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Background: The Dual Erb B Inhibition in Oesophago-gastric Cancer (DEBIOC) trial reported an acceptable safety profile for neoadjuvant oxaliplatin and capecitabine (Xelox) ± AZD8931 in oesophageal adenocarcinoma (OAC) but limited efficacy. We evaluated the impact of neoadjuvant Xelox ± AZD8931, a novel small-molecule inhibitor with equipotent activity against epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER)2 and HER3, on biological pathways using a unique software-driven solution., Patients and Methods: Transcriptomic profiles from 25 pre-treatment formalin-fixed paraffin-embedded OAC biopsies and 18 matched resection specimens, treated with Xelox + AZD8931 (n = 16) and Xelox alone (n = 9), were analysed using the Almac clara T total mRNA report analysing 92 gene signatures, 100 unique single-gene drug targets and 7337 single genes across 10 hallmarks of cancer. Gene-set enrichment analysis (GSEA) was utilised to investigate pathways governing pathological response. Tumour-infiltrating lymphocytes (TILs) were assessed digitally using the QuPath software., Results: Hierarchical clustering identified three molecular subgroups classified by activation of innate immune signalling. The immune-high subgroup was associated with HER2 positivity, increased pathological response and a marked reduction in immune signalling and TILs following neoadjuvant therapy. The immune-low cluster was predominantly HER2/EGFR-negative, and EGFR positivity was associated with the immune-mixed subgroup. Treatment with neoadjuvant therapy induced common resistance mechanisms, such as angiogenesis and epithelial-mesenchymal transition signalling, and a reduction in DNA repair signatures. Addition of AZD8931 was associated with reduction of expression of EGFR, HER2 and AKT pathways and also promoted an immunosuppressive microenvironment. GSEA showed that patients with a pathological response to treatment had increased immune signalling, whereas non-responders to neoadjuvant therapy were enriched for nucleotide repair and cellular growth through the action of E2F transcription factors., Conclusion: OAC may be subdivided into three immune-related subgroups which undergo modulation in response to neoadjuvant therapy with marked suppression of the immune microenvironment in HER2-positive/immune-high tumours., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Patients with class I V600EBRAF-mutant (MT) colorectal cancer (CRC) have a poor prognosis and their response to combined anti-BRAF/EGFR inhibition remains limited. There is clearly an unmet need in further understanding the biology of V600EBRAFMT CRC. We have used differential gene expression of BRAFWT and MT CRC cells to identify pathways underpinning BRAFMT CRC. We tested a panel of molecularly/genetically subtyped CRC cells for their sensitivity to the Unfolded Protein Response (UPR) activator BOLD-100. To identify novel combination strategies for BOLD-100, we performed RNA sequencing and high-throughput drug screening. Pathway enrichment analysis identified that the UPR and DNA repair pathways were significantly enriched in BRAFMT CRC. We found that oncogenic BRAF plays a crucial role in mediating response to BOLD-100. Using a systems biology approach, we identified V600EBRAFMT-dependent activation of the replication stress response kinase ATR as a key mediator of resistance to BOLD-100. Further analysis identified acute increases in BRAFMT-dependent-reactive oxygen species (ROS) levels following treatment with BOLD-100 that was demonstrated to promote ATR/CHK1 activation and apoptosis. Furthermore, activation of ROS/ATR/CHK1 following BOLD-100 was found to be mediated through the AHR transcription factor and CYP1A1. Importantly, pharmacological blockade of this resistance pathway with ATR inhibitors synergistically increased BOLD-100-induced apoptosis and growth inhibition in BRAFMT models. These results unveil possible novel therapeutic opportunity for BRAFMT CRC. Implications: BOLD-100 induces BRAFMT-dependent replication stress, and targeted strategies against replication stress (eg. by using ATR inhibitors) in combination with BOLD-100 may serve as a potential novel therapeutic strategy for clinically aggressive BRAFMT CRC.

Novel covalent inhibitors of KRASG12C have shown limited response rates in patients with KRASG12C-mutant (MT) colorectal cancer. Thus, novel KRASG12C inhibitor combination strategies that can achieve deep and durable responses are needed. Small-molecule KRASG12C inhibitors AZ'1569 and AZ'8037 were used. To identify novel candidate combination strategies for AZ'1569, we performed RNA sequencing, siRNA, and high-throughput drug screening. Top hits were validated in a panel of KRASG12CMT colorectal cancer cells and in vivo. AZ'1569-resistant colorectal cancer cells were generated and characterized. We found that response to AZ'1569 was heterogeneous across the KRASG12CMT models. AZ'1569 was ineffective at inducing apoptosis when used as a single agent or combined with chemotherapy or agents targeting the EGFR/KRAS/AKT axis. Using a systems biology approach, we identified the antiapoptotic BH3-family member BCL2L1/Bcl-xL as a top hit mediating resistance to AZ'1569. Further analyses identified acute increases in the proapoptotic protein BIM following AZ'1569 treatment. ABT-263 (navitoclax), a pharmacologic Bcl-2 family inhibitor that blocks the ability of Bcl-xL to bind and inhibit BIM, led to dramatic and universal apoptosis when combined with AZ'1569. Furthermore, this combination also resulted in dramatically attenuated tumor growth in KRASG12CMT xenografts. Finally, AZ'1569-resistant cells showed amplification of KRASG12C, EphA2/c-MET activation, increased proinflammatory chemokine profile and cross-resistance to several targeted agents. Importantly, KRAS amplification and AZ'1569 resistance were reversible upon drug withdrawal, arguing strongly for the use of drug holidays in the case of KRAS amplification. Taken together, combinatorial targeting of Bcl-xL and KRASG12C is highly effective, suggesting a novel therapeutic strategy for patients with KRASG12CMT colorectal cancer., (©2022 The Authors; Published by the American Association for Cancer Research.)

Expression of the deubiquitinase USP17 is induced by multiple stimuli, including cytokines (IL-4/6), chemokines (IL-8, SDF1), and growth factors (EGF), and several studies indicate it is required for cell proliferation and migration. However, the mechanisms via which USP17 impacts upon these cellular functions are unclear. Here, we demonstrate that USP17 depletion prevents peripheral lysosome positioning, as well as trafficking of lysosomes to the cell periphery in response to EGF stimulation. Overexpression of USP17 also increases secretion of the lysosomal protease cathepsin D. In addition, USP17 depletion impairs plasma membrane repair in cells treated with the pore-forming toxin streptolysin O, further indicating that USP17 is required for lysosome trafficking to the plasma membrane. Finally, we demonstrate that USP17 can deubiquitinate p62, and we propose that USP17 can facilitate peripheral lysosome trafficking by opposing the E3 ligase RNF26 to untether lysosomes from the ER and facilitate lysosome peripheral trafficking, lysosome protease secretion, and plasma membrane repair., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Cancer cells' ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells, and quantification of 18 cell lineage and apoptosis proteins. We identified an enrichment for BCL2 in immune, and BAK, SMAC, and XIAP in cancer cells. Ordinary differential equation-based modeling of apoptosis sensitivity at single-cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. Systems modeling at single-cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, but showed significant inter- and intra-tumor heterogeneity., (© 2021. The Author(s).)

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVE TM ) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as 'high', 'moderate' or 'low', for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1-) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1- Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1-. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes., (© 2021. The Author(s).)

Motivation: Multiplexed immunofluorescence bioimaging of single-cells and their spatial organization in tissue holds great promise to the development of future precision diagnostics and therapeutics. Current multiplexing pipelines typically involve multiple rounds of immunofluorescence staining across multiple tissue slides. This introduces experimental batch effects that can hide underlying biological signal. It is important to have robust algorithms that can correct for the batch effects while not introducing biases into the data. Performance of data normalization methods can vary among different assay pipelines. To evaluate differences, it is critical to have a ground truth dataset that is representative of the assay., Results: A new immunoFLuorescence Image NOrmalization method is presented and evaluated against alternative methods and workflows. Multiround immunofluorescence staining of the same tissue with the nuclear dye DAPI was used to represent virtual slides and a ground truth. DAPI was restained on a given tissue slide producing multiple images of the same underlying structure but undergoing multiple representative tissue handling steps. This ground truth dataset was used to evaluate and compare multiple normalization methods including median, quantile, smooth quantile, median ratio normalization and trimmed mean of the M-values. These methods were applied in both an unbiased grid object and segmented cell object workflow to 24 multiplexed biomarkers. An upper quartile normalization of grid objects in log space was found to obtain almost equivalent performance to directly normalizing segmented cell objects by the middle quantile. The developed grid-based technique was then applied with on-slide controls for evaluation. Using five or fewer controls per slide can introduce biases into the data. Ten or more on-slide controls were able to robustly correct for batch effects., Availability and Implementation: The data underlying this article along with the FLINO R-scripts used to perform the evaluation of image normalizations methods and workflows can be downloaded from https://github.com/GE-Bio/FLINO., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press.)

Clinical trials for MET inhibitors have demonstrated limited success for their use in colon cancer (CC). However, clinical efficacy may be obscured by a lack of standardisation in MET assessment for patient stratification. In this study, we aimed to determine the molecular context in which MET is deregulated in CC using a series of genomic and proteomic tests to define MET expression and identify patient subgroups that should be considered in future studies with MET-targeted agents. To this aim, orthogonal expression analysis of MET was conducted in a population-representative cohort of stage II/III CC patients (n = 240) diagnosed in Northern Ireland from 2004 to 2008. Targeted sequencing was used to determine the relative incidence of MET R970C and MET T992I mutations within the cohort. MET amplification was assessed using dual-colour dual-hapten brightfield in situ hybridisation (DDISH). Expression of transcribed MET and c-MET protein within the cohort was assessed using digital image analysis on MET RNA in situ hybridisation (ISH) and c-MET immunohistochemistry (IHC) stained slides. We found that less than 2% of the stage II/III CC patient population assessed demonstrated a genetic MET aberration. Determination of a high MET RNA-ISH/low c-MET IHC protein subgroup was found to be associated with poor 5-year cancer-specific outcomes compared to patients with concordant MET RNA-ISH and c-MET IHC protein expression (HR 2.12 [95%CI: 1.27-3.68]). The MET RNA-ISH/c-MET IHC protein biomarker paradigm identified in this study demonstrates that subtyping of MET expression may be required to identify MET-addicted malignancies in CC patients who will truly benefit from MET inhibition., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Entosis is a form of nonphagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumors show evidence of entosis; however, factors controlling entosis remain to be elucidated. Here, we find that besides inducing apoptosis, TRAIL signaling is a potent activator of entosis in colon cancer cells. Initiation of both apoptosis and entosis requires TRAIL receptors DR4 and DR5; however, induction of apoptosis and entosis diverges at caspase-8 as its structural presence is sufficient for induction of entosis but not apoptosis. Although apoptosis and entosis are morphologically and biochemically distinct, knockout of Bax and Bak, or inhibition of caspases, also inhibits entotic cell death and promotes survival and release of inner cells. Analysis of colorectal cancer tumors reveals a significant association between TRAIL signaling and CIC structures. Finally, the presence of CIC structures in the invasive front regions of colorectal tumors shows a strong correlation with adverse patient prognosis., (© 2021 Bozkurt et al.)

Resistance to chemotherapy-induced cell death is a major barrier to effective treatment of solid tumours such as colorectal cancer, CRC. Herein, we present a study aimed at developing a proteomics-based predictor of response to standard-of-care (SoC) chemotherapy in combination with antagonists of IAPs (inhibitors of apoptosis proteins), which have been implicated as mediators of drug resistance in CRC. We quantified the absolute expression of 19 key apoptotic proteins at baseline in a panel of 12 CRC cell lines representative of the genetic diversity seen in this disease to identify which proteins promote resistance or sensitivity to a model IAP antagonist [birinapant (Bir)] alone and in combination with SoC chemotherapy (5FU plus oxaliplatin). Quantitative western blotting demonstrated heterogeneous expression of IAP interactome proteins across the CRC cell line panel, and cell death analyses revealed a widely varied response to Bir/chemotherapy combinations. Baseline protein expression of cIAP1, caspase-8 and RIPK1 expression robustly correlated with response to Bir/chemotherapy combinations. Classifying cell lines into 'responsive', 'intermediate' and 'resistant' groups and using linear discriminant analysis (LDA) enabled the identification of a 12-protein signature that separated responders to Bir/chemotherapy combinations in the CRC cell line panel with 100% accuracy. Moreover, the LDA model was able to predict response accurately when cells were cocultured with Tumour necrosis factor-alpha to mimic a pro-inflammatory tumour microenvironment. Thus, our study provides the starting point for a proteomics-based companion diagnostic that predicts response to IAP antagonist/SoC chemotherapy combinations in CRC., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The present study shows the putative antiproliferative mechanism of action of the previously analytically characterized nudibranch extract ( Dolabella auricularia, NB) and its different effects in colon cancer cells vs. nontumor colon cells. NB extract increased the accumulation of reactive oxygen species (ROS) and increased endoplasmic reticulum (ER) stress via stimulation of the unfolded protein response. Stress scavengers, N-acetylcysteine (NAC) and 4-phenylbutyric acid (4-PBA), decreased the stress induced by NB. The results showed that NB extract increased ER stress through overproduction of ROS in superinvasive colon cancer cells, decreased their resistance threshold, and produced a nonreturn level of ER stress, causing DNA damage and cell cycle arrest, which prevented them from achieving hyperproliferative capacity and migrating to and invading other tissues. On the contrary, NB extract had a considerably lower effect on nontumor human colon cells, suggesting a selective effect related to stress balance homeostasis. In conclusion, our results confirm that the growth and malignancy of colon cancer cells can be decreased by marine compounds through the modification of one of the most potent resistance mechanisms present in tumor cells; this characteristic differentiates cancer cells from nontumor cells in terms of stress balance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ruiz-Torres, Forsythe, Pérez-Sánchez, Van Schaeybroeck, Barrajón-Catalán and Micol.)

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and a notable cause of cancer mortality when it metastasises. The unfolded protein response (UPR) can be cytoprotective but when acutely activated can lead to cell death. In this study, we sought to enhance the acute activation of the UPR using radiation and ONC201, an UPR activator. Treating PCa cells with ONC201 quickly increased the expression of all the key regulators of the UPR and reduced the oxidative phosphorylation, with cell death occurring 72 h later. We exploited this time lag to sensitize prostate cancer cells to radiation through short-term treatment with ONC201. To understand how priming occurred, we performed RNA-Seq analysis and found that ONC201 suppressed the expression of cell cycle and DNA repair factors. In conclusion, we have shown that ONC201 can prime enhanced radiation response.

Background: Antibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC., Methods: In vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens., Results: Site-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells., Conclusions: Together, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.

The phenotypic spectrum of colorectal cancer (CRC) is remarkably diverse, with seemingly endless variations in cell shape, mitotic figures and multicellular configurations. Despite this morphological complexity, histological grading of collective phenotype patterns provides robust prognostic stratification in CRC. Although mechanistic understanding is incomplete, previous studies have shown that the cortical protein ezrin controls diversification of cell shape, mitotic figure geometry and multicellular architecture, in 3D organotypic CRC cultures. Because ezrin is a substrate of Src tyrosine kinase that is frequently overexpressed in CRC, we investigated Src regulation of ezrin and morphogenic growth in 3D CRC cultures. Here we show that Src perturbations disrupt CRC epithelial spatial organisation. Aberrant Src activity suppresses formation of the cortical ezrin cap that anchors interphase centrosomes. In CRC cells with a normal centrosome number, these events lead to mitotic spindle misorientation, perturbation of cell cleavage, abnormal epithelial stratification, apical membrane misalignment, multilumen formation and evolution of cribriform multicellular morphology, a feature of low-grade cancer. In isogenic CRC cells with centrosome amplification, aberrant Src signalling promotes multipolar mitotic spindle formation, pleomorphism and morphological features of high-grade cancer. Translational studies in archival human CRC revealed associations between Src intensity, multipolar mitotic spindle frequency and high-grade cancer morphology. Collectively, our study reveals Src regulation of CRC morphogenic growth via ezrin-centrosome engagement and uncovers combined perturbations underlying transition to high-grade CRC morphology. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

The anti-Epidermal Growth Factor Receptor (EGFR) antibody Cetuximab (CTX) has demonstrated limited anti-cancer efficacy in cells overexpressing EGFR due to activating mutations in RAS in solid tumours, such as pancreatic cancer. The utilisation of antibodies as targeting components of antibody-drug conjugates, such as trastuzumab emtansine (Kadcyla), demonstrates that antibodies may be repurposed to direct therapeutic agents to antibody-resistant cancers. Here we investigated the use of CTX as a targeting agent for camptothecin (CPT)-loaded polymeric nanoparticles (NPs) directed against KRAS mutant CTX-resistant cancer cells. CPT was encapsulated within poly(lactic-co-glycolic acid) (PLGA) NPs using the solvent evaporation method. CTX conjugation improved NP binding and delivery of CPT to CTX-resistant cancer cell lines. CTX successfully targeted CPT-loaded NPs to mutant KRAS PANC-1 tumours in vivo and reduced tumour growth. This study highlights that CTX can be repurposed as a targeting agent against CTX-resistant cancers and that antibody repositioning may be applicable to other antibodies restricted by resistance.

Purpose: Dynamic network models predict clinical prognosis and inform therapeutic intervention by elucidating disease-driven aberrations at the systems level. However, the personalization of model predictions requires the profiling of multiple model inputs, which hampers clinical translation., Patients and Methods: We applied APOPTO-CELL, a prognostic model of apoptosis signaling, to showcase the establishment of computational platforms that require a reduced set of inputs. We designed two distinct and complementary pipelines: a probabilistic approach to exploit a consistent subpanel of inputs across the whole cohort (Ensemble) and a machine learning approach to identify a reduced protein set tailored for individual patients (Tree). Development was performed on a virtual cohort of 3,200,000 patients, with inputs estimated from clinically relevant protein profiles. Validation was carried out in an in-house stage III colorectal cancer cohort, with inputs profiled in surgical resections by reverse phase protein array (n = 120) and/or immunohistochemistry (n = 117)., Results: Ensemble and Tree reproduced APOPTO-CELL predictions in the virtual patient cohort with 92% and 99% accuracy while decreasing the number of inputs to a consistent subset of three proteins (40% reduction) or a personalized subset of 2.7 proteins on average (46% reduction), respectively. Ensemble and Tree retained prognostic utility in the in-house colorectal cancer cohort. The association between the Ensemble accuracy and prognostic value (Spearman ρ = 0.43; P = .02) provided a rationale to optimize the input composition for specific clinical settings. Comparison between profiling by reverse phase protein array (gold standard) and immunohistochemistry (clinical routine) revealed that the latter is a suitable technology to quantify model inputs., Conclusion: This study provides a generalizable framework to optimize the development of network-based prognostic assays and, ultimately, to facilitate their integration in the routine clinical workflow.

Background: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs)., Methods: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed., Results: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib., Conclusions: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

Purpose: Transcriptomic profiling of colorectal cancer (CRC) has led to identification of four consensus molecular subtypes (CMS1-4), which have prognostic value in stage II/III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors. However, the clinical value of these classifications in predicting response to standard-of-care adjuvant chemotherapy remains unknown., Patients and Methods: Using samples from 4 European sites, we assembled a novel stage II/III CRC patient cohort and performed transcriptomic profiling on 156 samples, targeted sequencing and generated a tissue microarray to enable integrated "multi-omics" analyses. We also accessed data from 2 published stage II/III CRC patient cohorts: GSE39582 and GSE14333 (479 and 185 samples respectively)., Results: The epithelial-rich CMS2 subtype of CRC benefitted significantly from adjuvant chemotherapy treatment in both stage II and III disease (p=0.02 and p<0.0001 respectively), while the CMS3 subtype significantly benefitted in stage III only (p=0.00073). Following CRIS sub-stratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from adjuvant chemotherapy in stage II and III disease (p=0.0081 and p<0.0001 respectively), while CRIS-D significantly benefitted in stage III only (p=0.0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk of relapse in both stage II and III disease (p=0.0031)., Conclusion: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying poor prognostic stage II/III patients who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

BRAF V600E mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAF MT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III BRAF MT (discovery set: n = 31; validation set: n = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the BRAF MT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the BRAF MT colorectal cancer subgroup with poorest outcome. We also found that oncogenic BRAF drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in BRAF MT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in BRAF MT in vitro and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic BRAF induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic BRAF MT colorectal cancer. Mol Cancer Ther; 17(6); 1280-90. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Objective: The mitochondrial apoptosis pathway is controlled by an interaction of multiple BCL-2 family proteins, and plays a key role in tumour progression and therapy responses. We assessed the prognostic potential of an experimentally validated, mathematical model of BCL-2 protein interactions (DR&#95;MOMP) in patients with stage III colorectal cancer (CRC)., Design: Absolute protein levels of BCL-2 family proteins were determined in primary CRC tumours collected from n=128 resected and chemotherapy-treated patients with stage III CRC. We applied DR&#95;MOMP to categorise patients as high or low risk based on model outputs, and compared model outputs with known prognostic factors (T-stage, N-stage, lymphovascular invasion). DR&#95;MOMP signatures were validated on protein of n=156 patients with CRC from the Cancer Genome Atlas (TCGA) project., Results: High-risk stage III patients identified by DR&#95;MOMP had an approximately fivefold increased risk of death compared with patients identified as low risk (HR 5.2, 95% CI 1.4 to 17.9, p=0.02). The DR&#95;MOMP signature ranked highest among all molecular and pathological features analysed. The prognostic signature was validated in the TCGA colon adenocarcinoma (COAD) cohort (HR 4.2, 95% CI 1.1 to 15.6, p=0.04). DR&#95;MOMP also further stratified patients identified by supervised gene expression risk scores into low-risk and high-risk categories. BCL-2-dependent signalling critically contributed to treatment responses in consensus molecular subtypes 1 and 3, linking for the first time specific molecular subtypes to apoptosis signalling., Conclusions: DR&#95;MOMP delivers a system-based biomarker with significant potential as a prognostic tool for stage III CRC that significantly improves established histopathological risk factors., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Data from many preclinical studies, including those using cellular models of colorectal, gastric, gastro-oesophageal and gastro-oesophageal junction cancers, indicate that the hepatocyte growth factor (HGF)-hepatocyte growth factor receptor (c-MET) pathway is vital for the growth, survival and invasive potential of gastrointestinal cancers. Following the availability of data from these various studies, and data on c-MET expression as a biomarker that indicates a poor prognosis in patients with gastrointestinal cancer and increased c-MET expression, inhibitors targeting this pathway have entered the clinic in the past decade. However, the design of clinical trials that incorporate the use of HGF/c-MET inhibitors in their most appropriate genetic and molecular context remains crucial. Recognizing and responding to this challenge, the European Commission funded Framework 7 MErCuRIC programme is running a biomarker-enriched clinical trial investigating the efficacy of combined c-MET/MEK inhibition in patients with RAS-mutant or RAS-wild-type metastatic colorectal cancer with aberrant c-MET expression. The design of this trial enables the continued refinement of the predictive biomarker and co-development of companion diagnostics. In this Review, we focus on advances in our understanding of inhibition of the HGF/c-MET pathway in patients with gastro-intestinal cancers, the prominent challenges facing the clinical translation and implementation of agents targeting HGF/c-MET, and discuss the various efforts, and associated obstacles to the discovery and validation of biomarkers that will enable patient stratification in this context.

Purpose: Apoptosis is essential for chemotherapy responses. In this discovery and validation study, we evaluated the suitability of a mathematical model of apoptosis execution (APOPTO-CELL) as a stand-alone signature and as a constituent of further refined prognostic stratification tools. Experimental Design: Apoptosis competency of primary tumor samples from patients with stage III colorectal cancer ( n = 120) was calculated by APOPTO-CELL from measured protein concentrations of Procaspase-3, Procaspase-9, SMAC, and XIAP. An enriched APOPTO-CELL signature (APOPTO-CELL-PC3) was synthesized to capture apoptosome-independent effects of Caspase-3. Furthermore, a machine learning Random Forest approach was applied to APOPTO-CELL-PC3 and available molecular and clinicopathologic data to identify a further enhanced signature. Association of the signature with prognosis was evaluated in an independent colon adenocarcinoma cohort (TCGA COAD, n = 136). Results: We identified 3 prognostic biomarkers ( P = 0.04, P = 0.006, and P = 0.0004 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively) with increasing stratification accuracy for patients with stage III colorectal cancer.The APOPTO-CELL-PC3 signature ranked highest among all features. The prognostic value of the signatures was independently validated in stage III TCGA COAD patients ( P = 0.01, P = 0.04, and P = 0.02 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively). The signatures provided further stratification for patients with CMS1-3 molecular subtype. Conclusions: The integration of a systems-biology-based biomarker for apoptosis competency with machine learning approaches is an appealing and innovative strategy toward refined patient stratification. The prognostic value of apoptosis competency is independent of other available clinicopathologic and molecular factors, with tangible potential of being introduced in the clinical management of patients with stage III colorectal cancer. Clin Cancer Res; 23(5); 1200-12. ©2016 AACR ., (©2016 American Association for Cancer Research.)

In recent times, our knowledge of the roles ubiquitin plays in multiple cellular processes has expanded exponentially, with one example being the role of ubiquitin in receptor endocytosis and trafficking. This has prompted a multitude of studies examining how the different machinery involved in the addition and removal of ubiquitin can influence this process. Multiple deubiquitylating enzymes (DUBs) have been implicated either in facilitating receptor endocytosis and lysosomal degradation or in rescuing receptor levels by preventing endocytosis and/or promoting recycling to the plasma membrane. In this review, we will discuss in detail what is currently known about the role of DUBs in regulating the endocytosis of various transmembrane receptors and ion channels. We will also expand upon the role DUBs play in receptor sorting at the multivesicular body to determine whether a receptor is recycled or trafficked to the lysosome for degradation. Finally, we will briefly discuss how the DUBs implicated in these processes may contribute to the pathogenesis of a range of diseases, and thus the potential these have as therapeutic targets., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

Purpose: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled., Experimental Design: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients., Results: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286-9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial-mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed., Conclusions: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095-104. ©2016 AACRSee related commentary by Morris and Kopetz, p. 3989., (©2016 American Association for Cancer Research.)

A recent phase II study of patients with metastatic colorectal carcinoma showed that mismatch repair gene status was predictive of clinical response to PD-1-targeting immune checkpoint blockade. Further examination revealed strong correlation between PD-L1 protein expression and microsatellite instability (MSI) in stage IV colorectal carcinoma, suggesting that the amount of PD-L1 protein expression could identify late-stage patients who might benefit from immunotherapy. To assess whether the clinical associations between PD-L1 gene expression and MSI identified in metastatic colorectal carcinoma are also present in stage II/III colorectal carcinoma, we used in silico analysis to elucidate the cell types expressing the PD-L1 gene. We found a statistically significant association of PD-L1 gene expression with MSI in early-stage colorectal carcinoma (P < 0.001) and show that, unlike in non-colorectal carcinoma tumors, PD-L1 is derived predominantly from the immune infiltrate. We demonstrate that PD-L1 gene expression has positive prognostic value in the adjuvant disease setting (PD-L1(low) vs. PD-L1(high) HR = 9.09; CI, 2.11-39.10). PD-L1 gene expression had predictive value, as patients with high PD-L1 expression appear to be harmed by standard-of-care treatment (HR = 4.95; CI, 1.10-22.35). Building on the promising results from the metastatic colorectal carcinoma PD-1-targeting trial, we provide compelling evidence that patients with PD-L1(high)/MSI/immune(high) stage II/III colorectal carcinoma should not receive standard chemotherapy. This conclusion supports the rationale to clinically evaluate this patient subgroup for PD-1 blockade treatment. Cancer Immunol Res; 4(7); 582-91. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear., Experimental Design: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models., Results: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells., Conclusions: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target., (©2015 American Association for Cancer Research.)

The recent discovery of oncogenic drivers and subsequent development of novel targeted strategies has significantly added to the therapeutic armamentarium of anti-cancer therapies. Targeting BCR-ABL in chronic myeloid leukemia (CML) or HER2 in breast cancer has led to practice-changing clinical benefits, while promising therapeutic responses have been achieved by precision medicine approaches in EGFR mutant lung cancer, colorectal cancer and BRAF mutant melanoma. However, although initial therapeutic responses to targeted therapies can be substantial, many patients will develop disease progression within 6-12 months. An increasing application of powerful omics-based approaches and improving preclinical models have enabled the rapid identification of secondary resistance mechanisms. Herein, we discuss how this knowledge has translated into rational, novel treatment strategies for relapsed patients in genomically selected cancer populations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mutations in the Ras-pathway occur in 40-45% of colorectal cancer patients and these are refractory to treatment with anti-EGFR-targeted therapies. With this in mind, we have studied novel guanidinium-based compounds with demonstrated ability to inhibit protein kinases. We have performed docking studies with several proteins involved in the Ras-pathway and evaluated 3,4'-bis-guanidinium derivatives as inhibitors of B-Raf. Compound 3, the most potent in this series, demonstrated strong cytotoxicity in (WT)B-Raf colorectal cancer cells and also cells with (V600E)B-Raf mutations. Cell death was induced by apoptosis, detected by cleavage of PARP. Compound 3 also potently inhibited ERK1/2 signalling, inhibited EGFR activation, as well as Src, STAT3 and AKT phosphorylation. Mechanistically, compound 3 did not inhibit ATP binding to B-Raf, but direct assay of B-Raf activity was inhibited in vitro. Summarizing, we have identified a novel B-Raf type-III inhibitor that exhibits potent cellular cytotoxicity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)