Title: Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

Abstract

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. We report experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not allmore » of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.« less

Authors:

Wiechmann, Svenja ^[1]; Maisonneuve, Pierre  ^[2];

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• Search DOE PAGES for ORCID "0000-0001-8292-5741"
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Grebbin, Britta M. ^[3]; Hoffmeister, Meike ^[4]; Kaulich, Manuel  ^[5];

• Search DOE PAGES for author "Kaulich, Manuel"
• Search DOE PAGES for ORCID "0000-0002-9528-8822"
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Clevers, Hans ^[6]; Rajalingam, Krishnaraj ^[7]; Kurinov, Igor  ^[8];

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• Search DOE PAGES for ORCID "0000-0001-5472-5574"
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Farin, Henner F.  ^[3];

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• Search DOE PAGES for ORCID "0000-0003-1558-5366"
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Sicheri, Frank ^[2]; Ernst, Andreas ^[1]

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+ Show Author Affiliations

1. Goethe Univ., Frankfurt (Germany). Univ. Hospital; Fraunhofer Inst. for Molecular Biology and Applied Ecology IME, Frankfurt (Germany)
2. Sinai Health System, Toronto, ON (Canada). Lunenfeld Tanenbaum Research Inst.
3. German Cancer Consortium (DKTK), Heidelberg (Germany); Georg-Speyer-Haus, Frankfurt (Germany). Inst. for Tumor Biology and Experimental Therapy; German Cancer Research Center (DKFZ), Heidelberg (Germany)
4. Goethe Univ., Frankfurt (Germany). Univ. Hospital; Brandenburg Medical School (MHB) (Germany)
5. Goethe Univ., Frankfurt (Germany). Univ. Hospital; Frankfurt Cancer Inst. (Germany)
6. Royal Netherlands Academy of Arts and Sciences (KNAW); Utrecht (The Netherlands); Univ. Medical Center Utrecht (The Netherlands)
7. Univ. Medical Center Mainz (Germany)
8. Argonne National Lab. (ANL), Argonne, IL (United States). Northeastern Collaborative Access Team (NE-CAT); Cornell Univ., Ithaca, NY (United States)

Publication Date:

2020-02-20

Research Org.:

Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Org.:

National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); LOEWE Ub-Net, Cluster of Excellence “Macromolecular Complexes”; Collaborative Research Center; Canadian Cancer Society Research Institute (CCSRI); Canadian Institutes of Health Research (CIHR); TD Bank; German Research Foundation (DFG)

OSTI Identifier:

1618482

Grant/Contract Number:  

P41 GM103403; S10 RR029205; 704116; FDN143277; RA1739/4-2

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 295; Journal Issue: 14; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; protein engineering; crystal structure; phage display; colorectal cancer; substrate specificity; GTPase; protein design; protein-protein interaction; cancer organoids; oncogenic Ras; CRAF RBD; Ras-binding domain; conformation-specific inhibitors; Ras signaling; MAPK pathway; organoids; cancer; cell signaling