Abstract
Therapeutic targeting of mutant KRAS pathways driving cancers is being actively investigated to identify feedback mechanisms responsible for the development of adaptive resistance to mutant KRAS inhibitors undergoing clinical trials. Here we report RASH3D19 as a mediator of RAS pathway activation through a positive feedback loop involving the KRAS–microRNA signalling axis. KRAS-induced miR-222 represses ETS1 expression and downstream transactivation of miR-301a leading to elevation of its target RASH3D19. RASH3D19 facilitates activation of RAS pathways by promoting dimerization and interaction of EGFR with the SOS2, GRB2, SHP2 and GAB1 complex. Genetic deletion of RASH3D19 in mutant KRAS-expressing cancer cells exhibits growth retardation in vitro, in vivo and sensitized pancreatic ductal adenocarcinoma and colorectal cancer cells, organoids and xenografts to mutant KRAS inhibitors, suppressing feedback reactivation of RAS pathways. Therapeutic targeting of RASH3D19 is expected to lead to tumour debulking and alleviating resistance to KRAS inhibitors in mutant KRAS-expressing cancers.
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Data availability
Previously published RNA–seq data that were reanalysed here are available under accession codes GSE41368, GSE62165 and GSE101448 in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). The human PDAC data were derived from the TCGA Research Network (https://www.cbioportal.org) and TCGA match with GTEx (http://gepia.cancer-pku.cn/). All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank M.-S. Tsao for sharing the HPDE cells generated in his laboratory and H. Clevers and J. van Es for getting us in touch with laboratory colleagues to perform the experiments with the CRC organoid. The services received from the MD Anderson Core Facilities for animal studies (DVMS) and research histology core facility (RHCL) for immunohistochemistry assay development supported by the NCI Cancer Center Support Grant (grant no. P30 CA016672) are gratefully acknowledged. This work was supported by NCI grants (grant no. U01 CA214263 and U01 CA252965) to S.S. The Sheikh Khalifa bin Zayed Foundation supports to A.M. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Authors and Affiliations
Contributions
W.T., H.K., D.S. and S.S. designed the study. W.T., H.K. and D.S. performed biochemical characterization. W.T., D.S. and T.N. performed cell-based characterization. W.T., D.S., M.-C.T. and T.N. performed miRNA and RNA expression analysis. S.R. and H.C. performed organoid experiments. W.T. and T.N. performed animal experiments. D.S., W.T., M.-C.T. and H.K. performed database analysis. K.H. and H.W. performed and analysed histology. W.Y., S.K.B., J.V.H. and S.K. provided cell lines and inhibitors. F.I.T., M.P.P., I.I.W., T.D.S., T.H. and A.M. analysed data and participated in discussions on content. W.T., H.K. and S.S. wrote the paper, with input from all authors.
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A.M. is a consultant for Tezcat Bioscience. A.M. is listed on a patent that had been licensed by Thrive Earlier Detection (an Exact Sciences Company). K.H. is funded by Nanostring Technology. The other authors declare no competing interests. A provisional patent on the clinically relevant findings of this study has been filed.
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Extended data
Extended Data Fig. 1 Mutant KRAS-induction of miR-222 downregulates miR-301a by targeting ETS1.
a,b, Expression levels of miR-222 (a) in Dox inducible KRAS G12D mutant AK196 and AK12282 mouse PDAC cells untreated and treated with Dox and (b) in AsPC1 and PANC1 cells transfected with control or pre-miR-222. c, Expression levels of ETS1 protein in Dox inducible KRAS G12D mutant AK192 and AK12282 mouse PDAC cells untreated and treated with Dox. Relative expression of ETS1 normalized to β-actin is shown. d, Expression levels of miR-222 (left) and indicated proteins (right) in AsPC1 and PANC1 cells transfected with siRNA targeting KRAS G12D and/or pre-miR-222. Relative expression of ETS1 normalized to β-actin is shown. e, ChIP assay of ETS1 enrichment at 5 different predicted regions (R1-R5) on miR-301 promoter in PANC1 cells. f, Expression levels of ETS1 protein in AsPC1 and PANC1 cells transfected with control siRNA or siRNAs targeting ETS1. g,h, miR-301a promoter activity in ETS1 silenced KRAS G12D mutant stably expressed HPNE cells compared to control siRNA (n=5) (g) and in Dox inducible KRAS G12D mutant HPNE cells untreated or treated with Dox (n=5) (h). i, miR-301a expression levels in KRAS G12D mutant inducible AK192 and AK196 mouse PDAC cells untreated or treated with Dox. All blots and qRT-PCR are representative of three independent experiments. (a-b,d-e,g-i); Bar graphs are represented as mean ± SD. P values were calculated using two-sided unpaired Student’s t-tests. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Extended Data Fig. 2 RASH3D19 overexpressed in PDAC downstream of mutant KRAS is a target of miR-301a.
a, Venn diagram identifying the targets of miR-301a. b, Pearson’s correlation of miR-301a vs RASH3D19 in PDAC patient tissues in TCGA (n=177) and PDAC cell lines with high miR-301a expression in CCLE database (n=32). R = Pearson’s correlation coefficient; P values were calculated for the statistical significance of the Pearson’s correlation coefficient. c, miR-301a expression levels in AsPC1 and PANC1 cells transfected with control or pre-miR-301a. Bar graphs are represented as mean ± SD. P values were calculated using two-sided unpaired Student’s t-tests. d, Conserved region of miR-301a binding site in RASH3D19 3′UTR. e, RASH3D19 expression levels in Dox-inducible KRAS G12D mutant AK196 and AK12282 cells untreated and treated with Dox. Relative expression of RASH3D19 normalized to β-actin is shown. f, Expression levels of miR-222 and miR-301a (left) and indicated proteins (right) in BxPC3 cells untreated with treated with KRAS inhibitor BI2865. Relative protein expressions of ETS1 and RASH3D19 normalized to β-actin are shown. Bar graphs are represented as mean ± SD. P values were calculated using two-sided unpaired Student’s t-tests. g,h, RASH3D19 expression levels in normal and PDAC patients in additional GSE (g) and TCGA match with GTEx (h) datasets. Box plots are represented as mean with minima to maxima values. P values were calculated using two-sided Wilcoxon rank-sum test. All in vitro assays are representative of three independent experiments. (c,f, g-h); ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Extended Data Fig. 3 RASH3D19 interacts with RAS regulatory proteins.
a,b,c, Immunoprecipitation (IP)/western blot analysis of RASH3D19 with EGFR, SHP2, SOS2 and SOS1 in PANC1 cells. d, Schematic representation of RASH3D19 full length and deletion mutants. Constructs were GST-tagged. Proline-rich region (PRR) and 5 SH3 domains (Domains A to E) are represented by orange and blue boxes, respectively. e, GST pull-down assay. Extract of 293T cells expressing EGFR, SOS2 or GRB2 was incubated with GST or GST-RASH3D19 full length immobilized on glutathione beads. Binding protein was detected with respective antibody. GST proteins were visualized by Coomassie brilliant blue (CBB) stain. f, GST pull-down assay. EGFR prepared in (e) was incubated with GST or GST-RASH3D19 full length in the presence or absence of GRB2. Binding protein was detected by western blot.
Extended Data Fig. 4 Mapping of RASH3D19 regions binding to SOS2 and GRB2, and the effect of RASH3D19 deletion in cell viability and cell cycle.
a,b, GST pull-down assay. SOS2 (a) or GRB2 (b) prepared in (Extended Data Fig. 3e) was incubated with GST, GST-RASH3D19 full length or deletion mutants. Binding proteins were detected by western blot. c, Schematic representation of GRB2 full length and deletion mutants. SH2 and SH3 domains are represented by light orange and blue boxes, respectively. d. GST pull-down assay. Extract of 293T cells expressing GFP-RASH3D19 was incubated with GST, GST-GRB2 full length or deletion mutants immobilized on glutathione beads. Binding proteins were detected by western blot. The dotted line indicates the boundary between the cropped images. e, Cell proliferation rate of sgControl and sgRASH3D19 AsPC1 and PANC1 cultured in 2D and 3D. Bar graphs are represented as mean ± SD. P values were calculated using two-sided unpaired Student’s t-tests. ns, not significant; *, P < 0.05; **, P < 0.01. f, Cell cycle distribution analysis of AsPC1 and PANC1 cell lines by quantitation of DNA content. Cells with either sgControl or sgRASH3D19 were cultured in 3D condition followed by staining with propidium iodide (PI) and DNA content was quantitated by flow cytometry. All the data are representative of three independent experiments.
Extended Data Fig. 5 RASH3D19 deletion in combination with KRAS mutant inhibition inhibits spheroid growth.
a,b, Time-lapse spheroid morphology images of sgControl and sgRASH3D19 AsPC1 (a) and PANC1 (b) cells untreated or treated with 10 nM MRTX1133. Pictures in red frame (Day 0 and Day 7 of both cell lines) were also represented in Fig. 3d.
Extended Data Fig. 6 RASH3D19 promotes adaptive response to RAS inhibitors through RAS activation.
a, RAS pull-down assay of sgControl and sgRASH3D19 MIAPaCa2 and BxPC3 cells. Cells were cultured in 3D for 24 h followed by serum starvation for additional 24 h. Then cells were treated with EGF for 10 min. Relative levels of RAS-GTP normalized to input RAS and β-actin are shown. b, EGF treated sgControl and sgRASH3D19 PANC1 cells as indicated in Fig. 4a were immunoprecipitated for GRB2 followed by western blotted with indicated antibodies. c, RASH3D19 expression levels in AsPC1, MIAPaCa2, HPAF-II cells untreated or treated with mutant KRAS inhibitor MRTX1133 or AMG510 for indicated time. d, Western blot analysis of cells prepared as in (c). Relative expression of RASH3D19 normalized to β-actin is shown. e, RASH3D19 expression levels in AsPC1, PANC1, MIAPaCa2, BxPC3 cells untreated or treated with pan-RAS inhibitor RMC7977 for indicated time. f, Western blot analysis of cells prepared as in (e). Relative expression of RASH3D19 normalized to β-actin is shown. All blots and in vitro assays are representative of three independent experiments. (c,e) Bar graphs are represented as mean ± SD. P values were calculated using two-sided unpaired Student’s t-tests. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Extended Data Fig. 7 Deletion of RASH3D19 sensitizes mutant KRAS expressing PDAC, CRC, and NSCLC cells to RAS inhibitors.
a, Cell viability assays of AsPC1 and MIAPaCa2 cells which were transfected with control sgRNA or sgRNAs targeting RASH3D19 cultured in 3D for 24 h and then treated with different concentrations of MRTX1133 or AMG510 for 3 days. b, RASH3D19 expression levels in SW837, H1972 and MIAPaCa2 cells prepared as in (a). Bar graphs are represented as mean ± SD. P values were calculated using two-sided unpaired Student’s t-tests. ***, P < 0.001. c, Cell viability assay of sgControl and sgRASH3D19 AsPC1 and PANC1 cells which were cultured in 3D for 24 h followed by treatment with different concentrations of RMC4550 for 3 days. All in vitro assays are representative of three independent experiments.
Extended Data Fig. 8 Deletion of RASH3D19 sensitizes mutant KRAS expressing PDAC and CRC organoids and xenografts to RAS inhibitors.
a,b, B40 and B106 PDAC-derived organoid cultures harboring KRAS G12D mutant, assayed for total area in percentage to control counted by Incucyte live cell imaging. Two PDAC organoids were pre-transfected with control siRNA or siRNAs targeting RASH3D19 for 6 h followed by treatment with MRTX1133 at 25 nM for 6 days. (a-b); Bar graphs are represented as percentage of organoid total area in RASH3D19 siRNA and/or MRTX1133 treated compared to untreated control siRNA from technical replicates (n=4) of two independent experiments. c,d, RASH3D19 expression levels (c) and relative survival (d) of sgControl and sgRASH3D19 CRC organoid, harboring KRAS G13D mutant, treated with DMSO or pan-RAS inhibitor RMC7997 25nM or 100 nM for 6 days compared to normal colon organoid (n=4). (c-d); Bar graphs are represented as mean ± SD. P values were calculated using unpaired two-sided Student’s t-tests. **, P < 0.01; ***, P < 0.001. e, In vivo tumor volume scatter plots on day 35 of sgControl and sgRASH3D19 PANC1 cells which were subcutaneously injected in nude mice (n=6 and 7 in the two groups) and treated daily with vehicle control or MRTX1133 at 3 mg/kg. f, In vivo tumor volume scatter plots on day 28 of sgControl and sgRASH3D19 MIAPaCa2 cells subcutaneously injected in nude mice (n=7 mice in each group) and treated daily with vehicle control or AMG510 at 3 mg/kg (e-f); Box plots are represented as mean with all data points from minima to maxima. P values were calculated using unpaired two-sided Student’s t-tests. ***, P < 0.001.
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Supplementary Table 1: List of oligonucleotides used in this study. Supplementary Table 2: List of antibodies used in this study. Supplementary Table 3: List of purchased plasmids used in this study.
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Treekitkarnmongkol, W., Katayama, H., Sankaran, D. et al. RASH3D19 mediates RAS activation through a positive feedback loop in KRAS-mutant cancer. Nat Cell Biol 28, 197–206 (2026). https://doi.org/10.1038/s41556-025-01816-5
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DOI: https://doi.org/10.1038/s41556-025-01816-5
