bims-pideca 2022-03-13 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2022‒03‒13
forty papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

PIK3CA-related overgrowth: silver bullets from the cancer arsenal?
miRNA-Dependent Regulation of AKT1 Phosphorylation.
Calcium/calmodulin-dependent protein kinase kinase 2 mediates pleiotropic effects of epidermal growth factor in cancer cells.
Genotypes and phenotypes heterogeneity in PIK3CA-related overgrowth spectrum and overlapping conditions: 150 novel patients and systematic review of 1007 patients with PIK3CA pathogenetic variants.
PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment.
Computation and visualization of cell-cell signaling topologies in single-cell systems data using Connectome.
Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.
Mouse model and human patient data reveal critical roles for Pten and p53 in suppressing POLE mutant tumor development.
Intertumor and intratumor heterogeneity of PIK3CA mutations in extramammary Paget's disease.
Fully-automated and ultra-fast cell-type identification using specific marker combinations from single-cell transcriptomic data.
A pan-tissue DNA methylation atlas enables in silico decomposition of human tissue methylomes at cell-type resolution.
PTEN/PI3K/Akt pathway alters sensitivity of T-cell acute lymphoblastic leukemia to L-asparaginase.
Investigation of the function of the PI3-Kinase / AKT signaling pathway for leukemogenesis and therapy of acute childhood lymphoblastic leukemia (ALL).
A non-canonical tricarboxylic acid cycle underlies cellular identity.
Myoepithelial progenitors as founder cells of hyperplastic human breast lesions upon PIK3CA transformation.
Enhanced Wild-Type MET Receptor Levels in Mouse Hepatocytes Attenuates Insulin-Mediated Signaling.
How we approach the use of sirolimus and new agents: Medical therapy to treat vascular anomalies.
OpenCell: Endogenous tagging for the cartography of human cellular organization.
Structural basis of phosphatidylinositol 3-kinase C2α function.
Therapy resistance: opportunities created by adaptive responses to targeted therapies in cancer.
Indirect targeting of MYC sensitizes pancreatic cancer cells to mechanistic target of rapamycin (mTOR) inhibition.
PTEN alterations in sporadic and BRCA1-associated triple negative breast carcinomas.
Using single-cell models to predict the functionality of synthetic circuits at the population scale.
Mapping the long road to cancer.
Confined migration promotes cancer metastasis through resistance to anoikis and increased invasiveness.
Mutations in the telomerase reverse transcriptase promoter and PIK3CA gene are common events in penile squamous cell carcinoma of Italian and Ugandan patients.
Optimizing the future: how mathematical models inform treatment schedules for cancer.
A saturation-mutagenesis analysis of the interplay between stability and activation in Ras.
Deep Hidden Physics Modeling of Cell Signaling Networks.
Urgent need for consistent standards in functional enrichment analysis.
PIK3CA Gene Mutations in HNSCC: Systematic Review and Correlations with HPV Status and Patient Survival.
Alevin-fry unlocks rapid, accurate and memory-frugal quantification of single-cell RNA-seq data.
Isoform-specific functions of PPARγ in gene regulation and metabolism.
Clinical implications of mosaicism: a 10-year retrospective review of 83 families in a university-affiliated genetics clinic.
Co-translational assembly orchestrates competing biogenesis pathways.
Optimal strategy and benefit of pulsed therapy depend on tumor heterogeneity and aggressiveness at time of treatment initiation.
Reproducibility: Has Cancer Biology Failed beyond Repair?
The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.
Host AKT-mediated phosphorylation of HIV-1 accessory protein Vif potentiates infectivity via enhanced degradation of the restriction factor APOBEC3G.
Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

Trends Mol Med. 2022 Mar 07. pii: S1471-4914(22)00050-8. [Epub ahead of print]

PIK3CA-related overgrowth: silver bullets from the cancer arsenal?

Ralitsa R Madsen, Robert K Semple.

  Mutations that activate growth factor signaling often drive cancer growth. Many also arise in isolation, causing developmental growth disorders. PIK3CA, that encodes a catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is a cardinal example of this paradigm. Recent exciting progress towards the key goal of cancer drug repurposing for PIK3CA-driven overgrowth is discussed.

Keywords:  CLOVES; PI3K; PIK3CA; PROS; alpelisib; overgrowth; phosphatidylinositol 3-kinase; sirolimus; trials

DOI:  https://doi.org/10.1016/j.molmed.2022.02.009
Cells. 2022 Feb 26. pii: 821. [Epub ahead of print]11(5):

miRNA-Dependent Regulation of AKT1 Phosphorylation.

Mallory I Frederick, Tarana Siddika, Pengcheng Zhang, Nileeka Balasuriya, Matthew A Turk, Patrick O'Donoghue, Ilka U Heinemann.

  The phosphoinositide-3-kinase (PI3K)/AKT pathway regulates cell survival and is over-activated in most human cancers, including ovarian cancer. Following growth factor stimulation, AKT1 is activated by phosphorylation at T308 and S473. Disruption of the AKT1 signaling pathway is sufficient to inhibit the epithelial-mesenchymal transition in epithelial ovarian cancer (EOC) cells. In metastatic disease, adherent EOC cells transition to a dormant spheroid state, characterized previously by low S473 phosphorylation in AKT1. We confirmed this finding and observed that T308 phosphorylation was yet further reduced in EOC spheroids and that the transition from adherent to spheroid growth is accompanied by significantly increased levels of let-7 miRNAs. We then used mechanistic studies to investigate the impact of let-7 miRNAs on AKT1 phosphorylation status and activity in cells. In growth factor-stimulated HEK 293T cells supplemented with let-7a, we found increased phosphorylation of AKT1 at T308, decreased phosphorylation at S473, and enhanced downstream AKT1 substrate GSK-3β phosphorylation. Let-7b and let-7g also deregulated AKT signaling by rendering AKT1 insensitive to growth factor simulation. We uncovered let-7a-dependent deregulation of PI3K pathway components, including PI3KC2A, PDK1, and RICTOR, that govern AKT1 phosphorylation and activity. Together, our data show a new role for miRNAs in regulating AKT signaling.

Keywords:  miRNA; oncogenic kinase; posttranslational modification; protein phosphorylation; signaling

DOI:  https://doi.org/10.3390/cells11050821
Biochim Biophys Acta Mol Cell Res. 2022 Mar 07. pii: S0167-4889(22)00043-X. [Epub ahead of print] 119252

Calcium/calmodulin-dependent protein kinase kinase 2 mediates pleiotropic effects of epidermal growth factor in cancer cells.

Shuhang Dai, Elisa Venturini, Saveg Yadav, Xiaoxuan Lin, Dylan Clapp, Martin Steckiewicz, Angela M Gocher-Demske, D Grahame Hardie, Arthur M Edelman.

  AIMS: Engagement of epidermal growth factor (EGF) with its receptor (EGFR) produces a broad range of cancer phenotypes. The overriding aim of this study was to understand EGFR signaling and its regulation by the Ca2+/calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) in cancer cells.RESULTS: In ovarian cancer cells and other cancer cell types, EGF-induced activation of oncogenic Akt is mediated by both the canonical PI3K-PDK1 pathway and by CaMKK2. Akt activation induced by EGF occurs by both calcium-dependent and calcium-independent mechanisms. In contrast to the canonical pathway, CaMKK2 neither binds to, nor is regulated by phosphoinositides but is activated by Ca2+/CaM. Akt activation at its primary activation site, T308 occurs by direct phosphorylation by CaMKK2, but activation at its secondary site (S473), is through an indirect mechanism requiring mTORC2. In cells in which another CaMKK2 target, 5'AMP-dependent protein kinase (AMPK) was deleted, Akt activation and calcium-dependency of activation were still observed. CaMKK2 accumulates in the nucleus in response to EGF and regulates transcription of phosphofructokinase platelet (PFKP) a glycolytic regulator. CaMKK2 is required for optimal PFK activity. CaMKK2 regulates transcription of plasminogen activator, urokinase (PLAU) a metastasis regulator. The EGFR inhibitor gefitinib synergizes with CaMKK2 inhibition in the regulation of cell survival and increases the dose-reduction index. CRISPR/Cas9 knockout of CaMKK2 leads to compensatory PTEN downregulation and upregulation of Akt activation.
CONCLUSIONS: CaMKK2-mediation of EGFR action may enable cancer cells to use intracellular calcium elevation as a signal for growth and survival.

Keywords:  Calcium; Calmodulin; Cancer; EGF; Kinase

DOI:  https://doi.org/10.1016/j.bbamcr.2022.119252
J Med Genet. 2022 Mar 07. pii: jmedgenet-2021-108093. [Epub ahead of print]

Genotypes and phenotypes heterogeneity in PIK3CA-related overgrowth spectrum and overlapping conditions: 150 novel patients and systematic review of 1007 patients with PIK3CA pathogenetic variants.

Alessandro Mussa, Chiara Leoni, Matteo Iacoviello, Diana Carli, Carlotta Ranieri, Antonino Pantaleo, Paola Sabrina Buonuomo, Rosanna Bagnulo, Giovanni Battista Ferrero, Andrea Bartuli, Daniela Melis, Silvia Maitz, Daria Carmela Loconte, Antonella Turchiano, Marilidia Piglionica, Annunziata De Luisi, Francesco Claudio Susca, Nenad Bukvic, Cinzia Forleo, Angelo Selicorni, Giuseppe Zampino, Roberta Onesimo, Gerarda Cappuccio, Livia Garavelli, Chiara Novelli, Luigi Memo, Carla Morando, Matteo Della Monica, Maria Accadia, Martina Capurso, Carmelo Piscopo, Anna Cereda, Marilena Carmela Di Giacomo, Veronica Saletti, Alessandro Mauro Spinelli, Patrizia Lastella, Romano Tenconi, Veronika Dvorakova, Alan D Irvine, Nicoletta Resta.

  BACKGROUND: Postzygotic activating PIK3CA variants cause several phenotypes within the PIK3CA-related overgrowth spectrum (PROS). Variant strength, mosaicism level, specific tissue involvement and overlapping disorders are responsible for disease heterogeneity. We explored these factors in 150 novel patients and in an expanded cohort of 1007 PIK3CA-mutated patients, analysing our new data with previous literature to give a comprehensive picture.METHODS: We performed ultradeep targeted next-generation sequencing (NGS) on DNA from skin biopsy, buccal swab or blood using a panel including phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway genes and GNAQ, GNA11, RASA1 and TEK. Additionally, 914 patients previously reported were systematically reviewed.
RESULTS: 93 of our 150 patients had PIK3CA pathogenetic variants. The merged PROS cohort showed that PIK3CA variants span thorough all gene domains, some were exclusively associated with specific PROS phenotypes: weakly activating variants were associated with central nervous system (CNS) involvement, and strongly activating variants with extra-CNS phenotypes. Among the 57 with a wild-type PIK3CA allele, 11 patients with overgrowth and vascular malformations overlapping PROS had variants in GNAQ, GNA11, RASA1 or TEK.
CONCLUSION: We confirm that (1) molecular diagnostic yield increases when multiple tissues are tested and by enriching NGS panels with genes of overlapping 'vascular' phenotypes; (2) strongly activating PIK3CA variants are found in affected tissue, rarely in blood: conversely, weakly activating mutations more common in blood; (3) weakly activating variants correlate with CNS involvement, strong variants are more common in cases without; (4) patients with vascular malformations overlapping those of PROS can harbour variants in genes other than PIK3CA.

Keywords:  genetic testing; genotype; molecular medicine; phenotype; sequence analysis, DNA

DOI:  https://doi.org/10.1136/jmedgenet-2021-108093
J Immunother Cancer. 2022 Mar;pii: e003402. [Epub ahead of print]10(3):

PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment.

Natalie B Collins, Rose Al Abosy, Brian C Miller, Kevin Bi, Qihong Zhao, Michael Quigley, Jeffrey J Ishizuka, Kathleen B Yates, Hans W Pope, Robert T Manguso, Yashaswi Shrestha, Marc Wadsworth, Travis Hughes, Alex K Shalek, Jesse S Boehm, William C Hahn, John G Doench, W Nicholas Haining.

  BACKGROUND: Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood.METHODS: We use an in vivo expression screen of known cancer-associated somatic mutations in mouse syngeneic tumor models treated with checkpoint blockade to identify oncogenes that promote immune evasion. We then validated candidates from this screen in vivo and analyzed the tumor immune microenvironment of tumors expressing mutant protein to identify mechanisms of immune evasion.
RESULTS: We found that expression of a catalytically active mutation in phospho-inositol 3 kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers a selective growth advantage to tumors treated with immunotherapy that is reversed by pharmacological PI3K inhibition. PIK3CA H1047R-expression in tumors decreased the number of CD8+ T cells but increased the number of inhibitory myeloid cells following immunotherapy. Inhibition of myeloid infiltration by pharmacological or genetic modulation of Ccl2 in PIK3CA H1047R tumors restored sensitivity to programmed cell death protein 1 (PD-1) checkpoint blockade.
CONCLUSIONS: PI3K activation enables tumor immune evasion by promoting an inhibitory myeloid microenvironment. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. Our data suggest that some oncogenes promote tumorigenesis by enabling tumor cells to avoid clearance by the immune system. Identification of those mechanisms can advance rational combination strategies to increase the efficacy of immunotherapy.

Keywords:  biomarkers; immune evation; immunotherapy; tumor

DOI:  https://doi.org/10.1136/jitc-2021-003402
Sci Rep. 2022 Mar 09. 12(1): 4187

Computation and visualization of cell-cell signaling topologies in single-cell systems data using Connectome.

Micha Sam Brickman Raredon, Junchen Yang, James Garritano, Meng Wang, Dan Kushnir, Jonas Christian Schupp, Taylor S Adams, Allison M Greaney, Katherine L Leiby, Naftali Kaminski, Yuval Kluger, Andre Levchenko, Laura E Niklason.

Single-cell RNA-sequencing data has revolutionized our ability to understand of the patterns of cell-cell and ligand-receptor connectivity that influence the function of tissues and organs. However, the quantification and visualization of these patterns in a way that informs tissue biology are major computational and epistemological challenges. Here, we present Connectome, a software package for R which facilitates rapid calculation and interactive exploration of cell-cell signaling network topologies contained in single-cell RNA-sequencing data. Connectome can be used with any reference set of known ligand-receptor mechanisms. It has built-in functionality to facilitate differential and comparative connectomics, in which signaling networks are compared between tissue systems. Connectome focuses on computational and graphical tools designed to analyze and explore cell-cell connectivity patterns across disparate single-cell datasets and reveal biologic insight. We present approaches to quantify focused network topologies and discuss some of the biologic theory leading to their design.

DOI: https://doi.org/10.1038/s41598-022-07959-x
Nat Commun. 2022 Mar 08. 13(1): 1204

Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.

Jianhang Yin, Rusen Lu, Changchang Xin, Yuhong Wang, Xinyu Ling, Dong Li, Weiwei Zhang, Mengzhu Liu, Wutao Xie, Lingyun Kong, Wen Si, Ping Wei, Bingbing Xiao, Hsiang-Ying Lee, Tao Liu, Jiazhi Hu.

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

DOI: https://doi.org/10.1038/s41467-022-28900-w
NAR Cancer. 2022 Mar;4(1): zcac004

Mouse model and human patient data reveal critical roles for Pten and p53 in suppressing POLE mutant tumor development.

Vivian S Park, Meijuan J S Sun, Wesley D Frey, Leonard G Williams, Karl P Hodel, Juliet D Strauss, Sydney J Wellens, James G Jackson, Zachary F Pursell.

Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole +/P286R;Trp53 +/- mice showed accelerated cancer mortality compared to Pole +/P286R;Trp53 +/+ mice. Cells from Pole +/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. However, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole +/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Re-analysis of human TCGA (The Cancer Genome Atlas) data showed that all PTEN-F341V mutations occurred in tumors with mutations in POLE. Taken together with recent published work, our results support the idea that development of POLE mutant tumors may involve disabling surveillance of nuclear DNA damage in addition to POLE-mediated hypermutagenesis.

DOI: https://doi.org/10.1093/narcan/zcac004
J Dermatol. 2022 Mar 06.

Intertumor and intratumor heterogeneity of PIK3CA mutations in extramammary Paget's disease.

Yudo Kusaba, Ikko Kajihara, Tselmeg Mijiddorj Myangat, Kenichiro Tanaka, Ryoko Sakamoto, Saki Maeda-Otsuka, Saori Yamada-Kanazawa, Soichiro Sawamura, Hisashi Kanemaru, Yuki Nishimura, Kayo Nakamura-Kashiwada, Katsunari Makino, Azusa Miyashita, Jun Aoi, Shinichi Masuguchi, Satoshi Fukushima.

  Although the prognosis of patients with extramammary Paget's disease (EMPD) treated with radical resection is good, the prognosis of EMPD with distant metastasis is very poor. PIK3CA mutations predict a good response to PIK3CA inhibitors. The aim of this study was to investigate the occurrence rate of PIK3CA mutations (including multiple mutations [MM]) related to the intertumor and intratumor heterogeneity in EMPD and to evaluate the correlation between these mutations and clinical parameters of EMPD. We performed droplet digital polymerase chain reaction to detect PIK3CA mutations (E542K, E545K, H1047R, and MM) in 68 patients with EMPD. In addition, we investigated the presence of PIK3CA mutations at multiple sites in 16 patients with PIK3CA mutations to assess the intratumor heterogeneity of PIK3CA mutations in EMPD. The frequency of one or more PIK3CA mutations in patients with EMPD was 30.8% (21/68). The frequency of E542K, E545K, H1047R, and MM were 10.2% (7/68), 13.2% (9/68), 11.7% (8/68), and 4.4% (3/68), respectively. No significant correlation was found between PIK3CA mutation patterns and clinical parameters. Of the 21 patients with PIK3CA mutations, 16 with tissue samples that could be analyzed at multiple sites were examined. The proportion of patients with the same PIK3CA mutations at all sites was 12.5% (2/16). The proportion of patients with the same PIK3CA mutations at least two or more sites, but not at all sites, was 31.2% (5/16). The proportion of patients with no PIK3CA mutations at other sites was 37.5% (6/16). The proportion of patients with other PIK3CA mutations at other sites was 18.7% (3/16). There is intertumor and intratumor heterogeneity of PIK3CA mutations. PIK3CA mutations in EMPD may be progressor mutations in EMPD.

Keywords:   PIK3CA ; droplet digital polymerase chain reaction; intertumor heterogeneity; intratumor heterogeneity; multiple mutations

DOI:  https://doi.org/10.1111/1346-8138.16343
Nat Commun. 2022 Mar 10. 13(1): 1246

Fully-automated and ultra-fast cell-type identification using specific marker combinations from single-cell transcriptomic data.

Aleksandr Ianevski, Anil K Giri, Tero Aittokallio.

Identification of cell populations often relies on manual annotation of cell clusters using established marker genes. However, the selection of marker genes is a time-consuming process that may lead to sub-optimal annotations as the markers must be informative of both the individual cell clusters and various cell types present in the sample. Here, we developed a computational platform, ScType, which enables a fully-automated and ultra-fast cell-type identification based solely on a given scRNA-seq data, along with a comprehensive cell marker database as background information. Using six scRNA-seq datasets from various human and mouse tissues, we show how ScType provides unbiased and accurate cell type annotations by guaranteeing the specificity of positive and negative marker genes across cell clusters and cell types. We also demonstrate how ScType distinguishes between healthy and malignant cell populations, based on single-cell calling of single-nucleotide variants, making it a versatile tool for anticancer applications. The widely applicable method is deployed both as an interactive web-tool ( https://sctype.app ), and as an open-source R-package.

DOI: https://doi.org/10.1038/s41467-022-28803-w
Nat Methods. 2022 Mar;19(3): 296-306

A pan-tissue DNA methylation atlas enables in silico decomposition of human tissue methylomes at cell-type resolution.

Tianyu Zhu, Jacklyn Liu, Stephan Beck, Sun Pan, David Capper, Matt Lechner, Chrissie Thirlwell, Charles E Breeze, Andrew E Teschendorff.

Bulk-tissue DNA methylomes represent an average over many different cell types, hampering our understanding of cell-type-specific contributions to disease development. As single-cell methylomics is not scalable to large cohorts of individuals, cost-effective computational solutions are needed, yet current methods are limited to tissues such as blood. Here we leverage the high-resolution nature of tissue-specific single-cell RNA-sequencing datasets to construct a DNA methylation atlas defined for 13 solid tissue types and 40 cell types. We comprehensively validate this atlas in independent bulk and single-nucleus DNA methylation datasets. We demonstrate that it correctly predicts the cell of origin of diverse cancer types and discovers new prognostic associations in olfactory neuroblastoma and stage 2 melanoma. In brain, the atlas predicts a neuronal origin for schizophrenia, with neuron-specific differential DNA methylation enriched for corresponding genome-wide association study risk loci. In summary, the DNA methylation atlas enables the decomposition of 13 different human tissue types at a high cellular resolution, paving the way for an improved interpretation of epigenetic data.

DOI: https://doi.org/10.1038/s41592-022-01412-7
Sci Rep. 2022 Mar 08. 12(1): 4043

PTEN/PI3K/Akt pathway alters sensitivity of T-cell acute lymphoblastic leukemia to L-asparaginase.

Katerina Hlozkova, Ivana Hermanova, Lucie Safrhansova, Natividad Alquezar-Artieda, Daniela Kuzilkova, Adela Vavrova, Kristyna Sperkova, Marketa Zaliova, Jan Stary, Jan Trka, Julia Starkova.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) still remains a therapeutic challenge due to relapses which are resistant to further treatment. L-asparaginase (ASNase) is a key therapy component in pediatric T-ALL and lower sensitivity of leukemia cells to this drug negatively influences overall treatment efficacy and outcome. PTEN protein deletion and/or activation of the PI3K/Akt signaling pathway leading to altered cell growth and metabolism are emerging as a common feature in T-ALL. We herein investigated the relationship amongst PTEN deletion, ASNase sensitivity and glucose metabolism in T-ALL cells. First, we found significant differences in the sensitivity to ASNase amongst T-ALL cell lines. While cell lines more sensitive to ASNase were PTEN wild type (WT) and had no detectable level of phosphorylated Akt (P-Akt), cell lines less sensitive to ASNase were PTEN-null with high P-Akt levels. Pharmacological inhibition of Akt in the PTEN-null cells rendered them more sensitive to ASNase and lowered their glycolytic function which then resembled PTEN WT cells. In primary T-ALL cells, although P-Akt level was not dependent exclusively on PTEN expression, their sensitivity to ASNase could also be increased by pharmacological inhibition of Akt. In summary, we highlight a promising therapeutic option for T-ALL patients with aberrant PTEN/PI3K/Akt signaling.

DOI: https://doi.org/10.1038/s41598-022-08049-8
Cell Signal. 2022 Mar 05. pii: S0898-6568(22)00062-6. [Epub ahead of print] 110301

Investigation of the function of the PI3-Kinase / AKT signaling pathway for leukemogenesis and therapy of acute childhood lymphoblastic leukemia (ALL).

Patrick Ehm, Astrid Grottke, Bettina Bettin, Manfred Jücker.

  Acute lymphoblastic leukemia is the most common cause of cancer-related death in children and, especially for patients in a high-risk group, still represents a poor prognosis. The PI3K/AKT/mTOR signaling pathway has been identified as a frequently constitutively activated switching point in the disease of ALL. Despite the knowledge of the therapeutic importance of the signaling pathway, the results of clinically effective treatment strategies have so far been extremely sobering. In particular, monotherapy approaches represent a major problem with regard to cell resistance. In this work, the PI3K/AKT/mTOR signaling pathway was examined as a therapeutic target for the treatment of childhood acute lymphoblastic leukemia (ALL) with a new therapeutic approach to avoid cell resistance. Therefore, we used a combined therapeutic approach with inhibitors directed against AKT (MK2206), mTOR (RAD001) and the most prominent and aberrantly activated tyrosine kinase. In case of BCR-ABL-positive B-ALL cells we used a combination with the classic inhibitor Imatinib and in case of MLL-AF4-positive B-ALL cells we used a combination with Quizartinib (directed against FLT3). We show, in particular compared to the monotherapies, a highly significant inhibition of the growth of these cells after this new specific triple combination therapy. Furthermore, we show that inhibiting AKT alone leads to a feedback mechanism and an upregulation of the phosphorylation of a number of receptor-tyrosine-kinases. After isoform-specific knockdown of the three AKT isoforms in ALL cells we identified that especially ErbB2/Her2 is most strongly phosphorylated in cells with AKT2 knockdown. AKT isoform 1 and 2 knockdown cells show, in contrast to AKT isoform 3 knockdown cells, a weak proliferation and are presumably kept alive among others by the increased phosphorylation of the receptor-tyrosine-kinase ErbB2. This work provides first indications for a new combination therapy of B-ALL cells, which is directed against AKT, mTOR and a predominantly highly activated kinase.

Keywords:  AKT-isoforms; Acute lymphoblastic leukemia; Imatinib; MK2206; PI3K/AKT/mTOR-signaling; RAD001

DOI:  https://doi.org/10.1016/j.cellsig.2022.110301
Nature. 2022 Mar 09.

A non-canonical tricarboxylic acid cycle underlies cellular identity.

Paige K Arnold, Benjamin T Jackson, Katrina I Paras, Julia S Brunner, Madeleine L Hart, Oliver J Newsom, Sydney P Alibeckoff, Jennifer Endress, Esther Drill, Lucas B Sullivan, Lydia W S Finley.

The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.

DOI: https://doi.org/10.1038/s41586-022-04475-w
Commun Biol. 2022 Mar 10. 5(1): 219

Myoepithelial progenitors as founder cells of hyperplastic human breast lesions upon PIK3CA transformation.

Nadine Goldhammer, Jiyoung Kim, René Villadsen, Lone Rønnov-Jessen, Ole William Petersen.

The myoepithelial (MEP) lineage of human breast comprises bipotent and multipotent progenitors in ducts and terminal duct lobular units (TDLUs). We here assess whether this heterogeneity impacts on oncogenic PIK3CA transformation. Single cell RNA sequencing (scRNA-seq) and multicolor imaging reveal that terminal ducts represent the most enriched source of cells with ductal MEP markers including α-smooth muscle actin (α-SMA), keratin K14, K17 and CD200. Furthermore, we find neighboring CD200high and CD200low progenitors within terminal ducts. When sorted and kept in ground state conditions, their CD200low and CD200high phenotypes are preserved. Upon differentiation, progenitors remain multipotent and bipotent, respectively. Immortalized progenitors are transduced with mutant PIK3CA on an shp53 background. Upon transplantation, CD200low MEP progenitors distinguish from CD200high by the formation of multilayered structures with a hyperplastic inner layer of luminal epithelial cells. We suggest a model with spatially distributed MEP progenitors as founder cells of biphasic breast lesions with implications for early detection and prevention strategies.

DOI: https://doi.org/10.1038/s42003-022-03161-x
Cells. 2022 Feb 24. pii: 793. [Epub ahead of print]11(5):

Enhanced Wild-Type MET Receptor Levels in Mouse Hepatocytes Attenuates Insulin-Mediated Signaling.

Patricia Rada, Fabienne Lamballe, Elena Carceller-López, Ana B Hitos, Celia Sequera, Flavio Maina, Ángela M Valverde.

  Compelling evidence points to the MET receptor tyrosine kinase as a key player during liver development and regeneration. Recently, a role of MET in the pathophysiology of insulin resistance and obesity is emerging. Herein, we aimed to determine whether MET regulates hepatic insulin sensitivity. To achieve this, mice in which the expression of wild-type MET in hepatocytes is slightly enhanced above endogenous levels (Alb-R26Met mice) were analyzed to document glucose homeostasis, energy balance, and insulin signaling in hepatocytes. We found that Alb-R26Met mice exhibited higher body weight and food intake when compared to R26stopMet control mice. Metabolic analyses revealed that Alb-R26Met mice presented age-related glucose and pyruvate intolerance in comparison to R26stopMet controls. Additionally, in Alb-R26Met mice, high MET levels decreased insulin-induced insulin receptor (IR) and AKT phosphorylation compared to control mice. These results were corroborated in vitro by analyzing IR and AKT phosphorylation in primary mouse hepatocytes from Alb-R26Met and R26stopMet mice upon insulin stimulation. Moreover, co-immunoprecipitation assays revealed MET-IR interaction under both basal and insulin stimulation conditions; this effect was enhanced in Alb-R26Met hepatocytes. Altogether, our results indicate that enhanced MET levels alter hepatic glucose homeostasis, which can be an early event for subsequent liver pathologies.

Keywords:  MET; glucose homeostasis; hepatocytes; insulin signaling

DOI:  https://doi.org/10.3390/cells11050793
Pediatr Blood Cancer. 2022 Mar 06. e29603

How we approach the use of sirolimus and new agents: Medical therapy to treat vascular anomalies.

Kristin A Shimano, Whitney Eng, Denise M Adams.

  Vascular anomalies (VAs) are a heterogeneous group of primarily congenital tumors and malformations. The International Society for the Study of Vascular Anomalies (ISSVA) has developed a standard classification of these disorders, creating a uniform approach to their diagnosis. Recent discoveries evaluating the genetic causes of VAs have revealed that they are due to mutations in cancer pathways, including the PI3K/AKT/mTOR and RAS/MAPK/MEK pathways. These discoveries have led to improved phenotype-genotype correlation and have expanded medical therapy for this group of unique disorders.

Keywords:  AKT inhibitor; MEK inhibitors; PIK3CA inhibitor; sirolimus; thalidomide; vascular anomalies

DOI:  https://doi.org/10.1002/pbc.29603
Science. 2022 Mar 11. 375(6585): eabi6983

OpenCell: Endogenous tagging for the cartography of human cellular organization.

Nathan H Cho, Keith C Cheveralls, Andreas-David Brunner, Kibeom Kim, André C Michaelis, Preethi Raghavan, Hirofumi Kobayashi, Laura Savy, Jason Y Li, Hera Canaj, James Y S Kim, Edna M Stewart, Christian Gnann, Frank McCarthy, Joana P Cabrera, Rachel M Brunetti, Bryant B Chhun, Greg Dingle, Marco Y Hein, Bo Huang, Shalin B Mehta, Jonathan S Weissman, Rafael Gómez-Sjöberg, Daniel N Itzhak, Loïc A Royer, Matthias Mann, Manuel D Leonetti.

Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.

DOI: https://doi.org/10.1126/science.abi6983
Nat Struct Mol Biol. 2022 Mar 07.

Structural basis of phosphatidylinositol 3-kinase C2α function.

Wen-Ting Lo, Yingyi Zhang, Oscar Vadas, Yvette Roske, Federico Gulluni, Maria Chiara De Santis, Andreja Vujicic Zagar, Heike Stephanowitz, Emilio Hirsch, Fan Liu, Oliver Daumke, Misha Kudryashev, Volker Haucke.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, viral replication, platelet formation and a role in mitosis. The molecular basis of these activities of PI3KC2α is poorly understood. Here, we report high-resolution crystal structures as well as a 4.4-Å cryogenic-electron microscopic (cryo-EM) structure of PI3KC2α in active and inactive conformations. We unravel a coincident mechanism of lipid-induced activation of PI3KC2α at membranes that involves large-scale repositioning of its Ras-binding and lipid-binding distal Phox-homology and C-C2 domains, and can serve as a model for the entire class II PI3K family. Moreover, we describe a PI3KC2α-specific helical bundle domain that underlies its scaffolding function at the mitotic spindle. Our results advance our understanding of PI3K biology and pave the way for the development of specific inhibitors of class II PI3K function with wide applications in biomedicine.

DOI: https://doi.org/10.1038/s41594-022-00730-w
Nat Rev Cancer. 2022 Mar 09.

Therapy resistance: opportunities created by adaptive responses to targeted therapies in cancer.

Marilyne Labrie, Joan S Brugge, Gordon B Mills, Ioannis K Zervantonakis.

Normal cells explore multiple states to survive stresses encountered during development and self-renewal as well as environmental stresses such as starvation, DNA damage, toxins or infection. Cancer cells co-opt normal stress mitigation pathways to survive stresses that accompany tumour initiation, progression, metastasis and immune evasion. Cancer therapies accentuate cancer cell stresses and invoke rapid non-genomic stress mitigation processes that maintain cell viability and thus represent key targetable resistance mechanisms. In this Review, we describe mechanisms by which tumour ecosystems, including cancer cells, immune cells and stroma, adapt to therapeutic stresses and describe three different approaches to exploit stress mitigation processes: (1) interdict stress mitigation to induce cell death; (2) increase stress to induce cellular catastrophe; and (3) exploit emergent vulnerabilities in cancer cells and cells of the tumour microenvironment. We review challenges associated with tumour heterogeneity, prioritizing actionable adaptive responses for optimal therapeutic outcomes, and development of an integrative framework to identify and target vulnerabilities that arise from adaptive responses and engagement of stress mitigation pathways. Finally, we discuss the need to monitor adaptive responses across multiple scales and translation of combination therapies designed to take advantage of adaptive responses and stress mitigation pathways to the clinic.

DOI: https://doi.org/10.1038/s41568-022-00454-5
Cancer Commun (Lond). 2022 Mar 07.

Indirect targeting of MYC sensitizes pancreatic cancer cells to mechanistic target of rapamycin (mTOR) inhibition.

Christian Schneeweis, Zonera Hassan, Katja Ascherl, Matthias Wirth, Stella Koutsouli, Felix Orben, Lukas Krauß, Carolin Schneider, Rupert Öllinger, Oliver H Krämer, Roland Rad, Maximilian Reichert, Maria S Robles, Dieter Saur, Günter Schneider.

DOI: https://doi.org/10.1002/cac2.12280
Cancer Genet. 2022 Feb 25. pii: S2210-7762(22)00014-X. [Epub ahead of print]264-265 8-15

PTEN alterations in sporadic and BRCA1-associated triple negative breast carcinomas.

Natalie Jones, Audrey Gros, Valérie Velasco, Valérie Dapremont, Véronique Brouste, Bernadette Gastaldello, Marc Debled, Christine Tunon de Lara, Françoise Bonnet, Emmanuelle Barouk-Simonet, Virginie Bubien, Laurence Venat, Gaëtan MacGrogan, Michel Longy, Nicolas Sevenet.

  The similarities between sporadic basal-like breast cancer (BLBC) and BRCA1-mutated breast tumours raise the possibility that deregulation of the same pathway may underlie these tumour types. The aim of this study was to determine if PTEN aberrations are characteristic of both BRCA1 tumours and sporadic TN breast carcinomas with low BRCA1 expression, and can thus be used to identify sporadic tumours potentially sensitive to PARP inhibitors. Twelve BRCA1 tumours, 19 non-BRCA familial breast tumours and 71 unselected TN breast carcinomas were screened for PTEN mutations and assessed for PTEN expression and BRCA1 mRNA expression. Loss of PTEN expression was observed in 67% of BRCA1 tumours and more specifically in 89% of TN BRCA1 tumours highlighting the link between PTEN loss and BLBC in the context of germline BRCA1 mutations. Regarding unselected TN tumours, 56% showed PTEN expression loss and 35% displayed low BRCA1 mRNA expression. Unlike familial breast cancers with low BRCA1 mRNA expression, no significant correlation was observed between the loss of PTEN expression and low BRCA1 mRNA expression in this unselected TN tumours panel. Our data suggest that, unlike the germinal context, PTEN and BRCA1 alterations in sporadic TN breast tumours are independent events.

Keywords:  BRCA1; Basal-like; Breast cancer; PTEN; Triple negative

DOI:  https://doi.org/10.1016/j.cancergen.2022.02.008
Proc Natl Acad Sci U S A. 2022 Mar 15. 119(11): e2114438119

Using single-cell models to predict the functionality of synthetic circuits at the population scale.

Chetan Aditya, François Bertaux, Gregory Batt, Jakob Ruess.

  SignificanceAt the single-cell level, biochemical processes are inherently stochastic. For many natural systems, the resulting cell-to-cell variability is exploited by microbial populations. In synthetic biology, however, the interplay of cell-to-cell variability and population processes such as selection or growth often leads to circuits not functioning as predicted by simple models. Here we show how multiscale stochastic kinetic models that simultaneously track single-cell and population processes can be obtained based on an augmentation of the chemical master equation. These models enable us to quantitatively predict complex population dynamics of a yeast optogenetic differentiation system from a specification of the circuit's components and to demonstrate how cell-to-cell variability can be exploited to purposefully create unintuitive circuit functionality.

Keywords:  chemical master equation; composability; optogenetics; population dynamics; synthetic differentiation circuits

DOI:  https://doi.org/10.1073/pnas.2114438119
Cell. 2022 Mar 02. pii: S0092-8674(22)00200-8. [Epub ahead of print]

Mapping the long road to cancer.

David M Cheek, Kamila Naxerova.

Every cell in our body accumulates mutations throughout life, and sometimes an unfortunate combination of mutations drives the initiation of cancer. A new study infers extraordinarily detailed timelines of pre-cancerous evolution by sequencing single-cell genomes in patients with blood malignancies-finding that key mutations can arrive decades before diagnosis.

DOI: https://doi.org/10.1016/j.cell.2022.02.020
Elife. 2022 Mar 08. pii: e73150. [Epub ahead of print]11

Confined migration promotes cancer metastasis through resistance to anoikis and increased invasiveness.

Deborah Fanfone, Zhichong Wu, Jade Mammi, Kevin Berthenet, David Neves, Kathrin Weber, Andrea Halaburkova, François Virard, Félix Bunel, Catherine Jamard, Hector Hernandez-Vargas, Stephen W G Tait, Ana Hennino, Gabriel Ichim.

  Mechanical stress is known to fuel several hallmarks of cancer, ranging from genome instability to uncontrolled proliferation or invasion. Cancer cells are constantly challenged by mechanical stresses not only in the primary tumour but also during metastasis. However, this latter has seldom been studied with regards to mechanobiology, in particular resistance to anoikis, a cell death programme triggered by loss of cell adhesion. Here, we show in vitro that migrating breast cancer cells develop resistance to anoikis following their passage through microporous membranes mimicking confined migration (CM), a mechanical constriction that cancer cells encounter during metastasis. This CM-induced resistance was mediated by Inhibitory of Apoptosis Proteins, and sensitivity to anoikis could be restored after their inhibition using second mitochondria-derived activator of caspase (SMAC) mimetics. Anoikis-resistant mechanically stressed cancer cells displayed enhanced cell motility and evasion from natural killer cell-mediated immune surveillance, as well as a marked advantage to form lung metastatic lesions in mice. Our findings reveal that CM increases the metastatic potential of breast cancer cells.

Keywords:  IAP; anoikis; cancer biology; caspases; cell biology; confinement; human; mechanobiology; metastasis; mouse

DOI:  https://doi.org/10.7554/eLife.73150
Int J Cancer. 2022 Mar 07.

Mutations in the telomerase reverse transcriptase promoter and PIK3CA gene are common events in penile squamous cell carcinoma of Italian and Ugandan patients.

Noemy Starita, Francesca Pezzuto, Sabrina Sarno, Nunzia Simona Losito, Sisto Perdonà, Luigi Buonaguro, Franco M Buonaguro, Maria Lina Tornesello.

  Penile carcinoma develops either through human papillomavirus (HPV) related or unrelated carcinogenic pathways. Genetic alterations and nucleotide changes in coding regions (i.e. TP53, CDKN2A, PIK3CA and NOTCH1) are main cancer driver events either in HPV positive or in HPV negative tumours. We investigated the presence of hotspot nucleotide mutations in TERT promoter (TERTp) and PIK3CA exon 9 and their relationship with HPV status in 69 penile cancer cases from Italian and Ugandan patients. Genetic variations and viral sequences have been characterized by end-point PCR and Sanger sequencing. The mutant allele frequencies (MAFs) of TERTp -124A/-146A and PIK3CA E545K have been determined by droplet digital PCR (ddPCR) assays. The results showed that TERTp mutations are highly prevalent in penile carcinoma (53.6%) and significantly more frequent in HPV negative (67.6%) than HPV positive (32.4%) cases (P=0.0482). PIK3CA mutations were similarly distributed in virus-related and unrelated cases (25.9% and 26.7%, respectively) and coexisted with TERTp changes in 15.8% of penile carcinoma samples. Notably, MAFs of co-occurring mutations were frequently discordant indicating that PIK3CA E545K nucleotide changes are subsequent genetic events occurring in sub-clones of TERTp mutated cells. The frequencies of TERTp and PIK3CA mutations were higher among Italian compared to Ugandan cases and inversely correlated with the HPV status. In conclusion, TERTp mutations are very common in penile carcinoma and their coexistence with PIK3CA in a substantial number of cases may represent a novel oncogenic synergy relevant for patient stratification and use of therapeutic strategies against new actionable targets. This article is protected by copyright. All rights reserved.

Keywords:  Digital droplet PCR, ddPCR; Human papillomavirus; Italy; Mutations; PIK3CA exon 9; Penile squamous cell carcinoma; TERT promoter; Uganda

DOI:  https://doi.org/10.1002/ijc.33990
Trends Cancer. 2022 Mar 08. pii: S2405-8033(22)00041-3. [Epub ahead of print]

Optimizing the future: how mathematical models inform treatment schedules for cancer.

Deepti Mathur, Ethan Barnett, Howard I Scher, Joao B Xavier.

  For decades, mathematical models have influenced how we schedule chemotherapeutics. More recently, mathematical models have leveraged lessons from ecology, evolution, and game theory to advance predictions of optimal treatment schedules, often in a personalized medicine manner. We discuss both established and emerging therapeutic strategies that deviate from canonical standard-of-care regimens, and how mathematical models have contributed to the design of such schedules. We first examine scheduling options for single therapies and review the advantages and disadvantages of various treatment plans. We then consider the challenge of scheduling multiple therapies, and review the mathematical and clinical support for various conflicting treatment schedules. Finally, we propose how a consilience of mathematical and clinical knowledge can best determine the optimal treatment schedules for patients.

Keywords:  clinical trials; mathematical models; treatment schedules

DOI:  https://doi.org/10.1016/j.trecan.2022.02.005
Elife. 2022 Mar 11. pii: e76595. [Epub ahead of print]11

A saturation-mutagenesis analysis of the interplay between stability and activation in Ras.

Frank Hidalgo, Laura M Nocka, Neel H Shah, Kent Gorday, Naomi R Latorraca, Pradeep Bandaru, Sage Templeton, David Lee, Deepti Karandur, Jeffrey G Pelton, Susan Marqusee, David Wemmer, John Kuriyan.

  Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

Keywords:  E. coli; ba/f3; cancer; cancer mutants; h-ras; high throughput screening; k-ras; molecular biophysics; ras; saturation-mutagenesis; signaling activation; structural biology

DOI:  https://doi.org/10.7554/eLife.76595
Curr Genomics. 2021 Dec 16. 22(4): 239-243

Deep Hidden Physics Modeling of Cell Signaling Networks.

Martin Seeger, James Longden, Edda Klipp, Rune Linding.

According to the WHO, cancer is the second most common cause of death worldwide. The social and economic damage caused by cancer is high and rising. In Europe, the annual direct medical expenses alone amount to more than €129 billion. This results in an urgent need for new and sustainable therapeutics, which has currently not been met by the pharmaceutical industry; only 3.4% of cancer drugs entering Phase I clinical trials get to market. Phosphorylation sites are parts of the core machinery of kinase signaling networks, which are known to be dysfunctional in all types of cancer. Indeed, kinases are the second most common drug target yet. However, these inhibitors block all functions of a protein, and they commonly lead to the development of resistance and increased toxicity. To facilitate global and mechanistic modeling of cancer and clinically relevant cell signaling networks, the community will have to develop sophisticated data-driven deep-learning and mechanistic computational models that generate in silico probabilistic predictions of molecular signaling network rearrangements causally implicated in cancer.

DOI: https://doi.org/10.2174/1389202922666210614131236
PLoS Comput Biol. 2022 Mar 09. 18(3): e1009935

Urgent need for consistent standards in functional enrichment analysis.

Kaumadi Wijesooriya, Sameer A Jadaan, Kaushalya L Perera, Tanuveer Kaur, Mark Ziemann.

Gene set enrichment tests (a.k.a. functional enrichment analysis) are among the most frequently used methods in computational biology. Despite this popularity, there are concerns that these methods are being applied incorrectly and the results of some peer-reviewed publications are unreliable. These problems include the use of inappropriate background gene lists, lack of false discovery rate correction and lack of methodological detail. To ascertain the frequency of these issues in the literature, we performed a screen of 186 open-access research articles describing functional enrichment results. We find that 95% of analyses using over-representation tests did not implement an appropriate background gene list or did not describe this in the methods. Failure to perform p-value correction for multiple tests was identified in 43% of analyses. Many studies lacked detail in the methods section about the tools and gene sets used. An extension of this survey showed that these problems are not associated with journal or article level bibliometrics. Using seven independent RNA-seq datasets, we show misuse of enrichment tools alters results substantially. In conclusion, most published functional enrichment studies suffered from one or more major flaws, highlighting the need for stronger standards for enrichment analysis.

DOI: https://doi.org/10.1371/journal.pcbi.1009935
Cancers (Basel). 2022 Mar 02. pii: 1286. [Epub ahead of print]14(5):

PIK3CA Gene Mutations in HNSCC: Systematic Review and Correlations with HPV Status and Patient Survival.

Daniela Cochicho, Susana Esteves, Miguel Rito, Fernanda Silva, Luís Martins, Pedro Montalvão, Mário Cunha, Miguel Magalhães, Rui M Gil da Costa, Ana Felix.

  PIK3CA mutations are believed to contribute to the pathogenesis of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC). This study aims to establish the frequency of PIK3CA mutations in a Portuguese HNSCC cohort and to determine their association with the HPV status and patient survival. A meta-analysis of scientific literature also revealed widely different mutation rates in cohorts from different world regions and a trend towards improved prognosis among patients with PIK3CA mutations. DNA samples were available from 95 patients diagnosed with HNSCC at the Portuguese Institute of Oncology in Lisbon between 2010 and 2019. HPV status was established based on viral DNA detected using real-time PCR. The evaluation of PIK3CA gene mutations was performed by real-time PCR for four mutations (H1047L; E542K, E545K, and E545D). Thirty-seven cases were found to harbour PIK3CA mutations (39%), with the E545D mutation (73%) more frequently detected. There were no significant associations between the mutational status and HPV status (74% WT and 68% MUT were HPV (+); p = 0.489) or overall survival (OS) (3-year OS: WT 54% and MUT 65%; p = 0.090). HPV status was the only factor significantly associated with both OS and disease-free survival (DFS), with HPV (+) patients having consistently better outcomes (3-year OS: HPV (+) 65% and HPV (-) 36%; p = 0.007; DFS HPV (+) 83% and HPV (-) 43%; p = 0.001). There was a statistically significant interaction effect between HPV status and PIK3CA mutation regarding DFS (Interaction test: p = 0.026). In HPV (+) patients, PIK3CA wild-type is associated with a significant 4.64 times increase in the hazard of recurrence or death (HR = 4.64; 95% CI 1.02-20.99; p = 0.047). Overall, PIK3CA gene mutations are present in a large number of patients and may help define patient subsets who can benefit from therapies targeting the PI3K pathway. The systematic assessment of PIK3CA gene mutations in HNSCC patients will require further methodological standardisation.

Keywords:  HNSCC; HPV; PIK3CA; p16 INK4a

DOI:  https://doi.org/10.3390/cancers14051286
Nat Methods. 2022 Mar;19(3): 316-322

Alevin-fry unlocks rapid, accurate and memory-frugal quantification of single-cell RNA-seq data.

Dongze He, Mohsen Zakeri, Hirak Sarkar, Charlotte Soneson, Avi Srivastava, Rob Patro.

The rapid growth of high-throughput single-cell and single-nucleus RNA-sequencing (scRNA-seq and snRNA-seq) technologies has produced a wealth of data over the past few years. The size, volume and distinctive characteristics of these data necessitate the development of new computational methods to accurately and efficiently quantify sc/snRNA-seq data into count matrices that constitute the input to downstream analyses. We introduce the alevin-fry framework for quantifying sc/snRNA-seq data. In addition to being faster and more memory frugal than other accurate quantification approaches, alevin-fry ameliorates the memory scalability and false-positive expression issues that are exhibited by other lightweight tools. We demonstrate how alevin-fry can be effectively used to quantify sc/snRNA-seq data, and also how the spliced and unspliced molecule quantification required as input for RNA velocity analyses can be seamlessly extracted from the same preprocessed data used to generate normal gene expression count matrices.

DOI: https://doi.org/10.1038/s41592-022-01408-3
Genes Dev. 2022 Mar 10.

Isoform-specific functions of PPARγ in gene regulation and metabolism.

Wenxiang Hu, Chunjie Jiang, Mindy Kim, Yang Xiao, Hannah J Richter, Dongyin Guan, Kun Zhu, Brianna M Krusen, Arielle N Roberts, Jessica Miller, David J Steger, Mitchell A Lazar.

  Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that is a vital regulator of adipogenesis, insulin sensitivity, and lipid metabolism. Activation of PPARγ by antidiabetic thiazolidinediones (TZD) reverses insulin resistance but also leads to weight gain that limits the use of these drugs. There are two main PPARγ isoforms, but the specific functions of each are not established. Here we generated mouse lines in which endogenous PPARγ1 and PPARγ2 were epitope-tagged to interrogate isoform-specific genomic binding, and mice deficient in either PPARγ1 or PPARγ2 to assess isoform-specific gene regulation. Strikingly, although PPARγ1 and PPARγ2 contain identical DNA binding domains, we uncovered isoform-specific genomic binding sites in addition to shared sites. Moreover, PPARγ1 and PPARγ2 regulated a different set of genes in adipose tissue depots, suggesting distinct roles in adipocyte biology. Indeed, mice with selective deficiency of PPARγ1 maintained body temperature better than wild-type or PPARγ2-deficient mice. Most remarkably, although TZD treatment improved glucose tolerance in mice lacking either PPARγ1 or PPARγ2, the PPARγ1-deficient mice were protected from TZD-induced body weight gain compared with PPARγ2-deficient mice. Thus, PPARγ isoforms have specific and separable metabolic functions that may be targeted to improve therapy for insulin resistance and diabetes.

Keywords:  PPARγ; adipocyte; diabetes; gene regulation; nuclear receptor; obesity

DOI:  https://doi.org/10.1101/gad.349232.121
Clin Dysmorphol. 2022 Mar 07.

Clinical implications of mosaicism: a 10-year retrospective review of 83 families in a university-affiliated genetics clinic.

Mianne Lee, Adrian C Y Lui, Christopher C Y Mak, Mandy H Y Tsang, Jasmine L F Fung, K S Yeung, Brian Hon Yin Chung.

Mosaicism refers to the coexistence of two or more genetically distinct cell populations in an individual from a single fertilized egg. We performed a retrospective analysis of all patients diagnosed with mosaic disorders between 2010 and 2021 in a university-affiliated genetics clinic, which attends to territory-wide genetic consultations. All patients with confirmed mosaic diagnoses through reproductive (n = 6), prenatal (n = 24), and postnatal (n = 53) testing were examined. We observed that mosaic 45, X (n = 31) and PIK3CA-related overgrowth spectrum (n = 16) disorders were among the most prevalent diagnoses in the clinic, and the total percentage of patients with mosaicism in our cohort was 2.0% (83/4157). A review of the diagnostic journey highlights the challenge in diagnosing mosaic disorders, whereby 38% of the subjects required more than one test sample, and 52% of the cases required more than one orthogonal method of detection to reach the correct diagnosis. While detection of mosaicism is passive through routine clinical testing, for example karyotyping in reproductive and prenatal care, in postnatal care, clinicians can more actively drive the detection of mosaicism. Therefore, we recommend a low threshold for additional genetic testing in suspected mosaicism for more accurate diagnosis and counselling.

DOI: https://doi.org/10.1097/MCD.0000000000000418
Nat Commun. 2022 Mar 09. 13(1): 1224

Co-translational assembly orchestrates competing biogenesis pathways.

Maximilian Seidel, Anja Becker, Filipa Pereira, Jonathan J M Landry, Nayara Trevisan Doimo de Azevedo, Claudia M Fusco, Eva Kaindl, Natalie Romanov, Janina Baumbach, Julian D Langer, Erin M Schuman, Kiran Raosaheb Patil, Gerhard Hummer, Vladimir Benes, Martin Beck.

During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examine structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally test candidate structural motifs and identify several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs may act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assemble co-translationally in only some but not all of the relevant biogenesis pathways. Our results highlight the regulatory complexity of assembly pathways.

DOI: https://doi.org/10.1038/s41467-022-28878-5
Mol Cancer Ther. 2022 Feb 28. pii: molcanther.MCT-21-0574-A.2021. [Epub ahead of print]

Optimal strategy and benefit of pulsed therapy depend on tumor heterogeneity and aggressiveness at time of treatment initiation.

Deepti Mathur, Bradford P Taylor, Walid K Chatila, Howard I Scher, Nikolaus Schultz, Pedram Razavi, Joao Xavier.

Therapeutic resistance is a fundamental obstacle in cancer treatment. Tumors that initially respond to treatment may have a pre-existing resistant subclone or acquire resistance during treatment, making relapse theoretically inevitable. Here, we investigate treatment strategies that may delay relapse using mathematical modeling. We find that for a single-drug therapy, pulse treatment - short, elevated doses followed by a complete break from treatment - delays relapse compared to continuous treatment with the same total dose over a length of time. For tumors treated with more than one drug, continuous combination treatment is only sometimes better than sequential treatment, while pulsed combination treatment or simply alternating between the two therapies at defined intervals delay relapse the longest. These results are independent of the fitness cost or benefit of resistance, and are robust to noise. Machine-learning analysis of simulations shows that the initial tumor response and heterogeneity at the start of treatment suffice to determine the benefit of pulsed or alternating treatment strategies over continuous treatment. Analysis of 8 tumor burden trajectories of breast cancer patients treated at MSK shows the model can predict time to resistance using initial responses to treatment and estimated pre-existing resistant populations. The model calculated that pulse treatment would delay relapse in all 8 cases. Overall, our results support that pulsed treatments optimized by mathematical models could delay therapeutic resistance.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0574
Clin Chem. 2022 Mar 09. pii: hvac030. [Epub ahead of print]

Reproducibility: Has Cancer Biology Failed beyond Repair?

John P A Ioannidis.

DOI: https://doi.org/10.1093/clinchem/hvac030
Am J Med Genet A. 2022 Mar 09.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

Maria I Kontaridis, Amy E Roberts, Lisa Schill, Lisa Schoyer, Beth Stronach, Gregor Andelfinger, Yoko Aoki, Marni E Axelrad, Annette Bakker, Anton M Bennett, Alberto Broniscer, Pau Castel, Caitlin A Chang, Lukas Cyganek, Tirtha K Das, Jeroen den Hertog, Emilia Galperin, Shruti Garg, Bruce D Gelb, Kristiana Gordon, Tamar Green, Karen W Gripp, Maxim Itkin, Maija Kiuru, Bruce R Korf, Jeff R Livingstone, Alejandro López-Juárez, Pilar L Magoulas, Sahar Mansour, Theresa Milner, Elisabeth Parker, Elizabeth I Pierpont, Kevin Plouffe, Katherine A Rauen, Suma P Shankar, Shane B Smith, David A Stevenson, Marco Tartaglia, Richard Van, Morgan E Wagner, Stephanie M Ware, Martin Zenker.

  RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

Keywords:  Costello syndrome; Noonan syndrome; RASopathy; cardiofaciocutaneus syndrome; neurofibromatosis; signaling

DOI:  https://doi.org/10.1002/ajmg.a.62716
J Biol Chem. 2022 Mar 05. pii: S0021-9258(22)00245-9. [Epub ahead of print] 101805

Host AKT-mediated phosphorylation of HIV-1 accessory protein Vif potentiates infectivity via enhanced degradation of the restriction factor APOBEC3G.

Rameez Raja, Chenyao Wang, Ritu Mishra, Arundhoti Das, Amjad Ali, Akhil C Banerjea.

  Human immunodeficiency virus-1 (HIV-1) encodes accessory proteins that neutralize anti-viral restriction factors to ensure its successful replication. One accessory protein, the HIV-1 viral infectivity factor (Vif), is known to promote ubiquitination and proteasomal degradation of the anti-viral restriction factor APOBEC3G, a cytosine deaminase which leads to hyper-mutations in the viral DNA and subsequent aberrant viral replication. We have previously demonstrated that the HIV-1 viral transcription mediator Tat activates the host pro-growth PI-3/AKT pathway, which in turn promotes HIV-1 replication. Because the HIV-1 Vif protein contains the putative AKT phosphorylation motif RMRINT, here we investigated whether AKT directly phosphorylates HIV-1 Vif to regulate its function. Co-immunoprecipitation experiments showed that AKT and Vif interact with each other, supporting this hypothesis. Using in vitro kinase assays, we further showed that AKT phosphorylates Vif at Thr20, which promotes its stability, as Vif becomes destabilized after this residue is mutated to alanine. Moreover, expression of dominant-negative kinase-deficient AKT (KD-AKT) as well as treatment with a chemical inhibitor of AKT (AKTi) increased K48-ubiquitination and proteasomal degradation of HIV-1 Vif. In contrast, constitutively active AKT (Myr-AKT) reduced K48-ubiquitination of Vif to promote its stability. Finally, inhibition of AKT function restored APOBEC3G levels, which subsequently reduced HIV-1 infectivity. Thus, our results establish a novel mechanism of HIV-1 Vif stabilization through AKT-mediated phosphorylation at Thr20, which reduces APOBEC3G levels and potentiates HIV-1 infectivity.

Keywords:  AKT; APOBEC3G; HIV-1 Vif; Phosphorylation; stabilization

DOI:  https://doi.org/10.1016/j.jbc.2022.101805
Nat Neurosci. 2022 Mar;25(3): 306-316

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

Samuel E Marsh, Alec J Walker, Tushar Kamath, Lasse Dissing-Olesen, Timothy R Hammond, T Yvanka de Soysa, Adam M H Young, Sarah Murphy, Abdulraouf Abdulraouf, Naeem Nadaf, Connor Dufort, Alicia C Walker, Liliana E Lucca, Velina Kozareva, Charles Vanderburg, Soyon Hong, Harry Bulstrode, Peter J Hutchinson, Daniel J Gaffney, David A Hafler, Robin J M Franklin, Evan Z Macosko, Beth Stevens.

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

DOI: https://doi.org/10.1038/s41593-022-01022-8