[en] Specific reactive oxygen species activate the GTPase Kirsten rat sarcoma virus (KRAS) by reacting with cysteine 118 (C118), leading to an electron transfer between C118 and nucleoside guanosine diphosphate (GDP), which causes the release of GDP. Here, we have mimicked permanent oxidation of human KRAS at C118 by replacing C118 with aspartic acid (C118D) in KRAS to show that oncogenic mutant KRAS is selectively inhibited via oxidation at C118, both in vitro and in vivo. Moreover, the combined treatment of hydrogen-peroxide-producing pro-oxidant paraquat and nitric-oxide-producing inhibitor N(ω)-nitro-l-arginine methyl ester selectively inhibits human mutant KRAS activity by inducing oxidization at C118. Our study shows for the first time the vulnerability of human mutant KRAS to oxidation, thereby paving the way to explore oxidation-based anti-KRAS treatments in humans.
Disciplines :
Oncology
Author, co-author :
Kramer-Drauberg, Maximilian; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
Petrini, Ettore; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
Mira, Alessia; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
Patrucco, Enrico; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
Scardaci, Rossella; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
Savinelli, Ilenia; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
Wang, Haiyun; School of Life Sciences and Technology, Tongji University, Shanghai, China
Qiao, Keying; School of Life Sciences and Technology, Tongji University, Shanghai, China
Carrà, Giovanna ; Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy
Nokin, Marie-Julie ; Université de Liège - ULiège > GIGA > GIGA Cancer - Tumors Biology and Development
Zhou, Zhiwei; Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX, USA ; Department of Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, TX, USA
Westover, Kenneth D; Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX, USA ; Department of Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, TX, USA
Santamaria, David; Molecular Mechanisms of Cancer Program, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Spain
Porporato, Paolo E ; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
Ambrogio, Chiara ; Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy
H2020 - 101001288 - KARMA - From the understanding of KRAS-RAF membrane dynamics to new therapeutic strategies in cancer
Funders :
Fondazione Umberto Veronesi ERC - European Research Council Worldwide Cancer Research AIRC - Associazione Italiana per la Ricerca sul Cancro Giovanni Armenise-Harvard Foundation
Funding text :
We would like to thank Valeria Malacarne and Alessia Brossa for their technical assistance with the flow cytometry and Alessio Menga for his technical support. This work was funded by the Giovanni Armenise\u2013Harvard Foundation, the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. [101001288]) and AIRC under IG 2021 \u2013 ID. 25737 project (to CA). MK\u2010D was supported by Fondazione Umberto Veronesi. CA is supported by the Zanon di Valgiurata family through Justus s.s.We would like to thank Valeria Malacarne and Alessia Brossa for their technical assistance with the flow cytometry and Alessio Menga for his technical support. This work was funded by the Giovanni Armenise\u2013Harvard Foundation, the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. [101001288]) and AIRC under IG 2021 \u2013 ID. 25737 project (to CA). MK-D was supported by Fondazione Umberto Veronesi. CA is supported by the Zanon di Valgiurata family through Justus s.s. Open access publishing facilitated by Universita degli Studi di Torino, as part of the Wiley - CRUI-CARE agreement.
Cox AD, Fesik SW, Kimmelman AC, Luo J, Der CJ. Drugging the undruggable RAS: mission possible? Nat Rev Drug Discov. 2014;13(11):828–851.
Prior IA, Hood FE, Hartley JL. The frequency of Ras mutations in cancer. Cancer Res. 2020;80:2969–2974.
Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401–404.
AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discov. 2017;7(8):818–831.
Hofmann MH, Gerlach D, Misale S, Petronczki M, Kraut N. Expanding the reach of precision oncology by drugging all KRAS mutants. Cancer Discov. 2022;12:924–937.
Hofmann MH, Gmachl M, Ramharter J, Savarese F, Gerlach D, Marszalek JR, et al. BI-3406, a potent and selective SOS1-KRAS interaction inhibitor, is effective in KRAS-driven cancers through combined MEK inhibition. Cancer Discov. 2021;11(1):142–157.
Janes MR, Zhang J, Li LS, Hansen R, Peters U, Guo X, et al. Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor. Cell. 2018;172(3):578–589.e17.
Wang X, Allen S, Blake JF, Bowcut V, Briere DM, Calinisan A, et al. Identification of MRTX1133, a noncovalent, potent, and selective KRAS(G12D) inhibitor. J Med Chem. 2022;65(4):3123–3133.
Holderfield M, Lee BJ, Jiang J, Tomlinson A, Seamon KJ, Mira A, et al. Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy. Nature. 2024;629:919–926.
Liu J, Kang R, Tang D. The KRAS-G12C inhibitor: activity and resistance. Cancer Gene Ther. 2022;29(7):875–878.
Awad MM, Liu S, Rybkin II, Arbour KC, Dilly J, Zhu VW, et al. Acquired resistance to KRAS(G12C) inhibition in cancer. N Engl J Med. 2021;384(25):2382–2393.
Negrao MV, Araujo HA, Lamberti G, Cooper AJ, Akhave NS, Zhou T, et al. Comutations and KRASG12C inhibitor efficacy in advanced NSCLC. Cancer Discov. 2023;13(7):1556–1571.
Chio IIC, Tuveson DA. ROS in cancer: the burning question. Trends Mol Med. 2017;23(5):411–429.
Sies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21:363–383.
Burska AN, Ilyassova B, Dildabek A, Khamijan M, Begimbetova D, Molnár F, et al. Enhancing an oxidative “trojan horse” action of vitamin C with arsenic trioxide for effective suppression of KRAS-mutant cancers: a promising path at the bedside. Cells. 2022;11(21):3454.
Weinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez M, et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci USA. 2010;107(19):8788–8793.
Kramer-Drauberg M, Ambrogio C. Discoveries in the redox regulation of KRAS. Int J Biochem Cell Biol. 2020;131:105901.
Heo J, Campbell SL. Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange. Biochemistry. 2004;43(8):2314–2322.
Raines KW, Bonini MG, Campbell SL. Nitric oxide cell signaling: S-nitrosation of Ras superfamily GTPases. Cardiovasc Res. 2007;75(2):229–239.
Foo CHJ, Pervaiz S. gRASping the redox lever to modulate cancer cell fate signaling. Redox Biol. 2019;25:101094.
Lander HM, Hajjar DP, Hempstead BL, Mirza UA, Chait BT, Campbell S, et al. A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction. J Biol Chem. 1997;272(7):4323–4326.
Hobbs GA, Bonini MG, Gunawardena HP, Chen X, Campbell SL. Glutathiolated Ras: characterization and implications for Ras activation. Free Radic Biol Med. 2013;57:221–229.
Huang L, Counter CM. Reduced HRAS G12V-driven tumorigenesis of cell lines expressing KRAS C118S. PLoS One. 2015;10(4):e0123918.
Lim KH, Ancrile BB, Kashatus DF, Counter CM. Tumour maintenance is mediated by eNOS. Nature. 2008;452(7187):646–649.
Heo J, Campbell SL. Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide. J Biol Chem. 2005;280(13):12438–12445.
Heo J, Campbell SL. Ras regulation by reactive oxygen and nitrogen species. Biochemistry. 2006;45(7):2200–2210.
Raines KW, Cao GL, Lee EK, Rosen GM, Shapiro P. Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity. Biochem J. 2006;397(2):329–336.
Adachi T, Pimentel DR, Heibeck T, Hou X, Lee YJ, Jiang B, et al. S-glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells. J Biol Chem. 2004;279(28):29857–29862.
Williams JG, Pappu K, Campbell SL. Structural and biochemical studies of p21Ras S-nitrosylation and nitric oxide-mediated guanine nucleotide exchange. Proc Natl Acad Sci USA. 2003;100(11):6376–6381.
Mott HR, Carpenter JW, Campbell SL. Structural and functional analysis of a mutant Ras protein that is insensitive to nitric oxide activation. Biochemistry. 1997;36(12):3640–3644.
Clavreul N, Adachi T, Pimental DR, Ido Y, Schöneich C, Cohen RA. S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells. FASEB J. 2006;20(3):518–520.
Huynh MV, Parsonage D, Forshaw TE, Chirasani VR, Hobbs GA, Wu H, et al. Oncogenic KRAS G12C: kinetic and redox characterization of covalent inhibition. J Biol Chem. 2022;298(8):102186.
Huang L, Carney J, Cardona DM, Counter CM. Decreased tumorigenesis in mice with a Kras point mutation at C118. Nat Commun. 2014;5:5410.
Kramer-Drauberg M, Liu JL, Desjardins D, Wang Y, Branicky R, Hekimi S. ROS regulation of RAS and vulva development in Caenorhabditis elegans. PLoS Genet. 2020;16(6):e1008838.
Drosten M, Dhawahir A, Sum EYM, Urosevic J, Lechuga CG, Esteban LM, et al. Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival. EMBO J. 2010;29(6):1091–1104.
Ambrogio C, Carmona FJ, Vidal A, Falcone M, Nieto P, Romero OA, et al. Modeling lung cancer evolution and preclinical response by orthotopic mouse allografts. Cancer Res. 2014;74(21):5978–5988.
Ambrogio C, Köhler J, Zhou ZW, Wang H, Paranal R, Li J, et al. KRAS dimerization impacts MEK inhibitor sensitivity and oncogenic activity of mutant KRAS. Cell. 2018;172(4):857–868.e15.
Li S, Liu S, Deng J, Akbay EA, Hai J, Ambrogio C, et al. Assessing therapeutic efficacy of MEK inhibition in a KRAS(G12C)-driven mouse model of lung cancer. Clin Cancer Res. 2018;24(19):4854–4864.
Baltanas FC, Kramer-Drauberg M, Garcia-Navas R, Patrucco E, Petrini E, Arnhof H, et al. Pharmacological SOS1 inhibitor BI-3406 demonstrates in vivo anti-tumor activity comparable to SOS1 genetic ablation in KRAS mutant tumors. bioRxiv. 2024. https://doi.org/10.1101/2024.09.18.613686
Nokin MJ, Mira A, Patrucco E, Ricciuti B, Cousin S, Soubeyran I, et al. RAS-ON inhibition overcomes clinical resistance to KRAS G12C-OFF covalent blockade. Nat Commun. 2024;15(1):7554.
Reczek CR, Birsoy K, Kong H, Martínez-Reyes I, Wang T, Gao P, et al. A CRISPR screen identifies a pathway required for paraquat-induced cell death. Nat Chem Biol. 2017;13(12):1274–1279.
Sayin VI, Ibrahim MX, Larsson E, Nilsson JA, Lindahl P, Bergo MO. Antioxidants accelerate lung cancer progression in mice. Sci Transl Med. 2014;6(221):221ra15.
Bera AK, Lu J, Lu C, Li L, Gondi S, Yan W, et al. GTP hydrolysis is modulated by Arg34 in the RASopathy-associated KRAS(P34R). Birth Defects Res. 2020;112(10):708–717.
Bera AK, Lu J, Wales TE, Gondi S, Gurbani D, Nelson A, et al. Structural basis of the atypical activation mechanism of KRAS(V14I). J Biol Chem. 2019;294(38):13964–13972.
Poulin EJ, Bera AK, Lu J, Lin YJ, Strasser SD, Paulo JA, et al. Tissue-specific oncogenic activity of KRAS(A146T). Cancer Discov. 2019;9(6):738–755.
Hunter JC, Manandhar A, Carrasco MA, Gurbani D, Gondi S, Westover KD. Biochemical and structural analysis of common cancer-associated KRAS mutations. Mol Cancer Res. 2015;13(9):1325–1335.
Paulsen CE, Carroll KS. Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery. Chem Rev. 2013;113(7):4633–4679.
Russell EG, Cotter TG. New insight into the role of reactive oxygen species (ROS) in cellular signal-transduction processes. Int Rev Cell Mol Biol. 2015;319:221–254.
Permyakov SE, Zernii EY, Knyazeva EL, Denesyuk AI, Nazipova AA, Kolpakova TV, et al. Oxidation mimicking substitution of conservative cysteine in recoverin suppresses its membrane association. Amino Acids. 2012;42(4):1435–1442.
Chung HS, Wang SB, Venkatraman V, Murray CI, van Eyk JE. Cysteine oxidative posttranslational modifications: emerging regulation in the cardiovascular system. Circ Res. 2013;112(2):382–392.
Paix A, Folkmann A, Rasoloson D, Seydoux G. High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics. 2015;201(1):47–54.
Messina S, De Simone G, Ascenzi P. Cysteine-based regulation of redox-sensitive Ras small GTPases. Redox Biol. 2019;26:101282.
Pershing NL, Yang CFJ, Xu M, Counter CM. Treatment with the nitric oxide synthase inhibitor L-NAME provides a survival advantage in a mouse model of Kras mutation-positive, non-small cell lung cancer. Oncotarget. 2016;7(27):42385–42392.
Krall J, Bagley AC, Mullenbach GT, Hallewell RA, Lynch RE. Superoxide mediates the toxicity of paraquat for cultured mammalian cells. J Biol Chem. 1988;263(4):1910–1914.
Fridovich I. Superoxide anion radical (O2-.), superoxide dismutases, and related matters. J Biol Chem. 1997;272(30):18515–18517.
Zhao C, Sethuraman M, Clavreul N, Kaur P, Cohen RA, O'Connor PB. Detailed map of oxidative post-translational modifications of human p21ras using Fourier transform mass spectrometry. Anal Chem. 2006;78(14):5134–5142.
Murphy E, Kohr M, Sun J, Nguyen T, Steenbergen C. S-nitrosylation: a radical way to protect the heart. J Mol Cell Cardiol. 2012;52(3):568–577.
Winter JJ, Anderson M, Blades K, Brassington C, Breeze AL, Chresta C, et al. Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation. J Med Chem. 2015;58(5):2265–2274.
