bims-pideca 2020-05-03 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2020‒05‒03
nineteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

How does mTOR sense glucose starvation? AMPK is the usual suspect.
Transforming growth factor β (TGF-β) receptor signaling regulates kinase networks and phosphatidylinositol metabolism during T-cell activation.
Small molecule H89 renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors.
Rheb1-Independent Activation of mTORC1 in Mammary Tumors Occurs through Activating Mutations in mTOR.
Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation.
Superior cancer preventive efficacy of low versus high dose of mTOR inhibitor in a mouse model of prostate cancer.
The duality of human oncoproteins: drivers of cancer and congenital disorders.
Buparlisib in combination with tamoxifen in pretreated patients with hormone receptor-positive, HER2-negative advanced breast cancer molecularly stratified for PIK3CA mutations and loss of PTEN expression.
Partial impairment of insulin receptor expression mimics fasting to prevent diet-induced fatty liver disease.
Targeted therapy for mTORC1-driven tumours through HDAC inhibition by exploiting innate vulnerability of mTORC1 hyper-activation.
PI3K Inhibitors and Their Role as Novel Agents for Targeted Therapy in Lymphoma.
Context specificity of the EMT transcriptional response.
Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1).
The mutational landscape of normal human endometrial epithelium.
Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids.
Redox Debt Leads to Metabolic Bankruptcy in Tumors.
Juvenile cataract in association with tuberous sclerosis complex.
Cell Atlas technologies and insights into tissue architecture.
A Direct Fluorometric Activity Assay for Lipid Kinases and Phosphatases.

Cell Death Discov. 2020 ;6 27

How does mTOR sense glucose starvation? AMPK is the usual suspect.

Gabriel Leprivier, Barak Rotblat.

  Glucose is a major requirement for biological life. Its concentration is constantly sensed at the cellular level, allowing for adequate responses to any changes of glucose availability. Such responses are mediated by key sensors and signaling pathway components that adapt cellular metabolism to glucose levels. One of the major hubs of these responses is mechanistic target of rapamycin (mTOR) kinase, which forms the mTORC1 and mTORC2 protein complexes. Under physiological glucose concentrations, mTORC1 is activated and stimulates a number of proteins and enzymes involved in anabolic processes, while restricting the autophagic process. Conversely, when glucose levels are low, mTORC1 is inhibited, in turn leading to the repression of numerous anabolic processes, sparing ATP and antioxidants. Understanding how mTORC1 activity is regulated by glucose is not only important to better delineate the biological function of mTOR, but also to highlight potential therapeutic strategies for treating diseases characterized by deregulated glucose availability, as is the case of cancer. In this perspective, we depict the different sensors and upstream proteins responsible of controlling mTORC1 activity in response to changes in glucose concentration. This includes the major energy sensor AMP-activated protein kinase (AMPK), as well as other independent players. The impact of such modes of regulation of mTORC1 on cellular processes is also discussed.

Keywords:  Cell biology; Cell signalling

DOI:  https://doi.org/10.1038/s41420-020-0260-9
J Biol Chem. 2020 May 01. pii: jbc.RA120.012572. [Epub ahead of print]

Transforming growth factor β (TGF-β) receptor signaling regulates kinase networks and phosphatidylinositol metabolism during T-cell activation.

Richard T Cattley, Mijoon Lee, William C Boggess, William F Hawse.

  The cytokine content in tissue microenvironments shapes the functional capacity of a T cell. This capacity depends on the integration of extracellular signaling through multiple receptors, including the T-cell receptor (TCR), co-receptors, and cytokine receptors. Transforming growth factor β (TGF-β) signals through its cognate receptor, TGFβR, to SMAD family member (SMAD) proteins and contributes to the generation of a transcriptional program that promotes regulatory T-cell differentiation. In addition to transcription, here we identified specific signaling networks that are regulated by TGFβR. Using an array of biochemical approaches, including immunoblotting, kinase assays, immunoprecipitation, and flow cytometry, we found that TGFβR signaling promotes the formation of a SMAD3/4-protein kinase A (PKA) complex that activates C-terminal Src kinase (CSK) and thereby down-regulates kinases involved in proximal TCR activation. Additionally, TGFβR signaling potentiated CSK phosphorylation of the P85 subunit in the P85-P110 phosphoinositide 3-kinase (PI3K) heterodimer, which reduced PI3K activity and down-regulated the activation of proteins that require phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) for their activation. Moreover, TGFβR-mediated disruption of the P85-P110 interaction enabled P85 binding to a lipid phosphatase, phosphatase and tensin homolog (PTEN), aiding in the maintenance of PTEN abundance and thereby promoting elevated PtdIns(4,5)P2 levels in response to TGFβR signaling. Taken together, these results highlight that TGF-β influences the trajectory of early T-cell activation by altering PI3K activity and PtdIns levels.

Keywords:  C-terminal Src kinase (CSK); Immunity; T-cell; T-cell biology; T-cell receptor (TCR); computer modeling; phosphatase and tensin homolog (PTEN); phosphatidylinositide 3-kinase (PI 3-kinase); phosphatidylinositol; phosphatidylinositol kinase (PI Kinase); phosphatidylinositol signaling; protein kinase A (PKA)

DOI:  https://doi.org/10.1074/jbc.RA120.012572
Biochem J. 2020 Apr 29. pii: BCJ20190958. [Epub ahead of print]

Small molecule H89 renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors.

Chase H Melick, Jenna Jewell.

  The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that comprises two complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 phosphorylates S6K1 at Thr 389, whereas mTORC2 phosphorylates AKT at Ser 473 to promote cell growth. As the mTOR name implies it is the target of natural product called rapamycin, a clinically approved drug used to treat human disease. Short-term rapamycin treatment inhibits the kinase activity of mTORC1 but not mTORC2. However, ATP-competitive catalytic mTOR inhibitor Torin1 was identified to inhibit the kinase activity of both mTORC1 and mTORC2. Here, we report that H89 (N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling.

Keywords:  kinase inhibitors; mechanistic target of rapamycin; nutrients

DOI:  https://doi.org/10.1042/BCJ20190958
Cell Rep. 2020 Apr 28. pii: S2211-1247(20)30520-9. [Epub ahead of print]31(4): 107571

Rheb1-Independent Activation of mTORC1 in Mammary Tumors Occurs through Activating Mutations in mTOR.

Bin Xiao, Dongmei Zuo, Alison Hirukawa, Robert D Cardiff, Richard Lamb, Nahum Sonenberg, William J Muller.

  Mechanistic target of rapamycin complex 1 (mTORC1) is a master modulator of cellular growth, and its aberrant regulation is recurrently documented within breast cancer. While the small GTPase Rheb1 is the canonical activator of mTORC1, Rheb1-independent mechanisms of mTORC1 activation have also been reported but have not been fully understood. Employing multiple transgenic mouse models of breast cancer, we report that ablation of Rheb1 significantly impedes mammary tumorigenesis. In the absence of Rheb1, a block in tumor initiation can be overcome by multiple independent mutations in Mtor to allow Rheb1-independent reactivation of mTORC1. We further demonstrate that the mTOR kinase is indispensable for tumor initiation as the genetic ablation of mTOR abolishes mammary tumorigenesis. Collectively, our findings demonstrate that mTORC1 activation is indispensable for mammary tumor initiation and that tumors acquire alternative mechanisms of mTORC1 activation.

Keywords:  Rheb; breast; cancer; mTOR; mutation; tumorigenesis

DOI:  https://doi.org/10.1016/j.celrep.2020.107571
Angiogenesis. 2020 Apr 30.

Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation.

Timothy D Le Cras, Jillian Goines, Nora Lakes, Patricia Pastura, Adrienne M Hammill, Denise M Adams, Elisa Boscolo.

  Capillary lymphatic venous malformations (CLVM) are complex vascular anomalies characterized by aberrant and enlarged lymphatic and blood vessels. CLVM appear during fetal development and enlarge after birth, causing life-long complications such as coagulopathy, pulmonary embolism, chronic pain, and disfigurement. Treatment includes surgical debulking, amputation, and recurrent sclerotherapy. Somatic, mosaic mutations in the 110-kD catalytic α-subunit of phosphoinositide-3-kinase (PIK3CA) gene have been previously identified in affected tissues from CLVM patients; however, the cell population harboring the mutation is still unknown. In this study, we hypothesized that endothelial cells (EC) carry the PIK3CA mutations and play a major role in the cellular origin of CLVM. We isolated EC from the lesions of seven patients with CLVM and identified PIK3CA hotspot mutations. The CLVM EC exhibited constitutive phosphorylation of the PI3K effector AKT as well as hyperproliferation and increased resistance to cell death compared to normal EC. Inhibitors of PIK3CA (BYL719) and AKT (ARQ092) attenuated the proliferation of CLVM EC in a dose-dependent manner. A xenograft model of CLVM was developed by injecting patient-derived EC into the flanks of immunocompromised mice. CLVM EC formed lesions with enlarged lymphatic and vascular channels, recapitulating the patient histology. EC subpopulations were further obtained by both immunomagnetic separation into lymphatic EC (LEC) and vascular EC (VEC) and generation of clonal populations. By sequencing these subpopulations, we determined that both LEC and VEC from the same patient express the PIK3CA mutation, exhibit increased AKT activation and can form lymphatic or vascular lesions in mouse.

Keywords:  AKT; Endothelial cell; Lymphatic malformation; PI3K; Patient-derived xenograft; Vascular; Vascular anomaly

DOI:  https://doi.org/10.1007/s10456-020-09722-0
Oncotarget. 2020 Apr 14. 11(15): 1373-1387

Superior cancer preventive efficacy of low versus high dose of mTOR inhibitor in a mouse model of prostate cancer.

Marina P Antoch, Michelle Wrobel, Bryan Gillard, Karen K Kuropatwinski, Ilia Toshkov, Anatoli S Gleiberman, Ellen Karasik, Michael T Moser, Barbara A Foster, Ekaterina L Andrianova, Olga V Chernova, Andrei V Gudkov.

  The mechanistic target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation and survival in response to the availability of energy sources and growth factors. Cancer development and progression is often associated with constitutive activation of the mTOR pathway, thus justifying mTOR inhibition as a promising approach to cancer treatment and prevention. However, development of previous rapamycin analogues has been complicated by their induction of adverse side effects and variable efficacy. Since mTOR pathway regulation involves multiple feedback mechanisms that may be differentially activated depending on the degree of mTOR inhibition, we investigated whether rapamycin dosing could be adjusted to achieve chemopreventive efficacy without side effects. Thus, we tested the efficacy of two doses of a novel, highly bioavailable nanoformulation of rapamycin, Rapatar, in a mouse prostate cancer model (male mice with prostate epithelium-specific Pten-knockout). We found that the highest efficacy was achieved by the lowest dose of Rapatar used in the study. While both doses tested were equally effective in suppressing proliferation of prostate epithelial cells, higher dose resulted in activation of feedback circuits that reduced the drug's tumor preventive efficacy. These results demonstrate that low doses of highly bioavailable mTOR inhibitor, Rapatar, may provide safe and effective cancer prevention.

Keywords:  PTEN; mTOR; prevention; prostate cancer; rapamycin

DOI:  https://doi.org/10.18632/oncotarget.27550
Nat Rev Cancer. 2020 Apr 27.

The duality of human oncoproteins: drivers of cancer and congenital disorders.

Pau Castel, Katherine A Rauen, Frank McCormick.

Human oncoproteins promote transformation of cells into tumours by dysregulating the signalling pathways that are involved in cell growth, proliferation and death. Although oncoproteins were discovered many years ago and have been widely studied in the context of cancer, the recent use of high-throughput sequencing techniques has led to the identification of cancer-associated mutations in other conditions, including many congenital disorders. These syndromes offer an opportunity to study oncoprotein signalling and its biology in the absence of additional driver or passenger mutations, as a result of their monogenic nature. Moreover, their expression in multiple tissue lineages provides insight into the biology of the proto-oncoprotein at the physiological level, in both transformed and unaffected tissues. Given the recent paradigm shift in regard to how oncoproteins promote transformation, we review the fundamentals of genetics, signalling and pathogenesis underlying oncoprotein duality.

DOI: https://doi.org/10.1038/s41568-020-0256-z
Cancer Med. 2020 Apr 30.

Buparlisib in combination with tamoxifen in pretreated patients with hormone receptor-positive, HER2-negative advanced breast cancer molecularly stratified for PIK3CA mutations and loss of PTEN expression.

Anja Welt, Marcel Wiesweg, Sarah Theurer, Wolfgang Abenhardt, Matthias Groschek, Lothar Müller, Jan Schröder, Mitra Tewes, Marco Chiabudini, Karin Potthoff, Agnes Bankfalvi, Norbert Marschner, Martin Schuler, Frank Breitenbücher.

  The PIKTAM study evaluated the efficacy and safety of the PI3K inhibitor buparlisib in combination with tamoxifen in hormone receptor-positive (HR+ ), HER2-negative advanced breast cancer patients after failure of prior endocrine therapy. In this open-label, single-arm phase II trial, 25 patients were enrolled in 11 sites in Germany. Patients were stratified according to PIK3CA mutation status (tissue and cfDNA from serum samples) and/or loss of PTEN expression. Patients received buparlisib (100 mg) and tamoxifen (20 mg) once daily on a continuous schedule (28-day cycle) until progression or unacceptable toxicity. Primary endpoint was overall 6-month progression-free survival (PFS) rate. Key secondary endpoints included the 6-month PFS rate in subpopulations, PFS, overall survival, overall response rate (ORR), disease control rate (DCR), and safety. Overall, the 6-month PFS rate was 33.3% (n/N = 7/21, one-sided 95% CI 16.8-100) and median PFS was 6.1 (CI 2.6-10.6) months. The ORR and DCR were 12.5% and 44%. The PIK3CA-mutated subgroup consistently showed the highest 6-month PFS rate (62.5%, n/N = 5/8), median PFS (8.7 months), ORR (40%), and DCR (80%). No new safety signals emerged. Most common adverse events were gastrointestinal disorders (56%), psychiatric/mood disorders (48%), skin rash/hypersensitivity (44%), cardiovascular (40%), and hepatic (32%) events. The trial was prematurely terminated due to the substantially altered risk-benefit profile of buparlisib. Nevertheless, PIK3CA mutations emerged as a clinically feasible and useful biomarker for combined PI3K inhibition and endocrine therapy in patients with HR+ breast cancer. Further biomarker-stratified studies with isoform-specific PI3K inhibitors are warranted. EudraCT No: 2014-000599-24.

Keywords:  PI3K; breast cancer; buparlisib; clinical trial; drug therapy; phase II

DOI:  https://doi.org/10.1002/cam4.3092
Nat Commun. 2020 Apr 29. 11(1): 2080

Partial impairment of insulin receptor expression mimics fasting to prevent diet-induced fatty liver disease.

Troy L Merry, Chris P Hedges, Stewart W Masson, Beate Laube, Doris Pöhlmann, Stephan Wueest, Michael E Walsh, Myrtha Arnold, Wolfgang Langhans, Daniel Konrad, Kim Zarse, Michael Ristow.

Excessive insulin signaling through the insulin receptor (IR) may play a role in the pathogenesis of diet-induced metabolic disease, including obesity and type 2 diabetes. Here we investigate whether heterozygous impairment of insulin receptor (IR) expression limited to peripheral, i.e. non-CNS, tissues of adult mice impacts the development of high-fat diet-induced metabolic deterioration. While exhibiting some features of insulin resistance, PerIRKO+/- mice display a hepatic energy deficit accompanied by induction of energy-sensing AMPK, mitochondrial biogenesis, PPARα, unexpectedly leading to protection from, and reversal of hepatic lipid accumulation (steatosis hepatis, NAFLD). Consistently, and unlike in control mice, the PPARα activator fenofibrate fails to further affect hepatic lipid accumulation in PerIRKO+/- mice. Taken together, and opposing previously established diabetogenic features of insulin resistance, incomplete impairment of insulin signaling may mimic central aspects of calorie restriction to limit hepatic lipid accumulation during conditions of metabolic stress.

DOI: https://doi.org/10.1038/s41467-020-15623-z
Br J Cancer. 2020 Apr 27.

Targeted therapy for mTORC1-driven tumours through HDAC inhibition by exploiting innate vulnerability of mTORC1 hyper-activation.

Fuchun Yang, Shaogang Sun, Chenran Wang, Michael Haas, Syn Yeo, Jun-Lin Guan.

BACKGOUND: The mechanistic target of rapamycin complex 1 (mTORC1) is important in the development and progression of many cancers. Targeted cancer therapy using mTORC1 inhibitors is used for treatment of cancers; however, their clinical efficacies are still limited.METHODS: We recently created a new mouse model for human lymphangiosarcoma by deleting Tsc1 in endothelial cells and consequent hyper-activation of mTORC1. Using Tsc1iΔEC tumour cells from this mouse model, we assessed the efficacies of histone deacetylase (HDAC) inhibitors as anti-tumour agents for mTORC1-driven tumours.
RESULTS: Unlike the cytostatic effect of mTORC1 inhibitors, HDAC inhibitors induced Tsc1iΔEC tumour cell death in vitro and their growth in vivo. Analysis of several HDAC inhibitors suggested stronger anti-tumour activity of class I HDAC inhibitor than class IIa or class IIb inhibitors, but these or pan HDAC inhibitor SAHA did not affect mTORC1 activation in these cells. Moreover, HDAC inhibitor-induced cell death required elevated autophagy, but was not affected by disrupting caspase-dependent apoptosis pathways. We also observed increased reactive oxygen species and endoplasmic reticulum stress in SAHA-treated tumour cells, suggesting their contribution to autophagic cell death, which were dependent on mTORC1 hyper-activation.
CONCLUSION: These studies suggest a potential new treatment strategy for mTORC1-driven cancers like lymphangiosarcoma through an alternative mechanism.

DOI: https://doi.org/10.1038/s41416-020-0839-1
Curr Treat Options Oncol. 2020 Apr 30. 21(6): 51

PI3K Inhibitors and Their Role as Novel Agents for Targeted Therapy in Lymphoma.

Vladimir Sapon-Cousineau, Sasha Sapon-Cousineau, Sarit Assouline.

  OPINION STATEMENT: Phosphatidylinositol 3-kinase (PI3K) inhibitors represent a novel class of agents targeting the key cellular regulatory PI3K/AKT/mTOR pathway involved in crucial functions such as cellular proliferation, cell cycle regulation, protein synthesis, and cell motility. This review starts with an overview of the PI3K pathway and the rationale for its targeting in lymphoma and potential on-target side effects of PI3K inhibition. With three agents now FDA approved for the treatment of relapsed and refractory (R/R) indolent non-Hodgkin lymphoma (iNHL), idelalisib, copanlisib, and duvelisib, we aim to review the pivotal trials leading to their approval as well as their clinical applications according to lymphoma subtypes. Important treatment-related adverse events are also reviewed and a perspective on the clinical role of these agents is provided, as well as some practical guidance on how to prevent, monitor, and manage potential adverse events in the clinic. PI3K inhibitors have an established role in the management of R/R iNHL, but their use and development are hampered by adverse events, particularly when used in combination with other anti-lymphoma therapies. Finally, this review highlights areas in need of more research in order to optimally use these agents in the care of patients with lymphoma.

Keywords:  Lymphoma; PI3K inhibitor; Phosphatidylinositol 3-kinase inhibitor; Phosphoinositide 3-kinase inhibitor; Treatment

DOI:  https://doi.org/10.1007/s11864-020-00746-8
Nat Commun. 2020 May 01. 11(1): 2142

Context specificity of the EMT transcriptional response.

David P Cook, Barbara C Vanderhyden.

Epithelial-mesenchymal plasticity contributes to many biological processes, including tumor progression. Various epithelial-mesenchymal transition (EMT) responses have been reported and no common, EMT-defining gene expression program has been identified. Here, we have performed a comparative analysis of the EMT response, leveraging highly multiplexed single-cell RNA sequencing (scRNA-seq) to measure expression profiles of 103,999 cells from 960 samples, comprising 12 EMT time course experiments and independent kinase inhibitor screens for each. We demonstrate that the EMT is vastly context specific, with an average of only 22% of response genes being shared between any two conditions, and over half of all response genes were restricted to 1-2 time course experiments. Further, kinase inhibitor screens revealed signaling dependencies and modularity of these responses. These findings suggest that the EMT is not simply a single, linear process, but is highly variable and modular, warranting quantitative frameworks for understanding nuances of the transition.

DOI: https://doi.org/10.1038/s41467-020-16066-2
J Biol Chem. 2020 Apr 29. pii: jbc.RA119.012425. [Epub ahead of print]

Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1).

Nileeka Balasuriya, Norman E Davey, Jared L Johnson, Huadong Liu, Kyle K Biggar, Lewis C Cantley, Shawn Shun-Cheng Li, Patrick O'Donoghue.

  Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 "known" substrates and two independent and larger oriented peptide array libraries (OPAL) of ~1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1Thr-308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.

Keywords:  Akt PKB; RNA metabolism; cell signaling; genetic code expansion; oriented peptide array library; peptide array; phosphoinositide- dependent kinase 1; phosphoseryl-tRNA synthetase; post-translational modification (PTM); protein phosphorylation; serine/threonine protein kinase; substrate specificity; tRNASep

DOI:  https://doi.org/10.1074/jbc.RA119.012425
Nature. 2020 Apr;580(7805): 640-646

The mutational landscape of normal human endometrial epithelium.

Luiza Moore, Daniel Leongamornlert, Tim H H Coorens, Mathijs A Sanders, Peter Ellis, Stefan C Dentro, Kevin J Dawson, Tim Butler, Raheleh Rahbari, Thomas J Mitchell, Francesco Maura, Jyoti Nangalia, Patrick S Tarpey, Simon F Brunner, Henry Lee-Six, Yvette Hooks, Sarah Moody, Krishnaa T Mahbubani, Mercedes Jimenez-Linan, Jan J Brosens, Christine A Iacobuzio-Donahue, Inigo Martincorena, Kourosh Saeb-Parsy, Peter J Campbell, Michael R Stratton.

All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium1,2. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.

DOI: https://doi.org/10.1038/s41586-020-2214-z
Cell Stem Cell. 2020 Apr 26. pii: S1934-5909(20)30142-9. [Epub ahead of print]

Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids.

Birgitta E Michels, Mohammed H Mosa, Barbara I Streibl, Tianzuo Zhan, Constantin Menche, Khalil Abou-El-Ardat, Tahmineh Darvishi, Ewelina Członka, Sebastian Wagner, Jan Winter, Hind Medyouf, Michael Boutros, Henner F Farin.

  Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor β (TGF-β) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC-/-;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.

Keywords:  clonal drift; colorectal cancer; human colonic stem cells; lentiviral barcoding; non-homologous end joining; patient-derived organoids; pooled-barcoded CRISPR-Cas9 screening; tumor microenvironment; tumor suppressor genes; unique molecular identifiers

DOI:  https://doi.org/10.1016/j.stem.2020.04.003
Trends Cancer. 2020 May;pii: S2405-8033(20)30073-X. [Epub ahead of print]6(5): 359-361

Redox Debt Leads to Metabolic Bankruptcy in Tumors.

Evan Quon, Madeleine L Hart, Lucas B Sullivan.

Lactate dehydrogenase (LDH) accounts for the fermentative component of aerobic glycolysis, a near ubiquitous metabolic alteration in cancer. Recently, Oshima et al. developed a bioavailable LDH inhibitor that decreases tumor growth in mice and functions synergistically with mitochondrial respiration inhibitors. These findings suggest a cooperative mechanism of action that targets redox homeostasis.

DOI: https://doi.org/10.1016/j.trecan.2020.02.012
Ophthalmic Genet. 2020 Apr 27. 1-5

Juvenile cataract in association with tuberous sclerosis complex.

A L Geffrey, K R Geenen, E Abati, S H Greenstein, D K VanderVeen, R L Levy, S L Davidson, M P McGarrey, E A Thiele, M E Aronow.

  Background: Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by benign hamartomas occurring in multiple organ systems including the brain, kidneys, heart, lungs, liver, skin, and the eyes. Typical retinal findings associated with TSC include astrocytic hamartoma and achromic patch. While rare cases of cataract occurring in the setting of TSC have been reported, this is the first analysis of a large series of individuals with TSC that aims to quantify the frequency of this finding and to describe its clinical and genetic associations.Materials and Methods: This is a retrospective chart review of 244 patients from the Herscot Center for Tuberous Sclerosis Complex at the Massachusetts General Hospital who underwent complete ophthalmic examination. We describe the clinical and genetic findings in five individuals with TSC and juvenile cataract.Results: Four of five cases (80%) were unilateral. The cataract was described as having an anterior subcapsular component in 3 of 5 cases (60%). Three individuals (60%) underwent lensectomy with intraocular lens (IOL) implant and two individuals (40%) were observed. Genetic testing revealed a known disease-causing mutation in TSC2 in 100% of cases.Conclusions: Recent evidence suggests that mTOR signaling may play a role in cataract formation which could explain the relatively high incidence of juvenile cataract in this population. Juvenile cataract is a potentially under-recognized ocular manifestation of TSC.

Keywords:  Tuberous sclerosis complex; cataract; juvenile

DOI:  https://doi.org/10.1080/13816810.2020.1755989
Biochem J. 2020 Apr 30. 477(8): 1427-1442

Cell Atlas technologies and insights into tissue architecture.

Anna Wilbrey-Clark, Kenny Roberts, Sarah A Teichmann.

  Since Robert Hooke first described the existence of 'cells' in 1665, scientists have sought to identify and further characterise these fundamental units of life. While our understanding of cell location, morphology and function has expanded greatly; our understanding of cell types and states at the molecular level, and how these function within tissue architecture, is still limited. A greater understanding of our cells could revolutionise basic biology and medicine. Atlasing initiatives like the Human Cell Atlas aim to identify all cell types at the molecular level, including their physical locations, and to make this reference data openly available to the scientific community. This is made possible by a recent technology revolution: both in single-cell molecular profiling, particularly single-cell RNA sequencing, and in spatially resolved methods for assessing gene and protein expression. Here, we review available and upcoming atlasing technologies, the biological insights gained to date and the promise of this field for the future.

Keywords:  cell atlasing; single-cell RNA sequencing; smFISH; spatial

DOI:  https://doi.org/10.1042/BCJ20190341
J Lipid Res. 2020 Apr 27. pii: jlr.D120000794. [Epub ahead of print]

A Direct Fluorometric Activity Assay for Lipid Kinases and Phosphatases.

Jiachen Sun, Indira Singaram, Mona Hoseini Soflaee, Wonhwa Cho.

  Lipid kinases and phosphatases play key roles in cell signaling and regulation, and are implicated in many human diseases, and are hence thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Due Because of to its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including Class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided the newdetailed mechanistic information about the product inhibition and substrate acyl acyl-chain selectivity of PI3K and allowed enabled rapid evaluation of its small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological functionThe assay also quantitatively determined the substrate specificity of PTEN, thereby providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.

Keywords:  Enzymology; Enzymology/Enzyme regulation; High-throughput screening; Lipid Kinases; PI3K; PTEN; Phosphatases/Lipid; Phosphoinositides; fluorescence assay

DOI:  https://doi.org/10.1194/jlr.D120000794