bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2020‒08‒23
fifteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute
  1. p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth.
  2. Insulin-like growth factor 1-receptor signaling stimulates GRP78 expression through the PI3K/AKT/mTOR/ATF4 axis.
  3. Rictor amplification promotes NSCLC cell proliferation through formation and activation of mTORC2 at the expense of mTORC1.
  4. Rictor promotes tumor progression of rapamycin-insensitive triple-negative breast cancer cells.
  5. Compassionate use of everolimus for refractory epilepsy in a patient with MTOR mosaic mutation.
  6. Anti-Insulin Receptor Antibodies Improve Hyperglycemia in a Mouse Model of Human Insulin Receptoropathy.
  7. Dual Inhibition of Autophagy and PI3K/AKT/MTOR Pathway as a Therapeutic Strategy in Head and Neck Squamous Cell Carcinoma.
  8. From Insulin to Akt: Time Delays and Dominant Processes.
  9. Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.
  10. Photothermogenetic inhibition of cancer stemness by near-infrared-light-activatable nanocomplexes.
  11. PIK3CA mutation inhibition in hormone receptor-positive breast cancer: time has come.
  12. Kinetics of receptor tyrosine kinase activation define ERK signaling dynamics.
  13. Integrating genetic and non-genetic determinants of cancer evolution by single-cell multi-omics.
  14. The Importance of Computational Modeling in Stem Cell Research.
  15. Insulin: too much of a good thing is bad.
  1. Nat Cell Biol. 2020 Aug 17.
    p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth.
    Ada Nowosad, Pauline Jeannot, Caroline Callot, Justine Creff, Renaud Thierry Perchey, Carine Joffre, Patrice Codogno, Stephane Manenti, Arnaud Besson.
      Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27-/- cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27-/- cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.
    DOI:  https://doi.org/10.1038/s41556-020-0554-4
  2. Cell Signal. 2020 Aug 14. pii: S0898-6568(20)30213-8. [Epub ahead of print]75 109736
    Insulin-like growth factor 1-receptor signaling stimulates GRP78 expression through the PI3K/AKT/mTOR/ATF4 axis.
    Dat P Ha, Amy S Lee.
      GRP78, a major molecular chaperone, is critical for the folding and maturation of membrane and secretory proteins and serves as the master regulator of the unfolded protein response. Thus, GRP78 is frequently upregulated in highly proliferative cells to cope with elevated protein synthesis and metabolic stress. IGF-1 is a potent regulator of cell growth, metabolism and survival. Previously we discovered that GRP78 is a novel downstream target of IGF-1 signaling by utilizing mouse embryonic fibroblast model systems where the IGF-1 receptor (IGF-1R) was either overexpressed (R+) or knockout (R-). Here we investigated the mechanisms whereby GRP78 is upregulated in the R+ cells. Our studies revealed that suppression of PI3K/AKT/mTOR downstream of IGF-1R signaling resulted in concurrent decrease in GRP78 and the transcription factor ATF4. Through knock-down and overexpression studies, we established ATF4 as the essential downstream nodal of the PI3K/AKT/mTOR signaling pathway critical for GRP78 transcriptional upregulation mediated by IGF-1R.
    Keywords:  ATF4; GRP78; Growth factor signaling; IGF-1; PI3K/AKT/mTOR
    DOI:  https://doi.org/10.1016/j.cellsig.2020.109736
  3. Mol Cancer Res. 2020 Aug 14. pii: molcanres.0262.2020. [Epub ahead of print]
    Rictor amplification promotes NSCLC cell proliferation through formation and activation of mTORC2 at the expense of mTORC1.
    Laura C Kim, Christopher H Rhee, Jin Chen.
      Non-small cell lung cancer (NSCLC) is characterized by genomic alterations, yet a targetable mutation has not been discovered in nearly half of all patients. Recent studies have identified amplification of RICTOR, an mTORC2-specific cofactor, as a novel actionable target in NSCLC. mTORC2 is one of two distinct mTOR complexes to sense environmental cues and regulate a variety of cellular processes including cell growth, proliferation, and metabolism, all of which promote tumorigenesis when aberrantly regulated. Interestingly, other components of mTORC2 are not co-amplified with RICTOR in human lung cancer, raising the question as to whether RICTOR amplification-induced changes are dependent on mTORC2 function. To model RICTOR amplification, we overexpressed Rictor using the Cas9 Synergistic Activator system. Overexpression of Rictor increased mTORC2 integrity and signaling, but at the expense of mTORC1, suggesting that overexpressed Rictor recruits common components away from mTORC1. Additionally, Rictor overexpression increases proliferation and growth of NSCLC 3D cultures and tumors in vivo. Conversely, knockout of RICTOR leads to decreased mTORC2 formation and activity, but increased mTORC1 function. Because Rictor has mTOR-dependent and independent functions, we also knocked out mLST8, a shared mTOR co-factor but is specifically required for mTORC2 function. Inducible loss of mLST8 in RICTOR-amplified NSCLC cells inhibited mTORC2 integrity and signaling, tumor cell proliferation, and tumor growth. Collectively, these data identify a mechanism for Rictor-driven tumor progression and provide further rationale for development of an mTORC2-specific inhibitor. Implications: RICTOR amplification drives NSCLC proliferation through formation of mTORC2, suggesting mTORC2-specific inhibition could be a beneficial therapeutic option.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0262
  4. Biochem Biophys Res Commun. 2020 Aug 17. pii: S0006-291X(20)31557-6. [Epub ahead of print]
    Rictor promotes tumor progression of rapamycin-insensitive triple-negative breast cancer cells.
    Risayo Watanabe, Mamiko Miyata, Chitose Oneyama.
      Triple-negative breast cancer (TNBC), characterized by decreased expression of hormone receptors and human epidermal growth factor type 2 receptor, has poor prognosis and lacks effective therapeutics. Recently, the mTOR inhibitor rapamycin and its analogs have attracted growing interests and evaluated as therapeutic agents against TNBC, in which the PI3K/AKT/mTOR pathway is often activated. However, some TNBCs are less sensitive to these drugs. In this study, we found that the sensitivity of TNBC cells to rapamycin was highly dependent on the expression level of rapamycin-insensitive companion of mTOR (Rictor), a key component of the mTOR complex 2. Repression of the Rictor expression strongly suppressed the growth of rapamycin-insensitive tumor cells. Furthermore, we showed that the suppression of Rictor expression was also effective in rapamycin-insensitive cells that had acquired resistance to mTOR kinase inhibitors. These findings indicate that Rictor can be a predictive marker for the use of rapamycin analogs in TNBC and highlight the need to develop therapeutics targeting Rictor in the treatment of TNBC.
    Keywords:  Rapamycin; Rictor; Triple-negative breast cancer
    DOI:  https://doi.org/10.1016/j.bbrc.2020.08.012
  5. Eur J Med Genet. 2020 Aug 14. pii: S1769-7212(20)30372-4. [Epub ahead of print] 104036
    Compassionate use of everolimus for refractory epilepsy in a patient with MTOR mosaic mutation.
    Nawale Hadouiri, Veronique Darmency, Laurent Guibaud, Alexis Arzimanoglou, Arthur Sorlin, Virginie Carmignac, Jean-Baptiste Rivière, Frédéric Huet, Maxime Luu, Marc Bardou, Christel Thauvin-Robinet, Pierre Vabres, Laurence Faivre.
      The MTOR gene encodes the mechanistic target of rapamycin (mTOR), which is a core component of the PI3K-AKT-mTOR signaling pathway. Postzygotic MTOR variants result in various mosaic phenotypes, referred to in OMIM as Smith-Kinsgmore syndrome or focal cortical dysplasia. We report here the case of a patient, with an MTOR mosaic gain-of-function variant (p.Glu2419Lys) in the DNA of 41% skin cells, who received compassionate off-label treatment with everolimus for refractory epilepsy. This 12-year-old-girl presented with psychomotor regression, intractable seizures, hypopigmentation along Blaschko's lines (hypomelanosis of Ito), asymmetric regional body overgrowth, and ocular anomalies, as well as left cerebral hemispheric hypertrophy with some focal underlying migration disorders. In response to the patient's increasingly frequent epileptic seizures, everolimus was initiated (after approval from the hospital ethics committee) at 5 mg/day and progressively increased to 12.5 mg/day. After 5 months of close monitoring (including neuropsychological and electroencephalographic assessment), no decrease in seizure frequency was observed. Though the physiopathological rationale was good, no significant clinical response was noticed under everolimus treatment. A clinical trial would be needed to draw conclusions, but, because the phenotype is extremely rare, it would certainly need to be conducted on an international scale.
    Keywords:  Everolimus; Mosaic mTOR mutation; Seizures; Therapy in rare diseases; mTOR
    DOI:  https://doi.org/10.1016/j.ejmg.2020.104036
  6. Diabetes. 2020 Aug 17. pii: db200345. [Epub ahead of print]
    Anti-Insulin Receptor Antibodies Improve Hyperglycemia in a Mouse Model of Human Insulin Receptoropathy.
    Gemma V Brierley, Hannah Webber, Eerika Rasijeff, Sarah Grocott, Kenneth Siddle, Robert K Semple.
      Loss-of-function mutations in both alleles of the human insulin receptor gene (INSR) cause extreme insulin resistance (IR) and usually death in childhood, with few effective therapeutic options. Bivalent anti-receptor antibodies can elicit insulin-like signaling by mutant INSR in cultured cells, but whether this translates into meaningful metabolic benefits in vivo, where dynamics of insulin signaling and receptor recycling are more complex, is unknown. To address this we adopted a strategy to model human insulin receptoropathy in mice, using Cre recombinase delivered by adeno-associated virus to knock out endogenous hepatic Insr acutely in floxed Insr mice (L-IRKO+GFP), before adenovirus-mediated 'add-back' of wild-type (WT) or mutant human INSR Two murine anti-INSR monoclonal antibodies, previously shown to be surrogate agonists for mutant INSR, were then tested by intraperitoneal injections. As expected, L-IRKO+GFP mice showed glucose intolerance and severe hyperinsulinemia, and this was fully corrected by add-back of WT but neither D734A nor S350L mutant INSR. Antibody injection improved glucose tolerance in D734A INSR-expressing mice and reduced hyperinsulinemia in both S350L and D734A INSR-expressing animals, and did not cause hypoglycemia in WT INSR-expressing mice. Antibody treatment also downregulated both wild-type and mutant INSR protein, attenuating its beneficial metabolic effects. Anti-INSR antibodies thus improve IR in an acute model of insulin receptoropathy, but these findings imply a narrow therapeutic window determined by competing effects of antibodies to stimulate receptors and induce their downregulation.
    DOI:  https://doi.org/10.2337/db20-0345
  7. Cancers (Basel). 2020 Aug 21. pii: E2371. [Epub ahead of print]12(9):
    Dual Inhibition of Autophagy and PI3K/AKT/MTOR Pathway as a Therapeutic Strategy in Head and Neck Squamous Cell Carcinoma.
    Monique Bernard, Guillaume B Cardin, Maxime Cahuzac, Tareck Ayad, Eric Bissada, Louis Guertin, Houda Bahig, Phuc Felix Nguyen-Tan, Edith Filion, Olivier Ballivy, Denis Soulieres, Francis Rodier, Apostolos Christopoulos.
      Genomic analyses of head and neck squamous cell carcinoma (HNSCC) have highlighted alterations in the phosphatidylinositol 3-kinase (PI3K) signaling pathway, presenting a therapeutic target for multiple ongoing clinical trials with PI3K or PI3K/MTOR inhibitors. However, these inhibitors can potentially increase autophagy in HNSCC and indirectly support cancer cell survival. Here, we sought to understand the relationship between the PI3K signaling pathway and autophagy during their dual inhibition in a panel of HNSCC cell lines. We used acridine orange staining, immunoblotting, and tandem sensor Red Fluorescent Protein- Green Fluorescent Protein-, microtubule-associated protein 1 light chain 3 beta (RFP-GFP-LC3B) expression analysis to show that PI3K inhibitors increase autophagosomes in HNSCC cells, but that chloroquine treatment effectively inhibits the autophagy that is induced by PI3K inhibitors. Using the Bliss independence model, we determined that the combination of chloroquine with PI3K inhibitors works in synergy to decrease cancer cell proliferation, independent of the PIK3CA status of the cell line. Our results indicate that a strategy focusing on autophagy inhibition enhances the efficacy of therapeutics already in clinical trials. Our results suggest a broader application for this combination therapy that can be promptly translated to in vivo studies.
    Keywords:  HNSCC; PI3K inhibitor; PI3K signaling pathway; autophagy; buparlisib; cancer; chloroquine; combination therapy; omipalisib; oral tongue
    DOI:  https://doi.org/10.3390/cancers12092371
  8. J Theor Biol. 2020 Aug 18. pii: S0022-5193(20)30309-X. [Epub ahead of print] 110454
    From Insulin to Akt: Time Delays and Dominant Processes.
    Catheryn W Gray, Adelle C F Coster.
      Akt/PKB regulates numerous processes in the mammalian cell, including cell survival and proliferation, and glucose uptake in response to insulin. Abnormalities in Akt signalling are linked to the development of Type 2 diabetes, cardio-vascular disease, and cancer. In the absence of insulin, Akt is predominantly found in the inactive state in the cytosol. Following insulin stimulation, Akt translocates to the plasma membrane, docks, and is phosphorylated to take on the active conformation. In turn, the activated Akt travels to and phosphorylates its many downstream substrates. Although crucial to the activation process, the translocation of Akt from the cytosol to the plasma membrane is currently not well understood. Here we detail the parameter optimisation of a mathematical model of Akt translocation to experimental data. We have quantified the time delay between the application of insulin and the downstream Akt translocation response, indicating the constraints on the timing of the intermediate processes. A delay of approximately 0.4 min prior to the Akt response was determined for the application of 1nM insulin to cells in the basal state, whereas it was found that a further transition from physiological insulin to higher stimuli did not incur a delay. Furthermore, our investigation indicates that the dominant processes regulating the appearance of Akt at the plasma membrane differ with the insulin level. For physiological insulin, the rate limiting step was the release of Akt to the plasma membrane in response to the insulin signal. In contrast, at high insulin levels, regulation of the recycling of Akt from the plasma membrane to the cytosol was also required.
    Keywords:  Akt/PKB; ODE model; Signalling; Signalling delays; Translocation processes
    DOI:  https://doi.org/10.1016/j.jtbi.2020.110454
  9. Nat Genet. 2020 Aug 17.
    Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.
    Hoon Kim, Nam-Phuong Nguyen, Kristen Turner, Sihan Wu, Amit D Gujar, Jens Luebeck, Jihe Liu, Viraj Deshpande, Utkrisht Rajkumar, Sandeep Namburi, Samirkumar B Amin, Eunhee Yi, Francesca Menghi, Johannes H Schulte, Anton G Henssen, Howard Y Chang, Christine R Beck, Paul S Mischel, Vineet Bafna, Roel G W Verhaak.
      Extrachromosomal DNA (ecDNA) amplification promotes intratumoral genetic heterogeneity and accelerated tumor evolution1-3; however, its frequency and clinical impact are unclear. Using computational analysis of whole-genome sequencing data from 3,212 cancer patients, we show that ecDNA amplification frequently occurs in most cancer types but not in blood or normal tissue. Oncogenes were highly enriched on amplified ecDNA, and the most common recurrent oncogene amplifications arose on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number-matched linear DNA, coupled with enhanced chromatin accessibility, and more frequently resulted in transcript fusions. Patients whose cancers carried ecDNA had significantly shorter survival, even when controlled for tissue type, than patients whose cancers were not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification is common in cancer, is different from chromosomal amplification and drives poor outcome for patients across many cancer types.
    DOI:  https://doi.org/10.1038/s41588-020-0678-2
  10. Nat Commun. 2020 Aug 17. 11(1): 4117
    Photothermogenetic inhibition of cancer stemness by near-infrared-light-activatable nanocomplexes.
    Yue Yu, Xi Yang, Sheethal Reghu, Sunil C Kaul, Renu Wadhwa, Eijiro Miyako.
      Strategies for eradicating cancer stem cells (CSCs) are urgently required because CSCs are resistant to anticancer drugs and cause treatment failure, relapse and metastasis. Here, we show that photoactive functional nanocarbon complexes exhibit unique characteristics, such as homogeneous particle morphology, high water dispersibility, powerful photothermal conversion, rapid photoresponsivity and excellent photothermal stability. In addition, the present biologically permeable second near-infrared (NIR-II) light-induced nanocomplexes photo-thermally trigger calcium influx into target cells overexpressing the transient receptor potential vanilloid family type 2 (TRPV2). This combination of nanomaterial design and genetic engineering effectively eliminates cancer cells and suppresses stemness of cancer cells in vitro and in vivo. Finally, in molecular analyses of mechanisms, we show that inhibition of cancer stemness involves calcium-mediated dysregulation of the Wnt/β-catenin signalling pathway. The present technological concept may lead to innovative therapies to address the global issue of refractory cancers.
    DOI:  https://doi.org/10.1038/s41467-020-17768-3
  11. ESMO Open. 2020 Aug;pii: e000890. [Epub ahead of print]5(4):
    PIK3CA mutation inhibition in hormone receptor-positive breast cancer: time has come.
    Leticia De Mattos-Arruda.
      
    Keywords:  PIK3CA mutation; alpelisib; breast cancer; endocrine therapy resistance; hormonal receptor
    DOI:  https://doi.org/10.1136/esmoopen-2020-000890
  12. Sci Signal. 2020 Aug 18. pii: eaaz5267. [Epub ahead of print]13(645):
    Kinetics of receptor tyrosine kinase activation define ERK signaling dynamics.
    Anatoly Kiyatkin, Iris K van Alderwerelt van Rosenburgh, Daryl E Klein, Mark A Lemmon.
      In responses to activation of receptor tyrosine kinases (RTKs), crucial cell fate decisions depend on the duration and dynamics of ERK signaling. In PC12 cells, epidermal growth factor (EGF) induces transient ERK activation that leads to cell proliferation, whereas nerve growth factor (NGF) promotes sustained ERK activation and cell differentiation. These differences have typically been assumed to reflect distinct feedback mechanisms in the Raf-MEK-ERK signaling network, with the receptors themselves acting as simple upstream inputs. We failed to confirm the expected differences in feedback type when investigating transient versus sustained signaling downstream of the EGF receptor (EGFR) and NGF receptor (TrkA). Instead, we found that ERK signaling faithfully followed RTK dynamics when receptor signaling was modulated in different ways. EGFR activation kinetics, and consequently ERK signaling dynamics, were switched from transient to sustained when receptor internalization was inhibited with drugs or mutations, or when cells expressed a chimeric receptor likely to have impaired dimerization. In addition, EGFR and ERK signaling both became more sustained when substoichiometric levels of erlotinib were added to reduce duration of EGFR kinase activation. Our results argue that RTK activation kinetics play a crucial role in determining MAP kinase cascade signaling dynamics and cell fate decisions, and that signaling outcome can be modified by activating a given RTK in different ways.
    DOI:  https://doi.org/10.1126/scisignal.aaz5267
  13. Nat Rev Genet. 2020 Aug 17.
    Integrating genetic and non-genetic determinants of cancer evolution by single-cell multi-omics.
    Anna S Nam, Ronan Chaligne, Dan A Landau.
      Cancer represents an evolutionary process through which growing malignant populations genetically diversify, leading to tumour progression, relapse and resistance to therapy. In addition to genetic diversity, the cell-to-cell variation that fuels evolutionary selection also manifests in cellular states, epigenetic profiles, spatial distributions and interactions with the microenvironment. Therefore, the study of cancer requires the integration of multiple heritable dimensions at the resolution of the single cell - the atomic unit of somatic evolution. In this Review, we discuss emerging analytic and experimental technologies for single-cell multi-omics that enable the capture and integration of multiple data modalities to inform the study of cancer evolution. These data show that cancer results from a complex interplay between genetic and non-genetic determinants of somatic evolution.
    DOI:  https://doi.org/10.1038/s41576-020-0265-5
  14. Trends Biotechnol. 2020 Aug 13. pii: S0167-7799(20)30201-8. [Epub ahead of print]
    The Importance of Computational Modeling in Stem Cell Research.
    Antonio Del Sol, Sascha Jung.
      The generation of large amounts of omics data is increasingly enabling not only the processing and analysis of large data sets but also the development of computational models in the field of stem cell research. Although computational models have been proposed in recent decades, we believe that the stem cell community is not fully aware of the potentiality of computational modeling in guiding their experimental research. In this regard, we discuss how single-cell technologies provide the right framework for computational modeling at different scales of biological organization in order to address challenges in the stem cell field and to guide experimentalists in the design of new strategies for stem cell therapies and treatment of congenital disorders.
    Keywords:  multiscale modeling; single-cell multiomics data; stem cell therapy
    DOI:  https://doi.org/10.1016/j.tibtech.2020.07.006
  15. BMC Med. 2020 Aug 21. 18(1): 224
    Insulin: too much of a good thing is bad.
    Hubert Kolb, Kerstin Kempf, Martin Röhling, Stephan Martin.
      BACKGROUND: Insulin shares a limited physiological concentration range with other endocrine hormones. Not only too low, but also too high systemic insulin levels are detrimental for body functions.MAIN BODY: The physiological function and clinical relevance of insulin are usually seen in association with its role in maintaining glucose homeostasis. However, insulin is an anabolic hormone which stimulates a large number of cellular responses. Not only too low, but also excess insulin concentrations are detrimental to the physiological balance. Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening the efficiency of insulin signaling ("insulin resistance"), this is not the case for most other hormonal actions of insulin, including the promotion of protein synthesis, de novo lipogenesis, and cell proliferation; the inhibition of lipolysis, of autophagy-dependent cellular turnover, and of nuclear factor E2-related factor-2 (Nrf2)-dependent antioxidative; and other defense mechanisms. Hence, there is no general insulin resistance but selective impairment of insulin signaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthase (eNOS). Because of the largely unrestricted insulin signaling, hyperinsulinemia increases the risk of obesity, type 2 diabetes, and cardiovascular disease and decreases health span and life expectancy. In epidemiological studies, high-dose insulin therapy is associated with an increased risk of cardiovascular disease. Randomized controlled trials of insulin treatment did not observe any effect on disease risk, but these trials only studied low insulin doses up to 40 IU/day. Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes from Mendelian randomization studies comparing individuals with genetically controlled low or high insulin production.
    CONCLUSIONS: The detrimental actions of prolonged high insulin concentrations, seen also in cell culture, argue in favor of a lifestyle that limits circadian insulin levels. The health risks associated with hyperinsulinemia may have implications for treatment regimens used in type 2 diabetes.
    Keywords:  Autophagy; Cardiovascular morbidity and mortality; Hyperinsulinemia; Inflammation; Insulin resistance; Nrf2; Obesity; Oxidative stress; Type 2 diabetes mellitus; eNOS
    DOI:  https://doi.org/10.1186/s12916-020-01688-6
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