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Statistical analysis of results shown in Figure 2.

Purpose:Approximately 20% of patients with RAS wild-type metastatic colorectal cancer (mCRC) experience objective responses to the anti-EGFR antibody cetuximab, but disease eradication is seldom achieved. The extent of tumor shrinkage correlates with long-term outcome. We aimed to find rational combinations that potentiate cetuximab efficacy by disrupting adaptive dependencies on antiapoptotic molecules (BCL2, BCL-XL, MCL1).Experimental Design:Experiments were conducted in patient-derived xenografts (PDX) and organoids (PDXO). Apoptotic priming was analyzed by BH3 profiling. Proapoptotic and antiapoptotic protein complexes were evaluated by co-immunoprecipitation and electroluminescence sandwich assays. The effect of combination therapies was assessed by caspase activation in PDXOs and by monitoring PDX growth.Results:A population trial in 314 PDX cohorts, established from as many patients, identified 46 models (14.6%) with appreciable (>50% tumor shrinkage) but incomplete response to cetuximab. From these models, 14 PDXOs were derived. Cetuximab primed cells for apoptosis, but only concomitant blockade of BCL-XL precipitated cell death. Mechanistically, exposure to cetuximab induced upregulation of the proapoptotic protein BIM and its sequestration by BCL-XL. Inhibition of BCL-XL resulted in displacement of BIM, which was not buffered by MCL1 and thereby became competent to induce apoptosis. In five PDX models, combination of cetuximab and a selective BCL-XL inhibitor triggered apoptosis and led to more pronounced tumor regressions and longer time to relapse after treatment discontinuation than cetuximab alone.Conclusions:In mCRC tumors that respond to cetuximab, antibody treatment confers a synthetic-lethal dependency on BCL-XL. Targeting this dependency unleashes apoptosis and increases the depth of response to cetuximab.

Supplementary Table S2

Generation and biological characterization of HER2-overexpressing DiFi and HDC-142 cells.

Purpose:Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available.Experimental Design:Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach.Results:XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion.Conclusions:The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.

Supplementary Legends and Figures

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Quantitations of phosphoproteins in cell lines and tumorgrafts treated with HER2/EGFR small-molecule inhibitors and antibodies.

Purpose: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities.Experimental Design: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays.Results: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib—or, importantly, monotherapy with afatinib—resulted in overt tumor shrinkage.Conclusions: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519–31. ©2015 AACR.

Purpose:Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers.Experimental Design:Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment.Results:Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance.Conclusions:Subtype classification systems represent canonical “termini a quo” (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.

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Purpose: Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti–EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC).Experimental Design: Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab.Results: At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment.Conclusions: The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses. Clin Cancer Res; 18(9); 2515–25. ©2012 AACR.

Supplementary Figures S1 - S8 and Table S1. Fig S1: Analysis of copy number burden in a PDX population trial of REG treated mCRC PDXs. Fig S2: Effect of REG on microvessel density and proliferation according to CNA cluster. Fig S3 - S6: BCL-2 family profiling of mCRC PDX models via quantitative Western blotting. Fig S7: RPPA protein scores indicating proteins' association with the PDX models' response to REG. Fig S8: Western blot validation of differential EPHA2 protein expression between REG post- and pre-treatment progressor and non-progressor PDX models. Table S1: List of antibodies and dilution factors used against desired targets in RPPA analysis.

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Patient-derived xenografts (PDXs) are tumour fragments engrafted into mice for preclinical studies. PDXs offer clear advantages over simplerin vitrocancer models - such as cancer cell lines (CCLs) and organoids - in terms of structural complexity, heterogeneity, and stromal interactions. We characterised 231 colorectal cancer PDXs at the genomic, transcriptomic, and epigenetic level and measured their response to cetuximab, an EGFR inhibitor in clinical use for metastatic colorectal cancer. After assessing PDXs’ quality, stability, and molecular concordance with publicly available patient cohorts, we trained, interpreted, and validated an integrated ensemble classifier (CeSta) which takes in input the PDXs’multi-omiccharacterisation and predicts their sensitivity to cetuximab treatment (AUROC > 0.9). Our study shows that large PDX collections can be used to train accurate, interpretable models of drug sensitivity, which 1) better recapitulate patient-derived therapeutic biomarkers than other models trained on CCL data, 2) can be robustly validated across independent PDX cohorts, and 3) can be used for the development of novel therapeutic biomarkers.

Purpose:Approximately 20% of patients with RAS wild-type metastatic colorectal cancer (mCRC) experience objective responses to the anti-EGFR antibody cetuximab, but disease eradication is seldom achieved. The extent of tumor shrinkage correlates with long-term outcome. We aimed to find rational combinations that potentiate cetuximab efficacy by disrupting adaptive dependencies on antiapoptotic molecules (BCL2, BCL-XL, MCL1).Experimental Design:Experiments were conducted in patient-derived xenografts (PDX) and organoids (PDXO). Apoptotic priming was analyzed by BH3 profiling. Proapoptotic and antiapoptotic protein complexes were evaluated by co-immunoprecipitation and electroluminescence sandwich assays. The effect of combination therapies was assessed by caspase activation in PDXOs and by monitoring PDX growth.Results:A population trial in 314 PDX cohorts, established from as many patients, identified 46 models (14.6%) with appreciable (>50% tumor shrinkage) but incomplete response to cetuximab. From these models, 14 PDXOs were derived. Cetuximab primed cells for apoptosis, but only concomitant blockade of BCL-XL precipitated cell death. Mechanistically, exposure to cetuximab induced upregulation of the proapoptotic protein BIM and its sequestration by BCL-XL. Inhibition of BCL-XL resulted in displacement of BIM, which was not buffered by MCL1 and thereby became competent to induce apoptosis. In five PDX models, combination of cetuximab and a selective BCL-XL inhibitor triggered apoptosis and led to more pronounced tumor regressions and longer time to relapse after treatment discontinuation than cetuximab alone.Conclusions:In mCRC tumors that respond to cetuximab, antibody treatment confers a synthetic-lethal dependency on BCL-XL. Targeting this dependency unleashes apoptosis and increases the depth of response to cetuximab.

Antibodies targeting the human epidermal growth factor receptor (EGFR) are used for the treatment of RAS wild-type metastatic colorectal cancer. A significant proportion of patients remains unresponsive to this therapy. Here, we performed a reverse phase protein array-based (phospho)protein analysis of 63 KRAS, NRAS, BRAF and PIK3CA wild-type metastatic CRC tumours. Responses of tumours to anti-EGFR therapy with cetuximab were recorded in patient-derived xenograft (PDX) models. Unsupervised hierarchical clustering of pre-treatment tumour tissue identified three clusters, of which cluster C3 was exclusively composed of responders. Clusters C1 and C2 showed mixed responses. None of the three protein clusters showed a significant correlation with transcriptome-based subtypes. Analysis of protein signatures across all PDXs identified 14 markers that discriminated cetuximab-sensitive and -resistant tumours: PDK1 (S241), Caspase-8, Shc (Y317), Stat3 (Y705), p27, GSK-3β (S9), HER3, PKC-α (S657), EGFR (Y1068), Akt (S473), S6 Ribosomal Protein (S240/244), HER3 (Y1289), NF-κB-p65 (S536) and Gab-1 (Y627). Least absolute shrinkage and selection operator and binominal logistic regression analysis delivered refined protein signatures for predicting response to cetuximab. (Phospo-)protein analysis of matched pre- and post-treated models furthermore showed significant reduction of Gab-1 (Y627) and GSK-3β (S9) exclusively in responding models, suggesting novel targets for treatment. This article is protected by copyright. All rights reserved.

Under the selective pressure of therapy, tumours dynamically evolve multiple adaptive mechanisms that make static interrogation of genomic alterations insufficient to guide treatment decisions. Clinical research does not enable the assessment of how various regulatory circuits in tumours are affected by therapeutic insults over time and space. Likewise, testing different precision oncology approaches informed by composite and ever-changing molecular information is hard to achieve in patients. Therefore, preclinical models that incorporate the biology and genetics of human cancers, facilitate analyses of complex variables and enable adequate population throughput are needed to pinpoint randomly distributed response predictors. Patient-derived xenograft (PDX) models are dynamic entities in which cancer evolution can be monitored through serial propagation in mice. PDX models can also recapitulate interpatient diversity, thus enabling the identification of response biomarkers and therapeutic targets for molecularly defined tumour subgroups. In this Review, we discuss examples from the past decade of the use of PDX models for precision oncology, from translational research to drug discovery. We elaborate on how and to what extent preclinical observations in PDX models have confirmed and/or anticipated findings in patients. Finally, we illustrate emerging methodological efforts that could broaden the application of PDX models by honing their predictive accuracy or improving their versatility.

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.

Purpose: Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers. Experimental Design: Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment. Results: Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance. Conclusions: Subtype classification systems represent canonical “termini a quo” (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.

Blockade of epidermal growth factor receptor (EGFR) causes tumor regressions in selected patients with metastatic colorectal cancer (mCRC); however, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts, we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns reminiscent of those displayed by a quiescent subpopulation of normal intestinal secretory precursors with Paneth-cell characteristics. These pseudodifferentiated remnants had reduced expression of EGFR-activating ligands, more pronounced activity of human epidermal growth factor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), and persistent signaling along the phosphatidylinositol-3-kinase (PI3K) pathway compared with untreated tumors. Clinically, residual disease traits were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming was mediated by inactivation of Yes-associated protein (YAP) – a master regulator of post-injury intestinal epithelium recovery – following EGFR neutralization. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. By showing that tolerance to EGFR inhibition is typified by the disengagement of an in-built lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis, our results shed light onto CRC lineage plasticity as an adaptive escape mechanism from therapeutic insults and suggest opportunities to pre-emptively target residual disease.

Resistance to chemotherapy often results from dysfunctional apoptosis, however multiple proteins with overlapping functions regulate this pathway. We sought to determine whether an extensively validated, deterministic apoptosis systems model, &lsquo, DR_MOMP&rsquo, could be used as a stratification tool for the apoptosis sensitiser and BCL-2 antagonist, ABT-199 in patient-derived xenograft (PDX) models of colorectal cancer (CRC). Through quantitative profiling of BCL-2 family proteins, we identified two PDX models which were predicted by DR_MOMP to be sufficiently sensitive to 5-fluorouracil (5-FU)-based chemotherapy (CRC0344), or less responsive to chemotherapy but sensitised by ABT-199 (CRC0076). Treatment with ABT-199 significantly improved responses of CRC0076 PDXs to 5-FU-based chemotherapy, but showed no sensitisation in CRC0344 PDXs, as predicted from systems modelling. 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) scans were performed to investigate possible early biomarkers of response. In CRC0076, a significant post-treatment decrease in mean standard uptake value was indeed evident only in the combination treatment group. Radiomic CT feature analysis of pre-treatment images in CRC0076 and CRC0344 PDXs identified features which could phenotypically discriminate between models, but were not predictive of treatment responses. Collectively our data indicate that systems modelling may identify metastatic (m)CRC patients benefitting from ABT-199, and that 18F-FDG-PET could independently support such predictions.

Purpose: Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available. Experimental Design: Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach. Results: XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion. Conclusions: The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

Introduction Anti-EGFR therapies with the monoclonal antibodies cetuximab and panitumumab have improved survival in colorectal cancer (CRC) patients; nevertheless, incomplete mass obliteration and eventual relapse are a common setback, even after a plateau of maximal response. Preclinical data suggest that tumour recurrence may be fueled by a reservoir of so-called ‘drug-tolerant persisters’ that engage non-mutational routes of adaptation to therapy. Yet, the molecular underpinnings that sustain residual disease, as well as the strategies to oppose it, are largely unexplored. Material and methods The effects of targeted therapies were evaluated in patient-derived xenografts. The biochemical and biological consequences of drug exposure were gauged by immunohistochemistry and morphometric analyses (in vivo), and by time-lapse imaging, Western Blot, Cell Titer-Glo and Caspase-Glo assays (in vitro). Transcriptional perturbations were assessed by microarray analysis and/or RT-qPCR. The activity of transcriptional modulators was measured by reporter assays in vitro. Results and discussions Residual tumours surviving cetuximab treatment exhibited a quiescent, Wnt-high, and secretory/Paneth cell-like state as a distinctive trait. This pattern outlines that of EGFR-inhibited quiescent stem cells of the normal intestine, suggesting that developmental trajectories are somehow coopted by cancer cells to face external insults. Such phenotype was reversible with drug suspension, pointing to non-genetic plasticity as a determinant of cancer cell reprogramming. Residual tumours also displayed lower expression of EGFR-activating ligands, congruent with reduced EGFR dependency, and showed rewired reliance on compensatory HER2/HER3 activity, as well as persistent PI3K signalling. Mechanistically, the acquisition of Paneth cell-like features was mediated, at least partly, by inactivation of YAP – a key driver of intestinal cell regeneration. Therapeutically, combined blockade of EGFR and PI3K/AKT lessened residual disease burden, but did not lead to long-term disease control. However, treatment with panHER, a mixture of antibodies concurrently targeting EGFR, HER2, and HER3, reduced tumour volumes and delayed tumour relapse after therapy cessation. Conclusion Drug tolerance in cetuximab-sensitive CRC models involves a switch towards a Paneth-cell like state typified by sustained HER2/HER3 and PI3K signalling. Treatment with panHER effectively exhausted residual tumour burden and impeded/delayed late relapse.

Stromal content heavily impacts the transcriptional classification of colorectal cancer (CRC), with clinical and biological implications. Lineage-dependent stromal transcriptional components could therefore dominate over more subtle expression traits inherent to cancer cells. Since in patient-derived xenografts (PDXs) stromal cells of the human tumour are substituted by murine counterparts, here we deploy human-specific expression profiling of CRC PDXs to assess cancer-cell intrinsic transcriptional features. Through this approach, we identify five CRC intrinsic subtypes (CRIS) endowed with distinctive molecular, functional and phenotypic peculiarities: (i) CRIS-A: mucinous, glycolytic, enriched for microsatellite instability or KRAS mutations; (ii) CRIS-B: TGF-β pathway activity, epithelial–mesenchymal transition, poor prognosis; (iii) CRIS-C: elevated EGFR signalling, sensitivity to EGFR inhibitors; (iv) CRIS-D: WNT activation, IGF2 gene overexpression and amplification; and (v) CRIS-E: Paneth cell-like phenotype, TP53 mutations. CRIS subtypes successfully categorize independent sets of primary and metastatic CRCs, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances., Stromal cells contribute to the gene expression profiles based on which colorectal cancer (CRC) molecular subtypes are classified. Here, patient-derived xenografts enable the authors to obtain cancer cell-specific transcriptomes by excluding transcripts from murine stromal cells, based on which they define CRC intrinsic subtypes (CRIS) and evaluate their prognostic and predictive potential.

Purpose: Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti–EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC). Experimental Design: Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab. Results: At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment. Conclusions: The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses. Clin Cancer Res; 18(9); 2515–25. ©2012 AACR.

Anti-cancer drugs designed to target specific molecular pathways have shown an excellent therapeutic potential but also very poor long-term durability of tumor responses, mainly due to the outbreak of resistant clones among the residual neoplastic cell population. For that reason, understanding the molecular mechanisms underlying the onset of anti-cancer drug resistance (ACDR) is one of the major goals of clinical research. ACDR has been widely studied by DNA/RNA sequencing of primary human samples and several culprits identified. We have previously developed an approach based on lentiviral vector (LV)-induced insertional mutagenesis that allowed to identify the genes involved in lapatinib and erlotinib resistance on HER2+ human breast cancer cell lines and EGFR+ pancreatic cell line respectively. Here we took advantage of this platform to investigate ACDR genes in colorectal cancer (CRC). Cetuximab, anti-EGFR monoclonal antibody, is used as first line therapy in metastatic CRC, which results in prolonged survival of treated patients. However, nearly all patients relapse due to ACDR. We thus selected CRC cells sensitive to cetuximab deriving either from five microsatellite stable cell lines or from eight Patient Derived Xenografts (PDX), primary human CRC cells implanted subcutaneously into immunodeficient mice (NSG). To induce insertional mutagenesis we generated a luciferase-expressing LV harboring the SFFV enhancer/promoter in the long terminal repeats able to perturb the expression of genes nearby the integration site. As control, we used a non-genotoxic SIN-LV. We set up a collagenase IV-based disaggregation protocol that allows single-cell suspension and a serum-free culture condition to maintain the stemness of in vitro cultured cells. This protocol allowed to efficiently disaggregate and expand CRC cells in vitro as well as reach a LV copy number per cell ranging from 0.25 to 5.6. Luciferase gene expression was stable and allowed live-animal monitoring for up to 30 weeks after transplant. CRC-0069 and -0077 PDXs and NCI-H508 and HDC82 cell lines were transduced ex vivo and kept in vitro and/or transplanted in NSG mice. After in vitro or in vivo expansion of the transduced CRCs cetuximab treatment was applied. After an initial shrinking of the tumor mass in mice we observed ACDR in 3 out of 10 mice transplanted with NCI-H508 cells transduced with SFFV-LV and in none of the controls. Genomic DNA from resistant cells is being used for insertion site (IS) analysis to identify common IS, ACDR gene candidates. IS obtained from SIN-LV groups will be used to filter LV integration biases, whereas IS from SFFV-LV transduced cells but not treated with cetuximab will be used to filter mutations that provide a proliferative advantage unrelated to cetuximab treatment. We will validate the most promising candidates by LV-mediated overexpression and knockdown techniques. This approach could pave the way to perform insertional mutagenesis-based forward genetics studies on primary human samples.

Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients' outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts ("xenopatients"), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.

Purpose: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities. Experimental Design: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays. Results: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib—or, importantly, monotherapy with afatinib—resulted in overt tumor shrinkage. Conclusions: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519–31. ©2015 AACR.

In the precision medicine era, multi-parametric interrogation of tumors in cancer patients has enabled rationally refined clinical trials and considerably improved response rates. Nevertheless, incomplete mass obliteration and late relapse invariably emerge. This also relates to metastatic colorectal cancer (mCRC), as massive tumor regression upon anti-EGFR therapy with cetuximab is relatively infrequent. Clinical evidence suggests that tumor recurrence may be endorsed by a population of lingering cancer cells that survive therapy and fuel residual disease through reversible, non-mutational cues, thus installing a “poised” state of drug tolerance that anticipates tumor relapse. The mechanisms underlying this resilience, and the strategies to tackle it, remain to be explored. In this study, we capitalized on different exploratory resources, including a proprietary collection of patient-derived xenografts, as well as CRC cell lines and primary cultures, to explore the mechanisms of cetuximab tolerance. Biochemical characterization of cetuximab persisters evidenced a dichotomous output, with full MAPK neutralization but persistent PI3K/AKT activity, suggesting that PI3K is engaged as a competitor route. Consistently, concomitant EGFR/PI3K blockade specifically unleashed apoptosis in cell lines, as opposed to the cytostatic activity of cetuximab alone, and prevented colosphere growth recovery after drug washout. In vivo, evaluation of residual cellularity confirmed the superior efficacy of dual EGFR/PI3K deactivation. Hence, both the number and fitness of tolerant cells were thwarted by EGFR/PI3K interception. Integrative morphologic analysis, immunohistochemistry, gene-expression profiling and reporter assays in vitro put forward increased beta-catenin activity and Paneth cell-like reprogramming as distinctive traits of residual cells surviving cetuximab treatment. These features are likely related to cetuximab-induced biochemical rewiring, as relieve of ERK-mediated beta-catenin repression in conjunction with persistent PI3K-AKT activity may converge on Wnt-dependent Paneth cell-like differentiation. Importantly, PI3K inhibition dampened cetuximab-triggered Paneth cell specification, thus intercepting a putative mechanism of drug-tolerance. Future studies will extricate the relative contribution of PI3K, MAPK, and Wnt-dependent networks to sustaining the reservoir of persisters, and how Paneth-like traits may foster residual disease. Citation Format: Barbara Lupo, Paolo Gagliardi, Francesco Sassi, Eugenia Rosalinda Zanella, Francesca Cottino, Andrea Bertotti, Livio Trusolino. Targeting the PI3K pathway to intercept cetuximab tolerance in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2017-LB-293

Colorectal cancer (CRC) is the third and second most commonly diagnosed cancer in males and females, and the second most common cause of cancer-related deaths in the developed world. Identifying the importance of epidermal growth factor (EGF) signalling pathways for the survival of CRC cells (Van Cutsem, Kohne et al. 2011) resulted in development of therapies that target EGF-receptors (EGFR). Late stage CRC patients are mainly treated with monoclonal anti-EGFR antibodies such as cetuximab. Although anti-EGFR therapies have significantly improved survival in CRC patients (Van Cutsem, Kohne et al. 2009), they are not effective in patients with activating KRAS, BRAF, NRAS and PI3KCA mutations (De Roock, Claes et al. 2010). Nevertheless, between 50-60% of patients will not benefit from the additional anti-EGFR treatment, even when these have a quadruple wild-type status (De Roock, De Vriendt et al. 2011), suggesting that novel biomarkers and targeted therapies are required. In this study, we investigate potential biomarkers for cetuximab response in a panel of 93 quadruple wild-type, liver metastatic CRC samples from patient derived xenografts (PDX) with known responses to cetuximab. Matching PDXs were used to measure changes in tumour volumes post cetuximab treatment (Bertotti, Papp et al. 2015) and were later categorised as regressing, progressing or stabilised. Protein expression levels were measured in matched PDXs, using a reverse phase protein array (RPPA) with a panel of 72 antibodies targeting key cancer related proteins as previously described). Statistical analysis of measured values showed significant correlation between high protein expression levels, such as Chk-1, PARP, HIAP-2 (cIAP-1), and PDX progression post cetuximab treatment. These proteins were significantly expressed lower in both regressing and stable PDXs. Interestingly, we found a high cross correlation between expression levels of these proteins across all the samples, giving them a great potential to act as predictive biomarkers for cetuximab response. Bioinformatics pathway enrichment of these proteins showed a significant enrichment of intrinsic apoptotic and cell cycle signalling pathways. These results have also been investigated in publically available patient data with cetuximab response and together will be used to construct a deterministic mathematical cell cycle model that will help to predict the outcome of cetuximab therapy in future. Citation Format: Naser Monsefi, Robert O’Byrne, Steven Carberry, Eugenia R. Zanella, Ana Barat, Mattia Cremona, Clare Morgan, Bryan T. Hennessy, Andrea Bertotti, Livio Trusolino, Jochen H.M. Prehn. Investigating potential molecular biomarkers for cetuximab response in metastatic colorectal cancer tissue using reverse phase protein array. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-354.

Purpose: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that co-targeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities. Experimental Design: The signaling consequences of HER2, HER3 and EGFR (HERs) blockade were evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting. HERs’ canonical downstream transducers were quantitatively gauged by multiplex phospho-proteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays for phosphoprotein analysis. Results: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was as effective as trastuzumab plus lapatinib in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib - or monotherapy with afatinib - resulted in overt tumor shrinkage. Conclusions: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Citation Format: Simonetta Maria Leto, Francesco Sassi, Irene Catalano, Giorgia Migliardi, Eugenia Rosalinda Zanella, Andrea Bertotti, Livio Trusolino. Sustained inhibition of HER3 and EGFR is necessary to induce regression of HER2-amplified gastrointestinal carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 659. doi:10.1158/1538-7445.AM2015-659

Only 10% of genetically unselected patients with chemorefractory metastatic colorectal cancer (mCRC) receive clinical benefit from therapy with the anti-EGFR antibodies cetuximab and panitumumab. Among non-responsive patients, 70% bear tumors harboring at least one alteration in KRAS, NRAS, BRAF and PIK3CA genes; therefore, the remaining 30% of “quadruple negative” cases display still-unidentified features that sustain primary resistance to EGFR-targeted therapies. By performing genotype-response correlations in a preclinical platform of patient-derived mCRC xenografts (xenopatients), our group has recently identified HER2 amplification as a new biomarker of resistance to cetuximab within a quadruple negative population. We found that the anti-HER2 monoclonal antibody trastuzumab, despite its demonstrated efficacy in patients with HER2-positive breast and gastric cancers, was completely ineffective in HER2-amplified mCRC xenopatients. Importantly, while the dual EGFR/HER2 small molecule inhibitor lapatinib induced disease stabilization, the combination of lapatinib with trastuzumab was synergistic, achieving rapid and long lasting tumor regressions. When the same regimens where applied to HER2-positive gastric cancer cells in vivo, trastuzumab and lapatinib as monotherapies induced disease stabilization, while the combination of both – similar to CRC – had stronger therapeutic effect. The aim of this study was to better characterize the molecular mechanisms underlying the synergistic effect of the combination therapy in HER2-positive gastric and colorectal cancers. We first generated three CRC cell lines (NCI-H508, DiFi and HDC-142) stably transfected with the HER2 oncogene and provided proof-of-concept of the causative role of HER2 in installing cetuximab resistance in mCRC. Indeed, while parental controls proved to be extremely sensitive to cetuximab, HER2 overexpressors displayed overt resistance, both in viability assays in vitro and in xenografts experiments in vivo. Moreover, NCI-H508-HER2 cells in vivo were poorly responsive to trastuzumab, while lapatinib induced disease stabilization and the combination of both achieved the strongest therapeutic effect. Overall, these responses were very similar to those observed for HER2-positive mCRC xenopatients, attesting to the reliability of this artificial cellular model for further mechanistic studies. We next analyzed the activation status of EGFR, HER2 and HER3 upon treatment with trastuzumab, lapatinib and combo and explored the signaling consequences of receptor blockade. Dose-response curves revealed that trastuzumab did not affect activation of EGFR and HER2, but was able to reduce HER3 phosphorylation; at variance, lapatinib and combo potently inhibited phosphorylation of all HERs in a similar manner, with no additive effect by combo. Interestingly, time-course experiments highlighted that treatment with lapatinib resulted in a paradoxical increase of HER3 expression and phosphorylation both in HER2-positive gastric cancer cells and in colorectal cancer cells. Importantly, adding trastuzumab to lapatinib completely neutralized lapatinib-induced HER3 hyper-phosphorylation, without affecting HER3 levels. Possibly, up-regulated HER3 could provide an alternate route to activate a “bypass track” signaling, attenuating the antitumor effects of lapatinib as monotherapy in these tumor settings. Full inhibition of a residual HER2 activity and consequent abolishment of HER3 phosphorylation achieved by the addition of trastuzumab could provide a mechanistic explanation of the synergic effect obtained with the combination therapy. Overall, these results provide new mechanistic insights into resistance and response to EGFR and HER2 targeted therapies and deserve further investigations to explore the key pathways involved in HER3 feedback up-regulation in the context of HER2-positive cancers. Citation Format: Simonetta Maria Leto, Francesco Sassi, Giorgia Migliardi, Eugenia Zanella, Irene Catalano, Andrea Bertotti, Livio Trusolino. HER2-amplified gastric and colorectal cancers: Mechanistic insights into HER2 targeting approaches. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B43.

Results Xenopatients retained the histological and genomic features of the original counterparts, responded to the anti-EGFR antibody cetuximab similarly to clinical observations, and could be prospectively stratified as responders or nonresponders based on predictive biomarkers. Genotype-response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/ NRAS/BRAF/PIK3CA wild-type cases. In this subset, combined HER2/EGFR inhibition induced long-lasting tumor regression. We also assessed the effects of MEK and PI3K/ mTor inhibitors (AZD6244 and BEZ235 respectively) in 40 specimens harboring KRAS/ NRAS/BRAF/PIK3CA mutations. Cotreatment of xenografts with AZD6244 +BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244. Triple therapy with cetuximab provided further advantage. The extent of disease control declined upon prolonged treatment.