Awardee 2016

Johannes C. Clevers

for his pioneering work in stem cell - and cancer research



Hans Clevers identified the crucial downstream component of the Wnt signaling cascade, TCF, and the mechanism by which Wnt signals activate specific TCF target genes. With these insights and in a collaboration with Bert Vogelstein, he proposed that in APC-deficient colon cancer, it is the inappropriate activation of the Wnt pathway that transforms cells. He was the first to link Wnt signaling with adult stem cell biology, when he showed that TCF4 gene disruption leads to the abolition of crypt stem cell compartments of the gut. He went on to show that the Tcf4-driven target gene program in colorectal cancer cells is the malignant counterpart of a physiological crypt stem cell program (1-5).

He then described the Wnt target Lgr5 as a marker for adult stem cells including those of crypts. By the creation of several ingenious Lgr5-based transgenic mice, he established the intestinal crypt as one of the pre-eminent models to visualize and study adult stem cells in mammals. He described several counter-intuitive characteristics for crypt stem cells: Lgr5 stem cells are abundant, they cycle rapidly, they divide symmetrically, and utilize their Paneth celldaughters as their niche (6-7).

Next, Hans Clevers identified the Wnt signal-enhancing Rspondins as ligands of Lgr5, and exploited the Rspondin/Lgr5 axis to develop a 3D organoid culture system for indefinite expansion of normal intestinal epithelium starting from a single adult Lgr5 stem cell. Similar results were then reported by him for multiple additional human and mouse tissues. This has opened ways to generate disease models directly from patients as well as avenues for regenerative medicine (8-15).

More detailed scientific description:

TCF1. Trained as immunologist, Hans Clevers cloned a T lymphocyte-specific transcription factor in 1991, that he termed TCF1 (1). Simultaneous with Walter Birchmeier, he discovered in 1996 that the TCF proteins constitute the effectors of the canonical Wnt pathway (2, and subsequent studies). For this, he redirected his lab towards developmental biology and employed model organisms such as frogs, zebrafish, flies and worms. He was the first to describe the correct molecular mechanism of Wnt-controlled gene transcription: stabilized ss-catenin binds and activates nuclear TCFs by providing a trans-activation domain (2,3). For these studies, he designed the widely used pTopflash Wnt reporters.

Wnt signaling in cancer. Previous work by Vogelstein, Polakis and others had unveiled that the tumor suppressor protein APC, mutant in most cases of colon cancer, binds ss-catenin and targets it for degradation. In a collaboration with Bert Vogelstein, he employed his reagents and insights in the role of ss-catenin and TCF to establish that the Wnt cascade is aberrantly activated in APC-deficient colon carcinoma cells. By showing that ss-catenin is constitutively complexed with the TCF family member TCF4 in APC-mutant cells, he provided a molecular explanation for the initiation of colon cancer (3).

Wnt signaling in adult stem cells. The Wnt signal transduction cascade was believed to play a role only during animal development. In 1998, Clevers was the first to link Wnt signaling with adult stem cell biology, when he showed that TCF4 gene disruption leads to the disappearance of crypts of the intestine (4). Clevers then refocused his lab again to become an intestinal stem cell lab. In the following years, studies by Clevers and many others confirmed a general, key role for Wnt signaling in the biology of adult stem cells and cancers derived thereof. When microarraying became first available, he demonstrated that the Tcf4-driven target gene program in human colorectal cancer cells represents the malignant counterpart of a physiological gene program active in self-renewing crypts (5, and subsequent studies).

Lgr5 as adult stem cell marker. While working his way through the Wnt target gene list shared between colon cancer and healthy crypts (5), he discovered that the Lgr5 gene uniquely marks small cycling cells at crypt bottoms. Nick Barker, then a postdoc in his lab, created several Lgr5-based knockmouse lines, now widely used. By lineage tracing driven from the Lgr5 locus, Barker and Clevers confirmed the all-butforgotten prediction of Cheng and Leblond from the 70s that these ‘crypt base columnar cells’ represent the epithelial stem cells of the small intestine and colon (6). Subsequent studies from his and other labs using these mouse models identified many other novel Lgr5+ adult tissue stem cell types, including the stomach, liver, pancreas, and hair follicle (Clevers and collaborators), taste buds, ovary and cochlea. It is now anticipated that all epithelia - be it ectodermal, mesodermal or endodermal - will use Lgr5 stem cells for physiological self-renewal/damage repair, and that Wnt is a major driving force for all these different types of stem cells.

Lgr5 stem cell biology. The fact that Lgr5 identifies stem cells as a single marker, allowed Clevers to study adult stem cell behavior in vivo and in vitro in ways that were previously not possible. He found that Lgr5 crypt stem cells behave in many unanticipated ways: Against common belief, they are abundant (and not extremely rare, as expected) and they divide constantly. In vivo, individual stem cells go through thousands of cell divisions, defying the Hayflick limit. Using multicolor lineage tracing he showed that stem cells numbers remain fixed because stem cells compete ‚neutrally‘ for niche space. Thus, they do not divide asymmetrically (7). These unexpected findings contrast starkly with the classical adult stem cell hierarchy as described for the hematopoietic system, yet they may hold for many other adult tissue stem cell types. Clevers argued recently in a Science Perspective that there is no good reason why evolution should have endowed us with a wide range of very different tissues -each with their own functions and challenges-, yet utilize the same stem cell strategy in all. “In the end, there may be no general rules as to how tissues are renewed, as there is no end to the inventive power of evolution.”

Lgr5 is the R-spondin receptor. Lgr5 is not simply a stem cell marker gene. Clevers showed that Lgr5 encodes a 7- transmembrane receptor that mediates signaling by a class of Wnt pathway agonists termed R-spondins (8), as independently demonstrated by Jim Liu. These observations explained the unique dependence of Lgr5 stem cells on R-spondins in vivo and in vitro. Subsequent independent studies from the Clevers’ and the Feng Cong labs provided a molecular mechanism for the Wnt-amplifying effects of R-spondin-Lgr5 interactions, involving negative regulation of two transmembrane E3 ligases RNF43/ ZNRF3 (9). Long-term clonal culturing of Lgr5 stem cells as self-organizing organoids In earlier studies, Clevers had found that Wnt signals intimately interact with the BMP and Notch cascades to drive proliferation and inhibit differentiation in intestinal crypts. By combining these insights with the observation that Lgr5 crypt stem cells appear to divide indefinitely in vivo, his lab established Lgr5/R-spondin-based culture systems that allow the outgrowth of single mouse Lgr5 stem cells into ever-expanding mini-guts that essentially retain all hallmarks of the healthy epithelium (10). In subsequent papers, he described modified culture versions for Lgr5 stem cells derived from a variety of healthy mouse and human organs including the stomach, the liver, theprostate and the pancreas. These epithelial organoid cultures are genetically and phenotypically extremely stable, allowing transplantation of the cultured offspring of a single stem cell (see for instance ref 10). In various collaborations, he demonstrated that in cancer (colorectal (11), pancreas (12) and prostate (13)) and in hereditary disease states such as cystic fibrosis and α1- anti-trypsin deficiency (10, 14), organoids directly generated from patient tissues allow disease modeling. Of note, the colon organoid-based swelling test for Cystic Fibrosis (14) has proven a remarkable ‘personalized’ predictor for the efficacy of the recently registered, very expensive CF drugs. Currently, a program is being implemented in Holland where a foundation established in Clevers’ Hubrecht institute (in collaboration with the major Dutch insurance companies and the Ministry of Health) will create a ‘living’ colon organoid biobank of all 1500 Dutch CF patients, to be tested against all current and future CF drugs. This will serve as a national pilot for the implementation of precision medicine. As proof-ofconcept of the utility of organoid technology for gene therapy, Clevers repaired the CFTR locus repaired in single gut stem cells from two Cystic Fibrosis patients, using CRISPR/ Cas9 technology in conjunction with homologous recombination. Repaired stem cells were clonally expanded into mini-guts and shown to contain a functional CFTR channel (14). In sum, a string of discoveries made by Clevers has established the crucial role of Wnt in the biology of adult stem cells and in cancer. The discovery of Lgr5 has led to the identification of multiple novel adult stem cells types, novel insights into adult stem cell behavior and their roles in disease, as well as in technologies to expand and manipulate Wnt-dependent Lgr5 stem cells in vitro. Clonal long-term expansion of primary adult Lgr5 stem cells in the form of organoids opens up a wide range of experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.


   
Curriculum vitae
   
  Born in Eindhoven, The Netherlands on March 27, 1957
Married, two children
   
Education
   
1982 M .Sc. („Doctoraal“) in Biology, University of Utrecht
1984 M .D. („Artsexamen“) University of Utrecht
1985 P h.D. („Promotie“) University of Utrecht
   
Scientific Training/Positions
   
1985-1989 Research Fellow in Pathology. Dana-Farber Cancer Institute, Harvard Medical School, Boston MA, USA
1989-1991 Universitair Docent, Department of Clinical Immunology, University of Utrecht
1991-2002 Professor and Chairman, Dept. of Immunology, Faculty of Medicine, University of Utrecht
2002-2012 Director of Hubrecht Institute, Developmental Biology and Stem Cell Research
2002 Professor in Molecular Genetics. The Academic Biomedical Centre, University of Utrecht
2002 Honorary Professor at Changsha-Hunan, China
2012-2015 President of the Royal Netherlands Academy of Sciences
   
Selected publications
   
1.   van de Wetering, M., Oosterwegel, M., Dooijes, D., and Clevers, H.C. Identification and cloning of TCF-1, a T cell-specific transcription factor containing a sequence-specific HMG box. EMBO J ., 10:123-132 (1991)
2.   Molenaar, M., Van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., DestreÅLe, O. And Clevers, H. Xtcf-3 Transcription factor mediates betacatenin-induced axis formation in xenopus embryos. Cell , 86, 391-399 (1996)
3.   Korinek, V, Barker, N., Morin, P.J., van Wichen, D., de Weger, R., Kinzler, K.W., Vogelstein, B., and Clevers, H. Constitutive Transcriptional Activation by a betacatenin- Tcf complex in APC -/- Colon Carcinoma. Science , 275: 1784-1787 (1997)
4.   Korinek, V., Barker, N., Moerer, P., van Donselaar, E., Huls, G., Peters, P.J. and Clevers, H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet 19(4): 379-383 (1998)
5.   Van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., Van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A-P., Tjon-Pon-Fong, M., Moerer, P., Van den Born, M., Soete, G., Pals, S., Eilers, M., Medema, R., Clevers, H. The beta-catenin/TCF4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111: 241-250 (2002)
6.   Barker, N, van Es, J.H., Kuipers, J., Kujala P., van den Born, M., Cozijnsen, M., Korving, J., Begthel, H., Peters, P.C., and Clevers, H. Identification of Stem Cells in Small Intestine and Colon by a Marker Gene LGR5. Nature, 449:1003-1007 (2007)
7.   S nippert, .J., van der Flier, L.G., Sato, T., van Es, J.H., van den Born, M., Kroon-Veenboer, C., Barker, N.,Klein, A.M., van Rheenen, J. Benjamin D. Simons, B.D. and Clevers, H. Intestinal Crypt Homeostasis results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells. Cell 143:134-44 (2010)
8.   de Lau, W., Barker, N., ... and Clevers, H. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signaling. Nature 476: 293-297 (2011)
9.   Koo, B-K., Spit, M. Jordens, I., Low, T.Y., Stange, D.E., van de Wetering, M., van Es, J.H., Mohammed, S., Heck, A.J.R., Maurice, M.M. and Hans Clevers. Tumour suppressor RNF43 is a stem cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488: 665-669 (2012)
10.   S ato, T., Vries, R., Snippert, H., van de Wetering, M., Barker, N., Stange, D., van Es, J., Abo, A., Kujala, P., Peters, P., and Clevers, H. Single lgr5 gut stem cells build crypt-villus structures in vitro without a stromal niche Nature 459 :262-5 (2009)
11.   Huch M, Gehart H, van Boxtel R, …, Cuppen E, Clevers H. Long-term culture of genomestable bipotent stem cells from adult human liver. Cell 160:299-312 (2015)
12.   van de Wetering, M., Francies, H.E., Francis, J.M., …, Clevers, H. Prospective derivation of a ‚Living Organoid Biobank‘ of colorectal cancer patients. Cell 161: 933-45 (2015)
13.   Boj, S.F., Hwang, C.I., Baker, L.A., ….. Clevers H, Tuveson DA. Organoid models of human and mouse ductal pancreatic cancer. Cell 160: 324-338 (2015)
14.   Karthaus, W.R., Iaquinta, P.J., Drost, J., Gracanin, A.., van Boxtel, R., Wongvipat, J., Dowling, C.M., Gao, D., Begthel, H., Sachs, N., Vries, R.G., Cuppen, E., Chen, Y., Sawyers, C.L., Clevers, H.C. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 159: 163-75 (2014).
15.   S chwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, Demircan T, Sasaki N, Boymans S, Cuppen E, van der Ent CK, Nieuwenhuis EE, Beekman JM and Clevers H. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13: 653-658 (2013)


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