bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2020‒12‒13
fourteen papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research


  1. Mater Today Bio. 2020 Sep;8 100082
    Landry MR, DuRoss AN, Neufeld MJ, Hahn L, Sahay G, Luxenhofer R, Sun C.
      Multimodal therapy is often used in oncology to overcome dosing limitations and chemoresistance. Recently, combination immunoradiotherapy has shown great promise in a select subset of patients with colorectal cancer (CRC). Furthermore, molecularly targeted agents delivered in tandem with immunotherapy regimens have been suggested to improve treatment outcomes and expand the population of responding patients. In this study, radiation-sensitizing small molecules niraparib (PARP inhibitor) and HS-173 (PI3K inhibitor) are identified as a novel combination that synergistically enhance toxicity and induce immunogenic cell death both in vitro and in vivo in a CRC model. These inhibitors were co-encapsulated in a polymer micelle to overcome solubility limitations while minimizing off-target toxicity. Mice bearing syngeneic colorectal tumors (CT26) were administered these therapeutic micelles in combination with X-ray irradiation and anti-CTLA-4 immunotherapy. This combination led to enhanced efficacy demonstrated by improved tumor control and increased tumor infiltrating lymphocytes. This report represents the first investigation of DNA damage repair inhibition combined with radiation to potentiate anti-CTLA-4 immunotherapy in a CRC model.
    Keywords:  Drug delivery; Immune checkpoint blockade; Nanomedicine; Poly(2-oxazoline); Radiation therapy
    DOI:  https://doi.org/10.1016/j.mtbio.2020.100082
  2. Molecules. 2020 Dec 03. pii: E5693. [Epub ahead of print]25(23):
    Zhang L, Ju Q, Sun J, Huang L, Wu S, Wang S, Li Y, Guan Z, Zhu Q, Xu Y.
      Concomitant inhibition of MAPK and PI3K signaling pathways has been recognized as a promising strategy for cancer therapy, which effectively overcomes the drug resistance of MAPK signaling pathway-related inhibitors. Herein, we report the scaffold-hopping generation of a series of 1H-pyrazolo[3,4-d]pyrimidine dual ERK/PI3K inhibitors. Compound 32d was the most promising candidate, with potent inhibitory activities against both ERK2 and PI3Kα which displays superior anti-proliferative profiles against HCT116 and HEC1B cancer cells. Meanwhile, compound 32d possessed acceptable pharmacokinetic profiles and showed more efficacious anti-tumor activity than GDDC-0980 and the corresponding drug combination (BVD-523 + GDDC-0980) in HCT-116 xenograft model, with a tumor growth inhibitory rate of 51% without causing observable toxic effects. All the results indicated that 32d was a highly effective anticancer compound and provided a promising basis for further optimization towards dual ERK/PI3K inhibitors.
    Keywords:  ERK inhibitor; PI3K inhibitor; cross-talk; dual ERK/PI3K inhibitors
    DOI:  https://doi.org/10.3390/molecules25235693
  3. Mol Cancer Res. 2020 Dec 07. pii: molcanres.0745.2020. [Epub ahead of print]
    Sapir T, Shifteh D, Pahmer M, Goel S, Maitra R.
      The ERK1/2 (RAS, RAF, MEK, ERK) and PI3K (PI3K, AKT, mTOR, PTEN) pathways are the chief signaling pathways for cellular proliferation, survival, and differentiation. Overactivation and hyperphosphorylation of the ERK1/2 & PI3K pathways is frequently observed in cancer and is associated with poor patient prognosis. While it is well known that genetic alterations lead to the dysregulation of the ERK1/2 & PI3K pathways, increasing evidence is showcasing that epigenetic alterations also play a major role in the regulation of the ERK1/2 & PI3K pathways. Protein Arginine Methyltransferase 5 (PRMT5) is a post-translational modifier for multiple cellular processes that is currently being tested as a therapeutic target for cancer. PRMT5 has been shown to be overexpressed in many types of cancers, as well as negatively correlated with patient survival. Numerous studies are indicating that as a post-translational modifier, PRMT5 is extensively involved in regulating the ERK1/2 & PI3K pathways. Additionally, a large number of in vitro and in vivo studies are demonstrating that PRMT5 inhibition, as well as PRMT5 and ERK1/2 & PI3K combination therapies, show significant therapeutic effects in many cancer types. In this review, we explore the vast interactions that PRMT5 has with the ERK1/2 & PI3K pathways, and we make the case for further testing of PRMT5 inhibition, as well as PRMT5 and ERK1/2 & PI3K combination therapies, for the treatment of cancer.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0745
  4. Nutr Cancer. 2020 Dec 07. 1-16
    Kalbkhani F, Pirnejad A, Sam S, Sam MR.
      Efforts to develop effective drugs targeting PI3K and KRAS signaling pathways in PIK3CA/KRAS-mutant colorectal cancer stem cells (CRCSCs) remain challenging. Finding safe compounds that can easily enter CRCSCs with the ability to target metastasis-driver gene CXCR4 and pluripotency network genes as key upstream and downstream effectors of both PI3K and KRAS signaling pathways may provide promising results. PIK3CA/KRAS-mutant CRCSCs display high expression of glucose transporters (GLUTs) on their cell membrane and a glycolytic phenotype providing an opportunity to deliver antiglycolytic compounds into these cells via the GLUTs. CRC patients with low levels of vitamin C in their plasma show a shorter survival suggesting the ability of this vitamin at the physiologic levels for caspase-3 activation and apoptosis in CRCSCs. Vitamin C in an oxidized form (L-dehydroascorbic acid; L-DHA) with antiglycolytic activity can be taken up into CRC cells via the GLUTs. This may provide selective toxicity on CRCSCs and affect CXCR4 and stemness markers genes expression in these cells. To this end, we treated PIK3CA/KRAS-mutant LS174T cells with high glycolytic activity as an attractive model for CRCSCs with L-DHA equal to the pharmacological levels of vitamin C in human plasma, after which cell numbers, metabolic activity, proliferation-rate, CXCR4 and pluripotency network genes expression, caspase-3 activity with apoptosis were evaluated. 48 h post-treatment with 100- to 1000 µM L-DHA, cell numbers were decreased and measured to be 70-47% control. L-DHA with selective toxicity on LS174T cells diminished metabolic activity and cell proliferation-rate to 1.4-0.8 (Control OD = 1.5) and 92-54.5% respectively with no toxicity on PBMCs. L-DHA decreased CXCR4, Bmi-1, Sox-2 and Oct-4 expression to 45%, 85%, 45% and 48% control respectively followed by caspase-3 reactivation by 2.5 to 4.9-fold increases and induction of apoptosis ranging from 0.5% to 58.3% for 100- to 1000 µM L-DHA. According to our data, CRC stem-like cells were highly sensitive to L-DHA in in-vitro. L-DHA selectively targeted LS174T cells and successfully reactivated caspase-3 and apoptosis in these cells. CXCR4, stemness marker genes and metabolic activity appear to be promising targets of L-DHA. Our results may provide a new therapeutic approach to target selectively GLUT-overexpressing PIK3CA/KRAS-mutant CRCSCs using L-DHA with no toxicity on normal cells.
    DOI:  https://doi.org/10.1080/01635581.2020.1856387
  5. Aging (Albany NY). 2020 Dec 03. 12
    Xu M, Wang J, Li H, Zhang Z, Cheng Z.
      Colorectal cancer (CRC) is a common malignant tumor and is one of the leading causes of cancer-related deaths worldwide. Absent in melanoma 2 (AIM2), as a member of the pyrin-HIN family proteins, plays contentious roles in different types of cancers. In the present work, we provide evidence that AIM2 was commonly downregulated in human CRC and loss of AIM2 significantly correlated with tumor size, depth of invasion, lymph node metastasis (LNM) and TNM (Tumor, Node, Metastases) stage in patients suffering from CRC. AIM2 knockdown promoted CRC cell proliferation, migration and epithelial-mesenchymal transition (EMT) progress, whereas AIM2 overexpression did the opposite. AIM2 inhibited glioma-associated oncogene-1 (Gli1) expression through Smoothened homolog (SMO)-independent pathway and regulated CRC cell proliferation and migration in a Gli1-dependent manner. Moreover, AIM2 could modulate Protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway and the increased Gli1 expression and EMT progress induced by AIM2 depletion was reversed after incubation with AKT inhibitor Ly294002 in CRC cells. In conclusion, our results define AIM2 as a novel regulator of Gli1 in CRC cell growth and metastasis, and suggest that the AIM2/AKT/mTOR/Gli1 signaling axis may serve as a potential target for treatment of CRC.
    Keywords:  AIM2; Gli1; colorectal cancer; migration; proliferation
    DOI:  https://doi.org/10.18632/aging.202226
  6. Cancer Cell Int. 2020 Dec 03. 20(1): 578
    Adam RS, van Neerven SM, Pleguezuelos-Manzano C, Simmini S, Léveillé N, de Groot NE, Holding AN, Markowetz F, Vermeulen L.
      BACKGROUND: Cancer results from the accumulation of mutations leading to the acquisition of cancer promoting characteristics such as increased proliferation and resistance to cell death. In colorectal cancer, an early mutation leading to such features usually occurs in the APC or CTNNB1 genes, thereby activating Wnt signalling. However, substantial phenotypic differences between cancers originating within the same organ, such as molecular subtypes, are not fully reflected by differences in mutations. Indeed, the phenotype seems to result from a complex interplay between the cell-intrinsic features and the acquired mutations, which is difficult to disentangle when established tumours are studied.METHODS: We use a 3D in vitro organoid model to study the early phase of colorectal cancer development. From three different murine intestinal locations we grow organoids. These are transformed to resemble adenomas after Wnt activation through lentiviral transduction with a stable form of β-Catenin. The gene expression before and after Wnt activation is compared within each intestinal origin and across the three locations using RNA sequencing. To validate and generalize our findings, we use gene expression data from patients.
    RESULTS: In reaction to Wnt activation we observe downregulation of location specific genes and differentiation markers. A similar effect is seen in patient data, where genes with significant differential expression between the normal left and right colon are downregulated in the cancer samples. Furthermore, the signature of Wnt target genes differs between the three intestinal locations in the organoids. The location specific Wnt signatures are dominated by genes which have been lowly expressed in the tissue of origin, and are the targets of transcription factors that are activated following enhanced Wnt signalling.
    CONCLUSION: We observed that the region-specific cell identity has a substantial effect on the reaction to Wnt activation in a simple intestinal adenoma model. These findings provide a way forward in resolving the distinct biology between left- and right-sided human colon cancers with potential clinical relevance.
    Keywords:  Cell of origin; Intestinal cancer; Wnt signalling
    DOI:  https://doi.org/10.1186/s12935-020-01661-6
  7. Cell Biol Int. 2020 Dec 10.
    de Araújo WM, Tanaka MN, Lima PHS, de Moraes CF, Leve F, Bastos LG, Rocha MR, Robbs BK, Viola JPB, Morgado-Diaz JA.
      Transforming growth factor-β (TGF-β) play a dual role acting as tumor promoter or suppressor. Along with cyclooxygenase-2 (COX-2) and oncogenic Ras, this multifunctional cytokine is deregulated in colorectal cancer. Despite of their individual abilities to promote tumor growth and invasion, the mechanisms of cross regulation between these pathways is still unclear. Here, we investigate the effects of TGF-β, Ras oncogene and COX-2 in the colorectal cancer context. We used colon adenocarcinoma cell line HT-29 and Ras-transformed IEC-6 cells, both treated with prostaglandin E2 (PGE2 ), TGF-β or a combined treatment with these agents. We demonstrated that PGE2 alters the subcellular localization of E-cadherin and β-catenin and enhanced the tumorigenic potential in HT-29 cells. This effect was inhibited by TGF-β indicating a tumor suppressor role. Conversely, in Ras-transformed IEC-6 cells, TGF-β induced COX-2 expression and increased invasiveness, acting as a tumor promoter. In IEC-6 Ras-transformed cells, TGF-β increased nuclear β-catenin and Wnt/β-catenin activation, opposite to what was seen in the PGE2 and TGF-β joint treatment in HT-29 cells. Together, our findings show that TGF-β increases COX-2 levels and induces invasiveness cooperating with Ras in a Wnt/β-catenin activation dependent manner. This shows TGF-β dual regulation over COX-2/PGE2 tumor promotion depending on the H-Ras and Wnt/ β-catenin pathways activation status in intestinal cancer cells. This article is protected by copyright. All rights reserved.
    Keywords:  Cell signaling; Colorectal cancer; E-cadherin; H-Ras ; Invasionβ; TGF-; Wnt/β-catenin pathway
    DOI:  https://doi.org/10.1002/cbin.11519
  8. Mol Cancer Res. 2020 Dec 10. pii: molcanres.0623.2020. [Epub ahead of print]
    Gao SP, Kiliti AJ, Zhang K, Vasani N, Mao N, Jordan E, Wise HC, Shrestha Bhattarai T, Hu W, Dorso M, Rodrigues JA, Kim K, Hanrahan AJ, Razavi P, Carver B, Chandarlapaty S, Reis-Filho JS, Taylor BS, Solit DB.
      Mutational activation of the PI3 kinase/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biologic consequences of AKT1 E17K mutation by developing an AKT1 E17K-mutant isogenic system in a TP53-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of ZEB1 transcription via altered β-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating PIK3CA mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in breast cancer patients could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent co-selection for CDH1 mutations in AKT1 mutated breast tumors. Implications: AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of β-catenin to the cell membrane leading to decreased ZEB1 transcription, resulting in increased E-cadherin expression and a reversal of epithelial-mesenchymal transition.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0623
  9. Oncologist. 2020 Dec 09.
    van Brummelen EMJ, Huijberts S, van Herpen C, Desar I, Opdam F, van Geel R, Marchetti S, Steeghs N, Monkhorst K, Thijssen B, Rosing H, Huitema A, Beijnen J, Bernards R, Schellens J.
      LESSONS LEARNED: Afatinib and selumetinib can be combined in continuous and intermittent dosing schedules, albeit at lower doses than approved for monotherapy. Maximum tolerated dose for continuous and intermittent schedules is afatinib 20 mg QD and selumetinib 25 mg BID. Since the anticancer activity was limited, we do not recommend to further develop this combination until better biomarkers for response and resistance are defined.BACKGROUND: Anti-tumor effects of MEK inhibitors are limited in KRAS-mutated tumors due to feedback activation of upstream epidermal growth factor receptors which reactivates the MAPK and the phosphoinositide 3-kinase (PI3K)-AKT pathway. Therefore, this phase I trial was initiated with the pan-HER inhibitor afatinib plus the MEK inhibitor selumetinib in patients with KRASmt, PIK3CA wild type tumors.
    METHODS: Afatinib and selumetinib were administered according to a 3+3 design in continuous and intermittent schedules. The primary objective was safety, and the secondary objective was clinical efficacy.
    RESULTS: Twenty-six patients were enrolled with colorectal cancer (n = 19), non-small cell lung cancer (NSCLC) (n = 6), and pancreatic cancer (n = 1). Dose-limiting toxicities occurred in six patients, including grade 3 diarrhea, dehydration, decreased appetite, nausea, vomiting, and mucositis. The RP2D was 20 mg afatinib QD and 25 mg selumetinib BID (21 days on/7 days off) for continuous afatinib dosing and for intermittent dosing with both drugs 5 days on/2 days off. Efficacy was limited with disease stabilization for 221 days in a patient with NSCLC as best response.
    CONCLUSION: Afatinib and selumetinib can be combined in continuous and intermittent schedules in patients with KRASmt tumors. Although target engagement was observed, the clinical efficacy was limited.
    Keywords:  Afatinib; Colorectal cancer; KRAS; Non-small cell lung cancer; Pancreatic cancer; Selumetinib
    DOI:  https://doi.org/10.1002/onco.13631
  10. Mol Cancer Res. 2020 Dec 07. pii: molcanres.0818.2020. [Epub ahead of print]
    Nussinov R, Zhang M, Tsai CJ, Jang H.
      PI3K and PTEN are the second and third most highly mutated proteins in cancer following only p53. Their actions oppose each other. PI3K phosphorylates signaling lipid PIP2 to PIP3. PTEN dephosphorylates it back. Driver mutations in both proteins accrue PIP3. PIP3 recruits Akt and PDK1 to the membrane, promoting cell cycle progression. Here we review phosphorylation events and mutations in autoinhibition in PI3K and PTEN from the structural standpoint. Our purpose is to clarify how they control the autoinhibited states. In autoinhibition a segment or a subunit of the protein occludes its functional site. Protein-protein interfaces are often only marginally stable, making them sensitive to changes in conditions in living cells. Phosphorylation can stabilize or destabilize the interfaces. Driver mutations commonly destabilize them. In analogy to 'passenger mutations', we coin 'passenger phosphorylation' to emphasize that the presence of a phosphorylation recognition sequence logo does not necessarily imply function. Rather, it may simply reflect a statistical occurrence. In both PI3K and PTEN autoinhibiting phosphorylation events are observed in the occluding 'piece'. In PI3Kα the 'piece' is the p85α subunit. In PTEN it is the C-terminal segment. In both enzymes the stabilized interface covers the domain that attaches to the membrane. Driver mutations that trigger rotation of the occluding piece or its deletion prompt activation. To date, both enzymes lack specific, potent drugs. We discuss the implications of detailed structural and mechanistic insight into oncogenic activation and how it can advance allosteric precision oncology.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0818
  11. Sci Rep. 2020 Dec 10. 10(1): 21762
    Fujimoto Y, Morita TY, Ohashi A, Haeno H, Hakozaki Y, Fujii M, Kashima Y, Kobayashi SS, Mukohara T.
      Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) are observed in 15-20% of breast cancers (HER2+ breast cancers), and anti-HER2 therapies have significantly improved prognosis of patients with HER2+ breast cancer. One resistance mechanism to anti-HER2 therapies is constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway. Combination therapy with small-molecule inhibitors of AKT and HER2 was conducted in HER2+ breast cancer cell lines with or without PIK3CA mutations, which lead to constitutive activation of the PI3K pathway. PIK3CA mutations played important roles in resistance to single-agent anti-HER2 therapy in breast cancer cell lines. Combination therapy of a HER2 inhibitor and an AKT inhibitor, as well as other PI3K pathway inhibitors, could overcome the therapeutic limitations associated with single-agent anti-HER2 treatment in PIK3CA-mutant HER2+ breast cancer cell lines. Furthermore, expression of phosphorylated 4E-binding protein 1 (p4EBP1) following the treatment correlated with the antiproliferative activities of the combination, suggesting that p4EBP1 may have potential as a prognostic and/or efficacy-linking biomarkers for these combination therapies in patients with HER2+ breast cancer. These findings highlight potential clinical strategies using combination therapy to overcome the limitations associated with single-agent anti-HER2 therapies in patients with HER2+ breast cancer.
    DOI:  https://doi.org/10.1038/s41598-020-78646-y
  12. Am J Cancer Res. 2020 ;10(11): 3666-3685
    Yin H, Xie J, Jiang P, Jiang X, Duan D, Qi J, Luo Z, Ma C, Hong H.
      Colorectal cancer (CRC) is one of the top three most deadly cancers despite using chemotherapy based on oxaliplatin or irinotecan combined with targeted therapy. Chiauranib has recently been identified to be a promising anticancer candidate with impressive efficacy and safety. However, the role and molecular mechanisms of Chiauranib in the treatment of CRC remain to be elucidated. Our study shows that Chiauranib inhibits cell proliferation and induces apoptosis in KRAS wild-type CRC cells in a dose- and time-dependent manner, but not mutation ones. Meanwhile, Chiauranib increases ROS production in KRAS wild-type CRC cells. Moreover, Chiauranib selectively suppresses KRAS wild-type CRC cells growth in vivo. Mechanistically, Chiauranib inhibits KRAS wild-type CRC cells by triggering ROS production via activating the p53 signaling pathway. Further, KRAS mutation CRC cells are resistant to Chiauranib by increasing Nrf2 to stably elevate the basal antioxidant program and thereby lower intracellular ROS induced by Chiauranib. Our findings provide the rationale for further clinical evaluation of Chiauranib as a therapeutic agent in treating KRAS wild-type CRC.
    Keywords:  Colorectal cancer; ROS; chiauranib
  13. Cancers (Basel). 2020 Dec 04. pii: E3633. [Epub ahead of print]12(12):
    Nicolazzo C, Barault L, Caponnetto S, Macagno M, De Renzi G, Gradilone A, Belardinilli F, Cortesi E, Di Nicolantonio F, Gazzaniga P.
      The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who "convert" to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1, ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2, ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3, 50% of wild-type RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples.
    Keywords:  EGFR inhibitors; RAS mutation clearance; circulating methylated DNA; circulating tumor DNA; liquid biopsy; metastatic colorectal cancer
    DOI:  https://doi.org/10.3390/cancers12123633
  14. Cell Res. 2020 Dec 09.
    Xie P, Peng Z, Chen Y, Li H, Du M, Tan Y, Zhang X, Lu Z, Cui CP, Liu CH, He F, Zhang L.
      PTEN tumor suppressor opposes the PI3K/Akt signaling pathway in the cytoplasm and maintains chromosomal integrity in the nucleus. Nucleus-cytoplasm shuttling of PTEN is regulated by ubiquitylation, SUMOylation and phosphorylation, and nuclear PTEN has been proposed to exhibit tumor-suppressive functions. Here we show that PTEN is conjugated by Nedd8 under high glucose conditions, which induces PTEN nuclear import without effects on PTEN stability. PTEN neddylation is promoted by the XIAP ligase and removed by the NEDP1 deneddylase. We identify Lys197 and Lys402 as major neddylation sites on PTEN. Neddylated PTEN accumulates predominantly in the nucleus and promotes rather than suppresses cell proliferation and metabolism. The nuclear neddylated PTEN dephosphorylates the fatty acid synthase (FASN) protein, inhibits the TRIM21-mediated ubiquitylation and degradation of FASN, and then promotes de novo fatty acid synthesis. In human breast cancer tissues, neddylated PTEN correlates with tumor progression and poor prognosis. Therefore, we demonstrate a previously unidentified pool of nuclear PTEN in the Nedd8-conjugated form and an unexpected tumor-promoting role of neddylated PTEN.
    DOI:  https://doi.org/10.1038/s41422-020-00443-z