Correction: Tusup et al. Evaluation of the Interplay between the ADAR Editome and Immunotherapy in Melanoma. Non-Coding RNA 2021, 7, 5.

In the original article [...].

RNA with chemotherapeutic base analogues as a dual-functional anti-cancer drug.

Nanoparticles of different sizes formulated with unmodified RNA and Protamine differentially engage Toll-like Receptors (TLRs) and activate innate immune responses in vitro. Here, we report that similar differential immunostimulation that depends on the nanoparticle sizes is induced in vivo in wild type as well as in humanized mice. In addition, we found that the schedule of injections strongly affects the magnitude of the immune response. Immunostimulating 130 nm nanoparticles composed of RNA and Protamine can promote lung metastasis clearance but provides no control of subcutaneous tumors in a CT26 tumor model. We further enhanced the therapeutic capacity of Protamine-RNA nanoparticles by incorporating chemotherapeutic base analogues in the RNA; we coined these immunochemotherapeutic RNAs (icRNAs). Protamine-icRNA nanoparticles were successful at controlling established subcutaneous CT26 and B16 tumors as well as orthotopic glioblastoma. These data indicate that icRNAs are promising cancer therapies, which warrants their further validation for use in the clinic.

The A to I editing landscape in melanoma and its relation to clinical outcome.

RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies.

Epitranscriptomics modifier pentostatin indirectly triggers Toll-like receptor 3 and can enhance immune infiltration in tumors.

The adenosine deaminase inhibitor 2'-deoxycoformycin (pentostatin, Nipent) has been used since 1982 to treat leukemia and lymphoma, but its mode of action is still unknown. Pentostatin was reported to decrease methylation of cellular RNA. We discovered that RNA extracted from pentostatin-treated cells or mice has enhanced immunostimulating capacities. Accordingly, we demonstrated in mice that the anticancer activity of pentostatin required Toll-like receptor 3, the type I interferon receptor, and T cells. Upon systemic administration of pentostatin, type I interferon is produced locally in tumors, resulting in immune cell infiltration. We combined pentostatin with immune checkpoint inhibitors and observed synergistic anti-cancer activities. Our work identifies pentostatin as a new class of an anticancer immunostimulating drug that activates innate immunity within tumor tissues and synergizes with systemic T cell therapies.

mRNA-Based Anti-TCR CDR3 Tumour Vaccine for T-Cell Lymphoma.

Efficient vaccination can be achieved by injections of in vitro transcribed mRNA (ivt mRNA) coding for antigens. This vaccine format is particularly versatile and allows the production of individualised vaccines conferring, T-cell immunity against specific cancer mutations. The CDR3 hypervariable regions of immune receptors (T-cell receptor, TCR or B-cell receptor, BCR) in the context of T- or B-cell leukaemia or lymphoma are targetable and specific sequences, similar to cancer mutations. We evaluated the functionality of an mRNA-based vaccine designed to trigger immunity against TCR CDR3 regions in an EL4 T-lymphoma cell line-derived murine in vivo model. Vaccination against the hypervariable TCR regions proved to be a feasible approach and allowed for protection against T-lymphoma, even though immune escape in terms of TCR downregulation paralleled the therapeutic effect. However, analysis of human cutaneous T-cell lymphoma samples indicated that, as is the case in B-lymphomas, the clonotypic receptor may be a driver mutation and is not downregulated upon treatment. Thus, vaccination against TCR CDR3 regions using customised ivt mRNA is a promising immunotherapy method to be explored for the treatment of patients with T-cell lymphomas.

Evaluation of the Interplay between the ADAR Editome and Immunotherapy in Melanoma.

BACKGROUND: RNA editing is a highly conserved posttranscriptional mechanism that contributes to transcriptome diversity. In mammals, it includes nucleobase deaminations that convert cytidine (C) into uridine (U) and adenosine (A) into inosine (I). Evidence from cancer studies indicates that RNA-editing enzymes promote certain mechanisms of tumorigenesis. On the other hand, recoding editing in mRNA can generate mutations in proteins that can participate in the Major Histocompatibility Complex (MHC) ligandome and can therefore be recognized by the adaptive immune system. Anti-cancer treatment based on the administration of immune checkpoint inhibitors enhance these natural anti-cancer immune responses. RESULTS: Based on RNA-Seq datasets, we evaluated the editome of melanoma cell lines generated from patients pre- and post-immunotherapy with immune checkpoint inhibitors. Our results reveal a differential editing in Arthrobacter luteus (Alu) sequences between samples pre-therapy and relapses during therapy with immune checkpoint inhibitors. CONCLUSION: These data pave the way towards the development of new diagnostics and therapies targeted to editing that could help in preventing relapses during immunotherapies.

Design of in vitro Transcribed mRNA Vectors for Research and Therapy.

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock.

The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncovered 240 transcripts with altered translation rate, and used linear regression analysis to extract 5' UTR features predictive of DENR dependence. Among core clock genes, we identified Clock as a DENR target. Using Clock 5' UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments revealed an alternative downstream start codon, likely representing the bona fide CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.

Epitranscriptomics of cancer.

The functional impact of modifications of cellular RNAs, including mRNAs, miRNAs and lncRNAs, is a field of intense study. The role of such modifications in cancer has started to be elucidated. Diverse and sometimes opposite effects of RNA modifications have been reported. Some RNA modifications promote, while others decrease the growth and invasiveness of cancer. The present manuscript reviews the current knowledge on the potential impacts of N6-Methyladenosine, Pseudouridine, Inosine, 2'O-methylation or methylcytidine in cancer's RNA. It also highlights the remaining questions and provides hints on research avenues and potential therapeutic applications, whereby modulating dynamic RNA modifications may be a new method to treat cancer.

Phase I study of a chloroquine-gemcitabine combination in patients with metastatic or unresectable pancreatic cancer.

PURPOSE: Following a previously published pre-clinical validation, this phase I study evaluated the safety, maximum tolerated dose, anti-tumour activity and immune status of a gemcitabine-chloroquine combination as a first- or late-line treatment in patients with metastatic or unresectable pancreatic cancer. METHODS: In this 3 + 3 dose escalation study, patients received a single weekly standard dose of intravenous gemcitabine, followed by single weekly oral intake of 100, 200 or 300 mg of chloroquine. Tumour response was assessed using the Response Evaluation Criteria in Solid Tumors version 1.1. Immune status was evaluated by RT-PCR to measure the relative expression of immune-related genes in peripheral blood mononuclear cells (PBMCs). RESULTS: Overall, nine patients [median age 72 years; interquartile range (IQR), 68-78 years] were treated. No dose-limiting toxicities as defined in the protocol were observed. Three patients experienced partial response, and two patients had stable disease. The median time to progression was 4 months (95% CI 0.8-7.2), and the median overall survival was 7.6 months (95% CI 5.3-9.9). Among 86 assayed immune genes, three were significantly differentially expressed in PBMCs from responding versus non-responding patients: interferon-gamma receptor-1, toll-like receptor 2, and beta-2 microglobulin. CONCLUSIONS: The addition of chloroquine to gemcitabine was well tolerated and showed promising effects on the clinical response to the anti-cancer chemotherapy. Based on these initial results, the efficacy of the gemcitabine-chloroquine combination should be further assessed.

Generation of Immunostimulating 130 nm Protamine-RNA nanoparticles.

Nanoparticles of defined size can be easily obtained by simply mixing Protamine, a pharmaceutical drug that is used to neutralize heparin after surgery, and RNA in the form of oligonucleotides or messenger RNA. Depending on the concentrations of the two reagents and their salt contents, homogenous nanoparticles with a mean diameter of 50 to more than 1000 nm can spontaneously be generated. RNA is a danger signal because it is an agonist of for example TLR-3, -7, and -8; therefore, Protamine-RNA nanoparticles are immunostimulating. We and others have shown in vitro that nanoparticle size and interferon-alpha production by human peripheral blood mononuclear cells (PBMCs) are inversely correlated. Conversely, nanoparticle size and TNF-alpha production by PBMCs are positively correlated (Rettig et al., Blood 115:4533-4541, 2010). Particles of less than 450 nm are most frequently used for research and clinical applications because they are very stable, remain polydispersed and induce interferon-alpha proteins, which are a natural antiviral and anticancer protein family with 12 members in humans. Herein, we describe a method to generate 130 nm nanoparticles as well as some of their physical and biological characteristics.

Proinflammatory cytokine responses in skin and epidermal cells following epicutaneous administration of anticoagulant rodenticide warfarin in rats.

CONTEXT: Dermal toxicity of coumarin anticoagulant rodenticides, such as warfarin, represents potential risk for workers handling these agents and for individuals applying easily available rodenticides in their households as well. OBJECTIVE: In this study, proinflammatory effects of repeated epicutaneous administration of warfarin in rats were explored by examining inflammatory cytokine skin responses. MATERIALS AND METHODS: Ex vivo production of IL-1ß, IL-6, TNF-α and IL-17 by skin explants and by epidermal cells isolated by enzyme (dispase/trypsin) digestion from skin repeatedly (once a day, three consecutive days) exposed to 10 µg of warfarin was measured 24 h and 72 h following the last warfarin application by ELISAs for respective rat cytokines. RESULTS: Warfarin treatment resulted in histological changes, but skin or epidermal cell viability were not compromised, judging by MTT reduction assay. Both skin and epidermal cells responded to administration of this agent by production of all examined inflammatory cytokines (skin explants by TNF-α and IL-17; epidermal cells by IL-1ß and TNF-α) except IL-6. DISCUSSION: Along with histomorphological changes, cytokines indicate functional consequences in treated skin. IL-1ß production, that precede production of TNF-α, might be responsible for production of the latter cytokine. Sustained production of IL-1ß suggests persistence of epidermal cell stimulation or existence of some amplification mechanisms. Requirements for T cells seem to exist concerning epidermal cell IL-17 production. CONCLUSION: Presented data provide additional new information concerning proinflammatory effects of warfarin.