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Tissue-Specific Oncogenic Activity of KRASA146T

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Tissue-Specific Oncogenic Activity of KRASA146T Published OnlineFirst April 5, 2019; DOI: 10.1158/2159-8290.CD-18-1220 RESEARCH ARTICLE Tissue-Specifi c Oncogenic Activity A146T of KRAS Emily J. Poulin 1 , 2 , Asim K. Bera 3 , Jia Lu 3 , Yi-Jang Lin 1 , 2 , Samantha Dale Strasser 1 , 4 , 5 , Joao A. Paulo 6 , Tannie Q. Huang7 , Carolina Morales 7 , Wei Yan 3 , Joshua Cook 1 , 2 , Jonathan A. Nowak 8 , Douglas K. Brubaker 1 , 2 , 4 , Brian A. Joughin9 , Christian W. Johnson 1 , 2 , Rebecca A. DeStefanis 1 , 2 , Phaedra C. Ghazi 1 , 2 , Sudershan Gondi 3 , Thomas E. Wales10 , Roxana E. Iacob 10 , Lana Bogdanova 7 , Jessica J. Gierut 1 , 2 , Yina Li 1 , 2 , John R. Engen 10 , Pedro A. Perez-Mancera11 , Benjamin S. Braun 7 , Steven P. Gygi 6 , Douglas A. Lauffenburger 4 , Kenneth D. Westover3 , and Kevin M. Haigis 1 , 2 , 12 ABSTRACT KRAS is the most frequently mutated oncogene. The incidence of specifi cKRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specifi c mutant KRAS proteins. We used a cross- disciplinary approach to compare KRAS G12D , a common mutant form, and KRAS A146T , a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor– induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRAS G12D and KRAS A146T exhibit distinct tissue-specifi c effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specifi c phenotypes result from allele-specifi c signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specifi c behaviors for KRAS , experimental evidence for allele-specifi c biological properties is limited. We combined struc- tural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specifi c KRAS mutants in human cancers from different tissues is due to their distinct signaling properties. See related commentary by Hobbs and Der, p. 696. 1 Cancer Research Institute, Beth Israel Deaconess Medical Center, Boston, Liverpool, Liverpool, UK. 12 Harvard Digestive Disease Center, Harvard Massachusetts. 2 Department of Medicine, Harvard Medical School, Boston, Medical School, Boston, Massachusetts. 3 Massachusetts. Departments of Biochemistry and Radiation Oncology, Note: Supplementary data for this article are available at Cancer Discovery The University of Texas Southwestern Medical Center at Dallas, Dallas, Online (http://cancerdiscovery.aacrjournals.org/). Texas. 4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts. 5 Department of Electrical Engi- A.K. Bera and J. Lu contributed equally to this work. neering and Computer Science, Massachusetts Institute of Technology, Corresponding Authors: Kevin M. Haigis, Beth Israel Deaconess Medical Cambridge, Massachusetts. 6 Department of Cell Biology, Harvard Medi- Center, 3 Blackfan Circle, CLS 409, Boston, MA 02115. Phone: 617-735- cal School, Boston, Massachusetts. 7 Department of Pediatrics and Helen 2056; E-mail: [email protected] ; and Kenneth D. Westover, UT South- Diller Family Comprehensive Cancer Center, University of California, San western Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390. Francisco, California. 8 Department of Pathology, Brigham and Women’s Phone: 214-645-0323; E-mail: [email protected] 9 Hospital, Boston, Massachusetts. Koch Institute for Integrative Cancer Cancer Discov 2019;9:738–55 Research, Massachusetts Institute of Technology, Cambridge, Massachu- setts. 10 Department of Chemistry and Chemical Biology, Northeastern doi: 10.1158/2159-8290.CD-18-1220 University, Boston, Massachusetts. 11 Department of Molecular and Clini- © 2019 American Association for Cancer Research. cal Cancer Medicine, Institute of Translational Medicine, University of 738 | CANCER DISCOVERY JUNE 2019 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst April 5, 2019; DOI: 10.1158/2159-8290.CD-18-1220 at glycine 12, and these impair GAP-induced hydrolysis (2). INTRODUCTION Studies of genetically engineered mouse models have revealed Effective implementation of precision medicine requires that expression of KRASG12D, the most common allele in a detailed understanding of the genetic, biochemical, bio- human cancers, is sufficient to disrupt homeostasis in a vari- logical, and clinical/epidemiologic properties of cancer genes ety of tissues (3–7). Some KRAS mutations are restricted to and their corresponding proteins. KRAS is the most fre- certain tumor types. For example, alanine 146 (A146) is the quently mutated oncogene in cancer. Its oncoprotein prod- fourth most common site of KRAS mutation across all cancer ucts, KRAS4A and KRAS4B, play an active role in tumor types, but A146 mutations are found almost exclusively in pathogenesis by engaging downstream signal transduction cancers of the intestinal tract and blood (8). Relative to other cascades to promote proliferation and survival. Efforts to mutant forms of KRAS, little is known about the biochemical target KRAS and its effector signaling pathways have largely and biological properties of KRAS mutated at A146. A146V, failed, making oncogenic KRAS a major barrier to precision the first mutant allele characterized, was originally identi- medicine for cancer. As a small GTPase, KRAS activity is mod- fied through random mutagenesis almost three decades ago ulated by its nucleotide binding state. Although wild-type and was determined to alter HRAS GTP binding affinity (WT) KRAS cycles between the GDP-bound (inactive) state and to increase the rate of intrinsic nucleotide exchange and the GTP-bound (active) state with the help of guanine (9). It was not until 2006 that A146 mutations (A146V nucleotide exchange factors (GEF) and GTPase activating and A146T) were identified as cancer-associated KRAS alleles proteins (GAP), somatic mutations found in cancer promote (10, 11). Although KRAS alleles are not typically differenti- the GTP-bound state (1). ated in clinical practice, epidemiologic studies suggest that KRAS can be activated by missense mutation at any of a cancers expressing different mutant forms of KRAS exhibit number of residues. Most cancer-associated mutations occur distinct clinical behaviors (1, 12). For example, in colorectal JUNE 2019 CANCER DISCOVERY | 739 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst April 5, 2019; DOI: 10.1158/2159-8290.CD-18-1220 RESEARCH ARTICLE Poulin et al. cancer, codon 146 mutations are associated with better over- We noted that SOS1 stimulated exchange more effectively for all survival relative to codon 12 mutations; however, like KRAS4BA146T than it did for KRAS4BWT or KRAS4BG12D, and codon 12 mutants, they promote resistance to anti–epidermal microscale thermophoresis (MST) demonstrated a roughly growth factor receptor (EGFR) therapy (11, 13, 14). Whether 100-fold increase in the affinity of SOS1 for KRAS4BA146T the biological properties of KRAS proteins activated through relative to the WT protein (Fig. 1C). Next, we measured the different biochemical mechanisms are similar or distinct, and rate of intrinsic and GAP-stimulated GTP hydrolysis. Intrinsic whether distinct clinical behaviors derive from distinct bio- rates were similar between KRAS4BA146T and KRAS4BG12D, but logical properties of KRAS alleles, is unresolved. In this study, reduced relative to KRAS4BWT (Fig. 1D), whereas GAP-stimu- we undertook a comprehensive, multifaceted approach to lated GTP hydrolysis was significantly impaired for KRASG12D, systematically analyze the similarities and differences between but only mildly impaired for KRAS4BA146T (Fig. 1E). We also mutant forms of KRAS in order to understand mechanisms measured the affinity of KRAS4B for its downstream effec- driving allelic selection in cancer. tor RAF by using an AlphaScreen-based assay (16). All pro- teins exhibited similar binding affinity, indicating that in its A146T RESULTS GTP-bound form, KRAS4B assumes a more typical closed conformation that is able to engage the RAS-binding domain A146T Promotes Nucleotide Exchange (RBD) of RAF (Supplementary Fig. S1). These data are con- Missense mutations activate RAS proteins by altering their sistent with a model in which KRAS4BA146T activation derives abilities to bind and/or hydrolyze GTP. To determine how the primarily from an increase in intrinsic and GEF-induced nucle- mutation of codon 146 affects the core biochemical properties otide exchange, not GAP insensitivity. of KRAS, we first measured GDP dissociation± SOS1, a RAS GEF (15). We found that KRAS4BA146T had a ∼12-fold higher Structural Basis of Rapid Nucleotide Exchange rate of intrinsic GDP dissociation than KRAS4BWT, which was To understand the molecular basis for the increased basal further increased by the addition of SOS1 (Fig. 1A and B). and GEF-stimulated exchange rates of KRASA146T, we solved K (µmol/L) A B ** C d WT WT + SOS1 WT 8.3 ± 0.6 A146T A146T + SOS1
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