KRAS4A Directly Regulates Hexokinase 1 Caroline R. Amendola 1,* , James P. Mahaffey 1,* , Seth J. Parker 1 , Ian M. Ahearn 1 , Wei-Ching Chen 2 , Mo Zhou 1 , Helen Court 1 , Jie Shi 1 , Sebastian L. Mendoza 1 , Michael Morten 1 , Eli Rothenberg 1 , Eyal Gottlieb 3 , Youssef Z. Wadghiri 1 , Richard Possemato 1 , Stevan R. Hubbard 1 , Allan Balmain 2 , Alec Kimmelman 1 , Mark R. Philips 1,§ 1 Perlmutter Cancer Center, NYU School of Medicine, New York, NY 2 Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco School of Medicine, San Francisco, CA 3 Technion Israel Institute of Technology, Haifa, Israel Abstract The most frequently mutated oncogene in cancer is KRAS, which utilizes alternative fourth exons to generate two gene products, KRAS4A and KRAS4B, that differ only in their C-terminal membrane-targeting region 1 . Because oncogenic mutations occur in exons 2 or 3, when KRAS is activated by mutation two constitutively active KRAS proteins are encoded, each capable of transforming cells 2 . No functional distinctions among the splice variants have been established. Oncogenic KRAS alters tumor metabolism 3 . Among these alterations is increased glucose uptake and glycolysis, even in the presence of abundant oxygen 4 (the Warburg Effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes 3–5 , direct regulation of metabolic enzymes has not been examined. We report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, establishing HK1 as an effector of KRAS4A. The interaction is unique to KRAS4A because the palmitoylation/depalmitoylation cycle of this RAS isoform permits co-localization with HK1 on the outer mitochondrial membrane (OMM). KRAS4A expression in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms § Correspondence to mark.philips@nyulangone.org. * These authors contributed equally to the work Author Contributions: C.R.A, J.P.M, and M.R.P. designed and interpreted all experiments and wrote the manuscript. Unless otherwise stipulated, J.P.M. and C.R.A. performed all experiments. W.C.H. generated the CRISPR/Cas9 engineered A549 and SUIT2 cells. M.Z. performed mitochondrial purifications. I.A. and H.C. performed the 2-DG growth inhibition studies. J.S. performed hexokinase activity assays. S.L.M. performed the PET CT studies. S.J.P. performed the Seahorse analysis and 13 C-glucose labeling. M.M. performed the super-resolution microscopy. E.G., A.K., Y.Z.W., R.P., S.R.H., E.R. and A.B. assisted with the interpretation of the results and edited the manuscript. Competing Interests: A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to KRAS regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as therapeutic approach, and the autophagic control of iron metabolism. A.C.K. is on the SAB of Rafael/Cornerstone Pharmaceuticals. Data Availability: The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. Supplemental Information: is linked to the online version of the paper at www.nature.com/nature. HHS Public Access Author manuscript Nature. Author manuscript; available in PMC 2020 June 11. Published in final edited form as: Nature. 2019 December ; 576(7787): 482–486. doi:10.1038/s41586-019-1832-9. Author Manuscript Author Manuscript Author Manuscript Author Manuscript