Mtorc1-mediated translational elongation limits intestinal tumour initiation and growth

ABSTRACT Inactivation of APC is a strongly predisposing event in the development of colorectal cancer1,2, prompting the search for vulnerabilities specific to cells that have lost APC

function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth3,4,5, and the current paradigm suggests that a critical function of

mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently

inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for

the proliferation of _Apc_-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected

increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of

eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the

mTORC1–S6K–eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically

approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer. Access through your institution Buy or subscribe

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BLOCKADE OF EIF5A HYPUSINATION LIMITS COLORECTAL CANCER GROWTH BY INHIBITING MYC ELONGATION Article Open access 10 December 2020 THE AMINO ACID TRANSPORTER SLC7A5 IS REQUIRED FOR EFFICIENT

GROWTH OF KRAS-MUTANT COLORECTAL CANCER Article 07 January 2021 AN UNANTICIPATED TUMOR-SUPPRESSIVE ROLE OF THE SUMO PATHWAY IN THE INTESTINE UNVEILED BY UBC9 HAPLOINSUFFICIENCY Article Open

access 18 September 2020 REFERENCES * Kinzler, K. W. & Vogelstein, B. Lessons from hereditary colorectal cancer. _Cell_ 87, 159–170 (1996) Article  CAS  Google Scholar  * Korinek, V. et

al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma. _Science_ 275, 1784–1787 (1997) Article  CAS  Google Scholar  * Ashton, G. H. et al.

Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling. _Dev. Cell_ 19, 259–269 (2010) Article  CAS  Google Scholar  * Fujishita,

T., Aoki, K., Lane, H. A., Aoki, M. & Taketo, M. M. Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in _Apc_Δ716 mice. _Proc. Natl Acad. Sci.

USA_ 105, 13544–13549 (2008) Article  ADS  CAS  Google Scholar  * Gulhati, P. et al. Targeted inhibition of mammalian target of rapamycin signaling inhibits tumorigenesis of colorectal

cancer. _Clin. Cancer Res._ 15, 7207–7216 (2009) Article  CAS  Google Scholar  * Pourdehnad, M. et al. Myc and mTOR converge on a common node in protein synthesis control that confers

synthetic lethality in Myc-driven cancers. _Proc. Natl Acad. Sci. USA_ 110, 11988–11993 (2013) Article  ADS  CAS  Google Scholar  * Martineau, Y. et al. Pancreatic tumours escape from

translational control through 4E-BP1 loss. _Oncogene_ 33, 1367–1374 (2014) Article  CAS  Google Scholar  * Jiang, Y. P., Ballou, L. M. & Lin, R. Z. Rapamycin-insensitive regulation of

4E-BP1 in regenerating rat liver. _J. Biol. Chem._ 276, 10943–10951 (2001) Article  CAS  Google Scholar  * Bach, S. P., Renehan, A. G. & Potten, C. S. Stem cells: the intestinal stem

cell as a paradigm. _Carcinogenesis_ 21, 469–476 (2000) Article  CAS  Google Scholar  * Bernal, N. P. et al. Evidence for active Wnt signaling during postresection intestinal adaptation. _J.

Pediatr. Surg._ 40, 1025–1029 (2005) Article  Google Scholar  * Ireland, H. et al. Inducible Cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of

β-catenin. _Gastroenterology_ 126, 1236–1246 (2004) Article  CAS  Google Scholar  * Muncan, V. et al. Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene

c-_Myc_. _Mol. Cell. Biol._ 26, 8418–8426 (2006) Article  CAS  Google Scholar  * Zoncu, R., Efeyan, A. & Sabatini, D. M. mTOR: from growth signal integration to cancer, diabetes and

ageing. _Nature Rev. Mol. Cell Biol._ 12, 21–35 (2011) Article  CAS  Google Scholar  * Yilmaz, Ö. H. et al. mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie

intake. _Nature_ 486, 490–495 (2012) Article  ADS  CAS  Google Scholar  * Farin, H. F., Van Es, J. H. & Clevers, H. Redundant sources of Wnt regulate intestinal stem cells and promote

formation of Paneth cells. _Gastroenterology_ 143, 1518–1529 (2012) Article  CAS  Google Scholar  * She, Q. B. et al. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK

signaling pathways that integrates their function in tumors. _Cancer Cell_ 18, 39–51 (2010) Article  CAS  Google Scholar  * Sato, T. et al. Single Lgr5 stem cells build crypt–villus

structures _in vitro_ without a mesenchymal niche. _Nature_ 459, 262–265 (2009) Article  ADS  CAS  Google Scholar  * Fresno, M., Jimenez, A. & Vazquez, D. Inhibition of translation in

eukaryotic systems by harringtonine. _Eur. J. Biochem._ 72, 323–330 (1977) Article  CAS  Google Scholar  * Schneider-Poetsch, T. et al. Inhibition of eukaryotic translation elongation by

cycloheximide and lactimidomycin. _Nature Chem. Biol._ 6, 209–217 (2010) Article  CAS  Google Scholar  * Hsieh, A. C. et al. The translational landscape of mTOR signalling steers cancer

initiation and metastasis. _Nature_ 485, 55–61 (2012) Article  ADS  CAS  Google Scholar  * Richter, J. D. & Sonenberg, N. Regulation of cap-dependent translation by eIF4E inhibitory

proteins. _Nature_ 433, 477–480 (2005) Article  ADS  CAS  Google Scholar  * Browne, G. J. & Proud, C. G. A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase

modulates the activity of the kinase and its binding to calmodulin. _Mol. Cell. Biol._ 24, 2986–2997 (2004) Article  CAS  Google Scholar  * Ryazanov, A. G., Shestakova, E. A. & Natapov,

P. G. Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translation. _Nature_ 334, 170–173 (1988) Article  ADS  CAS  Google Scholar  * Gorshtein, A. et al. Mammalian

target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells _in vitro_. _Endocr. Relat. Cancer_ 16, 1017–1027

(2009) Article  CAS  Google Scholar  * Gutzkow, K. B. et al. Cyclic AMP inhibits translation of cyclin D3 in T lymphocytes at the level of elongation by inducing eEF2-phosphorylation. _Cell.

Signal._ 15, 871–881 (2003) Article  CAS  Google Scholar  * Firczuk, H. et al. An _in vivo_ control map for the eukaryotic mRNA translation machinery. _Mol. Syst. Biol._ 9, 635 (2013)

Article  Google Scholar  * Hussey, G. S. et al. Identification of an mRNP complex regulating tumorigenesis at the translational elongation step. _Mol. Cell_ 41, 419–431 (2011) Article  CAS 

Google Scholar  * Nakamura, J. et al. Overexpression of eukaryotic elongation factor eEF2 in gastrointestinal cancers and its involvement in G2/M progression in the cell cycle. _Int. J.

Oncol._ 34, 1181–1189 (2009) CAS  PubMed  Google Scholar  * Din, F. V. et al. Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase, and induces autophagy in colorectal

cancer cells. _Gastroenterology_ 142, 1504–1515 (2012) Article  CAS  Google Scholar  * Baan, B. et al. 5-Aminosalicylic acid inhibits cell cycle progression in a phospholipase D dependent

manner in colorectal cancer. _Gut_ 61, 1708–1715 (2012) Article  CAS  Google Scholar  * El Marjou, F. et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium.

_Genesis_ 39, 186–193 (2004) Article  CAS  Google Scholar  * Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene _Lgr5_. _Nature_ 449, 1003–1007 (2007)

Article  ADS  CAS  Google Scholar  * Shibata, H. et al. Rapid colorectal adenoma formation initiated by conditional targeting of the _Apc_ gene. _Science_ 278, 120–123 (1997) Article  CAS 

Google Scholar  * Moser, A. R., Pitot, H. C. & Dove, W. F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. _Science_ 247, 322–324 (1990) Article  ADS

  CAS  Google Scholar  * de Alboran, I. M. et al. Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. _Immunity_ 14, 45–55 (2001) Article  CAS  Google Scholar

  * Polak, P. et al. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration. _Cell Metab._ 8, 399–410 (2008) Article  CAS  Google Scholar  * Luche,

H., Weber, O., Nageswara Rao, T., Blum, C. & Fehling, H. J. Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage

tracing studies. _Eur. J. Immunol._ 37, 43–53 (2007) Article  CAS  Google Scholar  * Tsukiyama-Kohara, K. et al. Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1.

_Nature Med._ 7, 1128–1132 (2001) Article  CAS  Google Scholar  * Banko, J. L. et al. The translation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity, and

memory in the hippocampus. _J. Neurosci._ 25, 9581–9590 (2005) Article  CAS  Google Scholar  * Shima, H. et al. Disruption of the p70s6k/p85s6k gene reveals a small mouse phenotype and a new

functional S6 kinase. _EMBO J._ 17, 6649–6659 (1998) Article  CAS  Google Scholar  * Ryazanov, A. G. Elongation factor-2 kinase and its newly discovered relatives. _FEBS Lett._ 514, 26–29

(2002) Article  CAS  Google Scholar  * Ruvinsky, I. et al. Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. _Genes Dev._ 19, 2199–2211 (2005)

Article  CAS  Google Scholar  * Sarbassov, D. D. et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. _Mol. Cell_ 22, 159–168 (2006) Article  CAS  Google Scholar  *

Sengupta, S., Peterson, T. R., Laplante, M., Oh, S. & Sabatini, D. M. mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. _Nature_ 468, 1100–1104 (2010) Article 

ADS  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS W.J.F. is funded by AICR. O.J.S. is funded by Cancer Research UK, European Research Council Investigator Grant (COLONCAN) and

the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement number 278568. M.B. is a Medical Research Council Senior Fellow. The authors acknowledge P. Cammareri, J.

Morton and C. Murgia for proofreading of the manuscript. AUTHOR INFORMATION Author notes * Thomas J. Jackson and John R. P. Knight: These authors contributed equally to this work. AUTHORS

AND AFFILIATIONS * Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK, William J. Faller, Rachel A. Ridgway, Thomas Jamieson, Saadia A. Karim, Sorina Radulescu, David J. Huels, Kevin

B. Myant, Helen A. Casey, Alessandro Scopelliti, Julia B. Cordero, Marcos Vidal & Owen J. Sansom * Medical Research Council Toxicology Unit, Leicester LE1 9HN, UK, Thomas J. Jackson, 

John R. P. Knight, Carolyn Jones, Kate M. Dudek, Martin Bushell & Anne E. Willis * Institut Necker-Enfants Malades, CS 61431, Paris, France Institut National de la Santé et de la

Recherche Médicale, U1151, F-75014 Paris, France Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France, Mario Pende * Department of Pharmacology, Rutgers The State University

of New Jersey, Robert Wood Johnson Medical School, Piscataway, 08854, New Jersey, USA Alexey G. Ryazanov * Department of Biochemistry and Goodman Cancer Research Center, McGill University,

Montreal, Québec H3A 1A3, Canada, Nahum Sonenberg * Department of Biochemistry and Molecular Biology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel, Oded

Meyuhas * Biozentrum, University of Basel, CH-4056 Basel, Switzerland, Michael N. Hall Authors * William J. Faller View author publications You can also search for this author inPubMed 

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inPubMed Google Scholar CONTRIBUTIONS O.J.S., A.E.W. and W.J.F. designed the project. W.J.F., R.A.R., T.J. and S.R. performed breeding and phenotypic analysis of mice; W.J.F., T.J.J. and

J.R.P.K. performed translational analysis; M.N.H., A.G.R., N.S., O.M., A.S., J.B.C., M.V., D.J.H., K.B.M., S.A.K., K.M.D., C.J., H.A.C. and M.P. provided advice and material; W.J.F., O.J.S.,

A.E.W. and M.B. wrote and edited the manuscript. CORRESPONDING AUTHOR Correspondence to Owen J. Sansom. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial

interests. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 MTORC1 IS ACTIVATED FOLLOWING WNT-SIGNAL AND ITS INHIBITION DOES NOT AFFECT HOMEOSTASIS. A, Representative IHC of

phospho-RPS6 (pS6) and phospho-4EBP1 (p4EBP1) show mTORC1 activity during intestinal regeneration, 72 h after 14 Gy γ-irradiation (representative of 5 biological replicates). B, C, Boxplots

demonstrating that 72 h of 10 mg kg−1 rapamycin treatment does not alter mitosis or apoptosis in normal intestinal crypts. Whiskers show maximum and minimum, black line shows median (_n_ = 4

per group). NS, not significant, Mann–Whitney _U_ test. D, Intestines imaged on OV100 microscope, 96 h after induction, for red fluorescent protein (RFP). Tissue without the ROSA-tdRFP

reporter (Neg control) show no RFP positivity, while the positive control (Pos control) and _Rptor_-deleted intestines show high RFP positivity (representative of 3 biological replicates).

E, F, Boxplot showing that _Rptor_ deletion does not affect mitosis or apoptosis rates in intestinal crypts, 96 h after induction. Whiskers show maximum and minimum, black line shows median

(_n_ = 4 per group). NS, not significant, Mann–Whitney _U_ test. Scale bars, 100 µm. EXTENDED DATA FIGURE 2 _RPTOR_ DELETION IS MAINTAINED IN THE SMALL INTESTINE. A, Representative IHC of

phospho-RPS6 (pS6) and phospho-4EBP1 (p4EBP1) shows maintained loss of mTORC1 signalling 400+ days after _Rptor_ deletion. Arrows indicate unrecombined escaper crypts that still show active

mTORC1 signalling (representative of 5 biological replicates). B, C, Boxplots showing that mitosis and apoptosis are unchanged 400+ days after _Rptor_ deletion. Mitosis and apoptosis were

counted on H&E sections and are quantified as percent mitosis or apoptosis per crypt. Whiskers show maximum and minimum, black line shows median (_n_ = 5 per group). NS, not significant,

Mann–Whitney _U_ test. Scale bars, 100 µm. EXTENDED DATA FIGURE 3 WNT SIGNALLING IS STILL ACTIVE AFTER _RPTOR_ DELETION AND RAPAMYCIN TREATMENT CAUSES REGRESSION OF ESTABLISHED TUMOURS. A,

B, Representative IHC of MYC and β-catenin showing high MYC levels and nuclear localization of β-catenin 96 h after _Apc_ and _Apc/Rptor_ deletion, demonstrating active Wnt signalling.

Nuclear staining (as opposed to membranous staining) of β-catenin is indicative of active Wnt signalling. Scale bar, 100 µm (representative of 3 biological replicates). C, Kaplan–Meyer

survival curve of _Apc__Min/+_ mice treated with rapamycin when showing signs of intestinal neoplasia. Rapamycin treatment (10 mg kg−1) started when mice showed signs of intestinal disease,

and was withdrawn after 30 days. Animals continued to be observed until signs of intestinal neoplasia. Death of animals in the rapamycin group almost always occurred after rapamycin

withdrawal (_n_ = 8 per group). ***_P_ value ≤ 0.001, log-rank test. D, Boxplot showing that 72 h 10 mg kg−1 rapamycin treatment causes an increase in lysozyme-positive cells in tumours.

Percentage lysozyme positivity within tumours was calculated using ImageJ software (http://imagej.nih.gov/ij/). Whiskers show maximum and minimum, black line shows median (10 tumours from

each of 5 mice per group were measured. **_P_ value ≤ 0.014, Mann–Whitney _U_ test. E, Boxplot showing that 72 h 10 mg kg−1 rapamycin treatment causes a decrease in BrdU positivity within

tumours. Percentage BrdU positivity within tumours was calculated using ImageJ software. Whiskers show maximum and minimum, black line shows median (10 tumours from each of 5 mice per group

were measured). **_P_ value ≤ 0.021, Mann–Whitney _U_ test. F, Representative IHC of lysozyme, showing a lack of lysozyme-positive paneth cells in remaining cystic tumours after 30 days of

10 mg kg−1 rapamycin treatment. Scale bars, 100 µm (representative of 5 biological replicates). EXTENDED DATA FIGURE 4 IHC AFTER RAPAMYCIN TREATMENT. A, Representative IHC of p21, p16 and

p53 after 6 h and 72 h of 10 mg kg−1 rapamycin treatment. Staining shows no induction of these proteins in tumours after rapamycin treatment (representative of 5 biological replicates). B,

Representative IHC for LGR5–GFP showing high numbers of LGR5-positive cells after 7 and 30 days of 10 mg kg−1 daily rapamycin treatment (representative of 5 biological replicates). Scale

bars, 100 µm. EXTENDED DATA FIGURE 5 _RPTOR_ DELETION IN THE INTESTINAL CRYPT IS LETHAL _IN VITRO_. A, Graph showing that _Rptor_ deletion prevents intestinal crypts from growing _ex vivo_.

Intestinal crypts were isolated and cultured as previously described17, 96 h after _Cre_ induction. Number of viable organoids was counted by eye 72 h after crypt isolation. WT, wild type.

Data are average ± standard deviation (_n_ = 3 biological replicates per group). EXTENDED DATA FIGURE 6 _APC_ DELETION INCREASES TRANSLATIONAL ELONGATION RATES AND CYCLOHEXIMIDE TREATMENT

PHENOCOPIES RAPAMYCIN TREATMENT. A, Representative polysome profiles from wild-type _ex vivo_ crypts incubated with harringtonine for 0 s (left) and 180 s (right) before harvest (_n_ = 3 per

time point). B, The areas under the sub-polysome (40S, 60S and 80S) and polysome sections as indicated by the dashed lines in A were quantified and expressed as a percentage of their sum.

Data in the bar graph are the average ± s.e.m. (_n_ = 3 per time point). C, D, Data are shown for _Apc_-deleted crypts, as for wild type in B and C (_n_ = 3 biological replicates). E,

Representative H&E staining showing that 35 mg kg−1 cycloheximide treatment phenocopies rapamycin treatment 96 h after _Apc_ deletion. Treatment began 24 h after induction (_n_ = 3

biological replicates). F, Representative IHC for BrdU showing a loss of proliferation in tumours after 72 h of 35 mg kg−1 cycloheximide treatment. (_n_ = 3 biological replicates). Arrow

highlights normal proliferating crypts. Scale bar, 100 µm. EXTENDED DATA FIGURE 7 _S6K_ DELETION DECREASES INTESTINAL REGENERATION. Graphical representation of findings, and boxplot showing

that murine intestinal regeneration after irradiation is dependent on S6K. Animals were exposed to 14 Gy γ-irradiation, and intestinal regeneration was calculated 72 h after exposure by

counting the number of viable crypts and multiplying that by the average size of the regenerating crypts. Relative regeneration was calculated by comparing each group to wild-type

regeneration. The rapamycin treatment arm is reproduced from Fig. 4 for visual clarity. Whiskers show maximum and minimum, black line shows median (_n_ = 4 per group). *_P_ value = 0.034,

Mann–Whitney _U_ test. EXTENDED DATA FIGURE 8 _EEF2K_ DELETION DRIVES RESISTANCE TO RAPAMYCIN. A, Representative IHC of phospho-eEF2 and phospho-RPS6 in wild-type (WT), _Apc_-deficient and

_Apc_- and _Eef2k_-deficient mice (with or without 72 h 10 mg kg−1 rapamycin (rapa) treatment) shows that rapamycin is unable to induce eEF2 phosphorylation in the absence of eEF2K (_n_ = 6

biological replicates). KO, knockout. Scale bars, 100 µm. EXTENDED DATA FIGURE 9 CYCLIN D3 IS REGULATED AT THE LEVEL OF ELONGATION. A, Representative IHC of _Apc_-deleted intestines with or

without _Eef2k_ deletion. Antibodies to eEF2K, phospho-RPS6 and cyclin D3 are shown (representative of 3 biological replicates). After _Eef2k_ knockout (KO), cyclin D3 levels are no longer

decreased upon 10 mg kg−1 rapamycin (rapa) treatment. B, Boxplot showing the number of cyclin-D3-positive cells per crypt, 96 h after _Apc_ deletion, with and without 10 mg kg−1 rapamycin

treatment. Graph shows that in _Eef2k_ knockout animals, rapamycin no longer reduced cyclin D3 levels (_n_ = 3 biological replicates per group). *_P_ value ≤ 0.05, Mann–Whitney _U_ test. C,

Western blot analysis of intestinal epithelial cells from _Apc_-deleted and _Apc_-deleted _Eef2k_ knockout, with and without 10 mg kg−1 rapamycin. Antibodies to eEF2K, phospho-RPS6, cyclin

D3 and β-actin are shown. Each well represents a different mouse from the relevant group. Cyclin D3 levels are no longer reduced after _Eef2k_ deletion. Scale bar, 100 µm. EXTENDED DATA

FIGURE 10 RIBOSOMES ELONGATE FASTER ON _CCND3_ AFTER _APC_ DELETION. The ribosome run-off rate of various messages was measured as in Fig. 3. Elongation of _Ccnd3_ was significantly

increased, while _Actb_, _Rps21_, _Rps6_ and _Ccnd1_ remain unchanged. Data are average ± s.e.m. (_n_ = 3 biological replicates per group). *_P_ value ≤ 0.05, Mann–Whitney _U_ test.

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ARTICLE CITE THIS ARTICLE Faller, W., Jackson, T., Knight, J. _et al._ mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. _Nature_ 517, 497–500 (2015).

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