Methylmalonic acid induces metabolic abnormalities and exhaustion in CD8+ T cells to suppress anti-tumor immunity.

Systemic levels of methylmalonic acid (MMA), a byproduct of propionate metabolism, increase with age and MMA promotes tumor progression via its direct effects in tumor cells. However, the role of MMA in modulating the tumor ecosystem remains to be investigated. The proliferation and function of CD8+ T cells, key anti-tumor immune cells, declines with age and in conditions of vitamin B12 deficiency, which are the two most well-established conditions that lead to increased systemic levels of MMA. Thus, we hypothesized that increased circulatory levels of MMA would lead to a suppression of CD8+ T cell immunity. Treatment of primary CD8+ T cells with MMA induced a dysfunctional phenotype characterized by robust immunosuppressive transcriptional reprogramming and marked increases in the expression of the exhaustion regulator, TOX. Accordingly, MMA treatment upregulated exhaustion markers in CD8+ T cells and decreased their effector functions, which drove the suppression of anti-tumor immunity in vitro and in vivo. Mechanistically, MMA-induced CD8+ T cell exhaustion was associated with a suppression of NADH-regenerating reactions in the TCA cycle and concomitant defects in mitochondrial function. Thus, MMA has immunomodulatory roles, thereby highlighting MMA as an important link between aging, immune dysfunction, and cancer.

The novel family of Warbicin® compounds inhibits glucose uptake both in yeast and human cells and restrains cancer cell proliferation.

Many cancer cells share with yeast a preference for fermentation over respiration, which is associated with overactive glucose uptake and breakdown, a phenomenon called the Warburg effect in cancer cells. The yeast tps1Δ mutant shows even more pronounced hyperactive glucose uptake and phosphorylation causing glycolysis to stall at GAPDH, initiation of apoptosis through overactivation of Ras and absence of growth on glucose. The goal of the present work was to use the yeast tps1Δ strain to screen for novel compounds that would preferentially inhibit overactive glucose influx into glycolysis, while maintaining basal glucose catabolism. This is based on the assumption that the overactive glucose catabolism of the tps1Δ strain might have a similar molecular cause as the Warburg effect in cancer cells. We have isolated Warbicin ® A as a compound restoring growth on glucose of the yeast tps1Δ mutant, showed that it inhibits the proliferation of cancer cells and isolated structural analogs by screening directly for cancer cell inhibition. The Warbicin ® compounds are the first drugs that inhibit glucose uptake by both yeast Hxt and mammalian GLUT carriers. Specific concentrations did not evoke any major toxicity in mice but increase the amount of adipose tissue likely due to reduced systemic glucose uptake. Surprisingly, Warbicin ® A inhibition of yeast sugar uptake depends on sugar phosphorylation, suggesting transport-associated phosphorylation as a target. In vivo and in vitro evidence confirms physical interaction between yeast Hxt7 and hexokinase. We suggest that reversible transport-associated phosphorylation by hexokinase controls the rate of glucose uptake through hydrolysis of the inhibitory ATP molecule in the cytosolic domain of glucose carriers and that in yeast tps1Δ cells and cancer cells reversibility is compromised, causing constitutively hyperactive glucose uptake and phosphorylation. Based on their chemical structure and properties, we suggest that Warbicin ® compounds replace the inhibitory ATP molecule in the cytosolic domain of the glucose carriers, preventing hexokinase to cause hyperactive glucose uptake and catabolism.

13C Tracer Analysis and Metabolomics in Dormant Cancer Cells.

Over the last two decades, major advances in the field of tumor dormancy have been made. Yet, it is not completely understood how dormant disseminated tumor cells survive and transition to a proliferative state to generate a metastatic lesion. On the other hand, metabolic rewiring has been shown to influence metastasis development through the modulation of both intracellular signaling and the crosstalk between metastatic cells and their microenvironment. Thus, studying the metabolic features of dormant disseminated tumor cells has gained importance in understanding the dormancy process. Here, we describe a method to perform metabolomics and 13C tracer analysis in 3D cultures of dormant breast cancer cells.

100 years of the Warburg effect: A cancer metabolism endeavor.

If you are a scientist and you only know one thing about tumor metabolism, it's likely the Warburg effect. But who was Otto Warburg, and how did his discoveries regarding the metabolism of tumors shape our current thinking about the metabolic needs of cancer cells?

Nucleotide metabolism in cancer cells fuels a UDP-driven macrophage cross-talk, promoting immunosuppression and immunotherapy resistance.

Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y6. Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDAhigh cancer cells match with T cell cytotoxicity dysfunction and P2RY6high TAMs. Overall, we suggest CDA and P2Y6 as potential targets for cancer immunotherapy.

mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.

Metabolic Signaling in Cancer Metastasis.

Metastases, which are the leading cause of death in patients with cancer, have metabolic vulnerabilities. Alterations in metabolism fuel the energy and biosynthetic needs of metastases but are also needed to activate cell state switches in cells leading to invasion, migration, colonization, and outgrowth in distant organs. Specifically, metabolites can activate protein kinases as well as receptors and they are crucial substrates for posttranslational modifications on histone and nonhistone proteins. Moreover, metabolic enzymes can have moonlighting functions by acting catalytically, mainly as protein kinases, or noncatalytically through protein-protein interactions. Here, we summarize the current knowledge on metabolic signaling in cancer metastasis. SIGNIFICANCE: Effective drugs for the prevention and treatment of metastases will have an immediate impact on patient survival. To overcome the current lack of such drugs, a better understanding of the molecular processes that are an Achilles heel in metastasizing cancer cells is needed. One emerging opportunity is the metabolic changes cancer cells need to undergo to successfully metastasize and grow in distant organs. Mechanistically, these metabolic changes not only fulfill energy and biomass demands, which are often in common between cancer and normal but fast proliferating cells, but also metabolic signaling which enables the cell state changes that are particularly important for the metastasizing cancer cells.

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Aging-accumulated methylmalonic acid serum levels at breast cancer diagnosis are not associated with distant metastases.

PURPOSE: Recent evidence suggests that age-accumulated methylmalonic acid (MMA) promotes breast cancer progression in mice. This study aims to investigate the association between baseline serum MMA concentrations in patients with breast cancer and the development of subsequent distant metastases. METHODS: We included 32 patients with early Luminal B-like breast cancer (LumB, median age 62.4y) and 52 patients with early triple-negative breast cancer (TNBC, median age 50.5y) who developed distant metastases within 5 years. They were matched to an equal number of early breast cancer patients (median age 62.2y for LumB and 50.5y for TNBC) who did not develop distant metastases with at least 5 years of follow-up. RESULTS: Baseline serum MMA levels at breast cancer diagnosis showed a positive correlation with age (P < 0.001) and a negative correlation with renal function and vitamin B12 (all P < 0.02), but no statistical association was found with BMI or tumor stage (P > 0.6). Between matched pairs, no significant difference was observed in MMA levels, after adjusting for kidney function and age (P = 0.19). Additionally, in a mouse model, a significant decline in MMA levels was observed in the tumor-bearing group compared to the group without tumors before and after tumor establishment or at identical times for the control group (P = 0.03). CONCLUSION: Baseline serum MMA levels in patients with breast cancer are not correlated with secondary distant metastasis. Evidence in the mouse model suggests that the presence of a tumor perturbates MMA levels.

Metabolic heterogeneity in cancer.

Cancer cells rewire their metabolism to survive during cancer progression. In this context, tumour metabolic heterogeneity arises and develops in response to diverse environmental factors. This metabolic heterogeneity contributes to cancer aggressiveness and impacts therapeutic opportunities. In recent years, technical advances allowed direct characterisation of metabolic heterogeneity in tumours. In addition to the metabolic heterogeneity observed in primary tumours, metabolic heterogeneity temporally evolves along with tumour progression. In this Review, we summarize the mechanisms of environment-induced metabolic heterogeneity. In addition, we discuss how cancer metabolism and the key metabolites and enzymes temporally and functionally evolve during the metastatic cascade and treatment.

Serum methylmalonic acid concentrations at breast cancer diagnosis significantly correlate with clinical frailty.

Methylmalonic acid (MMA), a by-product of propionate metabolism, is known to increase with age. This study investigates the potential of serum MMA concentrations as a biomarker for age-related clinical frailty in older patients with breast cancer. One hundred nineteen patients ≥ 70 years old with early-stage breast cancer were included (median age 76 years). G8 screening, full geriatric assessment, clinical parameters (i.e., estimated glomerular filtration rate (eGFR) and body mass index (BMI)), and serum sample collection were collected at breast cancer diagnosis before any therapy was administered. MMA concentrations were measured via liquid chromatography with tandem mass spectrometry. MMA concentrations significantly increased with age and eGFR (all P < 0.001) in this older population. The group with an abnormal G8 (≤ 14, 51% of patients) had significantly higher MMA levels than the group with normal G8 (> 14, 49%): 260 nmol/L vs. 188 nmol/L, respectively (P = 0.0004), even after correcting for age and eGFR (P = 0.001). Furthermore, in the detailed assessment, MMA concentrations correlated most with mobility (Eastern Cooperative Oncology Group (ECOG) Performance Status and Activities of Daily Living (ADL) tools, all P ≤ 0.02), comorbidity (Charlson Comorbidity Index (CCI) tool, P = 0.005), and polypharmacy (P < 0.001), whereas no significant associations were noted for instrumental ADL (IADL), Mini-Mental State Examination (MMSE), Geriatric Depression Scale-15 (GDS15), Mini Nutritional Assessment-Short Form (MNA-SF), and pain (all P > 0.1). In addition, our results showed that higher MMA levels correlate with poor overall survival in breast cancer patients (P = 0.003). Elevated serum MMA concentrations at initial diagnosis are significantly associated, not only with age but also independently with clinical frailty, suggesting a possible influence of MMA on clinical frailty in older patients with early-stage breast cancer.

Challenges and opportunities in targeting metabolism.

Over the past decade or two, targeting metabolism has been effective in the treatment of many diseases and disorders, particularly cancer. In a metabolism focus issue in Cell Chemical Biology, this Voices piece asks researchers from a range of backgrounds: what are some major challenges and opportunities facing the field in the coming years?

Loss of attachment promotes proline accumulation and excretion in cancer cells.

Previous studies have revealed a role for proline metabolism in supporting cancer development and metastasis. In this study, we show that many cancer cells respond to loss of attachment by accumulating and secreting proline. Detached cells display reduced proliferation accompanied by a general decrease in overall protein production and de novo amino acid synthesis compared to attached cells. However, proline synthesis was maintained under detached conditions. Furthermore, while overall proline incorporation into proteins was lower in detached cells compared to other amino acids, there was an increased production of the proline-rich protein collagen. The increased excretion of proline from detached cells was also shown to be used by macrophages, an abundant and important component of the tumor microenvironment. Our study suggests that detachment induced accumulation and secretion of proline may contribute to tumor progression by supporting increased production of extracellular matrix and providing proline to surrounding stromal cells.

Spatial metabolomics principles and application to cancer research.

Mass spectrometry imaging (MSI) is an emerging technology in cancer metabolomics. Desorption electrospray ionization (DESI) and matrix-assisted laser desorption ionization (MALDI) MSI are complementary techniques to identify hundreds of metabolites in space with close to single-cell resolution. This technology leap enables research focusing on tumor heterogeneity, cancer cell plasticity, and the communication signals between cancer and stromal cells in the tumor microenvironment (TME). Currently, unprecedented knowledge is generated using spatial metabolomics in fundamental cancer research. Yet, also translational applications are emerging, including the assessment of spatial drug distribution in organs and tumors. Moreover, clinical research investigates the use of spatial metabolomics as a rapid pathology tool during cancer surgeries. Here, we summarize MSI applications, the knowledge gained by this technology in space, future directions, and developments needed.

Combining the antianginal drug perhexiline with chemotherapy induces complete pancreatic cancer regression in vivo.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the human cancers with the poorest prognosis. Interestingly, we found that mitochondrial respiration in primary human PDAC cells depends mainly on the fatty acid oxidation (FAO) to meet basic energy requirements. Therefore, we treated PDAC cells with perhexiline, a well-recognized FAO inhibitor used in cardiac diseases. Some PDAC cells respond efficiently to perhexiline, which acts synergistically with chemotherapy (gemcitabine) in vitro and in two xenografts in vivo. Importantly, perhexiline in combination with gemcitabine induces complete tumor regression in one PDAC xenograft. Mechanistically, this co-treatment causes energy and oxidative stress promoting apoptosis but does not exert inhibition of FAO. Yet, our molecular analysis indicates that the carnitine palmitoyltransferase 1C (CPT1C) isoform is a key player in the response to perhexiline and that patients with high CPT1C expression have better prognosis. Our study reveals that repurposing perhexiline in combination with chemotherapy is a promising approach to treat PDAC.

Pharmacological induction of membrane lipid poly-unsaturation sensitizes melanoma to ROS inducers and overcomes acquired resistance to targeted therapy.

BACKGROUND: One of the key limitations of targeted cancer therapies is the rapid onset of therapy resistance. Taking BRAF-mutant melanoma as paradigm, we previously identified the lipogenic regulator SREBP-1 as a central mediator of resistance to MAPK-targeted therapy. Reasoning that lipogenesis-mediated alterations in membrane lipid poly-unsaturation lie at the basis of therapy resistance, we targeted fatty acid synthase (FASN) as key player in this pathway to evoke an exquisite vulnerability to clinical inducers of reactive oxygen species (ROS), thereby rationalizing a novel clinically actionable combination therapy to overcome therapy resistance. METHODS: Using gene expression analysis and mass spectrometry-based lipidomics of BRAF-mutant melanoma cell lines, melanoma PDX and clinical data sets, we explored the association of FASN expression with membrane lipid poly-unsaturation and therapy-resistance. Next, we treated therapy-resistant models with a preclinical FASN inhibitor TVB-3664 and a panel of ROS inducers and performed ROS analysis, lipid peroxidation tests and real-time cell proliferation assays. Finally, we explored the combination of MAPK inhibitors, TVB-3664 and arsenic trioxide (ATO, as a clinically used ROS-inducer) in Mel006 BRAF mutant PDX as a gold model of therapy resistance and assessed the effect on tumor growth, survival and systemic toxicity. RESULTS: We found that FASN expression is consistently increased upon the onset of therapy resistance in clinical melanoma samples, in cell lines and in Mel006 PDX and is associated with decreased lipid poly-unsaturation. Forcing lipid poly-unsaturation in therapy-resistant models by combining MAPK inhibition with FASN inhibition attenuated cell proliferation and rendered cells exquisitely sensitive to a host of ROS inducers. In particular, the triple combination of MAPK inhibition, FASN inhibition, and the clinical ROS-inducing compound ATO dramatically increased survival of Mel006 PDX models from 15 to 72% with no associated signs of toxicity. CONCLUSIONS: We conclude that under MAPK inhibition the direct pharmacological inhibition of FASN evokes an exquisite vulnerability to inducers of ROS by increasing membrane lipid poly-unsaturation. The exploitation of this vulnerability by combining MAPK and/or FASN inhibitors with inducers of ROS greatly delays the onset of therapy resistance and increases survival. Our work identifies a clinically actionable combinatorial treatment for therapy-resistant cancer.

Diet-induced loss of adipose hexokinase 2 correlates with hyperglycemia.

Chronically high blood glucose (hyperglycemia) leads to diabetes and fatty liver disease. Obesity is a major risk factor for hyperglycemia, but the underlying mechanism is unknown. Here, we show that a high-fat diet (HFD) in mice causes early loss of expression of the glycolytic enzyme Hexokinase 2 (HK2) specifically in adipose tissue. Adipose-specific knockout of Hk2 reduced glucose disposal and lipogenesis and enhanced fatty acid release in adipose tissue. In a non-cell-autonomous manner, Hk2 knockout also promoted glucose production in liver. Furthermore, we observed reduced hexokinase activity in adipose tissue of obese and diabetic patients, and identified a loss-of-function mutation in the hk2 gene of naturally hyperglycemic Mexican cavefish. Mechanistically, HFD in mice led to loss of HK2 by inhibiting translation of Hk2 mRNA. Our findings identify adipose HK2 as a critical mediator of local and systemic glucose homeostasis, and suggest that obesity-induced loss of adipose HK2 is an evolutionarily conserved mechanism for the development of selective insulin resistance and thereby hyperglycemia.

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-κB signaling.

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.