The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf protooncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal–regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)–GTPase–activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65–1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.
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Funding and additional information—This research was supported
Acknowledgments—We thank the patient and family for donating the patient’s remains for the advancement of cancer research, with the hope that this work will be of benefit to future patients. We also thank Dr. Christine Henzler for guidance regarding bioinformatic analysis, and Michael Franklin for assistance with editorial review of the manuscript. We thank members of the FNLCR Protein Expression Laboratory for help with cloning (Jennifer Mehalko, Vanessa Wall, Dom Esposito), protein expression (John-Paul Den-son, Jennifer Mehalko, Nitya Ramakrishnan, Bill Gillette), protein purification (John-Paul Denson, Peter Frank, Jose Sanchez Hernan-dez, Shelley Perkins), and QC (Matt Drew, Peter Frank). We also thank Dr. Simon Messing for the generous gift of PIK3CG(144-1102)-His6. We are thankful to Albert Chan, Timothy Tran, and Timothy Waybright for help with the structural and biochemical studies. Structural work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by NIGMS, National Institutes of Health Grant P30 GM124165. The Eiger 16M detector on the 24-ID-E beam line is funded by NIH-ORIP HEI Grant S10OD021527. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
This research was supported in part by contributions from the patient?s parents, and research funding from the Department of Laboratory Medicine and Pathology, University of Minnesota (to A. C. N.); Eastern Star Scholars, University of Minnesota Masonic Cancer Center (to A. C. N.); American Cancer Society Clinical Scientist Development Grant CSDG-18-139-01-CSM (to A. C. N.); the Mezin-Koats Colon Cancer Research Award (to E. L.); The Randy Shaver Cancer Research and Community Fund (to E. L.); the Litman Family Fund for Cancer Research; a Central Society for Clinical and Translational Research Early Career Development Award (to E. L.); Institutional Research Grant 118198-IRG-58-001-52-IRG94 from the American Cancer Society (to E. L.); Minnesota Masonic Charities; Minnesota Medical Foundation/University of Minnesota Foundation (to E. L.); the Masonic Cancer Center and Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota (to E. L.); and the National Institutes of Health Clinical and Translational Science KL2 Scholar Award 8UL1TR000114 (to E. L.). This project was funded in whole or in part with federal funds from the National Cancer Institute, NIH Contract HHSN261200800001E. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, and the mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government.
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PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't
- Research Support, U.S. Gov't, Non-P.H.S.