Inulin-gel-based oral immunotherapy remodels the small intestinal microbiome and suppresses food allergy.

Despite the potential of oral immunotherapy against food allergy, adverse reactions and loss of desensitization hinder its clinical uptake. Dysbiosis of the gut microbiota is implicated in the increasing prevalence of food allergy, which will need to be regulated to enable for an effective oral immunotherapy against food allergy. Here we report an inulin gel formulated with an allergen that normalizes the dysregulated ileal microbiota and metabolites in allergic mice, establishes allergen-specific oral tolerance and achieves robust oral immunotherapy efficacy with sustained unresponsiveness in food allergy models. These positive outcomes are associated with enhanced allergen uptake by antigen-sampling dendritic cells in the small intestine, suppressed pathogenic type 2 immune responses, increased interferon-γ+ and interleukin-10+ regulatory T cell populations, and restored ileal abundances of Eggerthellaceae and Enterorhabdus in allergic mice. Overall, our findings underscore the therapeutic potential of the engineered allergen gel as a suitable microbiome-modulating platform for food allergy and other allergic diseases.

Comprehensive sampling of the lung microbiome in early-stage non-small cell lung cancer.

Objectives: Data are scarce on whether the composition of the lung microbiome (extending from the nasopharynx to the peripheral lung tissue) varies according to histology or grade of non-small cell lung cancer. We hypothesized that the composition of the lung microbiome would vary according to the histology and the grade of non-small cell lung cancer. Methods: We collected naso-oral and central lobar (cancer affected, ipsilateral unaffected, and contralateral unaffected) bronchoalveolar lavage fluid and brushing samples from patients with clinical early-stage lung cancer between July 2018 and February 2020 at a single academic center. We performed bacterial 16S rRNA sequencing and then compared clinical and pathologic findings with microbiome signatures. Results: Samples were collected from 28 patients. Microbial composition in affected lobes displayed unique enrichment of oropharyngeal bacterial species that was significantly different compared with that from the unaffected contralateral lobes; patients with chronic obstructive pulmonary disease had similar diversity to those without chronic obstructive pulmonary disease (P = .1312). The lung microbiome diversity in patients with adenocarcinoma was similar to those with squamous cell cancer (P = .27). There were no differences in diversity or composition in the unaffected lobes of patients with adenocarcinoma versus squamous cell cancer. There was a trend toward lower lung microbial diversity in poorly differentiated adenocarcinomas compared with well-differentiated adenocarcinomas (P = .08). Conclusions: The lung microbiota differs between cancer affected and unaffected lobes in the same patient. Furthermore, poorly differentiated lung cancers were associated with lower microbial diversity. Larger studies will be required to confirm these findings.

Rewiring of cortical glucose metabolism fuels human brain cancer growth.

The brain avidly consumes glucose to fuel neurophysiology. Cancers of the brain, such as glioblastoma (GBM), lose aspects of normal biology and gain the ability to proliferate and invade healthy tissue. How brain cancers rewire glucose utilization to fuel these processes is poorly understood. Here we perform infusions of 13 C-labeled glucose into patients and mice with brain cancer to define the metabolic fates of glucose-derived carbon in tumor and cortex. By combining these measurements with quantitative metabolic flux analysis, we find that human cortex funnels glucose-derived carbons towards physiologic processes including TCA cycle oxidation and neurotransmitter synthesis. In contrast, brain cancers downregulate these physiologic processes, scavenge alternative carbon sources from the environment, and instead use glucose-derived carbons to produce molecules needed for proliferation and invasion. Targeting this metabolic rewiring in mice through dietary modulation selectively alters GBM metabolism and slows tumor growth. Significance: This study is the first to directly measure biosynthetic flux in both glioma and cortical tissue in human brain cancer patients. Brain tumors rewire glucose carbon utilization away from oxidation and neurotransmitter production towards biosynthesis to fuel growth. Blocking these metabolic adaptations with dietary interventions slows brain cancer growth with minimal effects on cortical metabolism.

Devimistat in Combination with Gemcitabine and Cisplatin in Biliary Tract Cancer: Preclinical Evaluation and Phase Ib Multicenter Clinical Trial (BilT-04).

PURPOSE: Devimistat (CPI-613) is a novel inhibitor of tumoral mitochondrial metabolism. We investigated the effect of devimistat in vitro and in a phase Ib clinical trial in patients with advanced biliary tract cancer (BTC). PATIENTS AND METHODS: Cell viability assays of devimistat ± gemcitabine and cisplatin (GC) were performed and the effect of devimistat on mitochondrial respiration via oxygen consumption rate (OCR) was evaluated. A phase Ib/II trial was initiated in patients with untreated advanced BTC. In phase Ib, devimistat was infused over 2 hours in combination with GC on days 1 and 8 every 21 days with a primary objective to determine the recommended phase II dose (RP2D). Secondary objectives included safety, overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: In vitro, devimistat with GC had a synergistic effect on two cell lines. Devimistat significantly decreased OCR at higher doses and in arms with divided dosing. In the phase Ib trial, 20 patients received a median of nine cycles (range, 3-19). One DLT was observed, and the RP2D of devimistat was determined to be 2,000 mg/m2 in combination with GC. Most common grade 3 toxicities included neutropenia (n = 11, 55%), anemia (n = 4, 20%), and infection (n = 3, 15%). There were no grade 4 toxicities. After a median follow-up of 15.6 months, ORR was 45% and median PFS was 10 months (95% confidence interval, 7.1-14.9). Median OS is not yet estimable. CONCLUSIONS: Devimistat in combination with GC is well tolerated and has an acceptable safety profile in patients with untreated advanced BTC.

Exploiting metabolic vulnerabilities after anti-VEGF antibody therapy in ovarian cancer.

Despite modest clinical improvement with anti-vascular endothelial growth factor antibody (AVA) therapy in ovarian cancer, adaptive resistance is ubiquitous and additional options are limited. A dependence on glutamine metabolism, via the enzyme glutaminase (GLS), is a known mechanism of adaptive resistance and we aimed to investigate the utility of a GLS inhibitor (GLSi). Our in vitro findings demonstrated increased glutamine abundance and a significant cytotoxic effect in AVA-resistant tumors when GLSi was administered in combination with bevacizumab. In vivo, GLSi led to a reduction in tumor growth as monotherapy and when combined with AVA. Furthermore, GLSi initiated after the emergence of resistance to AVA therapy resulted in a decreased metabolic conversion of pyruvate to lactate as assessed by hyperpolarized magnetic resonance spectroscopy and demonstrated robust antitumor effects with a survival advantage. Given the increasing population of patients receiving AVA therapy, these findings justify further development of GLSi in AVA resistance.

Therapeutic implications of mitochondrial stress-induced proteasome inhibitor resistance in multiple myeloma.

The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response-dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11, and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress-induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.

Metabolic collateral lethal target identification reveals MTHFD2 paralogue dependency in ovarian cancer.

Recurrent loss-of-function deletions cause frequent inactivation of tumour suppressor genes but often also involve the collateral deletion of essential genes in chromosomal proximity, engendering dependence on paralogues that maintain similar function. Although these paralogues are attractive anticancer targets, no methodology exists to uncover such collateral lethal genes. Here we report a framework for collateral lethal gene identification via metabolic fluxes, CLIM, and use it to reveal MTHFD2 as a collateral lethal gene in UQCR11-deleted ovarian tumours. We show that MTHFD2 has a non-canonical oxidative function to provide mitochondrial NAD+, and demonstrate the regulation of systemic metabolic activity by the paralogue metabolic pathway maintaining metabolic flux compensation. This UQCR11-MTHFD2 collateral lethality is confirmed in vivo, with MTHFD2 inhibition leading to complete remission of UQCR11-deleted ovarian tumours. Using CLIM's machine learning and genome-scale metabolic flux analysis, we elucidate the broad efficacy of targeting MTHFD2 despite distinct cancer genetic profiles co-occurring with UQCR11 deletion and irrespective of stromal compositions of tumours.

Radiotherapy-induced metabolic hallmarks in the tumor microenvironment.

Radiation is frequently administered for cancer treatment, but resistance or remission remains common. Cancer cells alter their metabolism after radiotherapy to reduce its cytotoxic effects. The influence of altered cancer metabolism extends to the tumor microenvironment (TME), where components of the TME exchange metabolites to support tumor growth. Combining radiotherapy with metabolic targets in the TME can improve therapy response. We review the metabolic rewiring of cancer cells following radiotherapy and put these observations in the context of the TME to describe the metabolic hallmarks of radiotherapy in the TME.

Targeting integrated epigenetic and metabolic pathways in lethal childhood PFA ependymomas.

Childhood posterior fossa group A ependymomas (PFAs) have limited treatment options and bear dismal prognoses compared to group B ependymomas (PFBs). PFAs overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3K27me3), similar to the oncohistone H3K27M. Integrated metabolic analyses in patient-derived cells and tumors, single-cell RNA sequencing of tumors, and noninvasive metabolic imaging in patients demonstrated enhanced glycolysis and tricarboxylic acid (TCA) cycle metabolism in PFAs. Furthermore, high glycolytic gene expression in PFAs was associated with a poor outcome. PFAs demonstrated high EZHIP expression associated with poor prognosis and elevated activating mark histone H3 lysine 27 acetylation (H3K27ac). Genomic H3K27ac was enriched in PFAs at key glycolytic and TCA cycle­related genes including hexokinase-2 and pyruvate dehydrogenase. Similarly, mouse neuronal stem cells (NSCs) expressing wild-type EZHIP (EZHIP-WT) versus catalytically attenuated EZHIP-M406K demonstrated H3K27ac enrichment at hexokinase-2 and pyruvate dehydrogenase, accompanied by enhanced glycolysis and TCA cycle metabolism. AMPKα-2, a key component of the metabolic regulator AMP-activated protein kinase (AMPK), also showed H3K27ac enrichment in PFAs and EZHIP-WT NSCs. The AMPK activator metformin lowered EZHIP protein concentrations, increased H3K27me3, suppressed TCA cycle metabolism, and showed therapeutic efficacy in vitro and in vivo in patient-derived PFA xenografts in mice. Our data indicate that PFAs and EZHIP-WT­expressing NSCs are characterized by enhanced glycolysis and TCA cycle metabolism. Repurposing the antidiabetic drug metformin lowered pathogenic EZHIP, increased H3K27me3, and suppressed tumor growth, suggesting that targeting integrated metabolic/epigenetic pathways is a potential therapeutic strategy for treating childhood ependymomas.

Generation of systemic antitumour immunity via the in situ modulation of the gut microbiome by an orally administered inulin gel.

The performance of immune-checkpoint inhibitors, which benefit only a subset of patients and can cause serious immune-related adverse events, underscores the need for strategies that induce T-cell immunity with minimal toxicity. The gut microbiota has been implicated in the outcomes of patients following cancer immunotherapy, yet manipulating the gut microbiome to achieve systemic antitumour immunity is challenging. Here we show in multiple murine tumour models that inulin-a widely consumed dietary fibre-formulated as a 'colon-retentive' orally administered gel can effectively modulate the gut microbiome in situ, induce systemic memory-T-cell responses and amplify the antitumour activity of the checkpoint inhibitor anti-programmed cell death protein-1 (α-PD-1). Orally delivered inulin-gel treatments increased the relative abundances of key commensal microorganisms and their short-chain-fatty-acid metabolites, and led to enhanced recall responses for interferon-γ+CD8+ T cells as well as to the establishment of stem-like T-cell factor-1+PD-1+CD8+ T cells within the tumour microenvironment. Gels for the in situ modulation of the gut microbiome may be applicable more broadly to treat pathologies associated with a dysregulated gut microbiome.

ITLN1 modulates invasive potential and metabolic reprogramming of ovarian cancer cells in omental microenvironment.

Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin's effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression.

Tumour-reprogrammed stromal BCAT1 fuels branched-chain ketoacid dependency in stromal-rich PDAC tumours.

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-ß-SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells.

Cellular Location of HNF4α is Linked With Terminal Liver Failure in Humans.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in hepatocyte function, and HNF4α-based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4α RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4α localization and the pathways involved in post-translational modification of HNF4α in human hepatocytes from patients with decompensated liver function. RNA-sequencing analysis revealed that AKT-related pathways, specifically phospho-AKT, is down-regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4α were significantly reduced, and cytoplasmic expression of HNF4α was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho-AKT to nuclear localization of HNF4α was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4α acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET-AKT pathway directly correlate with HNF4α localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4α and pathways involved in HNF4α posttranslational modification may restore hepatocyte function in patients with terminal liver failure.

Electron transport chain activity is a predictor and target for venetoclax sensitivity in multiple myeloma.

The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the 'primed' state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy.

Quantifying Metabolic Transfer Mediated by Extracellular Vesicles Using Exo-MFA: An Integrated Empirical and Computational Platform.

Extracellular vesicles (EVs) are ubiquitous nanoscale particles released from many different types of cells. They have been shown to contain proteins, DNA, RNA, miRNA, and, most recently, metabolites. These particles can travel through the intercellular space and bloodstream to have regulatory effects on distant recipients. When an EV reaches a target cell, it is taken up and degraded to release its contents for utilization within the cell. In addition to regulatory effects, EVs have been shown to supplement the high metabolic demands of recipient cells in a nutrient-deprived tumor microenvironment. We developed an integrated empirical and computational platform to quantify metabolic contribution of source cell-derived EVs to recipient cells. The versatile Exo-MFA software tool utilizes 13C stable-isotope tracing data to quantify the metabolic contributions of EVs from a source cell type on a recipient cell type. This is accomplished by creating EV-depleted culture medium, producing isotope-labeled EVs from the source cells, isolating the labeled EVs from the culture supernatant, culturing the recipient cells in the presence of the labeled EVs, and measuring the resulting metabolite levels across several time points.

Synthetic lethality: a step forward for personalized medicine in cancer.

Synthetic lethality occurs between two genes when silencing of either gene alone enables viable outcomes but inhibition of both is lethal. Understanding context-dependent functioning of synthetic lethality allows therapeutic targeting in cancer. Furthermore, the paradigm shift toward precision medicine to treat cancer necessitates the establishment of biomarkers to help determine which patient populations might respond to specific drug combinations. Elucidating synthetically lethal gene combinations in cancer could establish clinically relevant drug combinations as well as biomarkers to better treat patients. Here, we have reviewed the recent synthetically lethal gene combinations in preclinical and clinical settings and discuss how this approach could help reveal potential biomarkers.

Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

Generation of Human Fatty Livers Using Custom-Engineered Induced Pluripotent Stem Cells with Modifiable SIRT1 Metabolism.

The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis and end-stage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 play a role in the development of fatty liver in inbred animals. The ability to perform similar studies using human tissue has been limited by the genetic variability in man. We generated human induced pluripotent stem cells (iPSCs) with controllable expression of SIRT1. By differentiating edited iPSCs into hepatocytes and knocking down SIRT1, we found increased fatty acid biosynthesis that exacerbates fat accumulation. To model human fatty livers, we repopulated decellularized rat livers with human mesenchymal cells, fibroblasts, macrophages, and human SIRT1 knockdown iPSC-derived hepatocytes and found that the human iPSC-derived liver tissue developed macrosteatosis, acquired proinflammatory phenotype, and shared a similar lipid and metabolic profiling to human fatty livers. Biofabrication of genetically edited human liver tissue may become an important tool for investigating human liver biology and disease.