Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant KRAS, selective isolation and analysis of transforming or tumor cells that produce the KRAS oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant KRASG12D that enables the "activation" of KRASG12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated KrasRR) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the KrasRR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (APC). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that KRASG12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRASG12D function is effectively suppressed by specific inhibitor MRTX1133.

Institute of Pathology 1St Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

Laboratory of Cell and Developmental Biology Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

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bioRxiv. 2024 Jul 26:2024.07.26.605291. doi: 10.1101/2024.07.26.605291. PubMed

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Amalfitano A (2004) Utilization of adenovirus vectors for multiple gene transfer applications. Methods 33:173–178. 10.1016/j.ymeth.2003.11.006 PubMed DOI

Bahar ME, Kim HJ, Kim DR (2023) Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies. Signal Transduct Target Ther 8:455. 10.1038/s41392-023-01705-z PubMed DOI PMC

Bahrami A et al (2018) Targeting RAS signaling pathway as a potential therapeutic target in the treatment of colorectal cancer. J Cell Physiol 233:2058–2066. 10.1002/jcp.25890 PubMed DOI

Buchholz F, Angrand PO, Stewart AF (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16:657–662. 10.1038/nbt0798-657 PubMed DOI

Calcagno SR, Li S, Colon M, Kreinest PA, Thompson EA, Fields AP, Murray NR (2008) Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon. Int J Cancer 122:2462–2470. 10.1002/ijc.23383 PubMed DOI PMC

Cancer Genome Atlas N (2012) Comprehensive molecular characterization of human colon and rectal cancer Nature 487:330–337. 10.1038/nature11252 PubMed PMC

Dunne PD, Arends MJ (2024) Molecular pathological classification of colorectal cancer-an update. Virchows Arch 484:273–285. 10.1007/s00428-024-03746-3 PubMed DOI PMC

DuPage M, Dooley AL, Jacks T (2009) Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 4:1064–1072. 10.1038/nprot.2009.95 PubMed DOI PMC

Fakih MG et al. (2022) Sotorasib for previously treated colorectal cancers with KRAS(G12C) mutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial Lancet Oncol 23:115–124. 10.1016/S1470-2045(21)00605-7 PubMed

Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P (1996) Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci USA 93:10887–10890. 10.1073/pnas.93.20.10887 PubMed DOI PMC

Ferrer I, Zugazagoitia J, Herbertz S, John W, Paz-Ares L, Schmid-Bindert G (2018) KRAS-mutant non-small cell lung cancer: from biology to therapy. Lung Cancer 124:53–64. 10.1016/j.lungcan.2018.07.013 PubMed DOI

Haigis KM (2017) KRAS alleles: the devil is in the detail. Trends Cancer 3:686–697. 10.1016/j.trecan.2017.08.006 PubMed DOI PMC

Haigis KM et al (2008) Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nat Genet 40:600–608. 10.1038/ng.115 PubMed DOI PMC

Hallin J et al (2022) Anti-tumor efficacy of a potent and selective non-covalent KRAS(G12D) inhibitor. Nat Med 28:2171–2182. 10.1038/s41591-022-02007-7 PubMed DOI

Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674. 10.1016/j.cell.2011.02.013 PubMed DOI

Hobbs GA, Der CJ, Rossman KL (2016) RAS isoforms and mutations in cancer at a glance. J Cell Sci 129:1287–1292. 10.1242/jcs.182873 PubMed DOI PMC

Hrckulak D et al (2018) Wnt effector TCF4 is dispensable for wnt signaling in human cancer cells. Genes (Basel). 10.3390/genes9090439 PubMed DOI PMC

Jackson EL et al (2001) Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15:3243–3248. 10.1101/gad.943001 PubMed DOI PMC

Janeckova L et al (2015) HIC1 tumor suppressor loss potentiates TLR2/NF-kappaB signaling and promotes tissue damage-associated tumorigenesis. Mol Cancer Res 13:1139–1148. 10.1158/1541-7786.MCR-15-0033 PubMed DOI

Kasparek P, Krausova M, Haneckova R, Kriz V, Zbodakova O, Korinek V, Sedlacek R (2014) Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases. FEBS Lett 588:3982–3988. 10.1016/j.febslet.2014.09.014 PubMed DOI

Kim D et al (2023) Pan-KRAS inhibitor disables oncogenic signalling and tumour growth. Nature 619:160–166. 10.1038/s41586-023-06123-3 PubMed DOI PMC

Kris MG et al (2003) Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 290:2149–2158. 10.1001/jama.290.16.2149 PubMed DOI

Kristianto J, Johnson MG, Zastrow RK, Radcliff AB, Blank RD (2017) Spontaneous recombinase activity of Cre-ERT2 in vivo. Transgenic Res 26:411–417. 10.1007/s11248-017-0018-1 PubMed DOI PMC

Kuraguchi M et al (2006) Adenomatous polyposis coli (APC) is required for normal development of skin and thymus. PLoS Genet 2:e146. 10.1371/journal.pgen.0020146 PubMed DOI PMC

Ledford H (2015) Cancer: the ras renaissance. Nature 520:278–280. 10.1038/520278a PubMed DOI

Lenart S et al (2022) TACSTD2 upregulation is an early reaction to lung infection. Sci Rep 12:9583. 10.1038/s41598-022-13637-9 PubMed DOI PMC

Lievre A et al (2006) KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 66:3992–3995. 10.1158/0008-5472.CAN-06-0191 PubMed DOI

Madisen L et al (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13:133–140. 10.1038/nn.2467 PubMed DOI PMC

Meinke G, Bohm A, Hauber J, Pisabarro MT, Buchholz F (2016) Cre recombinase and other tyrosine recombinases. Chem Rev 116:12785–12820. 10.1021/acs.chemrev.6b00077 PubMed DOI

Ng K et al (2013) Phase II study of everolimus in patients with metastatic colorectal adenocarcinoma previously treated with bevacizumab-, fluoropyrimidine-, oxaliplatin-, and irinotecan-based regimens. J Clin Cancer Res 19:3987–3995. 10.1158/1078-0432.CCR-13-0027 PubMed DOI PMC

Nuevo-Tapioles C, Philips MR (2022) The role of KRAS splice variants in cancer biology. Front Cell Dev Biol 10:1033348. 10.3389/fcell.2022.1033348 PubMed DOI PMC

O’Gorman S, Fox DT, Wahl GM (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251:1351–1355. 10.1126/science.1900642 PubMed DOI

Ostrem JM, Peters U, Sos ML, Wells JA, Shokat KM (2013) K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503:548–551. 10.1038/nature12796 PubMed DOI PMC

Pospichalova V et al (2011) Generation of two modified mouse alleles of the Hic1 tumor suppressor gene. Genesis 49:142–151. 10.1002/dvg.20719 PubMed DOI

Provost E, Rhee J, Leach SD (2007) Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos. Genesis 45:625–629. 10.1002/dvg.20338 PubMed DOI

Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D (2011) RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 11:761–774. 10.1038/nrc3106 PubMed DOI PMC

Raymond CS, Soriano P (2007) High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS ONE 2:e162. 10.1371/journal.pone.0000162 PubMed DOI PMC

Rodriguez CI et al (2000) High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat Genet 25:139–140. 10.1038/75973 PubMed DOI

Ryan DP, Hong TS, Bardeesy N (2014) Pancreatic adenocarcinoma. N Engl J Med 371:1039–1049. 10.1056/NEJMra1404198 PubMed DOI

Sansom OJ et al (2006) Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo. Proc Natl Acad Sci U S A 103:14122–14127. 10.1073/pnas.0604130103 PubMed DOI PMC

Sato T et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265. 10.1038/nature07935 PubMed DOI

Sato T et al (2011) Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469:415–418. 10.1038/nature09637 PubMed DOI PMC

Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85:5166–5170. 10.1073/pnas.85.14.5166 PubMed DOI PMC

Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. 10.1038/nmeth.2019 PubMed DOI PMC

Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909. 10.1038/nmeth819 PubMed DOI

Shaw AT et al (2007) Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis. Genes Dev 21:694–707. 10.1101/gad.1526207 PubMed DOI PMC

Snippert HJ, Schepers AG, van Es JH, Simons BD, Clevers H (2014) Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion. EMBO Rep 15:62–69. 10.1002/embr.201337799 PubMed DOI PMC

Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR (2015) Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10:1709–1727. 10.1038/nprot.2015.085 PubMed DOI

Tuveson DA et al (2004) Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell 5:375–387. 10.1016/s1535-6108(04)00085-6 PubMed DOI

Yaeger R et al (2023) Adagrasib with or without cetuximab in colorectal cancer with mutated KRAS G12C. N Engl J Med 388:44–54. 10.1056/NEJMoa2212419 PubMed DOI PMC

Yang Y, Zhang H, Huang S, Chu Q (2023) KRAS mutations in solid tumors: characteristics, current therapeutic strategy, and potential treatment exploration. J Clin Med. 10.3390/jcm12020709 PubMed DOI PMC

Young NP, Crowley D, Jacks T (2011) Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis. Cancer Res 71:4040–4047. 10.1158/0008-5472.CAN-10-4563 PubMed DOI PMC