bims-pideca 2020-08-02 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2020–08–02
twenty-two papers selected by
Ralitsa Radostinova Madsen, University College London

Insulin signalling requires glucose to promote lipid anabolism in adipocytes.
PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice rescues molecular defects and reduces epilepsy-associated phenotypes.
The role of PI3K/Akt/mTOR signaling in dose-dependent biphasic effects of glycine on vascular development.
MTOR-initiated metabolic switch and degeneration in the retinal pigment epithelium.
Oncogenic Smurf1 promotes PTEN wild-type glioblastoma growth by mediating PTEN ubiquitylation.
Dihydroxyacetone phosphate signals glucose availability to mTORC1.
MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B.
TGFb acts through PDGFRb to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.
Ovariectomy uncouples lifespan from metabolic health and reveals a sex-hormone-dependent role of hepatic mTORC2 in aging.
PTEN suppresses epithelial-mesenchymal transition and cancer stem cell activity by downregulating Abi1.
Comprehensive characteristics of somatic mutations in the normal tissues of patients with cancer and existence of somatic mutant clones linked to cancer development.
Systems-Level Properties of EGFR-RAS-ERK Signaling Amplify Local Signals to Generate Dynamic Gene Expression Heterogeneity.
Glutamine deprivation alters the origin and function of cancer cell exosomes.
The rapamycin analog Everolimus reversibly impairs male germ cell differentiation and fertility in the mouse1.
Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters.
An integrative ENCODE resource for cancer genomics.
The Possibilities of Immunotherapy for Children with Primary Immunodeficiencies Associated with Cancers.
Mitochondrial spongiotic brain disease: astrocytic stress and harmful rapamycin and ketosis effect.
A spoonful of DHAP keeps mTORC1 running on sugars.
Mapping Gene Circuits Essential for Germ Layer Differentiation via Loss-of-Function Screens in Haploid Human Embryonic Stem Cells.
Molecular engineering of safe and efficacious oral basal insulin.
In vitro characterization of the effects of chronic insulin stimulation in mouse 3T3-L1 and human SGBS adipocytes.

J Biol Chem. 2020 Jul 28. pii: jbc.RA120.014907. [Epub ahead of print]

Insulin signalling requires glucose to promote lipid anabolism in adipocytes.

James R Krycer, Lake-Ee Quek, Deanne Francis, Armella Zadoorian, Fiona C Weiss, Kristen C Cooke, Marin E Nelson, Alexis Diaz-Vegas, Sean J Humphrey, Richard Scalzo, Akiyoshi Hirayama, Satsuki Ikeda, Futaba Shoji, Kumi Suzuki, Kevin Huynh, Corey Giles, Bianca Varney, Shilpa R Nagarajan, Andrew J Hoy, Tomoyoshi Soga, Peter J Meikle, Gregory J Cooney, Daniel J Fazakerley, David E James.

  Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilisation based on nutritional status. This is coordinated by insulin, which triggers kinase signalling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3'-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signalling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilises protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

Keywords:  Drosophila; adipocyte; cell metabolism; fat tissue; fatty acid; glucose; insulin; kinase signaling; lipid; metabolic regulation

DOI:  https://doi.org/10.1074/jbc.RA120.014907
Neurobiol Dis. 2020 Jul 24. pii: S0969-9961(20)30301-6. [Epub ahead of print]144 105026

PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice rescues molecular defects and reduces epilepsy-associated phenotypes.

Angela R White, Durgesh Tiwari, Molly C MacLeod, Steve C Danzer, Christina Gross.

  Epilepsy affects all ages, races, genders, and socioeconomic groups. In about one third of patients, epilepsy is uncontrolled with current medications, leaving a vast need for improved therapies. The causes of epilepsy are diverse and not always known but one gene mutated in a small subpopulation of patients is phosphatase and tensin homolog (PTEN). Moreover, focal cortical dysplasia, which constitutes a large fraction of refractory epilepsies, has been associated with signaling defects downstream of PTEN. So far, most preclinical attempts to reverse PTEN deficiency-associated neurological deficits have focused on mTOR, a signaling hub several steps downstream of PTEN. Phosphoinositide 3-kinases (PI3Ks), by contrast, are the direct enzymatic counteractors of PTEN, and thus may be alternative treatment targets. PI3K activity is mediated by four different PI3K catalytic isoforms. Studies in cancer, where PTEN is commonly mutated, have demonstrated that inhibition of only one isoform, p110β, reduces progression of PTEN-deficient tumors. Importantly, inhibition of a single PI3K isoform leaves critical functions of general PI3K signaling throughout the body intact. Here, we show that this disease mechanism-targeted strategy borrowed from cancer research rescues or ameliorates neuronal phenotypes in male and female mice with neuron-specific PTEN deficiency. These phenotypes include cell signaling defects, protein synthesis aberrations, seizures, and cortical dysplasia. Of note, p110β is also dysregulated and a promising treatment target in the intellectual disability Fragile X syndrome, pointing towards a shared biological mechanism that is therapeutically targetable in neurodevelopmental disorders of different etiologies. Overall, this work advocates for further assessment of p110β inhibition not only in PTEN deficiency-associated neurodevelopmental diseases but also other brain disorders characterized by defects in the PI3K/mTOR pathway.

Keywords:  Epilepsy; Mouse model; Neurodevelopmental disorder; PI3K; PTEN deficiency; Protein synthesis; Seizure; Signal transduction; p110β

DOI:  https://doi.org/10.1016/j.nbd.2020.105026
Biochem Biophys Res Commun. 2020 Aug 27. pii: S0006-291X(20)31303-6. [Epub ahead of print]529(3): 596-602

The role of PI3K/Akt/mTOR signaling in dose-dependent biphasic effects of glycine on vascular development.

Kiyomi Tsuji-Tamura, Mari Sato, Misato Fujita, Masato Tamura.

  Glycine, a non-essential amino acid, exerts concentration-dependent biphasic effects on angiogenesis. Low-doses of glycine promote angiogenesis, whereas high-doses cause anti-angiogenesis. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling participates in angiogenesis of both physiological development, and pathological events including tumor and inflammation. We assessed the role of PI3K/Akt/mTOR signaling in vascular development, and the interaction with glycine, using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos expressing fluorescent proteins in vascular endothelial cells. Treatment with inhibitors of mTORC1 (rapamycin and everolimus), mTORC1/mTORC2 (KU0063794), PI3K (LY29400), and Akt (Akt inhibitor) decreased the development of intersegmental vessels (ISVs). These inhibitors cancelled the angiogenic effects of a low-dose of glycine, while acted synergistically with a high-dose of glycine in anti-angiogenesis. mTOR signaling regulates the gene expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and nitric oxide (NO) synthase (NOS), an enzyme for the synthesis of an angiogenic mediator NO. Expressions of VEGF and NOS were consistent with the vascular features induced by glycine and an mTOR inhibitor. Our results suggest that PI3K/Akt/mTOR signaling may interact with dose-dependent biphasic effects of exogenous glycine on in vivo angiogenesis. mTOR signaling is a key target for cancer therapy, thus, the combining mTOR inhibitors with glycine may be a potential approach for controlling angiogenesis.

Keywords:  Akt; Angiogenesis; Glycine; PI3K; Zebrafish; mTOR

DOI:  https://doi.org/10.1016/j.bbrc.2020.06.085
FASEB J. 2020 Jul 28.

MTOR-initiated metabolic switch and degeneration in the retinal pigment epithelium.

Young-Mi Go, Jing Zhang, Jolyn Fernandes, Christopher Litwin, Rui Chen, Theodore G Wensel, Dean P Jones, Jiyang Cai, Yan Chen.

  The retinal pigment epithelium (RPE) is a particularly vulnerable tissue to age-dependent degeneration. Over the life span, the RPE develops an expanded endo-lysosomal compartment to maintain the high efficiency of phagocytosis and degradation of photoreceptor outer segments (POS) necessary for photoreceptor survival. As the assembly and activation of the mechanistic target of rapamycin complex 1 (mTORC1) occur on the lysosome surface, increased lysosome mass with aging leads to higher mTORC1 activity. The functional consequences of hyperactive mTORC1 in the RPE are unclear. In the current study, we used integrated high-resolution metabolomic and genomic approaches to examine mice with RPE-specific deletion of the tuberous sclerosis 1 (Tsc1) gene which encodes an upstream suppressor of mTORC1. Our data show that RPE cells with constitutively high mTORC1 activity were reprogramed to be hyperactive in glucose and lipid metabolism. Lipolysis was suppressed, mitochondrial carnitine shuttle was inhibited, while genes involved in fatty acid (FA) biosynthesis were upregulated. The metabolic changes occurred prior to structural changes of RPE and retinal degeneration. These findings have revealed cellular events and intrinsic mechanisms that contribute to lipid accumulation in the RPE cells during aging and age-related degeneration.

Keywords:  AMD; Mtor; aging; lipid; metabolism

DOI:  https://doi.org/10.1096/fj.202000612R
Oncogene. 2020 Jul 31.

Oncogenic Smurf1 promotes PTEN wild-type glioblastoma growth by mediating PTEN ubiquitylation.

Qin Xia, Hanwen Zhang, Pei Zhang, Yang Li, Mengchuan Xu, Xiaobo Li, Xuejun Li, Lei Dong.

PI3K/Akt/mTOR signaling pathway activity is highly elevated in glioblastoma (GBM). Although rapamycin is known to inhibit this pathway, GBM patients are resistant to rapamycin monotherapy. This may be related to mutations of tumor suppressor phosphatase and tensin homolog (PTEN). Here, we show that higher expression of E3 ligase Smad ubiquitylation regulatory factor 1 (Smurf1) in GBM is correlated with poor prognosis. Smurf1 promotes cell growth and colony formation by accelerating cell cycle and aberrant signaling pathways. In addition, we show that Smurf1 ubiquitylates and degrades PTEN. We further demonstrate that the oncogenic role of Smurf1 is dependent on PTEN. Upregulated Smurf1 impairs PTEN activity, leading to consistent activation of PI3K/Akt/mTOR signaling pathway; and depletion of Smurf1 dramatically inhibits cell proliferation and tumor growth. Moreover, loss of Smurf1 abolishes the aberrant regulation of PTEN, causing negative feedback on PI3K/Akt/mTOR signaling pathway, and thus leading to rescue of tumor sensitivity to rapamycin in an orthotopic GBM model. Taken together, we show that Smurf1 promotes tumor progression via PTEN, and combined treatment of Smurf1 knockdown with mammalian target of rapamycin (mTOR) inhibition reduces tumor progression. These results identify a unique role of Smurf1 in mTOR inhibitor resistance and provide a strong rationale for combined therapy targeting GBM.

DOI: https://doi.org/10.1038/s41388-020-01400-1
Nat Metab. 2020 Jul 27.

Dihydroxyacetone phosphate signals glucose availability to mTORC1.

Jose M Orozco, Patrycja A Krawczyk, Sonia M Scaria, Andrew L Cangelosi, Sze Ham Chan, Tenzin Kunchok, Caroline A Lewis, David M Sabatini.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.

DOI: https://doi.org/10.1038/s42255-020-0250-5
EMBO J. 2020 Jul 27. e105696

MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B.

Laura Cinque, Chiara De Leonibus, Maria Iavazzo, Natalie Krahmer, Daniela Intartaglia, Francesco Giuseppe Salierno, Rossella De Cegli, Chiara Di Malta, Maria Svelto, Carmela Lanzara, Marianna Maddaluno, Luca Giorgio Wanderlingh, Antje K Huebner, Marcella Cesana, Florian Bonn, Elena Polishchuk, Christian A Hübner, Ivan Conte, Ivan Dikic, Matthias Mann, Andrea Ballabio, Francesca Sacco, Paolo Grumati, Carmine Settembre.

  Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.

Keywords:   TFEB ; ER-phagy; FGF signaling; Fam134B; IRS1/PI3K signaling

DOI:  https://doi.org/10.15252/embj.2020105696
J Biol Chem. 2020 Jul 30. pii: jbc.RA120.014994. [Epub ahead of print]

TGFb acts through PDGFRb to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.

Soumya Maity, Falguni Das, Balakuntalam S Kasinath, Nandini Ghosh-Choudhury, Goutam Ghosh Choudhury.

  Interaction of TGFβ-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of TGFβ receptor involved in the noncanonical signaling is unknown. Here, we show that TGFβ increased the catalytic loop phosphorylation of platelet-derived growth factor receptor beta (PDGFRβ), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFβ increased phosphorylation of the PI 3 kinase-interacting Tyr-751 residue of PDGFRβ, thus activating Akt. Inhibition of PDGFRβ using a pharmacological inhibitor and siRNAs blocked TGFβ-stimulated phosphorylation of PRAS40, an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active Myr-Akt reversed the siPDGFRβ-mediated inhibition of mTORC1 activity; however, co-expression of phospho-deficient mutant of PRAS40 inhibited the effect of Myr-Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRβ in mesangial cells. Additionally, we demonstrate that PDGFRβ-initiated phosphorylation of PRAS40 is required for TGFβ-induced mesangial cell hypertrophy and, fibronectin and collagen I (α2) production. Increased activating phosphorylation of PDGFRβ is also associated with enhanced TGFβ expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rat, respectively. Thus, pursuing the TGFβ noncanonical signaling, we identified how TGFβ receptor I achieves mTORC1 activation through PDGFRβ-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRβ in TGFβ-driven renal fibrosis.

Keywords:  Akt PKB; PDGFRb; diabetic nephropathy; kidney; mTOR complex (mTORC); transforming growth factor beta (TGF-B)

DOI:  https://doi.org/10.1074/jbc.RA120.014994
Elife. 2020 Jul 28. pii: e56177. [Epub ahead of print]9

Ovariectomy uncouples lifespan from metabolic health and reveals a sex-hormone-dependent role of hepatic mTORC2 in aging.

Sebastian I Arriola Apelo, Amy Lin, Jacqueline A Brinkman, Emma Meyer, Mark Morrison, Jay L Tomasiewicz, Cassidy P Pumper, Emma L Baar, Nicole E Richardson, Mohammed Alotaibi, Dudley W Lamming.

  Inhibition of mTOR (mechanistic Target Of Rapamycin) signaling by rapamycin promotes healthspan and longevity more strongly in females than males, perhaps because inhibition of hepatic mTORC2 (mTOR Complex 2) specifically reduces the lifespan of males. Here, we demonstrate using gonadectomy that the sex-specific impact of reduced hepatic mTORC2 is not reversed by depletion of sex hormones. Intriguingly, we find that ovariectomy uncouples lifespan from metabolic health, with ovariectomized females having improved survival despite paradoxically having increased adiposity and decreased control of blood glucose levels. Further, ovariectomy unexpectedly promotes midlife survival of female mice lacking hepatic mTORC2, significantly increasing the survival of those mice that do not develop cancer. In addition to identifying a sex hormone-dependent role for hepatic mTORC2 in female longevity, our results demonstrate that metabolic health is not inextricably linked to lifespan in mammals, and highlight the importance of evaluating healthspan in mammalian longevity studies.

Keywords:  aging; genetics; genomics; healthspan; human biology; mTOR; mTORC2; medicine; mouse; ovariectomy; sex

DOI:  https://doi.org/10.7554/eLife.56177
Sci Rep. 2020 Jul 29. 10(1): 12685

PTEN suppresses epithelial-mesenchymal transition and cancer stem cell activity by downregulating Abi1.

Yanmei Qi, Jie Liu, Joshua Chao, Mark P Scheuerman, Saum A Rahimi, Leonard Y Lee, Shaohua Li.

The epithelial-mesenchymal transition (EMT) is an embryonic program frequently reactivated during cancer progression and is implicated in cancer invasion and metastasis. Cancer cells can also acquire stem cell properties to self-renew and give rise to new tumors through the EMT. Inactivation of the tumor suppressor PTEN has been shown to induce the EMT, but the underlying molecular mechanisms are less understood. In this study, we reconstituted PTEN-deficient breast cancer cells with wild-type and mutant PTEN, demonstrating that restoration of PTEN expression converted cancer cells with mesenchymal traits to an epithelial phenotype and inhibited cancer stem cell (CSC) activity. The protein rather than the lipid phosphatase activity of PTEN accounts for the reversal of the EMT. PTEN dephosphorylates and downregulates Abi1 in breast cancer cells. Gain- and loss-of-function analysis indicates that upregulation of Abi1 mediates PTEN loss-induced EMT and CSC activity. These results suggest that PTEN may suppress breast cancer invasion and metastasis via dephosphorylating and downregulating Abi1.

DOI: https://doi.org/10.1038/s41598-020-69698-1
J Med Genet. 2020 Jul 27. pii: jmedgenet-2020-106905. [Epub ahead of print]

Comprehensive characteristics of somatic mutations in the normal tissues of patients with cancer and existence of somatic mutant clones linked to cancer development.

Ji-Hye Oh, Chang Ohk Sung.

   BACKGROUND: Somatic mutations are a major driver of cancer development and many have now been identified in various cancer types, but the comprehensive somatic mutation status of the normal tissues matched to tumours has not been revealed.
METHOD: We analysed the somatic mutations of whole exome sequencing data in 392 patient tumour and normal tissue pairs based on the corresponding blood samples across 10 tumour types.
RESULTS: Many of the mutations involved in oncogenic pathways such as PI3K, NOTCH and TP53, were identified in the normal tissues. The ageing-related mutational signature was the most prominent contributing signature found and the mutations in the normal tissues were frequently in genes involved in late replication time (p<0.0001). Variants were rarely overlapping across tissue types but shared variants between normal and matched tumour tissue were present. These shared variants were frequently pathogenic when compared with non-shared variants (p=0.001) and showed a higher variant-allele-fraction (p<0.0001). Normal tissue-specific mutated genes were frequently non-cancer-associated (p=0.009). PIK3CA mutations were identified in 6 normal tissues and were harboured by all of the matched cancer tissues. Multiple types of PIK3CA mutations were found in normal breast and matched cancer tissues. The PIK3CA mutations exclusively present in normal tissue may indicate clonal expansions unrelated to the tumour. In addition, PIK3CA mutation was appeared that they arose before the occurrence of the allelic imbalance.
CONCLUSION: Our current results suggest that somatic mutant clones exist in normal tissues and that their clonal expansion could be linked to cancer development.

Keywords:  genetic research; mutation

DOI:  https://doi.org/10.1136/jmedgenet-2020-106905
Cell Syst. 2020 Jul 22. pii: S2405-4712(20)30247-7. [Epub ahead of print]

Systems-Level Properties of EGFR-RAS-ERK Signaling Amplify Local Signals to Generate Dynamic Gene Expression Heterogeneity.

Alexander E Davies, Michael Pargett, Stefan Siebert, Taryn E Gillies, Yongin Choi, Savannah J Tobin, Abhineet R Ram, Vaibhav Murthy, Celina Juliano, Gerald Quon, Mina J Bissell, John G Albeck.

  Intratumoral heterogeneity is associated with aggressive tumor behavior, therapy resistance, and poor patient outcomes. Such heterogeneity is thought to be dynamic, shifting over periods of minutes to hours in response to signaling inputs from the tumor microenvironment. However, models of this process have been inferred from indirect or post-hoc measurements of cell state, leaving the temporal details of signaling-driven heterogeneity undefined. Here, we developed a live-cell model system in which microenvironment-driven signaling dynamics can be directly observed and linked to variation in gene expression. Our analysis reveals that paracrine signaling between two cell types is sufficient to drive continual diversification of gene expression programs. This diversification emerges from systems-level properties of the EGFR-RAS-ERK signaling cascade, including intracellular amplification of amphiregulin-mediated paracrine signals and differential kinetic filtering by target genes including Fra-1, c-Myc, and Egr1. Our data enable more precise modeling of paracrine-driven transcriptional variation as a generator of gene expression heterogeneity. A record of this paper's transparent peer review process is included in the Supplemental Information.

Keywords:  EGFR; ERK; MAPK; RAS; basal-like breast cancer; computational modeling; microenvironment; plasticity; scRNA-seq; systems biology

DOI:  https://doi.org/10.1016/j.cels.2020.07.004
EMBO J. 2020 Jul 28. e103009

Glutamine deprivation alters the origin and function of cancer cell exosomes.

Shih-Jung Fan, Benjamin Kroeger, Pauline P Marie, Esther M Bridges, John D Mason, Kristie McCormick, Christos E Zois, Helen Sheldon, Nasullah Khalid Alham, Errin Johnson, Matthew Ellis, Maria Irina Stefana, Cláudia C Mendes, Stephen Mark Wainwright, Christopher Cunningham, Freddie C Hamdy, John F Morris, Adrian L Harris, Clive Wilson, Deborah Ci Goberdhan.

  Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of Rab11-positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.

Keywords:  Rab11(a); exosome; extracellular vesicle; mechanistic Target of Rapamycin; multivesicular body

DOI:  https://doi.org/10.15252/embj.2019103009
Biol Reprod. 2020 Jul 27. pii: ioaa130. [Epub ahead of print]

The rapamycin analog Everolimus reversibly impairs male germ cell differentiation and fertility in the mouse1.

Oleksandr Kirsanov, Randall H Renegar, Jonathan T Busada, Nicholas D Serra, Ellen V Harrington, Taylor A Johnson, Christopher B Geyer.

  Sirolimus, also known as rapamycin, and its closely-related rapamycin analog (rapalog) Everolimus inhibit 'mammalian target of rapamycin complex 1' (mTORC1), whose activity is required for spermatogenesis. Everolimus is FDA-approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P > 60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus approximately 6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration- and dose-dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring.

Keywords:  KIT; ZBTB16; mTOR; spermatogenesis; testis

DOI:  https://doi.org/10.1093/biolre/ioaa130
Nat Biotechnol. 2020 Jul 27.

Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters.

Christian Umkehrer, Felix Holstein, Laura Formenti, Julian Jude, Kimon Froussios, Tobias Neumann, Shona M Cronin, Lisa Haas, Jesse J Lipp, Thomas R Burkard, Michaela Fellner, Thomas Wiesner, Johannes Zuber, Anna C Obenauf.

We developed a functional lineage tracing tool termed CaTCH (CRISPRa tracing of clones in heterogeneous cell populations). CaTCH combines precise clonal tracing of millions of cells with the ability to retrospectively isolate founding clones alive before and during selection, allowing functional experiments. Using CaTCH, we captured rare clones representing as little as 0.001% of a population and investigated the emergence of resistance to targeted melanoma therapy in vivo.

DOI: https://doi.org/10.1038/s41587-020-0614-0
Nat Commun. 2020 Jul 29. 11(1): 3696

An integrative ENCODE resource for cancer genomics.

Jing Zhang, Donghoon Lee, Vineet Dhiman, Peng Jiang, Jie Xu, Patrick McGillivray, Hongbo Yang, Jason Liu, William Meyerson, Declan Clarke, Mengting Gu, Shantao Li, Shaoke Lou, Jinrui Xu, Lucas Lochovsky, Matthew Ung, Lijia Ma, Shan Yu, Qin Cao, Arif Harmanci, Koon-Kiu Yan, Anurag Sethi, Gamze Gürsoy, Michael Rutenberg Schoenberg, Joel Rozowsky, Jonathan Warrell, Prashant Emani, Yucheng T Yang, Timur Galeev, Xiangmeng Kong, Shuang Liu, Xiaotong Li, Jayanth Krishnan, Yanlin Feng, Juan Carlos Rivera-Mulia, Jessica Adrian, James R Broach, Michael Bolt, Jennifer Moran, Dominic Fitzgerald, Vishnu Dileep, Tingting Liu, Shenglin Mei, Takayo Sasaki, Claudia Trevilla-Garcia, Su Wang, Yanli Wang, Chongzhi Zang, Daifeng Wang, Robert J Klein, Michael Snyder, David M Gilbert, Kevin Yip, Chao Cheng, Feng Yue, X Shirley Liu, Kevin P White, Mark Gerstein.

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

DOI: https://doi.org/10.1038/s41467-020-14743-w
Biomolecules. 2020 Jul 28. pii: E1112. [Epub ahead of print]10(8):

The Possibilities of Immunotherapy for Children with Primary Immunodeficiencies Associated with Cancers.

Frederic Baleydier, Fanette Bernard, Marc Ansari.

  Many primary immunodeficiencies (PIDs) are recognised as being associated with malignancies, particularly lymphoid malignancies, which represent the highest proportion of cancers occurring in conjunction with this underlying condition. When patients present with genetic errors of immunity, clinicians must often reflect on whether to manage antitumoral treatment conventionally or to take a more personalised approach, considering possible existing comorbidities and the underlying status of immunodeficiency. Recent advances in antitumoral immunotherapies, such as monoclonal antibodies, antigen-specific adoptive cell therapies or compounds with targeted effects, potentially offer significant opportunities for optimising treatment for those patients, especially with lymphoid malignancies. In cases involving PIDs, variable oncogenic mechanisms exist, and opportunities for antitumoral immunotherapies can be considered accordingly. In cases involving a DNA repair defect or genetic instability, monoclonal antibodies can be proposed instead of chemotherapy to avoid severe toxicity. Malignancies secondary to uncontrolled virus-driven proliferation or the loss of antitumoral immunosurveillance may benefit from antivirus cell therapies or allogeneic stem cell transplantation in order to restore the immune antitumoral caretaker function. A subset of PIDs is caused by gene defects affecting targetable signalling pathways directly involved in the oncogenic process, such as the constitutive activation of phosphoinositol 3-kinase/protein kinase B (PI3K/AKT) in activated phosphoinositide 3-kinase delta syndrome (APDS), which can be settled with PI3K/AKT inhibitors. Therefore, immunotherapy provides clinicians with interesting antitumoral therapeutic weapons to treat malignancies when there is an underlying PID.

Keywords:  cancers; immunotherapies; primary immunodeficiencies

DOI:  https://doi.org/10.3390/biom10081112
Life Sci Alliance. 2020 Sep;pii: e202000797. [Epub ahead of print]3(9):

Mitochondrial spongiotic brain disease: astrocytic stress and harmful rapamycin and ketosis effect.

Olesia Ignatenko, Joni Nikkanen, Alexander Kononov, Nicola Zamboni, Gulayse Ince-Dunn, Anu Suomalainen.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a group of severe, tissue-specific diseases of childhood with unknown pathogenesis. Brain-specific MDS manifests as devastating spongiotic encephalopathy with no curative therapy. Here, we report cell type-specific stress responses and effects of rapamycin treatment and ketogenic diet (KD) in mice with spongiotic encephalopathy mimicking human MDS, as these interventions were reported to improve some mitochondrial disease signs or symptoms. These mice with astrocyte-specific knockout of Twnk gene encoding replicative mtDNA helicase Twinkle (TwKOastro) show wide-spread cell-autonomous astrocyte activation and mitochondrial integrated stress response (ISRmt) induction with major metabolic remodeling of the brain. Mice with neuronal-specific TwKO show no ISRmt Both KD and rapamycin lead to rapid deterioration and weight loss of TwKOastro and premature trial termination. Although rapamycin had no robust effects on TwKOastro brain pathology, KD exacerbated spongiosis, gliosis, and ISRmt Our evidence emphasizes that mitochondrial disease treatments and stress responses are tissue- and disease specific. Furthermore, rapamycin and KD are deleterious in MDS-linked spongiotic encephalopathy, pointing to a crucial role of diet and metabolism for mitochondrial disease progression.

DOI: https://doi.org/10.26508/lsa.202000797
Nat Metab. 2020 Jul 27.

A spoonful of DHAP keeps mTORC1 running on sugars.

Gerta Hoxhaj, Jason W Locasale, Issam Ben-Sahra.

DOI: https://doi.org/10.1038/s42255-020-0246-1
Cell Stem Cell. 2020 Jul 28. pii: S1934-5909(20)30290-3. [Epub ahead of print]

Mapping Gene Circuits Essential for Germ Layer Differentiation via Loss-of-Function Screens in Haploid Human Embryonic Stem Cells.

Atilgan Yilmaz, Carmel Braverman-Gross, Anna Bialer-Tsypin, Mordecai Peretz, Nissim Benvenisty.

  Pluripotent stem cells can differentiate into all embryonic germ layers, yet the genes essential for these cell fate transitions in human remain elusive. Here, we mapped the essential genes for the differentiation of human pluripotent stem cells (hPSCs) into the three germ layers by using a genome-wide loss-of-function library established in haploid hPSCs. Strikingly, we observed a high fraction of essential genes associated with plasma membrane, highlighting signaling pathways needed for each lineage differentiation. Interestingly, analysis of all hereditary neurological disorders uncovered high essentiality among microcephaly-causing genes. Furthermore, we demonstrated lineage-specific hierarchies among essential transcription factors and a set of Golgi- and endoplasmic reticulum-related genes needed for the differentiation into all germ layers. Our work sheds light on the gene networks regulating early gastrulation events in human by defining essential drivers of specific embryonic germ layer fates and essential genes for the exit from pluripotency.

Keywords:  differentiation; essential genes; exit from pluripotency; genetic screening; germ layers; haploid cells; human embryonic stem cells

DOI:  https://doi.org/10.1016/j.stem.2020.06.023
Nat Commun. 2020 Jul 27. 11(1): 3746

Molecular engineering of safe and efficacious oral basal insulin.

Frantisek Hubálek, Hanne H F Refsgaard, Sanne Gram-Nielsen, Peter Madsen, Erica Nishimura, Martin Münzel, Christian Lehn Brand, Carsten Enggaard Stidsen, Christian Hove Claussen, Erik Max Wulff, Lone Pridal, Ulla Ribel, Jonas Kildegaard, Trine Porsgaard, Eva Johansson, Dorte Bjerre Steensgaard, Lars Hovgaard, Tine Glendorf, Bo Falck Hansen, Maja Kirkegaard Jensen, Peter Kresten Nielsen, Svend Ludvigsen, Susanne Rugh, Patrick W Garibay, Mary Courtney Moore, Alan D Cherrington, Thomas Kjeldsen.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.

DOI: https://doi.org/10.1038/s41467-020-17487-9
Adipocyte. 2020 Dec;9(1): 415-426

In vitro characterization of the effects of chronic insulin stimulation in mouse 3T3-L1 and human SGBS adipocytes.

A Rossi, M Eid, J Dodgson, G Davies, B Musial, M Wabitsch, C Church, D C Hornigold.

  Hyperinsulinemia is the hallmark of the development of insulin resistance and precedes the diagnosis of type 2 diabetes. Here we evaluated the effects of prolonged exposure (≥4 days) to high insulin doses (150 nM) in vitro in two adipose cell types, mouse 3T3-L1 and human SGBS. Chronic insulin treatment significantly decreased lipid droplet size, insulin signalling and insulin-stimulated glucose uptake. 3T3-L1 displayed an increased basal glucose internalization following chronic insulin treatment, which was associated with increased GLUT1 expression. In addition, both cells showed increased basal lipolysis. In conclusion, we report the effects of prolonged hyperinsulinemia in 3T3-L1 and SGBS, highlighting similarities and discrepancies between the cell types, to be considered when using these cells to model insulin-induced insulin resistance.

Keywords:  3T3-L1; SGBS; glucose uptake; hyperinsulinemia; lipolysis

DOI:  https://doi.org/10.1080/21623945.2020.1798613