR-T efficacy.

Vitamin B6 Metabolism Determines T Cell Anti-Tumor Responses.

Targeting T cell metabolism is an established method of immunomodulation. Following activation, T cells engage distinct metabolic programs leading to the uptake and processing of nutrients that determine cell proliferation and differentiation. Redirection of T cell fate by modulation of these metabolic programs has been shown to boost or suppress immune responses in vitro and in vivo. Using publicly available T cell transcriptomic and proteomic datasets we identified vitamin B6-dependent transaminases as key metabolic enzymes driving T cell activation and differentiation. Inhibition of vitamin B6 metabolism using the pyridoxal 5’-phosphate (PLP) inhibitor, aminoxyacetic acid (AOA), suppresses CD8+ T cell proliferation and effector differentiation in a dose-dependent manner. We show that pyridoxal phosphate phosphatase (PDXP), a negative regulator of intracellular vitamin B6 levels, is under the control of the hypoxia-inducible transcription factor (HIF1), a central driver of T cell metabolism. Furthermore, by adoptive transfer of CD8 T cells into a C57BL/6 mouse melanoma model, we demonstrate the requirement for vitamin B6-dependent enzyme activity in mediating effective anti-tumor responses. Our findings show that vitamin B6 metabolism is required for CD8+ T cell proliferation and effector differentiation in vitro and in vivo. Targeting vitamin B6 metabolism may therefore serve as an immunodulatory strategy to improve anti-tumor immunotherapy.

Clinically relevant T cell expansion media activate distinct metabolic programs uncoupled from cellular function

Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables, including proliferation, differentiation, function, activation, and mitochondrial phenotypes. The extent of proliferation and function depended on the culture media rather than stimulation conditions. Moreover, the expanded T cell end products adapted their metabolism when switched to a different media formulation, as shown by glucose and glutamine uptake and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Expanded T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. Thus, ex vivo T cell expansion media have profound impacts on metabolism and function.

TAMI-70. METABOLIC VULNERABILITY TO GPX4 INHIBITION AND FERROPTOSIS OF QUIESCENT ASTROCYTE-LIKE GLIOMA CELL POPULATIONS

Abstract Diversity is a key feature in the glioma ecosystem. Adaptation to a changing tumor microenvironment is achieved through cellular and metabolic plasticity. Here we show that slow-cycling, astrocyte-like glioma cell subpopulations activate distinct metabolic programs, rendering them susceptible to novel treatments. We performed multi-omics analysis on transgenic murine glioma models to characterize cellular heterogeneity. Bulk RNAseq on targeted time-dependent biopsies combined with scRNAseq uncovered distinct tumor cell populations, including a quiescent, astrocyte-like population relatively insensitive to conventional chemotherapy targeting proliferating cells. Using scRNAseq, we identified a persistently conserved astrocytic population in human IDH1-mt/wt high-grade gliomas. This astrocytic tumor population was more abundant in mouse models with constitutive Notch activation, however it was associated with alterations in several other transcriptional programs, suggesting that targeted therapies would likely be ineffective at eradicating it. Gene ontology analysis revealed enrichment in mitochondrial genes specifically regulating oxidative phosphorylation and tricarboxylic acid cycle. Energetic, lipidomic and metabolomic analyses revealed significant mitochondrial β-fatty acid oxidation and lipid catabolism, with less effective oxygen consumption rate and higher basal oxidative stress. Furthermore, this astrocytic tumor population had depleted levels of basal GSH and was more sensitive to reactive oxygen species. Leveraging this metabolic vulnerability, we performed drug screens and found that therapeutic inhibition of complex I or GPX4 was highly effective and synergistic. GPX4 inhibition induced ferroptosis, a newly-discovered form of programmed non-necroptotic cell death mediated by iron-driven lipid peroxidation. Using scRNAseq and RNAscope on ex vivo slice cultures from murine and human gliomas, we found that GPX4 inhibition and ferroptosis induction in the glioma microenvironment selectively eradicated the quiescent astrocytic subpopulation whereas proliferating glioma were less sensitive. Our data therefore supports a novel treatment paradigm, employing metabolic strategies, such as ferroptosis, in conjunction with chemotherapy and RT to target distinct tumor cell populations with different therapeutic vulnerabilities.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

Distinct metabolic programs established in the thymus control effector functions of γδ T cell subsets in tumor microenvironments.

Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin-17 (IL-17) have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development, and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage, and were expanded in obesity, and in tumors of obese mice. Conversely, glucose supplementation enhanced the anti-tumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.