Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2′-methoxyethyl (2′MOE) modifications. Through a screen of 192 2′OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2′OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.
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.
Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2′OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2′OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2′OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.
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.
Upper Tract Urothelial Carcinomas (UTUC) are extremely aggressive tumors of ureter or renal pelvis. UTUC present less tumor mutational burden and low tumor immune infiltrate compared to bladder cancer. Despite this they are treated with the same protocol than bladder cancer with more than 50% of relapses justifying the need of new therapeutic options. To improve patient care, we suggest stimulating the immune system by platinum-based chemotherapies (Cisplatin-Gemcitabine (CisGem) or Carboplatin-Gemcitabine (CarboGem)) in order to potentiate the effect of an anti-PD-L1, the Durvalumab. We launched a phase II clinical trial called “iNDUCT” which studies these combinations in UTUC patients. In parallel, we conduct an in vitro project that aims to determine if chemotherapies could transform cold tumor into a hot tumor, and if so by which mechanisms? Using UTUC cell lines (UM-UC-14,UCC03,UCC17,UCC14,UCC47) we have evaluated the cytotoxicity effects of the chemotherapies combinations in 2D and 3D cell cultures. We have assessed their potential (i) to induce DNA damage using image cytometry, (ii) to induce PD-L1 expression using flow cytometry, (iii) to induce immune cell death using ELISA kits, (iv) to activate the cGAS/STING pathway using qPCR and Western-Blot, and finally (v) to attract immune cells by using heterotypic spheroids model (tumoral cells+PBMCs).Our results demonstrate that CisGem and CarboGem present synergistic effects in UTUC spheroid cultures. These treatments also induce DNA damage pathway demonstrated by an increase of γH2AX, P-ATM, P-CHK1 and P-CHK2 positive cells. We found an increase of PD-L1 membrane expression after treatment in UTUC cell lines. RNA Seq analyses indicates that the major pathways upregulated by these combinations are inflammatory pathways (TNF-α signaling via NFkB, interferon alpha response, inflammatory response, interferon gamma response). We could observe an immune cell death induction demonstrated by an increase of ATP and HMGB1 release and calreticulin translocation. We showed cGAS/STING pathway activation as evidenced by an increase of P-IRF3 and interferon stimulated genes (ISGs) expression. We demonstrated an inhibition of the ISGs induction after treatment by our chemotherapies when cells are treated with an ATM inhibitor or in UMUC-14 deleted for STING. And we finally showed that CisGem and CarboGem can increase immune infiltration in the heterotypic tumor spheroids. These results indicate that the combination of platinum salts + gemcitabine induces inflammatory pathways via a non-canonical STING pathway dependent on ATM activation in UTUC model. Furthermore, these combination induce an upregulation of PD-L1 expression and allow immune cells attraction at the tumor. All these data support that a combination CisGem or CarboGem with an anti-PD-L1 will be efficient for UTUC patients. Citation Format: Alexandra Fauvre, Nadia Vie, Mathilde Robin, Clara Taffoni, Nadine Laguette, Julien Faget, Laurent Gros, Aurélie Garcin, Celine Gongora, Nadine Houede. Platinum-based chemotherapy immunomodulatory effects and immunotherapy association in upper tract urothelial carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1294.
DNA mimicry has been the subject of intensive research and resulted in the development of DNA analogues such as PNAs and LNAs. There are also examples of proteins with structural and/or charge distribution analogies with respect to the DNA double helix which allow them to interfere with other DNA-binding proteins and modulate the biological processes in which they are involved. We previously characterized a new class of DNA-surface mimic molecules constituted by repetitions of dimeric units of 8-amino-2-quinolinecarboxylic acid (Q) and 8-aminomethyl-2-quinolinecarboxylic acid (mQ). The helical folding of these entities can mimic a B-DNA molecule, displaying a minor and a major groove that can be modulated depending on the dimers and of the nature of their side chains. In vitro, these DNA mimics could inhibit, in a relative selective manner, the catalytic activity of DNA topoisomerase I (Top1), whereas they had no effect on the activity of DNA polymerases or DNAses. Inhibition of Top1-mediated relaxation of supercoiled DNA plasmid increased with the length of the DNA mimics. Here, we further characterized the mechanism of Top1 inhibition by these DNA mimics. We found that, conversely to camptothecin (CPT) and its derivatives that poison Top1 via the inhibition or the re-ligation step of the reaction, DNA mimics inhibited Top1-mediated DNA cleavage in vitro by preventing the binding of the enzyme to its substrate, a mechanism of Top1 competitive inhibition that was formerly referred to as catalytic inhibition. We also found that co-incubation of DNA mimics with CPT had an additive effect on the inhibition of Top1-mediated relaxation of supercoiled DNA, further suggesting a mechanism that is different from CPT. Because transfection of DNA mimics could inhibit the growth of various cancer cell lines, we further investigated whether Top1 could play a role in this cytotoxicity. We found that Top1 knock-down in OVCAR4 ovarian cancer cells resulted in decreased sensitivity to the (mQQ4)8 DNA mimic as compared to OVCAR4 control cells, suggesting that Top1 is a target of DNA mimics in cells and is involved in their cytotoxic effects. Conversely to CPT, transfection of HCT116 cells with the (mQQ4)8 DNA mimic was not associated with an increase in γH2AX, suggesting that DNA mimics do not induce DNA breakage. We are currently analyzing whether the (mQQ4)8 can have an impact on CPT-induced Top1-DNA complexes formation. Interestingly, we also showed that several SN38-resistant HCT116 cell clones characterized by specific Top1 mutations were still sensitive to the (mQQ4)8 DNA mimic. Together our results demonstrate that DNA mimics can be considered as a new class of competitive inhibitors of Top1. Further studies are ongoing to identify the structural features that are essential for Top1 inhibition in order to generate more potent derivatives that could be used to counteract resistance to CPT derivatives used in the clinic. Citation Format: Aurélie Garcin, Valentina Corvaglia, Madeleine Bossaert, Marie-Jeanne Pillaire, Ivan Huc, Sébastien Britton, Vincent Parissi, Philippe Pourquier. Foldamers mimicking the B-DNA surface as a new class of DNA topoisomerase I inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4930.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.