Demonstration of lethal interactions by phenotypic screening in the context of resistance to colorectal cancer therapies

Aujourd’hui, les traitements du cancer colorectal métastatique ont évolué grâce à la combinaison de chimiothérapies conventionnelles à base de 5-FU, oxaliplatine et/ou Irinotécan et de thérapies ciblées dirigées contre le récepteur de l’EGF ou le VEGF. Malgré un taux de survie amélioré par la combinaison de ces drogues, la résistance innée et acquise aux traitements est une cause fréquente d'échec thérapeutique.Dans le but de découvrir de nouvelles cibles thérapeutiques nous avons effectué plusieurs criblages phénotypiques en utilisant des modèles cellulaires de résistance acquises aux chimiothérapies (oxaliplatine et irinotécan) générés au laboratoire ainsi que la lignée HCT116 qui présente une résistance innée aux thérapies anti-EGFR (cétuximab, panitumumab, Erlotinib). Le but final de ce projet étant de révéler des gènes, dont l’inhibition permet de rétablir la sensibilité à l’un de ces traitements, affichant ainsi une interaction létale avec le médicament.Une fois les kinases potentiellement impliquées dans la résistance aux thérapies du CCR identifiées, une inhibition spécifique par shRNA et/ou un inhibiteur spécifique a été effectuée afin de confirmer les potentielles cibles thérapeutiques et/ou biomarqueurs de réponse aux traitements. La cible la plus prometteuse, identifiée comme un déterminant de la résistance à l’oxaliplatine est la protéine ATR (Ataxia-telangiectasia mutated and rad3 related). Une protéine jouant un rôle clé dans la réparation de l'ADN et qui est activée en réponse à la présence d'ADN simple brin persistant (ssDNA) ou de stress réplicatif, pouvant être généré par certaines thérapies anticancéreuses.L’inhibition ATR via son inhibiteur pharmacologique VE-822 (VX-970) combinée à l’oxaliplatine a alors été étudiée par l’utilisation de tests cytotoxiques complétés par une étude d’additivité. Ainsi, nous avons démontré que l’inhibition d’ATR combinée avec l’oxaliplatine entraine une forte synergie dans la lignée HCT116-R1 à la fois en 2D et en 3D. Cet effet est également retrouvé dans d’autres lignées clonales résistantes à l’oxaliplatine (HCT116-R2, SW48-R1) ainsi que dans les lignées cellulaires à l’origine de ces dernières (HCT116, SW48). Nous avons également montré que l'effet synergique de l’oxaliplatine et du VE-822 dans la lignée HCT116-R1 s'accompagne d'une augmentation de la présence d’ADN simple brins suivie de nombreuses cassures double brins de l’ADN, d'un arrêt de la prolifération et d'une induction de l'apoptose. L'apparition de ces dommages à l'ADN est également corrélée avec l'activation de la voie ATM, de p53 et l'inhibition de l'activité CDK2. De plus, in vitro le double traitement provoque une induction des signaux moléculaires à l’origine de la mort immunogène équivalente ou bien supérieure aux traitements par l’oxaliplatine seul. Enfin, l'association d'oxaliplatine + VE-822 est également efficace in vivo, sur des souris immunodéprimées xénogreffées avec les cellules HCT116-R1 ainsi que sur des souris immunologiquement compétentes, avec un effet synergique plus élevé indiquant que la mort immunitaire (ICD) fait partie du mécanisme de cette combinaison de médicaments. En conclusion, toutes ces données confirment l’intérêt du criblage phénotypique dans la découverte de nouvelles cibles thérapeutiques en démontrant pour la première fois le rôle fonctionnel de l'ATR dans la sensibilité à l’oxaliplatine.Today, treatments for metastatic colorectal cancer have evolved through the combination of conventional chemotherapy 5-FU, oxaliplatin and / or Irinotecan and targeted therapies directed against the EGF receptor or VEGF. Despite an improved survival rate through the combination of these drugs, innate and acquired resistance to treatment is a common cause of therapeutic failure.In order to discover new therapeutic targets we carried out several phenotypic screenings using cellular resistance models acquired to chemotherapies (oxaliplatin and irinotecan) generated in the laboratory as well as the HCT116 line which exhibits an innate resistance to anti-EGFR therapies (cetuximab , panitumumab, Erlotinib). The ultimate goal of this project is to reveal genes, whose inhibition restores sensitivity to one of these treatments, thus displaying a lethal interaction with the drug.Once the kinases potentially involved in resistance to CCR therapies identified, specific inhibition by shRNA and / or a specific inhibitor was performed to confirm the potential therapeutic targets and / or biomarkers for response to treatments. The most promising target, identified as a determinant of resistance to oxaliplatin is the ATR protein (Ataxia-telangiectasia mutated and rad3 related). A protein that plays a key role in DNA repair and is activated in response to the presence of persistent single stranded DNA (ssDNA) or replicative stress, which can be generated by certain anti-cancer therapies.The inhibition of ATR via its pharmacological inhibitor VE-822 (VX-970) combined with oxaliplatin was then studied by the use of cytotoxic tests supplemented by an additivity study. Thus, we demonstrated that the inhibition of ATR combined with oxaliplatin leads to a strong synergy in the HCT116-R1 cell line in both 2D and 3D. This effect is also found in other oxaliplatin resistant clonal lines (HCT116-R2, SW48-R) as well as in the cell lines originating from them (HCT116, SW48).We have also shown that the synergistic effect of oxaliplatin and VE-822 in the HCT116-R1 line is accompanied by an increase in the presence of single-stranded DNA followed by numerous double-stranded DNA breaks, stopping proliferation and inducing apoptosis. The occurrence of this damage to DNA is also correlated with activation of the ATM pathway, p53 and inhibition of CDK2 activity. Moreover, in vitro the double treatment causes an induction of the molecular signals triggering the immunogenic cell death equivalent or superior to the treatments by oxaliplatin alone.Finally, the combination of oxaliplatin + VE-822 is also effective in vivo in immunodeficient mice xenografted with HCT116-R1 cells as well as in immunologically competent mice with a higher synergistic effect indicating that immune death (ICD ) is part of the mechanism of this combination of drugs.In conclusion, all these data confirm the interest of phenotypic screening in the discovery of new therapeutic targets by demonstrating for the first time the functional role of ATR in sensitivity to oxaliplatin

Mise en évidence d’intéractions létales par criblage phénotypique dans le contexte de la résistance aux thérapies du cancer colorectal

Today, treatments for metastatic colorectal cancer have evolved through the combination of conventional chemotherapy 5-FU, oxaliplatin and / or Irinotecan and targeted therapies directed against the EGF receptor or VEGF. Despite an improved survival rate through the combination of these drugs, innate and acquired resistance to treatment is a common cause of therapeutic failure.In order to discover new therapeutic targets we carried out several phenotypic screenings using cellular resistance models acquired to chemotherapies (oxaliplatin and irinotecan) generated in the laboratory as well as the HCT116 line which exhibits an innate resistance to anti-EGFR therapies (cetuximab , panitumumab, Erlotinib). The ultimate goal of this project is to reveal genes, whose inhibition restores sensitivity to one of these treatments, thus displaying a lethal interaction with the drug.Once the kinases potentially involved in resistance to CCR therapies identified, specific inhibition by shRNA and / or a specific inhibitor was performed to confirm the potential therapeutic targets and / or biomarkers for response to treatments. The most promising target, identified as a determinant of resistance to oxaliplatin is the ATR protein (Ataxia-telangiectasia mutated and rad3 related). A protein that plays a key role in DNA repair and is activated in response to the presence of persistent single stranded DNA (ssDNA) or replicative stress, which can be generated by certain anti-cancer therapies.The inhibition of ATR via its pharmacological inhibitor VE-822 (VX-970) combined with oxaliplatin was then studied by the use of cytotoxic tests supplemented by an additivity study. Thus, we demonstrated that the inhibition of ATR combined with oxaliplatin leads to a strong synergy in the HCT116-R1 cell line in both 2D and 3D. This effect is also found in other oxaliplatin resistant clonal lines (HCT116-R2, SW48-R) as well as in the cell lines originating from them (HCT116, SW48).We have also shown that the synergistic effect of oxaliplatin and VE-822 in the HCT116-R1 line is accompanied by an increase in the presence of single-stranded DNA followed by numerous double-stranded DNA breaks, stopping proliferation and inducing apoptosis. The occurrence of this damage to DNA is also correlated with activation of the ATM pathway, p53 and inhibition of CDK2 activity. Moreover, in vitro the double treatment causes an induction of the molecular signals triggering the immunogenic cell death equivalent or superior to the treatments by oxaliplatin alone.Finally, the combination of oxaliplatin + VE-822 is also effective in vivo in immunodeficient mice xenografted with HCT116-R1 cells as well as in immunologically competent mice with a higher synergistic effect indicating that immune death (ICD ) is part of the mechanism of this combination of drugs.In conclusion, all these data confirm the interest of phenotypic screening in the discovery of new therapeutic targets by demonstrating for the first time the functional role of ATR in sensitivity to oxaliplatin.Aujourd’hui, les traitements du cancer colorectal métastatique ont évolué grâce à la combinaison de chimiothérapies conventionnelles à base de 5-FU, oxaliplatine et/ou Irinotécan et de thérapies ciblées dirigées contre le récepteur de l’EGF ou le VEGF. Malgré un taux de survie amélioré par la combinaison de ces drogues, la résistance innée et acquise aux traitements est une cause fréquente d'échec thérapeutique.Dans le but de découvrir de nouvelles cibles thérapeutiques nous avons effectué plusieurs criblages phénotypiques en utilisant des modèles cellulaires de résistance acquises aux chimiothérapies (oxaliplatine et irinotécan) générés au laboratoire ainsi que la lignée HCT116 qui présente une résistance innée aux thérapies anti-EGFR (cétuximab, panitumumab, Erlotinib). Le but final de ce projet étant de révéler des gènes, dont l’inhibition permet de rétablir la sensibilité à l’un de ces traitements, affichant ainsi une interaction létale avec le médicament.Une fois les kinases potentiellement impliquées dans la résistance aux thérapies du CCR identifiées, une inhibition spécifique par shRNA et/ou un inhibiteur spécifique a été effectuée afin de confirmer les potentielles cibles thérapeutiques et/ou biomarqueurs de réponse aux traitements. La cible la plus prometteuse, identifiée comme un déterminant de la résistance à l’oxaliplatine est la protéine ATR (Ataxia-telangiectasia mutated and rad3 related). Une protéine jouant un rôle clé dans la réparation de l'ADN et qui est activée en réponse à la présence d'ADN simple brin persistant (ssDNA) ou de stress réplicatif, pouvant être généré par certaines thérapies anticancéreuses.L’inhibition ATR via son inhibiteur pharmacologique VE-822 (VX-970) combinée à l’oxaliplatine a alors été étudiée par l’utilisation de tests cytotoxiques complétés par une étude d’additivité. Ainsi, nous avons démontré que l’inhibition d’ATR combinée avec l’oxaliplatine entraine une forte synergie dans la lignée HCT116-R1 à la fois en 2D et en 3D. Cet effet est également retrouvé dans d’autres lignées clonales résistantes à l’oxaliplatine (HCT116-R2, SW48-R1) ainsi que dans les lignées cellulaires à l’origine de ces dernières (HCT116, SW48). Nous avons également montré que l'effet synergique de l’oxaliplatine et du VE-822 dans la lignée HCT116-R1 s'accompagne d'une augmentation de la présence d’ADN simple brins suivie de nombreuses cassures double brins de l’ADN, d'un arrêt de la prolifération et d'une induction de l'apoptose. L'apparition de ces dommages à l'ADN est également corrélée avec l'activation de la voie ATM, de p53 et l'inhibition de l'activité CDK2. De plus, in vitro le double traitement provoque une induction des signaux moléculaires à l’origine de la mort immunogène équivalente ou bien supérieure aux traitements par l’oxaliplatine seul. Enfin, l'association d'oxaliplatine + VE-822 est également efficace in vivo, sur des souris immunodéprimées xénogreffées avec les cellules HCT116-R1 ainsi que sur des souris immunologiquement compétentes, avec un effet synergique plus élevé indiquant que la mort immunitaire (ICD) fait partie du mécanisme de cette combinaison de médicaments. En conclusion, toutes ces données confirment l’intérêt du criblage phénotypique dans la découverte de nouvelles cibles thérapeutiques en démontrant pour la première fois le rôle fonctionnel de l'ATR dans la sensibilité à l’oxaliplatine

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

International audienceThe 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

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

International audienceSpleen 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

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

Abstract 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

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

International audienceResistance 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

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

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Telomere length, ATM mutation status and cancer risk in Ataxia-Telangiectasia families.

Recent studies have linked constitutive telomere length (TL) to aging-related diseases including cancer at different sites. ATM participates in the signaling of telomere erosion, and inherited mutations in ATM have been associated with increased risk of cancer, particularly breast cancer. The goal of this study was to investigate whether carriage of an ATM mutation and TL interplay to modify cancer risk in ataxia-telangiectasia (A-T) families.The study population consisted of 284 heterozygous ATM mutation carriers (HetAT) and 174 non-carriers (non-HetAT) from 103 A-T families. Forty-eight HetAT and 14 non-HetAT individuals had cancer, among them 25 HetAT and 6 non-HetAT were diagnosed after blood sample collection. We measured mean TL using a quantitative PCR assay and genotyped seven single-nucleotide polymorphisms (SNPs) recurrently associated with TL in large population-based studies.HetAT individuals were at increased risk of cancer (OR = 2.3, 95%CI = 1.2-4.4, P = 0.01), and particularly of breast cancer for women (OR = 2.9, 95%CI = 1.2-7.1, P = 0.02), in comparison to their non-HetAT relatives. HetAT individuals had longer telomeres than non-HetAT individuals (P = 0.0008) but TL was not associated with cancer risk, and no significant interaction was observed between ATM mutation status and TL. Furthermore, rs9257445 (ZNF311) was associated with TL in HetAT subjects and rs6060627 (BCL2L1) modified cancer risk in HetAT and non-HetAT women.Our findings suggest that carriage of an ATM mutation impacts on the age-related TL shortening and that TL per se is not related to cancer risk in ATM carriers. TL measurement alone is not a good marker for predicting cancer risk in A-T families