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Condensed droplets of protein regulate many cellular functions, yet the physiological conditions regulating their formation remain largely unexplored. Increasing our understanding of these mechanisms is paramount, as failure to control condensate formation and dynamics can lead to many diseases. Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo. We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol concentrations, but not glucose concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacological and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer-a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo, others are auxotrophic and therefore reliant on serine uptake. Importantly, despite several transporters being known to be capable of transporting serine, the transporters that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (SLC1A5) as a major contributor to serine uptake in cancer cells. ASCT2 is well known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that estrogen receptor α (ERα) promotes serine uptake by directly activating SLC1A5 transcription. Collectively, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target., Competing Interests: Declaration of interests P.K.S. is a member of the SAB or BOD for Applied Biomath, RareCyte. Nanostring, Glencoe Software, and Montai; he is consultant for Merck. None of these activities impact the content of this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that arise from neural tissues and carry a poor prognosis. Previously, we found that the glutamine amidotransferase inhibitor JHU395 partially impeded tumor growth in preclinical models of MPNST. JHU395 inhibits de novo purine synthesis in human MPNST cells and murine tumors with partial decreases in purine monophosphates. On the basis of prior studies showing enhanced efficacy when glutamine amidotransferase inhibition was combined with the antimetabolite 6-mercaptopurine (6-MP), we hypothesized that such a combination would be efficacious in MPNST. Given the known toxicity associated with 6-MP, we set out to develop a more efficient and well-tolerated drug that targets the purine salvage pathway. Here, we report the discovery of Pro-905, a phosphoramidate protide that delivered the active nucleotide antimetabolite thioguanosine monophosphate (TGMP) to tumors over 2.5 times better than equimolar 6-MP. Pro-905 effectively prevented the incorporation of purine salvage substrates into nucleic acids and inhibited colony formation of human MPNST cells in a dose-dependent manner. In addition, Pro-905 inhibited MPNST growth and was well-tolerated in both human patient-derived xenograft (PDX) and murine flank MPNST models. When combined with JHU395, Pro-905 enhanced the colony formation inhibitory potency of JHU395 in human MPNST cells and augmented the antitumor efficacy of JHU395 in mice. In summary, the dual inhibition of the de novo and purine salvage pathways in preclinical models may safely be used to enhance therapeutic efficacy against MPNST., (©2023 The Authors; Published by the American Association for Cancer Research.)

Nucleotides are the foundational elements of life. Proliferative cells acquire nutrients for energy production and the synthesis of macromolecules, including proteins, lipids, and nucleic acids. Nucleotides are continuously replenished through the activation of the nucleotide synthesis pathways. Despite the importance of nucleotides in cell physiology, there is still much to learn about how the purine and pyrimidine synthesis pathways are regulated in response to intracellular and exogenous signals. Over the past decade, evidence has emerged that several signaling pathways [Akt, mechanistic target of rapamycin complex I (mTORC1), RAS, TP53, and Hippo-Yes-associated protein (YAP) signaling] alter nucleotide synthesis activity and influence cell function. Here, we examine the mechanisms by which these signaling networks affect de novo nucleotide synthesis in mammalian cells. We also discuss how these molecular links can be targeted in diseases such as cancers and immune disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me 2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases, including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity toward increased purine synthesis in MLL3/4-KO mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA-Seq identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4-KO cells. Mechanistically, we demonstrated that compensation by MLL1/COMPASS was underlying these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vitro and in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.

During therapy, adaptations driven by cellular plasticity are partly responsible for driving the inevitable recurrence of glioblastoma (GBM). To investigate plasticity-induced adaptation during standard-of-care chemotherapy temozolomide (TMZ), we performed in vivo single-cell RNA sequencing in patient-derived xenograft (PDX) tumors of GBM before, during, and after therapy. Comparing single-cell transcriptomic patterns identified distinct cellular populations present during TMZ therapy. Of interest was the increased expression of ribonucleotide reductase regulatory subunit M2 ( RRM2 ), which we found to regulate dGTP and dCTP production vital for DNA damage response during TMZ therapy. Furthermore, multidimensional modeling of spatially resolved transcriptomic and metabolomic analysis in patients' tissues revealed strong correlations between RRM2 and dGTP. This supports our data that RRM2 regulates the demand for specific dNTPs during therapy. In addition, treatment with the RRM2 inhibitor 3-AP (Triapine) enhances the efficacy of TMZ therapy in PDX models. We present a previously unidentified understanding of chemoresistance through critical RRM2-mediated nucleotide production.

BACKGROUNDElevated circulating branched chain amino acids (BCAAs), measured at a single time point in middle life, are strongly associated with an increased risk of developing type 2 diabetes mellitus (DM). However, the longitudinal patterns of change in BCAAs through young adulthood and their association with DM in later life are unknown.METHODSWe serially measured BCAAs over 28 years in the Coronary Artery Risk Development in Young Adults (CARDIA) study, a prospective cohort of apparently healthy Black and White young adults at baseline. Trajectories of circulating BCAA concentrations from years 2-30 (for prevalent DM) or years 2-20 (for incident DM) were determined by latent class modeling.RESULTSAmong 3,081 apparently healthy young adults, trajectory analysis from years 2-30 revealed 3 distinct BCAA trajectory groups: low-stable (n = 1,427), moderate-stable (n = 1,384), and high-increasing (n = 270) groups. Male sex, higher body mass index, and higher atherogenic lipid fractions were more common in the moderate-stable and high-increasing groups. Higher risk of prevalent DM was associated with the moderate-stable (OR = 2.59, 95% CI: 1.90-3.55) and high-increasing (OR = 6.03, 95% CI: 3.86-9.43) BCAA trajectory groups in adjusted models. A separate trajectory group analysis from years 2-20 for incident DM after year 20 showed that moderate-stable and high-increasing trajectory groups were also significantly associated with higher risk of incident DM, after adjustment for clinical variables and glucose levels.CONCLUSIONBCAA levels track over a 28-year span in most young adults, but serial clinical metabolomic measurements identify subpopulations with rising levels associated with high risk of DM in later life.FUNDINGThis research was supported by the NIH, under grants R01 HL146844 (JTW) and T32 HL069771 (MRC). The CARDIA study is conducted and supported by the NIH National Heart, Lung, and Blood Institute in collaboration with the University of Alabama at Birmingham (HHSN268201800005I and HHSN268201800007I), Northwestern University (HHSN268201800003I), the University of Minnesota (HHSN268201800006I), and Kaiser Foundation Research Institute (HHSN268201800004I).

Aims: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of pulmonary hypertension (PH). Proliferative cells utilize purine bases from the de novo purine synthesis (DNPS) pathways for nucleotide synthesis; however, it is unclear whether DNPS plays a critical role in VSMC proliferation during development of PH. The last two steps of DNPS are catalysed by the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC). This study investigated whether ATIC-driven DNPS affects the proliferation of pulmonary artery smooth muscle cells (PASMCs) and the development of PH., Methods and Results: Metabolites of DNPS in proliferative PASMCs were measured by liquid chromatography-tandem mass spectrometry. ATIC expression was assessed in platelet-derived growth factor-treated PASMCs and in the lungs of PH rodents and patients with pulmonary arterial hypertension. Mice with global and VSMC-specific knockout of Atic were utilized to investigate the role of ATIC in both hypoxia- and lung interleukin-6/hypoxia-induced murine PH. ATIC-mediated DNPS at the mRNA, protein, and enzymatic activity levels were increased in platelet-derived growth factor-treated PASMCs or PASMCs from PH rodents and patients with pulmonary arterial hypertension. In cultured PASMCs, ATIC knockdown decreased DNPS and nucleic acid DNA/RNA synthesis, and reduced cell proliferation. Global or VSMC-specific knockout of Atic attenuated vascular remodelling and inhibited the development and progression of both hypoxia- and lung IL-6/hypoxia-induced PH in mice., Conclusion: Targeting ATIC-mediated DNPS compromises the availability of purine nucleotides for incorporation into DNA/RNA, reducing PASMC proliferation and pulmonary vascular remodelling and ameliorating the development and progression of PH., Competing Interests: Conflict of interest statement All authors declare no conflict of interest for this contribution., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The mechanistic target of rapamycin complex 1 (mTORC1) senses cellular leucine levels through the GATOR1/2-Rag axis. Jiang et al. show that the Ring domains of GATOR2 subunits maintain the integrity of the complex and promote ubiquitination and inhibition of GATOR1, thereby leading to mTORC1 activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Background: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arterial diseases, especially in arterial restenosis after angioplasty or stent placement. VSMCs reprogram their metabolism to meet the increased requirements of lipids, proteins, and nucleotides for their proliferation. De novo purine synthesis is one of critical pathways for nucleotide synthesis. However, its role in proliferation of VSMCs in these arterial diseases has not been defined., Methods: De novo purine synthesis in proliferative VSMCs was evaluated by liquid chromatography-tandem mass spectrometry. The expression of ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase), the critical bifunctional enzyme in the last 2 steps of the de novo purine synthesis pathway, was assessed in VSMCs of proliferative arterial neointima. Global and VSMC-specific knockout of Atic mice were generated and used for examining the role of ATIC-associated purine metabolism in the formation of arterial neointima and atherosclerotic lesions., Results: In this study, we found that de novo purine synthesis was increased in proliferative VSMCs. Upregulated purine synthesis genes, including ATIC, were observed in the neointima of the injured vessels and atherosclerotic lesions both in mice and humans. Global or specific knockout of Atic in VSMCs inhibited cell proliferation, attenuating the arterial neointima in models of mouse atherosclerosis and arterial restenosis., Conclusions: These results reveal that de novo purine synthesis plays an important role in VSMC proliferation in arterial disease. These findings suggest that targeting ATIC is a promising therapeutic approach to combat arterial diseases.

Bicarbonate (HCO 3 - ) ions maintain pH homeostasis in eukaryotic cells and serve as a carbonyl donor to support cellular metabolism. However, whether the abundance of HCO 3 - is regulated or harnessed to promote cell growth is unknown. The mechanistic target of rapamycin complex 1 (mTORC1) adjusts cellular metabolism to support biomass production and cell growth. We find that mTORC1 stimulates the intracellular transport of HCO 3 - to promote nucleotide synthesis through the selective translational regulation of the sodium bicarbonate cotransporter SLC4A7. Downstream of mTORC1, SLC4A7 mRNA translation required the S6K-dependent phosphorylation of the translation factor eIF4B. In mTORC1-driven cells, loss of SLC4A7 resulted in reduced cell and tumor growth and decreased flux through de novo purine and pyrimidine synthesis in human cells and tumors without altering the intracellular pH. Thus, mTORC1 signaling, through the control of SLC4A7 expression, harnesses environmental bicarbonate to promote anabolic metabolism, cell biomass, and growth., Competing Interests: Declaration of interests The authors declare no conflict of interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Liver cancer (LC) is the fourth leading cause of death from cancer malignancies. Recently, a putative fifth hexokinase, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in LC compared to healthy liver tissue. Using a combination of in vitro and in vivo tools, we examined the role of HKDC1 in LC development and progression. Importantly, HKDC1 ablation stops LC development and progression via its action at the mitochondria by promoting metabolic reprogramming and a shift of glucose flux away from the TCA cycle. HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy, which cannot be compensated by enhanced glucose uptake. Moreover, we show that the interaction of HKDC1 with the mitochondria is essential for its role in LC progression, and without this interaction, mitochondrial dysfunction occurs. As HKDC1 is highly expressed in LC cells, but only to a minimal degree in hepatocytes under normal conditions, targeting HKDC1, specifically its interaction with the mitochondria, may represent a highly selective approach to target cancer cells in LC., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.

Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pregnancy is associated with substantial physiological changes of the heart, and disruptions in these processes can lead to peripartum cardiomyopathy (PPCM). The molecular processes that cause physiological and pathological changes in the heart during pregnancy are not well characterized. Here, we show that mTORc1 was activated in pregnancy to facilitate cardiac enlargement that was reversed after delivery in mice. mTORc1 activation in pregnancy was negatively regulated by the mRNA-destabilizing protein ZFP36L2 through its degradation of Mdm2 mRNA and P53 stabilization, leading to increased SESN2 and REDD1 expression. This pathway impeded uncontrolled cardiomyocyte hypertrophy during pregnancy, and mice with cardiac-specific Zfp36l2 deletion developed rapid cardiac dysfunction after delivery, while prenatal treatment of these mice with rapamycin improved postpartum cardiac function. Collectively, these data provide what we believe to be a novel pathway for the regulation of mTORc1 through mRNA stabilization of a P53 ubiquitin ligase. This pathway was critical for normal cardiac growth during pregnancy, and its reduction led to PPCM-like adverse remodeling in mice.

Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration., (© 2022. The Author(s).)

The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism., Methods: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor, rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4 KO ) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4 KO mice., Results: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver, in a hepatocyte-intrinsic manner by insulin in response to feeding. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of nonessential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine., Conclusions: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in the liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of patients with BRAF V600E cutaneous metastatic melanoma (CMM) eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming that profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase, the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafenib-sensitive and vemurafenib-resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro . Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers. SIGNIFICANCE: Glycolytic and purine synthesis pathways are often deregulated in therapy-resistant tumors and can be targeted by the covalent inhibitor described in this study, suggesting its broad application for overcoming resistance in cancer., (©2021 American Association for Cancer Research.)

The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient, growth, and oncogenic signals. We found that mTORC1 stimulates the synthesis of the major methyl donor, S-adenosylmethionine (SAM), through the control of methionine adenosyltransferase 2 alpha (MAT2A) expression. The transcription factor c-MYC, downstream of mTORC1, directly binds to intron 1 of MAT2A and promotes its expression. Furthermore, mTORC1 increases the protein abundance of Wilms' tumor 1-associating protein (WTAP), the positive regulatory subunit of the human N 6 -methyladenosine (m 6 A) RNA methyltransferase complex. Through the control of MAT2A and WTAP levels, mTORC1 signaling stimulates m 6 A RNA modification to promote protein synthesis and cell growth. A decline in intracellular SAM levels upon MAT2A inhibition decreases m 6 A RNA modification, protein synthesis rate, and tumor growth. Thus, mTORC1 adjusts m 6 A RNA modification through the control of SAM and WTAP levels to prime the translation machinery for anabolic cell growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) is the most divergent member of UBR box-containing E3 ubiquitin ligases/recognins that mediate the proteasomal degradation of its substrates through the N-end rule. Here, we used a proteomic approach and found phospho r ibosyl pyrophosphate synthetases (PRPSs), the essential enzymes for nucleotide biosynthesis, as strong interacting partners of UBR7. UBR7 stabilizes PRPS catalytic subunits by mediating the polyubiquitination-directed degradation of PRPS-associated protein (PRPSAP), the negative regulator of PRPS. Loss of UBR7 leads to nucleotide biosynthesis defects. We define UBR7 as a transcriptional target of NOTCH1 and show that UBR7 is overexpressed in NOTCH1-driven T cell acute lymphoblastic leukemia (T-ALL). Impaired nucleotide biosynthesis caused by UBR7 depletion was concomitant with the attenuated cell proliferation and oncogenic potential of T-ALL. Collectively, these results establish UBR7 as a critical regulator of nucleotide metabolism through the regulation of the PRPS enzyme complex and uncover a metabolic vulnerability in NOTCH1-driven T-ALL., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Declining tissue nicotinamide adenine dinucleotide (NAD) levels are linked to ageing and its associated diseases. However, the mechanism for this decline is unclear. Here, we show that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues, including visceral white adipose tissue and liver, during ageing and acute responses to inflammation. These M1-like macrophages express high levels of the NAD-consuming enzyme CD38 and have enhanced CD38-dependent NADase activity, thereby reducing tissue NAD levels. We also find that senescent cells progressively accumulate in visceral white adipose tissue and liver during ageing and that inflammatory cytokines secreted by senescent cells (the senescence-associated secretory phenotype, SASP) induce macrophages to proliferate and express CD38. These results uncover a new causal link among resident tissue macrophages, cellular senescence and tissue NAD decline during ageing and offer novel therapeutic opportunities to maintain NAD levels during ageing.

The RAS-ERK/MAPK (RAS-extracellular signal-regulated kinase/mitogen-activated protein kinase) pathway integrates growth-promoting signals to stimulate cell growth and proliferation, at least in part, through alterations in metabolic gene expression. However, examples of direct and rapid regulation of the metabolic pathways by the RAS-ERK pathway remain elusive. We find that physiological and oncogenic ERK signaling activation leads to acute metabolic flux stimulation through the de novo purine synthesis pathway, thereby increasing building block availability for RNA and DNA synthesis, which is required for cell growth and proliferation. We demonstrate that ERK2, but not ERK1, phosphorylates the purine synthesis enzyme PFAS (phosphoribosylformylglycinamidine synthase) at T619 in cells to stimulate de novo purine synthesis. The expression of nonphosphorylatable PFAS (T619A) decreases purine synthesis, RAS-dependent cancer cell-colony formation, and tumor growth. Thus, ERK2-mediated PFAS phosphorylation facilitates the increase in nucleic acid synthesis required for anabolic cell growth and proliferation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although tumorigenesis is dependent on the reprogramming of cellular metabolism, the metabolic pathways engaged in the formation of metastases remain largely unknown. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pleiotropic role in the control of cancer cell metabolism and has been associated with a good prognosis in prostate cancer. Here, we show that PGC1α represses the metastatic properties of prostate cancer cells via modulation of the polyamine biosynthesis pathway. Mechanistically, PGC1α inhibits the expression of c-MYC and ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme for polyamine synthesis. Analysis of in vivo metastases and clinical data from patients with prostate cancer support the proposition that the PGC1α/c-MYC/ODC1 axis regulates polyamine biosynthesis and prostate cancer aggressiveness. In conclusion, downregulation of PGC1α renders prostate cancer cells dependent on polyamine to promote metastasis. SIGNIFICANCE: These findings show that a major regulator of mitochondrial metabolism controls polyamine synthesis and prostate cancer aggressiveness, with potential applications in therapy and identification of new biomarkers., (©2019 American Association for Cancer Research.)

Cancer cells exhibit a dynamic metabolic landscape and require a sufficient supply of nucleotides and other macromolecules to grow and proliferate. To meet the metabolic requirements for cell growth, cancer cells must stimulate de novo nucleotide synthesis to obtain adequate nucleotide pools to support nucleic acid and protein synthesis along with energy preservation, signaling activity, glycosylation mechanisms, and cytoskeletal function. Both oncogenes and tumor suppressors have recently been identified as key molecular determinants for de novo nucleotide synthesis that contribute to the maintenance of homeostasis and the proliferation of cancer cells. Inactivation of tumor suppressors such as TP53 and LKB1 and hyperactivation of the mTOR pathway and of oncogenes such as MYC , RAS , and AKT have been shown to fuel nucleotide synthesis in tumor cells. The molecular mechanisms by which these signaling hubs influence metabolism, especially the metabolic pathways for nucleotide synthesis, continue to emerge. Here, we focus on the current understanding of the molecular mechanisms by which oncogenes and tumor suppressors modulate nucleotide synthesis in cancer cells and, based on these insights, discuss potential strategies to target cancer cell proliferation.

Nicotinamide adenine dinucleotide phosphate (NADP + ) is essential for producing NADPH, the primary cofactor for reductive metabolism. We find that growth factor signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces acute synthesis of NADP + and NADPH. Akt phosphorylates NAD kinase (NADK), the sole cytosolic enzyme that catalyzes the synthesis of NADP + from NAD + (the oxidized form of NADH), on three serine residues (Ser 44 , Ser 46 , and Ser 48 ) within an amino-terminal domain. This phosphorylation stimulates NADK activity both in cells and directly in vitro, thereby increasing NADP + production. A rare isoform of NADK (isoform 3) lacking this regulatory region exhibits constitutively increased activity. These data indicate that Akt-mediated phosphorylation of NADK stimulates its activity to increase NADP + production through relief of an autoinhibitory function inherent to its amino terminus., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During wound injury, efferocytosis fills the macrophage with a metabolite load nearly equal to the phagocyte itself. A timely question pertains to how metabolic phagocytic signaling regulates the signature anti-inflammatory macrophage response. Here we report the metabolome of activated macrophages during efferocytosis to reveal an interleukin-10 (IL-10) cytokine escalation that was independent of glycolysis yet bolstered by apoptotic cell fatty acids and mitochondrial β-oxidation, the electron transport chain, and heightened coenzyme NAD + . Loss of IL-10 due to mitochondrial complex III defects was remarkably rescued by adding NAD + precursors. This activated a SIRTUIN1 signaling cascade, largely independent of ATP, that culminated in activation of IL-10 transcription factor PBX1. Il-10 activation by the respiratory chain was also important in vivo, as efferocyte mitochondrial dysfunction led to cardiac rupture after myocardial injury. These findings highlight a new paradigm whereby macrophages leverage efferocytic metabolites and electron transport for anti-inflammatory reprogramming that culminates in organ repair., (Copyright © 2018 Elsevier Inc. All rights reserved.)