bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2022‒08‒21
forty-five papers selected by
Christian Frezza
University Hospital Cologne

  1. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01015-4. [Epub ahead of print]40(7): 111198
      The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate.
    Keywords:  CP: Cell biology; CP: Metabolism; Drp1; Mfn1; Mfn2; Opa1; amino acid sensing; arginine; dynamics; fusion; glutamine; hyperfusion; leucine; mitochondria; stable isotope tracer
  2. Cell Metab. 2022 Aug 11. pii: S1550-4131(22)00311-4. [Epub ahead of print]
      How environmental nutrient availability impacts T cell metabolism and function remains poorly understood. Here, we report that the presence of physiologic carbon sources (PCSs) in cell culture medium broadly impacts glucose utilization by CD8+ T cells, independent of transcriptional changes in metabolic reprogramming. The presence of PCSs reduced glucose contribution to the TCA cycle and increased effector function of CD8+ T cells, with lactate directly fueling the TCA cycle. In fact, CD8+ T cells responding to Listeria infection preferentially consumed lactate over glucose as a TCA cycle substrate in vitro, with lactate enhancing T cell bioenergetic and biosynthetic capacity. Inhibiting lactate-dependent metabolism in CD8+ T cells by silencing lactate dehydrogenase A (Ldha) impaired both T cell metabolic homeostasis and proliferative expansion in vivo. Together, our data indicate that carbon source availability shapes T cell glucose metabolism and identifies lactate as a bioenergetic and biosynthetic fuel for CD8+ effector T cells.
    Keywords:  (13)C tracing; T cells; TCA cycle; immunometabolism; lactate; metabolic programming; metabolomics
  3. Cell Stress Chaperones. 2022 Aug 17.
      The molecular chaperone TNF-receptor-associated protein-1 (TRAP1) controls mitochondrial respiration through regulation of Krebs cycle and electron transport chain activity. Post-translational modification (PTM) of TRAP1 regulates its activity, thereby controlling global metabolic flux. O-GlcNAcylation is one PTM that is known to impact mitochondrial metabolism, however the major effectors of this regulatory PTM remain inadequately resolved. Here we demonstrate that TRAP1-O-GlcNAcylation decreases TRAP1 ATPase activity, leading to increased mitochondrial metabolism. O-GlcNAcylation of TRAP1 occurs following mitochondrial import and provides critical regulatory feedback, as the impact of O-GlcNAcylation on mitochondrial metabolism shows TRAP1-dependence. Mechanistically, loss of TRAP1-O-GlcNAcylation decreased TRAP1 binding to ATP, and interaction with its client protein succinate dehydrogenase (SDHB). Taken together, TRAP1-O-GlcNAcylation serves to regulate mitochondrial metabolism by the reversible attenuation of TRAP1 chaperone activity.
    Keywords:  GlcNAcylation; Metabolism; Molecular chaperone; Post-translational modification; TRAP1
  4. Cell Metab. 2022 Aug 11. pii: S1550-4131(22)00310-2. [Epub ahead of print]
      Uncoupling protein 1 (UCP1)-mediated adaptive thermogenesis protects mammals against hypothermia and metabolic dysregulation. Whether and how mitochondrial calcium regulates this process remains unclear. Here, we show that mitochondrial calcium uniporter (MCU) recruits UCP1 through essential MCU regulator (EMRE) to form an MCU-EMRE-UCP1 complex upon adrenergic stimulation. This complex formation increases mitochondrial calcium uptake to accelerate the tricarboxylic acid cycle and supply more protons that promote uncoupled respiration, functioning as a thermogenic uniporter. Mitochondrial calcium uptake 1 (MICU1) negatively regulates thermogenesis probably through inhibiting thermogenic uniporter formation. Accordingly, the deletion of Mcu or Emre in brown adipocytes markedly impairs thermogenesis and exacerbates obesity and metabolic dysfunction. Remarkably, the enhanced assembly of the thermogenic uniporter via Micu1 knockout or expressing linked EMRE-UCP1 results in opposite phenotypes. Thus, we have uncovered a "thermoporter" that provides a driving force for the UCP1 operation in thermogenesis, which could be leveraged to combat obesity and associated metabolic disorders.
    Keywords:  UCP1; brown adipose tissue; metabolic dysfunction; mitochondrial calcium uniporter; obesity; thermogenesis
  5. Mol Cell. 2022 Aug 12. pii: S1097-2765(22)00703-1. [Epub ahead of print]
      Proliferating cells exhibit a metabolic phenotype known as "aerobic glycolysis," which is characterized by an elevated rate of glucose fermentation to lactate irrespective of oxygen availability. Although several theories have been proposed, a rationalization for why proliferating cells seemingly waste glucose carbon by excreting it as lactate remains elusive. Using the NCI-60 cell lines, we determined that lactate excretion is strongly correlated with the activity of mitochondrial NADH shuttles, but not proliferation. Quantifying the fluxes of the malate-aspartate shuttle (MAS), the glycerol 3-phosphate shuttle (G3PS), and lactate dehydrogenase under various conditions demonstrated that proliferating cells primarily transform glucose to lactate when glycolysis outpaces the mitochondrial NADH shuttles. Increasing mitochondrial NADH shuttle fluxes decreased glucose fermentation but did not reduce the proliferation rate. Our results reveal that glucose fermentation, a hallmark of cancer, is a secondary consequence of MAS and G3PS saturation rather than a unique metabolic driver of cellular proliferation.
    Keywords:  NADH shuttles; aerobic glycolysis; cancer metabolism; glycerol 3-phosphate shuttle; isotope-tracer analysis; malate-aspartate shuttle; metabolic flux; metabolomics; the Warburg effect
  6. Nat Chem Biol. 2022 Aug 15.
      Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis. Even in fermenting S. cerevisiae that is net releasing ethanol, media 13C-ethanol rapidly enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing reveals that ethanol is also a major source of both NADH and NADPH high-energy electrons, and this role is augmented under oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapidly reversible reactions is a conserved feature of eukaryotic metabolism.
  7. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01035-X. [Epub ahead of print]40(7): 111218
      Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.
    Keywords:  AMPK; CP: Cancer; CP: Metabolism; Cdk5; PRKAG2; SDHB; Warburg effect; cancer bioenergetics; neuroendocrine tumor; p53; pheochromocytoma; senescence
  8. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01010-5. [Epub ahead of print]40(7): 111193
      Succinate dehydrogenase (SDH) loss-of-function mutations drive succinate accumulation in tumor microenvironments, for example in the neuroendocrine tumors pheochromocytoma (PC) and paraganglioma (PG). Control of innate immune cell activity by succinate is described, but effects on T cells have not been interrogated. Here we report that exposure of human CD4+ and CD8+ T cells to tumor-associated succinate concentrations suppresses degranulation and cytokine secretion, including of the key anti-tumor cytokine interferon-γ (IFN-γ). Mechanistically, this is associated with succinate uptake-partly via the monocarboxylate transporter 1 (MCT1)-inhibition of succinyl coenzyme A synthetase activity and impaired glucose flux through the tricarboxylic acid cycle. Consistently, pharmacological and genetic interventions restoring glucose oxidation rescue T cell function. Tumor RNA-sequencing data from patients with PC and PG reveal profound suppression of IFN-γ-induced genes in SDH-deficient tumors compared with those with other mutations, supporting a role for succinate in modulating the anti-tumor immune response in vivo.
    Keywords:  CP: immunology; CP: metabolism; T cell; metabolism; metabolite; succinate; tumor microenvironment
  9. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01050-6. [Epub ahead of print]40(7): 111233
      5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches.
    Keywords:  5-FU resistance; CP: Cancer; DNA damage response; Serine metabolism; colorectal cancer; mitochondrial metabolism; nucleotide metabolism; one-carbon metabolism (OCM)
  10. Nat Commun. 2022 Aug 17. 13(1): 4848
      Mammalian cells can acquire exogenous amino acids through endocytosis and lysosomal catabolism of extracellular proteins. In amino acid-replete environments, nutritional utilization of extracellular proteins is suppressed by the amino acid sensor mechanistic target of rapamycin complex 1 (mTORC1) through an unknown process. Here, we show that mTORC1 blocks lysosomal degradation of extracellular proteins by suppressing V-ATPase-mediated acidification of lysosomes. When mTORC1 is active, peripheral V-ATPase V1 domains reside in the cytosol where they are stabilized by association with the chaperonin TRiC. Consequently, most lysosomes display low catabolic activity. When mTORC1 activity declines, V-ATPase V1 domains move to membrane-integral V-ATPase Vo domains at lysosomes to assemble active proton pumps. The resulting drop in luminal pH increases protease activity and degradation of protein contents throughout the lysosomal population. These results uncover a principle by which cells rapidly respond to changes in their nutrient environment by mobilizing the latent catabolic capacity of lysosomes.
  11. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01021-X. [Epub ahead of print]40(7): 111204
      Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.
    Keywords:  CP: Metabolism; CYTB; SEPs; SMIM4; UQCC1; UQCC2; complex III; electron transport chain; microproteins; nuclear-mitochondrial coordination; smORFs
  12. Cell Death Discov. 2022 Aug 13. 8(1): 357
      Proliferating cancer cells are dependent on glutamine metabolism for survival when challenged with oxidative stresses caused by reactive oxygen species, hypoxia, nutrient deprivation and matrix detachment. ATF4, a key stress responsive transcription factor, is essential for cancer cells to sustain glutamine metabolism when challenged with these various types of stress. While it is well documented how the ATF4 transcript is translated into protein as a stress response, an important question concerns how the ATF4 message levels are sustained to enable cancer cells to survive the challenges of nutrient deprivation and damaging reactive oxygen species. Here, we now identify the pathway in triple negative breast cancer cells that provides a sustained ATF4 response and enables their survival when encountering these challenges. This signaling pathway starts with mTORC2, which upon sensing cellular stresses arising from glutamine deprivation or an acute inhibition of glutamine metabolism, initiates a cascade of events that triggers an increase in ATF4 transcription. Surprisingly, this signaling pathway is not dependent on AKT activation, but rather requires the mTORC2 target, PKC, which activates the transcription factor Nrf2 that then induces ATF4 expression. Additionally, we identify a sirtuin family member, the NAD+-dependent de-succinylase Sirt5, as a key transcriptional target for ATF4 that promotes cancer cell survival during metabolic stress. Sirt5 plays fundamental roles in supporting cancer cell metabolism by regulating various enzymatic activities and by protecting an enzyme essential for glutaminolysis, glutaminase C (GAC), from degradation. We demonstrate that ectopic expression of Sirt5 compensates for knockdowns of ATF4 in cells exposed to glutamine deprivation-induced stress. These findings provide important new insights into the signaling cues that lead to sustained ATF4 expression as a general stress-induced regulator of glutamine metabolism, as well as highlight Sirt5 an essential effector of the ATF4 response to metabolic stress.
  13. Cancer Cell. 2022 Aug 12. pii: S1535-6108(22)00324-5. [Epub ahead of print]
      Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.
    Keywords:  DHODH; IDH; cancer metabolism; genetically engineered mouse model; glioma; isocitrate dehydrogenase; pyrimidine nucleotides
  14. PLoS One. 2022 ;17(8): e0273080
      Ulcerative colitis (UC) is a complex, multifactorial disease driven by a dysregulated immune response against host commensal microbes. Despite rapid advances in our understanding of host genomics and transcriptomics, the metabolic changes in UC remain poorly understood. We thus sought to investigate distinguishing metabolic features of the UC colon (14 controls and 19 patients). Metabolomics analyses revealed inflammation state as the primary driver of metabolic variation rather than diagnosis, with multiple metabolites differentially regulated between inflamed and uninflamed tissues. Specifically, inflamed tissues were characterized by reduced levels of nicotinamide adenine dinucleotide (NAD+) and enhanced levels of nicotinamide (NAM) and adenosine diphosphate ribose (ADPr). The NAD+/NAM ratio, which was reduced in inflamed patients, served as an effective classifier for inflammation in UC. Mitochondria were also structurally altered in UC, with UC patient colonocytes displaying reduced mitochondrial density and number. Together, these findings suggest a link between mitochondrial dysfunction, inflammation, and NAD+ metabolism in UC.
  15. J Cell Sci. 2022 Aug 17. pii: jcs.259778. [Epub ahead of print]
      In yeast, proteasomes are enriched in cell nuclei where they execute important cellular functions. Nutrient-stress can change this localization indicating proteasomes respond to the cell's metabolic state. However, the signals that connect these processes remain poorly understood. Carbon starvation triggers a reversible translocation of proteasomes to cytosolic condensates known as proteasome storage granules (PSGs). Surprisingly, we observed strongly reduced levels of proteasome granules when cells had active cellular respiration prior to starvation. This suggests the mitochondrial activity of cells is a determining factor in the response of proteasomes to carbon starvation. Consistent with this, upon inhibition of mitochondrial function we observed proteasomes relocalize to granules. These links between proteasomes and metabolism involve specific signaling pathways, as we identified a MAP kinase cascade that is critical to the formation of proteasome granules after respiratory growth but not following glycolytic growth. Furthermore, the yeast homolog of AMP kinase, Snf1, is important for proteasome granule formation induced by mitochondrial inhibitors, while dispensable for granule formation following carbon starvation. We propose a model where mitochondrial activity promotes proteasome nuclear localization.
    Keywords:  MAP kinases; Mitochondrial inhibition; Mitochondrial respiration; Proteaphagy; Proteasome; Proteasome storage granules (PSG)
  16. Proc Natl Acad Sci U S A. 2022 Aug 23. 119(34): e2120157119
      Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to electron transport chain (ETC) toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here, we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with ETC toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Importantly, we observed that the PDK4-mediated regulation of mitochondrial fission was independent of its canonical function, i.e., inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Phosphoproteomic screen for PDK4 substrates, followed by nonphosphorylatable and phosphomimetic mutations of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. Conversely, inhibition of the PDK4-SEPT2 axis could restore the balance in mitochondrial dynamics and reinvigorates cellular respiration in mitochondrial fusion factor, mitofusin 2-deficient cells. Furthermore, PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.
    Keywords:  OCR; dynamin-related protein 1; mitochondrial fission; pyruvate dehydrogenase kinase 4; septin 2
  17. J Biol Chem. 2022 Aug 17. pii: S0021-9258(22)00830-4. [Epub ahead of print] 102387
      Isocitrate dehydrogenase 3 (IDH3) is a key enzyme in the mitochondrial tricarboxylic acid (TCA) cycle, which catalyzes the decarboxylation of isocitrate into α-ketoglutarate (αKG) and concurrently converts NAD+ into NADH. Dysfunction of IDH3B, the β subunit of IDH3, has been previously correlated with retinal degeneration and male infertility in humans, but tissue-specific effects of IDH3 dysfunction are unclear. Here, we generated Idh3b-knockout (KO) mice and found that IDH3B is essential for IDH3 activity in multiple tissues. We determined loss of Idh3b in mice causes substantial accumulation of isocitrate and its precursors in the TCA cycle, particularly in the testes, whereas the levels of the downstream metabolites remain unchanged or slightly increased. However, the Idh3b-KO mice did not fully recapitulate the defects observed in humans. Global deletion of Idh3b only causes male infertility but not retinal degeneration in mice. Our investigation showed that loss of Idh3b causes an energetic deficit and disrupts the biogenesis of acrosome and flagellum, resulting in spermiogenesis arrestment in sperm cells. Together, we demonstrate IDH3B controls its substrate levels in the TCA cycle, and it is required for sperm mitochondrial metabolism and spermiogenesis, highlighting the importance of the tissue-specific function of the ubiquitous TCA cycle.
    Keywords:  IDH3B; TCA cycle; male infertility; metabolism; retinal degeneration; spermatogenesis; spermatozoa; spermiogenesis
  18. Biochem Biophys Res Commun. 2022 Aug 05. pii: S0006-291X(22)01109-3. [Epub ahead of print]625 167-173
      Cancer cells rely on glycolysis to generate ATP for survival. However, inhibiting glycolysis is insufficient for the eradication of cancer cells because glycolysis-suppressed cells undergo metabolic reprogramming toward mitochondrial oxidative phosphorylation. We previously described that upon glycolytic suppression in pancreatic cancer cells, intracellular glycometabolism is shifted toward mitochondrial oxidative phosphorylation in an autophagy-dependent manner for cellular survival. Here, we hypothesized that mitophagy, which selectively degrades mitochondria via autophagy, is involved in mitochondrial activation under metabolic reprogramming. We revealed that glycolytic suppression notably increased mitochondrial membrane potential and mitophagy in a pancreatic cancer cell model (PANC-1). PTEN-induced kinase 1 (PINK1), a ubiquitin kinase that regulates mitophagy in healthy cells, regulated mitochondrial activation through mitophagy by glycolytic suppression. However, Parkin, a ubiquitin ligase regulated by PINK1 in healthy cells to induce mitophagy, was not involved in the PINK1-dependent mitophagy of the cancer glycometabolism. These results imply that cancer cells and healthy cells have different regulatory pieces of machinery for mitophagy, and inhibition of cancer-specific mechanisms may be a potential strategy for cancer therapy targeting metabolic reprogramming.
    Keywords:  Glycometabolism; Mitophagy; PINK1; Pancreatic cancer; Parkin
  19. Nat Commun. 2022 Aug 17. 13(1): 4836
      The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.
  20. J Cell Biol. 2022 Sep 05. pii: e202112107. [Epub ahead of print]221(9):
      Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the endoplasmic reticulum (ER). The ER protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at mitochondria-associated ER membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulates seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis and maturation at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at the membrane contact sites.
  21. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01051-8. [Epub ahead of print]40(7): 111234
      Spermidine is essential for cellular growth and acts as a prerequisite of hypusination, a post-translational modification of eukaryotic initiation factor 5A (eIF5A), allowing the translation of polyproline-containing proteins. Here, we show that oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) increases spermidine synthesis and eIF5A hypusination to enhance expression of polyproline-containing latency-associated nuclear antigen (LANA) for viral episomal maintenance. KSHV upregulates intracellular spermidine levels by dysregulating polyamine metabolic pathways in three-dimensional (3D) culture and 2D de novo infection conditions. Increased intracellular spermidine leads to increased eIF5A hypusination, ultimately enhancing LANA expression. In contrast, inhibition of spermidine synthesis or eIF5A hypusination alleviates LANA expression, decreasing viral episomal maintenance and KSHV-infected cell proliferation in vitro and in vivo, which is reversed by spermidine supplement. This demonstrates that KSHV hijacks spermidine synthesis and eIF5A hypusination pathways to enhance LANA expression for viral episomal maintenance, suggesting polyamine metabolism and eIF5A hypusination as therapeutic targets for KSHV-induced tumorigenesis.
    Keywords:  CP: Microbiology; KSHV; Kaposi’s sarcoma-associated herpesvirus; LANA; cancer metabolism; eIF5A hypusination; polyamine metabolism
  22. Cell Stress. 2022 Jul;6(7): 68-71
      Brown adipocytes react to temperature and nutritional challenges by ramping up their metabolism and generating heat. This adaptation to changes in the environment is crucial for defending organismal homeostasis, but is impaired in obesity and during aging. Writing in Nature, Niemann et al. show that brown adipocytes become apoptotic under thermoneutral conditions and release ATP, which in turn is converted extracellularly into inosine. They further present evidence that pharmacological and genetic manipulations that enhance signalling of this purine metabolite stimulates thermogenesis in brown adipocytes and promotes metabolic health.
    Keywords:  Adipose tissue thermogenesis; Apoptosis; ENT1; Inosine; Obesity
  23. Front Immunol. 2022 ;13 949451
      In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.
    Keywords:  EVs; STING; cGAS; ectosomes; exosomes; inflammation; microvesicles
  24. Diabetes. 2022 Aug 19. pii: db220256. [Epub ahead of print]
      The innate immune kinase TBK1 (TANK-binding kinase 1) responds to microbial-derived signals to initiate responses against viral and bacterial pathogens. More recent work implicates TBK1 in metabolism and tumorigenesis. The kinase mTOR (mechanistic target of rapamycin) integrates diverse environmental cues to control fundamental cellular processes. Our prior work demonstrated in cells that TBK1 phosphorylates mTOR (on S2159) to increase mTORC1 and mTORC2 catalytic activity and signaling. Here we investigate a role for TBK1-mTOR signaling in control of glucose metabolism in vivo. We find that diet induced obese (DIO) but not lean mice bearing a whole-body "TBK1 resistant" Mtor S2159A knockin allele (MtorA/A) display exacerbated hyperglycemia and systemic insulin resistance with no change in energy balance. Mechanistically, Mtor S2159A knockin in DIO mice reduces mTORC1 and mTORC2 signaling in response to insulin and innate immune agonists, reduces anti-inflammatory gene expression in adipose tissue, and blunts anti-inflammatory macrophage M2 polarization, phenotypes shared by mice with tissue-specific inactivation of TBK1 or mTOR complexes. Tissues from DIO mice display elevated TBK1 activity and mTOR S2159 phosphorylation relative to lean mice. We propose a model whereby obesity-associated signals increase TBK1 activity and mTOR phosphorylation, which boosts mTORC1 and mTORC2 signaling in parallel to the insulin pathway, thereby attenuating insulin resistance to improve glycemic control during diet-induced obesity.
  25. Nat Protoc. 2022 Aug 19.
      Identifying metabolites and delineating their immune-regulatory contribution in the tumor microenvironment is an area of intense study. Interrogating metabolites and metabolic networks among immune cell subsets and host cells from resected tissues and fluids of human patients presents a major challenge, owing to the specialized handling of samples for downstream metabolomics. To address this, we first outline the importance of collaborating with a biobank for coordinating and streamlining workflow for point of care, sample collection, processing and cryopreservation. After specimen collection, we describe our 60-min rapid bead-based cellular enrichment method that supports metabolite analysis between T cells and tumor cells by mass spectrometry. We also describe how the metabolic data can be complemented with metabolic profiling by flow cytometry. This protocol can serve as a foundation for interrogating the metabolism of cell subsets from primary human ovarian cancer.
  26. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01024-5. [Epub ahead of print]40(7): 111207
      Iron is essential for deoxyribonucleotides production and for enzymes containing an Fe-S cluster involved in DNA replication and repair. How iron bioavailability and DNA metabolism are coordinated remains poorly understood. NCOA4 protein mediates autophagic degradation of ferritin to maintain iron homeostasis and inhibits DNA replication origin activation via hindrance of the MCM2-7 DNA helicase. Here, we show that iron deficiency inhibits DNA replication, parallel to nuclear NCOA4 stabilization. In iron-depleted cells, NCOA4 knockdown leads to unscheduled DNA synthesis, with replication stress, genome instability, and cell death. In mice, NCOA4 genetic inactivation causes defective intestinal regeneration upon dextran sulfate sodium-mediated injury, with DNA damage, defective cell proliferation, and cell death; in intestinal organoids, this is fostered by iron depletion. In summary, we describe a NCOA4-dependent mechanism that coordinates iron bioavailability and DNA replication. This function prevents replication stress, maintains genome integrity, and sustains high rates of cell proliferation during tissue regeneration.
    Keywords:  CP: Metabolism; DNA replication; NCOA4; ferritin; genome stability; iron metabolism
  27. Cancer Cell. 2022 Aug 15. pii: S1535-6108(22)00325-7. [Epub ahead of print]
      Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify druggable targets for DMG, we conducted a genome-wide CRISPR screen that reveals a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. This metabolic vulnerability reflects an elevated rate of uridine/uracil degradation that depletes DMG cells of substrates for the alternate salvage pyrimidine biosynthesis pathway. A clinical stage inhibitor of DHODH (rate-limiting enzyme in the de novo pathway) diminishes uridine-5'-phosphate (UMP) pools, generates DNA damage, and induces apoptosis through suppression of replication forks-an "on-target" effect, as shown by uridine rescue. Matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy imaging demonstrates that this DHODH inhibitor (BAY2402234) accumulates in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts, highlighting BAY2402234 as a promising therapy against DMGs.
    Keywords:  ATR; BAY2402234; DHODH; DPYD; de novo pyrimidine synthesis; diffuse intrinsic pontine glioma; diffuse midline glioma; elimusertib; pyrimidine degradation; replication stress
  28. Methods Mol Biol. 2022 ;2540 401-414
      The rather recent development of genetically encoded metabolite sensors has changed the way we can study metabolism in living cells, ex vivo tissues, and in vivo immensely. In recent years, these sensors have also been adapted for use in Drosophila tissues. Here, we describe a standard protocol to image such sensors in ex vivo Drosophila larval brains using the glucose sensor FLII12Pglu-700μδ6. The protocol, however, can be adapted for the use of other sensors, tissues, and can even be used in vivo.
    Keywords:  Carbohydrate transport; FRET; Genetically encoded metabolite sensors; Live imaging; Metabolism
  29. Science. 2022 Aug 18. eabn0478
      Drug resistance in cancer is often linked to changes in tumor cell state or lineage, but the molecular mechanisms driving this plasticity remain unclear. Using murine organoid and genetically engineered mouse models, we investigated the causes of lineage plasticity in prostate cancer and its relationship to antiandrogen resistance. We found that plasticity initiates in an epithelial population defined by mixed luminal-basal phenotype and that it depends on elevated JAK and FGFR activity. Organoid cultures from patients with castration-resistant disease harboring mixed-lineage cells reproduce the dependency observed in mice, by upregulating luminal gene expression upon JAK and FGFR inhibitor treatment. Single-cell analysis confirms the presence of mixed lineage cells with elevated JAK/STAT and FGFR signaling in a subset of patients with metastatic disease, with implications for stratifying patients for clinical trials.
  30. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01018-X. [Epub ahead of print]40(7): 111201
      Stimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of "poised" cDC1s within stromal sheets is unlikely to simply reflect passive exclusion from tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling generates developmentally conserved cell fate cues that regulate cDC1 behavior. We find that, in human T cell-inflamed tumors, CD8+ T cells penetrate tumor nests, whereas cDC1s are confined within adjacent stroma that recurrently displays site-specific proteolysis of the matrix proteoglycan versican (VCAN), an essential organ-sculpting modification in development. VCAN is necessary, and its proteolytic fragment (matrikine) versikine is sufficient for cDC1 accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive cDC1 activation program conferring exquisite sensitivity to DNA sensing, supported by atypical innate lymphoid cells. Thus, peritumoral stroma mimicking embryonic provisional matrix remodeling regulates cDC1 abundance and activity to elicit T cell-inflamed tumor microenvironments.
    Keywords:  CD40; CP: Cancer; CP: Immunology; cDC1; checkpoint inhibitors; dendritic cells; immunotherapy; proteoglycans; tumor matrix; tumor stroma; versican; versikine
  31. Cancer Immunol Res. 2022 Aug 15. pii: CIR-21-0813. [Epub ahead of print]
      Chronic T-cell receptor (TCR) signaling in the tumor microenvironment is known to promote T-cell dysfunction. However, we reasoned that poorly immunogenic tumors may also compromise T cells by impairing their metabolism. To address this, we assessed temporal changes in T-cell metabolism, fate, and function in models of ¬B-cell lymphoma driven by Myc, a promoter of energetics and repressor of immunogenicity. Increases in lymphoma burden most significantly impaired CD4+ T-cell function and promoted regulatory T-cell (Treg) and Th1-cell differentiation. Metabolomic analyses revealed early reprogramming of CD4+ T-cell metabolism, reduced glucose uptake, and impaired mitochondrial function, which preceded changes in T-cell fate. In contrast, B-cell lymphoma metabolism remained robust during tumor progression. Finally, mitochondrial functions were impaired in CD4+ and CD8+ T cells in lymphoma-transplanted OT-II and OT-I transgenic mice, respectively. These findings support a model, whereby early, TCR-independent, metabolic interactions with developing lymphomas limits T cell-mediated immune surveillance.
  32. Life Sci Alliance. 2022 Nov;pii: e202201413. [Epub ahead of print]5(11):
      Mitochondrial respiration and metabolism play a key role in the pathogenesis and progression of colon adenocarcinoma (COAD). Here, we report a functional pool of FKBP4, a co-chaperone protein, in the mitochondrial intermembrane space (IMS) of colon cancer cells. We found that IMS-localized FKBP4 is essential for the maintenance of mitochondrial respiration, thus contributing to the sensitivity of COAD cells to 5-fluorouracil (5-FU). Mechanistically, FKBP4 interacts with COA6 and controls the assembly of the mitochondrial COA6/SCO1/SCO2 complex, thereby governing COA6-regulated biogenesis and activity of mitochondrial cytochrome c oxidase (complex IV). Thus, our data reveal IMS-localized FKBP4 as a novel regulator of 5-FU sensitivity in COAD, linking mitochondrial respiration to 5-FU sensitivity in COAD.
  33. Nucleic Acids Res. 2022 Aug 18. pii: gkac690. [Epub ahead of print]
      Cells are constantly challenged by genotoxic stresses that can lead to genome instability. The integrity of the nuclear genome is preserved by the DNA damage response (DDR) and repair. Additionally, these stresses can induce mitochondria to transiently hyperfuse; however, it remains unclear whether canonical DDR is linked to these mitochondrial morphological changes. Here, we report that the abolition of mitochondrial fusion causes a substantial defect in the ATM-mediated DDR signaling. This deficiency is overcome by the restoration of mitochondria fusion. In cells with fragmented mitochondria, genotoxic stress-induced activation of JNK and its translocation to DNA lesion are lost. Importantly, the mitochondrial fusion machinery of MFN1/MFN2 associates with Sab (SH3BP5) and JNK, and these interactions are indispensable for the Sab-mediated activation of JNK and the ATM-mediated DDR signaling. Accordingly, the formation of BRCA1 and 53BP1 foci, as well as homology and end-joining repair are impaired in cells with fragmented mitochondria. Together, these data show that mitochondrial fusion-dependent JNK signaling is essential for the DDR, providing vital insight into the integration of nuclear and cytoplasmic stress signals.
  34. Nature. 2022 Aug 17.
      Although p53 inactivation promotes genomic instability1 and presents a route to malignancy for more than half of all human cancers2,3, the patterns through which heterogenous TP53 (encoding human p53) mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases-Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications-each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53-the 'guardian of the genome'-is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours.
  35. Nat Metab. 2022 Aug 15.
      Measurement of oxygen consumption is a powerful and uniquely informative experimental technique. It can help identify mitochondrial mechanisms of action following pharmacologic and genetic interventions, and characterize energy metabolism in physiology and disease. The conceptual and practical benefits of respirometry have made it a frontline technique to understand how mitochondrial function can interface with-and in some cases control-cell physiology. Nonetheless, an appreciation of the complexity and challenges involved with such measurements is required to avoid common experimental and analytical pitfalls. Here we provide a practical guide to oxygen consumption measurements covering the selection of experimental models and instrumentation, as well as recommendations for the collection, interpretation and normalization of data. These guidelines are provided with the intention of aiding experimental design and enhancing the overall reputability, transparency and reliability of oxygen consumption measurements.
  36. Microbiol Spectr. 2022 Aug 16. e0289122
      Succinate dehydrogenase (SDH), also known as respiratory chain complex II, plays a crucial role in energy production in which SdhC functions as an anchored subunit in the inner membrane of mitochondria. In this study, domain annotation analyses revealed that two SdhC domain-containing proteins were present in the filamentous insect-pathogenic fungus Beauveria bassiana, and they were named BbSdhC1 and BbSdhC2, respectively. Only BbSdhC1 localized to mitochondria; hence, this protein is considered the ortholog of SdhC in B. bassiana. Ablation of BbSdhC1 led to significantly reduced vegetative growth on various nutrients. The ΔBbsdhc1 mutant displayed the significantly reduced ATP synthesis and abnormal differentiation under aerial and submerged conditions. Notably, the BbSdhC1 loss resulted in enhanced intracellular levels of reactive oxygen species (ROS) and impaired growth of mycelia under oxidative stress. Finally, insect bioassays (via cuticle and intrahemocoel injection infection) revealed that disruption of BbSdhC1 significantly attenuated fungal virulence against the insect hosts. These findings indicate that BbSdhC1 contributes to vegetative growth, resistance to oxidative stress, differentiation, and virulence of B. bassiana due to its roles in energy generation and maintaining the homeostasis of the intracellular ROS levels. IMPORTANCE The electron transport chain (ETC) is critical for energy supply by mediating the electron flow along the mitochondrial membrane. Succinate dehydrogenase (SDH) is also known as complex II in the ETC, in which SdhC is a subunit anchored in mitochondrial membrane. However, the physiological roles of SdhC remain enigmatic in filamentous fungi. In filamentous insect-pathogenic fungus B. bassiana, SdhC is required for maintaining mitochondrial functionality, which is critical for fungal stress response, development, and pathogenicity. These findings improve our understanding of physiological mechanisms of ETC components involved in pathogenicity of the entomopathogenic fungi.
    Keywords:  development; entomopathogenic fungus; oxidative stress; succinate dehydrogenase; virulence
  37. Nature. 2022 Aug 17.
      Compelling evidence shows that brown and beige adipose tissue are protective against metabolic diseases1,2. PR domain-containing 16 (PRDM16) is a dominant activator of the biogenesis of beige adipocytes by forming a complex with transcriptional and epigenetic factors and is therefore an attractive target for improving metabolic health3-8. However, a lack of knowledge surrounding the regulation of PRDM16 protein expression hampered us from selectively targeting this transcriptional pathway. Here we identify CUL2-APPBP2 as the ubiquitin E3 ligase that determines PRDM16 protein stability by catalysing its polyubiquitination. Inhibition of CUL2-APPBP2 sufficiently extended the half-life of PRDM16 protein and promoted beige adipocyte biogenesis. By contrast, elevated CUL2-APPBP2 expression was found in aged adipose tissues and repressed adipocyte thermogenesis by degrading PRDM16 protein. Importantly, extended PRDM16 protein stability by adipocyte-specific deletion of CUL2-APPBP2 counteracted diet-induced obesity, glucose intolerance, insulin resistance and dyslipidaemia in mice. These results offer a cell-autonomous route to selectively activate the PRDM16 pathway in adipose tissues.
  38. Cell Syst. 2022 Aug 15. pii: S2405-4712(22)00313-1. [Epub ahead of print]
      Small cell lung cancer (SCLC) tumors comprise heterogeneous mixtures of cell states, categorized into neuroendocrine (NE) and non-neuroendocrine (non-NE) transcriptional subtypes. NE to non-NE state transitions, fueled by plasticity, likely underlie adaptability to treatment and dismal survival rates. Here, we apply an archetypal analysis to model plasticity by recasting SCLC phenotypic heterogeneity through multi-task evolutionary theory. Cell line and tumor transcriptomics data fit well in a five-dimensional convex polytope whose vertices optimize tasks reminiscent of pulmonary NE cells, the SCLC normal counterparts. These tasks, supported by knowledge and experimental data, include proliferation, slithering, metabolism, secretion, and injury repair, reflecting cancer hallmarks. SCLC subtypes, either at the population or single-cell level, can be positioned in archetypal space by bulk or single-cell transcriptomics, respectively, and characterized as task specialists or multi-task generalists by the distance from archetype vertex signatures. In the archetype space, modeling single-cell plasticity as a Markovian process along an underlying state manifold indicates that task trade-offs, in response to microenvironmental perturbations or treatment, may drive cell plasticity. Stifling phenotypic transitions and plasticity may provide new targets for much-needed translational advances in SCLC. A record of this paper's Transparent Peer Review process is included in the supplemental information.
    Keywords:  RNA velocity; dynamical systems; gene regulatory networks; heterogeneity; phenotypic plasticity; single cell; small cell lung cancer
  39. Nat Commun. 2022 Aug 16. 13(1): 4827
      Although cellular senescence drives multiple age-related co-morbidities through the senescence-associated secretory phenotype, in vivo senescent cell identification remains challenging. Here, we generate a gene set (SenMayo) and validate its enrichment in bone biopsies from two aged human cohorts. We further demonstrate reductions in SenMayo in bone following genetic clearance of senescent cells in mice and in adipose tissue from humans following pharmacological senescent cell clearance. We next use SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from human and murine bone marrow/bone scRNA-seq data. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Using this senescence panel, we are able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways. SenMayo also represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.
  40. Front Oncol. 2022 ;12 926437
      Metabolic reprogramming, due in part to the overexpression of metabolic enzymes, is a key hallmark of cancer cells. Lactate dehydrogenase (LDHA), a metabolic enzyme that catalyzes the interconversion of lactate and pyruvate, is overexpressed in a wide variety of cancer types, including pancreatic ductal adenocarcinoma (PDAC). Furthermore, the genetic or pharmacological inhibition of LDHA suppresses cancer growth, demonstrating a cancer-promoting role for this enzyme. Therefore, several pharmacological LDHA inhibitors are being developed and tested as potential anti-cancer therapeutic agents. Because cancer cells are known to rapidly adapt and become resistant to anti-cancer therapies, in this study, we modeled the adaptation of cancer cells to LDHA inhibition. Using PDAC as a model system, we studied the molecular aspects of cells resistant to the competitive LDHA inhibitor sodium oxamate. We performed unbiased RNA-sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), and metabolomics analyses of parental and oxamate-resistant PDAC cells treated with and without oxamate to identify the transcriptional, chromatin, and metabolic landscapes of these cells. We found that oxamate-resistant PDAC cells were significantly different from parental cells at the levels of mRNA expression, chromatin accessibility, and metabolites. Additionally, an integrative analysis combining the RNA-seq and ATAC-seq datasets identified a subset of differentially expressed mRNAs that directly correlated with changes in chromatin accessibility. Finally, functional analysis of differentially expressed metabolic genes in parental and oxamate-resistant PDAC cells treated with and without oxamate, together with an integrative analysis of RNA-seq and metabolomics data, revealed changes in metabolic enzymes that might explain the changes in metabolite levels observed in these cells. Collectively, these studies identify the transcriptional, chromatin, and metabolic landscapes of LDHA inhibitor resistance in PDAC cells. Future functional studies related to these changes remain necessary to reveal the direct roles played by these changes in the development of LDHA inhibitor resistance and uncover approaches for more effective use of LDHA inhibitors in cancer therapy.
    Keywords:  LDHA; cancer metabolism; chromatin accessibility; metabolomics; transcriptomics
  41. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)00993-7. [Epub ahead of print]40(7): 111180
      Intratumor heterogeneity (ITH) represents a major challenge for anticancer therapies. An integrated, multidimensional, multiregional approach dissecting ITH of the clear cell renal cell carcinoma (ccRCC) tumor microenvironment (TME) is employed at the single-cell level with mass cytometry (CyTOF), multiplex immunofluorescence (MxIF), and single-nucleus RNA sequencing (snRNA-seq) and at the bulk level with whole-exome sequencing (WES), RNA-seq, and methylation profiling. Multiregional analyses reveal unexpected conservation of immune composition within each individual patient, with profound differences among patients, presenting patient-specific tumor immune microenvironment signatures despite underlying genetic heterogeneity from clonal evolution. Spatial proteogenomic TME analysis using MxIF identifies 14 distinct cellular neighborhoods and, conversely, demonstrated architectural heterogeneity among different tumor regions. Tumor-expressed cytokines are identified as key determinants of the TME and correlate with clinical outcome. Overall, this work signifies that spatial ITH occurs in ccRCC, which may drive clinical heterogeneity and warrants further interrogation to improve patient outcomes.
    Keywords:  CP: Cancer; ccRCC; multiomic profiling; multiplex imaging; multiregional biopsies; single-cell proteogenomics; spatial heterogeneity; tumor heterogeneity; tumor microenvironment
  42. Nat Commun. 2022 Aug 15. 13(1): 4765
      Autosomal dominant polycystic kidney disease (ADPKD), among the most common human genetic conditions and a frequent etiology of kidney failure, is primarily caused by heterozygous PKD1 mutations. Kidney cyst formation occurs when PKD1 dosage falls below a critical threshold. However, no framework exists to harness the remaining allele or reverse PKD1 decline. Here, we show that mRNAs produced by the noninactivated PKD1 allele are repressed via their 3'-UTR miR-17 binding element. Eliminating this motif (Pkd1∆17) improves mRNA stability, raises Polycystin-1 levels, and alleviates cyst growth in cellular, ex vivo, and mouse PKD models. Remarkably, Pkd2 is also inhibited via its 3'-UTR miR-17 motif, and Pkd2∆17-induced Polycystin-2 derepression retards cyst growth in Pkd1-mutant models. Moreover, acutely blocking Pkd1/2 cis-inhibition, including after cyst onset, attenuates murine PKD. Finally, modeling PKD1∆17 or PKD2∆17 alleles in patient-derived primary ADPKD cultures leads to smaller cysts, reduced proliferation, lower pCreb1 expression, and improved mitochondrial membrane potential. Thus, evading 3'-UTR cis-interference and enhancing PKD1/2 mRNA translation is a potentially mutation-agnostic ADPKD-arresting approach.
  43. Metabol Open. 2022 Sep;15 100205
    Keywords:  Mitochondrial DNA; Mitochondrion; Nucleoid; Phase separation; Transcription
  44. Proc Natl Acad Sci U S A. 2022 Aug 23. 119(34): e2207392119
      Regulatory relationships between transcription factors (TFs) and their target genes lie at the heart of cellular identity and function; however, uncovering these relationships is often labor-intensive and requires perturbations. Here, we propose a principled framework to systematically infer gene regulation for all TFs simultaneously in cells at steady state by leveraging the intrinsic variation in the transcriptional abundance across single cells. Through modeling and simulations, we characterize how transcriptional bursts of a TF gene are propagated to its target genes, including the expected ranges of time delay and magnitude of maximum covariation. We distinguish these temporal trends from the time-invariant covariation arising from cell states, and we delineate the experimental and technical requirements for leveraging these small but meaningful cofluctuations in the presence of measurement noise. While current technology does not yet allow adequate power for definitively detecting regulatory relationships for all TFs simultaneously in cells at steady state, we investigate a small-scale dataset to inform future experimental design. This study supports the potential value of mapping regulatory connections through stochastic variation, and it motivates further technological development to achieve its full potential.
    Keywords:  gene regulation; single-cell transcriptomics; transcriptional bursting