bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2020–09–20
thirteen papers selected by
Ralitsa Radostinova Madsen, University College London
  1. Quantitative genetic screening reveals a Ragulator-FLCN feedback loop that regulates the mTORC1 pathway.
  2. mTOR-mediated cancer drug resistance suppresses autophagy and generates a druggable metabolic vulnerability.
  3. Structural Features that Distinguish Inactive and Active PI3K Lipid Kinases.
  4. Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer.
  5. SWELL1 regulates skeletal muscle cell size, intracellular signalling, adiposity and glucose metabolism.
  6. PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition.
  7. Targeting phosphatidylinositol 3 kinase-β and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma.
  8. PIK3CA mutations matter for cancer in dogs.
  9. mTOR Promotes Tissue Factor Expression and Activity in EGFR-Mutant Cancer.
  10. The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease.
  11. Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence.
  12. Persistent Rheb-induced mTORC1 activation in spinal cord neurons induces hypersensitivity in neuropathic pain.
  13. Phenotypic profiling with a living biobank of primary rhabdomyosarcoma unravels disease heterogeneity and AKT sensitivity.
  1. Sci Signal. 2020 Sep 15. pii: eaba5665. [Epub ahead of print]13(649):
    Quantitative genetic screening reveals a Ragulator-FLCN feedback loop that regulates the mTORC1 pathway.
    Erica De Zan, Ruud van Stiphout, Bianca V Gapp, Vincent A Blomen, Thijn R Brummelkamp, Sebastian M B Nijman.
      Forward genetic screens in mammalian cell lines, such as RNAi and CRISPR-Cas9 screens, have made major contributions to the elucidation of diverse signaling pathways. Here, we exploited human haploid cells as a robust comparative screening platform and report a set of quantitative forward genetic screens for identifying regulatory mechanisms of mTORC1 signaling, a key growth control pathway that senses diverse metabolic states. Selected chemical and genetic perturbations in this screening platform, including rapamycin treatment and genetic ablation of the Ragulator subunit LAMTOR4, revealed the known core mTORC1 regulatory signaling complexes and the intimate interplay of the mTORC1 pathway with lysosomal function, validating the approach. In addition, we identified a differential requirement for LAMTOR4 and LAMTOR5 in regulating the mTORC1 pathway under fed and starved conditions. Furthermore, we uncovered a previously unknown "synthetic-sick" interaction between the tumor suppressor folliculin and LAMTOR4, which may have therapeutic implications in cancer treatment. Together, our study demonstrates the use of iterative "perturb and observe" genetic screens to uncover regulatory mechanisms driving complex mammalian signaling networks.
    DOI:  https://doi.org/10.1126/scisignal.aba5665
  2. Nat Commun. 2020 Sep 17. 11(1): 4684
    mTOR-mediated cancer drug resistance suppresses autophagy and generates a druggable metabolic vulnerability.
    Niklas Gremke, Pierfrancesco Polo, Aaron Dort, Jean Schneikert, Sabrina Elmshäuser, Corinna Brehm, Ursula Klingmüller, Anna Schmitt, Hans Christian Reinhardt, Oleg Timofeev, Michael Wanzel, Thorsten Stiewe.
      Cancer cells have a characteristic metabolism, mostly caused by alterations in signal transduction networks rather than mutations in metabolic enzymes. For metabolic drugs to be cancer-selective, signaling alterations need to be identified that confer a druggable vulnerability. Here, we demonstrate that many tumor cells with an acquired cancer drug resistance exhibit increased sensitivity to mechanistically distinct inhibitors of cancer metabolism. We demonstrate that this metabolic vulnerability is driven by mTORC1, which promotes resistance to chemotherapy and targeted cancer drugs, but simultaneously suppresses autophagy. We show that autophagy is essential for tumor cells to cope with therapeutic perturbation of metabolism and that mTORC1-mediated suppression of autophagy is required and sufficient for generating a metabolic vulnerability leading to energy crisis and apoptosis. Our study links mTOR-induced cancer drug resistance to autophagy defects as a cause of a metabolic liability and opens a therapeutic window for the treatment of otherwise therapy-refractory tumor patients.
    DOI:  https://doi.org/10.1038/s41467-020-18504-7
  3. J Mol Biol. 2020 Sep 09. pii: S0022-2836(20)30532-5. [Epub ahead of print]
    Structural Features that Distinguish Inactive and Active PI3K Lipid Kinases.
    Mingzhen Zhang, Hyunbum Jang, Ruth Nussinov.
      PI3K lipid kinases signal through the PI3K/Akt pathway, regulating cell growth and proliferation. While the structural features that distinguish between the active and inactive states of protein kinases are well established, that has not been the case for lipid kinases, and neither was the structural mechanism controlling the switch between the two states. Class I PI3Ks are obligate heterodimers with catalytic and regulatory subunits. Here, we analyze PI3K crystal structures. Structures with the nSH2 (inactive state) are featured by collapsed activation loop (a-loop) and an IN kinase domain helix 11 (kα11). In the active state, the a-loop is extended and kα11 in the OUT conformation. Our analysis suggests that the nSH2 domain in the regulatory subunit regulates activation, catalysis and autoinhibition through the a-loop. Inhibition, activation and catalytic scenarios are shared by class IA PI3Ks; the activation is mimicked by oncogenic mutations and the inhibition offers an allosteric inhibitor strategy.
    Keywords:  Catalytic and regulatory subunits; PI3K mutations; PI3Kα; Ras; nSH2
    DOI:  https://doi.org/10.1016/j.jmb.2020.09.002
  4. Mol Cell. 2020 Sep 09. pii: S1097-2765(20)30580-3. [Epub ahead of print]
    Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer.
    Xinyu Chen, Majd M Ariss, Gopalakrishnan Ramakrishnan, Veronique Nogueira, Catherine Blaha, William Putzbach, Abul B M M K Islam, Maxim V Frolov, Nissim Hay.
      Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.
    Keywords:  Akt1; Akt2; breast cancer; insulin; metastasis; neutrophils; therapy
    DOI:  https://doi.org/10.1016/j.molcel.2020.08.017
  5. Elife. 2020 Sep 15. pii: e58941. [Epub ahead of print]9
    SWELL1 regulates skeletal muscle cell size, intracellular signalling, adiposity and glucose metabolism.
    Ashutosh Kumar, Litao Xie, Chau My Ta, Antentor J Hinton, Susheel K Gunasekar, Rachel A Minerath, Karen Shen, Joshua M Maurer, Chad E Grueter, E Dale Abel, Gretchen Meyer, Rajan Sah.
      Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism, however the molecular mechanosensor remains unknown. Here, we show that SWELL1 (Lrrc8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. LRRC8A over-expression in Lrrc8a KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle targeted Lrrc8a KO mice have smaller myofibers, generate less force ex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to WT mice. These results reveal that the LRRC8 complex regulates insulin-PI3K-AKT-mTOR signalling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size, muscle function, adiposity and systemic metabolism in vivo.
    Keywords:  cell biology; mouse
    DOI:  https://doi.org/10.7554/eLife.58941
  6. Proc Natl Acad Sci U S A. 2020 Sep 14. pii: 202000060. [Epub ahead of print]
    PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition.
    Jennifer M Spangle, Thanh Von, Dean C Pavlick, Arina Khotimsky, Jean J Zhao, Thomas M Roberts.
      PIK3CA hotspot mutation is well established as an oncogenic driver event in cancer and its durable and efficacious inhibition is a focus in the development and testing of clinical cancer therapeutics. However, hundreds of cancer-associated PIK3CA mutations remain uncharacterized, their sensitivity to PI3K inhibitors unknown. Here, we describe a series of PIK3CA C-terminal mutations, primarily nucleotide insertions, that produce a frame-shifted protein product with an extended C terminus. We report that these mutations occur at a low frequency across multiple cancer subtypes, including breast, and are sufficient to drive oncogenic transformation in vitro and in vivo. We demonstrate that the oncogenicity of these mutant p110α proteins is dependent on p85 but not Ras association. P110α-selective pharmacologic inhibition blocks transformation in cells and mammary tumors characterized by PIK3CA C-terminal mutation. Taken together, these results suggest patients with breast and other tumors characterized by PIK3CA C-terminal frameshift mutations may derive benefit from p110α-selective inhibitors, including the recently FDA-approved alpelisib.
    Keywords:  PI3K; cancer; signal transduction
    DOI:  https://doi.org/10.1073/pnas.2000060117
  7. Blood Adv. 2020 Sep 22. 4(18): 4382-4392
    Targeting phosphatidylinositol 3 kinase-β and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma.
    Neeraj Jain, Satishkumar Singh, Georgios Laliotis, Amber Hart, Elizabeth Muhowski, Kristyna Kupcova, Tereza Chrbolkova, Tamer Khashab, Sayan Mullick Chowdhury, Anuvrat Sircar, Fazal Shirazi, Ram Kumar Singh, Lapo Alinari, Jiangjiang Zhu, Ondrej Havranek, Philip Tsichlis, Jennifer Woyach, Robert Baiocchi, Felipe Samaniego, Lalit Sehgal.
      Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse after a complete response or are refractory to therapy. To survive, the activated B-cell (ABC) subtype of DLBCL relies upon B-cell receptor signaling, which can be modulated by the activity of Bruton tyrosine kinase (BTK). Targeting BTK with ibrutinib, an inhibitor, provides a therapeutic approach for this subtype of DLBCL. However, non-Hodgkin lymphoma is often resistant to ibrutinib or acquires resistance soon after exposure. We explored how this resistance develops. We generated 3 isogenic ibrutinib-resistant DLBCL cell lines and investigated the deregulated pathways known to be associated with tumorigenic properties. Reduced levels of BTK and enhanced phosphatidylinositol 3-kinase (PI3K)/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of PI3K-β expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-β/δ. Treatment with the selective PI3K-β/δ dual inhibitor KA2237 reduced both tumorigenic properties and survival-based PI3K/AKT/mTOR signaling of these ibrutinib-resistant cells. In addition, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited metabolic growth. This study elucidates the compensatory upregulated PI3K/AKT axis that emerges in ibrutinib-resistant cells.
    DOI:  https://doi.org/10.1182/bloodadvances.2020001685
  8. Res Vet Sci. 2020 Sep 08. pii: S0034-5288(20)31002-X. [Epub ahead of print]133 39-41
    PIK3CA mutations matter for cancer in dogs.
    Jong Hyuk Kim.
      Cancer is a genetic disease that arises from the accumulation of genetic mutations within a cell. Mutations in PIK3CA gene are frequently observed in human solid cancers, and also appear to occur in canine tumors. Specifically, recurrent somatic PIK3CA variants identified in canine mammary tumors and hemangiosarcomas are comparable to human hotspot mutations such as H1047R. PIK3CA mutations found in canine tumors encode functional proteins that may alter downstream PI3K/Akt/mTOR signaling pathway. Therefore, PI3K inhibitors have potential in cancer therapy for dogs. This article concisely reviews the emerging evidence concerning the genetic and molecular properties of PIK3CA mutations to discuss future perspectives in veterinary and comparative oncology.
    Keywords:  Canine; Comparative oncology; Hemangiosarcoma; Mammary tumor; Mutation; PIK3CA
    DOI:  https://doi.org/10.1016/j.rvsc.2020.09.001
  9. Front Oncol. 2020 ;10 1615
    mTOR Promotes Tissue Factor Expression and Activity in EGFR-Mutant Cancer.
    Ying Cong, Qingrou Li, Xuesai Zhang, Yaqing Chen, Ker Yu.
      Mechanistic target of rapamycin (mTOR) signaling pathway mediates the function of oncogenic receptor tyrosine kinases (RTKs). We aimed to elucidate new role of mTOR in EGFR-mutant (EGFR-mut) non-small cell lung cancer (NSCLC) and glioblastoma (GBM) with a focus on tumor microenvironments. Here, we report a novel regulatory link between mTOR complexes (mTORCs) and tissue factor (TF), an initiator of tumor-derived thrombosis. TF is elevated in EGFR-mut NSCLC/GBM cell lines and tumors from patients with poor prognosis. Application of mTORC1/2 inhibitors (AZD8055, WYE-125132, MTI-31, and rapamycin) or genetic mTORC-depletion all reduced TF expression, which appeared to be differentially mediated depending on cellular context. In U87MG and HCC827 cells, mTORC1 exerted a dominant role via promoting TF mRNA transcription. In EGFR-TKI-resistant H1975 and PC9 cells, it was mTORC2 that played a major role in specific repression of lysosomal-targeted TF protein degradation. Successful inhibition of TF expression was demonstrated in AZD8055- or MTI-31-treated H1975 and U87MG tumors in mice, while a TF-targeted antibody antagonized TF activity without reducing TF protein. Both the mTOR- and TF-targeted therapy induced a multifaceted remodeling of tumor microenvironment reflecting not only a diminished hypercoagulopathy state (fibrin level) but also a reduced stromal fibrosis (collagen distribution), compromised vessel density and/or maturity (CD31 and/or α-SMA) as well as a substantially decreased infiltration of immune-suppressive M2-type tumor-associated macrophages (CD206/F4/80 ratio). Thus, our results have identified TF as a functional biomarker of mTOR. Downregulation of mTOR-TF axis activity likely contributes to the therapeutic mechanism of mTORC1/2- and TF-targeted agents in EGFR-mut advanced NSCLC and GBM.
    Keywords:  EGFR mutant cancer; mTOR inhibitor; mTOR-TF axis; tissue factor; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2020.01615
  10. Kidney Int. 2020 Sep 05. pii: S0085-2538(20)30687-6. [Epub ahead of print]
    The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease.
    Marine Berquez, Jonathan R Gadsby, Beatrice Paola Festa, Richard Butler, Stephen P Jackson, Valeria Berno, Alessandro Luciani, Olivier Devuyst, Jennifer L Gallop.
      Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.
    Keywords:  cytoskeleton; endocytosis; lipids; proximal tubule; renal Fanconi syndrome
    DOI:  https://doi.org/10.1016/j.kint.2020.05.040
  11. J Clin Med. 2020 Sep 11. pii: E2934. [Epub ahead of print]9(9):
    Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence.
    Salihanur Darici, Hazem Alkhaldi, Gillian Horne, Heather G Jørgensen, Sandra Marmiroli, Xu Huang.
      Acute myeloid leukemia (AML) is a highly heterogeneous hematopoietic malignancy characterized by excessive proliferation and accumulation of immature myeloid blasts in the bone marrow. AML has a very poor 5-year survival rate of just 16% in the UK; hence, more efficacious, tolerable, and targeted therapy is required. Persistent leukemia stem cell (LSC) populations underlie patient relapse and development of resistance to therapy. Identification of critical oncogenic signaling pathways in AML LSC may provide new avenues for novel therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway, is often hyperactivated in AML, required to sustain the oncogenic potential of LSCs. Growing evidence suggests that targeting key components of this pathway may represent an effective treatment to kill AML LSCs. Despite this, accruing significant body of scientific knowledge, PI3K/Akt/mTOR inhibitors have not translated into clinical practice. In this article, we review the laboratory-based evidence of the critical role of PI3K/Akt/mTOR pathway in AML, and outcomes from current clinical studies using PI3K/Akt/mTOR inhibitors. Based on these results, we discuss the putative mechanisms of resistance to PI3K/Akt/mTOR inhibition, offering rationale for potential candidate combination therapies incorporating PI3K/Akt/mTOR inhibitors for precision medicine in AML.
    Keywords:  AML; LSC; PI3K/Akt; combination treatment strategy; drug resistance; mTOR; targeted therapy
    DOI:  https://doi.org/10.3390/jcm9092934
  12. Cell Death Dis. 2020 Sep 12. 11(9): 747
    Persistent Rheb-induced mTORC1 activation in spinal cord neurons induces hypersensitivity in neuropathic pain.
    Xiaqing Ma, Wenjie Du, Wenying Wang, Limin Luo, Min Huang, Haiyan Wang, Raozhou Lin, Zhongping Li, Haibo Shi, Tifei Yuan, Wei Jiang, Paul F Worley, Tao Xu.
      The small GTPase Ras homolog enriched in the brain (Rheb) can activate mammalian target of rapamycin (mTOR) and regulate the growth and cell cycle progression. We investigated the role of Rheb-mediated mTORC1 signaling in neuropathic pain. A chronic constriction injury (CCI) model was dopted. CCI induced obvious spinal Rheb expression and phosphorylation of mTOR, S6, and 4-E-BP1. Blocking mTORC1 signal with rapamycin alleviated the neuropathic pain and restored morphine efficacy in CCI model. Immunofluoresence showed a neuronal co-localization of CCI-induced Rheb and pS6. Rheb knockin mouse showed a similar behavioral phenotype as CCI. In spinal slice recording, CCI increased the firing frequency of neurons expressing HCN channels; inhibition of mTORC1 with rapamycin could reverse the increased spinal neuronal activity in neuropathic pain. Spinal Rheb is induced in neuropathic pain, which in turn active the mTORC1 signaling in CCI. Spinal Rheb-mTOR signal plays an important role in regulation of spinal sensitization in neuropathic pain, and targeting mTOR may give a new strategy for pain management.
    DOI:  https://doi.org/10.1038/s41419-020-02966-0
  13. Nat Commun. 2020 09 15. 11(1): 4629
    Phenotypic profiling with a living biobank of primary rhabdomyosarcoma unravels disease heterogeneity and AKT sensitivity.
    Gabriele Manzella, Leonie D Schreck, Willemijn B Breunis, Jan Molenaar, Hans Merks, Frederic G Barr, Wenyue Sun, Michaela Römmele, Luduo Zhang, Joelle Tchinda, Quy A Ngo, Peter Bode, Olivier Delattre, Didier Surdez, Bharat Rekhi, Felix K Niggli, Beat W Schäfer, Marco Wachtel.
      Cancer therapy is currently shifting from broadly used cytotoxic drugs to patient-specific precision therapies. Druggable driver oncogenes, identified by molecular analyses, are present in only a subset of patients. Functional profiling of primary tumor cells could circumvent these limitations, but suitable platforms are unavailable for most cancer entities. Here, we describe an in vitro drug profiling platform for rhabdomyosarcoma (RMS), using a living biobank composed of twenty RMS patient-derived xenografts (PDX) for high-throughput drug testing. Optimized in vitro conditions preserve phenotypic and molecular characteristics of primary PDX cells and are compatible with propagation of cells directly isolated from patient tumors. Besides a heterogeneous spectrum of responses of largely patient-specific vulnerabilities, profiling with a large drug library reveals a strong sensitivity towards AKT inhibitors in a subgroup of RMS. Overall, our study highlights the feasibility of in vitro drug profiling of primary RMS for patient-specific treatment selection in a co-clinical setting.
    DOI:  https://doi.org/10.1038/s41467-020-18388-7
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