The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells.

YAP and TAZ, the Hippo pathway terminal transcriptional activators, are frequently upregulated in cancers. In tumor cells, they have been mainly associated with increased tumorigenesis controlling different aspects from cell cycle regulation, stemness, or resistance to chemotherapies. In fewer cases, they have also been shown to oppose cancer progression, including by promoting cell death through the action of the p73/YAP transcriptional complex, in particular after chemotherapeutic drug exposure. Using HCT116 cells, we show here that oxaliplatin treatment led to core Hippo pathway down-regulation and nuclear accumulation of TAZ. We further show that TAZ was required for the increased sensitivity of HCT116 cells to oxaliplatin, an effect that appeared independent of p73, but which required the nuclear relocalization of TAZ. Accordingly, Verteporfin and CA3, two drugs affecting the activity of YAP and TAZ, showed antagonistic effects with oxaliplatin in co-treatments. Importantly, using several colorectal cell lines, we show that the sensitizing action of TAZ to oxaliplatin is dependent on the p53 status of the cells. Our results support thus an early action of TAZ to sensitize cells to oxaliplatin, consistent with a model in which nuclear TAZ in the context of DNA damage and p53 activity pushes cells towards apoptosis.

Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells.

BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma.

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

Targeting the splicing isoforms of spleen tyrosine kinase affects the viability of colorectal cancer cells.

Spleen tyrosine kinase (Syk) expression have been both positively and negatively associated with tumorigenesis. Our goal was to evaluate the contribution of Syk and its two splice variants, full length Syk (L) and short isoform Syk (S), in the tumor biology of colorectal cancer cells (CRC). The analysis of Syk expression in primary human colorectal tumors, as well as the analysis of TCGA database, revealed a high Syk mRNA expression score in colorectal cancer tumors, suggesting a tumor promotor role of Syk in CRC. Our analysis showed that Syk (L) isoform is highly expressed in the majority of the tumor tissues and that it remains expressed in tumors in which global Syk expression is downregulated, suggesting the dependence of tumors to Syk (L) isoform. We also identified a small cluster of tumor tissues, which express a high proportion of Syk (S) isoform. This specific cluster is associated with overexpressed genes related to translation and mitochondria, and down regulated genes implicated in the progression of mitosis. For our functional studies, we used short hairpin RNA tools to target the expression of Syk in CRC cells bearing the activating K-Ras (G13D) mutation. Our results showed that while global Syk knock down increases cell proliferation and cell motility, Syk (L) expression silencing affects the viability and induces the apoptosis of the cells, confirming the dependence of cells on Syk (L) isoform for their survival. Finally, we report the promising potential of compound C-13, an original non-enzymatic inhibitor of Syk isolated in our group. In vitro studies showed that C-13 exerts cytotoxic effects on Syk-positive CRC cells by inhibiting their proliferation and their motility, and by inducing their apoptosis, while Syk-negative cell lines viability was not affected. Moreover, the oral and intraperitoneal administration of C-13 reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo.

Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry.

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.

Pharmacokinetic Variability Drives Palbociclib-Induced Neutropenia in Metastatic Breast Cancer Patients: Drug-Drug Interactions Are the Usual Suspects.

Palbociclib is a good candidate for therapeutic drug monitoring (TDM) due to its narrow therapeutic range and frequency of toxicities, particularly high-grade neutropenia. In this prospective, bicentric clinical trial, we evaluated the palbociclib exposure−toxicity relationship and determined the relevant sources of palbociclib pharmacokinetic variability, including drug−drug interactions (DDI). We followed 58 patients (mean age: 62.9 years) for 1 year. The geometric median of palbociclib plasma trough concentration (Ctrough) was 74.1 ng/mL. Neutropenia occurred in 70.7% of patients (high grade in 67.2% of patients). High-grade neutropenia occurrence during the first two palbociclib cycles was higher in patients with lower neutrophil count at initiation (p = 0.002). Palbociclib plasma Ctrough was correlated with high-grade neutropenia occurrence during the first two cycles (p = 0.024, OR 5.51). Co-treatment with agents that may interfere with palbociclib PK significantly influenced palbociclib Ctrough (p < 0.05). CYP3A4/P-glycoprotein inhibitors increased by 25% palbociclib Ctrough (p = 0.035), while antacids reduced it by 20% (p = 0.036). However, DDI did not have any significant effect on high-grade neutropenia occurrence (p > 0.05). This study confirms the major role of TDM to manage palbociclib safe use from the first week of treatment, particularly the significant incidence of hematological toxicity. Moreover, this first dedicated prospective study confirmed the importance of characterizing co-treatments to limit the DDI risk with oral-targeted therapies.

Anticancer Drugs for Intra-Arterial Treatment of Colorectal Cancer Liver Metastases: In-Vitro Screening after Short Exposure Time.

To treat colorectal liver metastases, intra-arterial chemotherapies may complete therapeutic arsenal. Drugs using intra-arterially are very heterogeneous. The aim of this study was to select the most efficient drug on a panel of colorectal cancer (CRC) cell lines (Caco-2, HCT 116, HT 29, SW 48, SW 480, SW 620) exposed for 30 min to 12 cytotoxic agents (doxorubicin, epirubicin, idarubicin, 5-FU, raltitrexed, gemcitabine, cisplatin, oxaliplatin, mitomycin C, irinotecan, streptozocin, paclitaxel) at different concentrations. The effect on cell viability was measured using the WST-1 cell viability assay. For each drug and cell line, the IC50 and IC90 were calculated, which respectively correspond to the drug concentration (mg/mL) required to obtain 50% and 90% of cell death. We also quantified the cytotoxic index (CyI90 = C Max/IC90) to compare drug efficacy. The main findings of this study are that idarubicin emerged as the most cytotoxic agent to most of the tested CRC cell lines (Caco-2, HT29, HCT116, SW620 and SW480). Gemcitabine seemed to be the most efficient chemotherapy for SW48. Interestingly, the most commonly used cytotoxic agents in the systemic and intra-arterial treatment of colorectal liver metastasis (CRLM) (oxaliplatin, 5-FU, irinotecan) showed very limited cytotoxicity to all the cell lines.

Internalization of Foldamer-Based DNA Mimics through a Site-Specific Antibody Conjugate to Target HER2-Positive Cancer Cells.

Inhibition of protein-DNA interactions represents an attractive strategy to modulate essential cellular functions. We reported the synthesis of unique oligoamide-based foldamers that adopt single helical conformations and mimic the negatively charged phosphate moieties of B-DNA. These mimics alter the activity of DNA interacting enzymes used as targets for cancer treatment, such as DNA topoisomerase I, and they are cytotoxic only in the presence of a transfection agent. The aim of our study was to improve internalization and selective delivery of these highly charged molecules to cancer cells. For this purpose, we synthesized an antibody-drug conjugate (ADC) using a DNA mimic as a payload to specifically target cancer cells overexpressing HER2. We report the bioconjugation of a 16-mer DNA mimic with trastuzumab and its functional validation in breast and ovarian cancer cells expressing various levels of HER2. Binding of the ADC to HER2 increased with the expression of the receptor. The ADC was internalized into cells and was more efficient than trastuzumab at inhibiting their growth in vitro. These results provide proof of concept that it is possible to site-specifically graft high molecular weight payloads such as DNA mimics onto monoclonal antibodies to improve their selective internalization and delivery in cancer cells.

PXR Modulates the Prostate Cancer Cell Response to Afatinib by Regulating the Expression of the Monocarboxylate Transporter SLC16A1.

Resistance to castration is a crucial issue in the treatment of metastatic prostate cancer. Kinase inhibitors (KIs) have been tested as potential alternatives, but none of them are approved yet. KIs are subject of extensive metabolism at both the hepatic and the tumor level. Here, we studied the role of PXR (Pregnane X Receptor), a master regulator of metabolism, in the resistance to KIs in a prostate cancer setting. We confirmed that PXR is expressed in prostate tumors and is more frequently detected in advanced forms of the disease. We showed that stable expression of PXR in 22Rv1 prostate cancer cells conferred a resistance to dasatinib and a higher sensitivity to erlotinib, dabrafenib, and afatinib. Higher sensitivity to afatinib was due to a ~ 2-fold increase in its intracellular accumulation and involved the SLC16A1 transporter as its pharmacological inhibition by BAY-8002 suppressed sensitization of 22Rv1 cells to afatinib and was accompanied with reduced intracellular concentration of the drug. We found that PXR could bind to the SLC16A1 promoter and induced its transcription in the presence of PXR agonists. Together, our results suggest that PXR could be a biomarker of response to kinase inhibitors in castration-resistant prostate cancers.

Anti-tumoral activity of the Pan-HER (Sym013) antibody mixture in gemcitabine-resistant pancreatic cancer models.

Chemoresistance, particularly to gemcitabine, is a major challenge in pancreatic cancer. The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptors 2 and 3 (HER2, HER3) are expressed in many tumors, and they are relevant therapeutic targets due to their synergistic interaction to promote tumor aggressiveness and therapeutic resistance. Cocktails of antibodies directed against different targets are a promising strategy to overcome these processes. Here, we found by immunohistochemistry that these three receptors were co-expressed in 11% of patients with pancreatic adenocarcinoma. We then developed gemcitabine-resistant pancreatic cancer cell models (SW-1990-GR and BxPC3-GR) and one patient-derived xenograft (PDX2846-GR) by successive exposure to increasing doses of gemcitabine. We showed that expression of EGFR, HER2 and HER3 was increased in these gemcitabine-resistant pancreatic cancer models, and that an antibody mixture against all three receptors inhibited tumor growth in mice and downregulated HER receptors. Finally, we demonstrated that the Pan-HER and gemcitabine combination has an additive effect in vitro and in mice xenografted with the gemcitabine-sensitive or resistant pancreatic models. The mixture of anti-EGFR, HER2 and HER3 antibodies is a good candidate therapeutic approach for gemcitabine-sensitive and -resistant pancreatic cancer.

BAG6 promotes PINK1 signaling pathway and is essential for mitophagy.

Bcl-2-associated athanogen-6 (BAG6) is a nucleocytoplasmic shuttling protein involved in protein quality control. We previously demonstrated that BAG6 is essential for autophagy by regulating the intracellular localization of the acetyltransferase EP300, and thus, modifying accessibility to its substrates (TP53 in the nucleus and autophagy-related proteins in the cytoplasm). Here, we investigated BAG6 localization and function in the cytoplasm. First, we demonstrated that BAG6 is localized in the mitochondria. Specifically, BAG6 is expressed in the mitochondrial matrix under basal conditions, and translocates to the outer mitochondrial membrane after mitochondrial depolarization with carbonyl cyanide m-chlorophenyl hydrazine, a mitochondrial uncoupler that induces mitophagy. In SW480 cells, the deletion of BAG6 expression abrogates its ability to induce mitophagy and PINK1 accumulation. On the reverse, its ectopic expression in LoVo colon cancer cells, which do not express endogenous BAG6, reduces the size of the mitochondria, induces mitophagy, leads to the activation of the PINK1/PARKIN pathway and to the phospho-ubiquitination of mitochondrial proteins. Finally, BAG6 contains two LIR (LC3-interacting Region) domains specifically found in receptors for selective autophagy and responsible for the interaction with LC3 and for autophagosome selectivity. Site-directed mutagenesis showed that BAG6 requires wild-type LIRs domains for its ability to stimulate mitophagy. In conclusion, we propose that BAG6 is a novel mitophagy receptor or adaptor that induces PINK1/PARKIN signaling and mitophagy in a LIR-dependent manner.

Synthesis and in vitro antitumour activity of carboplatin analogues containing functional handles compatible for conjugation to drug delivery systems.

We describe herein the synthesis of a series of carboplatin derivatives with different functional groups at position 3 of the cyclobutane ring. This pharmacomodulation approach aims at facilitating the vectorisation of these analogues, via their subsequent conjugation to a drug delivery system. Five different derivatives bearing a hydroxy, keto, iodo, azido or amino function at position 3 were synthesised. One of these compounds was coupled to a bifunctional maleimide-containing linker. All compounds were tested in vitro for their cytotoxicity on four different cell lines including two platinum-resistant colorectal cancer cell line (SK-OV-3, HCT116, D3E2, D5B7) using an MTS assay. Overall, the tested compounds were up to six times more potent than carboplatin, especially on D5B7 human colorectal cancer cells. We demonstrated that these modifications led to potent analogues which are compatible with conjugation to a drug delivery system.

The Anti-Cancer Drug Dabrafenib Is a Potent Activator of the Human Pregnane X Receptor.

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in both the development of multidrug resistance and enhanced cancer cells aggressiveness. Moreover, its unintentional activation by pharmaceutical drugs can mediate drug-drug interactions and cause severe adverse events. In that context, the potential of the anticancer BRAF inhibitor dabrafenib suspected to activate hPXR and the human constitutive androstane receptor (hCAR) has not been thoroughly investigated yet. Using different reporter cellular assays, we demonstrate that dabrafenib can activate hPXR as efficiently as its reference agonist SR12813, whereas it does not activate mouse or zebrafish PXR nor hCAR. We also showed that dabrafenib binds to recombinant hPXR, induces the expression of hPXR responsive genes in colon LS174T-hPXR cancer cells and human hepatocytes and finally increases the proliferation in LS174T-hPXR cells. Our study reveals that by using a panel of different cellular techniques it is possible to improve the assessment of hPXR agonist activity for new developed drugs.

Liquid chromatography-tandem mass spectrometric assay for the quantification of CDK4/6 inhibitors in human plasma in a clinical context of drug-drug interaction.

The CDK4/6 inhibitors palbociclib and ribociclib are kinase inhibitors used in association with hormonal therapy for the management of patients with metastatic breast cancer. Like most kinase inhibitors, therapeutic drug monitoring may be used for personalize their dosage. To this aim, we developed and validated a sensitive and specific HPLC-MS/MS method for palbociclib and ribociclib quantification in blood samples. We then quantified exposure to palbociclib (plasma trough concentration; Ctrough) in a real-life cohort of patients with locally invasive or metastatic breast cancer (n = 18) at day 15 of the first cycle of palbociclib treatment to characterize palbociclib concentration at steady state (Clinicaltrials.gov identifier NCT04025541, IdRCB n° 2018-A00064-51, 03/07/2018). The geometric mean (± standard deviation [min-max]) of palbociclib plasma Ctrough was 88.58 ng/mL (± 26.4 [46.5 ng/mL - 133 ng/mL]) at day 15. Some covariates, such as drug-drug interactions, could explain the concentration variations observed in our Caucasian cohort. These first results in real-life settings obtained with our HPLC-MS/MS method give important information on palbociclib monitoring and pharmacokinetic variability.

High Content Screening Using New U2OS Reporter Cell Models Identifies Harmol Hydrochloride as a Selective and Competitive Antagonist of the Androgen Receptor.

Prostate cancer is the most commonly diagnosed malignancy in men. Its growth mainly relies on the activity of the androgen receptor (AR), justifying the use of androgen deprivation therapy as a gold standard treatment for the metastatic disease. Inhibition of the androgen axis using second generation antagonists has improved patients' survival, but is systematically confronted to resistance mechanisms, leading to a median survival that does not exceed 5 years. Counteracting this resistance has been the object of a large number of investigations, with a particular emphasis towards the identification of new AR inhibitors, whether they antagonize the receptor by a competitive or a non-competitive binding. To this end, many high content screens have been performed, to identify new non-steroidal AR antagonists, using a variety of approaches, but reported somewhat controversial results, depending on the approach and on the cell model that was used for screening. In our study, we used the U2OS osteosarcoma cells stably transfected with AR or ARv7 and a luciferase reporter as a previously validated model to screen the Prestwick Phytochemical library. The results of our screen identified ellipticine, harmol, and harmine hydrochloride as confirmed hits. Surprisingly, we could demonstrate that harmol hydrochloride, previously identified as a non-competitive inhibitor of AR or a weak inhibitor of androgen signaling, was actually a competitive antagonist of AR, which inhibits the growth of VCaP prostate cancer line, at concentrations for which it did not affect the growth of the AR negative DU145 and PC3 cells. Interestingly, we also report for the first time that harmol hydrochloride was selective for AR, as it could not alter the activity of other nuclear receptors, such as the glucocorticoid receptor (GR), the progesterone receptor (PR), or the mineralocorticoid receptor (MR). Additionally, we demonstrate that, conversely to enzalutamide, harmol hydrochloride did not show any agonistic activity towards the pregnane X receptor (PXR), a master regulator of drug metabolism. Together, our results shed light on the importance of the cellular context for the screening of new AR antagonists. They further indicate that some of the potential hits that were previously identified may have been overlooked.

Inhibition of Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer.

Although many patients with colorectal cancer initially respond to the chemotherapeutic agent oxaliplatin, acquired resistance to this treatment remains a major challenge to the long-term management of this disease. To identify molecular targets of oxaliplatin resistance in colorectal cancer, we performed an shRNA-based loss-of-function genetic screen using a kinome library. We found that silencing of ataxia-telangiectasia mutated and RAD3-related (ATR), a serine/threonine protein kinase involved in the response to DNA stress, restored oxaliplatin sensitivity in a cellular model of oxaliplatin resistance. Combined application of the ATR inhibitor VE-822 and oxaliplatin resulted in strong synergistic effects in six different colorectal cancer cell lines and their oxaliplatin-resistant subclones, promoted DNA single- and double-strand break formation, growth arrest, and apoptosis. This treatment also increased replicative stress, cytoplasmic DNA, and signals related to immunogenic cell death such as calreticulin exposure and HMGB1 and ATP release. In a syngeneic colorectal cancer mouse model, combined administration of VE-822 and oxaliplatin significantly increased survival by promoting antitumor T-cell responses. Finally, a DNA repair gene signature discriminated sensitive from drug-resistant patients with colorectal cancer. Overall, our results highlight the potential of ATR inhibition combined with oxaliplatin to sensitize cells to chemotherapy as a therapeutic option for patients with colorectal cancer. SIGNIFICANCE: These findings demonstrate that resistance to oxaliplatin in colorectal cancer cells can be overcome with inhibitors of ATR and that combined treatment with both agents exerts synergistic antitumor effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2933/F1.large.jpg.

Claudin gene expression profiles and clinical value in colorectal tumors classified according to their molecular subtype.

PURPOSE: Colorectal cancer (CRC) is a heterogeneous disease that can be classified into distinct molecular subtypes. The aims of this study were 1) to compare claudin (CLDN) gene expression in CRC samples and normal colon mucosa, and then in the different CRC molecular subtypes, and 2) to assess their prognostic value. PATIENTS AND METHODS: CLDN expression in CRC samples was analyzed using gene expression data for a cohort of 143 primary CRC samples, and compared in the same CRC samples classified into different molecular subtypes (C1 to C6 according to the Marisa's classification, and CMS1 to CMS4 of the consensus classification). Comparison of CLDN expression in normal and tumor colon samples was also made on a smaller number of samples. Then, the relationship between CLDN expression profiles and overall survival (OS) and progression-free survival was examined. RESULTS: Compared with normal mucosa, CLDN1 and CLDN2 were upregulated, whereas CLDN5, 7, 8, and 23 were downregulated in CRC samples. Variations in CLDN expression profiles were observed mainly in the CMS2/C1 and CMS4/C4 subtypes. Overall, expression of CLDN2 or CLDN4 alone had a strong prognostic value that increased when they were associated. In the CMS4/C4 subtypes, lower expressions of CLDN11, CLDN12, and CLDN23 were associated with longer OS. Conversely, in the CMS2 and C1 subtypes, low CLDN23 expression was associated with shorter OS and progression-free survival, suggesting a dual role for CLDN23 as a tumor suppressor/promoter in CRC. CLDN6 and CLDN11 had a prognostic value in the CMS2 and C4 subtypes, respectively. CONCLUSION: This analysis of CLDN gene expression profiles and prognostic value in CRC samples classified according to their molecular subtype shows that CRC heterogeneity must be taken into account when assessing CLDN potential value as prognostic markers or therapeutic targets.

Modulating PCAF/GCN5 Immune Cell Function through a PROTAC Approach.

P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAF-deficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multidomain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.

Rational development of synergistic combinations of chemotherapy and molecular targeted agents for colorectal cancer treatment.

BACKGROUND: The irinotecan-induced phosphokinome changes in colorectal cancer (CRC) cells were used to guide the selection of targeted agents to be tested in combination with irinotecan. METHODS: Phosphokinome profiling with peptide arrays of tumour samples from nude mice xenografted with HT29 cells and treated or not with an effective dose of irinotecan was used to identify signalling pathways activated by irinotecan treatment. Then, drugs targeting these pathways were combined in vitro with irinotecan to test potential synergistic effect. The interactions between these drug combinations were assessed by a dose matrix approach. Confirmation of the most potential combination has been confirmed in vivo in xenografted mice. RESULTS: Irinotecan induced in vivo the activation of AKT and MEK1 phosphorylation. The dose matrix approach showed that BKM120 (PI3K inhibitor) and MEK162 (MEK inhibitor) are synergistic in vitro and in vivo with a cytostatic and cytotoxic effect, while combination of BKM120 and irinotecan or MEK162 and irinotecan are only additive or even antagonistic. However, the triple combination of SN38, BKM120 and MEK162 showed a better synergistic effect that BKM120 and MEK162, indicating that the cells need to inhibit both AKT and ERK pathways to become more sensitive to irinotecan-based chemotherapies. CONCLUSION: Analysis of chemotherapy-induced phosphokinome changes helps to elucidate the mechanisms of drug resistance and to guide the selection of targets for combination therapies with synergistic activity.

Targeting the NRG1/HER3 pathway in tumor cells and cancer-associated fibroblasts with an anti-neuregulin 1 antibody inhibits tumor growth in pre-clinical models of pancreatic cancer.

Neuregulin 1 (NRG1), a ligand for HER3 and HER4 receptors, is secreted by both pancreatic tumor cells (PC) and cancer-associated fibroblasts (CAFs), the latter representing the most abundant compound of pancreatic stroma. This desmoplastic stroma contributes to Pancreatic Ductal Adenocarcinoma (PDAC) aggressiveness and therapeutic failure by promoting tumor progression, invasion and resistance to chemotherapies. In the present work, we aimed at disrupting the complex crosstalk between PC and CAF in order to prevent tumor cell proliferation. To do so, we demonstrated the promising tumor growth inhibitory effect of the 7E3, an original antibody directed to NRG1. This antibody promotes antibody dependent cellular cytotoxicity in NRG1-positive PC and CAFs and inhibits NRG1-associated signaling pathway induction, by blocking NRG1-mediated HER3 activation. Moreover, 7E3 inhibits migration and growth of pancreatic cancer cells co-cultured with CAFs, both in vitro and in vivo using orthotopic pancreatic tumor xenografts. Our preclinical results demonstrate that the anti-NRG1 antibody 7E3 could represent a promising approach to target pancreatic stroma and cancer cells, thereby providing novel therapeutic options for PDAC.