bims-pideca 2022-08-21 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2022–08–21
twenty-two papers selected by
Ralitsa Radostinova Madsen, University College London

Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.
Case Report of Fibro-Adipose Vascular Anomaly (FAVA) with Activating Somatic PIK3CA Mutation.
PI3K inhibitors in trastuzumab-resistant HER2-positive breast cancer cells with PI3K pathway alterations.
TBK1-mTOR Signaling Attenuates Obesity-Linked Hyperglycemia and Insulin Resistance.
PH domain-mediated autoinhibition and oncogenic activation of Akt.
ALPELISIB - INDUCED HYPERGLYCEMIA.
Early diagnosis enabling precision medicine treatment in a young boy with PIK3R1-related overgrowth.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy.
AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.
Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly.
S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.
PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors.
A lymphatic-absorbed multi-targeted kinase inhibitor for myelofibrosis therapy.
Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.
Saturation of the mitochondrial NADH shuttles drives aerobic glycolysis in proliferating cells.
Treg-driven tumour control by PI3Kδ inhibition limits myeloid-derived suppressor cell expansion.
The efficacy of receptor tyrosine kinase EphA2 autophosphorylation increases with EphA2 oligomer size.
Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector.
Developing Bottom-Up Induced Pluripotent Stem Cell Derived Solid Tumor Models Using Precision Genome Editing Technologies.
A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements.
Insulin increases placental triglyceride as a potential mechanism for fetal adiposity in maternal obesity.
Fluorescence Imaging of 3D Cell Models with Subcellular Resolution.

J Biol Chem. 2022 Aug 13. pii: S0021-9258(22)00822-5. [Epub ahead of print] 102379

Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.

Amelia K Luciano, Ekaterina Korobkina, Scott P Lyons, John A Haley, Shelagh Fluharty, Su Myung Jung, Arminja N Kettenbach, David A Guertin.

  Mechanistic Target of Rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2 interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain- and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.

Keywords:  ADP ribosylation factor (ARF); Akt/PKB; BioID2; RICTOR; cell signaling; growth factor; mTORC2; mammalian target of rapamycin (mTOR); proximity labeling

DOI:  https://doi.org/10.1016/j.jbc.2022.102379
Case Rep Genet. 2022 ;2022 9016497

Case Report of Fibro-Adipose Vascular Anomaly (FAVA) with Activating Somatic PIK3CA Mutation.

Jordan H Driskill, Helena Hwang, Alexandra K Callan, Dwight Oliver.

Fibro-adipose vascular anomaly (FAVA) is a recently described complex and painful benign lesion found in young adults and the pediatric population composed of intramuscular vascular, fibrous, and adipose tissues. A previous report has identified the presence of somatic mosaic mutations in the gene for the catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA) in cases of FAVA. Herein, we present a case of FAVA found in a 23-year-old male patient who presented with chronic wrist pain associated with a mass, and we identified an associated somatic activating mutation (H1047R) in PIK3CA. We briefly review the relevant literature surrounding the identification and histology of FAVA, the known mutational spectrum, downstream signaling pathways, and relevant treatment modalities. Our case highlights the association between FAVA and somatic mosaic activating PIK3CA mutations.

DOI: https://doi.org/10.1155/2022/9016497
Am J Cancer Res. 2022 ;12(7): 3067-3082

PI3K inhibitors in trastuzumab-resistant HER2-positive breast cancer cells with PI3K pathway alterations.

Wei-Pang Chung, Wei-Lun Huang, Chun-Hui Lee, Hui-Ping Hsu, Wan-Ling Huang, You-Yu Liu, Wu-Chou Su.

The activation of the PI3K signaling pathway resulting from genetic alterations induces carcinogenesis and resistance to anticancer therapies. Breast cancer is a major malignancy that is associated with dysregulation of the PI3K signaling pathway. PIK3CA mutations and PTEN loss occur in every subtype of breast cancer. PI3K inhibitors are being evaluated in breast cancer after the success of an alpha isoform-specific PI3K inhibitor in estrogen receptor (ER)-positive/HER2-negative metastatic breast cancer. Some preclinical data indicate the potential for PI3K/mTOR targeting in combination with trastuzumab for HER2-positive breast cancer with or without expression of the estrogen receptor. However, the role of this therapy in HER2-positive breast cancer with PIK3CA mutations and/or PTEN loss remains unclear. We examined three HER2-positive, ER-negative breast cancer cell lines to determine the efficacy of a novel alpha isoform-specific PI3K inhibitor in combination with trastuzumab. The results indicated that this combination was effective in PIK3CA-mutant or PTEN-deficient breast cancer cells by inducing apoptosis and inhibiting the expression of downstream proteins. PTEN loss by siRNA modulation in parental HER2-positive cancer cells with PI3K signaling pathway alterations could not confer resistance to alpelisib or GDC-0077 plus trastuzumab. We selected the CK-MB-1 cell line without alterations in the PI3K pathway to demonstrate that PI3K inhibitors plus trastuzumab represented a biomarker-specific treatment. In vivo effects of alpelisib plus trastuzumab were tested and confirmed in a mouse model, showing the combination strategy offered the best opportunity to achieve tumor volume reduction. With known safety profiles, this cytotoxic chemotherapy-free regimen warrants further attention as a biomarker-driven strategy for treating HER2-positive breast cancer.

Keywords: PI3K inhibitor; PIK3CA; PTEN; alpha-isoform; breast cancer; trastuzumab
Diabetes. 2022 Aug 19. pii: db220256. [Epub ahead of print]

TBK1-mTOR Signaling Attenuates Obesity-Linked Hyperglycemia and Insulin Resistance.

Cagri Bodur, Dubek Kazyken, Kezhen Huang, Aaron Seth Tooley, Kae Won Cho, Tammy M Barnes, Carey N Lumeng, Martin G Myers, Diane C Fingar.

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.

DOI: https://doi.org/10.2337/db22-0256
Elife. 2022 Aug 15. pii: e80148. [Epub ahead of print]11

PH domain-mediated autoinhibition and oncogenic activation of Akt.

Hwan Bae, Thibault Viennet, Eunyoung Park, Nam Chu, Antonieta Salguero, Michael J Eck, Haribabu Arthanari, Philip A Cole.

  Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.80148
Acta Endocrinol (Buchar). 2022 Jan-Mar;18(1):18(1): 115-117

ALPELISIB - INDUCED HYPERGLYCEMIA.

P S Ekanayake, J Gerwer, K Mccowen.

   Context: Phosphoinositide-3-kinase (PI3K) pathway inhibitors are increasingly used as targeted therapy in malignancies. We discuss here three cases of PI3K inhibitor induced hyperglycemia and discuss the mechanism of action of these medications and treatment of this class side effect.
Objectives: Alpelisib (Piqray) is the newest PI3K inhibitor used in conjunction with Fulvestrant to treat specific types of breast cancer. Since PI3K is a critical mediator of insulin signaling, hyperglycemia is an on-target, unfortunate side effect of this treatment. We present a case series of severe hyperglycemia induced by the alpelisib in three women without a history of diabetes.
Design: All three women in this study had hormone receptor (HR) positive, human epidermal growth factor receptor 2 (Her2) negative, PI3K mutated breast cancer. They were referred to our clinic by Oncology for alpelisib-induced hyperglycemia.
Subjects and Methods: Review of laboratory values and glucometer values were conducted during each visit allowing treatment decisions. Two of these women are actively managed by us for their diabetes. One woman recently died due to progression of malignancy.
Results: All three women presented with new onset of severe hyperglycemia after the initiation of PI3K inhibitor, alpelisib. At least one case noted maximal glucose elevation in the hours following drug ingestion. In another, cessation of Alpelisib reversed the hyperglycemia within the span of one week.
Conclusion: Hyperglycemia induced by PI3K inhibitors can be recalcitrant and might necessitate interruption of chemotherapy. Optimal glucose-lowering therapy remains unclear as exogenous insulin has the theoretical potential to overcome PI3K inhibition.

Keywords:  Alpelisib; Phosphoinositide-3-kinase; breast cancer; hyperglycemia

DOI:  https://doi.org/10.4183/aeb.2022.115
Eur J Med Genet. 2022 Aug 11. pii: S1769-7212(22)00171-9. [Epub ahead of print]65(10): 104590

Early diagnosis enabling precision medicine treatment in a young boy with PIK3R1-related overgrowth.

Bitten Schönewolf-Greulich, Helena Gásdal Karstensen, Tina D Hjortshøj, Finn Stener Jørgensen, Katja M Harder, Susanne Frevert, Hanne Hove, Birgitte R Diness.

  Mosaic PIK3R1 variants have recently been demonstrated in patients with complex vascular malformations and overgrowth in a syndrome resembling PIK3CA-related overgrowth syndrome (PROS). The PIK3CA-inhibitor, alpelisib, seems to be a promising treatment option for PROS patients. We describe a young boy with overgrowth and a pathogenic mosaic variant in PIK3R1; c.1699A > G, p.(Lys567Glu). He was prenatally suspected of a syndrome on the presence of unusual transient fluctuating subcutaneous edemas and lymphedema of his left shoulder. The pathogenic variant, later found to be causative, was below detection threshold in whole-genome sequencing (WGS) analysis of amniotic fluid. Upon delivery a mosaic pathogenic PIK3R1 variant, was identified by whole-exome sequencing (WES) of a skin biopsy. With no proven treatment options available, and based on the theoretical disease mechanism, alpelisib therapy was initiated at nine months of age. In the first year of treatment growth normalized and the affected vascular and lymphatic tissue regressed. No side effects have been observed. This report underlines the importance of early variant detection in children suspected of having severe mosaic overgrowth, and proves that prenatal diagnosis is possible, enabling prompt treatment. Furthermore, it demonstrates the promising effects of alpelisib in this patient group.

Keywords:  Alpelisib; Congenital vascular malformation; Mosaicism; PIK3R1; Personalized medicine; Segmental overgrowth

DOI:  https://doi.org/10.1016/j.ejmg.2022.104590
EMBO J. 2022 Aug 15. e110398

Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy.

Samuel J Rodgers, Emily I Jones, Senthil Arumugam, Sabryn A Hamila, Jill Danne, Rajendra Gurung, Matthew J Eramo, Randini Nanayakkara, Georg Ramm, Meagan J McGrath, Christina A Mitchell.

  Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co-ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient-rich conditions, is unknown. Here, we identify an endosome-dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα-derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation-induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5-kinase, PIKfyve, to generate PI(3,5)P2 . The SNX-BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve-dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient-rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P2 -dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.

Keywords:  INPP4B; PI3Kα; PIKfyve; autophagy; lysosome

DOI:  https://doi.org/10.15252/embj.2021110398
Nat Commun. 2022 Aug 17. 13(1): 4836

AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.

George K E Umanah, Leire Abalde-Atristain, Mohammed Repon Khan, Jaba Mitra, Mohamad Aasif Dar, Melissa Chang, Kavya Tangella, Amy McNamara, Samuel Bennett, Rong Chen, Vasudha Aggarwal, Marisol Cortes, Paul F Worley, Taekjip Ha, Ted M Dawson, Valina L Dawson.

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.

DOI: https://doi.org/10.1038/s41467-022-32365-2
Nat Commun. 2022 Aug 17. 13(1): 4848

Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly.

Edoardo Ratto, S Roy Chowdhury, Nora S Siefert, Martin Schneider, Marten Wittmann, Dominic Helm, Wilhelm Palm.

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.

DOI: https://doi.org/10.1038/s41467-022-32515-6
Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01011-7. [Epub ahead of print]40(7): 111194

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.

Yan Sun, Huan Zhang, Junpeng Meng, Feng Guo, Dianyun Ren, Heshui Wu, Xin Jin.

  Sorafenib is currently the first-line treatment for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a significant challenge. Aberrant AKT signaling activation is a crucial mechanism driving sorafenib resistance in HCC. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital role in antitumor immune responses. In this study, we demonstrate that aberrant PCSK9 upregulation promotes cell proliferation and sorafenib resistance in HCC by inducing AKT-S473 phosphorylation. After palmitoylation at cysteine 600, the binding affinity between PCSK9 and tensin homolog (PTEN) is dramatically increased, inducing lysosome-mediated PTEN degradation and subsequent AKT activation. We identify zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) as a palmitoyltransferase that promotes PCSK9 palmitoylation at cysteine 600. We also develop a biologically active PCSK9-derived peptide that competitively inhibits PCSK9 palmitoylation, suppressing AKT phosphorylation and augmenting the antitumor effects of sorafenib in HCC.

Keywords:  AKT; CP: Cancer; CP: Molecular biology; PCSK9; PTEN; ZDHHC16; liver cancer; palmitoylation; sorafenib resistance

DOI:  https://doi.org/10.1016/j.celrep.2022.111194
Elife. 2022 Aug 17. pii: e67954. [Epub ahead of print]11

PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors.

Divya Khattar, Sharlene Fernandes, John Snowball, Minzhe Guo, Matthew C Gillen, Suchi Singh Jain, Debora Sinner, William Zacharias, Daniel T Swarr.

  The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+ progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+ progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development in Mus musculus. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in mouse resulted in an expansion of the SOX9+ EPC population and impaired airway epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA and ATAC seq in defining regulatory networks in development.

Keywords:  developmental biology; genetics; genomics; mouse

DOI:  https://doi.org/10.7554/eLife.67954
Nat Commun. 2022 Aug 17. 13(1): 4730

A lymphatic-absorbed multi-targeted kinase inhibitor for myelofibrosis therapy.

Brian D Ross, Youngsoon Jang, Amanda Welton, Christopher A Bonham, Dilrukshika S W Palagama, Kevin Heist, Jagadish Boppisetti, Kasun P Imaduwage, Tanner Robison, Leah R King, Edward Z Zhang, Cyrus Amirfazli, Kathryn E Luker, Winston Y Lee, Gary D Luker, Thomas L Chenevert, Marcian E Van Dort.

Activation of compensatory signaling nodes in cancer often requires combination therapies that are frequently plagued by dose-limiting toxicities. Intestinal lymphatic drug absorption is seldom explored, although reduced toxicity and sustained drug levels would be anticipated to improve systemic bioavailability. A potent orally bioavailable multi-functional kinase inhibitor (LP-182) is described with intrinsic lymphatic partitioning for the combined targeting of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways without observable toxicity. We demonstrate selectivity and therapeutic efficacy through reduction of downstream kinase activation, amelioration of disease phenotypes, and improved survival in animal models of myelofibrosis. Our further characterization of synthetic and physiochemical properties for small molecule lymphatic uptake will support continued advancements in lymphatropic therapy for altering disease trajectories of a myriad of human disease indications.

DOI: https://doi.org/10.1038/s41467-022-32486-8
Elife. 2022 Aug 17. pii: e73747. [Epub ahead of print]11

Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.

Scott D Hansen, Albert A Lee, Benjamin R Duewell, Jay T Groves.

  The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2 containing membranes and dimerize in a protein density dependent manner. Although dispensable for cooperative PI(4,5)P2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane bound kinase.

Keywords:  biochemistry; chemical biology; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.73747
Mol Cell. 2022 Aug 12. pii: S1097-2765(22)00703-1. [Epub ahead of print]

Saturation of the mitochondrial NADH shuttles drives aerobic glycolysis in proliferating cells.

Yahui Wang, Ethan Stancliffe, Ronald Fowle-Grider, Rencheng Wang, Cheng Wang, Michaela Schwaiger-Haber, Leah P Shriver, Gary J Patti.

  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

DOI:  https://doi.org/10.1016/j.molcel.2022.07.007
Br J Cancer. 2022 Aug 19.

Treg-driven tumour control by PI3Kδ inhibition limits myeloid-derived suppressor cell expansion.

Sarah N Lauder, Kathryn Smart, Valentina M T Bart, Ana Pires, Jake Scott, Stefan Milutinovic, Andrew Godkin, Bart Vanhaesebroeck, Awen Gallimore.

BACKGROUND: Recent studies have demonstrated that blocking the PI3Kδ signalling enzyme (by administering a small molecule inhibitor, PI-3065) can potently improve the anti-tumour T-cell response through direct inhibition of Tregs. This treatment also has a negative impact on MDSC numbers but the primary mechanism driving this effect has remained unclear.
METHODS: The 4T1 breast cancer mouse model was used in combination with PI-3065 to gain insights into the effect of PI3Kδ inhibition on MDSCs.
RESULTS: PI-3065 treatment resulted in a concomitant reduction in MDSC expansion and tumour size. However, targeting Tregs independent of PI-3065 was also associated with reduced tumour volume and MDSC numbers. Surgical removal of tumours resulted in a rapid and significant decline in MDSC numbers, whilst ex vivo studies using cells from PI-3065-treated mice demonstrated no direct effect of the inhibitor on MDSC activity.
CONCLUSIONS: Our data suggest that MDSCs are not inhibited directly by PI-3065 treatment but that their reduced recruitment and immunosuppression within the tumour microenvironment is an indirect consequence of PI3Kδ-inhibition-driven tumour control. This indicates that PI3Kδ inhibition drives tumour immunity by breaking down multiple immunosuppressive pathways through both direct mechanisms (on Treg) and indirect mechanisms, secondary to tumour control (on MDSCs).

DOI: https://doi.org/10.1038/s41416-022-01917-0
J Biol Chem. 2022 Aug 12. pii: S0021-9258(22)00813-4. [Epub ahead of print] 102370

The efficacy of receptor tyrosine kinase EphA2 autophosphorylation increases with EphA2 oligomer size.

Elmer Zapata-Mercado, Gabriel Biener, Daniel M McKenzie, William C Wimley, Elena B Pasquale, Valerica Raicu, Kalina Hristova.

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms higher order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used Fluorescence Intensity Fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

DOI: https://doi.org/10.1016/j.jbc.2022.102370
Cell Death Discov. 2022 Aug 13. 8(1): 357

Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector.

Ruizhi Li, Kristin F Wilson, Richard A Cerione.

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.

DOI: https://doi.org/10.1038/s41420-022-01156-5
CRISPR J. 2022 Aug;5(4): 517-535

Developing Bottom-Up Induced Pluripotent Stem Cell Derived Solid Tumor Models Using Precision Genome Editing Technologies.

Kelsie L Becklin, Garrett M Draper, Rebecca A Madden, Mitchell G Kluesner, Tomoyuki Koga, Miller Huang, William A Weiss, Logan G Spector, David A Largaespada, Branden S Moriarity, Beau R Webber.

Advances in genome and tissue engineering have spurred significant progress and opportunity for innovation in cancer modeling. Human induced pluripotent stem cells (iPSCs) are an established and powerful tool to study cellular processes in the context of disease-specific genetic backgrounds; however, their application to cancer has been limited by the resistance of many transformed cells to undergo successful reprogramming. Here, we review the status of human iPSC modeling of solid tumors in the context of genetic engineering, including how base and prime editing can be incorporated into "bottom-up" cancer modeling, a term we coined for iPSC-based cancer models using genetic engineering to induce transformation. This approach circumvents the need to reprogram cancer cells while allowing for dissection of the genetic mechanisms underlying transformation, progression, and metastasis with a high degree of precision and control. We also discuss the strengths and limitations of respective engineering approaches and outline experimental considerations for establishing future models.

DOI: https://doi.org/10.1089/crispr.2022.0032
Nat Metab. 2022 Aug 15.

A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements.

Ajit S Divakaruni, Martin Jastroch.

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.

DOI: https://doi.org/10.1038/s42255-022-00619-4
Mol Metab. 2022 Aug 12. pii: S2212-8778(22)00143-0. [Epub ahead of print] 101574

Insulin increases placental triglyceride as a potential mechanism for fetal adiposity in maternal obesity.

Anika K Anam, Katherine M Cooke, Milana Bochkur Dratver, Jane V O'Bryan, Lauren E Perley, Seth M Guller, Janice J Hwang, Hugh S Taylor, Leigh Goedeke, Harvey J Kliman, Daniel F Vatner, Clare A Flannery.

   OBJECTIVE: Maternal obesity increases the incidence of excess adiposity in newborns, resulting in lifelong diabetes risk. Elevated intrauterine fetal adiposity has been attributed to maternal hyperglycemia; however, this hypothesis does not account for the increased adiposity seen in newborns of mothers with obesity who have euglycemia. We aimed to explore the placental response to maternal hyperinsulinemia and the effect of insulin-like growth factor 2 (IGF-2) in promoting fetal adiposity by increasing storage and availability of nutrients to the fetus.
METHODS: We used placental villous explants and isolated trophoblasts from normal weight and obese women to assess the effect of insulin and IGF-2 on triglyceride content and insulin receptor signaling. Stable isotope tracer methods were used ex vivo to determine effect of hormone treatment on de novo lipogenesis (DNL), fatty acid uptake, fatty acid oxidation, and esterification in the placenta.
RESULTS: Here we show that placentae from euglycemic women with normal weight and obesity both have abundant insulin receptor. Placental depth and triglyceride were greater in women with obesity compared with normal weight women. In syncytialized placental trophoblasts and villous explants, insulin and IGF-2 activate insulin receptor, induce expression of lipogenic transcription factor SREBP-1 (sterol regulatory element-binding protein 1), and stimulate triglyceride accumulation. We demonstrate elevated triglyceride is attributable to increased esterification of fatty acids, without contribution from DNL and without an acceleration of fatty acid uptake.
CONCLUSIONS: Our work reveals that obesity-driven aberrations in maternal metabolism, such as hyperinsulinemia, alter placental metabolism in euglycemic conditions, and may explain the higher prevalence of excess adiposity in the newborns of obese women.

Keywords:  esterification; fatty acids; insulin; macrosomia; obesity

DOI:  https://doi.org/10.1016/j.molmet.2022.101574
Bio Protoc. 2022 Jul 20. pii: e4469. [Epub ahead of print]12(14):

Fluorescence Imaging of 3D Cell Models with Subcellular Resolution.

Indra Van Zundert, Nina Maenhoudt, Silke De Vriendt, Hugo Vankelecom, Beatrice Fortuni, Susana Rocha.

  Over the past years, research has made impressive breakthroughs towards the development and implementation of 3D cell models for a wide range of applications, such as drug development and testing, organogenesis, cancer biology, and personalized medicine. Opposed to 2D cell monolayer culture systems, advanced 3D cell models better represent the in vivo physiology. However, for these models to deliver scientific insights, appropriate investigation techniques are required. Despite the potential of fluorescence microscopy to visualize these models with high spatial resolution, sample preparation and imaging assays are not straightforward. Here, we provide different protocols of sample preparation for fluorescence imaging, for both matrix-embedded and matrix-free models ( e.g ., organoids and spheroids, respectively). Additionally, we provide detailed guidelines for imaging 3D cell models via confocal multi-photon fluorescence microscopy. We show that using these protocols, images of 3D cell culture systems can be obtained with sub-cellular resolution. Graphical abstract.

Keywords:   3D cell culture systems ; Fluorescence Microscopy ; Multi-Photon Fluorescence Microscopy ; Organoids ; Spheroids

DOI:  https://doi.org/10.21769/BioProtoc.4469