bims-pideca 2022-09-11 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2022–09–11
27 papers selected by
Ralitsa Radostinova Madsen, University College London

Phospholipid Scramblase 4 (PLSCR4) Regulates Adipocyte Differentiation via PIP3-Mediated AKT Activation.
Double Deletion of PI3K and PTEN Modifies Lens Postnatal Growth and Homeostasis.
mTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.
Alpelisib for the treatment of PIK3CA-related head and neck lymphatic malformations and overgrowth.
Long-term treatment with low dose rapamycin extends lifespan and delays tumours in cancer-prone germline PTEN mutant mice.
High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER+ breast cancer.
Hyperactivation of mTOR and AKT in a cardiac hypertrophy animal model of Friedreich ataxia.
Akt isoforms in the immune system.
Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates.
Genetically manipulating endogenous Kras levels and oncogenic mutations in vivo influences tissue patterning of murine tumorigenesis.
Comparative roadmaps of reprogramming and oncogenic transformation identify Bcl11b and Atoh8 as broad regulators of cellular plasticity.
The crosstalk between MYC and mTORC1 during osteoclastogenesis.
Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.
Integrating temporal single-cell gene expression modalities for trajectory inference and disease prediction.
The potential application of branch-PCR assembled PTEN gene nanovector in lung cancer gene therapy.
Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks.
Modelling metabolic diseases and drug response using stem cells and organoids.
How we approach complex vascular anomalies and overgrowth syndromes.
Modelling aggressive prostate cancers of young men in immune-competent mice, driven by isogenic Trp53 alterations and Pten loss.
Integrating and formatting biomedical data as pre-calculated knowledge graph embeddings in the Bioteque.
Live cold to grow old? Thermogenesis to fight cancer.
Highly efficient generation of isogenic pluripotent stem cell models using prime editing.
Large-scale genomic analysis of human iPSCs identifies recurrent somatic driver mutations.
Sustained potentiation of bystander killing via PTEN-loss driven macropinocytosis targeted peptide-drug conjugate therapy in metastatic triple-negative breast cancer.
Virtual screening for small-molecule pathway regulators by image-profile matching.
An R-based reproducible and user-friendly preprocessing pipeline for CyTOF data.
Multiple ciliary localization signals control INPP5E ciliary targeting.

Int J Mol Sci. 2022 Aug 29. pii: 9787. [Epub ahead of print]23(17):

Phospholipid Scramblase 4 (PLSCR4) Regulates Adipocyte Differentiation via PIP3-Mediated AKT Activation.

Lisa A G Barth, Michèle Nebe, Hermann Kalwa, Akhil Velluva, Stephanie Kehr, Florentien Kolbig, Patricia Prabutzki, Wieland Kiess, Diana Le Duc, Antje Garten, Anna S Kirstein.

  Phospholipid scramblase 4 (PLSCR4) is a member of a conserved enzyme family with high relevance for the remodeling of phospholipid distribution in the plasma membrane and the regulation of cellular signaling. While PLSCR1 and -3 are involved in the regulation of adipose-tissue expansion, the role of PLSCR4 is so far unknown. PLSCR4 is significantly downregulated in an adipose-progenitor-cell model of deficiency for phosphatase and tensin homolog (PTEN). PTEN acts as a tumor suppressor and antagonist of the growth and survival signaling phosphoinositide 3-kinase (PI3K)/AKT cascade by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (PIP3). Patients with PTEN germline deletion frequently develop lipomas. The underlying mechanism for this aberrant adipose-tissue growth is incompletely understood. PLSCR4 is most highly expressed in human adipose tissue, compared with other phospholipid scramblases, suggesting a specific role of PLSCR4 in adipose-tissue biology. In cell and mouse models of lipid accumulation, we found PLSCR4 to be downregulated. We observed increased adipogenesis in PLSCR4-knockdown adipose progenitor cells, while PLSCR4 overexpression attenuated lipid accumulation. PLSCR4 knockdown was associated with increased PIP3 levels and the activation of AKT. Our results indicated that PLSCR4 is a regulator of PI3K/AKT signaling and adipogenesis and may play a role in PTEN-associated adipose-tissue overgrowth and lipoma formation.

Keywords:  PIP3; PLSCR4; PTEN; adipogenesis; lipoma; scramblase

DOI:  https://doi.org/10.3390/ijms23179787
Cells. 2022 Aug 30. pii: 2708. [Epub ahead of print]11(17):

Double Deletion of PI3K and PTEN Modifies Lens Postnatal Growth and Homeostasis.

Caterina Sellitto, Leping Li, Thomas W White.

  We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth.

Keywords:  Akt; PI3K; PTEN; knockout; lens; mouse

DOI:  https://doi.org/10.3390/cells11172708
Molecules. 2022 Aug 24. pii: 5414. [Epub ahead of print]27(17):

mTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.

Lucia Jimenez, Carlos Amenabar, Victor Mayoral-Varo, Thomas A Mackenzie, Maria C Ramos, Andreia Silva, Giampaolo Calissi, Inês Grenho, Carmen Blanco-Aparicio, Joaquin Pastor, Diego Megías, Bibiana I Ferreira, Wolfgang Link.

  Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.

Keywords:  FOXO; aging; cancer; chemical biology; high content screening; kinases; mTOR

DOI:  https://doi.org/10.3390/molecules27175414
Genet Med. 2022 Sep 06. pii: S1098-3600(22)00891-7. [Epub ahead of print]

Alpelisib for the treatment of PIK3CA-related head and neck lymphatic malformations and overgrowth.

Tara L Wenger, Sheila Ganti, Catherine Bull, Erika Lutsky, James T Bennett, Kaitlyn Zenner, Dana M Jensen, Victoria Dmyterko, Ezgi Mercan, Giri M Shivaram, Seth D Friedman, Michael Bindschadler, Madeleine Drusin, Jonathan N Perkins, Ada Kong, Randall A Bly, John P Dahl, Juliana Bonilla-Velez, Jonathan A Perkins.

   PURPOSE: PIK3CA-related overgrowth spectrum (PROS) conditions of the head and neck are treatment challenges. Traditionally, these conditions require multiple invasive interventions, with incomplete malformation removal, disfigurement, and possible dysfunction. Use of the PI3K inhibitor alpelisib, previously shown to be effective in PROS, has not been reported in PIK3CA-associated head and neck lymphatic malformations (HNLMs) or facial infiltrating lipomatosis (FIL). We describe prospective treatment of 5 children with PIK3CA-associated HNLMs or head and neck FIL with alpelisib monotherapy.
METHODS: A total of 5 children with PIK3CA-associated HNLMs (n = 4) or FIL (n = 1) received alpelisib monotherapy (aged 2-12 years). Treatment response was determined by parental report, clinical evaluation, diary/questionnaire, and standardized clinical photography, measuring facial volume through 3-dimensional photos and magnetic resonance imaging.
RESULTS: All participants had reduction in the size of lesion, and all had improvement or resolution of malformation inflammation/pain/bleeding. Common invasive therapy was avoided (ie, tracheotomy). After 6 or more months of alpelisib therapy, facial volume was reduced (range 1%-20%) and magnetic resonance imaging anomaly volume (range 0%-23%) were reduced, and there was improvement in swallowing, upper airway patency, and speech clarity.
CONCLUSION: Individuals with head and neck PROS treated with alpelisib had decreased malformation size and locoregional overgrowth, improved function and symptoms, and fewer invasive procedures.

Keywords:  Alpelisib; Infiltrating lipomatosis; Lymphatic malformation; PIK3CA-related overgrowth spectrum (PROS); Precision medicine

DOI:  https://doi.org/10.1016/j.gim.2022.07.026
J Pathol. 2022 Sep 08.

Long-term treatment with low dose rapamycin extends lifespan and delays tumours in cancer-prone germline PTEN mutant mice.

Priyanka Tibarewal, Victoria Rathbone, Georgia Constantinou, Wayne Pearce, Mahreen Adil, Zofia Varyova, Lisa Folkes, Alix Hampson, Gala Anastasia Electra Classen, Adriana Alves, Sara Carvalho, Cheryl L Scudamore, Bart Vanhaesebroeck.

  PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underly the PTEN Hamartoma Tumour Syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI-3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralize the impact of PTEN loss, rendering this a more druggable context compared to those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6-weeks onwards to mice with heterozygous germline PTEN loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well-tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay, but not full blockade of the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor This article is protected by copyright. All rights reserved.

Keywords:  PHTS; PI 3-kinase; PTEN; cancer prevention; drug; hamartoma; kinase inhibitor; mTORC1; rapamycin; rare disease; syndrome

DOI:  https://doi.org/10.1002/path.6009
Nat Commun. 2022 Sep 07. 13(1): 5258

High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER+ breast cancer.

Marta Palafox, Laia Monserrat, Meritxell Bellet, Guillermo Villacampa, Abel Gonzalez-Perez, Mafalda Oliveira, Fara Brasó-Maristany, Nusaibah Ibrahimi, Srinivasaraghavan Kannan, Leonardo Mina, Maria Teresa Herrera-Abreu, Andreu Òdena, Mònica Sánchez-Guixé, Marta Capelán, Analía Azaro, Alejandra Bruna, Olga Rodríguez, Marta Guzmán, Judit Grueso, Cristina Viaplana, Javier Hernández, Faye Su, Kui Lin, Robert B Clarke, Carlos Caldas, Joaquín Arribas, Stefan Michiels, Alicia García-Sanz, Nicholas C Turner, Aleix Prat, Paolo Nuciforo, Rodrigo Dienstmann, Chandra S Verma, Nuria Lopez-Bigas, Maurizio Scaltriti, Monica Arnedos, Cristina Saura, Violeta Serra.

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies.

DOI: https://doi.org/10.1038/s41467-022-32828-6
Heliyon. 2022 Aug;8(8): e10371

Hyperactivation of mTOR and AKT in a cardiac hypertrophy animal model of Friedreich ataxia.

Wing-Hang Tong, Hayden Ollivierre, Audrey Noguchi, Manik C Ghosh, Danielle A Springer, Tracey A Rouault.

  Cardiomyopathy is a primary cause of death in Friedreich ataxia (FRDA) patients with defective iron-sulfur cluster (ISC) biogenesis due to loss of functional frataxin and in rare patients with functional loss of other ISC biogenesis factors. The mechanistic target of rapamycin (mTOR) and AKT signaling cascades that coordinate eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors, are crucial regulators of cardiovascular growth and homeostasis. We observed increased phosphorylation of AKT and dysregulation of multiple downstream effectors of mTORC1, including S6K1, S6, ULK1 and 4EBP1, in a cardiac/skeletal muscle specific FRDA conditional knockout (cKO) mouse model and in human cell lines depleted of ISC biogenesis factors. Knockdown of several mitochondrial metabolic proteins that are downstream targets of ISC biogenesis, including lipoyl synthase and subunit B of succinate dehydrogenase, also resulted in activation of mTOR and AKT signaling, suggesting that mTOR and AKT hyperactivations are part of the metabolic stress response to ISC deficiencies. Administration of rapamycin, a specific inhibitor of mTOR signaling, enhanced the survival of the Fxn cKO mice, providing proof of concept for the potential of mTOR inhibition to ameliorate cardiac disease in patients with defective ISC biogenesis. However, AKT phosphorylation remained high in rapamycin-treated Fxn cKO hearts, suggesting that parallel mTOR and AKT inhibition might be necessary to further improve the lifespan and healthspan of ISC deficient individuals.

Keywords:  AKT; Cardiac hypertrophy; FXN; Frataxin; Friedreich ataxia; ISCU; Iron-sulfur cluster biogenesis; Metabolic stress; mTOR

DOI:  https://doi.org/10.1016/j.heliyon.2022.e10371
Front Immunol. 2022 ;13 990874

Akt isoforms in the immune system.

Mireia Guerau-de-Arellano, Zayda L Piedra-Quintero, Philip N Tsichlis.

  Akt is a PI3K-activated serine-threonine kinase that exists in three distinct isoforms. Akt's expression in most immune cells, either at baseline or upon activation, reflects its importance in the immune system. While Akt is most highly expressed in innate immune cells, it plays crucial roles in both innate and adaptive immune cell development and/or effector functions. In this review, we explore what's known about the role of Akt in innate and adaptive immune cells. Wherever possible, we discuss the overlapping and distinct role of the three Akt isoforms, namely Akt1, Akt2, and Akt3, in immune cells.

Keywords:  Akt; Akt1; Akt2; Akt3; immune cells

DOI:  https://doi.org/10.3389/fimmu.2022.990874
Sci Rep. 2022 Sep 05. 12(1): 15095

Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates.

Che-Fan Huang, Cara J Gottardi, Milan Mrksich.

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts β-catenin from cell-cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant β-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations.

DOI: https://doi.org/10.1038/s41598-022-19133-4
Elife. 2022 Sep 07. pii: e75715. [Epub ahead of print]11

Genetically manipulating endogenous Kras levels and oncogenic mutations in vivo influences tissue patterning of murine tumorigenesis.

Özgün Le Roux, Nicole L K Pershing, Erin Kaltenbrun, Nicole J Newman, Jeffrey I Everitt, Elisa Baldelli, Mariaelena Pierobon, Emanuel F Petricoin, Christopher M Counter.

  Despite multiple possible oncogenic mutations in the proto-oncogene KRAS, unique subsets of these mutations are detected in different cancer types. As KRAS mutations occur early, if not being the initiating event, these mutational biases are ostensibly a product of how normal cells respond to the encoded oncoprotein. Oncogenic mutations can impact not only the level of active oncoprotein, but also engagement with proteins. To attempt to separate these two effects, we generated four novel Cre-inducible (LSL) Kras alleles in mice with the biochemically distinct G12D or Q61R mutations and encoded by native (nat) rare or common (com) codons to produce low or high protein levels. While there were similarities, each allele also induced a distinct transcriptional response shortly after activation in vivo. At one end of the spectrum, activating the KrasLSL-natG12D allele induced transcriptional hallmarks suggestive of an expansion of multipotent cells, while at the other end, activating the KrasLSL-comQ61R allele led to hallmarks of hyperproliferation and oncogenic stress. Evidence suggests that these changes may be a product of signaling differences due to increased protein expression as well as the specific mutation. To determine the impact of these distinct responses on RAS mutational patterning in vivo, all four alleles were globally activated, revealing that hematolymphopoietic lesions were permissive to the level of active oncoprotein, squamous tumors were permissive to the G12D mutant, while carcinomas were permissive to both these features. We suggest that different KRAS mutations impart unique signaling properties that are preferentially capable of inducing tumor initiation in a distinct cell-specific manner.

Keywords:  RAS; cancer; cancer biology; genetically engineered mice; mouse; oncogenes

DOI:  https://doi.org/10.7554/eLife.75715
Nat Cell Biol. 2022 Sep 08.

Comparative roadmaps of reprogramming and oncogenic transformation identify Bcl11b and Atoh8 as broad regulators of cellular plasticity.

A Huyghe, G Furlan, J Schroeder, E Cascales, A Trajkova, M Ruel, F Stüder, M Larcombe, Y Bo Yang Sun, F Mugnier, L De Matteo, A Baygin, J Wang, Y Yu, N Rama, B Gibert, J Kielbassa, L Tonon, P Wajda, N Gadot, M Brevet, M Siouda, P Mulligan, R Dante, P Liu, H Gronemeyer, M Mendoza-Parra, J M Polo, F Lavial.

Coordinated changes of cellular plasticity and identity are critical for pluripotent reprogramming and oncogenic transformation. However, the sequences of events that orchestrate these intermingled modifications have never been comparatively dissected. Here, we deconvolute the cellular trajectories of reprogramming (via Oct4/Sox2/Klf4/c-Myc) and transformation (via Ras/c-Myc) at the single-cell resolution and reveal how the two processes intersect before they bifurcate. This approach led us to identify the transcription factor Bcl11b as a broad-range regulator of cell fate changes, as well as a pertinent marker to capture early cellular intermediates that emerge simultaneously during reprogramming and transformation. Multiomics characterization of these intermediates unveiled a c-Myc/Atoh8/Sfrp1 regulatory axis that constrains reprogramming, transformation and transdifferentiation. Mechanistically, we found that Atoh8 restrains cellular plasticity, independent of cellular identity, by binding a specific enhancer network. This study provides insights into the partitioned control of cellular plasticity and identity for both regenerative and cancer biology.

DOI: https://doi.org/10.1038/s41556-022-00986-w
Front Cell Dev Biol. 2022 ;10 920683

The crosstalk between MYC and mTORC1 during osteoclastogenesis.

Seyeon Bae, Brian Oh, Jefferson Tsai, Peter Sang Uk Park, Matthew Blake Greenblatt, Eugenia G Giannopoulou, Kyung-Hyun Park-Min.

  Osteoclasts are bone-resorbing cells that undergo extensive changes in morphology throughout their differentiation. Altered osteoclast differentiation and activity lead to changes in pathological bone resorption. The mammalian target of rapamycin (mTOR) is a kinase, and aberrant mTOR complex 1 (mTORC1) signaling is associated with altered bone homeostasis. The activation of mTORC1 is biphasically regulated during osteoclastogenesis; however, the mechanism behind mTORC1-mediated regulation of osteoclastogenesis and bone resorption is incompletely understood. Here, we found that MYC coordinates the dynamic regulation of mTORC1 activation during osteoclastogenesis. MYC-deficiency blocked the early activation of mTORC1 and also reversed the decreased activity of mTORC1 at the late stage of osteoclastogenesis. The suppression of mTORC1 activity by rapamycin in mature osteoclasts enhances bone resorption activity despite the indispensable role of high mTORC1 activation in osteoclast formation in both mouse and human cells. Mechanistically, MYC induces Growth arrest and DNA damage-inducible protein (GADD34) expression and suppresses mTORC1 activity at the late phase of osteoclastogenesis. Taken together, our findings identify a MYC-GADD34 axis as an upstream regulator of dynamic mTORC1 activation in osteoclastogenesis and highlight the interplay between MYC and mTORC1 pathways in determining osteoclast activity.

Keywords:  GADD34 (PPP1R15A); MYC (c-myc); bone resorption; mTORC1 (mechanistic target of rapamycin complex 1); osteoclast (OC)

DOI:  https://doi.org/10.3389/fcell.2022.920683
Nat Commun. 2022 Sep 09. 13(1): 5317

Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.

Eva C Freckmann, Emma Sandilands, Erin Cumming, Matthew Neilson, Alvaro Román-Fernández, Konstantina Nikolatou, Marisa Nacke, Tamsin R M Lannagan, Ann Hedley, David Strachan, Mark Salji, Jennifer P Morton, Lynn McGarry, Hing Y Leung, Owen J Sansom, Crispin J Miller, David M Bryant.

Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.

DOI: https://doi.org/10.1038/s41467-022-32958-x
Genome Biol. 2022 Sep 05. 23(1): 186

Integrating temporal single-cell gene expression modalities for trajectory inference and disease prediction.

Jolene S Ranek, Natalie Stanley, Jeremy E Purvis.

   BACKGROUND: Current methods for analyzing single-cell datasets have relied primarily on static gene expression measurements to characterize the molecular state of individual cells. However, capturing temporal changes in cell state is crucial for the interpretation of dynamic phenotypes such as the cell cycle, development, or disease progression. RNA velocity infers the direction and speed of transcriptional changes in individual cells, yet it is unclear how these temporal gene expression modalities may be leveraged for predictive modeling of cellular dynamics.
RESULTS: Here, we present the first task-oriented benchmarking study that investigates integration of temporal sequencing modalities for dynamic cell state prediction. We benchmark ten integration approaches on ten datasets spanning different biological contexts, sequencing technologies, and species. We find that integrated data more accurately infers biological trajectories and achieves increased performance on classifying cells according to perturbation and disease states. Furthermore, we show that simple concatenation of spliced and unspliced molecules performs consistently well on classification tasks and can be used over more memory intensive and computationally expensive methods.
CONCLUSIONS: This work illustrates how integrated temporal gene expression modalities may be leveraged for predicting cellular trajectories and sample-associated perturbation and disease phenotypes. Additionally, this study provides users with practical recommendations for task-specific integration of single-cell gene expression modalities.

Keywords:  Data integration; Multi-omics; RNA velocity; Single-cell prediction; Trajectory inference

DOI:  https://doi.org/10.1186/s13059-022-02749-0
Chembiochem. 2022 Sep 08.

The potential application of branch-PCR assembled PTEN gene nanovector in lung cancer gene therapy.

Liqing Lu, Tian Fang, Tuo Pang, Ziyi Chen, Longhuai Cheng, Dejun Ma, Zhen Xi.

  Gene therapy offers an alternative and promising avenue to lung cancer treatment. Here, we used dibenzocyclooctyne (DBCO)-branched primers to construct a kind of PTEN gene nanovector (NP-PTEN) through branch-PCR. NP-PTEN showed the nanoscale structure with the biocompatible size (84.7 ± 11.2 nm) and retained the improved serum stability compared to linear DNA. When transfected into NCI-H1299 cancer cells, NP-PTEN could overexpress PTEN protein to restore PTEN function through the deactivation of PI3K-AKT-mTOR signaling pathway to inhibit cell proliferation and induce cell apoptosis. The apoptosis rate of NCI-H1299 cancer cells could reach up to 54.5% ± 4.6% when the transfection concentration of NP-PTEN was 4.0 μg/mL. In mice bearing NCI-H1299 tumor xenograft intratumorally administrated with NP-PTEN, the average tumor volume and tumor weight was separately reduced by 61.7% and 63.9% compared with the PBS group on the 18 th day of administration. The anticancer efficacy of NP-PTEN in NCI-H1299 tumor xenograft suggested the promising therapeutic potential of this branch-PCR assembled PTEN gene nanovectors in lung cancer gene therapy and also provided more opportunities to introduce two or more tumor suppressor genes as the all-in-one gene nanovectors for multiple gene-based cancer gene therapy.

Keywords:  Gene therapy, PTEN, NSCLC, Branch-PCR

DOI:  https://doi.org/10.1002/cbic.202200387
Nat Commun. 2022 Sep 08. 13(1): 5295

Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks.

Bert van de Kooij, Alex Kruswick, Haico van Attikum, Michael B Yaffe.

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

DOI: https://doi.org/10.1038/s41467-022-32743-w
Nat Rev Endocrinol. 2022 Sep 07.

Modelling metabolic diseases and drug response using stem cells and organoids.

Wenxiang Hu, Mitchell A Lazar.

Metabolic diseases, including obesity, diabetes mellitus and cardiovascular disease, are a major threat to health in the modern world, but efforts to understand the underlying mechanisms and develop rational treatments are limited by the lack of appropriate human model systems. Notably, advances in stem cell and organoid technology allow the generation of cellular models that replicate the histological, molecular and physiological properties of human organs. Combined with marked improvements in gene editing tools, human stem cells and organoids provide unprecedented systems for studying mechanisms of metabolic diseases. Here, we review progress made over the past decade in the generation and use of stem cell-derived metabolic cell types and organoids in metabolic disease research, especially obesity and liver diseases. In particular, we discuss the limitations of animal models and the advantages of stem cells and organoids, including their application to metabolic diseases. We also discuss mechanisms of drug action, understanding the efficacy and toxicity of existing therapies, screening for new treatments and pursuing personalized therapies. We highlight the potential of combining stem cell-derived organoids with gene editing and functional genomics to revolutionize the approach to finding treatments for metabolic diseases.

DOI: https://doi.org/10.1038/s41574-022-00733-z
Pediatr Blood Cancer. 2022 Aug;69 Suppl 3 e29273

How we approach complex vascular anomalies and overgrowth syndromes.

Katherine D Watson, Kyung R Kim, Julie Blatt.

  Vascular anomalies, both vascular tumors and vascular malformations, can occur in isolation or as part of syndromes including those which feature phenotypic overgrowth. To update what is known about vascular anomalies associated with overgrowth, PubMed was searched for "overgrowth syndromes and vascular anomalies or malformations." PubMed, OMIM, and the Rare Disease Database also were searched for specific diagnoses. We review individual overgrowth syndromes, provide a case-based approach to the clinical, radiographic, pathologic, and genetic basis for diagnosis, to complications of both the vascular anomalies and the overgrowth, and emphasize the need for a multidisciplinary approach to care.

Keywords:  PIK3CA; overgrowth; vascular malformations

DOI:  https://doi.org/10.1002/pbc.29273
Cell Death Dis. 2022 Sep 08. 13(9): 777

Modelling aggressive prostate cancers of young men in immune-competent mice, driven by isogenic Trp53 alterations and Pten loss.

Javier Octavio Mejía-Hernández, Simon P Keam, Reem Saleh, Fenella Muntz, Stephen B Fox, David Byrne, Arielle Kogan, Lokman Pang, Jennifer Huynh, Cassandra Litchfield, Franco Caramia, Guillermina Lozano, Hua He, James M You, Shahneen Sandhu, Scott G Williams, Ygal Haupt, Sue Haupt.

Understanding prostate cancer onset and progression in order to rationally treat this disease has been critically limited by a dire lack of relevant pre-clinical animal models. We have generated a set of genetically engineered mice that mimic human prostate cancer, initiated from the gland epithelia. We chose driver gene mutations that are specifically relevant to cancers of young men, where aggressive disease poses accentuated survival risks. An outstanding advantage of our models are their intact repertoires of immune cells. These mice provide invaluable insight into the importance of immune responses in prostate cancer and offer scope for studying treatments, including immunotherapies. Our prostate cancer models strongly support the role of tumour suppressor p53 in functioning to critically restrain the emergence of cancer pathways that drive cell cycle progression; alter metabolism and vasculature to fuel tumour growth; and mediate epithelial to mesenchymal-transition, as vital to invasion. Importantly, we also discovered that the type of p53 alteration dictates the specific immune cell profiles most significantly disrupted, in a temporal manner, with ramifications for disease progression. These new orthotopic mouse models demonstrate that each of the isogenic hotspot p53 amino acid mutations studied (R172H and R245W, the mouse equivalents of human R175H and R248W respectively), drive unique cellular changes affecting pathways of proliferation and immunity. Our findings support the hypothesis that individual p53 mutations confer their own particular oncogenic gain of function in prostate cancer.

DOI: https://doi.org/10.1038/s41419-022-05211-y
Nat Commun. 2022 Sep 09. 13(1): 5304

Integrating and formatting biomedical data as pre-calculated knowledge graph embeddings in the Bioteque.

Adrià Fernández-Torras, Miquel Duran-Frigola, Martino Bertoni, Martina Locatelli, Patrick Aloy.

Biomedical data is accumulating at a fast pace and integrating it into a unified framework is a major challenge, so that multiple views of a given biological event can be considered simultaneously. Here we present the Bioteque, a resource of unprecedented size and scope that contains pre-calculated biomedical descriptors derived from a gigantic knowledge graph, displaying more than 450 thousand biological entities and 30 million relationships between them. The Bioteque integrates, harmonizes, and formats data collected from over 150 data sources, including 12 biological entities (e.g., genes, diseases, drugs) linked by 67 types of associations (e.g., 'drug treats disease', 'gene interacts with gene'). We show how Bioteque descriptors facilitate the assessment of high-throughput protein-protein interactome data, the prediction of drug response and new repurposing opportunities, and demonstrate that they can be used off-the-shelf in downstream machine learning tasks without loss of performance with respect to using original data. The Bioteque thus offers a thoroughly processed, tractable, and highly optimized assembly of the biomedical knowledge available in the public domain.

DOI: https://doi.org/10.1038/s41467-022-33026-0
Cell Res. 2022 Sep 09.

Live cold to grow old? Thermogenesis to fight cancer.

Henning T Langer, Lewis C Cantley, Marcus D Goncalves.

DOI: https://doi.org/10.1038/s41422-022-00723-w
Elife. 2022 Sep 07. pii: e79208. [Epub ahead of print]11

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner.

  The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multi-component editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair (HDR) of site-specific nuclease-induced double-strand breaks (DSBs). Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs (ngRNAs) improved editing efficiencies up to 13-fold compared to transfecting the prime editing components as plasmids or ribonucleoprotein particles (RNPs). Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

Keywords:  genetics; genomics; human; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.79208
Nat Genet. 2022 Sep 05.

Large-scale genomic analysis of human iPSCs identifies recurrent somatic driver mutations.

DOI: https://doi.org/10.1038/s41588-022-01169-x
Biomaterials. 2022 Sep 02. pii: S0142-9612(22)00423-9. [Epub ahead of print]289 121783

Sustained potentiation of bystander killing via PTEN-loss driven macropinocytosis targeted peptide-drug conjugate therapy in metastatic triple-negative breast cancer.

Young Seok Cho, Ha Rin Kim, Seong Jin Park, Seung Woo Chung, Yoon Gun Ko, Joo Hye Yeo, Jinu Lee, Sang Kyoon Kim, Jeong Uk Choi, Sang Yoon Kim, Youngro Byun.

  While conventional approaches for PTEN-loss cancers mainly focus on turning off growth promoting process through modulation of PI3K/AKT pathways, no effective therapeutic treatments that target PTEN-loss cancer cells have yielded results. Moreover, conventional targeted therapies, which are potent against only a subset of cancer cells with limited specificity, bring on temporary response. Here, we report the development of albumin-binding caspase-3 cleavable peptide-drug conjugate (PDC), which utilizes the enhanced albumin metabolism pathway in PTEN-loss cancer cells to enhance the intracellular delivery of chemotherapeutic payload that could exert a bystander killing effect. Albumin metabolism-mediated apoptosis triggered expression of caspase-3 allows the continuous activation of the PDC, accumulation of payloads, sustained upregulation of tumoral caspase-3, and intensified in-situ apoptosis. Importantly, PDC strategy exerts potent therapeutic efficacy against PTEN-loss metastatic triple-negative breast cancer, the highly aggressive and heterogenous nature of which remains a challenge conventional targeted therapies need to overcome. This study thus presents a conceptually novel approach to treat PTEN-loss cancer and creates new translational perspectives of exploiting PTEN-loss for providing an avenue to advance current targeted therapy.

Keywords:  Bystander killing; Caspase-3; Macropinocytosis; Metastatic triple-negative breast cancer; PTEN-Loss; Peptide-drug conjugate

DOI:  https://doi.org/10.1016/j.biomaterials.2022.121783
Cell Syst. 2022 Sep 01. pii: S2405-4712(22)00330-1. [Epub ahead of print]

Virtual screening for small-molecule pathway regulators by image-profile matching.

Mohammad H Rohban, Ashley M Fuller, Ceryl Tan, Jonathan T Goldstein, Deepsing Syangtan, Amos Gutnick, Ann DeVine, Madhura P Nijsure, Megan Rigby, Joshua R Sacher, Steven M Corsello, Grace B Peppler, Marta Bogaczynska, Andrew Boghossian, Gabrielle E Ciotti, Allison T Hands, Aroonroj Mekareeya, Minh Doan, Jennifer P Gale, Rik Derynck, Thomas Turbyville, Joel D Boerckel, Shantanu Singh, Laura L Kiessling, Thomas L Schwarz, Xaralabos Varelas, Florence F Wagner, Ran Kafri, T S Karin Eisinger-Mathason, Anne E Carpenter.

  Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

Keywords:  Hippo/Yap1 pathway; computational drug discovery; drug screening; image-based profiling; virtual screening

DOI:  https://doi.org/10.1016/j.cels.2022.08.003
F1000Res. 2020 ;9 1263

An R-based reproducible and user-friendly preprocessing pipeline for CyTOF data.

Tumor Profiler Consortium

  Mass cytometry (CyTOF) has become a method of choice for in-depth characterization of tissue heterogeneity in health and disease, and is currently implemented in multiple clinical trials, where higher quality standards must be met. Currently, preprocessing of raw files is commonly performed in independent standalone tools, which makes it difficult to reproduce. Here, we present an R pipeline based on an updated version of CATALYST that covers all preprocessing steps required for downstream mass cytometry analysis in a fully reproducible way. This new version of CATALYST is based on Bioconductor's SingleCellExperiment class and fully unit tested. The R-based pipeline includes file concatenation, bead-based normalization, single-cell deconvolution, spillover compensation and live cell gating after debris and doublet removal. Importantly, this pipeline also includes different quality checks to assess machine sensitivity and staining performance while allowing also for batch correction. This pipeline is based on open source R packages and can be easily be adapted to different study designs. It therefore has the potential to significantly facilitate the work of CyTOF users while increasing the quality and reproducibility of data generated with this technology.

Keywords:  Batch correction; Compensation; CyTOF; Debarcoding; Gating; Normalization; Preprocessing; Reproducibility

DOI:  https://doi.org/10.12688/f1000research.26073.1
Elife. 2022 09 05. pii: e78383. [Epub ahead of print]11

Multiple ciliary localization signals control INPP5E ciliary targeting.

Dario Cilleros-Rodriguez, Raquel Martin-Morales, Pablo Barbeito, Abhijit Deb Roy, Abdelhalim Loukil, Belen Sierra-Rodero, Gonzalo Herranz, Olatz Pampliega, Modesto Redrejo-Rodriguez, Sarah C Goetz, Manuel Izquierdo, Takanari Inoue, Francesc R Garcia-Gonzalo.

  Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoes.

Keywords:  INPP5E; Joubert syndrome; biochemistry; cell biology; chemical biology; cilia; ciliopathies; none; phosphatase; phosphoinositides

DOI:  https://doi.org/10.7554/eLife.78383