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Somatic mutations are a major source of cancer development, and many driver mutations have been identified in protein coding regions. However, the function of mutations located in miRNA and their target binding sites throughout the human genome remains largely unknown. Here, we built detailed cancer-specific miRNA regulatory networks across 30 cancer types to systematically analyze the effect of mutations in miRNAs and their target sites in 3′ untranslated region (3′ UTR), coding sequence (CDS), and 5′ UTR regions. A total of 3,518,261 mutations from 9,819 samples were mapped to miRNA–gene interactions (mGI). Mutations in miRNAs showed a mutually exclusive pattern with mutations in their target genes in almost all cancer types. A linear regression method identified 148 candidate driver mutations that can significantly perturb miRNA regulatory networks. Driver mutations in 3′UTRs played their roles by altering RNA binding energy and the expression of target genes. Finally, mutated driver gene targets in 3′ UTRs were significantly downregulated in cancer and functioned as tumor suppressors during cancer progression, suggesting potential miRNA candidates with significant clinical implications. A user-friendly, open-access web portal (mGI-map) was developed to facilitate further use of this data resource. Together, these results will facilitate novel noncoding biomarker identification and therapeutic drug design targeting the miRNA regulatory networks. Significance: A detailed miRNA–gene interaction map reveals extensive miRNA-mediated gene regulatory networks with mutation-induced perturbations across multiple cancers, serving as a resource for noncoding biomarker discovery and drug development.

Supplementary Figures and Supplementary Table S1

Somatic mutations are a major source of cancer development, and many driver mutations have been identified in protein coding regions. However, the function of mutations located in miRNA and their target binding sites throughout the human genome remains largely unknown. Here, we built detailed cancer-specific miRNA regulatory networks across 30 cancer types to systematically analyze the effect of mutations in miRNAs and their target sites in 3′ untranslated region (3′ UTR), coding sequence (CDS), and 5′ UTR regions. A total of 3,518,261 mutations from 9,819 samples were mapped to miRNA–gene interactions (mGI). Mutations in miRNAs showed a mutually exclusive pattern with mutations in their target genes in almost all cancer types. A linear regression method identified 148 candidate driver mutations that can significantly perturb miRNA regulatory networks. Driver mutations in 3′UTRs played their roles by altering RNA binding energy and the expression of target genes. Finally, mutated driver gene targets in 3′ UTRs were significantly downregulated in cancer and functioned as tumor suppressors during cancer progression, suggesting potential miRNA candidates with significant clinical implications. A user-friendly, open-access web portal (mGI-map) was developed to facilitate further use of this data resource. Together, these results will facilitate novel noncoding biomarker identification and therapeutic drug design targeting the miRNA regulatory networks.Significance:A detailed miRNA–gene interaction map reveals extensive miRNA-mediated gene regulatory networks with mutation-induced perturbations across multiple cancers, serving as a resource for noncoding biomarker discovery and drug development.

Malignancies can become reliant on glutamine as an alternative energy source and as a facilitator of aberrant DNA methylation, thus implicating glutaminase (GLS) as a potential therapeutic target. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective GLS inhibitor, when combined with azacytidine (AZA), in vitro and in vivo, followed by a phase Ib/II study of the combination in patients with advanced MDS. Treatment with telaglenastat/AZA led to an ORR of 70% with CR/mCRs in 53% patients and a median overall survival of 11.6 months. scRNAseq and flow cytometry demonstrated a myeloid differentiation program at the stem cell level in clinical responders. Expression of non-canonical glutamine transporter, SLC38A1, was found to be overexpressed in MDS stem cells; was associated with clinical responses to telaglenastat/AZA and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of a combined metabolic and epigenetic approach in MDS.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib's antileukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. In addition, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Finally, we found that the glutaminase inhibitor CB-839 enhanced antileukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD–positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.

Obesity is a major contributing factor to the pathogenesis of Type 2 diabetes. Multiple human genetics studies suggest that high activity of the low molecular weight protein tyrosine phosphatase (LMPTP) promotes metabolic syndrome in obesity. We reported that LMPTP is a critical promoter of insulin resistance in obesity by regulating liver insulin receptor signaling and that inhibition of LMPTP reverses obesity-associated diabetes in mice. Since LMPTP is expressed in adipose tissue but little is known about its function, here we examined the role of LMPTP in adipocyte biology. Using conditional knockout mice, we found that selective deletion of LMPTP in adipocytes impaired obesity-induced subcutaneous adipocyte hypertrophy. We assessed the role of LMPTP in adipogenesis in vitro, and found that LMPTP deletion or knockdown substantially impaired differentiation of primary preadipocytes and 3T3-L1 cells into adipocytes, respectively. Inhibition of LMPTP in 3T3-L1 preadipocytes also reduced adipogenesis and expression of proadipogenic transcription factors peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha. Inhibition of LMPTP increased basal phosphorylation of platelet-derived growth factor receptor alpha (PDGFRα) on activation motif residue Y849 in 3T3-L1, resulting in increased activation of the mitogen-associated protein kinases p38 and c-Jun N-terminal kinase and increased PPARγ phosphorylation on inhibitory residue S82. Analysis of the metabolome of differentiating 3T3-L1 cells suggested that LMPTP inhibition decreased cell glucose utilization while enhancing mitochondrial respiration and nucleotide synthesis. In summary, we report a novel role for LMPTP as a key driver of adipocyte differentiation via control of PDGFRα signaling.

BACKGROUND Hypoxia is a major driver of invasiveness and resistance in glioblastoma (GBM). We hypothesize that GBM adapts to chronic hypoxic conditions through utilization of peroxisomal fatty acid oxidation (FAO). We explored the contribution of acyl coenzyme A oxidase 1 (ACOX-1), a rate-limiting enzyme for peroxisomal FAO, in metabolic adaptation to hypoxia and as a potential therapeutic target. METHODS A primary cell line from a bevacizumab-resistant tumor (012015) was cultured under chronic hypoxia (2%O2) or normoxia (21% O2). We used UPLC-MS/MS and GC-MS to determine lipid and polar metabolites. RNA seq was performed on U251 ACOX-1 (KO) cells under normoxic and hypoxic conditions. Differential gene expression was obtained using TopHat2 (genome alignment), HTSeq, and DEseq. Gene set enrichment analysis was used to compare biological pathways between conditions. ACOX-1 was deleted using CRISPR-Cas9 and orthotopically implanted in NCr/SCID nude mice. Animals were treated with 30 mg/kg of pazopanib or vehicle daily through oral gavage. RESULTS In 012015 cells we observed altered carnitine metabolites, increase in specific triglycerides, and S-lactoylglutathione indicating dysfunctional mitochondrial FAO, and increase in oxidative stress between hypoxic and normoxic conditions. Also, decrease in citric acid cycle metabolites, increase in glycolytic metabolites were seen in hypoxia. Further RNA seq on U251 ACOX-1 (KO) cells showed downregulated genes such as PCK1, SCD, SREBF1, PIPOX (p< 1E-8) and significant KEGG pathways such as unsaturated fatty acid (p= 0.009), peroxisome (p= 0.025) and PPAR (p= 0.016) between hypoxia and normoxia suggesting increased dependence of these cells on peroxisomal FAO. ACOX-1 (KO) mice showed increased survival when treated (p= 6.7E-04) with pazopanib. CONCLUSION In ACOX-1 (KO) model, we observed metabolomic reprogramming with downregulated genes, dysregulated metabolites, and pathways in peroxisomal FAO in hypoxia. These identified genes could be potential targets for therapy in combination with anti-angiogenic therapies that increase hypoxia in GBM.

While targeting oxidative phosphorylation (OXPHOS) is a rational anticancer strategy, patient benefit with OXPHOS inhibitors in the clinic has yet to be achieved. Based on promising preclinical data, we advanced IACS-010759, a highly potent and selective small-molecule inhibitor of mitochondrial complex I, into two phase I trials in patients with acute myeloid leukemia (NCT02882321) or advanced solid tumors (NCT03291938). Clinical findings revealed that IACS-010759 had a narrow therapeutic index with emergent dose-limiting toxicities that included elevated blood lactate and neurotoxicity, obstructing efforts to maintain target plasma exposure. Consequently, only modest on-target inhibition and limited antitumor activity were observed. Follow-up reverse translational studies uncovered that IACS-010759 reduced oxygen consumption rates in neurons and damaged myelin. Further, IACS-010759-treated mice displayed behaviors indictive of neuropathy, which were minimized with the co-administration of a histone deacetylase 6 inhibitor. Our findings urge caution in the continued development of complex I inhibitors as antitumor agents.

Exploiting metabolic vulnerabilities of cancer cells with nontoxic, plant derived compounds constitutes a novel strategy for both chemoprevention and treatment. A high-throughput screening approach was used to evaluate a library of natural products to determine the most synergistic combination in precursor-B cell acute lymphoblast leukemia. Dimethylaminoparthenolide and shikonin effectively inhibited proliferation resulting in cell death in primary and immortalized leukemia cells, while having negligible effects on normal cells. Dimethylaminoparthenolide and shikonin have been shown separately to inhibit cell survival and proliferative signaling and activate tumor suppressors and proapoptotic pathways. Untargeted metabolomics and metabolic flux analysis with stable isotopically labeled glucose and glutamine exhibited a global shift in metabolism following treatment. Pathway analysis indicated significant differences in amino acid, antioxidant, tricarboxylic acid cycle, and nucleotide metabolism. Together, dimethylaminoparthenolide and shikonin reduced the shunting of glycolytic intermediates into the pentose phosphate pathway for biosynthetic purposes. Similarly, the incorporation of glutamine and glutamine-derived metabolites into purine and pyrimidine synthesis was inhibited by the combination of dimethylaminoparthenolide and shikonin, effectively impeding biosynthetic pathways critical for leukemia cell survival. This approach demonstrates that a synergistic pair of compounds with malignant cell specificity can effectively target metabolic pathways crucial to leukemia cell proliferation and induce apoptosis.

Fatty acid profiling on gas chromatography–mass spectrometry (GC–MS) platforms is typically performed offline by manually derivatizing and analyzing small batches of samples. A GC–MS system with a fully integrated robotic autosampler can significantly improve sample handling, standardize data collection, and reduce the total hands-on time required for sample analysis. In this study, we report an optimized high-throughput GC–MS-based methodology that utilizes trimethyl sulfonium hydroxide (TMSH) as a derivatization reagent to convert fatty acids into fatty acid methyl esters. An automated online derivatization method was developed, in which the robotic autosampler derivatizes each sample individually and injects it into the GC–MS system in a high-throughput manner. This study investigated the robustness of automated TMSH derivatization by comparing fatty acid standards and lipid extracts, derivatized manually in batches and online automatically from four biological matrices. Automated derivatization improved reproducibility in 19 of 33 fatty acid standards, with nearly half of the 33 confirmed fatty acids in biological samples demonstrating improved reproducibility when compared to manually derivatized samples. In summary, we show that the online TMSH-based derivatization methodology is ideal for high-throughput fatty acid analysis, allowing rapid and efficient fatty acid profiling, with reduced sample handling, faster data acquisition, and, ultimately, improved data reproducibility.&nbsp

The inferior cure rate of T-cell acute lymphoblastic leukemia (T-ALL) is associated with inherent drug resistance. The activating NOTCH1 gene mutations have been reported to cause chemoresistance at the stem cell level1. Direct NOTCH1 inhibition has failed in clinical trials due to a narrow therapeutic window but targeting key oncogenic and metabolic pathways downstream of mutated NOTCH1 may offer novel approaches. We previously reported that rapid transformation of thymocytes at the DN3 differentiation stage into preleukemic stem cells (pre-LSC) requires elevated Notch1 in addition to the presence of Scl/Lmo11. Notably, we showed that cellular metabolism of NOTCH1-mutated T-ALLs depends on Oxidative Phosphorylation (OxPhos) and that OxPhos inhibition using the complex I inhibitor IACS-010759 (OxPhos-i) is efficacious in NOTCH1-mutated T-ALL patient derived xenografts (PDXs)2. Here, we investigated the link between NOTCH1-mutated chemoresistance and OxPhos in pre-leukemic and leukemic cells, utilizing comprehensive molecular and functional assays. We hypothesized that chemotherapy aided by OxPhos-i overcomes chemoresistance, depletes LSCs and combats T-ALL. First, we analyzed the role of OxPhos in downstream Notch1 targets at the pre- and leukemic stage considering four stages of thymocyte differentiation (D1-D4), in a mouse model of human T-ALL1. Gene set enrichment analysis (GSEA) implicated increased expression of Notch1 target genes starting at DN1, and OxPhos target genes were the highest-ranked gene set at DN3. Next, activation of Notch1 by its ligand DL4 and inhibition of OxPhos reduced viability of pre-LSCs, indicating that ligand-dependent activation of Notch1 signaling upregulates the OxPhos pathway and sensitizes pre-LSCs to OxPhos-i. To clarify the role of Notch1 signaling, we examined the effect of IACS-010759 on pre-leukemic thymocytes harboring LMO1, SCL-LMO1, NOTCH1, LMO1-NOTCH1 and SCL-LMO1-NOTCH1 with and without DL4 stimulation. We found that in the absence of DL4, only thymocytes harboring the Notch1 oncogene responded to OxPhos-i, whereas all DL4-stimulated thymocytes responded regardless of Notch1 status (Fig. 1a). In addition, at the leukemic stage, we found elevation of the OxPhos pathway driven by oncogenic Notch1 when we compared transcriptomes of SCL-LMO1 induced T-ALL in the presence or absence of the NOTCH1 oncogene. In line with the murine T-ALL NOTCH1 model, we performed transcriptome analysis of two independent T-ALL patient cohorts prior to chemotherapy, COG TARGET ALL (n=263) and AALL1231 (n=75), comparing transcriptomes of NOTCH1-mutated vs NOTCH1-wt T-ALLs. We found co-segregation of NOTCH1 mutations with significant upregulation of OxPhos and TCA cycle genes and downregulation of apoptosis signaling. Aiming to reverse the NOTCH1-controlled anti-apoptotic program and chemoresistance, we next tested the combination of Vincristine, Dexamethasone and L-Asparaginase (VXL) with IACS-010759. When compared to vehicle, OxPhos-i or VXL alone, only the VXL-OxPhos-i treatment caused an energetic crisis indicated by decreased OCR and ECAR (Seahorse), which translated to a profound reduction of viability (CTG, flow cytometry) in T-ALL cell lines (n=9) and primary T-ALL samples (n=5). Additionally, the IACS-VXL combination in vivo resulted in pan-metabolic blockade, which caused metabolic shut-down and triggered early induction of apoptosis in leukemic cells in peripheral blood, spleen and bone marrow (Fig. 1b). Single cell Proteomic analysis (CyTOF) of spleen showed reduced expression of cell proliferation marker -ki67, c-myc, ERK and p38 proteins, and reduction in number of leukemic cells. Finally, this combination therapy resulted in reduced leukemia burden and extension of overall survival across all three aggressive NOTCH1-mutated T-ALL PDX models (p Disclosures Jabbour: Pfizer: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding. Teachey:Sobi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; La Roche: Consultancy. Rezvani:Takeda: Other: Licensing agreement; GemoAb: Membership on an entity's Board of Directors or advisory committees; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Virogen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Other: Educational grant; Affimed: Other: Educational grant; Formula Pharma: Membership on an entity's Board of Directors or advisory committees. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Lorenzi:Precision Pathways: Consultancy. Konopleva:Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Rafael Pharmaceutical: Research Funding; Eli Lilly: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Agios: Research Funding; AstraZeneca: Research Funding; Ablynx: Research Funding; Forty-Seven: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Ascentage: Research Funding.

Stereospecific recognition of metabolites plays a significant role in the detection of potential disease biomarkers thereby providing new insights in diagnosis and prognosis. D-Hdroxy/amino acids are recognized as potential biomarkers in several metabolic disorders. Despite continuous advances in metabolomics technologies, the simultaneous measurement of different classes of enantiomeric metabolites in a single analytical run remains challenging. Here, we develop a novel strategy for untargeted chiral metabolomics of hydroxy/amine groups (-OH/-NH2) containing metabolites, including all hydroxy acids (HAs) and amino acids (AAs), by chiral derivatization coupled with liquid chromatography-high resolution tandem mass spectrometry (LC-HR-MS/MS). Diacetyl-tartaric anhydride (DATAN) was used for the simultaneous derivatization of-OH/-NH2 containing metabolites as well as the resulting diastereomers, and all the derivatized metabolites were resolved in a single analytical run. Data independent MS/MS acquisition (DIA) was applied to positively identify DATAN-labeled metabolites based on reagent specific diagnostic fragment ions. We discriminated chiral from achiral metabolites based on the reversal of elution order of D and L isomers derivatized with the enantiomeric pair (±) of DATAN in an untargeted manner. Using the developed strategy, a library of 301 standards that consisted of 214 chiral and 87 achiral metabolites were separated and detected in a single analytical run. This approach was then applied to investigate the enantioselective metabolic profile of the bone marrow (BM) and peripheral blood (PB) plasma samples from patients with acute myeloid leukemia (AML) at diagnosis and following completion of the induction phase of chemotherapeutic treatment. The sensitivity and selectivity of the developed method enabled the detection of trace levels of the D-enantiomer of HAs and AAs in primary plasma patient samples. Several of these metabolites were significantly altered in response to chemotherapy. The developed LC-HR-MS method entails a valuable step forward in chiral metabolomics.

Acute myeloid leukemia (AML) with a FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib’s anti-leukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. Additionally, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Lastly, we found that the glutaminase inhibitor CB-839 enhanced anti-leukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD-positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.

Simple Summary Increasing numbers of cancer patients are turning to complementary and alternative medicines (CAM) to facilitate or replace their cancer treatments, or they are obtaining natural products in their diet. This is concerning, as there is evidence of molecular interactions between these bioactive compounds and cancer drugs that can impede or reverse their efficacy and prevent cancer regression. High-throughput drug screening and deep learning techniques have successfully been applied in the past to evaluate synergistic cancer drug and natural product combinations. However, these techniques should be applied more commonly in the context of drug antagonism to uncover potentially harmful interactions and drive safer recommendations for cancer patients. In this review, we evaluate the antagonistic interactions between natural products and chemotherapeutics and highlight how the application of high-throughput screening and deep learning techniques can strengthen this area of research. Abstract Natural products have been used for centuries to treat various human ailments. In recent decades, multi-drug combinations that utilize natural products to synergistically enhance the therapeutic effects of cancer drugs have been identified and have shown success in improving treatment outcomes. While drug synergy research is a burgeoning field, there are disagreements on the definitions and mathematical parameters that prevent the standardization and proper usage of the terms synergy, antagonism, and additivity. This contributes to the relatively small amount of data on the antagonistic effects of natural products on cancer drugs that can diminish their therapeutic efficacy and prevent cancer regression. The ability of natural products to potentially degrade or reverse the molecular activity of cancer therapeutics represents an important but highly under-emphasized area of research that is often overlooked in both pre-clinical and clinical studies. This review aims to evaluate the body of work surrounding the antagonistic interactions between natural products and cancer therapeutics and highlight applications for high-throughput screening (HTS) and deep learning techniques for the identification of natural products that antagonize cancer drug efficacy.

Metabolic reprogramming is recognized as one of the key hallmarks in acquiring aggressive phenotype and chemoresistance in solid tumors and hematologic malignancies. We have previously demonstrated that T-ALL are characterized by significant dependency on oxidative phosphorylation (OxPhos) with ability to utilize glutamine either in oxidative or reductive directions of TCA cycle, when mitochondria are blocked by Complex I Inhibitor (Baran N, et al. ASH 2020). To survive upon Complex I blockade leukemic cells require functional monocarboxylate transporter MCT1, that enables excretion of lactate and permissive pyruvate flux (Fig.1 a). Here we show that metabolic intervention utilizing OxPhos blockade can be potentiated by targeting MCT1 transporter and propose a novel metabolic synthetic lethality that could be exploited to eradicate T-ALL and other OxPhos-dependent malignancies. We first demonstrated that Complex I inhibition leads to increased MCT1 expression; on the contrary, MCT1 transporter blockade forces cells to increase OxPhos. In turn, the combinatorial therapy with Complex I inhibitor (IACS-010759) and MCT1 inhibitor (AZD3965) causes loss of ATP content (Fig. 1b), significant reduction of cell number and massive induction of apoptosis. Mechanistically, the combination treatment further reduced oxygen consumption rate (OCR) (Fig. 1c) and increased extracellular acidification rate, as measured by Seahorse. In concert with those results, dual inhibition led to TCA blockade, accumulation of intracellular lactate and depletion of glutamine, cystathionine and glutathione, indicating severe disruption of redox balance as measured by mass spectrometry and confirmed by significant accumulation of intracellular and mitochondrial reactive oxygen species (ROS) (Fig. 1d), loss of mitochondrial membrane potential (ΔΨ) (Fig. 1e) and subsequent mitochondria swelling. RNAseq data showed simultaneous upregulation of glycolysis and glutathione-related processes as possible mechanisms of metabolic compensation, yet strong upregulation of genes regulating apoptosis related to mitochondria dysfunction (Fig. 1f). Real-time hyperpolarized MRI based metabolic imaging studies with [1-13C]-pyruvate in patient-derived xenografts in vivo revealed significant decrease of lactate-to-pyruvate ratio in mice treated with AZD3965 or IACS-010759 alone, and in mice treated with drug combination. [13C]-Glucose isotope tracing analysis in patient-derived xenografts in vivo revealed an increased intracellular trapping of lactate as a marker of treatment effectiveness in mice subjected to dual blockade. While MCT1 inhibition induced only moderate reduction of leukemia growth in vitro and tumor burden in vivo, combination with IACS-010759 depleted significantly both, circulating and marrow/spleen/liver resident leukemia cells. Mechanistically, inhibition of MCT1 by AZD3965 therapy in leukemia-bearing mice led to lactate accumulation, OCR increase, moderate ROS production and mitochondrial membrane hyperpolarization, while Complex I blockade resulted in upregulation of MCT-1, reduction of OCR, lactate production and increase of ROS ; consequently, combinatorial therapy caused complete mitochondria shut-down and drastic inhibition of tumor growth both in vitro and in vivo in two xenografts models and led to significant extension of overall survival (p Figure 1 Figure 1. Disclosures Skwarska: Halilovich E, Wang Y, Morris E, Konopleva M, Skwarska A.: Patents & Royalties: Combination of a MCL-1 inhibitor and midostaurin, uses and pharmaceutical composition thereof.. Konopleva: Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Ascentage: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Cellectis: Other: grant support; Sanofi: Other: grant support, Research Funding; KisoJi: Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support.

Despite advances in surgery and adjuvant therapy, brain tumours represent one of the leading causes of cancer-related mortality and morbidity in both adults and children. Gliomas constitute about 60% of all cerebral tumours, showing varying degrees of malignancy. They are difficult to treat due to dismal prognosis and limited therapeutics. Metabolomics is the untargeted and targeted analyses of endogenous and exogenous small molecules, which characterizes the phenotype of an individual. This emerging “omics” science provides functional readouts of cellular activity that contribute greatly to the understanding of cancer biology including brain tumour biology. Metabolites are highly informative as a direct signature of biochemical activity; therefore, metabolite profiling has become a promising approach for clinical diagnostics and prognostics. The metabolic alterations are well-recognized as one of the key hallmarks in monitoring disease progression, therapy and revealing new molecular targets for effective therapeutic intervention. Taking advantage of the latest high-throughput analytical technologies, i.e. nuclear magnetic resonance spectroscopy and mass spectrometry, metabolomics is now a promising field for precision medicine and drug discovery. In the present report, we review the application of metabolomics and in vivo metabolic profiling in the context of adult gliomas and paediatric brain tumours. Analytical platforms such as high-resolution nuclear magnetic resonance, in vivo magnetic resonance spectroscopic imaging and high- and low-resolution mass spectrometry are discussed. Moreover, the relevance of metabolic studies in the development of new therapeutic strategies for treatment of gliomas are reviewed.

Acute myeloid leukemia (AML) cells are highly dependent on mitochondrial function for survival 1. We have recently reported a novel oxidative phosphorylation (OXPHOS) inhibitor IACS-010759 that potently inhibits mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells in vitro and in vivo2. In this study, we aimed to identify chemotherapeutic agents that synergistically deplete AML cells when administered in combination with IACS-010759. We performed a high-throughput screening of a drug library (289 anti-cancer compounds) administered either individually or in combination with IACS-010759 on two leukemia cell lines (OCI-AML3, MOLM-13) and three bone marrow stromal cell lines (HS-5, HS-27A, MSC) in both hypoxia (1% O2) and normoxia conditions. Based on the cell viability datasets, we selected top candidates for combinations based on the following criteria: either bliss index > 0.1 (synergy of the combination treatment; red in Fig. 1A&B), or high cytotoxicity to leukemia cells (relative cell viability < 0.5, blue in Fig. 1A&B), as well as low toxicity against normal cells (relative cell viability in normal cells > 0.8, yellow in Fig. 1A&B). Twenty-four compounds satisfied the selection criteria above, either in normoxia or hypoxia, or both. Out of the 24 compounds, 5 agents (Fig. 1C) are known FLT3 (FMS-like tyrosine kinase 3) inhibitors, including AC220 (quizartinib), dovitinib, nintedanib, SGI-1776, and rebastinib, pointing to a molecular target of great potential interest in the design of synergistic drug combinations with IACS-010759. Thus, we investigated more in-depth the synergism between IACS-010759 (10nM) and 13 FLT3 inhibitors, all currently in clinical trials (AC220, sorafenib, gilteritinib, sunitinib, ponatinib, midostaurin, ibrutinib, TP-0903, crenolanib, tandutinib, FF-10101, lestaurtinib, and KW-2449; 0.0128:5x:5000nM), in AML cell lines (FLT3-wt KG-1, U937, OCI-AML2, OCI-AML3; and FLT3-mutant MOLM-13 and MOLM-14). Among the 13 FLT3 inhibitors, only AC220 combined with IACS-010759 showed concentration windows with bliss index higher than 0.1 across different lines. Next, we further characterized the synergism between AC220 and IACS-010759 in AML cell lines (U937 and OCI-AML3) under hypoxic conditions using metabolic flux analysis (MFA) to trace the incorporation of 13C5,15N2-glutamine and 1,2-13C2-glucose and study the metabolic modulation associated with the synergy. Leukemia cells were incubated with unlabeled/labeled medium for 24h and concurrently treated with 5nM IACS-010759 and/or 500nM AC220. While both individual agents modulate glutamine consumption and TCA cycle dynamics, by far the most dramatic metabolic effects on TCA cycle intermediates are observed following administration of the combined treatment. Severe drops in the levels of TCA cycle metabolites, (Fig. 1D) point to a reduced mitochondrial activity following the combined treatment, which is also validated by the increased ratio of oxidized/reduced forms of nicotinamide adenine dinucleotide (NAD/NADH). Interestingly, the total pool of the oncometabolite 2-hydroxyglutarate, while increasing following the individual treatments, significantly dropped to very low levels in response to the combined treatment. The significantly reduced metabolite levels as well as the glucose-derived enrichment fractions of glucose 6-phosphate, fructose bisphosphate, phosphoenolpyruvate and ribose 5-phosphate in the AC220-containing treatment groups (significantly more pronounced in the combined treatment) point to impaired glycolysis /pentose phosphate pathway (Fig. 1E). In turn this results in lower de novo nucleotide biosynthesis (based on the decreased glutamine and glucose incorporation). Similar results were observed in OCI-AML3 cells. Overall, the combinatorial treatment with IACS-010759 and AC220 impaired AML cell metabolism tremendously and to a much greater extent than any of the individual treatments alone. Influx inhibition of both the two main carbon sources, glucose and glutamine, was observed leading to impairment of the TCA cycle and glycolysis for energy production, as well as pentose phosphate pathway and de novo nucleotide biosynthesis. In conclusion, we identified a novel drug combination AC220 and IACS-010759 which synergistically inhibits AML cell growth regardless of FLT3 mutation at least by metabolism disruption. Disclosures Konopleva: Kisoji: Consultancy; Agios: Research Funding; Amgen: Consultancy; Cellectis: Research Funding; Eli Lilly: Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Sanofi: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; AstraZeneca: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding.

Despite recent approval of hypomethylating agent/venetoclax (HMA+VEN) therapy for older patients (pt) with AML unfit for induction chemotherapy, their outcomes remain suboptimal. Such pts have a median overall survival of only 14 months and only approximately 35% of pts enjoying long-term survival (DiNardo, EHA 2020). Approximately 20-40% of newly diagnosed older pts with AML do not respond to the HMA+VEN regimens, with higher rates of refractory disease as well as early relapse in high-risk pts. Metabolic reprogramming and dependence on mitochondrial oxidative phosphorylation (OxPhos) is a core feature of AML leukemia stem cells (LSC). Recent reports have shown that upregulation of OxPhos confers intrinsic and acquired resistance to VEN in AML, multiple myeloma and lymphoid malignancies, which can be reversed by disrupting OxPhos; mutated TP53 has been shown to confer intrinsic resistance to venetoclax through increased OxPhos (Nechiporuk T, et al. Cancer Discovery 2019). We hypothesized that combined blockade of mitochondrial fitness by OxPhos inhibitors and of BCL-2 with VEN/azacitidine (AZA) will perform synergistically in pre-clinical AML models. To test this hypothesis, we utilized a novel complex I inhibitor IACS-010759 that effectively inhibits cell respiration and leukemia progression in the in vitro and in vivo AML pre-clinical models (Molina, Nat Med 2018). Priming of MOLM-13 cells with 20nM VEN for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while single agents reduced OCR by 70%) of viable cell numbers in several AML cell lines tested (OCI-AML2, MV-4-11, and MOLM-13) (Figure 1). By co-immunoprecipitation VEN disrupted interaction of BCL-2 with the mitochondrial protein VDAC, known to regulate ADP/ATP exchange during electron transport across mitochondria membrane; this resulted in dramatic reduction of the intracellular ATP and CTP levels measured by MS-based metabolomics. Further, VEN increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of VEN and IACS-010759, possibly because of RNA degradation. In primary AML samples (n=3) and AML PDX cells (n=4) cultured ex vivo, combined VEN and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. We next tested the efficacy of the "triple" combination of VEN, AZA and IACS-010759 in the in vivo AML PDX models. We injected AML PDX cells 3871344 (with no mutations identified by targeted sequencing) and 4404778 (harboring IDH1, NPM1, FLT3-ITD mutations) into NRG mice and upon engraftment, randomized mice into 4 groups to receive 2 cycles of treatment with 3 weeks interruption between cycles: vehicle, VEN (50mg/kg daily, 5 days on/2 days off, day 1-21) with AZA (2.5 mg/kg daily, day 1-7), IACS-010759 (1mg/kg ?daily, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. In the less aggressive model 3871344, all therapies reduced circulating leukemia burden, and the triple treatment achieved best efficacy, with average circulating tumor burden at 10 weeks after cycle 2 of 90.8%, 38.7%, 68.8% and 7.4% in vehicle, VEN+AZA, IACS-010759, and triple-therapy cohorts, respectively. In the aggressive model 4404778, the treatments were less effective, but the combination offered highest activity, with average circulating tumor burden of 71.0%, 52.3%, 88.7% and 39.9% in vehicle, IACS-010759, VEN+AZA and triple-therapy cohorts, respectively (Figure 1). Analysis of survival and additional PDX models are ongoing and will be reported. In summary, our study demonstrates that BCL-2 modulates mitochondrial respiration and mitochondrial ATP generation in addition to its established anti-apoptotic role. VEN disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitro and in vivo. Disclosures Konopleva: Ascentage: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Kisoji: Consultancy; AstraZeneca: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding.

BACKGROUND Metabolic adaptation to hypoxia is a crucial consideration in combating anti-angiogenic resistance. We previously explored the potential of targeting the peroxisomal fatty acid oxidation (FAO) pathway and observed higher potency to peroxisomal FAO inhibitor thioridazine in hypoxia in vitro. In this study we further examine the effects of peroxisomal FAO enzyme ACOX1, looking at differences in mitochondrial and peroxisomal FAO in hypoxia between subtypes, and explore additional metabolic pathways that can be potentially further studied. METHODS We utilized a CRISPR doxycycline-inducible U251 knockout cell line for the peroxisomal FAO gene ACOX1 and examined growth and viability in 2 weeks hypoxia (2% O2). We analyzed the gene expression of 13 peroxisomal and mitochondrial FAO enzymes in six different patient-derived cell lines. Finally, we characterized serum metabolites that are associated with tumor hypoxic volume, progression-free survival, and overall survival in patients undergoing clinical trial for TH302 and bevacizumab with bevacizumab refractory tumors. RESULTS We observed decreased cell growth and viability (p < .03) specifically in hypoxia but not normoxia with ACOX1 knock out. We saw some changes in gene expression (|ΔΔCt| >1) in hypoxia for all genes, which differed between cell lines. ACOX1 and CPT1A expression were strongly decreased (|ΔΔCt| >2) while ACADSB expression was strongly increased for the proneural cell line. ACOX1 expression was strongly decreased in our most thioridazine-sensitive cell line. ACOX2, ACADVL, CPT1A, CPT1C, and DECR2 expression were strongly increased in one of the proneural cell lines. Twelve polar metabolites were positively or negatively correlated (|r| >.4) with both hypoxic volume and either overall survival or progression-free survival. CONCLUSION Peroxisomal FAO and other metabolic pathways may be essential to target in order combat metabolic resistance during anti-angiogenic therapy. Better understanding differences in metabolism in different tumor environments will help determine which targets will be most therapeutically useful.

Metabolomics is increasingly applied to investigate different individuals and time-dependent responses to environmental stimuli. Rapid data acquisition and improved detection limits of direct infusion mass spectrometry (DIMS) are paving the way for applications of metabolomics in preclinical screening, opening new opportunities in drug discovery and personalized medicine. Three-dimensional (3D) cell culture systems, which mimic the in vivo cell microenvironment, are well recognized as tissue and organ substitutes. Here, we investigated cell viability and induction of reactive oxygen species (ROS) in stromal cells cultured in various 3D systems as well as the standard monolayer culture to evaluate which system provides the most favorable growing conditions. The selected 3D system was then tested for use in 3D co-culture of leukemia and stromal cells for DIMS-based high-throughput/high-content metabolic drug screens. The NanobioMatrix-poly(e-caprolactone) (NBM-PCL) scaffold resulted in the lowest ROS production, supported rapid cell proliferation, and was suitable for the 96- and 384-well plate formats. Doxorubicin treatment in leukemia co-cultured with stromal cells induced some unique metabolic responses that drastically differed from those observed in leukemia cells alone. The DIMS results also showed that the drug-induced metabolic modulations in both normal and cancer cells were weakened by co-culturing even at high treatment doses, thereby demonstrating the value of the 3D co-culture high-content metabolic drug screen. In conclusion, we optimized a high sample throughput method for 3D co-culture with a DIMS-based high-content metabolic drug screen and drug development.

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase, which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages and microglia through induction of the choline transporter CTL1. Inhibition of CTL1 expression or choline phosphorylation attenuated NLRP3 inflammasome activation and IL-1β and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis, and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, choline kinase inhibitors ameliorated acute and chronic models of IL-1β-dependent inflammation.

Objective Up-regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast-like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage. Methods Synovium and FLS were obtained from patients with RA and patients with osteoarthritis (OA). The rate of glycolysis after stimulation of FLS with lipopolysaccharide and platelet-derived growth factor BB was measured using glycolysis stress test technology. FLS function was evaluated using a glycolysis inhibitor, 2-deoxy-d-glucose (2-DG). After stimulation of the FLS, a migration scratch assay, MTT assay, and enzyme-linked immunosorbent assay were performed to measure the effect of 2-DG on FLS migration, viability of the FLS, and cytokine secretion, respectively. IRDye 800CW 2-DG was used to assess glucose uptake in the arthritic joints and stromal cells of mice after K/BxN mouse serum transfer. The mice were injected daily, intraperitoneally, with 3-bromopyruvate (BrPa; 5 mg/kg) to assess the effect of inhibition of glycolysis in vivo. Results Compared to human OA FLS, the balance between glycolysis and oxidative phosphorylation was shifted toward glycolysis in RA FLS. Glucose transporter 1 (GLUT1) messenger RNA (mRNA) expression correlated with baseline functions of the RA FLS. Glucose deprivation or incubation of the FLS with glycolytic inhibitors impaired cytokine secretion and decreased the rate of proliferation and migration of the cells. In a mouse model of inflammatory arthritis, GLUT1 mRNA expression in the synovial lining cells was observed, and increased levels of glucose uptake and glycolytic gene expression were detected in the stromal compartment of the arthritic mouse joints. Inhibition of glycolysis by BrPa, administered in vivo, significantly decreased the severity of arthritis in this mouse model. Conclusion Targeting metabolic pathways is a novel approach to understanding the mechanisms of disease. Inhibition of glycolysis may directly modulate synoviocyte-mediated inflammatory functions and could be an effective treatment strategy for arthritis.

A major hallmark of cancer is the metabolic reprogramming of cancer cells to fuel tumor growth and proliferation. Various plant-derived bioactive compounds efficiently target the metabolic vulnerabilities of cancer cells and exhibit potential as emerging therapeutic agents. Due to their safety and common use as dietary components, they are also ideal for cancer prevention. However, to render their use as efficient as possible, the mechanism of action of these phytochemicals needs to be well characterized. Stable isotope tracing is an essential technology to study the molecular mechanisms by which nutraceuticals modulate and target cancer metabolism. The use of positionally labeled tracers as exogenous nutrients and the monitoring of their downstream metabolites labeling patterns enable the analysis of the specific metabolic pathway activity, via the relative production and consumption of the labeled metabolites. Although stable isotope tracing metabolomics is a powerful tool to investigate the molecular activity of bioactive compounds as well as to design synergistic nutraceutical combinations, this methodology is still underutilized. This review aims to investigate the research efforts and potentials surrounding the use of stable isotope tracing metabolomics to examine the metabolic alterations mediated by bioactive compounds in cancer.

BACKGROUND: Peroxisomal fatty acid oxidation (FAO) offers some unique metabolic advantages compared to mitochondrial FAO that may be important to cancer cells adapting to chronic hypoxia exacerbated by anti-angiogenic treatment. When peroxisomal FAO is dominant, the resulting low FADH(2)/NADH ratio allows for more efficient oxygen utilization and lower oxidative stress production during oxidative phosphorylation. Our previous findings suggest cells from tumors treated with bevacizumab are more sensitive to peroxisomal FAO inhibition. The current study further examines changes in peroxisomes, mitochondria, and their FAO pathways, and their roles in adaptation to hypoxia and anti-angiogenic treatment. METHODS: Peroxisomal number was determined by electron microscopy. Gene expression was determined by QT-PCR. RESULTS: Metabolomic analysis by NMR/MRS revealed glioblastoma cells under chronic hypoxia (2–5 weeks) likely utilize less mitochondrial FAO and showed signs of increased oxidative stress. In some cell lines we observed that chronic hypoxic cells were more sensitive to treatment with a peroxisomal FAO inhibitor, particularly in an anti-angiogenic resistant tumor cell line. Increased expression (RQ>2) of the genes ACADVL, ACADS, and DECR2, suggests an increase in mitochondrial FAO for very long and short fatty acids and increase in peroxisomal FAO for unsaturated fatty acids. There were higher overall levels of peroxisomes in the anti-angiogenic resistant tumor cell line and lower levels of mitochondria in hypoxia compared to normoxia for cell lines which were sensitive to peroxisomal FAO inhibition (p

Background Metabolomics is an emerging field of biomedical research that may offer a better understanding of the mechanisms of underlying conditions including inflammatory arthritis. Perturbations caused by inflamed synovial tissue can lead to correlated changes in concentrations of certain metabolites in the synovium and thereby function as potential biomarkers in blood. Here, we explore the hypothesis of whether characterization of patients’ metabolomic profiles in blood, utilizing 1H-nuclear magnetic resonance (NMR), predicts synovial marker profiling in rheumatoid arthritis (RA). Methods Nineteen active, seropositive patients with RA, on concomitant methotrexate, were studied. One of the involved joints was a knee or a wrist appropriate for arthroscopy. A Bruker Avance 700 MHz spectrometer was used to acquire NMR spectra of serum samples. Gene expression in synovial tissue obtained by arthroscopy was analyzed by real-time PCR. Data processing and statistical analysis were performed in Python and SPSS. Results Analysis of the relationships between each synovial marker-metabolite pair using linear regression and controlling for age and gender revealed significant clustering within the data. We observed an association of serine/glycine/phenylalanine metabolism and aminoacyl-tRNA biosynthesis with lymphoid cell gene signature. Alanine/aspartate/glutamate metabolism and choline-derived metabolites correlated with TNF-α synovial expression. Circulating ketone bodies were associated with gene expression of synovial metalloproteinases. Discriminant analysis identified serum metabolites that classified patients according to their synovial marker levels. Conclusion The relationship between serum metabolite profiles and synovial biomarker profiling suggests that NMR may be a promising tool for predicting specific pathogenic pathways in the inflamed synovium of patients with RA. Electronic supplementary material The online version of this article (10.1186/s13075-018-1655-3) contains supplementary material, which is available to authorized users.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.

High-throughput screening of a natural compound library was performed to identify the most efficacious combinatorial treatment on prostate cancer. Ursolic acid, curcumin and resveratrol were selected for further analyses and administered in vivo via the diet, either alone or in combination, in a mouse allograft model of prostate cancer. All possible combinations of these natural compounds produced synergistic effects on tumor size and weight, as predicted in the screens. A subsequent untargeted metabolomics and metabolic flux analysis using isotopically labeled glutamine indicated that the compound combinations modulated glutamine metabolism. In addition, ASCT2 levels and STAT3, mTORC1 and AMPK activity were modulated to a greater extent by the combinations compared to the individual compounds. Overall, this approach can be useful for identifying synergistic combinations of natural compounds for chemopreventive and therapeutic interventions. Combinations of two molecules found naturally in edible plants synergistically help reduce tumor growth in a mouse model of prostate cancer. Stefano Tiziani and John DiGiovanni from the University of Texas at Austin, and colleagues screened a library of 142 natural compounds for the effects of each molecule, alone or in combination, on the viability of cells from mouse and human prostate cancer cell lines. Amongst other promising combinations, the researchers identified ursolic acid and curcumin as the most promising combination for inhibiting tumor growth. (These compounds are found naturally in apple peels and turmeric, respectively.) In mice with implanted prostate tumors, the two compounds synergistically reduced tumor volume and weight, while in cell culture the researchers showed that the compound-combination strategy modulated metabolism of a critical amino acid and other cell signaling pathways.