bims-pideca 2020-12-06 papers

on Class IA PI3K signalling in development and cancer

Issue of 2020‒12‒06
twenty-four papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus.
PTEN and Other PtdIns(3,4,5)P3 Lipid Phosphatases in Breast Cancer.
RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.
miR-29 Sustains B Cell Survival and Controls Terminal Differentiation via Regulation of PI3K Signaling.
mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.
mTORC1 Overactivation Leads to Abnormalities in Skeletal Development.
CDK1, the Other 'Master Regulator' of Autophagy.
Managing toxicities of phosphatidylinositol-3-kinase (PI3K) inhibitors.
Phosphatidylinositol 3-Kinase δ Inhibitor-Induced Immunomodulation and Secondary Opportunistic Infection in the Cynomolgus Monkey (Macaca fascicularis).
Isoform- and Paralog-Switching in IR-Signaling: When Diabetes Opens the Gates to Cancer.
Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region.
mTORC1-mediated amino acid signaling is critical for cell fate determination under transplant-induced stress.
Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities.
Imaging phenotype correlation with molecular and molecular pathway defects in malformations of cortical development.
Leveraging multi-way interactions for systematic prediction of pre-clinical drug combination effects.
Phase 2 Study of the Safety and Efficacy of Umbralisib in Patients with CLL Who Are Intolerant to BTK or PI3Kδ Inhibitor Therapy.
Transomics analysis reveals allosteric and gene regulation axes for altered hepatic glucose-responsive metabolism in obesity.
Metabolomics revealed the influence of breast cancer on lymphatic endothelial cell metabolism, metabolic crosstalk, and lymphangiogenic signaling in co-culture.
Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.
Loss of Furin in β cells Induces an mTORC1-ATF4 Anabolic Pathway that Leads to β cell Dysfunction.
Implication of integrins α3β1 and α5β1 in invasion and anoikis of SK-Mel-147 human melanoma cells: non-canonical functions of protein kinase Akt.
Accurate prediction of kinase-substrate networks using knowledge graphs.
The effect of a ketogenic diet and synergy with rapamycin in a mouse model of breast cancer.
Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stress.

Nat Commun. 2020 11 30. 11(1): 6088

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus.

Xin Zhou, Yanghao Zhong, Olivia Molinar-Inglis, Maya T Kunkel, Mingyuan Chen, Tengqian Sun, Jiao Zhang, John Y-J Shyy, JoAnn Trejo, Alexandra C Newton, Jin Zhang.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.

DOI: https://doi.org/10.1038/s41467-020-19937-w
Int J Mol Sci. 2020 Dec 02. pii: E9189. [Epub ahead of print]21(23):

PTEN and Other PtdIns(3,4,5)P3 Lipid Phosphatases in Breast Cancer.

Mariah P Csolle, Lisa M Ooms, Antonella Papa, Christina A Mitchell.

  The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. PtdIns(3,4,5)P3 can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P2. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced PIPP expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although PIPP depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression.

Keywords:  AKT; Src homology 2-containing inositol phosphatase 2 (SHIP2); breast cancer; inositol polyphosphate phosphatases; phosphatase tensin homolog deleted on chromosome 10 (PTEN); phosphoinositide 3-kinase (PI3K); proline rich inositol polyphosphate 5-phosphatase (PIPP); synaptojanin 2 (SYNJ2)

DOI:  https://doi.org/10.3390/ijms21239189
Mol Cell. 2020 Nov 23. pii: S1097-2765(20)30786-3. [Epub ahead of print]

RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.

Ayaz Najafov, Hoang Son Luu, Adnan K Mookhtiar, Lauren Mifflin, Hong-Guang Xia, Palak P Amin, Alban Ordureau, Huibing Wang, Junying Yuan.

  The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.

Keywords:  AMPK; CASP8; MLKL; RIPK1; RIPK3; TSC2; lysosome; mTORC1; neonatal lethality

DOI:  https://doi.org/10.1016/j.molcel.2020.11.008
Cell Rep. 2020 Dec 01. pii: S2211-1247(20)31425-X. [Epub ahead of print]33(9): 108436

miR-29 Sustains B Cell Survival and Controls Terminal Differentiation via Regulation of PI3K Signaling.

Marcus J Hines, Maryaline Coffre, Tenny Mudianto, Marisella Panduro, Eric J Wigton, Cosmin Tegla, Victoria Osorio-Vasquez, Robin Kageyama, David Benhamou, Oriana Perez, Sofia Bajwa, Michael T McManus, K Mark Ansel, Doron Melamed, Sergei B Koralov.

  The phosphatidylinositol 3-kinase (PI3K) signaling cascade downstream of the B cell receptor (BCR) signalosome is essential for B cell maturation. Proper signaling strength is maintained through the PI3K negative regulator phosphatase and tensin homolog (PTEN). Although a role for microRNA (miRNA)-dependent control of the PTEN-PI3K axis has been described, the contribution of individual miRNAs to the regulation of this crucial signaling modality in mature B lymphocytes remains to be elucidated. Our analyses reveal that ablation of miR-29 specifically in B lymphocytes results in an increase in PTEN expression and dampening of the PI3K pathway in mature B cells. This dysregulation has a profound impact on the survival of B lymphocytes and results in increased class switch recombination and decreased plasma cell differentiation. Furthermore, we demonstrate that ablation of one copy of Pten is sufficient to ameliorate the phenotypes associated with miR-29 loss. Our data suggest a critical role for the miR-29-PTEN-PI3K regulatory axis in mature B lymphocytes.

Keywords:  B cell; B lymphocyte; CSR; Class Switch Recombination; PI3K; PTEN; Plasma Cell; Terminal Differentiation; miR-29; miRNA

DOI:  https://doi.org/10.1016/j.celrep.2020.108436
J Biol Chem. 2020 Dec 03. pii: jbc.RA120.014960. [Epub ahead of print]

mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.

Roberta L Beauchamp, Serkan Erdin, Luke Witt, Justin T Jordan, Scott R Plotkin, James F Gusella, Vijaya Ramesh.

  Meningiomas (MN) arise from the arachnoid/meningeal layer and are non-responsive to chemotherapies, with ~50-60% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 signaling, leading to clinical trials for NF2 and meningioma. Recently our 'omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in both NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly NRG1/neuregulin 1, and confirm increased NRG1 secretion and activation of ERBB3 receptor tyrosine kinase to which NRG1 binds. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2 and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated basal mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a PDK1-dependent signaling mechanism independent of NRG1-ERBB3, but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of another receptor tyrosine kinase, IGF1R Drug treatment co-targeting mTORC1/2 and IGF1R/IR in NF2-null cells attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion of NRG1 and activation of ERBB3. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/IR, suggesting that co-targeting these pathways may prove effective for treatment of NF2-deficient meningioma.

Keywords:  Akt PKB; NF2; NRG1-ERBB3; brain tumor; dual mTORC1/mTORC2 inhibition; insulin-like growth factor (IGF) receptor; mammalian target of rapamycin (mTOR); meningioma; signaling; tumor suppressor gene

DOI:  https://doi.org/10.1074/jbc.RA120.014960
Biol Pharm Bull. 2020 ;43(12): 1983-1986

mTORC1 Overactivation Leads to Abnormalities in Skeletal Development.

Sayuki Iwahashi, Kazuya Tokumura, Gyujin Park, Shinsuke Ochiai, Yasuka Okayama, Hiroki Fusawa, Kaname Ohta, Kazuya Fukasawa, Takashi Iezaki, Eiichi Hinoi.

  The mechanistic/mammalian target of rapamycin complex-1 (mTORC1) integrates multiple signaling pathways and regulates various cellular processes. Tuberous sclerosis complex 1 (Tsc1) and complex 2 (Tsc2) are critical negative regulators of mTORC1. Mouse genetic studies, including ours, have revealed that inactivation of mTORC1 in undifferentiated mesenchymal cells and chondrocytes leads to severe skeletal abnormalities, indicating a pivotal role for mTORC1 in skeletogenesis. Here, we show that hyperactivation of mTORC1 influences skeletal development through its expression in undifferentiated mesenchymal cells at the embryonic stage. Inactivation of Tsc1 in undifferentiated mesenchymal cells by paired-related homeobox 1 (Prx1)-Cre-mediated recombination led to skeletal abnormalities in appendicular skeletons. In contrast, Tsc1 deletion in chondrocytes using collagen type II α1 (Col2a1)-Cre or in osteoprogenitors using Osterix (Osx)-Cre did not result in skeletal defects in either appendicular or axial skeletons. These findings indicate that Tsc complex-mediated chronic overactivation of mTORC1 influences skeletal development at the embryonic stage through its expression in undifferentiated mesenchymal cells but not in chondrocytes or osteoprogenitors.

Keywords:  mechanistic/mammalian target of rapamycin complex-1 (mTORC1); skeletogenesis; tuberous sclerosis complex 1; undifferentiated mesenchymal cell

DOI:  https://doi.org/10.1248/bpb.b20-00619
Trends Cell Biol. 2020 Nov 30. pii: S0962-8924(20)30226-9. [Epub ahead of print]

CDK1, the Other 'Master Regulator' of Autophagy.

Richard I Odle, Oliver Florey, Nicholas T Ktistakis, Simon J Cook.

  Autophagy and cap-dependent mRNA translation are tightly regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signalling complex in response to nutrient availability. However, the regulation of these processes, and mTORC1 itself, is different during mitosis, and this has remained an area of significant controversy; for example, studies have argued that autophagy is either repressed or highly active during mitosis. Recent studies have shown that autophagy initiation is repressed, and cap-dependent mRNA translation is maintained during mitosis despite mTORC1 activity being repressed. This is achieved in large part by a switch from mTORC1- to cyclin-dependent kinase 1 (CDK1)-mediated regulation. Here, we review the history and recent advances and seek to present a unifying model to inform the future study of autophagy and mTORC1 during mitosis.

Keywords:  CDK1; autophagy; mTORC1; mitosis; translation

DOI:  https://doi.org/10.1016/j.tcb.2020.11.001
Hematology Am Soc Hematol Educ Program. 2020 Dec 04. 2020(1): 346-356

Managing toxicities of phosphatidylinositol-3-kinase (PI3K) inhibitors.

Ashley Hanlon, Danielle M Brander.

Despite the proven effective approach to targeting the phosphatidylinositol-3-kinase (PI3K) pathway in B-cell malignancies, the approved PI3K inhibitors idelalisib and duvelisib have been less commonly selected for patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), given the availability of other more tolerable agents. However, patients with CLL/SLL can experience a disease course that is multiply relapsed, refractory, or intolerant to treatment, and PI3K inhibitors can achieve meaningful responses. This article reviews the common early- and late-onset (considered immune-mediated) toxicities with PI3K inhibitors, including infections, hepatotoxicity, diarrhea and/or colitis, and pneumonitis. Data on pretreatment considerations, toxicity management, and drug rechallenge are presented. In addition, next-generation PI3K inhibitors and novel treatment approaches with PI3K inhibitors, including combinations, time-limited treatments, and intermittent dosing, are highlighted.

DOI: https://doi.org/10.1182/hematology.2020000119
Toxicol Pathol. 2020 Nov 28. 192623320966238

Phosphatidylinositol 3-Kinase δ Inhibitor-Induced Immunomodulation and Secondary Opportunistic Infection in the Cynomolgus Monkey (Macaca fascicularis).

Jonathan A Werner, Katsu Ishida, John Wisler, Christine Karbowski, Jackson Kalanzi, Jeanine Bussiere, Thomas M Monticello.

  Phosphatidylinositol 3-kinases (PI3Ks) regulate intracellular signaling events for multiple cell surface receptors. Phosphatidylinositol 3-kinase δ, 1 of 4 class I PI3K isoforms, is primarily found in leukocytes and regulates immune cell functions. Here, we report changes in the immune and digestive systems that were associated with AMG2519493, a highly selective small-molecule PI3Kδ inhibitor. Following 1- or 3-month oral repeat dosing in the cynomolgus monkey, changes were observed in circulating B cells, lymphoid tissues (spleen, lymph nodes, gut-associated lymphoid tissue, tonsil), and the digestive tract. Decreased circulating B cells and lymphoid cellularity in B cell-rich zones in lymphoid tissues were attributed to the intended pharmacologic activity of AMG2519493. Dose- and duration-dependent digestive system toxicity was characterized by inflammation in the large intestine and secondary opportunistic infections restricted to the digestive tract. Digestive tract changes were associated with moribundity and mortality at high-dose levels, and the effect level decreased with increased duration of exposure. These observations demonstrate the role of PI3Kδ in regulation of the immune system and of host resistance to opportunistic infections of the digestive tract.

Keywords:  cynomolgus monkey; immunomodulation; opportunistic infection; phosphatidylinositol 3-kinase

DOI:  https://doi.org/10.1177/0192623320966238
Biomolecules. 2020 Nov 30. pii: E1617. [Epub ahead of print]10(12):

Isoform- and Paralog-Switching in IR-Signaling: When Diabetes Opens the Gates to Cancer.

Pierluigi Scalia, Antonio Giordano, Caroline Martini, Stephen J Williams.

  Insulin receptor (IR) and IR-related signaling defects have been shown to trigger insulin-resistance in insulin-dependent cells and ultimately to give rise to type 2 diabetes in mammalian organisms. IR expression is ubiquitous in mammalian tissues, and its over-expression is also a common finding in cancerous cells. This latter finding has been shown to associate with both a relative and absolute increase in IR isoform-A (IR-A) expression, missing 12 aa in its EC subunit corresponding to exon 11. Since IR-A is a high-affinity transducer of Insulin-like Growth Factor-II (IGF-II) signals, a growth factor is often secreted by cancer cells; such event offers a direct molecular link between IR-A/IR-B increased ratio in insulin resistance states (obesity and type 2 diabetes) and the malignant advantage provided by IGF-II to solid tumors. Nonetheless, recent findings on the biological role of isoforms for cellular signaling components suggest that the preferential expression of IR isoform-A may be part of a wider contextual isoform-expression switch in downstream regulatory factors, potentially enhancing IR-dependent oncogenic effects. The present review focuses on the role of isoform- and paralog-dependent variability in the IR and downstream cellular components playing a potential role in the modulation of the IR-A signaling related to the changes induced by insulin-resistance-linked conditions as well as to their relationship with the benign versus malignant transition in underlying solid tumors.

Keywords:  HIF: hypoxia-inducible factor; IGF: insulin-like growth factor; IR: insulin receptor; Isoform: for the scope of this review, the term isoform is restricted to products of alternatively spliced coding genes; MAPK-ERK: Mitogen-activated protein Kinase-Extracellular-signal-regulated Kinase; Paralog: the product of gene variants with high sequence similarity encoded by duplicated genes in the genome

DOI:  https://doi.org/10.3390/biom10121617
PLoS One. 2020 ;15(11): e0242819

Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region.

Guido Plotz, Laura A Lopez-Garcia, Angela Brieger, Stefan Zeuzem, Ricardo M Biondi.

Three AKT serine/threonine kinase isoforms (AKT1/AKT2/AKT3) mediate proliferation, metabolism, differentiation and anti-apoptotic signals. AKT isoforms are activated downstream of PI3-kinase and also by PI3-kinase independent mechanisms. Mutations in the lipid phosphatase PTEN and PI3-kinase that increase PIP3 levels increase AKT signaling in a large proportion of human cancers. AKT and other AGC kinases possess a regulatory mechanism that relies on a conserved hydrophobic motif (HM) C-terminal to the catalytic core. In AKT, the HM is contiguous to the serine 473 and two other newly discovered (serine 477 and tyrosine 479) regulatory phosphorylation sites. In AKT genes, this regulatory HM region is encoded in the final exon. We identified a splice variant of AKT2 (AKT2-13a), which contains an alternative final exon and lacks the HM regulatory site. We validated the presence of mRNA for this AKT2-13a splice variant in different tissues, and the presence of AKT2-13a protein in extracts from HEK293 cells. When overexpressed in HEK293 cells, AKT2-13a is phosphorylated at the activation loop and at the zipper/turn motif phosphorylation sites but has reduced specific activity. Analysis of the human transcriptome corresponding to other AGC kinases revealed that all three AKT isoforms express alternative transcripts lacking the HM regulatory motif, which was not the case for SGK1-3, S6K1-2, and classical, novel and atypical PKC isoforms. The transcripts of splice variants of Akt1-3 excluding the HM regulatory region could lead to expression of deregulated forms of AKT.

DOI: https://doi.org/10.1371/journal.pone.0242819
FEBS Lett. 2020 Nov 29.

mTORC1-mediated amino acid signaling is critical for cell fate determination under transplant-induced stress.

Xiaoyan Cheng, Maolin Ge, Shouhai Zhu, Dan Li, Ruiheng Wang, Qiongyu Xu, Zhihong Chen, Shufeng Xie, Han Liu.

  Transplantation of in vitro-manipulated cells is widely used in hematology. While transplantation is well recognized to impose severe stress on transplanted cells, the nature of transplant-induced stress remains elusive. Here we propose that the lack of amino acids in serum is the major cause of transplant-induced stress. Mechanistically, amino acid deficiency decreases protein synthesis and nutrient consummation. However, in cells with overactive AKT and ERK, mTORC1 is not inhibited and protein synthesis remains relatively high. This impaired signaling causes nutrient depletion, cell cycle block, and eventually autophagy and cell death, which can be inhibited by cycloheximide or mTORC1 inhibitors. Thus, mTORC1-mediated amino acid signaling is critical in cell fate determination under transplant-induced stress, and protein synthesis inhibition can improve transplantation efficiency.

Keywords:  amino acid; leukemia; mTORC1; metabolism; transplant-induced stress

DOI:  https://doi.org/10.1002/1873-3468.14008
Sci Rep. 2020 Dec 01. 10(1): 20908

Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities.

Mohammad A Mohammad, John Greenman, Anthony Maraveyas, Camille Ettelaie.

Tissue factor (TF) signalling has been associated with alterations in Akt activity influencing cellular survival and proliferation. TF is also shown to induce signalling through activation of the protease activated receptor (PAR)2. Seven cell lines were exposed to recombinant-TF (rec-TF), or activated using a PAR2-agonist peptide and the phosphorylation state of PTEN, and the activities of PTEN and Akt measured. Furthermore, by measuring the association of PTEN with MAGI proteins a mechanism for the induction of signalling by TF was proposed. Short term treatment of cells resulted in de-phosphorylation of PTEN, increased lipid-phosphatase activity and reduced Akt kinase activity in most of the cell lines examined. In contrast, continuous exposure to rec-TF up to 14 days, resulted in lower PTEN antigen levels, enhanced Akt activity and increased rate of cell proliferation. To explore the mechanism of activation of PTEN by TF, the association of "membrane-associated guanylate kinase-with inverted configuration" (MAGI)1-3 proteins with PTEN was assessed using the proximity ligation assay and by co-immunoprecipitation. The interaction of PTEN with all three MAGI proteins was transiently reduced following PAR2 activation and explains the changes in PTEN activity. Our data is first to show that PAR2 activation directly, or through exposure of cells to TF releases PTEN from MAGI proteins and is concurrent with increases in PTEN phosphatase activity. However, prolonged exposure to TF results in the reduction in PTEN antigen with concurrent increase in Akt activity which may explain the aberrant cell survival, proliferation and invasion associated with TF during chronic diseases.

DOI: https://doi.org/10.1038/s41598-020-77963-6
Pediatr Radiol. 2020 Dec;50(13): 1974-1987

Imaging phenotype correlation with molecular and molecular pathway defects in malformations of cortical development.

Carolina V A Guimaraes, Hisham M Dahmoush.

  The increase in understanding of molecular biology and recent advances in genetic testing have caused rapid growth in knowledge of genetic causes of malformations of cortical development. Imaging diagnosis of malformations of cortical development can be made prenatally in a large subset of fetuses based on the presence of specific deviations from the normal pattern of development, characteristic imaging features, and associated non-central-nervous-system (CNS) abnormalities. In this review the authors discuss the role of four key cell molecules/molecular pathways in corticogenesis that are frequently implicated in complex prenatally diagnosed malformations of cortical development. The authors also list the currently described genes causing defects in these molecules/molecular pathways when mutated, and the constellation of imaging findings resultant of such defects.

Keywords:  Alpha (α)-dystroglycanopathies; Cerebral cortex; Ciliopathies; Fetus; Magnetic resonance imaging; Malformation of cortical development; PI3K/AKT/mTOR pathway; Tubulinopathies

DOI:  https://doi.org/10.1007/s00247-020-04674-5
Nat Commun. 2020 12 01. 11(1): 6136

Leveraging multi-way interactions for systematic prediction of pre-clinical drug combination effects.

Heli Julkunen, Anna Cichonska, Prson Gautam, Sandor Szedmak, Jane Douat, Tapio Pahikkala, Tero Aittokallio, Juho Rousu.

We present comboFM, a machine learning framework for predicting the responses of drug combinations in pre-clinical studies, such as those based on cell lines or patient-derived cells. comboFM models the cell context-specific drug interactions through higher-order tensors, and efficiently learns latent factors of the tensor using powerful factorization machines. The approach enables comboFM to leverage information from previous experiments performed on similar drugs and cells when predicting responses of new combinations in so far untested cells; thereby, it achieves highly accurate predictions despite sparsely populated data tensors. We demonstrate high predictive performance of comboFM in various prediction scenarios using data from cancer cell line pharmacogenomic screens. Subsequent experimental validation of a set of previously untested drug combinations further supports the practical and robust applicability of comboFM. For instance, we confirm a novel synergy between anaplastic lymphoma kinase (ALK) inhibitor crizotinib and proteasome inhibitor bortezomib in lymphoma cells. Overall, our results demonstrate that comboFM provides an effective means for systematic pre-screening of drug combinations to support precision oncology applications.

DOI: https://doi.org/10.1038/s41467-020-19950-z
Blood. 2020 Dec 01. pii: blood.2020007376. [Epub ahead of print]

Phase 2 Study of the Safety and Efficacy of Umbralisib in Patients with CLL Who Are Intolerant to BTK or PI3Kδ Inhibitor Therapy.

Anthony R Mato, Nilanjan Ghosh, Stephen J Schuster, Nicole Lamanna, John M Pagel, Ian W Flinn, Jacqueline Barrientos, Kanti R Rai, James A Reeves, Bruce D Cheson, Paul M Barr, Suman Kambhampati, Frederick Lansigan, Jeffrey J Pu, Alan P Skarbnik, Lindsey Elizabeth Roeker, Gustavo Fonseca, Andrea Sitlinger, Issam S Hamadeh, Colleen Dorsey, Nicole LaRatta, Hanna Weissbrot, Eline T Luning Prak, Patricia Y Tsao, Dana Paskalis, Peter Sportelli, Hari P Miskin, Michael S Weiss, Jakub Svoboda, Danielle M Brander.

PURPOSE: Intolerance is the most common reason for kinase inhibitor (KI) discontinuation in CLL. Umbralisib a novel, highly selective PI3Kδ/CK1ε inhibitor, is active and well tolerated in CLL patients. This phase 2 trial evaluated umbralisib in CLL patients who are intolerant to prior BTK or PI3K inhibitor therapy.PATIENTS AND METHODS: In this phase 2 trial (NCT02742090), umbralisib was initiated at 800 mg oral daily in CLL patients requiring therapy per investigator discretion who were intolerant to prior BTK or PI3K inhibitor therapy, until progression or toxicity. Primary endpoint was progression-free survival (PFS). Secondary endpoints included time to treatment failure and umbralisib safety profile. DNA isolated from buccal swabs was genotyped for polymorphisms in CYP3A4, CYP3A5 and CYP2D6.
RESULTS: Fifty-one patients were enrolled (44 BTKi and 7 PI3Kδi intolerant). Median age was 70 years (range 48-96), median of 2 prior lines of therapy (1-7), 24% had del17p and/or TP53 mutation, and 65% were IGHV unmutated. Most common AEs leading to prior KI discontinuation were rash (27%), arthralgia (18%), and atrial fibrillation (16%). Median progression free survival (PFS) was 23.5 months (95% CI 13.1-not estimable). 58% of patients were on umbralisib for a longer duration than prior KI. Most common (≥5%) grade ≥3 AEs on umbralisib (all causality) were neutropenia (18%), leukocytosis (14%), thrombocytopenia (12%), pneumonia (12%), and diarrhea (8%). Six patients (12%) discontinued umbralisib due to an AE. Eight patients (16%) had dose reductions and were successfully re-challenged.
CONCLUSIONS: Umbralisib is safe and effective in this BTK and alternate PI3K inhibitor intolerant CLL population. These are the first prospective data to confirm that switching from a BTK or alternate PI3K inhibitor to umbralisib can result in durable, well tolerated responses.

DOI: https://doi.org/10.1182/blood.2020007376
Sci Signal. 2020 Dec 01. pii: eaaz1236. [Epub ahead of print]13(660):

Transomics analysis reveals allosteric and gene regulation axes for altered hepatic glucose-responsive metabolism in obesity.

Toshiya Kokaji, Atsushi Hatano, Yuki Ito, Katsuyuki Yugi, Miki Eto, Keigo Morita, Satoshi Ohno, Masashi Fujii, Ken-Ichi Hironaka, Riku Egami, Akira Terakawa, Takaho Tsuchiya, Haruka Ozaki, Hiroshi Inoue, Shinsuke Uda, Hiroyuki Kubota, Yutaka Suzuki, Kazutaka Ikeda, Makoto Arita, Masaki Matsumoto, Keiichi I Nakayama, Akiyoshi Hirayama, Tomoyoshi Soga, Shinya Kuroda.

Impaired glucose tolerance associated with obesity causes postprandial hyperglycemia and can lead to type 2 diabetes. To study the differences in liver metabolism in healthy and obese states, we constructed and analyzed transomics glucose-responsive metabolic networks with layers for metabolites, expression data for metabolic enzyme genes, transcription factors, and insulin signaling proteins from the livers of healthy and obese mice. We integrated multiomics time course data from wild-type and leptin-deficient obese (ob/ob) mice after orally administered glucose. In wild-type mice, metabolic reactions were rapidly regulated within 10 min of oral glucose administration by glucose-responsive metabolites, which functioned as allosteric regulators and substrates of metabolic enzymes, and by Akt-induced changes in the expression of glucose-responsive genes encoding metabolic enzymes. In ob/ob mice, the majority of rapid regulation by glucose-responsive metabolites was absent. Instead, glucose administration produced slow changes in the expression of carbohydrate, lipid, and amino acid metabolic enzyme-encoding genes to alter metabolic reactions on a time scale of hours. Few regulatory events occurred in both healthy and obese mice. Thus, our transomics network analysis revealed that regulation of glucose-responsive liver metabolism is mediated through different mechanisms in healthy and obese states. Rapid changes in allosteric regulators and substrates and in gene expression dominate the healthy state, whereas slow changes in gene expression dominate the obese state.

DOI: https://doi.org/10.1126/scisignal.aaz1236
Sci Rep. 2020 Dec 04. 10(1): 21244

Metabolomics revealed the influence of breast cancer on lymphatic endothelial cell metabolism, metabolic crosstalk, and lymphangiogenic signaling in co-culture.

Suehelay Acevedo-Acevedo, Douglas C Millar, Aaron D Simmons, Peter Favreau, Paulo F Cobra, Melissa Skala, Sean P Palecek.

Breast cancer metastasis occurs via blood and lymphatic vessels. Breast cancer cells 'educate' lymphatic endothelial cells (LECs) to support tumor vascularization and growth. However, despite known metabolic alterations in breast cancer, it remains unclear how lymphatic endothelial cell metabolism is altered in the tumor microenvironment and its effect in lymphangiogenic signaling in LECs. We analyzed metabolites inside LECs in co-culture with MCF-7, MDA-MB-231, and SK-BR-3 breast cancer cell lines using [Formula: see text] nuclear magnetic resonance (NMR) metabolomics, Seahorse, and the spatial distribution of metabolic co-enzymes using optical redox ratio imaging to describe breast cancer-LEC metabolic crosstalk. LECs co-cultured with breast cancer cells exhibited cell-line dependent altered metabolic profiles, including significant changes in lactate concentration in breast cancer co-culture. Cell metabolic phenotype analysis using Seahorse showed LECs in co-culture exhibited reduced mitochondrial respiration, increased reliance on glycolysis and reduced metabolic flexibility. Optical redox ratio measurements revealed reduced NAD(P)H levels in LECs potentially due to increased NAD(P)H utilization to maintain redox homeostasis. [Formula: see text]-labeled glucose experiments did not reveal lactate shuttling into LECs from breast cancer cells, yet showed other [Formula: see text] signals in LECs suggesting internalized metabolites and metabolic exchange between the two cell types. We also determined that breast cancer co-culture stimulated lymphangiogenic signaling in LECs, yet activation was not stimulated by lactate alone. Increased lymphangiogenic signaling suggests paracrine signaling between LECs and breast cancer cells which could have a pro-metastatic role.

DOI: https://doi.org/10.1038/s41598-020-76394-7
Nat Commun. 2020 12 03. 11(1): 6194

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Francesco Trovato, Riccardo Parra, Enrico Pracucci, Silvia Landi, Olga Cozzolino, Gabriele Nardi, Federica Cruciani, Vinoshene Pillai, Laura Mosti, Andrzej W Cwetsch, Laura Cancedda, Laura Gritti, Carlo Sala, Chiara Verpelli, Andrea Maset, Claudia Lodovichi, Gian Michele Ratto.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

DOI: https://doi.org/10.1038/s41467-020-19864-w
Diabetes. 2020 Dec 04. pii: db200474. [Epub ahead of print]

Loss of Furin in β cells Induces an mTORC1-ATF4 Anabolic Pathway that Leads to β cell Dysfunction.

Bas Brouwers, Ilaria Coppola, Katlijn Vints, Bastian Dislich, Nathalie Jouvet, Leentje Van Lommel, Charlotte Segers, Natalia V Gounko, Lieven Thorrez, Frans Schuit, Stefan F Lichtenthaler, Jennifer L Estall, Jeroen Declercq, Bruno Ramos-Molina, John W M Creemers.

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, yet its specific role in pancreatic β cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, while PCs that potentially could provide redundancy are expressed at considerably lower levels. β cell-specific Furin knockout (βFurKO) mice are glucose intolerant, due to smaller islets with lower insulin content and abnormal dense core secretory granule morphology. mRNA expression analysis and differential proteomics on βFurKO islets revealed activation of Activating Transcription Factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). βFurKO cells show impaired cleavage or shedding of the V-ATPase subunits Ac45 and prorenin receptor (PRR), respectively, and impaired lysosomal acidification. Blocking the V-ATPase pharmacologically in β cells increases mTORC1 activity, suggesting the involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in β cells lacking Furin, which causes β cell dysfunction.

DOI: https://doi.org/10.2337/db20-0474
Aging (Albany NY). 2020 Dec 01. 12

Implication of integrins α3β1 and α5β1 in invasion and anoikis of SK-Mel-147 human melanoma cells: non-canonical functions of protein kinase Akt.

Nadezhda I Kozlova, Galina E Morozevich, Nina M Gevorkian, Albert E Berman.

  Downregulation of integrins α3β1 and α5β1 strongly decreased cell colony formation and in vitro invasion and markedly enhanced anoikis in SK-Mel-147 human melanoma cells. These modifications were accompanied by a marked increase in the levels of active Akt protein kinase, which indicated it played a non-canonical function in the melanoma cells. Pharmacological inhibition of Akt1, an Akt isozyme, in cells depleted of α3β1 or α5β1 restored their invasive activity, while inhibition of the Akt 2 isoform did not cause a visible effect. Similar to our previous results with the α2β1 integrin, this finding suggested that in signaling pathways initiated by α3β1 and α5β1, the Akt1 isoform performs a non-canonical function in regulating invasive phenotype of melanoma cells. In contrast, when the effects of Akt inhibitors on anoikis of the melanoma cells were compared, the Akt2 isoform demonstrated a non-canonical activity in which Akt2 suppression led to a significant attenuation of apoptosis in cells with downregulated α3β1 or α5β1. Our results were the first evidence that, in the same tumor cells, different integrins can control various manifestations of tumor progression through distinct signaling pathways that are both common to various integrins and specific to a particular receptor.

Keywords:  anoikis; integrins; proteinkinases; tumor invasion

DOI:  https://doi.org/10.18632/aging.202243
PLoS Comput Biol. 2020 Dec 03. 16(12): e1007578

Accurate prediction of kinase-substrate networks using knowledge graphs.

Vít Nováček, Gavin McGauran, David Matallanas, Adrián Vallejo Blanco, Piero Conca, Emir Muñoz, Luca Costabello, Kamalesh Kanakaraj, Zeeshan Nawaz, Brian Walsh, Sameh K Mohamed, Pierre-Yves Vandenbussche, Colm Ryan, Walter Kolch, Dirk Fey.

Phosphorylation of specific substrates by protein kinases is a key control mechanism for vital cell-fate decisions and other cellular processes. However, discovering specific kinase-substrate relationships is time-consuming and often rather serendipitous. Computational predictions alleviate these challenges, but the current approaches suffer from limitations like restricted kinome coverage and inaccuracy. They also typically utilise only local features without reflecting broader interaction context. To address these limitations, we have developed an alternative predictive model. It uses statistical relational learning on top of phosphorylation networks interpreted as knowledge graphs, a simple yet robust model for representing networked knowledge. Compared to a representative selection of six existing systems, our model has the highest kinome coverage and produces biologically valid high-confidence predictions not possible with the other tools. Specifically, we have experimentally validated predictions of previously unknown phosphorylations by the LATS1, AKT1, PKA and MST2 kinases in human. Thus, our tool is useful for focusing phosphoproteomic experiments, and facilitates the discovery of new phosphorylation reactions. Our model can be accessed publicly via an easy-to-use web interface (LinkPhinder).

DOI: https://doi.org/10.1371/journal.pcbi.1007578
PLoS One. 2020 ;15(12): e0233662

The effect of a ketogenic diet and synergy with rapamycin in a mouse model of breast cancer.

Yiyu Zou, Susan Fineberg, Alexander Pearlman, Richard D Feinman, Eugene J Fine.

BACKGROUND: The effects of diet in cancer, in general, and breast cancer in particular, are not well understood. Insulin inhibition in ketogenic, high fat diets, modulate downstream signaling molecules and are postulated to have therapeutic benefits. Obesity and diabetes have been associated with higher incidence of breast cancer. Addition of anti-cancer drugs together with diet is also not well studied.METHODS: Two diets, one ketogenic, the other standard mouse chow, were tested in a spontaneous breast cancer model in 34 mice. Subgroups of 3-9 mice were assigned, in which the diet were implemented either with or without added rapamycin, an mTOR inhibitor and potential anti-cancer drug.
RESULTS: Blood glucose and insulin concentrations in mice ingesting the ketogenic diet (KD) were significantly lower, whereas beta hydroxybutyrate (BHB) levels were significantly higher, respectively, than in mice on the standard diet (SD). Growth of primary breast tumors and lung metastases were inhibited, and lifespans were longer in the KD mice compared to mice on the SD (p<0.005). Rapamycin improved survival in both mouse diet groups, but when combined with the KD was more effective than when combined with the SD.
CONCLUSIONS: The study provides proof of principle that a ketogenic diet a) results in serum insulin reduction and ketosis in a spontaneous breast cancer mouse model; b) can serve as a therapeutic anti-cancer agent; and c) can enhance the effects of rapamycin, an anti-cancer drug, permitting dose reduction for comparable effect. Further, the ketogenic diet in this model produces superior cancer control than standard mouse chow whether with or without added rapamycin.

DOI: https://doi.org/10.1371/journal.pone.0233662
Nat Cell Biol. 2020 Dec;22(12): 1460-1470

Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stress.

Noa Lamm, Mark N Read, Max Nobis, David Van Ly, Scott G Page, V Pragathi Masamsetti, Paul Timpson, Maté Biro, Anthony J Cesare.

Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.

DOI: https://doi.org/10.1038/s41556-020-00605-6