This file contains Supplementary Text, Supplementary Figures S1-S13 and Supplementary Tables S1, S3-S5, S9-S10, S14-S15, S18-S19. Supplementary Fig. S1. Validation of prognostic associations and biological properties of the CMS groups in primary CRCs Supplementary Fig. S2. The original CMS classifier fails to accurately identify the CMS groups in CRC cell lines Supplementary Fig. S3. Principal components analysis of cell lines based on gene expression Supplementary Fig. S4. Assessment of misclassification risk of CRC cell lines Supplementary Fig. S5. Additional template gene filter for classifier genes expressed in the tumor stroma Supplementary Fig. S6. Biological characteristics of the CMS groups are recapitulated in PDX models of CRC Supplementary Fig. S7. CRC cell lines analyzed by drug screening are representative with respect to gene expression and CMS groups Supplementary Fig. S8. Principal components analysis of in vitro drug screen data Supplementary Fig. S9. Validation of strong relative activity of EGFR inhibition in CMS2 Supplementary Fig. S10. Drug response analyses between CMS1/4 and CMS2/3 cell lines Supplementary Fig. S11. In vitro validation of strong relative sensitivity to HSP90 inhibition in CMS1 and CMS4 cell lines in published data Supplementary Fig. S12. Up-regulation of heat shock response in cell lines treated with luminespib Supplementary Fig. S13. Poor response to fluoropyrimidines in CMS4 cell lines Supplementary Table S1. Clinicopathological and molecular characteristics of consecutive patient series (n = 409) Supplementary Table S3. Enrichment of molecular characteristics among CMS groups in primary CRC Supplementary Table S4. Enrichment of clinicopathological characteristics among CMS groups in primary CRC Supplementary Table S5. Multivariable survival analyses Supplementary Table S9. Classification concordance between the original and new cancer cell-adapted CMS classifier in CRC cell lines Supplementary Table S10. Enrichment of molecular characteristics among CMS groups of CRC cell lines Supplementary Table S14. Overview of the molecular target classes for the highthroughput drug screening library Supplementary Table S15. Drugs with strong effect in MSI+ versus MSS cell lines Supplementary Table S18. Drugs with strong relative activity in CMS2 Supplementary Table S19. Differential gene expression in response to luminespib treatment

This file contains Supplementary Tables S2, S6-S8, S11-S13, and S16-S17. Supplementary Table S2. Overview and CMS groups of CRC cell line Supplementary Table S6. Gene set analysis among CMS groups in primary CRCs Supplementary Table S7. Potential template genes and filtering steps Supplementary Table S8. Template genes for the adapted CMS classifier Supplementary Table S11. Gene set analyses among CMS groups in CRC cell lines Supplementary Table S12. Assessment of misclassification risk in CRC cell lines Supplementary Table S13. CMS classification of PDX models by adapted CMS classifier Supplementary Table S16. Differential drug sensitivity between cell lines of individual CMS groups Supplementary Table S17. Drug response data for 27 selected drugs in 29 individual cell lines

Cell–cell and cell–matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial‐to‐mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E‐cadherin (ECAD), integrin β4 (ITGB4), zonula occludens 1 (ZO‐1), and cytokeratins in a single‐hospital series of Norwegian patients with colorectal cancer (CRC) stages I–IV (n = 922) using multiplex fluorescence‐based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO‐1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E‐cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.

Tumor heterogeneity is a primary cause of treatment failure. However, changes in drug sensitivity over time are not well mapped in cancer. Patient-derived organoids (PDOs) may predict clinical drug responses ex vivo and offer an opportunity to evaluate novel treatment strategies in a personalized fashion. Here we have evaluated spatio-temporal functional and molecular dynamics of five PDO models established after hepatic re-resections and neoadjuvant combination chemotherapies in a patient with microsatellite stable and KRAS mutated metastatic rectal cancer. Histopathological differentiation phenotypes of the PDOs corresponded with the liver metastases, and ex vivo drug sensitivities generally reflected clinical responses and selection pressure, assessed in comparison to a reference data set of PDOs from metastatic colorectal cancers. PDOs from the initial versus the two recurrent metastatic settings showed heterogeneous cell morphologies, protein marker expression, and drug sensitivities. Exploratory analyses of a drug screen library of 33 investigational anticancer agents showed the strongest ex vivo sensitivity to the SMAC mimetic LCL161 in PDOs of recurrent disease compared to those of the initial metastasis. Functional analyses confirmed target inhibition and apoptosis induction in the LCL161 sensitive PDOs from the recurrent metastases. Gene expression analyses indicated an association between LCL161 sensitivity and tumor necrosis factor alpha signaling and RIPK1 gene expression. In conclusion, LCL161 was identified as a possible experimental therapy of a metastatic rectal cancer that relapsed after hepatic resection and standard systemic treatment.

Gene expression-based subtypes of colorectal cancer have clinical relevance, but the representativeness of primary tumors and the consensus molecular subtypes (CMS) for metastatic cancers is not well known. We investigated the metastatic heterogeneity of CMS. The best approach to subtype translation was delineated by comparisons of transcriptomic profiles from 317 primary tumors and 295 liver metastases, including multi-metastatic samples from 45 patients and 14 primary-metastasis sets. Associations were validated in an external data set ( n = 618). Projection of metastases onto principal components of primary tumors showed that metastases were depleted of CMS1-immune/CMS3-metabolic signals, enriched for CMS4-mesenchymal/stromal signals, and heavily influenced by the microenvironment. The tailored CMS classifier (available in an updated version of the R package CMScaller) therefore implemented an approach to regress out the liver tissue background. The majority of classified metastases were either CMS2 or CMS4. Nonetheless, subtype switching and inter-metastatic CMS heterogeneity were frequent and increased with sampling intensity. Poor-prognostic value of CMS1/3 metastases was consistent in the context of intra-patient tumor heterogeneity.

A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR , GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.

Background Gene expression-based subtyping has the potential to form a new paradigm for stratified treatment of colorectal cancer. However, current frameworks are based on the transcriptomic profiles of primary tumors, and metastatic heterogeneity is a challenge. Here we aimed to develop a de novo metastasis-oriented framework. Methods In total, 829 transcriptomic profiles from patients with colorectal cancer were analyzed, including primary tumors, liver metastases, and non-malignant liver samples. High-resolution microarray gene expression profiling was performed of 283 liver metastases from 171 patients treated by hepatic resection, including multiregional and/or multi-metastatic samples from each of 47 patients. A single randomly selected liver metastasis sample from each patient was used for unsupervised subtype discovery by nonnegative matrix factorization, and a random forest prediction model was trained to classify multi-metastatic samples, as well as liver metastases from two independent series of 308 additional patients. Results Initial comparisons with non-malignant liver samples and primary colorectal tumors showed a highly variable degree of influence from the liver microenvironment in metastases, which contributed to inter-metastatic transcriptomic heterogeneity, but did not define subtype distinctions. The de novo liver metastasis subtype (LMS) framework recapitulated the main distinction between epithelial-like and mesenchymal-like tumors, with a strong immune and stromal component only in the latter. We also identified biologically distinct epithelial-like subtypes originating from different progenitor cell types. LMS1 metastases had several transcriptomic features of cancer aggressiveness, including secretory progenitor cell origin, oncogenic addictions, and microsatellite instability in a microsatellite stable background, as well as frequent RAS/TP53 co-mutations. The poor-prognostic association of LMS1 metastases was independent of mutation status, clinicopathological variables, and current subtyping frameworks (consensus molecular subtypes and colorectal cancer intrinsic subtypes). LMS1 was also the least heterogeneous subtype in comparisons of multiple metastases per patient, and tumor heterogeneity did not confound the prognostic value of LMS1. Conclusions We report the first large study of multi-metastatic gene expression profiling of colorectal cancer. The new metastasis-oriented subtyping framework showed potential for clinically relevant transcriptomic classification in the context of metastatic heterogeneity, and an LMS1 mini-classifier was constructed to facilitate prognostic stratification and further clinical testing.

Abstract Patients with metastatic colorectal cancer (CRC) have few targeted treatment options compared with other major malignancies, and both over- and undertreatment with cytostatic drugs remain a challenge. Recent studies show that patient-derived organoids (PDOs) can predict clinical responses to systemic therapies in a personalized manner, but tumor heterogeneity may limit the clinical benefit of anticancer therapies, as well as the accuracy of preclinical predictions. PDO lineage establishment from multiple distinct lesions per patient presents a novel opportunity for preclinical investigation of intrapatient pharmacotranscriptomic heterogeneity. From September 2017 until November 2019, we have established a living biobank of 107 PDOs from 53 patients who underwent resection of CRC liver metastases at Oslo University Hospital, Norway, 31 of whom had multiple (2-5) metastases. All PDOs were screened for sensitivity to 40 anticancer agents, including clinically relevant targeted drugs and conventional chemotherapies. Molecular profiling by gene expression and mutation analyses is ongoing and currently completed for 27 PDOs. Recapitulation of known pharmacogenomic/transcriptomic associations was confirmed in PDOs for EGFR inhibition and RAS/BRAFV600E mutation status, as well as TP53-MDM2 inhibition and TP53 mutation status and TP53 transcriptional activity. Antimetabolites such as gemcitabine and methotrexate, or small-molecule inhibitors in late-stage clinical development targeting Aurora A, PLK1, and HSP90, showed strong differential activities, enabling identification of sensitive subgroups. Strong sensitivity to HSP90 inhibition was associated with low protein expression of Heat Shock Transcription Factor 1, which had a homogenous intrapatient intermetastatic expression pattern. Principal component analyses revealed a clear patient-wise separation of PDOs based on both their pharmacologic and transcriptomic profiles separately, indicating only modest intrapatient heterogeneity among distinct metastatic lesions. Accurate prediction of clinical responses to 5-FU and oxaliplatin was shown in PDOs from one patient treated for recurrent liver metastases. Additionally, PDOs from recurrent liver metastases showed an increased sensitivity to off-label drugs compared to PDOs from the first liver resection, supporting therapy repurposing in later lines of treatment. In summary, patient-derived models of CRC liver metastases reveal modest intrapatient pharmacotranscriptomic heterogeneity—an encouraging result for further efforts to develop personalized drug repurposing strategies for this poor-prognosis patient group. Citation Format: Kushtrim Kryeziu, Jarle Bruun, Peter W. Eide, Seyed H. Moosavi, Ina A. Eilertsen, Jonas Langerud, Bård Røsok, Tuva H. Brunsell, Marianne G. Guren, Andreas Abildgaard, Arild Nesbakken, Bjørn Atle Bjørnbeth, Anita Sveen, Ragnhild A. Lothe. Modeling intrapatient pharmacotranscriptomic heterogeneity with organoids derived from colorectal cancer liver metastases [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A19.

Background: PARP inhibitors are active in various tumour types beyond BRCA-mutant cancers, but their activity and molecular correlates in colorectal cancer (CRC) are not well studied. Methods: Mutations and genome-wide mutational patterns associated with homologous recombination deficiency (HRD) were investigated in 255 primary CRCs with whole-exome sequencing and/or DNA copy number data. Efficacy of five PARP inhibitors and their molecular correlates were evaluated in 93 CRC cell lines partly annotated with mutational-, DNA copy number-, and/or gene expression profiles. Post-treatment gene expression profiling and specific protein expression analyses were performed in two pairs of PARP inhibitor sensitive and resistant cell lines. Findings: A subset of microsatellite stable (MSS) CRCs had truncating mutations in homologous recombination-related genes, but these were not associated with genomic signatures of HRD. Eight CRC cell lines (9%) were sensitive to PARP inhibition, but sensitivity was not predicted by HRD-related genomic and transcriptomic signatures. In contrast, drug sensitivity in MSS cell lines was strongly associated with TP53 wild-type status (odds ratio 15.7, p = 0.023) and TP53-related expression signatures. Increased downstream TP53 activity was among the primary response mechanisms, and TP53 inhibition antagonized the effect of PARP inhibitors. Wild-type TP53-mediated suppression of RAD51 was identified as a possible mechanism of action for sensitivity to PARP inhibition. Interpretation: PARP inhibitors are active in a subset of CRC cell lines and preserved TP53 function may increase the likelihood of response.

AbstractPurpose:Molecular tumor heterogeneity may have important implications for the efficacy of targeted therapies in metastatic cancers. Inter-metastatic heterogeneity of sensitivity to anticancer agents has not been well explored in colorectal cancer.Experimental Design:We established a platform for ex vivo pharmacogenomic profiling of patient-derived organoids (PDO) from resected colorectal cancer liver metastases. Drug sensitivity testing (n = 40 clinically relevant agents) and gene expression profiling were performed on 39 metastases from 22 patients.Results:Three drug–response clusters were identified among the colorectal cancer metastases, based primarily on sensitivities to EGFR and/or MDM2 inhibition, and corresponding with RAS mutations and TP53 activity. Potentially effective therapies, including off-label use of drugs approved for other cancer types, could be nominated for eighteen patients (82%). Antimetabolites and targeted agents lacking a decisive genomic marker had stronger differential activity than most approved chemotherapies. We found limited intra-patient drug sensitivity heterogeneity between PDOs from multiple (2–5) liver metastases from each of ten patients. This was recapitulated at the gene expression level, with a highly proportional degree of transcriptomic and pharmacological variation. One PDO with a multi-drug resistance profile, including resistance to EGFR inhibition in a RAS-mutant background, showed sensitivity to MEK plus mTOR/AKT inhibition, corresponding with low-level PTEN expression.Conclusions:Intra-patient inter-metastatic pharmacological heterogeneity was not pronounced and ex vivo drug screening may identify novel treatment options for metastatic colorectal cancer. Variation in drug sensitivities was reflected at the transcriptomic level, suggesting potential to develop gene expression–based predictive signatures to guide experimental therapies.

Abstract Introduction: NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), a member of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases, has been shown to be an important regulator of multiple proteins involved in cancer development, including the tumor suppressor PTEN and the proto-oncogene MDM2. Furthermore, NEDD4 has been shown to be overexpressed and act as an oncogene in multiple cancer types. We have previously shown that NEDD4 is overexpressed in colorectal cancer (CRC) and that it can promote growth of colon cancer cells independently of PTEN and PI3K/AKT signaling. In the present study, we investigated the role of NEDD4 in regulating the PTEN/PI3K pathway and the MDM2/p53 axis in CRC. Materials and Methods: The CRISPR/Cas9 system was applied to generate a Caco2 NEDD4 knockout cell line, and NEDD4 knockdown was performed in LS174T and SW480 cells. Gene expression profiles of 412 primary CRCs, 51 normal mucosa samples, 38 CRC cell lines, as well as the Caco2 parental and NEDD4 knockout cell lines, were generated using exon-resolution Affymetrix Human Exon Arrays or Human Transcriptome Arrays. The NEDD4 expression level was correlated with the mutation status of KRAS, PTEN, TP53, PIK3CA, BRAF and NRAS. Western blotting was used to detect and quantify NEDD4, PTEN and MDM2 protein levels. Results and Discussions: Gene expression analysis of the patient material confirmed our previous study that NEDD4 is significantly upregulated in CRC as compared to normal colonic mucosa. There was no correlation between NEDD4 expression and mutations in KRAS, BRAF, NRAS, PTEN, PIK3CA and TP53. CRISPR/Cas9-mediated NEDD4 knockout in Caco2 cells resulted in a reduction (P <0.01) in both the PTEN and MDM2 protein levels as compared to control cells. Gene set analysis showed that PI3K/AKT/MTOR signaling was upregulated in the NEDD4 knockout cells as compared to control cells. siRNA-mediated depletion of NEDD4 in LS174T cells was associated with reduced levels of MDM2, but did not affect the PTEN protein level. In SW480 cells, depletion of NEDD4 affected neither the MDM2 nor the PTEN protein level. By analyzing the expression of NEDD4, PTEN and MDM2 in 38 CRC cell lines by Western blotting, a positive correlation (P<0.01) was observed between NEDD4 and PTEN protein, while there was no significant correlation between NEDD4 and MDM2. Conclusion: The data show that NEDD4 is significantly upregulated in CRC, and that NEDD4 expression correlates with PTEN expression. The data further suggest that NEDD4 has the ability to regulate the PTEN and MDM2 protein levels in colon cancer cells in a cell line-specific manner. Citation Format: Lars M. Knudsen, Anita Sveen, Christer A. Andreassen, Christian H. Bergsland, Ina A. Eilertsen, Nikoline L. Rasmussen, Max Z. Totland, Peter W. Eide, Jarle Bruun, Ragnhild A. Lothe, Edward Leithe. Role of the E3 ubiquitin ligase NEDD4 in the regulation of PTEN and MDM2 in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1433.

Background Accumulating evidence suggests immunomodulatory and context-dependent effects of TP53 mutations in cancer. We performed an exploratory analysis of the transcriptional, immunobiological and prognostic associations of TP53 mutations within the gene expression-based consensus molecular subtypes (CMSs) of colorectal cancer (CRC). Materials and methods In a single-hospital series of 401 stage I-IV primary CRCs, we sequenced the whole coding region of TP53 and analysed CMS-dependent transcriptional consequences of the mutations by gene expression profiling. Immunomodulatory associations were validated by multiplex, fluorescence-based immunohistochemistry of immune cell markers. Prognostic associations of TP53 mutations were analysed in an aggregated series of 635 patients classified according to CMS, including publicly available data from a French multicentre cohort (GSE39582). Results TP53 mutations were found in 60% of the CRCs. However, gene set enrichment analyses indicated that their transcriptional consequences varied among the CMSs and were most pronounced in CMS1-immune and CMS4-mesenchymal. Subtype specificity was primarily seen as an upregulation of gene sets reflecting cell cycle progression in CMS4 and a downregulation of T cell activity in CMS1. The subtype-dependent immunomodulatory associations were reinforced by significant depletion of several immune cell populations in mutated tumours compared with wild-type (wt) tumours exclusively in CMS1, including cytotoxic lymphocytes (adjusted p value in CMS1=0.002 and CMS2-4>0.9, Microenvironment Cell Populations (MCP)-counter algorithm). This was validated by immunohistochemistrybased quantification of tumour infiltrating CD8+ cells. Within CMS1, the immunomodulatory association of TP53 mutations was strongest among microsatellite stable (MSS) tumours, and this translated into a propensity for metastatic disease and poor prognostic value of the mutations specifically in the CMS1/MSS subtype (both series overall survival: TP53 mutation vs wt: HR 5.52, p=0.028). Conclusions Integration of TP53 mutation status with the CMS framework in primary CRC suggested subtype-dependent immunobiological associations with prognostic and potentially immunotherapeutic implications, warranting independent validation.

Elevated miR-31 expression is associated with poor outcome in colorectal cancer (CRC). Whether the prognostic information is independent of known molecular subgroups and gene expression-based consensus molecular subtypes (CMS) is currently unknown. To investigate this, we analyzed nearly 2000 CRC biopsies and preclinical models. The expression of miR-31-5p and its host transcript, long noncoding RNA MIR31HG, was strongly correlated (Spearman's ρ > 0.80). MIR31HG outlier expression was observed in 158/1265 (12%) of pCRCs and was associated with depletion of CMS2-canonical subgroup (odds ratio = 0.21 [0.11-0.35]) and shorter relapse-free survival (RFS) in multivariable analysis (adjusted hazard ratio = 2.2 [1.6-3.0]). For stage II disease, 5-year RFS for patients with MIR31HG outlier status was 49% compared to 77% for those with normal-like expression. MIR31HG outlier status was associated with inferior outcome also within clinical high risk groups and within the poor prognostic CMS4-mesenchymal gene expression subtype specifically. Preclinical models with MIR31HG outlier expression were characterized by reduced expression of MYC targets as well as elevated epithelial-mesenchymal transition, TNF-α/NFκB, TGF-β, and IFN-α/γ gene expression signatures, indicating cancer cell-intrinsic properties resembling the CMS4 subgroup-associations which were recapitulated in patient biopsies. Moreover, the prognostic value of MIR31HG outlier status was independent of cytotoxic T lymphocyte and fibroblast infiltration. We here present evidence that MIR31HG expression provides clinical stratification beyond major gene expression phenotypes and tumor immune and stromal cell infiltration and propose a model where increased expression is an indicator of a cellular state conferring intrinsic invasive and/or immuno-evasive capabilities.

TP53 mutations are common in colorectal cancer (CRC). Most TP53 sequencing studies have been restricted to coding regions, but recent studies have revealed that splice mutations can generate transcript variants with distinct tumorigenic and prognostic properties. Here, we performed unrestricted sequencing of all coding sequences and splice regions of TP53 in a single-hospital series of 401 primary CRCs. TP53 splice mutations were detected in 4% of the cases (N = 16), considerably more frequent than reported in major databases, and they were mutually exclusive to exon mutations. RNA sequencing revealed high-level expression of aberrant transcript variants in the majority of splice mutated tumors (75%). Most variants were predicted to produce truncated TP53 proteins, including one sample expressing the potentially oncogenic and druggable p53ψ isoform. Despite heterogeneous transcript structures, downstream transcriptional profiling revealed that TP53 splice mutations had similar effects on TP53 target gene expression and pathway activity as exonic mutations. Intriguingly, TP53 splice mutations were associated with worse 5-year relapse-free survival in stage II disease, compared to both TP53 wild-type and exon mutations (P = 0.007). These data highlight the importance of including splice regions when examining the biological and clinical consequences of TP53 mutations in CRC.

Introduction Colorectal cancer (CRC) is a global health challenge. There is limited response to standard oncological treatment and few stratified treatment options based on prognostic and/or predictive factors. The four biologically distinct and gene expression-based consensus molecular subtypes (CMS) provide prognostic stratification independent of cancer stage, and represent a new potential paradigm for stratified treatment. In this project, we are analysing the biomarker and drug target potential of members of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases in CRC. Material and methods A total of 412 primary CRCs and 51 normal colonic mucosa samples were subjected to genome wide expression analysis at exon level resolution using either the Affymetrix Human Exon Array or the Affymetrix Human Transcriptome Array. The CRISPR/Cas9 system was applied to generate NEDD4 knock-out Caco2 cell lines. Results and discussions We identified NEDD4 (neural precursor cell-expressed developmentally down-regulated 4) as significantly overexpressed in CRC, while the NEDD4 homolog NEDD4L was significantly down regulated. Furthermore, we found that NEDD4 is differentially expressed between the different CMS classes, with high expression particularly in the epithelial and canonical CMS2 group. CRISPR/Cas9-mediated knock-out of NEDD4 in the CRC cell line Caco2 was found to result in altered growth characteristics. Conclusion The data indicate that NEDD4 and NEDD4L are differently expressed in CRC samples compared to normal colonic mucosa, and that knock-out of NEDD4 in Caco2 cells affects their growth characteristics.

We aimed to refine the value of CDX2 as an independent prognostic and predictive biomarker in colorectal cancer (CRC) according to disease stage and chemotherapy sensitivity in preclinical models. CDX2 expression was evaluated in 1045 stage I–IV primary CRCs by gene expression (n = 403) or immunohistochemistry (n = 642) and in relation to 5‐year relapse‐free survival (RFS), overall survival (OS), and chemotherapy. Pharmacogenomic associations between CDX2 expression and 69 chemotherapeutics were assessed by drug screening of 35 CRC cell lines. CDX2 expression was lost in 11.6% of cases and showed independent poor prognostic value in multivariable models. For individual stages, CDX2 was prognostic only in stage IV, independent of chemotherapy. Among stage I–III patients not treated in an adjuvant setting, CDX2 loss was associated with a particularly poor survival in the BRAF‐mutated subgroup, but prognostic value was independent of microsatellite instability status and the consensus molecular subtypes. In stage III, the 5‐year RFS rate was higher among patients with loss of CDX2 who received adjuvant chemotherapy than among patients who did not. The CDX2‐negative cell lines were significantly more sensitive to chemotherapeutics than CDX2‐positive cells, and the multidrug resistance genes MDR1 and CFTR were significantly downregulated both in CDX2‐negative cells and in patient tumors. Loss of CDX2 in CRC is an adverse prognostic biomarker only in stage IV disease and appears to be associated with benefit from adjuvant chemotherapy in stage III. Early‐stage patients not qualifying for chemotherapy might be reconsidered for such treatment if their tumor has loss of CDX2 and mutated BRAF.

Abstract Purpose: Response to standard oncologic treatment is limited in colorectal cancer. The gene expression–based consensus molecular subtypes (CMS) provide a new paradigm for stratified treatment and drug repurposing; however, drug discovery is currently limited by the lack of translation of CMS to preclinical models. Experimental Design: We analyzed CMS in primary colorectal cancers, cell lines, and patient-derived xenografts (PDX). For classification of preclinical models, we developed an optimized classifier enriched for cancer cell–intrinsic gene expression signals, and performed high-throughput in vitro drug screening (n = 459 drugs) to analyze subtype-specific drug sensitivities. Results: The distinct molecular and clinicopathologic characteristics of each CMS group were validated in a single-hospital series of 409 primary colorectal cancers. The new, cancer cell–adapted classifier was found to perform well in primary tumors, and applied to a panel of 148 cell lines and 32 PDXs, these colorectal cancer models were shown to recapitulate the biology of the CMS groups. Drug screening of 33 cell lines demonstrated subtype-dependent response profiles, confirming strong response to EGFR and HER2 inhibitors in the CMS2 epithelial/canonical group, and revealing strong sensitivity to HSP90 inhibitors in cells with the CMS1 microsatellite instability/immune and CMS4 mesenchymal phenotypes. This association was validated in vitro in additional CMS-predicted cell lines. Combination treatment with 5-fluorouracil and luminespib showed potential to alleviate chemoresistance in a CMS4 PDX model, an effect not seen in a chemosensitive CMS2 PDX model. Conclusions: We provide translation of CMS classification to preclinical models and uncover a potential for targeted treatment repurposing in the chemoresistant CMS4 group. Clin Cancer Res; 24(4); 794–806. ©2017 AACR.

Colorectal cancers (CRCs) can be divided into four gene expression-based biologically distinct consensus molecular subtypes (CMS). This classification provides a potential framework for stratified treatment, but to identify novel CMS-drug associations, translation of the subtypes to pre-clinical models is essential. The currently available classifier is dependent on gene expression signals from the immune and stromal compartments of tumors and fails to identify the poor-prognostic CMS4-mesenchymal group in immortalized cell lines, patient-derived organoids and xenografts. To address this, we present a novel CMS classifier based on a filtered set of cancer cell-intrinsic, subtype-enriched gene expression markers. This new classifier, referred to as CMScaller, recapitulated the subtypes in both in vitro and in vivo models (551 in total). Importantly, by analyzing public drug response data from patient-derived xenografts and cell lines, we show that the subtypes are predictive of response to standard CRC drugs. CMScaller is available as an R package.