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.

Synthesis of Benzo[c][2,7]naphthyridinones and Benzo[c][2,6]naphthyridinones via Ruthenium-Catalyzed [2+2+2] Cycloaddition between 1,7-Diynes and Cyanamides.

A convenient method for the ruthenium-catalyzed synthesis of benzo[c]naphthyridinone derivatives is reported. The [2+2+2] cycloaddition from various mono- and disubstituted 1,7-diynes and cyanamides provided benzo[c][2,7]naphthyridinones as major products and benzo[c][2,6]naphthyridinones as minor ones in yields of ≤79% and regioselectivities of ≤99:1. This method is amenable to internal and terminal diynes and a number of cyanamides with diverse functional group tolerance.

Dual intra- and extracellular release of monomethyl auristatin E from a neutrophil elastase-sensitive antibody-drug conjugate.

Antibody-drug conjugates (ADCs) are targeted therapies, mainly used in oncology, consisting in a three-component molecular architecture combining a highly potent drug conjugated via a linker onto a monoclonal antibody (mAb), designed for the selective delivery of the drug to the tumor site. The linker is a key component, defining the ADC stability and mechanism of action, and particularly the drug release strategy. In this study, we have developed and synthesized a cleavable linker, which possesses an Asn-Pro-Val (NPV) sequence sensitive to the human neutrophil elastase (HNE), overexpressed in the tumor microenvironment. This linker permitted the site-specific conjugation of the cell-permeable drug, monomethyl auristatin E (MMAE), onto trastuzumab, using a disulfide re-bridging technology. The resulting ADC was then evaluated in vitro. This conjugate demonstrated retained antigen (Ag) binding affinity and exhibited high subnanomolar potency against Ag-positive tumor cells after internalization, suggesting an intracellular mechanism of linker cleavage. While no internalization and cytotoxic activity of this ADC was observed on Ag-negative cells in classical conditions, the supplementation of exogenous HNE permitted to restore a nanomolar activity on these cells, suggesting an extracellular mechanism of drug release in these conditions. This in vitro proof of concept tends to prove that the NPV sequence could allow a dual intra- and extracellular mechanism of drug release. This work represents a first step in the design of original ADCs with a new dual intra- and extracellular drug delivery system and opens the way to further experimentations to evaluate their full potential in vivo.

A double-triggered self-immolative spacer for increased selectivity of molecular release.

A self-immolative spacer based on dissymmetrical N,N'-bis-carbamate aniline is introduced to liberate a substrate from a precursor after dual activation. The proof of principle of its exclusive selectivity for substrate liberation has been conducted on a profluorophore.

Impact of Maleimide Disubstitution on Chemical and Biological Characteristics of HER2 Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are the spearhead of targeted therapies. According to the technology used, the conjugation of a cytotoxic drug to an antibody can produce suboptimal heterogeneous species, impacting the overall efficacy. Herein, we describe the synthesis of HER2-targeting ADCs with three disulfide rebridging heads, allowing homogeneous and site-specific bioconjugation: dibromomaleimide (DBM), dithiomaleimide (DTM), and hybrid thio-bromomaleimide (TBM) chemical bricks to combine the properties of both previously used heads. The primary purpose of this study was to compare the reactivity of these three chemical bricks in the bioconjugation process. Then, the resulting ADCs were evaluated in terms of physicochemical stability, binding, and biological activity. We have demonstrated that the higher percentage of a drug-to-antibody ratio of 4 was obtained with TBM. Additionally, the reaction time was drastically reduced with TBM in comparison to DTM. The three ADCs showed good binding to HER2 and in vitro cytotoxicity, which validate the TBM structure as an attractive alternative scaffold for rebridging bioconjugation.

A chemically encoded timer for dual molecular delivery at tailored ranges and concentrations.

Spatiotemporal control of molecular distribution is much in demand in many fields of chemistry. To address this goal, we exploit a low molecular weight branched self-immolative architecture, which acts as a triggerable chemically encoded timer for autonomous sequential release of two chemicals. Using a light-activated model liberating two distinct fluorophores, we generated a tunable spatially contrasted molecular distribution.

Syntheses and kinetic studies of cyclisation-based self-immolative spacers.

Kinetic analysis of the disassembly of self-immolative spacers based on cyclisation processes was performed. Five compounds were synthesized belonging to two different series, and their kinetic constants were determined. Electron-donating substituents gave a slight acceleration but the main effect was steric, and the Thorpe-Ingold effect was indeed particularly effective. Comparison with the self-immolative spacers based on elimination processes showed that cyclisations gave comparable or lower rate, but the corresponding spacers are more difficult to modulate.