bims-pideca 2021-06-27 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2021‒06‒27
23 papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

mTORC1 Activation is Not Sufficient to Suppress Hepatic PPARα Signaling or Ketogenesis.
Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, oncogenesis, and HER2 inhibitor sensitivity.
mTOR Regulation of AGC Kinases: New Twist to an Old Tail.
Universal principles of lineage architecture and stem cell identity in renewing tissues.
Targeting therapy-resistant lung cancer stem cells via disruption of the AKT/TSPYL5/PTEN positive-feedback loop.
Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.
Akt scaffold proteins: the key to controlling specificity of Akt signaling.
The Transcriptional Co-Regulator CITED2 Suppresses Expression of IRS-2 and Impairs Insulin Signaling in Endothelial Cells.
Phosphatidylinositol 3-kinase signalling and immune regulation: insights into disease pathogenesis and clinical implications.
Single cell derived mRNA signals across human kidney tumors.
Clinical and molecular data in case of prenatal localized overgrowth disorders: major implication of genetic variants in the PI3K-AKT-mTOR signaling pathway.
CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function.
Lymphatic Malformations: Genetics, Mechanisms and Therapeutic Strategies.
PHENSIM: Phenotype Simulator.
Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment.
Targeting the HIF-1α-IGFBP2 axis therapeutically reduces IGF1-AKT signaling and blocks the growth and metastasis of relapsed anaplastic Wilms tumor.
A Novel Controlled PTEN-Knockout Mouse Model for Prostate Cancer Study.
Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.
Josep Baselga (1959-2021).
A community challenge to evaluate RNA-seq, fusion detection, and isoform quantification methods for cancer discovery.
Generation of locus-specific degradable tag knock-ins in mouse and human cell lines.
Potential for Long-Term Disease Control with Alpelisib Plus Fulvestrant Spans Patient Subgroups in HR+ PIK3CA-Mutated Advanced Breast Cancer.
Multi-omic analysis of lung tumors defines pathways activated in neuroendocrine transformation.

J Biol Chem. 2021 Jun 16. pii: S0021-9258(21)00684-0. [Epub ahead of print] 100884

mTORC1 Activation is Not Sufficient to Suppress Hepatic PPARα Signaling or Ketogenesis.

Ebru S Selen, Michael J Wolfgang.

  The mechanistic target of rapamycin (mTOR) is often referred to as a master regulator of cellular metabolism that can integrate growth factor and nutrient signaling. Fasting suppresses hepatic mTORC1 activity via the activity of the Tuberous Sclerosis Complex (TSC), a negative regulator of mTORC1, in order to suppress anabolic metabolism. The loss of TSC1 in the liver locks the liver in a constitutively anabolic state even during fasting, which was suggested to regulate PPARα signaling and ketogenesis, but the molecular determinants of this regulation are unknown. Here, we examined if the activation of the mTORC1 complex in mice by the liver-specific deletion of TSC1 (TSC1L-/-) is sufficient to suppress PPARα signaling and therefore ketogenesis in the fasted state. We found that the activation of mTORC1 in the fasted state is not sufficient to repress PPARα-responsive genes or ketogenesis. Further, we examined whether the activation of the anabolic program mediated by mTORC1 complex activation in the fasted state could suppress the robust catabolic programming and enhanced PPARα transcriptional response of mice with a liver-specific defect in mitochondrial long-chain fatty acid oxidation using Cpt2L-/- mice. We generated liver-specific Cpt2L-/-; Tsc1L-/- double knockout mice and showed that the activation of mTORC1 by deletion of TSC1 could not suppress the catabolic PPARα-mediated phenotype of Cpt2L-/- mice. These data demonstrate that the activation of mTORC1 by the deletion of TSC1 is not sufficient to suppress a PPARα transcriptional program or ketogenesis following fasting.

Keywords:  Ketogenesis; carnitine palmitoyltransferase 2 (Cpt2); fatty acid oxidation; mTOR; metabolism; peroxisome proliferator-activated receptor alpha (PPARα); β-hydroxybutyrate (βHB)

DOI:  https://doi.org/10.1016/j.jbc.2021.100884
Cancer Cell. 2021 Jun 22. pii: S1535-6108(21)00284-1. [Epub ahead of print]

Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, oncogenesis, and HER2 inhibitor sensitivity.

Ariella B Hanker, Benjamin P Brown, Jens Meiler, Arnaldo Marín, Harikrishna S Jayanthan, Dan Ye, Chang-Ching Lin, Hiroaki Akamatsu, Kyung-Min Lee, Sumanta Chatterjee, Dhivya R Sudhan, Alberto Servetto, Monica Red Brewer, James P Koch, Jonathan H Sheehan, Jie He, Alshad S Lalani, Carlos L Arteaga.

  Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.

Keywords:  HER2; HER3; PI3K; Rosetta; breast cancer; molecular dynamics; neratinib; personalized structural biology; precision oncology

DOI:  https://doi.org/10.1016/j.ccell.2021.06.001
Mol Pharmacol. 2021 Jun 21. pii: MOLPHARM-MR-2021-000310. [Epub ahead of print]

mTOR Regulation of AGC Kinases: New Twist to an Old Tail.

Timothy R Baffi, Alexandra C Newton.

  The family of AGC kinases not only regulate cellular biology by phosphorylating substrates, but are themselves controlled by phosphorylation. Phosphorylation generally occurs at two conserved regions in these kinases: a loop near the entrance to the active site, termed the activation loop, that correctly aligns residues for catalysis, and a C-terminal tail whose phosphorylation at a site termed the hydrophobic motif stabilizes the active conformation. Whereas phosphorylation of the activation loop is well established to be catalyzed by the phosphoinositide-dependent kinase 1 (PDK1), the mechanism of phosphorylation of the C-tail hydrophobic motif has been controversial. For a subset of AGC kinases, which includes most protein kinase C (PKC) isozymes and Akt, phosphorylation of the hydrophobic motif in cells was shown to depend on mTORC2 over 15 years ago, yet whether by direct phosphorylation or by another mechanism has remained elusive. The recent identification of a novel and evolutionarily conserved phosphorylation site on the C-tail termed the TOR-Interaction Motif (TIM) has finally unraveled the mystery of how mTORC2 regulates its client kinases. mTORC2 does not directly phosphorylate the hydrophobic motif, rather it converts kinases such as PKC and Akt into a conformation that can ultimately autophosphorylate at the hydrophobic motif. Identification of the direct mTOR phosphorylation that facilitates auto-regulation of the C-tail hydrophobic motif revises the activation mechanisms of mTOR-regulated AGC kinases. This new twist to an old tail opens avenues for therapeutic intervention. Significance Statement The enzyme mTORC2 has been an enigmatic regulator of AGC kinases such as protein kinase C (PKC) and Akt. The recent discovery of a motif named the TOR Interaction Motif in the C-tail of these kinases solves the mystery: mTORC2 marks these kinases for maturity by, ultimately, facilitating autophosphorylation another C-tail site, the hydrophobic motif.

Keywords:  AKT; Protein Kinase C (PKC); mTOR

DOI:  https://doi.org/10.1124/molpharm.121.000310
Development. 2021 Apr 15. pii: dev.194399. [Epub ahead of print]

Universal principles of lineage architecture and stem cell identity in renewing tissues.

Philip Greulich, Ben D MacArthur, Cristina Parigini, Rubén J Sánchez-García.

  Adult tissues in multicellular organisms typically contain a variety of stem, progenitor and differentiated cell types arranged in a lineage hierarchy that regulates healthy tissue turnover. Lineage hierarchies in disparate tissues often exhibit common features, yet the general principles regulating their architecture are not known. Here, we provide a formal framework for understanding the relationship between cell molecular 'states' and cell 'types', based on the topology of admissible cell state trajectories. We show that a self-renewing cell type - if defined as suggested by this framework - must reside at the top of any homeostatic renewing lineage hierarchy, and only there. This architecture arises as a natural consequence of homeostasis, and indeed is the only possible way that lineage architectures can be constructed to support homeostasis in renewing tissues. Furthermore, under suitable feedback regulation, for example from the stem cell niche, we show that the property of 'stemness' is entirely determined by the cell environment, in accordance with the notion that stem cell identities are contextual and not determined by hard-wired, cell-intrinsic, characteristics.

Keywords:  Cell state; Cell trajectories; Cell type; Self-renewal; Stem cell fate choice; Stem cell lineage

DOI:  https://doi.org/10.1242/dev.194399
Commun Biol. 2021 Jun 23. 4(1): 778

Targeting therapy-resistant lung cancer stem cells via disruption of the AKT/TSPYL5/PTEN positive-feedback loop.

In-Gyu Kim, Jei-Ha Lee, Seo-Yeon Kim, Chang-Kyu Heo, Rae-Kwon Kim, Eun-Wie Cho.

Cancer stem cells (CSCs) are regarded as essential targets to overcome tumor progression and therapeutic resistance; however, practical targeting approaches are limited. Here, we identify testis-specific Y-like protein 5 (TSPYL5) as an upstream regulator of CSC-associated genes in non-small cell lung cancer cells, and suggest as a therapeutic target for CSC elimination. TSPYL5 elevation is driven by AKT-dependent TSPYL5 phosphorylation at threonine-120 and stabilization via inhibiting its ubiquitination. TSPYL5-pT120 also induces nuclear translocation and functions as a transcriptional activator of CSC-associated genes, ALDH1 and CD44. Also, nuclear TSPYL5 suppresses the transcription of PTEN, a negative regulator of PI3K signaling. TSPYL5-pT120 maintains persistent CSC-like characteristics via transcriptional activation of CSC-associated genes and a positive feedback loop consisting of AKT/TSPYL5/PTEN signaling pathway. Accordingly, elimination of TSPYL5 by inhibiting TSPYL5-pT120 can block aberrant AKT/TSPYL5/PTEN cyclic signaling and TSPYL5-mediated cancer stemness regulation. Our study suggests TSPYL5 be an effective target for therapy-resistant cancer.

DOI: https://doi.org/10.1038/s42003-021-02303-x
Nat Chem Biol. 2021 Jun 24.

Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

Bianca J Lee, Jacob A Boyer, G Leslie Burnett, Arun P Thottumkara, Nidhi Tibrewal, Stacy L Wilson, Tientien Hsieh, Abby Marquez, Edward G Lorenzana, James W Evans, Laura Hulea, Gert Kiss, Hui Liu, Dong Lee, Ola Larsson, Shannon McLaughlan, Ivan Topisirovic, Zhengping Wang, Zhican Wang, Yongyuan Zhao, David Wildes, James B Aggen, Mallika Singh, Adrian L Gill, Jacqueline A M Smith, Neal Rosen.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.

DOI: https://doi.org/10.1038/s41589-021-00813-7
Am J Physiol Cell Physiol. 2021 06 23.

Akt scaffold proteins: the key to controlling specificity of Akt signaling.

Fan Bao, Peiqi Hao, Su An, Yang Yang, Ying Liu, Qian Hao, Ejaz Mubashir, Xiao-Xi Guo, Tian-Rui Xu.

  The PI3K-Akt signaling pathway plays an essential role in regulating cell proliferation and apoptosis. Akt kinase is at the center of this signaling pathway and interacts with a variety of proteins. Overexpression of Akt has been found in almost 80% of tumors, however, inhibiting Akt has serious clinical side effects so is not a suitable treatment for cancer. During recent years, Akt scaffold proteins have received increasing attention for their ability to regulate Akt signaling and have emerged as potential targets for cancer therapy. In this paper, we categorize AKT kinase scaffold proteins into four groups based on their cellular location: membrane-bound activator and inhibitor, cytoplasm, and endosome. We describe how these scaffolds interact with Akt kinase, how they affect AKT activity, and how they regulate the specificity of Akt signaling. We also discuss the clinical application of Akt-scaffold proteins as targets for cancer therapy.

Keywords:  Akt/PKB; cancer; interactome; phosphorylation; scaffold protein

DOI:  https://doi.org/10.1152/ajpcell.00146.2020
Am J Physiol Endocrinol Metab. 2021 06 21.

The Transcriptional Co-Regulator CITED2 Suppresses Expression of IRS-2 and Impairs Insulin Signaling in Endothelial Cells.

Britta Kunkemoeller, Kuangyang Chen, Sam M Lockhart, Xuanchun Wang, Christian Rask-Madsen.

  Endothelial cell insulin resistance contributes to the development of vascular complications in diabetes. Hypoxia-inducible factors (HIF) modulate insulin sensitivity, and we have previously shown that a negative regulator of HIF activity, CBP/p300 interacting transactivator-2 (CITED2), is increased in the vasculature of people with type 2 diabetes. Therefore, we examined whether CITED2 regulates endothelial insulin sensitivity. In endothelial cells isolated from mice with a "floxed" mutation in the Cited2 gene, loss of CITED2 markedly enhanced insulin-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation. Similarly, insulin-stimulated Akt phosphorylation was increased in aortas of mice with endothelial-specific deletion of CITED2. Consistent with these observations, loss of CITED2 in endothelial cells increased insulin-stimulated endothelial nitric oxide synthase phosphorylation, Vegfa expression, and cell proliferation. Endothelial cells lacking CITED2 exhibited an increase in insulin receptor substrate (IRS)-2 protein, a key mediator of the insulin signaling cascade, while IRS-1 was unchanged. Conversely, overexpression of CITED2 in endothelial cells decreased IRS-2 protein by 55% without altering IRS-1, resulting in impaired insulin-stimulated Akt phosphorylation and Vegfa expression. Overexpression of HIF-2α significantly increased activity of the Irs2 promoter and co-expression of CITED2 abolished this increase. Moreover, ChIP showed that loss of CITED2 increased occupancy of p300, a key component of the HIF transcriptional complex, on the Irs2 promoter. Together, these results show that CITED2 selectively inhibits endothelial insulin signaling and action through the PI3K/Akt pathway via repression of HIF-dependent IRS-2 expression. CITED2 is thus a promising target to improve endothelial insulin sensitivity and prevent the vascular complications of diabetes.

Keywords:  endothelium; hypoxia-inducible factor; insulin resistance; insulin signaling

DOI:  https://doi.org/10.1152/ajpendo.00435.2020
Expert Rev Clin Immunol. 2021 Jun 22.

Phosphatidylinositol 3-kinase signalling and immune regulation: insights into disease pathogenesis and clinical implications.

Tina Nguyen, Elissa K Deenick, Stuart G Tangye.

  INTRODUCTION: Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that plays a key role in fundamental aspects of cell biology including survival, metabolism, proliferation and differentiation. Thus, balanced PI3K signalling is critical for multiple aspects of human health. The initial discovery that germline variants in genes in the PI3K pathway resulted in inborn errors of immunity highlighted the non-redundant role of these signalling proteins in the human immune system. The subsequent identification and characterisation of >250 individuals with a novel immune dysregulatory disorder, termed activated PI3K-delta syndrome (APDS), has reinforced the status of PI3K as a key pathway regulating immune function. Studies of APDS have demonstrated that dysregulated PI3K function is disruptive for many immune cell processes including development, activation, differentiation, effector function and self-tolerance, which are all important in supporting effective, long-term immune responses.AREAS COVERED: In this review, we recount recent findings regarding humans with germline variants in PI3K genes and discuss the underlying cellular and molecular pathologies, with a focus on the implications of these findings for therapy in APDS patients.
EXPERT OPINION: Fine tuning immune signalling by modulating PI3K offers opportunities for therapeutic interventions in settings of immunodeficiency, autoimmunity and malignancy, but also highlights potential adverse events that may result from overt pharmacological or intrinsic inhibition of PI3K function.

Keywords:  B-cell development; PI3K; autoimmunity; human B cells; immune dysregulation; immunodeficiency; phosphatidylinositol-3-kinase; therapeutics; transitional B cells

DOI:  https://doi.org/10.1080/1744666X.2021.1945443
Nat Commun. 2021 06 23. 12(1): 3896

Single cell derived mRNA signals across human kidney tumors.

Matthew D Young, Thomas J Mitchell, Lars Custers, Thanasis Margaritis, Francisco Morales-Rodriguez, Kwasi Kwakwa, Eleonora Khabirova, Gerda Kildisiute, Thomas R W Oliver, Ronald R de Krijger, Marry M van den Heuvel-Eibrink, Federico Comitani, Alice Piapi, Eva Bugallo-Blanco, Christine Thevanesan, Christina Burke, Elena Prigmore, Kirsty Ambridge, Kenny Roberts, Felipe A Vieira Braga, Tim H H Coorens, Ignacio Del Valle, Anna Wilbrey-Clark, Lira Mamanova, Grant D Stewart, Vincent J Gnanapragasam, Dyanne Rampling, Neil Sebire, Nicholas Coleman, Liz Hook, Anne Warren, Muzlifah Haniffa, Marcel Kool, Stefan M Pfister, John C Achermann, Xiaoling He, Roger A Barker, Adam Shlien, Omer A Bayraktar, Sarah A Teichmann, Frank C Holstege, Kerstin B Meyer, Jarno Drost, Karin Straathof, Sam Behjati.

Tumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer.

DOI: https://doi.org/10.1038/s41467-021-23949-5
Ultrasound Obstet Gynecol. 2021 Jun 25.

Clinical and molecular data in case of prenatal localized overgrowth disorders: major implication of genetic variants in the PI3K-AKT-mTOR signaling pathway.

N Bourgon, V Carmignac, A Sorlin, Y Duffourd, C Philippe, C Thauvin-Robinet, L Guibaud, L Faivre, P Vabres, P Kuentz, .

  OBJECTIVES: To describe clinical and molecular findings in fetuses with localized overgrowth disorders (LOD) involving the brain (BO) and/or a limb (LO) diagnosed on prenatal imaging (ultrasonography or MRI), suggesting involvement of the PI3K-AKT-mTOR signaling pathway.METHODS: We retrospectively analyzed data from 21 fetuses with BO and/or LO referred for next-generation sequencing (NGS) molecular diagnosis of PI3K-AKT-mTOR pathway genes on affected tissue obtained on fetal autopsy. We assessed the diagnostic yield on amniotic fluid.
RESULTS: Among 17 fetuses with BO, 6 had megalencephaly (MEG) and 11 had hemimegalencephaly (HMEG). In MEG, germline variants were identified in 4 cases, either in PIK3R2, AKT3, or MTOR, and a postzygotic PIK3R2 variant was found in 2 cases. In HMEG, a postzygotic PIK3CA variant was found in 3 fetuses with extra-cerebral features of PIK3CA-related overgrowth spectrum (PROS), and in 7 fetuses with "isolated HMEG". Four fetuses with LO also had lymphatic malformations (LM) and harbored a postzygotic PIK3CA variant. NGS on cultured amniocytes performed in 10 cases showed variants in 5 cases, either in PIK3CA, PIK3R2, or AKT3.
CONCLUSIONS: Isolated MEG or HMEG led to the identification of genetic variants in the PI3K-AKT-mTOR signaling pathway. Prenatally diagnosed cases of LO and LM were associated with PIK3CA variants. This article is protected by copyright. All rights reserved.

Keywords:  Localized overgrowth disorders; PI3K-AKT-mTOR; cultured amniocytes; genetic counselling; postzygotic; prenatal diagnosis

DOI:  https://doi.org/10.1002/uog.23715
Nat Commun. 2021 06 23. 12(1): 3906

CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function.

Saumil Sethna, Patrick A Scott, Arnaud P J Giese, Todd Duncan, Xiaoying Jian, Sheikh Riazuddin, Paul A Randazzo, T Michael Redmond, Steven L Bernstein, Saima Riazuddin, Zubair M Ahmed.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling-a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to 'nucleotide empty' or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

DOI: https://doi.org/10.1038/s41467-021-24056-1
Circ Res. 2021 Jun 25. 129(1): 136-154

Lymphatic Malformations: Genetics, Mechanisms and Therapeutic Strategies.

Taija Mäkinen, Laurence M Boon, Miikka Vikkula, Kari Alitalo.

  Lymphatic vessels maintain tissue fluid homeostasis by returning to blood circulation interstitial fluid that has extravasated from the blood capillaries. They provide a trafficking route for cells of the immune system, thus critically contributing to immune surveillance. Developmental or functional defects in the lymphatic vessels, their obstruction or damage, lead to accumulation of fluid in tissues, resulting in lymphedema. Here we discuss developmental lymphatic anomalies called lymphatic malformations and complex lymphatic anomalies that manifest as localized or multifocal lesions of the lymphatic vasculature, respectively. They are rare diseases that are caused mostly by somatic mutations and can present with variable symptoms based upon the size and location of the lesions composed of fluid-filled cisterns or channels. Substantial progress has been made recently in understanding the molecular basis of their pathogenesis through the identification of their genetic causes, combined with the elucidation of the underlying mechanisms in animal disease models and patient-derived lymphatic endothelial cells. Most of the solitary somatic mutations that cause lymphatic malformations and complex lymphatic anomalies occur in genes that encode components of oncogenic growth factor signal transduction pathways. This has led to successful repurposing of some targeted cancer therapeutics to the treatment of lymphatic malformations and complex lymphatic anomalies. Apart from the mutations that act as lymphatic endothelial cell-autonomous drivers of these anomalies, current evidence points to superimposed paracrine mechanisms that critically contribute to disease pathogenesis and thus provide additional targets for therapeutic intervention. Here, we review these advances and discuss new treatment strategies that are based on the recently identified molecular pathways.

Keywords:  capillaries; lymphangiogenesis; mutation; neoplasms; sirolimus

DOI:  https://doi.org/10.1161/CIRCRESAHA.121.318142
PLoS Comput Biol. 2021 Jun;17(6): e1009069

PHENSIM: Phenotype Simulator.

Salvatore Alaimo, Rosaria Valentina Rapicavoli, Gioacchino P Marceca, Alessandro La Ferlita, Oksana B Serebrennikova, Philip N Tsichlis, Bud Mishra, Alfredo Pulvirenti, Alfredo Ferro.

Despite the unprecedented growth in our understanding of cell biology, it still remains challenging to connect it to experimental data obtained with cells and tissues' physiopathological status under precise circumstances. This knowledge gap often results in difficulties in designing validation experiments, which are usually labor-intensive, expensive to perform, and hard to interpret. Here we propose PHENSIM, a computational tool using a systems biology approach to simulate how cell phenotypes are affected by the activation/inhibition of one or multiple biomolecules, and it does so by exploiting signaling pathways. Our tool's applications include predicting the outcome of drug administration, knockdown experiments, gene transduction, and exposure to exosomal cargo. Importantly, PHENSIM enables the user to make inferences on well-defined cell lines and includes pathway maps from three different model organisms. To assess our approach's reliability, we built a benchmark from transcriptomics data gathered from NCBI GEO and performed four case studies on known biological experiments. Our results show high prediction accuracy, thus highlighting the capabilities of this methodology. PHENSIM standalone Java application is available at https://github.com/alaimos/phensim, along with all data and source codes for benchmarking. A web-based user interface is accessible at https://phensim.tech/.

DOI: https://doi.org/10.1371/journal.pcbi.1009069
Nat Protoc. 2021 Jun 25.

Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment.

Stefan J Tekel, Nicholas Brookhouser, Kylie Standage-Beier, Xiao Wang, David A Brafman.

Deaminase fused-Cas9 base editing technologies have enabled precise single-nucleotide genomic editing without the need for the introduction of damaging double-stranded breaks and inefficient homology-directed repair. However, current methods to isolate base-edited cell populations are ineffective, especially when utilized with human pluripotent stem cells, a cell type resistant to genome modification. Here, we outline a series of methods that employ transient reporters of editing enrichment (TREE) to facilitate the highly efficient single-base editing of human cells at precise genomic loci. Briefly, these transient reporters of editing enrichment based methods employ a transient episomal fluorescent reporter that allows for the real-time, flow-cytometry-based enrichment of cells that have had single nucleotide changes at precise genomic locations. This protocol details how these approaches can enable the rapid (~3-4 weeks) and efficient (clonal editing efficiencies >80%) generation of biallelic or multiplexed edited isogenic hPSC lines using adenosine and cytosine base editors.

DOI: https://doi.org/10.1038/s41596-021-00552-y
Oncogene. 2021 Jun 21.

Targeting the HIF-1α-IGFBP2 axis therapeutically reduces IGF1-AKT signaling and blocks the growth and metastasis of relapsed anaplastic Wilms tumor.

Yan Liu, Marie V Nelson, Christopher Bailey, Peng Zhang, Pan Zheng, Jeffrey S Dome, Yang Liu, Yin Wang.

For patients with anaplastic Wilms tumor (WiT), metastasis and recurrence are common, and prognosis is generally poor. Novel therapies are needed to improve outcomes for patients with this high-risk WiT. A potential contributor to WiT development is constitutive activation of AKT by insulin-like growth factor 1 (IGF1) and its receptor (IGF1R) signaling pathway, but the complete underlying mechanism remains unclear. Here, we demonstrate that the hypoxia-inducible factor 1α (HIF-1α)-IGF binding protein 2 (IGFBP2) axis and the tumor-specific IGF1A are key players for constitutive activation of IGF1-AKT signaling leading to the tumor malignancy. HIF-1α and IGFBP2 are highly expressed in a majority of WiT patient samples. Deficiency of either HIF-1α or IGFBP2 or IGF1 in the tumor cells significantly impairs tumor growth and nearly abrogates metastasis in xenografted mice. Pharmacologic targeting of HIF-1α by echinomycin delivered via nanoliposomes can efficiently restrain growth and metastasis of patient-derived relapsed anaplastic WiT xenografts. Liposomal echinomycin is more potent and effective in inhibiting WiT growth than vincristine in an anaplastic WiT mouse model, and eliminates metastasis by suppressing HIF-1α targets and the HIF-1α-IGFBP2 axis, which governs IGF1-AKT signaling.

DOI: https://doi.org/10.1038/s41388-021-01907-1
Front Mol Biosci. 2021 ;8 696537

A Novel Controlled PTEN-Knockout Mouse Model for Prostate Cancer Study.

Sen Liu, Bing Zhang, Brian G Rowan, S Michal Jazwinski, Asim B Abdel-Mageed, Chad Steele, Alun R Wang, Oliver Sartor, Tianhua Niu, Qiuyang Zhang.

  Prostate cancer (PCa) is associated with advanced age, but how age contributes to prostate carcinogenesis remains unknown. The prostate-specific Pten conditional knockout mouse model closely imitates human PCa initiation and progression. To better understand how age impacts PCa in an experimental model, we have generated a spatially and temporally controlled Pten-null PCa murine model at different ages (aged vs. non-aged) of adult mice. Here, we present a protocol to inject the Cre-expressing adenovirus with luciferin tag, intraductally, into the prostate anterior lobes of Pten-floxed mice; Pten-loss will be triggered post-Cre expression at different ages. In vivo imaging of luciferin signal following viral infection confirmed successful delivery of the virus and Cre activity. Immunohistochemical staining confirmed prostate epithelial-specific expression of Cre recombinase and the loss of Pten and activation of P-Akt, P-S6, and P-4E-BP1. The Cre-expression, Pten ablation, and activated PI3K/AKT/mTOR pathways were limited to the prostate epithelium. All mice developed prostatic epithelial hyperplasia within 4 weeks after Pten ablation and prostatic intraepithelial neoplasia (PIN) within 8 weeks post-Pten ablation. Some PINs had progressed to invasive adenocarcinoma at 8-16 weeks post-Pten ablation. Aged mice exhibited significantly accelerated PI3K/AKT/mTOR signaling and increased PCa onset and progression compared to young mice. The viral infection success rate is ∼80%. This model will be beneficial for investigations of cancer-related to aging.

Keywords:  Pten; age; cre-expressing adenovirus; mouse models; prostate cancer

DOI:  https://doi.org/10.3389/fmolb.2021.696537
Nat Biotechnol. 2021 Jun 21.

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.

Ping Xu, Zhiheng Liu, Ying Liu, Huazheng Ma, Yiyuan Xu, Ying Bao, Shiyou Zhu, Zhongzheng Cao, Zeguang Wu, Zhuo Zhou, Wensheng Wei.

Canonical CRISPR-knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR-KO screens in various settings.

DOI: https://doi.org/10.1038/s41587-021-00944-1
Cell. 2021 Jun 24. pii: S0092-8674(21)00686-3. [Epub ahead of print]184(13): 3349-3351

Josep Baselga (1959-2021).

Pau Castel, Eneda Toska.

DOI: https://doi.org/10.1016/j.cell.2021.05.034
Cell Syst. 2021 Jun 16. pii: S2405-4712(21)00207-6. [Epub ahead of print]

A community challenge to evaluate RNA-seq, fusion detection, and isoform quantification methods for cancer discovery.

Allison Creason, David Haan, Kristen Dang, Kami E Chiotti, Matthew Inkman, Andrew Lamb, Thomas Yu, Yin Hu, Thea C Norman, Alex Buchanan, Jeltje van Baren, Ryan Spangler, M Rick Rollins, Paul T Spellman, Dmitri Rozanov, Jin Zhang, Christopher A Maher, Cristian Caloian, John D Watson, Sebastian Uhrig, Brian J Haas, Miten Jain, Mark Akeson, Mehmet Eren Ahsen, , Gustavo Stolovitzky, Justin Guinney, Paul C Boutros, Joshua M Stuart, Kyle Ellrott.

  The accurate identification and quantitation of RNA isoforms present in the cancer transcriptome is key for analyses ranging from the inference of the impacts of somatic variants to pathway analysis to biomarker development and subtype discovery. The ICGC-TCGA DREAM Somatic Mutation Calling in RNA (SMC-RNA) challenge was a crowd-sourced effort to benchmark methods for RNA isoform quantification and fusion detection from bulk cancer RNA sequencing (RNA-seq) data. It concluded in 2018 with a comparison of 77 fusion detection entries and 65 isoform quantification entries on 51 synthetic tumors and 32 cell lines with spiked-in fusion constructs. We report the entries used to build this benchmark, the leaderboard results, and the experimental features associated with the accurate prediction of RNA species. This challenge required submissions to be in the form of containerized workflows, meaning each of the entries described is easily reusable through CWL and Docker containers at https://github.com/SMC-RNA-challenge. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords:  Cancer; Cloud compute; DREAM Challenge; RNA fusion; RNA-seq; benchmark; crowd-sourced; evaluation; isoform quantification

DOI:  https://doi.org/10.1016/j.cels.2021.05.021
STAR Protoc. 2021 Jun 18. 2(2): 100575

Generation of locus-specific degradable tag knock-ins in mouse and human cell lines.

Helene Damhofer, Aliaksandra Radzisheuskaya, Kristian Helin.

  Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol for the efficient generation of biallelic degradation tag knock-ins in mouse and human cell lines using CRISPR-Cas9. We use knockin of an endogenous Kansl3 degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types. For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021).

Keywords:  CRISPR; Genetics; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2021.100575
Oncologist. 2021 Jun 26.

Potential for Long-Term Disease Control with Alpelisib Plus Fulvestrant Spans Patient Subgroups in HR+ PIK3CA-Mutated Advanced Breast Cancer.

DOI: https://doi.org/10.1002/onco.13873
Cancer Discov. 2021 Jun 21. pii: candisc.1863.2021. [Epub ahead of print]

Multi-omic analysis of lung tumors defines pathways activated in neuroendocrine transformation.

Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yingqian A Zhan, Maysun M Hasan, Shweta S Chavan, Fanli Meng, Fathema Uddin, Parvathy Manoj, Mark T A Donoghue, Helen H Won, Joseph M Chan, Metamia Ciampricotti, Andrew Chow, Michael Offin, Jason C Chang, Jordana Ray-Kirton, Sam E Tischfield, Jacklynn Egger, Umesh K Bhanot, Irina Linkov, Marina Asher, Sonali Sinha, Joachim Silber, Christine A Iacobuzio-Donahue, Michael H Roehrl, Travis J Hollmann, Helena A Yu, Juan Qiu, Elisa de Stanchina, Marina K Baine, Natasha Rekhtman, John T Poirier, Brian Loomis, Richard P Koche, Charles M Rudin, Triparna Sen.

Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUADs) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre-/post-transformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre-/post-transformation samples, support that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacological inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of neuroendocrine transformation in lung cancer.

DOI: https://doi.org/10.1158/2159-8290.CD-20-1863