Ribosome biogenesis as a potential therapeutic target in KRAS mutant colorectal cancer

Domain: chemistry
Published: 2025-12-27
Editorial outcome: major_revision

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.

Introduction
Colorectal cancer (CRC) is among the most commonly diagnosed cancers and the third most common cause of cancer-related deaths worldwide. Owing to the high prevalence of KRAS-activating mutations, the RAS pathway is considered a promising target for CRC treatment1. Specific inhibitors of RAS pathway molecules and mutant allele-specific inhibitors have been successfully developed2–5. However, resistance inevitably occurs, and the insufficient efficacy of these agents in trials limit their use in clinical practice as monotherapies.

Reactivation of signaling and pathway bypass confer therapeutic resistance to several types of cancers6–8. In CRC, combinational inhibition of the RAS pathway and epidermal growth factor receptor has been tested in RAS-mutant CRC. However, despite the improvement in patient prognosis using combination chemotherapies, their efficacy is insufficient, warranting further improvement for CRC treatment3,9,10. Tumor tissues are hierarchically organized with heterogeneous cells, and their plasticity is a possible cause of drug resistance11,12. Cancer stem cells are characterized by their self-renewal and differentiation capacities, and cell stemness is a key feature responsible for the drug resistance of multiple cancer types13. Initially, cancer stem cells were thought to be slow-replicating cells that were dormant in several cancer types, which contributed to their drug resistance14. However, their proliferative properties vary depending on the cancer type, and low levels of tumor dormancy have been reported in CRC cells15. Cancer stem cells have unique cellular properties, such as cellular metabolism, energy consumption, and epithelial-mesenchymal transition16. Ribosome biogenesis determines the stemness of embryonic and adult stem cells17. In line with this, transcription of ribosomal DNA defines the stem cell hierarchy in CRC18. Selective pressures imposed by chemotherapeutic agents eliminate the non-cancer stem cells, leading to an enrichment of cancer stem cells19,20. Therefore, specific characteristics of the cellular states and vulnerabilities of drug-resistant cells need to be elucidated to enhance the efficacy of chemotherapeutic agents. Here, we show KRAS inhibition induces a cellular state marked by ribosome biogenesis, which is vulnerable to RNA polymerase I inhibition.

Results
Mitogen-activated protein kinase kinase (MEK) inhibition promotes a cellular state with elevated ribosomal biosynthesis and reduced protein synthesis resembling the ground state of ESCs

We previously showed that 75% of KRAS-mutated CRC PDOs are resistant to the MEK inhibitor, trametinib21. To elucidate the molecular basis of this resistance, we analyzed a previously characterized trametinib-resistant PDO (HCT24-8) carrying a KRASG12A mutation. The PDOs was treated with 10nM trametinib for 24h or left them untreated and obtained the multiplexed single-cell expression profiles. Cells were divided into five groups via unbiased clustering and visualized using uniform manifold approximation and projection (UMAP; Fig. 1a, left panel). Each cluster was initially annotated by calculating the p-value of overlaps using Fisher’s exact test between the differentially expressed genes (DEGs) of each cluster and CRC markers reported previously22. Two absorptive cell clusters, C2 and C3, were further characterized using a single-cell transcriptomic atlas (Supplementary Fig. 1b)23. Notably, trametinib treatment increased the proportion of C1 cells (Supplementary Fig. 1a), which were annotated as intestinal crypt stem cells of the colon, and decreased the proportion of C2 cells, which were enriched in mature enterocyte markers (Fig. 1b). C3 and C4 were annotated as absorptive cells and transit-amplifying cells, respectively, with their proportions remaining relatively unchanged. C5 was annotated as goblet cells, which were absent under untreated conditions but was induced following trametinib treatment.

Gene set enrichment analysis of the Gene Ontology cellular components (GOCC) revealed that C1 cells were enriched in multiple ribosome-related signatures (Fig. 1c), including ribosome gene sets (normalized enrichment score [NES]=8.41, padj=0.1; Fig. 1c and Supplementary Fig. 1c). Further analysis of hallmark genes in the molecular signatures database (MsigDB) revealed Myc target V1 as the most enriched gene set (NES=3.84, padj=0.04; Fig. 1d and Supplementary Fig. 1d). As Myc plays a critical role in the coordinated transcription of ribosome genes in various types of stem cells17,24, these observations suggest that cells in C1 exhibit elevated Myc-dependent transcription of ribosome genes. Protein levels of three ribosome components, PABP1, RPL13A and RPL23, identified through enrichment analysis were upregulated following trametinib treatment by 1.7±0.2, 1.5±0.09 and 1.4±0.1 fold (mean±SEM, N=3 biological replicates), respectively, thereby confirming the transcriptome analysis (Fig. 1e).

Embryonic stem cells (ESCs) are pluripotent stem cells derived from epiblasts, and two phases of cell state have been defined25. Ground state represents cells that produce a fully unrestricted population, whereas the primed state exhibits a restricted pluripotency configuration (Fig. 1f). The cellular properties of ground state ESCs are maintained by Myc-dependent ribosome biogenesis26. Notably, MEK inhibition promotes the reprogramming of mouse ESCs from the primed state to the ground state27. As trametinib promoted the cell state with elevated ribosome biogenesis and Myc-dependent transcription in CRC PDOs, we compared their transcriptome profile with that of ground-state mouse ESCs. The gene expression profile of C1 was highly enriched in the ground state mouse ESC signature (NES=4.12, padj<0.001; Fig. 1f, lower panel and Supplementary Fig. 1c)28. Next, we compared the expression profiles of trametinib-treated CRC cells with those of two states of mouse epiblasts, as the pluripotent naïve ground state and primed state of ESCs represent the pre- and post-implantation epiblasts, respectively (Fig. 1g, upper panel). This analysis revealed that the expression profile of C1 was positively (NES=2.17, padj=0.017) and negatively (NES=−0.92, padj=0.97) enriched in pre-implanted ground and post-implanted primed epiblasts, respectively (Fig. 1g, lower panes, and Supplementary Fig. 1d)29.

We next investigate the expression profile of the genes enriched in the ground state ESCs (Fig. 1f) and epiblast (Fig. 1g). Ten genes representing higher expression levels in ground state mouse epiblast were significantly highly expressed in C1 (adj-p-value<0.05) (Fig. 1h and Supplementary Table 1). Interestingly, seven genes were also highly expressed in C5. Given that the proportion of C1 and C5 was increased by trametinib treatment, these results indicate that trametinib induces a cell population with the expression profile similar to the ground state mouse epiblast in CRC organoids. Elevated ribosome biogenesis in C1 were confirmed by calculating the gene score of ribosome biogenesis feature (Supplementary Fig. 1e).

To further evaluate the trametinib-induced cell state of CRC organoids, we analyzed the expression profile of human ESCs. The expression levels of ribosome subunits vary among distinct tissues, and some of them are highly expressed in ESCs30. We found RPS13A, RPS18, RPL7A and RPL36A, which are highly expressed in human ESCs and decreased upon differentiation, were highly expressed in C1 (adj-p-value<1e10−62) (Fig. 1i and Supplementary Table 2)31. RPL13A is known as a stem cell marker in human ESCs and was highly expressed in C1 and the trametinib-treated fraction of C332. These observations highlight the shared expression profiles of ribosome-related genes between CRC organoids and human ESCs.

Low global translation rates, despite high levels of ribosome biogenesis, have been well-documented in pluripotent naïve ground-state ESCs33. Here, we evaluated these rates via immunofluorescence analysis. Fibrillarin, a methyltransferase for ribosome RNA processing in nucleoli that is necessary for the maintenance of pluripotency, is highly expressed in pluripotent ES cells34. We found that trametinib significantly elevated the fibrillarin content in CRC organoids (p=5.46E-5; Fig. 1j). Moreover, trametinib significantly reduced the protein synthesis rate in CRC organoids, as measured via O-propargylpuromycin (OPP) incorporation (p=5.73E-5; Fig. 1k)35. 5-ethylnyl-2’-deoxyuridine (EdU) incorporation was partially reduced by trametinib treatment, resembling pluripotent ESCs with remarkable global suppression of DNA replication (p=4.54E-3; Fig. 1l)36. Collectively, these observations suggest that trametinib induces a cellular state in KRAS-mutant CRC PDOs in which Myc-dependent ribosome biogenesis is upregulated.

Trametinib reduces the phosphorylation of RNA-binding molecules

Next, we focused on the phosphorylation profile of PDOs to systematically dissect the network that controls the cell state of CRC cells. We first compared the KRAS-mutant PDO, HCT24-8, with the isogenic KRAS wild-type PDO, HCT24-14 (Fig. 2a). Trametinib treatment significantly reduced the viability of HCT24-14 cells, whereas HCT24-8 cells were resistant, confirming the role of KRAS mutations in trametinib response (Fig. 2b)21. Using mass spectrometry-based phospho-proteomics analysis optimized for Matrigel-embedded organoids37, we identified 3043 proteins and 8354 phosphorylation sites (Supplementary Fig. 2a). Phosphorylation was upregulated at 4181 sites and downregulated at 4173 sites in KRAS-mutant PDOs compared to wild-type PDOs. Ingenuity pathway analysis (IPA) identified ERK/MAPK signaling as the most significantly associated gene set (p=2.51E-11; Fig. 2c). Additionally, multiple RAS-mediated signals were highly enriched, including molecular mechanisms of cancer (p=6.73E-11), AMPK signaling (p=2.34E-09), and integrin signaling (p=2.72E-09), demonstrating the validity of the analysis. Kyoto Encyclopedia of Genes and Genomes analysis of the differentially phosphorylated proteins identified terms related to cellular proliferation, including cell cycle, base excision repair, and DNA replication (Supplementary Fig. 2b). Additionally, processes associated with cell adhesion, including tight and adherens junctions, were positively enriched in the KRAS mutation status (Supplementary Fig. 2b), providing evidence that trametinib counteracted the effects of KRAS-activating mutations.

Comparative analysis of trametinib treatment and KRAS mutation demonstrated that the phosphorylation sites altered by trametinib treatment were inversely correlated with those altered by KRAS mutation in a time-dependent manner (Fig. 2e). The most significant correlation was observed 72h after treatment (Spearman’s coefficient=−0.42, p=2.2E-16), supporting the notion that the altered phosphorylation induced by trametinib treatment was mostly caused by the perturbation of KRAS signaling. The proteins that were differentially phosphorylated at 24 and 72h were highly correlated (Spearman’s coefficient=0.88, p-value=2.2E-16). Interestingly, gene ontology analysis of molecular function identified RNA binding as the most enriched term, regardless of the up- or downregulation of phosphorylation (Fig. 2f). Furthermore, two molecules involved in ribosome biogenesis, Nucleophosmin 1 and Nucleolin, were highly phosphorylated in the KRAS-mutant and remained unphosphorylated after trametinib treatment (Fig. 2g). The phosphorylation of these ribosome binding proteins drives liquid-liquid phase separation and controls rDNA transcription and rRNA maturation42,43. These observations suggest that phosphorylation signaling is involved in trametinib-regulated ribosome biogenesis.

Trametinib alters the phosphorylation of cyclin-dependent kinase (CDK) substrates

Next, we performed an enrichment analysis of the post-translational modification signature database (PTMsigDB) to depict site-centric molecular signatures (Fig. 2h)44. The strongest positive enrichment was detected for the perturbation signature of the MEK inhibitor PERT-PSP_U0126. A high enrichment score was observed at 6h (false discovery rate [FDR] <0.001, NES=6.16) and persisted for 72h (FDR<0.001, NES=4.44), demonstrating the validity of this analysis. Conversely, two kinase-substrate signatures, KINASE-PSP-CDK1 and −2, were negatively enriched after trametinib treatment (FDR<0.01, NES=−8.4). This result demonstrated a reduced proliferation rate in trametinib-treated cells, as these phosphor-signaling are hallmark of cell cycle progression44. The phosphorylation levels of RB1 at Ser807/Ser811 were reduced in the enrichment analysis, which was further confirmed by immunoblot (Supplementary Fig. 2e, f). Overall, these results demonstrated the validity of our phosphoproteomics analysis of trametinib-treated CRC organoids.

Two mTOR-related pathways, including p90RSK/RPS6KA1 signature and RSK2/RPS6KA3 signature, were identified to be negatively enriched pathways (FDR<0.01, NES=−5.5 and FDR<0.01, NES=−4.4, respectively). We focused on RPS6, because its phosphorylation is commonly used as readout of the mTOR signaling and found that phosphorylation levels at Ser235 and Ser240 were reduced in a time-dependent manner (Supplementary Fig. 2e). The reduced phosphorylation levels of two molecules linked to mTOR, including HSF145,46 and STAT147,48 were also reduced. These alterations of phosphorylation were confirmed by immunoblot (Supplementary Fig. 2f). Given that mTOR plays central roles in protein synthesis, these observations further validated the phosphoproteomics analysis to explore the response of CRC organoids to trametinib.

The signature sets of PTMsigDB were divided into two categories based on their kinetics. The first group exhibited transient negative enrichment at 6h, including the perturbation signatures of IL-1B (FDR<0.01, NES=−4.2) and TNF (FDR<0.01, NES=−4.1) and the signature of the molecular signaling pathways of LEPTIN (FDR<0.01, NES=−5.2) and NOTCH (FDR<0.01, NES=−4.2) (Fig. 2h, outline in blue). The other group showed sustained enrichment over time (Fig. 2h, outline in red). Interestingly, this group included multiple kinase substrate signatures of CDKs, including CDK4, 6, 7 and 9. These results shed light on the ability of trametinib to regulate the substrate sites of other kinase inhibitors.

Dinaciclib exerts synergistic effects with trametinib

As trametinib affects the substrate phosphorylation of multiple CDKs, we elucidated its biological significance. We hypothesized that the trametinib-induced phosphorylation of CDKs substrates is involved in the resistance of KRAS-mutated CRC. To test this hypothesis, we treated HCT24-8 cells with various CDK inhibitors, and their combination effect was evaluated by the median-effect principle49 and its extension, the combination index equation50. We tested seven CDK inhibitors, and their median-effect plots for both single agents and their combination with trametinib indicated that the dose-effect data conformed to the median-effect principle of the mass-action law (Supplementary Fig. 3a). Among them, dinaciclib synergized most effectively with trametinib in reducing the viability of HCT24-8 cells (combination index <1)50 (Fig. 3a). This result was further confirmed by a nonconstant ratio combination experiment (Fig. 3b). Dinaciclib is a CDK inhibitor that targets transcription-related CDKs51. Transcriptome analysis revealed that the DEGs of dinaciclib-treated organoids were highly enriched in multiple ribosome-related gene sets in GOCC, and large and cytoplasmic ribosome subunits were the most significantly enriched (p-value=3.31E-14; Fig. 3c). The volcano plot indicated that the expression levels of the ribosomal genes were mostly decreased by dinaciclib treatment (Fig. 3d). This reduced expression of ribosome-related genes was further confirmed by IPA Canonical pathway analysis, in which eukaryotic initiation factor 2 (EIF2) signaling was identified as the most significantly enriched pathway (p.val=6.3E-26; Supplementary Fig. 3c, d). These results suggested that dinaciclib preferentially perturbed the transcription of ribosome-related genes. Ribosomal protein genes contain a polypyrimidine initiator called the TCT motif, encompassing the transcription start sites, which confers a specialized transcription system for these genes52. Furthermore, RNA pol II preferentially regulates ribosomal protein expression53. These observations support the hypothesis that dinaciclib preferentially perturbs ribosome-related gene expression by inhibiting transcription-related CDKs. Notably, GOCC terms that were negatively enriched in dinaciclib-treated PDOs were positively enriched following trametinib treatment (Fig. 1c). This inverse correlation highlights ribosomal biogenesis as a key process related to trametinib tolerance in CRC PDOs.

RNA polymerase I inhibitor, CX-5461, synergizes with trametinib

In addition to transcription-related CDKs, dinaciclib perturbs cell cycle-associated CDKs, which may contribute to the synergistic effects of trametinib. To clarify the role of ribosome biogenesis in the synergistic effect of trametinib, we targeted RNA pol I because its critical role in rRNA transcription is a rate-limiting step in ribosome biogenesis54. We used CX-5461, which is widely accepted as the first in-human inhibitor of RNA pol I55. Indeed, CX-5461 significantly suppressed production of nascent rRNA as detected by 5-ethynyluridine (EU) incorporation (Fig. 3i, j) and reduced the amount of ribosome RNA rRNA (Supplementary Fig. 3e, f) expression, confirming its effects on CRC PDOs. Dinaciclib also reduced the rRNA levels, which is consistent with previous reports that rRNA transcription is coupled with ribosomal protein gene expression56,57.

The median-effect plot demonstrated the conformity of CX-5461, both as a single agent and in combination with trametinib, to the median-effect principle of the mass-action law (Supplementary Fig. 3b). CX-5461 exhibited a synergistic effect on PDOs with trametinib (Fig. 3e), and its effects were more potent than those of dinaciclib over a wider range of concentrations (Fig. 3f). Interestingly, CX-5461 treatment strongly inhibited the expression of ribosomal protein genes (Fig. 3g, h) and EIF2 signaling gene sets (Supplementary Fig. 3g, h). Reduction in the transcription of ribosome-related genes by CX-5461 was expected, as RNA pol II-mediated transcription of ribosomal protein genes is tightly coordinated with the RNA pol I-dependent production of rRNA24,58,59. Collectively, these observations indicate that CRC characterized by a high ribosome biogenesis induced by trametinib are vulnerable to the inhibition of ribosome biogenesis.

Next, anti-tumor activities of CX-5461 and trametinib were evaluated in a mouse model of intestinal tumors. Autochthonous tumors were induced via the subcutaneous injection of 4-hydroxytamoxifen (4-OHT) into Apcflox/flox, KrasLSL-G12D/+, Lgr5-CreERT2/+ mice (Fig. 4a). Trametinib significantly elevated the fibrillarin levels, recapitulating the in vitro response of PDOs to trametinib (Fig. 4b, c). No significant changes were observed in normal intestines after trametinib treatment (Fig. 4d, e).

Trametinib, CX-5461, and their combination were administrated at 13 days after 4-OHT injection once a day for 5 days, and tumor was evaluated by immunostaining of β-catenin (Fig. 4f). In control mice, 19.1% of area was identified as tumor region (Supplementary Fig. 4a, b). Trametinib and CX-5461 reduced tumor area by 8.4% and 8.2%, respectively (Fig. 4g, h). Further reduction was achieved with their combination, and the tumor was reduced to 1.3%. CX-5461 did not affect EdU incorporation, but treatment with trametinib and the combination of CX-5461 and trametinib resulted in a decrease in EdU incorporation by 2.7% and 2.3% respectively (Fig. 4i, j). No significant changes in size or in EdU incorporation were observed in the normal intestines of WT mice (Fig. 4k–n) of mutant mice (Supplementary Fig. 4c–f). Notably, the combination of CX-5461 and trametinib significantly suppressed weight loss in mouse model of intestinal tumor, while having no significant effect on the body weight of wild-type mice (Fig. 4o).

In this analysis, experiments were conducted using 0.1 mg/kg of CX-5461, which is a lower concentration compared to the previously reported 35–50 mg/kg used to assess the efficacy of monotherapy60,61. The human equivalent dose (HED) of 0.28 mg/m262. In a dose-escalation study of CX-5461 in phase I studies, patients with hematologic malignancies and solid tumors were treated at doses ranging from 25 to 450 mg/m2 and 50 to 650 mg/m2, respectively55,63. Given that the HED of CX-5461 used in combination with trametinib was markedly lower than the doses used in human clinical studies, these findings provide in vivo evidence that CX-5461 synergistically suppresses tumor growth when combined with trametinib.

Intrinsic ribosome biogenesis is correlated with AMG510 sensitivity

AMG510 is a small compound that specifically inhibits the KRASG12C protein and shows significant anti-tumor effects in non-small cell lung cancer64. However, the objective response rate for patients with CRC was limited, and its modest efficacy warrants further evaluation as a treatment option for combination therapy10,64. To explore the role of ribosomal biogenesis in response to AMG510 in CRC, we established four PDOs from adenocarcinomas harboring the KRASG12C mutation (Supplementary Table 3). Exome sequencing confirmed the KRASG12C mutation and various other mutations frequently observed in CRC (Supplementary Fig. 5). We found that AMG510 efficiently reduced the viability of HCT191-1T, but had only a modest effect on other PDOs (Fig. 5a). UMAP visualization of single-cell expression was used to divide the cells into five clusters, and HCT191-1T contained a higher abundance of cells in C2 than other PDOs (Fig. 5b, c). Interestingly, the enrichment profile of C2 resembled that of the trametinib-induced cluster in HCT24-8 (Fig. 5d); it was highly enriched in multiple ribosome-related gene sets in GOCC and Myc targets V1 in the Hallmark datasets. The heatmap of genes listed in the cytosolic ribosome of GOCC revealed most of them are highly expressed in HCT191-1T (Fig. 5e). The gene score calculated using the cytoplasmic ribosome gene set in GOCC showed a high value for C2 (p=4.3E-153) (Fig. 5f). The gene score for C5 was also significantly high (p=4.9E-9). Considering that HCT191-1T had a high proportion of both C2 and C5 (Fig. 5c), these results supported the idea that the ribosome biogenesis of this organoid was elevated.

The expression profile of C2 was significantly enriched with the gene signature of naïve ground-state ESC (NES=2.76, padj=0.01; Fig. 5g)28. Furthermore, it was positively enriched in the pre-implanted ground state epiblasts (NES=1.25, padj=0.66) but negatively enriched in the implanted primed state epiblasts (NES=−1.09, padj=0.09; Fig. 5h)29. Overall, PDOs sensitive to AMG510 contained a high fraction of cells with an expression profile resembling that of ground-state ESCs and epiblast.

To further evaluate differences in ribosome biogenesis among CRC organoids, we analyzed the transcription of ribosome DNA. EU incorporation assay was employed to quantify newly synthesized RNA. Most of the EU label was detected in nucleolar, indicating that it was primarily incorporated into nascent ribosomal RNA (Fig. 5i). 3D imaging of organoids, followed by quantification of EU intensity in individual cells, revealed that HCT191-1T exhibited significantly higher incorporation compared to other organoids (Fig. 5j). Immunofluorescence staining of fibrillarin demonstrated that its localization in nucleoli, indicating the involvement in the modification of ribosome RNA in PDOs derived from advanced CRC (Fig. 5k). 3D imaging and quantification of individual cells revealed their higher fibrillarin intensity in HCT191-1T compared to other organoids (Fig. 5l). These observations indicated that, in addition to the elevated expression of ribosomal proteins, transcriptional levels of ribosome RNA and its modification are up-regulated. Taken together, it appears that HCT191-1T has enhanced ribosome biogenesis.

CX-5461 synergizes with the allele-specific KRAS inhibitor, AMG510

Next, we analyzed the response of KRASG12C mutants to AMG510. AMG510 reduced EdU incorporation after treatment, and HCT191-1T was most significantly affected, consistent with the results indicating high sensitivity (Fig. 6a and Supplementary Fig. 6a). They also reduced the global protein synthesis rate as revealed by OPP incorporation (Fig. 6b and Supplementary Fig. 6b). Fibrillarin was accumulated in nuclei upon AMG510 treatment (Fig. 6c and Supplementary Fig. 6c). These observations suggested that AMG treatment reduced the global protein synthesis and upregulated the modification.

Transcriptome analysis demonstrated that AMG510 led to the enrichment of the ribosome signature in resistant clones, whereas the sensitive clone HCT191-1T was negatively enriched (Fig. 6d). Conversely, all clones exhibited positive enrichment of the MYC TARGET V1 gene sets after AMG510 treatment. Overall, similar to the trametinib treatment, AMG510-tolerant PDOs adopted a cell state with high ribosome biogenesis, yet the global protein synthesis rate is low. Interestingly, PDOs sensitive to AMG510 had a highly intrinsic ribosome signature and did not show enhanced expression in response to AMG510 treatment. These findings highlight the role of ribosome biogenesis in the response to AMG510, although the correlation needs to be carefully evaluated using a large number of PDOs with KRASG12C mutations.

Finally, we evaluated the synergistic effects of CX-5461. CX-5461 reduced the incorporation of EU (Supplementary Fig. 6d). The CRC organoids exhibited different sensitivities to CX-5461 (Fig. 6f). Nevertheless, it exhibited a synergistic effect on AMG-510-tolerant clones (Fig. 6g). Nonconstant CI estimation confirmed their robust synergistic effects at various concentrations (Fig. 6h). Taken together, these results suggest ribosome biogenesis as a promising target for combination therapy with AMG510 for patients with CRC with the KRASG12C mutation.

Methods
Establishment of colorectal tumor organoids

HCT24-8 and -14 PDOs were established and characterized in a previous study21. To establish PDOs harboring the KRASG12C mutation, CRC specimens were collected via surgical resection and histological diagnosis was performed by an experienced pathologist. PDOs were established as previously reported93. Briefly, surgical specimens were washed with phosphate-buffered saline (PBS) (Thermo Fisher Scientific) and minced into 1mm3 fragments using surgical scissors. The fragments were digested with a digestion buffer (Dulbecco’s modified Eagle’s medium [DMEM], fetal bovine serum, penicillin/streptomycin, collagenase, and dispase) (Thermo Fisher Scientific) at 37°C for 60min. Isolated tumor cells were embedded in Matrigel droplets and overlaid on a culture medium, as previously described93. Advanced DMEM/F12 was supplemented with penicillin/streptomycin, 10mM HEPES, 2mM GlutaMAX, 1× B27 (Thermo Fisher Scientific), 10nM gastrin I (Sigma), and 1mM N-acetylcysteine (Wako) to prepare the basal culture medium. The ENR (EGF, Noggin, and R-spondin1) medium was prepared by supplementing the basal culture medium with the following niche factors: 50ng/mL of mouse recombinant EGF (Thermo Fisher Scientific), 10% Noggin conditional medium94, and 1mg/mL of human recombinant R-spondin1 (R&D). The medium was changed every 3–4 days.

Exome sequencing

Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen), and its quality was validated using the Qubit system (Thermo Fisher Scientific). Exome sequencing was performed by Macrogen Japan Corp. (Tokyo, Japan). Genomic DNA was fragmented and used to prepare 151 bp paired-end libraries using the SureSelect Human All Exon V6 kit (Agilent). Sequencing was performed using the Illumina HiSeq 2500 system. Fastq files were analyzed using the Genomon pipeline provided by the Human Genome Center, Institute of Medical Science, University of Tokyo.

PDO viability assay

Trametinib and AMG510 were purchased from Sellec (Kanagawa, Japan). Organoids were digested with TrypLE Express (Thermo Fisher Scientific) supplemented with 10μmol/L Y27632 at 37°C for 15min with pipetting every 5min. The cells were suspended in a basal medium, and clumps were removed by passing the suspension through a 40-μm cell strainer. The suspension was centrifuged at 1000×g for 5min, and the cells were resuspended in the basal medium. Cells were counted and adjusted to 2.0×105 cells/mL in the Matrigel. Then, 10mL of cells suspension were dispensed into each well of U-bottom 96-well microplates (Thermo Fisher Scientific). PDOs were cultured for three days to allow the formation of organoids and then treated with drugs at the indicated doses for 7 days. The medium was changed every 3–4 days. We generated 8-step, ten-fold drug matrices in technical quadruples. Cell viability was measured using a CellTiter-Glo 3D Cell Viability Assay Kit (Promega). The readings were obtained using a Mithras LB940 luminometer (Berthold Technologies). In the 96-well plate, the average cell viability at each drug concentration was calculated using DMSO-treated cells as controls. Data were analyzed using the GraphPad Prism 9 software (GraphPad).

Titrations to determine drug synergy were performed by plating the PDOs in 96-well plates as described above. After 3 days, PDOs were treated with a combination of trametinib and various CDK inhibitors, or CX-5461 (Fig. 3), or AMG510 and CX-5461 (Fig. 5). The drugs were titrated in a seven-dose manner, ranging from four times the IC50 concentration to one-sixteenth of the IC50 concentration. To determine synergy in a nonconstant ratio, drugs were titrated in a four-dose manner, ranging from twice the IC50 concentration to one-fourth the IC50 concentration. Cell viability was determined using the CellTiter-Glo 3D Cell Viability Assay, as described above. The synergistic effects were determined using the median effect principle proposed by Chou and Tallalay95. R package was obtained from the following website: https://rdrr.io/github/snowoflondon/CIcomputeR/.

RNA sequencing

PDOs were treated with 100nM AMG510 for 24h. Total RNA was extracted using an RNeasy mini kit (Qiagen) and quantified using 2100 Expert Bioanalyzer software (Agilent). RNA-seq analysis was outsourced to Macrogen Japan Corp. (Tokyo, Japan). Following the manufacturer’s instructions, total RNA with an RIN>7 was used to generate a barcode-labeled library using an Illumina TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). Sequencing of 150 bp paired-end reads was performed using the Illumina NovaSeq 6000 system (Illumina).

Salmon was used for quantifying transcript abundance from RNA-seq reads96. Tximport package was used to import transcript-level abundances and summarizes abundances, counts, and transcript lengths to gene-level matrix (https://github.com/thelovelab/tximport). Gene enrichment analysis was performed using the fgsea package with nperm=1000 97 (https://bioconductor.org/packages/release/bioc/html/fgsea.html). Tidy format gene sets were retrieved using msigdbr package (https://cran.r-project.org/web/packages/msigdbr/vignettes/msigdbr-intro.html), and the rnk file was generated using wilcoxauc function in the presto package (https://rdrr.io/github/immunogenomics/presto/man/wilcoxauc.html).

Single-cell RNA sequencing. HCT24-8 PDOs were cultured for 3 days. PDOs harboring the KRAS G12C mutation were cultured for 3 days, followed by treatment with 10nM trametinib for 24h, or left untreated. To analyze KRAS G12C mutant PDOs, cells were collected 3 days after splitting. Multiplexing was performed using the 10X Genomics 3’ Cell Plex Kit (PN-1000261). Organoids were collected, and single cells were prepared using TrypLE (Thermo Fisher Scientific). Cells were suspended in PBS containing 0.25% BSA and centrifuged at 1000×g for 10min. After removing the supernatant, cells were resuspended in 100µL of Cell Multiplexing Oligo and incubated for 5min at RT. Cells were then washed with PBS containing 0.25% BSA three times. Cells were then resuspended with 100µL Cell Plex Oligo and incubated for 5min. Three multiplex pools were then generated. Pool 1 contained trametinib-treated and untreated HCT24-8. Pool 2 contained HCT191-1T and HCT-215. Pool 3 included HCT128-1T and HCT201-1T. The Chromium Nex GEM Single Cell 3’ Kit v. 3.1 was used to encapsulate single cells. To target 20,000-cell recovery for each pool, cells were concentrated to approximately 800 cells/µl. Pooled cells were loaded into one lane of the Chromium chip, and the single-cell RNA sequencing (scRNA-seq) library was prepared using the 10X Genomics Chromium protocol. Libraries were sequenced using an Illumina NextSeq 550 platform (Illumina) with a 150-bp paired-end run.

Analysis of scRNA-seq data

Sequence data were performed using Cell Ranger (v. 6.1.1), and Seurat was used for downstream analysis98. The data were filtered to remove cells with fewer than 500 and over 9000 unique genes per cell, and over 20% of mitochondrial expression. The filtered data were processed with the Seurat pipeline using SCTransform, followed by principal component analysis using RunPCA. Cell clustering was conducted using the FindNeighbors function with 1:30 dimensions and the FindClusters function with a resolution of 0.2. The cells were projected onto the UMAP embedding space using RunUMAP at 1:30 dimensions. To obtain cluster-specific expressed genes, we used the FindAllMarkers function with the options of only pos=TRUE, min.pct=0.1, and logfc.threshold=0.1. AddModuleScore was used to calculate gene score.

To annotate clusters, gene overlap analysis between cluster-specific expressed genes and large intestine markers, developed using the single-cell transcriptomic atlas of humans23 was performed using the newGeneOverlap function in the GeneOverlap package (https://bioconductor.org/packages/release/bioc/html/GeneOverlap.html). Gene enrichment analysis was performed as described in the RNA-seq section.

Phospho-proteomics analysis

Cells were dissolved in PTS buffer (12mM sodium deoxycholate, 12mM sodium lauroyl sarcosinate, and 50mM ammonium bicarbonate). The samples were reduced with 10mM TCEP, alkylated with 20mM iodoacetamide, and quenched with 21mM of L-cysteine, following digestion with trypsin (protein weight: 1/50) and Lys-C (protein weight: 1/50) for 16h at 37°C. Samples were acidified with 1% TFA and centrifuged at 20,000×g for 10min at 4C. Supernatants were desalted, and an IMAC column was used for phosphopeptide enrichment99. The phosphopeptides were labeled by the TMT 11plex reagent (Thermo Fisher Scientific) and quenched with hydroxylamine. The TMT-labeled peptides were mixed, desalted, and fractionated on C18-SCX Stage Tips100. Phosphotyrosine enrichment was performed using the pY1000 antibody as previously reported101,102. The time course experiment was performed in biological duplicate. Other experiments were performed in triplicate.

The nano-LC gradient was performed at 280nL/min and consisted of a linear gradient of buffer B from 5 to 30% B over 135min for phosphoproteome analysis and 45min for phosphotyrosine proteome analysis. The Q Exactive or Q Exactive Plus instrument was operated in a data-dependent mode100. Phosphopeptide identification was conducted using MaxQuant 1.5.1.2 or 1.6.3.3, and the UniPort Human Database (01/2017 release) was supported by the Andromeda search engine. Phosphorylated and non-phosphorylated peptides were distinguished by the mass shift corresponding to the phosphorylation modification (molecular weight=79.966) at the MS1 level. Additionally, MS2 scans (fragmented ionic products of phosphopeptides) were used to determine the localization of phosphorylation in the phosphopeptide sequence. The PIF filter was set at 75%. The search results were filtered to a maximum (FDR) of 0.01 at the protein, peptide-spectrum match, and post translational modification (PTM) site levels. Kinase activity prediction was performed using site-centric post-translational modification-signature enrichment analysis (PTM-SEA)44, a seven-amino-acid sequence flanking the phosphosite as an identifier and the human kinase/pathway definitions of PTMsigDB (v. 1.9.0) according to the following parameters: (gene.set.database= “ptm.sig.db.all.flanking.human.v1.9.0. gmt”, sample.norm.type=“rank, “weight=0.75, statistic=“area.under. RES”, output.score.type=”NES, “nperm=1000, global.fdr=TRUE, min.overlap=3, correl.type=“z.score”).

Whole-mount immunofluorescence analysis of organoids

Organoids were embedded into Matrigel, and 20µl of droplets were placed into 48-well plate. After three days of culture in ENR medium, the organoids were treated with 10nM trametinib or 100nM AMG510 for 24h. The Matrigel was removed by gentle pipetting in a cell recovery solution (Corning) and replated onto an optically clear flat-bottom 96-well plate (PhenoPlate, PerkinElmer). After centrifugation at 1000×g for 5min, organoids were fixed in 4% PFA in PBS for 30min at room temperature and permeabilized with 0.5% Triton X-100 in PBS for 1h. Rabbit anti-fibrillarin (1:500, ab5821; Abcam) was used as the primary antibody, and cy3 conjugated goat anti-rabbit IgG (1:1000, AP132C; Merck Millipore) was used as the secondary antibody. Cell nuclei were stained with 10µg/mL 4’6-daamidino-2-phenylindole (DAPI; Sigma) in PBS for 15min. For EdU and OPP staining, we used the Click-iT EdU cell proliferation kit and Click-iT Plus OPP protein synthesis kit, respectively, with Alexa Fluor 488 dye (Thermo Fisher Scientific) according to the manufacturer’s instructions. For EU staining, we used the Click-iT Nascent RNA Capture kit with Alex Fluor 594 dye (Thermo Fisher Scientific) according to the manufacturer’s instructions.

High-throughput imaging was performed on a Yokogawa CV8000 instrument equipped with a CSU-W1 spinning disk. Nine fields were scanned for each well with a x4 objective lens, and the images were analyzed using custom-written Cell Pafhfiner software (Yokogawa) to obtain individual organoid positions. Coordinated fields were imaged with x40 objective lens, and three dimension imaging were acquired with 100µm of z-plane and 2µm z-steps.

Immunoblot analysis

Organoids seeded in 24-well plates were harvested after 6-h incubation with trametinib in PTS buffer supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche), followed by sonication (Branson Sonifier, Heinemann). Protein concentrations of supernatants were measured using a BCA assay kit (Thermo Fisher Scientific). Lysates were mixed with an SDS-loading buffer and heated to 100°C for 5min. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% (w/v) skim milk in PBS. Antibodies against PABP1 (x1000, Proteintech 10970-1) and RPL13A (x1000, Proteintech 14633-1-AP), RPL23 (x1000, Proteintech 16086-1-AP), and β-actin (x1000, Sigma A1978) were used. Goat anti-rabbit HRP conjugate (x1000, BioRad 1705046) or Goat anti-mouse HRP conjugate (x1000, BioRad 1705047) were used as secondary antibody. ECL Western blotting substrate (BioRad 1705061) was used for visualization of bands.

Animal procedures

To generate Lgr5-CreERT2；Apc580S/S; KrasG12D/+ double conditional knockouts mice, Apc580S/S; KrasG12D/+ mice were crossed with Lgr5-CreERT2; Apc580S/S mice. For genotyping, DNA was extracted from tail tips, and alleles were genotyped using standard PCR with Taq DNA polymerase (Millipore Sigma). Allele-specific primers are shown in Supplementary Table 4. All mice were 10 weeks old at the start of the experiment. Sex was not considered in the study design or analysis, as preliminary experiments showed no significant sex-dependent differences in the measured outcomes. In vivo recombination was induced by subcutaneous injection of 4-hydroxytamoxifen (4OHT, Sigma-Aldrich) at 40mg/kg/day dissolved in corn oil, followed by three consecutive injections at 20mg/kg/day. Mice were assigned to control group or treated with trametinib (1mg/kg), CX-5461 (100µg/kg) or their combination. Treatment with chemotherapeutic drugs was initiated 13 days after recombination by injection of 40HT and was administered for 5 days until the day before the mice were killed. If the body weight loss exceeded 20% or abnormal behavior was observed, the experiment was terminated in accordance with the institutional guidelines.

For histological analysis, intestines were fixed in 10% neutral-buffered formalin (Wako) for 24–48h and stored in 100% ethanol for 24h. Swiss rolls were dehydrated via successive incubation with 100% ethanol for 2h 7 times and then with xylene for 2h 2 times. Subsequently, they were embedded in paraffin, and sections were cut at 3µm with microtome.

For Immunofluorescence analysis, slides were deparaffinized by successive incubation in 100% xylene four times and were subsequently rehydrated by following incubation; 100% ethanol six times. For heat-induced epitope retrieval, slides were cooked in 10mM sodium citrate (pH 6.0) in a microwave oven for 5min, cooled gently, and rinsed with water. Slides were incubated with rabbit anti-fibrillarin (1:500, ab5821; Abcam) or rat anti-CD44v6 antibody (1:400, MCA1967; BioRad) for overnight at 4°C. Subsequently, the slides were incubated with Cy3-conjugated goat anti-rabbit IgG (1:200, AP132C; Millipore) or Cy5-conjugated anti-rat IgG (1:200, 712-175-153; Jackson) for 1h at room temperature. Cell nuclei were stained with 10µg/mL DAPI in PBS for 30min. For EdU staining, 200µg of EdU was intraperitoneally injected 1h before killing, and the Click-iT EdU cell proliferation kit for imaging, Alexa Fluor 647 dye (Thermo Fisher Scientific) was used to detect EdU-incorporated cells.

For histological analysis, slides were deparaffinized, rehydrated as described above, and stained with H&E for histological analysis. For immunohistochemistry, epitopes retrieval was performed as described above, and slides were incubated with mouse anti-β-catenin (1:1000, 610154, BD Biosciences) overnight at 4°C. The primary antibodies were visualized using the Envision+ system-HRP labeled polymer (DAKO) and 3.3’-diaminobenzidine (DAKO) according to the manufacturer’s instructions. To score the number and size of the adenomatous lesions, images were obtained using an Axio Imager M2 microscope (Zeiss), and tiled images were created and scored using the MosaiX and AutoMeasure Modules provided with the AxioVision 4 program (Zeiss).

Statistical analyses

Drug responses and the results of the linear regression model and Pearson correlation analyses of phosphorylated proteins were analyzed using GraphPad Prism (GraphPad Software, Inc.) and R software packages. Data for each experimental PDO are expressed as the mean±standard error of the mean. Confidence intervals of 95% or more were considered significant. For further statistical details, please refer to the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an excellent manuscript that identifies ribosome biogenesis and inhibitors of RNA polymerase as a novel therapeutic targets for addressing resistance to Ras targeted therapies. Using patient derived organoids the authors convincingly demonstrate that MEK inhibition results in upregulation of ribosome components yet reduced protein synthesis analogous to the phenotype of embryonic stem cells. Various experiments in cells demonstrate synergistic effects of Ras pathway + RNA polymerase inhibition that importantly are confirmed in a mouse model of colorectal cancer in Figure 3k. They also provide some interesting data correlating AMG510 sensitivity to ribosome biogenesis that may provide the basis for future triaging of patients for AMG510 treatment. The interpretation of the results and the discussion were fair and highlighted weaknesses and areas of future work. THe entire manuscript was was written and presented. I have a couple of minor comments:\n1. Drug dosing - the authors need to provide context about whether the doses used are equivalent to those being used in clinical trials.\n2. Provide more detail in Figure 3 legend about the genetics of the Ras driven colorectal cancer model.\n3. Different Ras mutations are not all equally capable of driving oncogenesis. The authors have used KG12A and KG12C patient derived organoids and KG12D mice. Did they observe any differences in the strength of the phenotype or responsiveness with these different mutations? A comment in the discussion would be good.", "reviewer_2": "In the present manuscript Tanaka and coworkers investigate the effect of selected compounds inhibiting MAP-kinase and K-Ras on the behavior and gene expression profile of colon cancer patient derived organioids. They found that these inhibitors strongly decrease cellular protein synthetic activity and upregulate ribosome biogenesis. The use of the selective Pol I inhibitor CX-5461 showed a synergistic effect on patient derived organoids survival.\nOverall this is a comprehensive manuscript using powerful experimental approaches, however, some additional analysis can strongly improve Authors' conclusions.\nMajor Points:\n- In Fig. 1, 2 and 3 the ascribed affects of the treatments on ribosome biogenesis is only implied indirectly. Namely increased levels of a series of mRNA not necessarily implies that a cellular process is unregulated, particularly if global protein synthesis is reduced.\nExperiments assessing the rate of ribosome production are missing. The evaluation of rRNA synthesis and/or of ribosomal protein expression (at the protein level) would strongly support Authors' conclusions.\n- Transcriptomic and phosphoproteomic data (Fig. 1, 2) are never validated, even few representative top deregulated hits or the results of the subsequent functional characterization analysis should be tested individually by independent technical approaches. As an example, in Fig. 2 Authors identify a signature of deregulated proteins at the phosphorylation levels including NOTCH, leptin etc. Confirming this result by Western blot would be relatively easy and would strongly strengthen the findings.\nMinor points:\nPag 2 line 82 POO should be PDO\nPage 4 line 99, a mention to Extended Figure 1A should be added here\nFigure 1 legend: panel G and H appear to be mislabeled. Grand state should spell ground state (check this throughout the manuscript).\nLine 199-200. post-transcriptional should sound post-translational\nline 230 refers to figure 3 b, which drug was used: trametinib or dinaciclib?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "KRAS mutant CRC are a real challenge as these tumoral cells do not respond to KRAS-targeting therapeutic. The manuscript by Tanaka et al. describe the pathways that are relevant when KRAS mut CRC tumours are treated by the MEK inhibitor, trametinib and allele-specific KRAS inhibitor, AMG510. By using PDOs the authors suggest that ribosome biogenesis and Myc expression induced by these treatments are resembling the pluripotent naive ground state of ESCs. The authors also show data of how these inhibitors can act when co-treated with a RNA pol I inhibitor, CX-5461.\nMajor points:\nFigure 1. It shows that a signature of myc-related ribosome biogenesis is induced when PDOs KRAS mut are being treated with trametinib. It is not convincing with these few experiments that is really resembling the pluripotent ground state.\nFigure 2. Phospho-proteomics was done but results are broadly mentioned and further experiments done in organoids is a must.\nFigure 3. Mouse experiments are not convincing and images quality is low and should be better described in the panel. The main question is if all the cells (healthy and cancer) are dying because of toxicity? the histology of both treatments in the intestine looks as fatal loss of intestinal homeostasis. Cancer cells are poorly detected even in all the images because of low quality.\nFigure 4. Data shown is poor considering what was collected using this methodology. This also need to be improved to be convincing and also to include biological data with PDOs showing increased ribosome biogenesis.\nFigure 5. Only one PDOs mutant was responding (1 out of 5?), what is happening to the non-responders? Synergistic effect data is only shown in 5e of this figure and the heatmaps do not show one treatment only, just both with different concentrations. This is not convincing and again points out that healthy cells are affected when both reagents are co-treated.\nAbstract needs to be re-written to describe what the paper data is showing.\nTitle is also not representing the data shown.\nMinor points:\n1. lane 33 and 34, the sentence \"KRAS targeting therapeutic agents are not very effective against CRC\", this seems very general and more rigorous statement needs to be written.\n2. lane 82, POOs instead of PDOs\n3. lane 95, \"previously reported CRC markers\", it is not clear how was it done.\n4. lane 100, what about the other clusters?\n5. lane 141, very broad writing \"CRC cells adopt an ...\", as only some type of CRC cells.\n6. lane 146, why first writing K-RAS mut and wt and after the HCT24-14 and HCT24-8? should always be called the same name.\n7. lane 164, what type of cell lines?\n8. lane 168, more information should be added about the hours that cells were treated.\n9. lane 192, complete name of NPM1 and NCL.\n10. lane 212 and 213, FDR and NES information to be added so it can be comparable to the ones mentioned before.\n11.lane 241, \"GOCC terms\" quite general, can be written some details?\n12. lane 256, PDOs were done here, so it can be written in the text.\n13. lane 295, the clone HCT248P needs to be introduced if was not done before.\n14. lane 330, only the inhibition was in Ras mutants.\n15. More details about how RNAseq was done in methods.\n16. Specify what is included in ENR medium.\n17. lane 571, the \"generated compound\", looks like is a typo."}, "author_response": "We thank the reviewers for their comments and insights, which have helped improve the manuscript.\nPoint-by-point rebuttal is presented below. Reviewers’ comments are in BLACK, and authors’\nresponse are in BLUE. The revisions in the manuscript are indicated in RED.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nThis is an excellent manuscript that identifies ribosome biogenesis and inhibitors of RNA polymerase\nas a novel therapeutic targets for addressing resistance to Ras targeted therapies. Using patient derived\norganoids the authors convincingly demonstrate that MEK inhibition results in upregulation of\nribosome components yet reduced protein synthesis analogous to the phenotype of embryonic stem\ncells. Various experiments in cells demonstrate synergistic effects of Ras pathway + RNA polymerase\ninhibition that importantly are confirmed in a mouse model of colorectal cancer in Figure 3k. They\nalso provide some interesting data correlating AMG510 sensitivity to ribosome biogenesis that may\nprovide the basis for future triaging of patients for AMG510 treatment. The interpretation of the results\nand the discussion were fair and highlighted weaknesses and areas of future work. THe entire\nmanuscript was was written and presented. I have a couple of minor comments:\nWe thank the reviewer for recognizing the significance of our work.\n1. Drug dosing - the authors need to provide context about whether the doses used are equivalent to\nthose being used in clinical trials.\nWe thank the reviewer for this insightful comment. We used a dose of 0.1mg/kg of CX-5461 to\nevaluate the combination effects with trametinib, which is a significantly lower concentration\ncompared to the previously reported 35-50mg/kg used to assess the efficacy of CX-5461 alone1,2.\nAccording to the reviewer’s suggestion, we calculated human equivalent dose (HED) using the\ninterspecies allometric scaling for dose conversion3, and found it to be equivalent to 0.28 mg/m2. In\nthe first-in-human dose-escalation study of CX-5461, patients were treated at doses ranging from 25\nto 450 mg/m24. In the multicenter phase I trial involving patients with solid tumors, doses ranged from\n50 to 650 mg/m25. Thus, it appears that the estimated HED are lower than those being used in clinical\ntrials, suggesting that a low dose of CX-5461 is sufficient for the combination therapy with trametinib.\nAlthough allometric scaling is an empirical approach that assumes unique characteristics in anatomical\nand physiological processes among species6,we believe that these results strengthen our claim that\nCX-5461 is a potential candidate for combination therapy with KRAS inhibition.\nIn the revised manuscript, we added these points in the discussion (page 11 line 338-345)\n2. Provide more detail in Figure 3 legend about the genetics of the Ras driven colorectal cancer model.\nWe thank the reviewer for pointing out the insufficient description. In response to Reviewer 4, figures\nrelated to the mouse model of colorectal cancer was moved to Fig. 4. Mouse genotypes were explicitly\nshown in the figures, and the genetics was described in the figure legends. They are conditional\nknockout mice, and the detailed experimental procedure for introducing mutations is described in\nmaterials and methods (page 22 line 676-678).\n3. Different Ras mutations are not all equally capable of driving oncogenesis. The authors have used\nKG12A and KG12C patient derived organoids and KG12D mice. Did they observe any differences in\nthe strength of the phenotype or responsiveness with these different mutations? A comment in the\ndiscussion would be good.\nWe agree with the reviewer that different RAS mutations exhibit varying strength of the phenotype\nand the responsiveness against chemotherapeutic agents. This is exemplified by KRAS G13D\nmutation, which has been clinically proven to be sensitive to cetuximab7. Two proteomic studies\nreported mutation specific phosphorylation signature between KRAS G12D and G13D8,9. However,\nin this study, we did not directory compare the differences among the distinct mutations. In the context\nof the recent development of mutation-specific and pan-mutant KRAS inhibitors, it is of particular\ninterests to clarify the different response among the mutations. We believe that establishing organoids\nwith different KRAS mutations and conducting comparative analyses will be highly important\nexperiment in the future.\nAs the reviewer suggested, we included these points in the discussion (page16 line483-491).\nReviewer #2 (Remarks to the Author):\nIn the present manuscript Tanaka and coworkers investigate the effect of selected compounds\ninhibiting MAP-kinase and K-Ras on the behavior and gene expression profile of colon cancer patient\nderived organioids. They found that these inhibitors strongly decrease cellular protein synthetic\nactivity and upregulate ribosome biogenesis. The use of the selective Pol I inhibitor CX-5461 showed\na synergistic effect on patient derived organoids survival.\nOverall this is a comprehensive manuscript using powerful experimental approaches, however, some\nadditional analysis can strongly improve Authors' conclusions.\nWe thank the reviewer for the overall positive feedback.\nMajor Points:\n- In Fig. 1, 2 and 3 the ascribed affects of the treatments on ribosome biogenesis is only implied\nindirectly. Namely increased levels of a series of mRNA not necessarily implies that a cellular process\nis unregulated, particularly if global protein synthesis is reduced.\nExperiments assessing the rate of ribosome production are missing. The evaluation of rRNA synthesis\nand/or of ribosomal protein expression (at the protein level) would strongly support Authors'\nconclusions.\nThank you for pointing out such highly relevant and important points, which we have followed to\nstrengthen our conclusion of ribosome biogenesis. In Fig. 1, we evaluated the protein levels of\nribosomal genes by immunoblot analysis, and demonstrated increased protein levels of genes\nidentified through GOCC enrichment analysis following trametinib treatment (new Fig. 1e, page4\nline118-121). According to the reviewer’s suggestion, we have performed 5-ethylnyluridine (EU)\nlabeling to detect newly transcribed RNA, and this new analysis clearly show that CX-5461 reduces\nrRNA synthesis in CRC organoids, as shown by substantial decrease of EU signal in the nucleoli (new\nFig. 3i and j, page10 line300-303). Overall, we believe these additional data validated the RNA-seq\ndata.\ncontroltrametinib\nPABP1\nRPL13A\nPRL23\nb-actin\nFig.1e Immunoblot analysis of ribosomal proteins. Protein levels of indicated proteins in\ntrametinib treated (trametinib) and untreated (control) PDOs were evaluated. b-actin was shown\nas the loading control.\ni j\n75\n50\n25\n0\ncontrol CX−5461\nEU/DAPI\nFig. 3. i. Reduced EU incorporation in CX-5461-treated CRC organoids. Representative images\nof EU (red) and nuclei (blue) in control and CX-5461-treated organoids are shown. High\nmagnification view of the white boxed regions are presented in the insets. Bar = 50µm, Bar in\ninset = 10µm. j. The percentage of EU-positive nuclei per organoid is shown in the boxplot.\nData of 20 PDOs obtained from four independent experiments are shown. **p < 0.01.\n- Transcriptomic and phosphoproteomic data (Fig. 1, 2) are never validated, even few representative\ntop deregulated hits or the results of the subsequent functional characterization analysis should be\ntested individually by independent technical approaches. As an example, in Fig. 2 Authors identify a\nsignature of deregulated proteins at the phosphorylation levels including NOTCH, leptin etc.\nConfirming this result by Western blot would be relatively easy and would strongly strengthen the\nfindings.\nWe validated the transcriptomic data of three representative up-regulated ribosome protein via\nimmunoblot as mentioned above (new Fig. 1e). Among 8354 phosphorylation sites identified in\nproteome analysis, only 489 sites have shown functional evidences. We assessed the phosphorylation\nstate of ribosomal protein S6, a commonly used read out of mTORC1 activity, because it is a major\nregulator of protein synthesis. Consistent with the observation indicating the reduced protein synthesis\nfollowing trametinib treatment (Fig. 1k), immunoblot analysis showed significant reduction of\nSer235/Ser236 and Ser240/244 confirming the phosphoproteomic data. Additionally, key regulator of\ncell proliferation, Ser807/Ser811 of RB1 and Ser727 of STAT1, as well as primary mediator of\ntranscriptional response to proteotoxic response, Ser303/Ser307 of HSF1, were decreased, further\nconfirming the phosphoproteomic data. Unfortunately, no antibodies against phosphorylation sites\nrelated to NOTCH or leptin were available. However, we believe that the consistency between the\nphosphorylation analysis by immunoblot and the phophoproteome analysis validate our findings.\nThe results of immunoblot and the heatmap showing phosphoproteomic data of corresponding\nphosphorylation sites were shown in Extended Data Fig. 2e and f, respectively, and text was amended\naccordingly (page8 line 245-page9 line256).\ne f\nRPS6_S235/S236\nRPS6_S240/S244\nRB1_S807/S811\nStat1_S727\nHSF1_S303/S307\nGAPDH\nExtended Data Fig. 2. e. Heatmap showing phosphorylation levels of representative proteins.\nf. Immunoblot analysis of the proteins shown in (e). Phosphorylation at the indicated sites\nwas evaluated using phospho-specific antibodies. GAPDH is shown as the loading control.\nMinor points:\nPag 2 line 82 POO should be PDO\nThank you for pointing out this typo. We amended the spelling.\nPage 4 line 99, a mention to Extended Figure 1A should be added here\n)%(\nsllec\nevitisop\nUE\nText was amended.\nFigure 1 legend: panel G and H appear to be mislabeled. Grand state should spell ground state (check\nthis throughout the manuscript).\nThank you for pointing out the mislabel. We fixed them in fig. 1j and k. We have confirmed that the\nspelling of “ground state” is consistent throughout the manuscript.\nLine 199-200. post-transcriptional should sound post-translational\nThank you for pointing out the important mistake. We have corrected it.\nline 230 refers to figure 3 b, which drug was used: trametinib or dinaciclib?\nThank you again for pointing out the important mistake. We have collected ‘trametinib’ to ‘dinaciclib’.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nKRAS mutant CRC are a real challenge as these tumoral cells do not respond to KRAS-targeting\ntherapeutic. The manuscript by Tanaka et al. describe the pathways that are relevant when KRAS mut\nCRC tumours are treated by the MEK inhibitor, trametinib and allele-specific KRAS inhibitor,\nAMG510. By using PDOs the authors suggest that ribosome biogenesis and Myc expression induced\nby these treatments are resembling the pluripotent naive ground state of ESCs. The authors also show\ndata of how these inhibitors can act when co-treated with a RNA pol I inhibitor, CX-5461.\nWe would like to thank the reviewer for critical reading of our manuscript.\nMajor points:\nFigure 1. It shows that a signature of myc-related ribosome biogenesis is induced when PDOs KRAS\nmut are being treated with trametinib. It is not convincing with these few experiments that is really\nresembling the pluripotent ground state.\nWe thank the reviewer for raising this critical point. We did not make any claims as to whether CRC\ncells have acquired pluripotency after trametinib treatment. We argue that there is a similarity in the\ngene expression profile between trametinib-treated CRC organoids and ground-state ESCs and\nepiblasts. In addition, the accumulation of fibrillarin in the nucleolus and the decrease in OPP\nincorporation reproduced two hallmark features of the ground state: enhanced ribosome biogenesis\nand decreased global protein synthesis. Based on these observations, we suggested the similarities in\nribosome biogenesis and translation, but did not assert the pluripotency. We revised the text to avoid\nexpressions that might give the misleading impression that CRC cells could acquire pluripotency\n(page4 line123, page4 line125 and page12 line371).\nIn response to the reviewer’s concern, we further evaluated the cell state using two approaches. First,\nwe analyzed the expression of genes that were significantly upregulated in the mouse ground state\nepiblast. In line with our hypothesis, these genes were confirmed to be significantly highly expressed\nin C1 (p_val_adj < 0.05) (new Fig. 1h, page5 line 141-143). Interestingly, this analysis identified three\ngenes, including SLC12A2, PDGFA and RPL34, were also found to be significantly highly expressed\nin C5 (p_val_adj < 0.05, page5 line143-147). Considering that C5 is absent in trametinib-free medium\nand is induced by trametinib treatment, these new gene-based analysis provided additional evidence\nindicating the similarity between trametinib-induced cell state of CRC and ground state epiblast. Based\non these results, we calculated the gene score for ribosome biogenesis feature, and found C1 and C5\nexhibited higher score compared to other clusters (new Extended Fig. 1e, page5 line 147-148).\nSecondly, we focused on ribosome-related genes that are highly expressed in human ESC10,11. The\nheatmap demonstrated that they were highly expressed in C1 (new Fig. 1i, page5 line149-157 ). We\nnoticed that RPL13A was also upregulated in trametinib treated fraction of C3. We believe these\ncomparative analyses of human ESCs further support the notion of the similarity in the cell state\nbetween trametinib treated CRC organoids and the ground state epiblast. Additionally, we\ndemonstrated that trametinib reduced the global protein synthesis (Fig. 1k) and reduced cell\nproliferation rate (Fig. 1l), which are hallmark of pre-implanted epiblast. We believe these\nobservations demonstrate the significant similarity between trametinib-induced cell state of CRC\norganoids and the ground state embryonic cells.\nh\ni\nFig. 1. h. Violin plot depicting the expression of ground state genes in each cluster in CRC\norganoids. The expression of gene sets identified in the enrichment analysis of ground state\nESCs (f) and epiblasts (g) were shown. i. Heatmap of genes known to have high expression\nlevels in ESC.\nFigure 2. Phospho-proteomics was done but results are broadly mentioned and further experiments\ndone in organoids is a must.\nAccording to the reviewer’s suggestion, we analyzed the phosphorylation state of key molecules\ninvolved in mTOR signaling, because it plays central roles in the protein translation. PTMsigDB\nanalysis identified two KINASE-PSP pathways, p90RSK/RPS6KA3 and RSK2/RPS6KA3, as\nnegatively enriched pathways in response to trametinib treatment (Fig. 2h). The phosphorylation of\nSer235/Ser236 and Ser240/Ser244 of RPS6, a commonly used as a readout of the mTOR signaling,\nwere negatively enriched in trametinib treated CRC organoids in phospho-proteomics analysis (new\nExtended Data Fig. 2e). Additionally, the phosphorylation of two mTOR-linked molecules including\nHSF1 Ser303/Ser307 and STAT1 S727 were also negatively enriched12,13. The reduced\nphosphorylation of these molecules was validated with immunoblot analysis using phospho-specific\nantibodies (new Extended Data Fig. 2f). These results explain the reduced global protein synthesis\nafter trametinib treatment. We described these results in the text (page8 line240-page9 line256)\nFigure 3. Mouse experiments are not convincing and images quality is low and should be better\ndescribed in the panel. The main question is if all the cells (healthy and cancer) are dying because of\ntoxicity? the histology of both treatments in the intestine looks as fatal loss of intestinal homeostasis.\nCancer cells are poorly detected even in all the images because of low quality.\nThank you again for the critical comments. We apologize the low quality of the provided images in\nthe manuscript, which is due to the significant deterioration of the images when they were consolidated\ninto a single file during the initial submission. In the revised manuscript, we reorganized mouse\nexperiments data into a new standalone Figure 4. Each panel was enlarged to create a clear and more\ncomprehensive illustration. In immunofluorescence images, tumor cells were identified by CD44v6\n(Fig. 4a and e) or b-catenin staining (Fig. 4c and g). The nucleolar staining of fibrillarin in tumor cells\nis shown in the insets (Fig. 4a).\nIn the initial submission, we only analyzed tumor tissues, because the primary aim of this work is to\npresent a finding on the previously unrecognized cellular state of intestinal tumor cells after RAS\ninhibition. However, we do agree that the toxic effects to healthy cells is a critical issue to consider for\nclinical applications, so we have tried to address this issue as much as possible. We additionally\nperformed the immunofluorescence analysis of normal tissues, and found trametinib treatment does\nnot significantly altered either fibrillarin intensity (new Fig. 4e and f) or EdU incorporation (new Fig.\n4g and h). These results may support the notion that normal tissues are less sensitive to trametinib\ntreatment than tumor tissue. We evaluated the effects of trametinib and CX-5461 by measuring size of\nvilli (new Fig. 4k). CX-5461 did not significantly affected, but trametinib and combination of\ntrametinib and CX-5461 reduced the size by 80.8% and 79.7%, respectively (new Fig. 4l). Thus,\ntrametinib monotherapy exhibit inhibitory effects on normal tissue, but no synergistic effect with CX-\n5461 was observed. Importantly, the combination of trametinib and CX-5461demonstrates\nsignificantly higher inhibitory effects on tumor tissues compared to normal tissue. We believe these\nresults support the effectiveness of the combination of trametinib an CX-5461 as a novel therapeutic\nstrategy for CRC (page 11 line 332-335).\ne\n11000\n10000\n9000\n8000\n7000\n6000\ncontrol trametinib\nFig.4, e,f. Fibrillarin expression in normal intestine. Representative images of fibrillarin (magenta),\nnuclei (cyan), and CD44v6 (gray) in normal intestine from control and trametinib-treated mice are\nshown. Bar =50 µm, Bar in insets = 10µm. The intensity of fibrillarin signals in each nucleus per\ntumors is shown in the boxplot. Data obtained from four independent experiments are shown. **p\n< 0.01, t. test. g,h. EdU incorporation in trametinib-treated mouse intestine. Representative\nimages of incorporated EdU (gray), b-catenin (green), and nuclei (blue) in normal intestine from\ncontrol and trametinib-treated mice are shown. Bar =100 µm. The percentage of EdU-positive nuclei\nper villus is shown in the boxplot. Data obtained from four independent experiments. **p < 0.01, t.\ntest. k-l. Evaluation of normal intestin in trametinib- and CX-5461-treated mice. Representative HE\nimages of mouse intestine treated with the indicated drugs are shown (k). Bar =100 µm. Villi size\nis shown in the bar plot (j). Average and standard deviations of six independent experiments are\nshown. **p < 0.01, *p < 0.05, Brown-Forsythe test.\nFigure 4. Data shown is poor considering what was collected using this methodology. This also need\nto be improved to be convincing and also to include biological data with PDOs showing increased\nribosome biogenesis.\nIn response to the reviewer’s criticism, we further analyzed scRNA-seq data. We included the\nproportion plot to highlight that the percentage of C2 cluster in HCT191-1T is higher than in other\norganoids (new Fig. 5c). The heatmap of genes identified in GOCC enrichment analysis clearly\nindicated the increased expression of ribosomal protein genes in HCT191-1T (new Fig. 5e).\nFurthermore, we calculated the gene score for ribosome biogenesis in each cell and, C2 showed a high\nvalue (new Fig. 5f). Interestingly, this analysis newly revealed that ribosome biogenesis is also\nenhanced in C5. Considering that the proportion of C5 is higher in HCT191-1T compared to other\norganoids (new Fig. 5c), these findings support the correlation between ribosome biogenesis and\n)tinu.bra(\nytisnetni\nbiF\nf g\ncontrol trametinib 8\n6\n4\n)%(\nsllec\nevitisop\nUdE\nh\ncontrol trametinib\nk l\ncontrol trametinib CX-5461 combo\n300\n200\n100\n0\ncontrol trametinibCX−5461 combo\ntreatment\n)mµ(\nilliv\nfo\nezis\n**\n** ** **\nresponse to AMG510 (page12 line362-368).\nAccording to the reviewer’s suggestion, we performed two biological experiments. 5-ethyluridine\n(EU) labeling detects increased transcription of ribosome DNA in HCT191-1T (new Fig. 5i and j).\nFibrillarin staining indicated its increased accumulation at nucleoli (Fig. 5k and l). Both experiments\ndemonstrated that HCT191-1T not only exhibit an increase expression of genes encoding ribosomal\nprotein, but also enhance ribosome RNA expression and its modification. We believe these additional\ndata further support our notion that HCT191-1T increased ribosome biogenesis (page12 line377-\npage13 line383).\ne\nc\nHCT191−1T HCT128−1T HCT202−1THCT215−1T\nC1 f ** **\nC2\nCCC 345\ni j k l\nHCT128-1T HCT191-1T HCT128-1T HCT191-1T\nHCT202-1T HCT215-1T HCT202-1T HCT215-1T\nEU DAPI Fib DAPI\nFig. 5. c. Proportion of cells in each cluster of CRC organoids. e, f. Heatmap (e) and boxplot (f)\ndepicting the different expression levels of genes listed in cytosplic ribosome of GOCC among\nCRC organoids. **p < 0.01 I and j. Different EU incorporation rate among CRC organoids.\nRepresentative images of EU (magenta) and nuclei (cyan) of indicated organoids are shown (i).\nBar = 10µm. The intensity of EU signals in each cells is shown in violin plot (j). Data of 2000\ncells obtained from four independent experiments are shown. **P < 0.01. k and l. accumulation\nof fibrillarin in nucleolus. Representative images of fibrillarin (magenta) and nuclei (cyan) of\nindicated organoids are shown (k). Bar = 10µm. The intensity of fibrillarin signals in each cells is\nshown in violin plot (l). Data of 2000 cells obtained from four independent experiments are\nshown. **P < 0.01.\nFigure 5. Only one PDOs mutant was responding (1 out of 5?), what is happening to the non-\nresponders? Synergistic effect data is only shown in 5e of this figure and the heatmaps do not show\none treatment only, just both with different concentrations. This is not convincing and again points out\nthat healthy cells are affected when both reagents are co-treated.\nAs noted by the reviewer, only one PDO responded to AMG510. In the first clinical trial of sotorasib,\na partial response was observed in three out of 42 CRC patients14. More recently, the objective\nresponse was observed in six out of 62 CRC patients15. Thus, the response rate of PDOs appears to\nreflect the efficacy of clinical trials.\nNon-responders exhibited reduced EdU incorporation, but the reduction was less pronounced\ncompared to responders (Fig. 6a). This difference is thought to reflect the distinct responses of CRC\norganoids to AMG510. Two hallmarks of ground state ESCs, including the increased transcription of\nribosome genes (Fig. 6d) and the decreased global protein synthesis (Fig. 6b), were observed in the\nnon-responders. To further evaluate the response of non-responders, we performed fibrillarin staining\nand found the elevated intensity (new Fig. 6c and new Extended data Fig. 6c). Based on these\nobservations, we concluded that AMG510 induced KRAS G12C-mutant CRC organoids into a cellular\nstate resembling ground-state ESCs, mirroring the effect observed in KRAS-mutant organoids treated\nwith trametinib, as shown in Fig. 1 (page13 line398-401).\nAccording to the reviewer’s comment, we have added the dose-effect curve for CX-5461 alone (new\nFig. 6f) and mentioned in the text (page 14 line 413-414). The dose-effect curves for AMG510 is\nshown in Figure 4a. Chou-Talalay methods is widely used in the evaluation of chemotherapeutic\nagents16. We presented the results by Fa-CI plot for the constant ratio evaluation and by heatmap for\nthe non-constant ratio evaluation, because this method is based on the median-effect equation and\nprovides common link between single entity and multiple entities, respectively.\nTo establish organoids, we adopted the protocol developed by Sato and Cleavers group, which have\nbeen widely used for establishing PDOs17,18. This culture condition is designed to exclude the growth\nof normal colorectal cells, ensuring that the readout of this analysis specifically represented the\nresponse of tumor cells. Unfortunately, normal organoids have not been established from these patients.\n800\n600\n400\ncontrol AMG510\nFig. 6. c. Increased fibrillarin expression in AMG510-treated KRAS G12C CRC organoids. The\nintensity of fibrillarin signals in each nuclei per organoids is swhon in the boxlot. Data of 20\norganoids obtaind from four independent experiments are shown. **p < 0.01, *p < 0.05. f. Response\nof KRAS G12C organoids to CX-5461. Cell viabilities of organoids were evaluated at 72 hours\nafter treatment with indicated concentarations of CX-5461. Mean ± standard deviation from four\nindependent experiments is shown.\nAbstract needs to be re-written to describe what the paper data is showing.\nTitle is also not representing the data shown.\nWe appreciate the feedback.\nWe substantially edited the abstract to improve the clarity of our findings. Title was revised to more\naccurately reflect the content of the manuscript.\nMinor points:\n1. lane 33 and 34, the sentence \"KRAS targeting therapeutic agents are not very effective against CRC\",\nthis seems very general and more rigorous statement needs to be written.\nTo provide a more specific description of the response rate for CRC, we cited the report of phase II\n)tinu.bra(\nytisnetni\nbiF\nHCT191−1T\n** 700 600\n500 400\n300\n200\ncontrol AMG510\n)tinu.bra(\nytisnetni\nbiF\nHCT128−1T\n* 500\n400\n300\n200\ncontrol AMG510\n)tinu.bra(\nytisnetni\nbiF\nc\nHCT215−1T\n** 600\n400\n200\n0\ncontrol AMG510\n)tinu.bra(\nytisnetni\nbiF\n1.5\nHCT202−1T\n** 1.0\n0.5\n0.0\n100 101 102 103 104\nconcentration(nM)\nytilibaiV\nlleC\nf\nHCT128-1T\nHCT191-1T\nHCT202-1T HCT215-1T\nc\nHCT128-1T HCT191-1T HCT202-1T HCT215-1T\ncontrol\nAMG510\nExtended Fig. 6c. Fibrillarin was visualized by immune fluorescent staining (c). Bar = 50 µm.\nclinical trial and included information on ORR and PFS as follows:\n“A phase 2 clinical trial of KRAS G12C allele specific inhibitor, AMG510, in colorectal cancer (CRC)\nreported an objective response rate of 9.7% and a median progression-free survival of 4.0 months15”\n2. lane 82, POOs instead of PDOs\nAmended.\n3. lane 95, \"previously reported CRC markers\", it is not clear how was it done.\nWe amended the sentence as below.\n“Each cluster was initially annotated by calculating the p-value of overlaps using Fisher’s exact test\nbetween the differentially expressed genes (DEGs) of each cluster and CRC markers reported\npreviously”.\n4. lane 100, what about the other clusters?\nWe added following sentences.\n“C3 and C4 were annotated as absorptive cells and transit-amplifying cells, respectively, with their\nproportions remaining relatively unchanged. C5 was annotated as goblet cells, which were absent\nunder untreated conditions but was induced following trametinib treatment.”\n5. lane 141, very broad writing \"CRC cells adopt an ...\", as only some type of CRC cells.\nWe amended the sentence as follows.\n“Collectively, these observations suggest that some trametinib-tolerant KRAS mutant CRC cells are\ninduced into an embryonic ground-state-like cellular state by trametinib treatment.”\n6. lane 146, why first writing K-RAS mut and wt and after the HCT24-14 and HCT24-8? should\nalways be called the same name.\nWe have corrected the points and standardized the terminology.\n7. lane 164, what type of cell lines?\nThey are isogenic established cell lines with various KRAS mutations. Specific cell names and genetic\nmutations have been included.\n8. lane 168, more information should be added about the hours that cells were treated.\nThe treatment time points have been included.\n9. lane 192, complete name of NPM1 and NCL.\nComplete names have been included.\n10. lane 212 and 213, FDR and NES information to be added so it can be comparable to the ones\nmentioned before.\nFDR and NES have been included.\n11.lane 241, \"GOCC terms\" quite general, can be written some details?\nGOCC (Gene ontology cellular components) is explained at its first mention on page 4 line110.\n12. lane 256, PDOs were done here, so it can be written in the text.\nWe added PDOs into the relevant section.\n13. lane 295, the clone HCT248P needs to be introduced if was not done before.\nThis is a typo for HCT24-8. We have corrected it.\n14. lane 330, only the inhibition was in Ras mutants.\nWe have added “in KRAS-mutant CRC organoids” to the relevant sentence.\n15. More details about how RNAseq was done in methods.\nThe details of RNA-seq analysis have been described (page19 line576-585)\n16. Specify what is included in ENR medium.\nENR has been explained in the first instance on page17 line528.\n17. lane 571, the \"generated compound\", looks like is a typo.\nThis is not typo, but it has been corrected as follows to more accurately represent the genotype of the\nmutant mouse.\n“The generated Apc580S/S, Kras G12D/+ double conditional knockouts bearing Lgr5-CreERT2,\nApc580S/S, Kras G12D/+ were crossed with a homozygous background of Apc580S/S bearing Lgr5-\nCreERT2.”\nReferences for point-by-point response\ntargeting of ribosome biogenesis. EMBO J 39, e105111 (2020).\nhttps://doi.org/10.15252/embj.2020105111\nand vulnerability to senolytics in colorectal cancer cells. Mol Oncol 16, 2788-2809 (2022).\nhttps://doi.org/10.1002/1878-0261.13265\nhuman. J Basic Clin Pharm 7, 27-31 (2016). https://doi.org/10.4103/0976-0105.177703\nPatients with Advanced Hematologic Cancers: Results of a Phase I Dose-Escalation Study.\nCancer Discov 9, 1036-1049 (2019). https://doi.org/10.1158/2159-8290.CD-18-1455\nadvanced solid tumors enriched for DNA-repair deficiencies. Nature communications 13,\n3607 (2022). https://doi.org/10.1038/s41467-022-31199-2\nusing experimental approaches during early drug discovery. Curr Opin Chem Biol 5, 452-\n463 (2001). https://doi.org/10.1016/s1367-5931(00)00228-3\nchemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 304,\n1812-1820 (2010). https://doi.org/10.1001/jama.2010.1535\ndistinct activating KRAS mutations. J Proteome Res 14, 1535-1546 (2015).\nhttps://doi.org/10.1021/pr501191a\nG12D and G13D Alleles. J Proteome Res 20, 670-683 (2021).\nhttps://doi.org/10.1021/acs.jproteome.0c00587\nEmbryonic Stem Cell Pluripotency and Differentiation. Cell stem cell 22, 543-558 e512\n(2018). https://doi.org/10.1016/j.stem.2018.03.002\nof human embryonic stem cells as defined by SAGE. Stem cells 22, 51-64 (2004).\nhttps://doi.org/10.1634/stemcells.22-1-51\nthe anabolic malignant state. Science 341, 1238303 (2013).\nhttps://doi.org/10.1126/science.1238303\nresponse to heat shock in mammalian cells. Nature 440, 556-560 (2006).\nhttps://doi.org/10.1038/nature04518\nEngl J Med 383, 1207-1217 (2020). https://doi.org/10.1056/NEJMoa1917239\nmutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial. Lancet\nOncol 23, 115-124 (2022). https://doi.org/10.1016/S1470-2045(21)00605-7\nTalalay method. Cancer Res 70, 440-446 (2010). https://doi.org/10.1158/0008-\n5472.CAN-09-1947\nmesenchymal niche. Nature 459, 262-265 (2009). https://doi.org/10.1038/nature07935\nadenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).\nhttps://doi.org/10.1053/j.gastro.2011.07.050"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have no further comments. The authors responded to all of my comments (Reviewer 1) with suitable edits made to the manuscript.", "reviewer_2": "The Authors responded to all my previous concerns, I don't have further requests.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I would like to thank the authors for their responses and the additional experiments conducted to address the queries. While the manuscript has been improved, there are still some concerns that have not been fully addressed.\nFig. 1\nThe statement that CRC cells resemble an embryo-ground state is not convincing. Are the analyses in Figure 1 conducted on HCT24-8 and HCT24-14, or on other CRC PDOs? How many were analyzed? This crucial information is missing. A significant conclusion is drawn from this analysis, but if it was performed on only one control and one RASmut PDO, the data would be insufficient to support such a claim. Additionally, while HCT191 cells were treated, only the Fib marker and ribosome biogenesis were assessed.\nThe gene analysis in Figure 1h is not convincing, are these differences statistically significant? The protein blots in Figure 1e also lack clarity. How many replicates were performed? Was there any quantification? Visually, the upregulation of PRL23 and RPL13A needs stronger validation.\nLastly, Figure 1i lacks any quantification visible or clear enough, making it an inadequate basis for drawing conclusions about gene regulation.\nSimilarly, the immunostainings in Figures 1j and 1l are not convincing. Can the authors provide representative images that correspond to the quantification shown below?\nFig. 2\nThe phospho-proteomics data has been improved in terms of signalling pathways; however, further refinements are needed to make the findings more convincing. Specifically, the data should more clearly demonstrate how trametinib (timing) affects ribosome biogenesis in both control and PDO mutant models. Experiments of treated cells (timing) are only shown in the PDO mutant.\nFig.3 and 4\nThe authors claim a synergistic effect of trametinib with dinaciclib and trametinib with CX-5461. However, they only test the PDO mutant and not the healthy control. Only Figures 3a and 3b address this statement for dinaciclib, and Figures 3e and 3f for CX-5461. The rest of the data focuses on single-compound treatments rather than their synergy, making the findings less novel. It remains possible that both compounds are generally toxic to all PDO types rather than specifically synergistic.\nThe synergy is more convincingly demonstrated in Figure 4, which presents in vivo data, but only for CX-5461—there is no in vivo data for dinaciclib. Were all wild-type and Apc-Ras-Lgr5 mice analyzed at day 5? Can the authors provide additional images of the combination-treated WT mice? Furthermore, could they examine embryonic-ground state markers in these in vivo experiments? It is unclear why the study focuses solely on villi size without assessing any markers related to ribosome biogenesis or the embryonic ground state, which would strengthen the conclusions.\nImmunostainings have been improved.\nFig. 5 and 6\nThe experiments presented focus on another mutant, G12C, but only include ribosome biogenesis data and Fib stainings, which are insufficient to support a definitive conclusion. The data for the combination of CX-5461 and AMG510 is limited to Figures 6g and 6h\nThe abstract and title are still not clear.\nTitle: CRC cells adopt the embryo-ground state in response to KRAS inhibition\nThe embryo-ground state was observed on few PDOs RAS mut\nThe embryo-ground state data can be improved.\nAbstract\n\"synergistic anti-tumor effect with trametinib and AMG510 in .. mouse model and human PDOs..\"\nonly was done in vivo with trametinib and CX.\n\"CRC cells adopt a cellular state..\"\na few PDOs RAS mut were done not CRC cells in general. It is confusing including all CRC cells\nembry-ground state was observed in few experiments.\n\"these observations demonstrated that CRC cells become tolerant to K-RAS inhibition by exploiting the cellular program of early embryogenesis..\"\nagain too general writing about CRC cells, as only was observed in PDOS RAS mut."}, "author_response": "We thank the reviewers for their comments and insights, which have helped improve the\nmanuscript. Point-by-point rebuttal is presented below. Reviewers’ comments are in\nBLACK, and authors’ response are in BLUE. The revisions in the manuscript are\nindicated in RED.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI have no further comments. The authors responded to all of my comments (Reviewer 1)\nwith suitable edits made to the manuscript.\nReviewer #2 (Remarks to the Author):\nThe Authors responded to all my previous concerns, I don't have further requests.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #4 (Remarks to the Author):\nI would like to thank the authors for their responses and the additional experiments\nconducted to address the queries. While the manuscript has been improved, there are\nstill some concerns that have not been fully addressed.\nWe would like to sincerely thank reviewer #4 for the thorough and thoughtful review.\nFig. 1\nThe statement that CRC cells resemble an embryo-ground state is not convincing. Are\nthe analyses in Figure 1 conducted on HCT24-8 and HCT24-14, or on other CRC\nPDOs? How many were analyzed? This crucial information is missing. A significant\nconclusion is drawn from this analysis, but if it was performed on only one control and\none RASmut PDO, the data would be insufficient to support such a claim. Additionally,\nwhile HCT191 cells were treated, only the Fib marker and ribosome biogenesis were\nassessed.\nGiven the substantial expense of scRNA-seq, we limited the analysis to HCT24-8. In\nFigure 6, we performed bulk RNA-seq on four PDOs and confirmed that KRAS\ninhibition led to increased Myc-dependent transcriptional activity. Furthermore,\nenrichment of the ribosome signature was observed in KRAS inhibitor-tolerant clones\nafter drug treatment, supporting our conclusions.\nNevertheless, we understand the reviewer’s concern regarding the limited number of\nsamples in this figure. Therefore, we have avoided to generalize the findings from this\nanalysis. We also revised the conclusions regarding the embryonic ground state and\namended the text as follows (page6, line 171-173) :\nCollectively, these observations suggest that trametinib induces a cellular state in\nKRAS-mutant colorectal cancer PDOs in which Myc-dependent ribosome biogenesis is\nupregulated.\nThe gene analysis in Figure 1h is not convincing, are these differences statistically\nsignificant? The protein blots in Figure 1e also lack clarity. How many replicates were\nperformed? Was there any quantification? Visually, the upregulation of PRL23 and\nRPL13A needs stronger validation.\nIn response to the reviewer’s comments, we have included the statistical analysis results\nfor Figure1h in supplementary Table1, which indicate a significantly different\nexpression of all genes in cluster C1 (p_val_adj ranging from 2.91E-198 to 0.00031).\nThe immunoblot presented in Figure 1e was conducted in triplicate, and the\ncorresponding quantification results are described in the text (page4, line118-121).\nLastly, Figure 1i lacks any quantification visible or clear enough, making it an\ninadequate basis for drawing conclusions about gene regulation.\nIn Figure 1i, we showed the result as a heatmap because it is commonly used to present\nthe quantification data of gene expression in single-cell analysis. In revision, we have\nadded the statistical data for this figure as a supplementary table 2 to demonstrate the\nvalidity of our conclusion from this analysis (adj_p_val < 1e10-62).\nSimilarly, the immunostainings in Figures 1j and 1l are not convincing. Can the authors\nprovide representative images that correspond to the quantification shown below?\nIn accordance with the reviewer’s suggestion, we have provided clear representative\nimages by excluding the bright-field images.\nFig. 2\nThe phospho-proteomics data has been improved in terms of signalling pathways;\nhowever, further refinements are needed to make the findings more convincing.\nSpecifically, the data should more clearly demonstrate how trametinib (timing) affects\nribosome biogenesis in both control and PDO mutant models. Experiments of treated\ncells (timing) are only shown in the PDO mutant.\nThe response of wild-type PDOs to trametinib is certainly intriguing. However, the\nphospho-proteomics analysis in the KRAS wild-type PDOs are technically difficult, as\nthey are eradicated by trametinib treatment, making it extremely difficult to distinguish\nbetween the response to KRAS inhibition and the secondary response associated with\ncell death. Accordingly, trametinib-induced changes were analyzed exclusively in\nKRAS-mutant PDOs, and no in wild-type ones.\nFig.3 and 4\nThe authors claim a synergistic effect of trametinib with dinaciclib and trametinib with\nCX-5461. However, they only test the PDO mutant and not the healthy control. Only\nFigures 3a and 3b address this statement for dinaciclib, and Figures 3e and 3f for CX-\n5461. The rest of the data focuses on single-compound treatments rather than their\nsynergy, making the findings less novel. It remains possible that both compounds are\ngenerally toxic to all PDO types rather than specifically synergistic.\nWe appreciate the reviewer for highlighting this important point. While the response of\nnormal cells is indeed an important issue, we have not established PDOs from normal\ntissue. To address this point, we have included a new analysis of wild-type mice in\nFigure 4.\nTo the best of our knowledge, this is the first report of a synergistic effect of dinaciclib\nor CX-5461 with trametinib. Therefore, we believe that elucidating the pharmacological\naction of each drug as single agent against CRC is of great significance.\nWe acknowledge the possibility that the observed effects may be due to general toxicity.\nHowever, our combination index (CI) analysis demonstrated CI values consistently <1,\nsuggesting a synergistic interaction rather than non-specific toxicity. To strengthen our\nunderstanding of the synergistic interaction, we analyzed the median-effect principle.\nThe resulting plot clearly demonstrated that the effect of each drug- both as single agent\nand in combination of trametinib- are consistent with the mass-action law. These new\nresults were shown as Supplemental Data Figure 3a and b, and described in page 9,\nline272-279 and page 10, line 317-319. We believe these results, together with the\npreviously presented combination index data, support that dinaciclib and CX-5461\nexhibit synergistic effect when combined with trametinib.\na b\nExtended Data Fig.3a and b. Median-effect plot of CDK inhibitors as a single\nagent (single) or as combination with trametinib (Combos) (a) and of\ncombination of CX-5461 and trametinib (b). The fraction of affected (Fa) versus\nthe drug concentration (D) was plotted in a log-log scale as log(Fa/Fu) versus\nlog(D), where Fu=1-Fa.\nThe synergy is more convincingly demonstrated in Figure 4, which presents in vivo\ndata, but only for CX-5461—there is no in vivo data for dinaciclib. Were all wild-type\nand Apc-Ras-Lgr5 mice analyzed at day 5? Can the authors provide additional images\nof the combination-treated WT mice? Furthermore, could they examine embryonic-\nground state markers in these in vivo experiments? It is unclear why the study focuses\nsolely on villi size without assessing any markers related to ribosome biogenesis or the\nembryonic ground state, which would strengthen the conclusions.\nWe did not perform in vivo analysis of dinaciclib, as it shows limited synergy with the\nKRAS inhibitor and affects pathways beyond ribosome biogenesis, rendering it\nunsuitable for further evaluation of synergistic effects with KRAS inhibition.\nIn response to the reviewer’s comments regarding the experimental protocol, we have\nprovided the illustration of the experimental timeline in Figure 4a and f. To analyze\nribosome biogenesis, we examined fibrillarin expression six hours after a single\nadministration of trametinib. To evaluate the antitumor effects of the drug treatment, we\nperformed the analysis 24 hours after the fifth administration. Additional mages of the\ncombination-treated WT mice were included in Figure 4k and m. The text has been\namended accordingly (page 11, line 330-351).\nWe have shown expression of fibrillarin to demonstrate the elevated ribosome\nbiogenesis after trametinib treatment. As the reviewer pointed out, evaluation of\nribosome biogenesis does refine our conclusions; however, establishing pathological\nmarkers requires considerable time and efforts. Therefore, we would like to leave this as\na task for future studies.\nImmunostainings have been improved.\nWe appreciate the favorable evaluation of the immunostainings.\nFig. 5 and 6\nThe experiments presented focus on another mutant, G12C, but only include ribosome\nbiogenesis data and Fib stainings, which are insufficient to support a definitive\nconclusion. The data for the combination of CX-5461 and AMG510 is limited to\nFigures 6g and 6h\nWe respectfully disagree with the reviewer’s comment that the data presented in Figure\n5 and 6 are insufficient. The GOCC and MSigDB gene sets that identified through\ncomprehensive analyses of bulk and scRNA-seq data consistently exhibited extremely\nhigh NES scores. The ground state gene signature of ESCs and epiblast are also\nsignificantly enriched. We believe these results strongly support our conclusions.\nWe consider Figures 6g and 6h to provide sufficient evidence for the synergistic effect\nbetween CX-5461 and AMG510. Additionally, the enrichment of ribosome biogenesis\nand the suppression of protein synthesis are demonstrated in Figure 6a-e, respectively.\nIn response to the reviewer’s concern, we performed an additional EU incorporation\nassay, which demonstrated that CX-5461 reduces nascent RNA synthesis. The results\nhave been shown in Extended Data Fig. 6d and the text has been amended accordingly\n(Page 14, line 430-431). We believe these results provide sufficient evidence to support\nour conclusions.\n80\n60\n40\n20\n0\ncontrol CX−5461\nExtended Data Fig. 6d. Reduced EU incorporation in CX-5461-treated CRC\norganoids. Representative images of EU (red) and nuclei (blue) in control and\nCX-5461-treated organoids are shown. High magnification views of the white\nboxed regions are shown in the insets. Bar = 50µm, Bar in inset = 10µm. The\npercentage of EU-positive nuclei per organoid is shown in the boxplot. Data of\n< 0.01.\nThe abstract and title are still not clear.\nTitle: CRC cells adopt the embryo-ground state in response to KRAS inhibition\nThe embryo-ground state was observed on few PDOs RAS mut\nThe embryo-ground state data can be improved.\nIn accordance with the reviewer’s comments, we tone down conclusions on the embryo-\nground state and focus on the potential of ribosome biogenesis as a therapeutic target in\nKRAS-mutant colorectal cancer. The revised title is as follows:\n‘Ribosome biogenesis as a potential therapeutic target in KRAS mutant colorectal\ncancer’.\nAbstract\nWe have made substantial revision to the abstract.\n\"synergistic anti-tumor effect with trametinib and AMG510 in .. mouse model and\nhuman PDOs..\"\nonly was done in vivo with trametinib and CX.\nAccording to the reviewer’s suggestion, we have amended the text.\n)%(\nsllec\nevitisop\nUE\nHCT128−1T*\n75\n50\n25\n0\ncontrol CX−5461\n)%(\nsllec\nevitisop\nUE\nHCT191−1T*\n60\n40\n20\n0\ncontrol CX−5461\n)%(\nsllec\nevitisop\nUE\nHCT202−1T*\n40\n30\n20\n10\n0\ncontrol CX−5461\n)%(\nsllec\nevitisop\nUE\nd\ncontrol CX-5461l control CX-5461l\nHCT191 HCT128\n-1T -1T\nHCT215−1T\n**\nHCT202 HCT215\n-1T -1T\nWe found that they were vulnerable to the inhibition of RNA polymerase I, and it\nexhibited synergistic anti-tumor effects with trametinib in an autochthonous mouse\nmodel of intestinal tumors and human patient-derived organoids (PDOs). (Page2,\nline45-48)\n\"CRC cells adopt a cellular state..\"\na few PDOs RAS mut were done not CRC cells in general. It is confusing including all\nCRC cells\nembry-ground state was observed in few experiments.\nAccording to the reviewer’s suggestion, we have removed the term ‘embryonic ground\nstate’, and revised this paragraph as follows:\nHere, we demonstrated that a subset of KRAS-mutant CRC cells transitions to a cellular\nstate characterized by enhanced ribosome biogenesis upon KRAS signaling inhibition.\n(Page2, line40-42)\n\"these observations demonstrated that CRC cells become tolerant to K-RAS inhibition\nby exploiting the cellular program of early embryogenesis..\"\nagain too general writing about CRC cells, as only was observed in PDOS RAS mut.\nWe have modified the text according to the reviewer’s suggestion.\nThese observations demonstrated that high ribosome biogenesis induced by KRAS\ninhibition is indispensable to maintain this cellular state and is a potential therapeutic\ntarget. (Page2, line48-52)\nReviewer #4 (Remarks to the Author):\nI would like to thank the authors for their efforts in providing more convincing data.\nThe manuscript has been improved.\nWe sincerely appreciate your positive feedback and are glad to hear that the revisions\nhave improved the manuscript.\nMajor Points:\n1. The authors have confirmed that the scRNA-seq data presented is derived from a\nsingle PDO. This should be clearly stated in both the main text, methodology and the\nfigure legend. In this revised version, the abstract and title have been updated. However,\nIt is clear that additional data will be necessary in the future to robustly support the\nclaim of embryo ground state phenotype acquisition.\nIn accordance with the reviewer’s suggestions, we have revised the text and figure\nlegends to clearly state that the analysis was conducted using a single PDO (page 3,\nline88 and figure 1a legend). In this study, we performed scRNA-seq analysis on a total\nof five PDOs, with the detailed methodology provided in the “Single-cell RNA\nsequencing” section.\n2. The western blot analyses are now shown to be statistically significant, which I\nconsider sufficient. It is ultimately up to the editor to decide whether this level of\nevidence is acceptable.\nWe appreciate the favorable evaluation of the western blot. We have prepared the\nmanuscript in accordance with the submission guidelines of Nature Communications.\n3. The in vivo data is more convincing; however, the previously raised concerns have\nbeen only partially addressed. Additional experiments would have helped strengthen the\nconclusions. For example, it is unclear why only a single marker, such as fibrillarin, was\nused in some of the experiments. Relying on a single marker can raise questions about\nthe robustness of the findings.\nWe appreciate your further comments. We consider that this in vivo analysis supports\nthe conclusions drawn from the scRNA-seq results. Nevertheless, as you have rightly\nnoted, the evidence relies on fibrillarin staining, and we will address this point in future\nstudies by elucidating the molecular mechanisms underlying ribosome biogenesis.\n4. The connection between ribosome biogenesis and colorectal cancer/PDOs is not\nnovel in the field. While this study includes PDO experiments and in vivo data, the\nmain message is now different to before in terms of novelty and significance.\nWe appreciate your comment regarding the existing knowledge on ribosome biogenesis\nin colorectal cancer and PDOs. While the connection may have been reported, our study\naims to contribute to the development of therapeutics involving KRAS inhibitors by\nutilizing single-cell RNA sequencing and proteome analysis combined with\ncomplementary in vivo experiments. We believe this integrative approach adds valuable\ndata to the ongoing research in this area.\nOverall, I am happy to leave it to the editor to judge the originality of the work and\nwhether the data presented is sufficient to support the findings.\nThank you for your thoughtful evaluation. We appreciate your consideration and will\nrespect the editor’s judgment regarding the originality of our work and the sufficiency of\nthe presented data."}, {"round": 3, "reviewer_comments": {"reviewer_4": "I would like to thank the authors for their efforts in providing more convincing data. The manuscript has been improved.\nMajor Points:\n1. The authors have confirmed that the scRNA-seq data presented is derived from a single PDO. This should be clearly stated in both the main text, methodology and the figure legend. In this revised version, the abstract and title have been updated. However, It is clear that additional data will be necessary in the future to robustly support the claim of embryo ground state phenotype acquisition.\n2. The western blot analyses are now shown to be statistically significant, which I consider sufficient. It is ultimately up to the editor to decide whether this level of evidence is acceptable.\n3. The in vivo data is more convincing; however, the previously raised concerns have been only partially addressed. Additional experiments would have helped strengthen the conclusions. For example, it is unclear why only a single marker, such as fibrillarin, was used in some of the experiments. Relying on a single marker can raise questions about the robustness of the findings.\n4. The connection between ribosome biogenesis and colorectal cancer/PDOs is not novel in the field. While this study includes PDO experiments and in vivo data, the main message is now different to before in terms of novelty and significance.\nOverall, I am happy to leave it to the editor to judge the originality of the work and whether the data presented is sufficient to support the findings."}, "author_response": ""}]

Decision letter