bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021–07–11
twenty-six papers selected by
Ralitsa Radostinova Madsen, University College London
  1. The INPP4B paradox: Like PTEN, but different.
  2. A review of mechanisms of disease across PIK3CA-related disorders with vascular manifestations.
  3. A standard of care for individuals with PIK3CA-related disorders: an international expert consensus statement.
  4. Reactivating PTEN promotes antitumour immunity.
  5. Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability.
  6. Phosphoinositide 3-Kinase p110 Delta Differentially Restrains and Directs Naïve Versus Effector CD8+ T Cell Transcriptional Programs.
  7. Redefining the PTEN promoter: Identification of novel upstream transcription start regions.
  8. Analysis of EGFR, KRAS, and PIK3CA gene mutation rates and clinical distribution in patients with different types of lung cancer.
  9. Active elimination of intestinal cells drives oncogenic growth in organoids.
  10. Endoscopic Findings in Patients With PTEN Hamartoma Tumor Syndrome Undergoing Surveillance.
  11. Combined Inhibition of Akt and mTOR Is Effective Against Non-Hodgkin Lymphomas.
  12. SpaceM reveals metabolic states of single cells.
  13. Transforming growth factor-β-regulated mTOR activity preserves cellular metabolism to maintain long-term T cell responses in chronic infection.
  14. Signaling in the crowded cell.
  15. Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells.
  16. Fractional response analysis reveals logarithmic cytokine responses in cellular populations.
  17. Splicing factor SRSF1 is indispensable for regulatory T cell homeostasis and function.
  18. The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila.
  19. mTOR Mediated Metabolic Rewiring and Ischemic Preconditioning, its Complicated.
  20. Metabolic reprogramming in cancer: mechanistic insights from Drosophila.
  21. Inborn errors of metabolism: Lessons from iPSC models.
  22. A synthetic circuit for buffering gene dosage variation between individual mammalian cells.
  23. Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect.
  24. Renal neoplasms in tuberous sclerosis mice are neurocristopathies.
  25. Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.
  26. MTORC1 inhibition drives crinophagic degradation of glucagon.
  1. Adv Biol Regul. 2021 Jun 16. pii: S2212-4926(21)00033-6. [Epub ahead of print]82 100817
    The INPP4B paradox: Like PTEN, but different.
    Sabryn A Hamila, Lisa M Ooms, Samuel J Rodgers, Christina A Mitchell.
      Cancer is a complex and heterogeneous disease marked by the dysregulation of cancer driver genes historically classified as oncogenes or tumour suppressors according to their ability to promote or inhibit tumour development and growth, respectively. Certain genes display both oncogenic and tumour suppressor functions depending on the biological context, and as such have been termed dual-role cancer driver genes. However, because of their context-dependent behaviour, the tumourigenic mechanism of many dual-role genes is elusive and remains a significant knowledge gap in our effort to understand and treat cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) is an emerging dual-role cancer driver gene, primarily known for its role as a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway. In response to growth factor stimulation, class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane. PtdIns(3,4,5)P3 can be hydrolysed by inositol polyphosphate 5-phosphatases to generate PtdIns(3,4)P2, which, together with PtdIns(3,4,5)P3, facilitates the activation of AKT to promote cell proliferation, survival, migration, and metabolism. Phosphatase and tensin homology on chromosome 10 (PTEN) and INPP4B are dual-specificity phosphatases that hydrolyse PtdIns(3,4,5)P3 and PtdIns(3,4)P2, respectively, and thus negatively regulate PI3K/AKT signalling. PTEN is a bona fide tumour suppressor that is frequently lost in human tumours. INPP4B was initially characterised as a tumour suppressor akin to PTEN, and has been implicated as such in a number of cancers, including prostate, thyroid, and basal-like breast cancers. However, evidence has since emerged revealing INPP4B as a paradoxical oncogene in several malignancies, with increased INPP4B expression reported in AML, melanoma and colon cancers among others. Although the tumour suppressive function of INPP4B has been mostly ascribed to its ability to negatively regulate PI3K/AKT signalling, its oncogenic function remains less clear, with proposed mechanisms including promotion of PtdIns(3)P-dependent SGK3 signalling, inhibition of PTEN-dependent AKT activation, and enhancing DNA repair mechanisms to confer chemoresistance. Nevertheless, research is ongoing to identify the factors that dictate the tumourigenic output of INPP4B in different human cancers. In this review we discuss the dualistic role that INPP4B plays in the context of cancer development, progression and treatment, drawing comparisons to PTEN to explore how their similarities and, importantly, their differences may account for their diverging roles in tumourigenesis.
    Keywords:  Cancer; Inositol polyphosphate 4-phosphatase (INPP4B); Oncogene; Phosphate and tensin homologue deleted on chromosome 10 (PTEN); Phosphoinositide signalling; Tumour suppressor
    DOI:  https://doi.org/10.1016/j.jbior.2021.100817
  2. Orphanet J Rare Dis. 2021 Jul 08. 16(1): 306
    A review of mechanisms of disease across PIK3CA-related disorders with vascular manifestations.
    Guillaume Canaud, Adrienne M Hammill, Denise Adams, Miikka Vikkula, Kim M Keppler-Noreuil.
       BACKGROUND: PIK3CA-related disorders include vascular malformations and overgrowth of various tissues that are caused by postzygotic, somatic variants in the gene encoding phosphatidylinositol-3-kinase (PI3K) catalytic subunit alpha. These mutations result in activation of the PI3K/AKT/mTOR signaling pathway. The goals of this review are to provide education on the underlying mechanism of disease for this group of rare conditions and to summarize recent advancements in the understanding of, as well as current and emerging treatment options for PIK3CA-related disorders.
    MAIN BODY: PIK3CA-related disorders include PIK3CA-related overgrowth spectrum (PROS), PIK3CA-related vascular malformations, and PIK3CA-related nonvascular lesions. Somatic activating mutations (predominantly in hotspots in the helical and kinase domains of PIK3CA, but also in other domains), lead to hyperactivation of the PI3K signaling pathway, which results in abnormal tissue growth. Diagnosis is complicated by the variability and overlap in phenotypes associated with PIK3CA-related disorders and should be performed by clinicians with the required expertise along with coordinated care from a multidisciplinary team. Although tissue mosaicism presents challenges for confirmation of PIK3CA mutations, next-generation sequencing and tissue selection have improved detection. Clinical improvement, radiological response, and patient-reported outcomes are typically used to assess treatment response in clinical studies of patients with PIK3CA-related disorders, but objective assessment of treatment response is difficult using imaging (due to the heterogeneous nature of these disorders, superimposed upon patient growth and development). Despite their limitations, patient-reported outcome tools may be best suited to gauge patient improvement. New therapeutic options are needed to provide an alternative or supplement to standard approaches such as surgery and sclerotherapy. Currently, there are no systemic agents that have regulatory approval for these disorders, but the mTOR inhibitor sirolimus has been used for several years in clinical trials and off label to address symptoms. There are also other agents under investigation for PIK3CA-related disorders that act as inhibitors to target different components of the PI3K signaling pathway including AKT (miransertib) and PI3K alpha (alpelisib).
    CONCLUSION: Management of patients with PIK3CA-related disorders requires a multidisciplinary approach. Further results from ongoing clinical studies of agents targeting the PI3K pathway are highly anticipated.
    Keywords:  Alpelisib; Miransertib; PI3K; PIK3CA; PROS; Sirolimus; Vascular malformation
    DOI:  https://doi.org/10.1186/s13023-021-01929-8
  3. Clin Genet. 2021 Jul 08.
    A standard of care for individuals with PIK3CA-related disorders: an international expert consensus statement.
    Sofia Douzgou, Myfanwy Rawson, Eulalia Baselga, Moise Danielpour, Laurence Faivre, Alon Kashanian, Kim M Keppler-Noreuil, Paul Kuentz, Grazia M S Mancini, Marie-Cecile Maniere, Victor Martinez-Glez, Victoria E Parker, Robert K Semple, Siddharth Srivastava, Pierre Vabres, Marie-Claire Y de Wit, John M Graham, Jill Clayton-Smith, Ghayda M Mirzaa, Leslie G Biesecker.
      Growth promoting variants in PIK3CA cause a spectrum of developmental disorders, depending on the developmental timing of the mutation and tissues involved. These phenotypically heterogeneous entities have been grouped as PIK3CA-Related Overgrowth Spectrum disorders (PROS). Deep sequencing technologies have facilitated detection of low-level mosaic, often necessitating testing of tissues other than blood. Since clinical management practices vary considerably among healthcare professionals and services across different countries, a consensus on management guidelines is needed. Clinical heterogeneity within this spectrum leads to challenges in establishing management recommendations, which must be based on patient-specific considerations. Moreover, as most of these conditions are rare, affected families may lack access to the medical expertise that is needed to help address the multi-system and often complex medical issues seen with PROS. In March 2019, macrocephaly-capillary malformation (M-CM) patient organizations hosted an expert meeting in Manchester, United Kingdom, to help address these challenges with regards to M-CM syndrome. We have expanded the scope of this project to cover PROS and developed this consensus statement on the preferred approach for managing affected individuals based on our current knowledge.
    Keywords:  PIK3CA-related overgrowth spectrum; clinical management; expert consensus; mosaic
    DOI:  https://doi.org/10.1111/cge.14027
  4. Nat Rev Drug Discov. 2021 Jul 07.
    Reactivating PTEN promotes antitumour immunity.
    Sarah Crunkhorn.
      
    DOI:  https://doi.org/10.1038/d41573-021-00119-2
  5. Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00703-8. [Epub ahead of print]36(1): 109327
    Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability.
    Yaxiong Cui, Yanxiao Wang, Xiaopeng Song, Huimin Ning, Yizhe Zhang, Yan Teng, Jun Wang, Xiao Yang.
      The low level of transcytosis is a unique feature of cerebrovascular endothelial cells (ECs), ensuring restrictive blood-brain barrier (BBB) permeability. Major facilitator superfamily domain-containing 2a (MFSD2A) is a key regulator of the BBB function by suppressing caveolae-mediated transcytosis. However, the mechanisms regulating MFSD2A at the BBB have been barely explored. Here, we show that cerebrovascular EC-specific deletion of Pten (phosphatase and tensin homolog) results in a dramatic increase in vesicular transcytosis by the reduction of MFSD2A, leading to increased transcellular permeability of the BBB. Mechanistically, AKT signaling inhibits E3 ubiquitin ligase NEDD4-2-mediated MFSD2A degradation. Consistently, cerebrovascular Nedd4-2 overexpression decreases MFSD2A levels, increases transcytosis, and impairs BBB permeability, recapitulating the phenotypes of Pten-deficient mice. Furthermore, Akt deletion decreases phosphorylated NEDD4-2 levels, restores MFSD2A levels, and normalizes BBB permeability in Pten-mutant mice. Altogether, our work reveals the essential physiological function of the PTEN/AKT/NEDD4-2/MFSD2A axis in the regulation of BBB permeability.
    Keywords:  MFSD2A; NEDD4-2; PTEN/AKT signaling; blood-brain barrier; brain endothelial cells; transcytosis
    DOI:  https://doi.org/10.1016/j.celrep.2021.109327
  6. Front Immunol. 2021 ;12 691997
    Phosphoinositide 3-Kinase p110 Delta Differentially Restrains and Directs Naïve Versus Effector CD8+ T Cell Transcriptional Programs.
    Laura Spinelli, Julia M Marchingo, Aneela Nomura, Marcos P Damasio, Doreen A Cantrell.
      Phosphoinositide 3-kinase p110 delta (PI3K p110δ) is pivotal for CD8+ T cell immune responses. The current study explores PI3K p110δ induction and repression of antigen receptor and cytokine regulated programs to inform how PI3K p110δ directs CD8+ T cell fate. The studies force a revision of the concept that PI3K p110δ controls metabolic pathways in T cells and reveal major differences in PI3K p110δ regulated transcriptional programs between naïve and effector cytotoxic T cells (CTL). These differences include differential control of the expression of cytolytic effector molecules and costimulatory receptors. Key insights from the work include that PI3K p110δ signalling pathways repress expression of the critical inhibitory receptors CTLA4 and SLAMF6 in CTL. Moreover, in both naïve and effector T cells the dominant role for PI3K p110δ is to restrain the production of the chemokines that orchestrate communication between adaptive and innate immune cells. The study provides a comprehensive resource for understanding how PI3K p110δ uses multiple processes mediated by Protein Kinase B/AKT, FOXO1 dependent and independent mechanisms and mitogen-activated protein kinases (MAPK) to direct CD8+ T cell fate.
    Keywords:  CD8+ T cells; PI3K; TCR signalling; chemokines; cytokines; p110δ; transcriptomics
    DOI:  https://doi.org/10.3389/fimmu.2021.691997
  7. Hum Mol Genet. 2021 Jul 03. pii: ddab175. [Epub ahead of print]
    Redefining the PTEN promoter: Identification of novel upstream transcription start regions.
    Dennis J Grencewicz, Todd Romigh, Stetson Thacker, Ata Abbas, Ritika Jaini, Donal Luse, Charis Eng.
      Germline mutation of PTEN is causally observed in Cowden syndrome (CS) and is one of the most common, penetrant risk genes for autism spectrum disorder (ASD). However, the majority of individuals who present with CS-like clinical features are PTEN-mutation negative. Reassessment of PTEN promoter regulation may help explain abnormal PTEN dosage, as only the minimal promoter and coding regions are currently included in diagnostic PTEN mutation analysis. Therefore, we reanalyzed the architecture of the PTEN promoter using next-generation sequencing datasets. Specifically, run-on sequencing assays identified two additional transcription start regions (TSRs) at -2053 and - 1906 basepairs from the canonical start of PTEN, thus extending the PTEN 5'UTR and redefining the PTEN promoter. We show that these novel upstream TSRs are active in cancer cell lines, human cancer, and normal tissue. Further, these TSRs can produce novel PTEN transcripts due to the introduction of new splice donors at -2041, -1826, and - 1355, which may allow for splicing out of the PTEN 5'UTR or the first and second exon in upstream-initiated transcripts. Combining ENCODE ChIP-seq and pertinent literature, we also compile and analyze all transcription factors (TFs) binding at the redefined PTEN locus. Enrichment analyses suggest that TFs bind specifically to the upstream TSRs may be implicated in inflammatory processes. Together, these data redefine the architecture of the PTEN promoter, an important step toward a comprehensive model of PTEN transcription regulation, a basis for future investigations into the new promoters' role in disease pathogenesis.
    DOI:  https://doi.org/10.1093/hmg/ddab175
  8. World J Surg Oncol. 2021 Jul 03. 19(1): 197
    Analysis of EGFR, KRAS, and PIK3CA gene mutation rates and clinical distribution in patients with different types of lung cancer.
    Shuo Li, Xinju Li.
       BACKGROUND: To analyze and evaluate EGFR, KRAS, and PIK3CA gene mutation rates and clinical distribution in patients with different types of lung cancer METHOD: A total of 221 lung cancer patients treated in our hospital between January 2016 and June 2019 were enrolled. Tissue and whole blood samples were collected and analyzed to determine the mutation status of EGFR, KRAS, and PIK3CA genes. The gene exon mutation rates were determined. Relevant clinical data, such as age, gender, tumor sample type, treatment method, pathologic type, and lung cancer stage were recorded and statistically analyzed.
    RESULTS: The EGFR gene mutation rates in exons E18-E21 were 2.3%, 17.6%, 3.6%, and 20.4%, respectively. E18, E19, and E20 mutations were commonly detected in adenosquamous carcinoma, and E21 mutations were commonly detected in adenocarcinoma. Mutations in exons E18-E21 were frequently detected in patients with lung cancer stages IA, IB, IIA, or IIB, respectively. The KRAS gene mutation rate in lung cancer patients in exon E2 was higher in whole blood and tissue samples than other exon mutations, while the KRAS gene mutation rate in exons E2 and E3 was significantly higher in patients with lung cancer stages IIB and IA, respectively. PIK3CA gene mutations in exons E9 and E20 occurred in patients < 60 years of age. Exon E9-positive mutations were more common in men or patients with squamous cell carcinoma, while exon E20-positive mutations were more common in females.
    CONCLUSION: The EGFR, KRAS, and PIK3CA gene exon mutation rates differ and were shown to be correlated with different clinical indicators, which have significance in clinical treatment.
    Keywords:  Clinic; EGFR; Gene mutation; KRAS; Lung cancer; PIK3CA
    DOI:  https://doi.org/10.1186/s12957-021-02315-1
  9. Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00683-5. [Epub ahead of print]36(1): 109307
    Active elimination of intestinal cells drives oncogenic growth in organoids.
    Ana Krotenberg Garcia, Arianna Fumagalli, Huy Quang Le, Rene Jackstadt, Tamsin Rosemary Margaret Lannagan, Owen James Sansom, Jacco van Rheenen, Saskia Jacoba Elisabeth Suijkerbuijk.
      Competitive cell interactions play a crucial role in quality control during development and homeostasis. Here, we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell-state transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. Jun N-terminal kinase (JNK) signaling is activated in competing cells and is required for cell-state change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell-state change in a JNK-dependent manner.
    Keywords:  JNK; cancer; cell competition; fetal-like; organoids; small intestine
    DOI:  https://doi.org/10.1016/j.celrep.2021.109307
  10. J Clin Gastroenterol. 2021 Jul 07.
    Endoscopic Findings in Patients With PTEN Hamartoma Tumor Syndrome Undergoing Surveillance.
    Anshika Khare, Carol A Burke, Brandie Heald, Margaret O'Malley, Lisa LaGuardia, Susan Milicia, Michael Cruise, Charis Eng, Gautam Mankaney.
       GOALS AND BACKGROUND: Phosphatase and tensin homolog hamartoma tumor syndrome (PHTS) is an inherited disorder that increases the risk for cancer in multiple organ systems, including breast, endometrial, thyroid, and the gastrointestinal tract. Surveillance is recommended however there lacks data to describe the change in polyposis phenotype and cancer incidence over surveillance. Our aim is to describe the polyposis phenotype and cancer incidence in PHTS patients undergoing endoscopic surveillance.
    STUDY: PHTS patients, ages 17 through 89, who underwent at least 2 esophagogastroduodenoscopy (EGDs) or colonoscopies were identified. Number and sizes of polyps were noted, from which 5 categories were recreated. Incidence of colorectal and gastric cancer was evaluated.
    RESULTS: Seventy patients were included. Patients were clustered and classified into 1 of 5 categories: no polyps, few small polyps (<1 cm, < 10 polyps), few large polyps (≥1 cm, < 10 polyps), many small polyps (<1 cm, ≥10 polyps), many large polyps (≥1 cm, ≥10 polyps). There was no significant difference in polyp number or size on EGD (P=0.47 and 0.83, respectively) or colonoscopy (P=0.49 and 0.10, respectively) over the surveillance period (4.8±3.9 y for stomach and 5.6±4.4 y for colon). The average interval between endoscopies was 28±24 months for EGDs and 29±23 months for colonoscopies. A stage II transverse colon adenocarcinoma and stage IV gastric adenocarcinoma were identified. Standardized incidence rates for gastric and colon cancers were 5427 (P=0.0002) and 353 (P=0.002), respectively.
    CONCLUSIONS: PTHS individuals can be classified into polyposis phenotypes which do not change over an endoscopic surveillance period. Two cancers were associated with a large size polyp phenotype. Surveillance intervals should be determined by polyp size ≥1 cm and pathology.
    DOI:  https://doi.org/10.1097/MCG.0000000000001580
  11. Front Oncol. 2021 ;11 670275
    Combined Inhibition of Akt and mTOR Is Effective Against Non-Hodgkin Lymphomas.
    Ricardo Rivera-Soto, Yi Yu, Dirk P Dittmer, Blossom Damania.
      Non-Hodgkin lymphoma (NHL) are a diverse group of hematological malignancies comprised of over 60 subtypes. These subtypes range from indolent to aggressive. The PI3K/Akt/mTOR pathway has been shown to contribute to cell survival and proliferation and is constitutively active in most NHL. MK-7075 (miransertib) and MK-4440 are small molecules that effectively inhibit Akt and have entered clinical development. Using in vitro and in vivo models of NHL, we explored targeting the kinase Akt with miransertib and MK-4440 alone or in combination with the mTORC1 inhibitor, rapamycin (sirolimus). Both Akt inhibitors inhibited the pathway and NHL proliferation in a subtype-dependent manner. However, these compounds had a minimal effect on the viability of primary B-cells. Importantly, the combination of miransertib and sirolimus synergistically reduced cell proliferation in NHL, including in one indolent subtype, e.g., follicular lymphoma (FL), and two aggressive subtypes, e.g., diffuse large B-cell lymphoma (DLBCL) and primary effusion lymphoma (PEL). To establish in vivo efficacy, we used several xenograft models of FL, DLBCL, and PEL. The results obtained in vivo were consistent with the in vitro studies. The FL xenograft was highly sensitive to the inhibition of Akt alone; however, the tumor burden of PEL xenografts was only significantly reduced when both Akt and mTORC1 were targeted. These data suggest that targeting the PI3K/Akt/mTOR pathway with Akt inhibitors such as miransertib in combination with mTOR inhibitors serves as a broadly applicable therapeutic in NHL.
    Keywords:  MK-4440; miransertib; non-Hodgkin lymphomas; sirolimus; viral lymphomas
    DOI:  https://doi.org/10.3389/fonc.2021.670275
  12. Nat Methods. 2021 Jul;18(7): 799-805
    SpaceM reveals metabolic states of single cells.
    Luca Rappez, Mira Stadler, Sergio Triana, Rose Muthoni Gathungu, Katja Ovchinnikova, Prasad Phapale, Mathias Heikenwalder, Theodore Alexandrov.
      A growing appreciation of the importance of cellular metabolism and revelations concerning the extent of cell-cell heterogeneity demand metabolic characterization of individual cells. We present SpaceM, an open-source method for in situ single-cell metabolomics that detects >100 metabolites from >1,000 individual cells per hour, together with a fluorescence-based readout and retention of morpho-spatial features. We validated SpaceM by predicting the cell types of cocultured human epithelial cells and mouse fibroblasts. We used SpaceM to show that stimulating human hepatocytes with fatty acids leads to the emergence of two coexisting subpopulations outlined by distinct cellular metabolic states. Inducing inflammation with the cytokine interleukin-17A perturbs the balance of these states in a process dependent on NF-κB signaling. The metabolic state markers were reproduced in a murine model of nonalcoholic steatohepatitis. We anticipate SpaceM to be broadly applicable for investigations of diverse cellular models and to democratize single-cell metabolomics.
    DOI:  https://doi.org/10.1038/s41592-021-01198-0
  13. Immunity. 2021 Jun 29. pii: S1074-7613(21)00251-X. [Epub ahead of print]
    Transforming growth factor-β-regulated mTOR activity preserves cellular metabolism to maintain long-term T cell responses in chronic infection.
    Sarah S Gabriel, Carlson Tsui, David Chisanga, Flora Weber, Manuela Llano-León, Patrick M Gubser, Laurent Bartholin, Fernando Souza-Fonseca-Guimaraes, Nicholas D Huntington, Wei Shi, Daniel T Utzschneider, Axel Kallies.
      Antigen-specific CD8+ T cells in chronic viral infections and tumors functionally deteriorate, a process known as exhaustion. Exhausted T cells are sustained by precursors of exhausted (Tpex) cells that self-renew while continuously generating exhausted effector (Tex) cells. However, it remains unknown how Tpex cells maintain their functionality. Here, we demonstrate that Tpex cells sustained mitochondrial fitness, including high spare respiratory capacity, while Tex cells deteriorated metabolically over time. Tpex cells showed early suppression of mTOR kinase signaling but retained the ability to activate this pathway in response to antigen receptor signals. Early transient mTOR inhibition improved long-term T cell responses and checkpoint inhibition. Transforming growth factor-β repressed mTOR signaling in exhausted T cells and was a critical determinant of Tpex cell metabolism and function. Overall, we demonstrate that the preservation of cellular metabolism allows Tpex cells to retain long-term functionality to sustain T cell responses during chronic infection.
    Keywords:  OXPHOS; T cell exhaustion; T cell function; TCF1; checkpoint inhibition; mitochondria; precursors of exhausted T cells; progenitor T cells; rapamycin; stem-like T cells
    DOI:  https://doi.org/10.1016/j.immuni.2021.06.007
  14. Curr Opin Struct Biol. 2021 Jul 01. pii: S0959-440X(21)00078-6. [Epub ahead of print]71 43-50
    Signaling in the crowded cell.
    Ruth Nussinov, Chung-Jung Tsai, Hyunbum Jang.
      High-resolution technologies have clarified some of the principles underlying cellular actions. However, understanding how cells receive, communicate, and respond to signals is still challenging. Questions include how efficient regulation of assemblies, which execute cell actions at the nanoscales, transmits productively at micrometer scales, especially considering the crowded environment, and how the cell organization makes it happen. Here, we describe how cells can navigate long-range diffusion-controlled signaling via association/dissociation of spatially proximal entities. Dynamic clusters can span the cell, engaging in most signaling steps. Effective local concentration, allostery, scaffolding, affinities, and the chemical and mechanical properties of the macromolecules and the environment play key roles. Signaling strength and duration matter, for example, deciding if a mutation promotes cancer or developmental syndromes.
    DOI:  https://doi.org/10.1016/j.sbi.2021.05.009
  15. Methods Mol Biol. 2021 ;2312 193-223
    Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells.
    Soh Ishiguro, Nozomu Yachie.
      CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated from different species and adopted for genome editing. Furthermore, various effector domains have been fused to these Cas proteins to expand their genome-editing abilities. Although the number of genome-editing tools has been rapidly increasing, the throughput of cell-based characterization of new genome-editing tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced by hundreds to thousands of different genome-editing reagents in mammalian cells. We have successful experiences of developing several key genome-editing tools using this protocol. The protocol also is designed to be compatible with robotic liquid handling systems for further scalability.
    Keywords:  Amplicon sequencing; Base editing; CRISPR–Cas9; Genome editing; High-throughput sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-1441-9_12
  16. Nat Commun. 2021 Jul 07. 12(1): 4175
    Fractional response analysis reveals logarithmic cytokine responses in cellular populations.
    Karol Nienałtowski, Rachel E Rigby, Jarosław Walczak, Karolina E Zakrzewska, Edyta Głów, Jan Rehwinkel, Michał Komorowski.
      Although we can now measure single-cell signaling responses with multivariate, high-throughput techniques our ability to interpret such measurements is still limited. Even interpretation of dose-response based on single-cell data is not straightforward: signaling responses can differ significantly between cells, encompass multiple signaling effectors, and have dynamic character. Here, we use probabilistic modeling and information-theory to introduce fractional response analysis (FRA), which quantifies changes in fractions of cells with given response levels. FRA can be universally performed for heterogeneous, multivariate, and dynamic measurements and, as we demonstrate, quantifies otherwise hidden patterns in single-cell data. In particular, we show that fractional responses to type I interferon in human peripheral blood mononuclear cells are very similar across different cell types, despite significant differences in mean or median responses and degrees of cell-to-cell heterogeneity. Further, we demonstrate that fractional responses to cytokines scale linearly with the log of the cytokine dose, which uncovers that heterogeneous cellular populations are sensitive to fold-changes in the dose, as opposed to additive changes.
    DOI:  https://doi.org/10.1038/s41467-021-24449-2
  17. Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00715-4. [Epub ahead of print]36(1): 109339
    Splicing factor SRSF1 is indispensable for regulatory T cell homeostasis and function.
    Takayuki Katsuyama, Vaishali R Moulton.
      The ability of regulatory T (Treg) cells to control the immune response and limit the development of autoimmune diseases is determined by distinct molecular processes, which are not fully understood. We show here that serine/arginine-rich splicing factor 1 (SRSF1), which is decreased in T cells from patients with systemic lupus erythematosus, is necessary for the homeostasis and proper function of Treg cells, because its conditional absence in these cells leads to profound autoimmunity and organ inflammation by elevating the glycolytic metabolism and mTORC1 activity and the production of proinflammatory cytokines. Our data reveal a molecular mechanism that controls Treg cell plasticity and offer insights into the pathogenesis of autoimmune disease.
    Keywords:  SRSF1; T cells; Treg; autoimmunity; cytokines; immune homeostasis; immune regulation; inflammation; mTOR pathway; splicing factor
    DOI:  https://doi.org/10.1016/j.celrep.2021.109339
  18. Front Physiol. 2021 ;12 690564
    The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila.
    Yuya Ohhara, Genki Hoshino, Kyosuke Imahori, Tomoya Matsuyuki, Kimiko Yamakawa-Kobayashi.
      Animals can sense internal nutrients, such as amino acids/proteins, and are able to modify their developmental programs in accordance with their nutrient status. In the fruit fly, Drosophila melanogaster, amino acid/protein is sensed by the fat body, an insect adipose tissue, through a nutrient sensor, target of rapamycin (TOR) complex 1 (TORC1). TORC1 promotes the secretion of various peptide hormones from the fat body in an amino acid/protein-dependent manner. Fat-body-derived peptide hormones stimulate the release of insulin-like peptides, which are essential growth-promoting anabolic hormones, from neuroendocrine cells called insulin-producing cells (IPCs). Although the importance of TORC1 and the fat body-IPC axis has been elucidated, the mechanism by which TORC1 regulates the expression of insulinotropic signal peptides remains unclear. Here, we show that an evolutionarily conserved molecular chaperone, heat shock protein 90 (Hsp90), promotes the expression of insulinotropic signal peptides. Fat-body-selective Hsp90 knockdown caused the transcriptional downregulation of insulinotropic signal peptides. IPC activity and systemic growth were also impaired in fat-body-selective Hsp90 knockdown animals. Furthermore, Hsp90 expression depended on protein/amino acid availability and TORC1 signaling. These results strongly suggest that Hsp90 serves as a nutrient-responsive gene that upregulates the fat body-IPC axis and systemic growth. We propose that Hsp90 is induced in a nutrient-dependent manner to support anabolic metabolism during the juvenile growth period.
    Keywords:  Drosophila; fat body; heat shock protein 90; insulin-like peptide; target of rapamycin
    DOI:  https://doi.org/10.3389/fphys.2021.690564
  19. Circ Res. 2021 Mar 05. 128(5): 652-654
    mTOR Mediated Metabolic Rewiring and Ischemic Preconditioning, its Complicated.
    E Dale Abel.
      
    Keywords:  Ischemic Precondition; Metabolism; Myocardial Ischemia; mTOR
    DOI:  https://doi.org/10.1161/circresaha.121.318874
  20. Dis Model Mech. 2021 Jul 01. 14(7): 1-17
    Metabolic reprogramming in cancer: mechanistic insights from Drosophila.
    Kenneth Kin Lam Wong, Esther M Verheyen.
      Cancer cells constantly reprogram their metabolism as the disease progresses. However, our understanding of the metabolic complexity of cancer remains incomplete. Extensive research in the fruit fly Drosophila has established numerous tumor models ranging from hyperplasia to neoplasia. These fly tumor models exhibit a broad range of metabolic profiles and varying nutrient sensitivity. Genetic studies show that fly tumors can use various alternative strategies, such as feedback circuits and nutrient-sensing machinery, to acquire and consolidate distinct metabolic profiles. These studies not only provide fresh insights into the causes and functional relevance of metabolic reprogramming but also identify metabolic vulnerabilities as potential targets for cancer therapy. Here, we review the conceptual advances in cancer metabolism derived from comparing and contrasting the metabolic profiles of fly tumor models, with a particular focus on the Warburg effect, mitochondrial metabolism, and the links between diet and cancer.
    Keywords:   Drosophila cancer models; Aerobic glycolysis; Metabolic reprogramming; Mitochondria
    DOI:  https://doi.org/10.1242/dmm.048934
  21. Rev Endocr Metab Disord. 2021 Jul 09.
    Inborn errors of metabolism: Lessons from iPSC models.
    Rubén Escribá, Raquel Ferrer-Lorente, Ángel Raya.
      The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs.
    Keywords:  CRISPR/Cas9; Disease modeling; Human induced pluripotent stem cells; Lysosomal storage disorders; Reprogramming; Targeted genome edition
    DOI:  https://doi.org/10.1007/s11154-021-09671-z
  22. Nat Commun. 2021 07 05. 12(1): 4132
    A synthetic circuit for buffering gene dosage variation between individual mammalian cells.
    Jin Yang, Jihwan Lee, Michelle A Land, Shujuan Lai, Oleg A Igoshin, François St-Pierre.
      Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits - Equalizers - that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.
    DOI:  https://doi.org/10.1038/s41467-021-23889-0
  23. Nat Metab. 2021 Jul 05.
    Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect.
    Elizabeth K Wiese, Sadae Hitosugi, Sharon T Loa, Annapoorna Sreedhar, Lindsey G Andres-Beck, Kiran Kurmi, Yuan-Ping Pang, Larry M Karnitz, Wilson I Gonsalves, Taro Hitosugi.
      Pharmacological activation of the glycolytic enzyme PKM2 or expression of the constitutively active PKM1 isoform in cancer cells results in decreased lactate production, a phenomenon known as the PKM2 paradox in the Warburg effect. Here we show that oxaloacetate (OAA) is a competitive inhibitor of human lactate dehydrogenase A (LDHA) and that elevated PKM2 activity increases de novo synthesis of OAA through glutaminolysis, thereby inhibiting LDHA in cancer cells. We also show that replacement of human LDHA with rabbit LDHA, which is relatively resistant to OAA inhibition, eliminated the paradoxical correlation between the elevated PKM2 activity and the decreased lactate concentration in cancer cells treated with a PKM2 activator. Furthermore, rabbit LDHA-expressing tumours, compared to human LDHA-expressing tumours in mice, displayed resistance to the PKM2 activator. These findings describe a mechanistic explanation for the PKM2 paradox by showing that OAA accumulates and inhibits LDHA following PKM2 activation.
    DOI:  https://doi.org/10.1038/s42255-021-00424-5
  24. iScience. 2021 Jul 23. 24(7): 102684
    Renal neoplasms in tuberous sclerosis mice are neurocristopathies.
    Uchenna Unachukwu, Takayuki Shiomi, Monica Goldklang, Kiran Chada, Jeanine D'Armiento.
      Tuberous sclerosis (TS) is a rare disorder exhibiting multi-systemic benign neoplasms. We hypothesized the origin of TS neoplastic cells derived from the neural crest given the heterogeneous ecto-mesenchymal phenotype of the most common TS neoplasms. To test this hypothesis, we employed Cre-loxP lineage tracing of myelin protein zero (Mpz)-expressing neural crest cells (NCCs) in spontaneously developing renal tumors of Tsc2 +/- /Mpz(Cre)/TdT fl/fl reporter mice. In these mice, ectopic renal tumor onset was detected at 4 months of age increasing in volume by 16 months of age with concomitant increase in the subpopulation of tdTomato+ NCCs from 0% to 6.45% of the total number of renal tumor cells. Our results suggest that Tsc2 +/- mouse renal tumors arise from domiciled proliferative progenitor cell populations of neural crest origin that co-opt tumorigenesis due to mutations in Tsc2 loci. Targeting neural crest antigenic determinants will provide a potential alternative therapeutic approach for TS pathogenesis.
    Keywords:  cancer;; molecular physiology; stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2021.102684
  25. Nat Methods. 2021 Jul;18(7): 747-756
    Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.
    Saleh Alseekh, Asaph Aharoni, Yariv Brotman, Kévin Contrepois, John D'Auria, Jan Ewald, Jennifer C Ewald, Paul D Fraser, Patrick Giavalisco, Robert D Hall, Matthias Heinemann, Hannes Link, Jie Luo, Steffen Neumann, Jens Nielsen, Leonardo Perez de Souza, Kazuki Saito, Uwe Sauer, Frank C Schroeder, Stefan Schuster, Gary Siuzdak, Aleksandra Skirycz, Lloyd W Sumner, Michael P Snyder, Huiru Tang, Takayuki Tohge, Yulan Wang, Weiwei Wen, Si Wu, Guowang Xu, Nicola Zamboni, Alisdair R Fernie.
      Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.
    DOI:  https://doi.org/10.1038/s41592-021-01197-1
  26. Mol Metab. 2021 Jul 02. pii: S2212-8778(21)00131-9. [Epub ahead of print] 101286
    MTORC1 inhibition drives crinophagic degradation of glucagon.
    Sangam Rajak, Sherwin Xie, Archana Tewari, Sana Raza, Wu Yajun, Boon-Huat Bay, Paul M Yen, Rohit A Sinha.
       OBJECTIVE: Crinophagy is a secretory granule-specific autophagic process that regulates hormone content and secretion in endocrine cells. However, despite being one of the earliest described autophagic processes, its mechanism of action and regulation in mammalian cells remains unclear.
    METHODS AND RESULTS: Here, we examined mammalian crinophagy and its modulation regulate hormone secretion in a glucagon-producing mouse pancreatic α-cell line, alpha TC1 clone 9 (αTC9) and in vivo. Western blot, electron microscopy and immunofluorescence analyses were performed to study crinophagy and glucagon secretion in αTC9 cells and C57BL/6 mice, in response to the mammalian target of rapamycin complex 1 (MTORC1) inhibitor rapamycin. Amino acid depletion and pharmacological inhibition of MTORC1 increased the shuttling of glucagon-containing secretory granules into lysosomes for crinophagic degradation to reduce glucagon secretion via a macroautophagy-independent mechanism. Furthermore, MTORC1 inhibition reduced both intracellular and secreted glucagon in rapamycin-treated mice, in response to hypoglycaemia.
    CONCLUSION: In summary, we have identified a novel crinophagic mechanism of intracellular glucagon turnover in pancreatic α-cells regulated by MTORC1 signaling.
    Keywords:  Autophagy; Crinophagy; Diabetes; Glucagon; Lysosomes; MTORC1; Rapamycin
    DOI:  https://doi.org/10.1016/j.molmet.2021.101286
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