TY - JOUR
T1 - Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids
AU - Wiechmann, Svenja
AU - Maisonneuve, Pierre
AU - Grebbin, Britta M.
AU - Hoffmeister, Meike
AU - Kaulich, Manuel
AU - Clevers, Hans
AU - Rajalingam, Krishnaraj
AU - Kurinov, Igor
AU - Farin, Henner F.
AU - Sicheri, Frank
AU - Ernst, Andreas
N1 - Funding Information:
Acknowledgments—We thank Mani Ravichandran from the Structural and Genomic Consortium (SGC) of Toronto for providing X-ray crystallography screening kits. Diffraction work conducted at the Northeastern Collaborative Access Team beamlines was funded by NIGMS, National Institutes of Health (NIH), Grant P41 GM103403 and by NIH-ORIP HEI Grant S10 RR029205. We thank Nancy R. Gough (BioSerendipity, LLC) for constructive comments and editorial assistance. We thank Jason Moffat (University of Toronto) for donation of inducible lentiviral plasmids. We thank Georg Tascher for help in analyzing MS data, Mateusz Putyrski for cloning lentiviral gateway constructs, and Suchithra Guntur and Andrew Vorobyov for assistance during the revision.
Funding Information:
This work was supported by LOEWE Ub-Net, Cluster of Excellence “Macromo-lecular Complexes” (DFG EXC115) and the Collaborative Research Center SFB 1177. This work was also supported by funds from Impact Grant 704116 (to F. S.) from the Canadian Cancer Society Research Institute and by operating fund Grant FDN143277 (to F. S.) from the Canadian Institutes for Health Research. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 Wiechmann et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/4/3
Y1 - 2020/4/3
N2 - 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. 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 all 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.
AB - 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. 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 all 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.
UR - http://www.scopus.com/inward/record.url?scp=85082964804&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA119.011025
DO - 10.1074/jbc.RA119.011025
M3 - Article
C2 - 32086379
AN - SCOPUS:85082964804
SN - 0021-9258
VL - 295
SP - 4526
EP - 4540
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -