Abstract
Molecular targeted therapies targeting KRAS signaling have significantly improved patient outcomes, but they have not achieved sufficient therapeutic efficacy in colorectal cancer (CRC). Here, we demonstrate that a subset of KRAS-mutant CRC cells transitions to a cellular state characterized by enhanced ribosome biogenesis upon KRAS signaling inhibition. The mitogen-activated protein kinase kinase inhibitor, trametinib, and AMG510 induce a cellular state characterized by a gene expression profile highly enriched for ribosome biogenesis. We find that they are vulnerable to the inhibition of RNA polymerase I, and they exhibit synergistic anti-tumor effects with trametinib in an autochthonous mouse model of intestinal tumors and human patient-derived organoids (PDOs). These observations demonstrate that high ribosome biogenesis induced by KRAS inhibition is indispensable to maintain this cellular state and is a potential therapeutic target. Overall, this study reveals novel mechanisms of drug tolerance to KRAS inhibition, thereby facilitating the development of new therapeutic strategies.
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Data availability
The raw RNA-seq data generated in this study have been deposited in the European Nucleotide Archive (ENA) database under accession code E-MTAB-16177 [sample entry: https://www.ebi.ac.uk/ena/browser/view/SAMEA120567684?show=reads] and E-MTAB-16244 [project entry: https://www.ebi.ac.uk/ena/browser/view/ERP185461]. The processed data from the scRNA-seq are available in the Gene Expression Omnibus (GEO) database under accession code GSE264485. The raw mass spectrometry (MS) data generated in this study have been deposited in the ProteomeXchange Consortium (https://www.proteomexchange.org/) via the Jpost partner repository under accession ID PXD052914/JPST003164 [https://repository.jpostdb.org/entry/JPST003164.0]. The raw exome-seq data are protected and are not available due to data privacy laws, and the processed data generated in this study are provided in the Supplementary Information file. Source data are provided with this paper. The data from this study are available from the corresponding author upon reasonable request.
References
De Roock, W. et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 11, 753–762 (2010).
Poulikakos, P. I. & Solit, D. B. Resistance to MEK inhibitors: should we co-target upstream? Sci. Signal 4, pe16 (2011).
Caunt, C. J., Sale, M. J., Smith, P. D. & Cook, S. J. MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road. Nat. Rev. Cancer 15, 577–592 (2015).
Kim, D., Xue, J. Y. & Lito, P. Targeting KRAS(G12C): from inhibitory mechanism to modulation of antitumor effects in patients. Cell 183, 850–859 (2020).
Kim, D. et al. Pan-KRAS inhibitor disables oncogenic signalling and tumour growth. Nature 619, 160–166 (2023).
Konieczkowski, D. J., Johannessen, C. M. & Garraway, L. A. A convergence-based framework for cancer drug resistance. Cancer Cell 33, 801–815 (2018).
Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010).
Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010).
Manchado, E. et al. A combinatorial strategy for treating KRAS-mutant lung cancer. Nature 534, 647–651 (2016).
Fakih, M. G. et al. 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 (2022).
Beck, B. & Blanpain, C. Unravelling cancer stem cell potential. Nat. Rev. Cancer 13, 727–738 (2013).
Valent, P. et al. Cancer stem cell definitions and terminology: the devil is in the details. Nat. Rev. Cancer 12, 767–775 (2012).
Phi, L. T. H. et al. Cancer stem cells (CSCs) in drug resistance and their therapeutic implications in cancer treatment. Stem Cells Int. 2018, 5416923 (2018).
Aguirre-Ghiso, J. A. Models, mechanisms and clinical evidence for cancer dormancy. Nat. Rev. Cancer 7, 834–846 (2007).
Wiecek, A. J. et al. Genomic hallmarks and therapeutic implications of G0 cell cycle arrest in cancer. Genome Biol. 24, 128 (2023).
De Conti, G., Dias, M. H. & Bernards, R. Fighting drug resistance through the targeting of drug-tolerant persister cells. Cancers 13, https://doi.org/10.3390/cancers13051118 (2021).
Saba, J. A., Liakath-Ali, K., Green, R. & Watt, F. M. Translational control of stem cell function. Nat. Rev. Mol. Cell Biol. 22, 671–690 (2021).
Morral, C. et al. Zonation of ribosomal DNA transcription defines a stem cell hierarchy in colorectal cancer. Cell Stem Cell 26, 845–861 (2020).
Pu, Y. et al. Drug-tolerant persister cells in cancer: the cutting edges and future directions. Nat. Rev. Clin. Oncol. 20, 799–813 (2023).
Dhanyamraju, P. K., Schell, T. D., Amin, S. & Robertson, G. P. Drug-tolerant persister cells in cancer therapy resistance. Cancer Res. 82, 2503–2514 (2022).
Sakahara, M. et al. IFN/STAT signaling controls tumorigenesis and the drug response in colorectal cancer. Cancer Sci. 110, 1293–1305 (2019).
Chen, B. et al. Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps. Cell 184, 6262–6280 (2021).
Tabula Sapiens, C. et al. The Tabula Sapiens: a multiple-organ, single-cell transcriptomic atlas of humans. Science 376, eabl4896 (2022).
Lempiainen, H. & Shore, D. Growth control and ribosome biogenesis. Curr. Opin. Cell Biol. 21, 855–863 (2009).
Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009).
Zaidi, S. K. et al. Expression of ribosomal RNA and protein genes in human embryonic stem cells is associated with the activating H3K4me3 histone mark. J. Cell. Physiol. 231, 2007–2013 (2016).
Weinberger, L., Ayyash, M., Novershtern, N. & Hanna, J. H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155–169 (2016).
Atlasi, Y. et al. The translational landscape of ground state pluripotency. Nat. Commun. 11, 1617 (2020).
Boroviak, T. et al. Lineage-specific profiling delineates the emergence and progression of naive pluripotency in mammalian embryogenesis. Dev. Cell 35, 366–382 (2015).
Li, D. & Wang, J. Ribosome heterogeneity in stem cells and development. J. Cell Biol. 219, https://doi.org/10.1083/jcb.202001108 (2020).
Corsini, N. S. et al. Coordinated control of mRNA and rRNA processing controls embryonic stem cell pluripotency and differentiation. Cell Stem Cell 22, 543–558 e512 (2018).
Richards, M., Tan, S. P., Tan, J. H., Chan, W. K. & Bongso, A. The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22, 51–64 (2004).
Sampath, P. et al. A hierarchical network controls protein translation during murine embryonic stem cell self-renewal and differentiation. Cell Stem Cell 2, 448–460 (2008).
Watanabe-Susaki, K. et al. Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells. Stem Cells 32, 3099–3111 (2014).
Morral, C., Stanisavljevic, J. & Batlle, E. Protocol for efficient protein synthesis detection by click chemistry in colorectal cancer patient-derived organoids grown in vitro. STAR Protoc. 1, 100103 (2020).
Bulut-Karslioglu, A. et al. Inhibition of mTOR induces a paused pluripotent state. Nature 540, 119–123 (2016).
Abe, Y. et al. Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids. Sci. Rep. 8, 11401 (2018).
Tahir, R. et al. Mutation-specific and common phosphotyrosine signatures of KRAS G12D and G13D alleles. J. Proteome Res. 20, 670–683 (2021).
Hammond, D. E. et al. Differential reprogramming of isogenic colorectal cancer cells by distinct activating KRAS mutations. J. Proteome Res. 14, 1535–1546 (2015).
Lackner, A. et al. Cooperative genetic networks drive embryonic stem cell transition from naive to formative pluripotency. EMBO J. 40, e105776 (2021).
Pieters, T. & van Roy, F. Role of cell-cell adhesion complexes in embryonic stem cell biology. J. Cell Sci. 127, 2603–2613 (2014).
Huai, Y. et al. How do RNA binding proteins trigger liquid-liquid phase separation in human health and diseases? Biosci. Trends 16, 389–404 (2022).
Okuwaki, M. et al. The stability of NPM1 oligomers regulated by acidic disordered regions controls the quality of liquid droplets. J. Biochem. 174, 461–476 (2023).
Krug, K. et al. A curated resource for phosphosite-specific signature analysis. Mol. Cell. Proteom. 18, 576–593 (2019).
Lu, W. C. et al. AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation. FEBS J. 289, 3876–3893 (2022).
Chou, S. D., Prince, T., Gong, J. & Calderwood, S. K. mTOR is essential for the proteotoxic stress response, HSF1 activation and heat shock protein synthesis. PLoS ONE 7, e39679 (2012).
Yang, L. et al. CCL2 regulation of MST1-mTOR-STAT1 signaling axis controls BCR signaling and B-cell differentiation. Cell Death Differ 28, 2616–2633 (2021).
Fielhaber, J. A. et al. Inactivation of mammalian target of rapamycin increases STAT1 nuclear content and transcriptional activity in alpha4- and protein phosphatase 2A-dependent fashion. J. Biol. Chem. 284, 24341–24353 (2009).
Chou, T. C. Derivation and properties of Michaelis-Menten type and Hill type equations for reference ligands. J. Theor. Biol. 59, 253–276 (1976).
Chou, T. C. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 70, 440–446 (2010).
Asghar, U., Witkiewicz, A. K., Turner, N. C. & Knudsen, E. S. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat. Rev. Drug Discov. 14, 130–146 (2015).
Parry, T. J. et al. The TCT motif, a key component of an RNA polymerase II transcription system for the translational machinery. Genes Dev. 24, 2013–2018 (2010).
Li, Y. et al. RNA pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits. Mol. Cell 83, 1280–1297 (2023).
Hannan, K. M., Hannan, R. D. & Rothblum, L. I. Transcription by RNA polymerase I. Front. Biosci. 3, d376–398 (1998).
Khot, A. et al. First-in-human RNA polymerase I transcription inhibitor CX-5461 in patients with advanced hematologic cancers: results of a phase I dose-escalation study. Cancer Discov. 9, 1036–1049 (2019).
Rodgers, M. L. & Woodson, S. A. Transcription increases the cooperativity of ribonucleoprotein assembly. Cell 179, 1370–1381.
Duss, O., Stepanyuk, G. A., Puglisi, J. D. & Williamson, J. R. Transient protein-RNA interactions guide nascent ribosomal RNA folding. Cell 179, 1357–1369 (2019).
Calo, E. et al. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature 518, 249–253 (2015).
Bohnsack, K. E., Yi, S., Venus, S., Jankowsky, E. & Bohnsack, M. T. Cellular functions of eukaryotic RNA helicases and their links to human diseases. Nat. Rev. Mol. Cell Biol. 24, 749–769 (2023).
Kusnadi, E. P. et al. Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis. EMBO J. 39, e105111 (2020).
Otto, C. et al. RNA polymerase I inhibition induces terminal differentiation, growth arrest, and vulnerability to senolytics in colorectal cancer cells. Mol. Oncol. 16, 2788–2809 (2022).
Nair, A. B. & Jacob, S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm. 7, 27–31 (2016).
Hilton, J. et al. Results of the phase I CCTG IND.231 trial of CX-5461 in patients with advanced solid tumors enriched for DNA-repair deficiencies. Nat. Commun. 13, 3607 (2022).
Hong, D. S. et al. KRAS(G12C) inhibition with sotorasib in advanced solid tumors. N. Engl. J. Med. 383, 1207–1217 (2020).
Cartwright, P. et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 132, 885–896 (2005).
van Riggelen, J., Yetil, A. & Felsher, D. W. MYC as a regulator of ribosome biogenesis and protein synthesis. Nat. Rev. Cancer 10, 301–309 (2010).
Sullivan, D. K. et al. MYC oncogene elicits tumorigenesis associated with embryonic, ribosomal biogenesis, and tissue-lineage dedifferentiation gene expression changes. Oncogene 41, 4960–4970 (2022).
Fuentealba, L. C. et al. Embryonic origin of postnatal neural stem cells. Cell 161, 1644–1655 (2015).
Blanco, S. et al. Stem cell function and stress response are controlled by protein synthesis. Nature 534, 335–340 (2016).
Signer, R. A., Magee, J. A., Salic, A. & Morrison, S. J. Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509, 49–54 (2014).
Baser, A. et al. Onset of differentiation is post-transcriptionally controlled in adult neural stem cells. Nature 566, 100–104 (2019).
Sanchez, C. G. et al. Regulation of ribosome biogenesis and protein synthesis controls germline stem cell differentiation. Cell Stem Cell 18, 276–290 (2016).
Zhang, Q., Shalaby, N. A. & Buszczak, M. Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage. Science 343, 298–301 (2014).
Figueiredo, V. C. & McCarthy, J. J. Targeting cancer via ribosome biogenesis: the cachexia perspective. Cell. Mol. Life Sci. 78, 5775–5787 (2021).
Pellegrino, S., Terrosu, S., Yusupova, G. & Yusupov, M. Inhibition of the eukaryotic 80S ribosome as a potential anticancer therapy: a structural perspective. Cancers 13, https://doi.org/10.3390/cancers13174392 (2021).
Elhamamsy, A. R., Metge, B. J., Alsheikh, H. A., Shevde, L. A. & Samant, R. S. Ribosome biogenesis: a central player in cancer metastasis and therapeutic resistance. Cancer Res. 82, 2344–2353 (2022).
Xu, Y. & Ruggero, D. The role of translation control in tumorigenesis and its therapeutic implications. Annu. Rev. Cancer Biol. 4, 437–457 (2020).
Lee, H. C. et al. RNA polymerase I inhibition with CX-5461 as a novel therapeutic strategy to target MYC in multiple myeloma. Br. J. Haematol. 177, 80–94 (2017).
Devlin, J. R. et al. Combination therapy targeting ribosome biogenesis and mRNA translation synergistically extends survival in MYC-driven lymphoma. Cancer Discov. 6, 59–70 (2016).
Bywater, M. J. et al. Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53. Cancer Cell 22, 51–65 (2012).
Tsoi, H., You, C. P., Leung, M. H., Man, E. P. S. & Khoo, U. S. Targeting ribosome biogenesis to combat tamoxifen resistance in ER+ve breast cancer. Cancers 14, https://doi.org/10.3390/cancers14051251 (2022).
Taylor, J. S. et al. Down-regulation of MYCN protein by CX-5461 leads to neuroblastoma tumor growth suppression. J. Pediatr. Surg. 54, 1192–1197 (2019).
Wang, S. et al. ZNF545 loss promotes ribosome biogenesis and protein translation to initiate colorectal tumorigenesis in mice. Oncogene https://doi.org/10.1038/s41388-021-01938-8 (2021).
Xu, H. et al. CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours. Nat. Commun. 8, 14432 (2017).
Pan, M. et al. The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma. Nat. Commun. 12, 6468 (2021).
Bruno, P. M. et al. The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning. Proc. Natl. Acad. Sci. USA 117, 4053–4060 (2020).
Koh, G. C. C. et al. The chemotherapeutic drug CX-5461 is a potent mutagen in cultured human cells. Nat. Genet. 56, 23–26 (2024).
De Roock, W. et al. Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 304, 1812–1820 (2010).
Hofmann, M. H., Gerlach, D., Misale, S., Petronczki, M. & Kraut, N. Expanding the reach of precision oncology by drugging all KRAS mutants. Cancer Discov. 12, 924–937 (2022).
Neumann, J., Zeindl-Eberhart, E., Kirchner, T. & Jung, A. Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathol. Res. Pract. 205, 858–862 (2009).
Holderfield, M. et al. Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy. Nature https://doi.org/10.1038/s41586-024-07205-6 (2024).
Jiang, J. et al. Translational and therapeutic evaluation of RAS-GTP inhibition by RMC-6236 in RAS-driven cancers. Cancer Discov. 14, 994–1017 (2024).
Okamoto, T. et al. Comparative analysis of patient-matched PDOs revealed a reduction in OLFM4-associated clusters in metastatic lesions in colorectal cancer. Stem Cell Rep. 16, 1–14 (2021).
Okamoto, T., Natsume, Y., Yamanaka, H., Fukuda, M. & Yao, R. A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids. STAR Protoc. 2, 100780 (2021).
Zhang, N., Fu, J. N. & Chou, T. C. Synergistic combination of microtubule targeting anticancer fludelone with cytoprotective panaxytriol derived from panax ginseng against MX-1 cells in vitro: experimental design and data analysis using the combination index method. Am. J. Cancer Res. 6, 97–104 (2016).
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Korotkevich, G. et al. Fast gene set enrichment analysis. Preprint at bioRxiv https://doi.org/10.1101/060012 (2021).
Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587 (2021).
Abe, Y. et al. Comprehensive characterization of the phosphoproteome of gastric cancer from endoscopic biopsy specimens. Theranostics 10, 2115–2129 (2020).
Adachi, J. et al. Improved proteome and phosphoproteome analysis on a cation exchanger by a combined acid and salt gradient. Anal. Chem. 88, 7899–7903 (2016).
Abe, Y., Nagano, M., Tada, A., Adachi, J. & Tomonaga, T. Deep phosphotyrosine proteomics by optimization of phosphotyrosine enrichment and MS/MS parameters. J. Proteome Res. 16, 1077–1086 (2017).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).
Acknowledgements
We are grateful to all participants of this study. We would like to thank all lab members and staff for their assistance with sample collection and completion of this study. This work was supported in part by MEXT/JSPS KAKENHI (grant numbers 21H02770 [R.Y.] and 22K19468) and AMED (grant number JP23ama221116).
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Y.N., H.O., E.S., and S.N. established the PDOs. Y.T., H.Y., Y.N., H.Y., and D.K. conducted the in vitro analysis of PDOs. M.S. conducted in vivo analysis. N.K., Y.A., and J.A. conducted proteomic analysis. K.K. and R.M. conducted data analysis and visualization of the transcriptome. K.O., S.M., and Y.K. supervised the project. Y.K., S.N., R.M., and R.Y. designed the study. R.Y. wrote and edited the manuscript.
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Tanaka, Y., Sakahara, M., Yamanaka, H. et al. Ribosome biogenesis as a potential therapeutic target in KRAS mutant colorectal cancer. Nat Commun (2025). https://doi.org/10.1038/s41467-025-67979-9
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DOI: https://doi.org/10.1038/s41467-025-67979-9
