Differential functionality of fluoropyrimidine nucleosides for safe cancer therapy.

Chemotherapies are standard care for most cancer types. Pyrimidine analogs including 5-fluorouracil, cytosine arabinoside, 5-azacytidine, and gemcitabine are effective drugs that are utilized as part of a number of anticancer regimens. However, their lack of cell-specificity results in severe side effects. Therefore, there is a capacity to improve the efficacy of such therapies, while decreasing unwanted side effects. Here, we report that while 5-fluorocytosine is not chemotherapeutic in itself, incorporated into a ribonucleoside and more importantly into an RNA oligonucleotide, it induces cytotoxic effects on cancer cells in vitro. Interestingly, these effects are rescued by both uridine and thymidine. Similarly, in-vitro 2'-deoxy-5-fluorocytidine inhibits the growth of tumor cells but has the advantage of being less toxic to human primary cells compared with 5-fluorocytidine, suggesting that the deoxyribonucleoside could exhibit less side-effects in vivo. Thus, this work indicates that the potency of 5-fluorocytidine and 2'-deoxy-5-fluorocytidine should be further explored. In particular, oligonucleotides incorporating 5-fluorocytosine could be novel chemotherapeutic drugs that could be formulated in cancer-specific particles for safe and efficacious cancer treatments.

Role of the NLRP1 inflammasome in skin cancer and inflammatory skin diseases.

Inflammasomes are cytoplasmic protein complexes that play a crucial role in protecting the host against pathogenic and sterile stressors by initiating inflammation. Upon activation, these complexes directly regulate the proteolytic processing and activation of proinflammatory cytokines interleukin (IL)-1ß and IL-18 to induce a potent inflammatory response, and induce a programmed form of cell death called pyroptosis to expose intracellular pathogens to the surveillance of the immune system, thus perpetuating inflammation. There are various types of inflammasome complexes, with the NLRP1 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-1) inflammasome being the first one identified and currently recognized as the predominant inflammasome sensor protein in human keratinocytes. Human NLRP1 exhibits a unique domain structure, containing both an N-terminal pyrin (PYD) domain and an effector C-terminal caspase recruitment domain (CARD). It can be activated by diverse stimuli, such as viruses, ultraviolet B radiation and ribotoxic stress responses. Specific mutations in NLRP1 or related genes have been associated with rare monogenic skin disorders, such as multiple self-healing palmoplantar carcinoma; familial keratosis lichenoides chronica; autoinflammation with arthritis and dyskeratosis; and dipeptidyl peptidase 9 deficiency. Recent research breakthroughs have also highlighted the involvement of dysfunctions in the NLRP1 pathway in a handful of seemingly unrelated dermatological conditions. These range from monogenic autoinflammatory diseases to polygenic autoimmune diseases such as vitiligo, psoriasis, atopic dermatitis and skin cancer, including squamous cell carcinoma, melanoma and Kaposi sarcoma. Additionally, emerging evidence implicates NLRP1 in systemic lupus erythematosus, pemphigus vulgaris, Addison disease, Papillon-Lefèvre syndrome and leprosy. The aim of this review is to shed light on the implications of pathological dysregulation of the NLRP1 inflammasome in skin diseases and investigate the potential rationale for targeting this pathway as a future therapeutic approach.

High p62 expression suppresses the NLRP1 inflammasome and increases stress resistance in cutaneous SCC cells.

NLRP1 is the primary inflammasome sensor in human keratinocytes. Sensing of UVB radiation by NLRP1 is believed to underlie the induction of sunburn. Although constitutive NLRP1 activation causes skin inflammation and predisposes patients to the development of cutaneous SCCs, the NLRP1 pathway is suppressed in established SCCs. Here, we identified high levels of the autophagy receptor p62 in SCC cells lines and SCC tumors. Increased NF-κB activity in SCC cells causes p62 up-regulation. Suppression of p62 expression rescues UVB-induced NLRP1 inflammasome activation in early-stage SCC cells. p62 expression protects SCC cells from cytotoxic drugs, whereas NLRP1 sensitizes them. In summary, we identify p62 as a novel negative regulator of the NLRP1 inflammasome in human cutaneous SCC cells, in which suppression of NLRP1 by increased levels of p62 supports stress resistance of skin cancer cells.

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.

Immunoproteasome inhibition attenuates experimental psoriasis.

Introduction: Psoriasis is an autoimmune skin disease associated with multiple comorbidities. The immunoproteasome is a special form of the proteasome expressed in cells of hematopoietic origin. Methods: The therapeutic use of ONX 0914, a selective inhibitor of the immunoproteasome, was investigated in Card14ΔE138+/- mice, which spontaneously develop psoriasis-like symptoms, and in the imiquimod murine model. Results: In both models, treatment with ONX 0914 significantly reduced skin thickness, inflammation scores, and pathological lesions in the analyzed skin tissue. Furthermore, immunoproteasome inhibition normalized the expression of several pro-inflammatory genes in the ear and significantly reduced the inflammatory infiltrate, accompanied by a significant alteration in the αß+ and γδ+ T cell subsets. Discussion: ONX 0914 ameliorated psoriasis-like symptoms in two different murine psoriasis models, which supports the use of immunoproteasome inhibitors as a therapeutic treatment in psoriasis.

Keratinocyte-intrinsic BCL10/MALT1 activity initiates and amplifies psoriasiform skin inflammation.

Psoriasis is a chronic inflammatory skin disease arising from poorly defined pathological cross-talk between keratinocytes and the immune system. BCL10 (B cell lymphoma/leukemia 10) and MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) are ubiquitously expressed inflammatory signaling proteins that can interact with the psoriasis susceptibility factor CARD14, but their functions in psoriasis are insufficiently understood. We report that although keratinocyte-intrinsic BCL10/MALT1 deletions completely rescue inflammatory skin pathology triggered by germline Card14 gain-of-function mutation in mice, the BCL10/MALT1 signalosome is unexpectedly not involved in the CARD14-dependent interleukin-17 receptor (IL-17R) proximal pathway. Instead, it plays a more pleiotropic role by amplifying keratinocyte responses to a series of inflammatory cytokines, including IL-17A, IL-1ß, and TNF. Moreover, selective keratinocyte-intrinsic activation of BCL10/MALT1 signaling with an artificial engager molecule is sufficient to initiate lymphocyte-mediated psoriasiform skin inflammation, and aberrant BCL10/MALT1 activity is frequently detected in the skin of human sporadic psoriasis. Together, these results establish that BCL10/MALT1 signalosomes can act as initiators and crucial amplifiers of psoriatic skin inflammation and indicate a critical function for this complex in sporadic psoriasis.

Protamine-Based Strategies for RNA Transfection.

Protamine is a natural cationic peptide mixture mostly known as a drug for the neutralization of heparin and as a compound in formulations of slow-release insulin. Protamine is also used for cellular delivery of nucleic acids due to opposite charge-driven coupling. This year marks 60 years since the first use of Protamine as a transfection enhancement agent. Since then, Protamine has been broadly used as a stabilization agent for RNA delivery. It has also been involved in several compositions for RNA-based vaccinations in clinical development. Protamine stabilization of RNA shows double functionality: it not only protects RNA from degradation within biological systems, but also enhances penetration into cells. A Protamine-based RNA delivery system is a flexible and versatile platform that can be adjusted according to therapeutic goals: fused with targeting antibodies for precise delivery, digested into a cell penetrating peptide for better transfection efficiency or not-covalently mixed with functional polymers. This manuscript gives an overview of the strategies employed in protamine-based RNA delivery, including the optimization of the nucleic acid's stability and translational efficiency, as well as the regulation of its immunostimulatory properties from early studies to recent developments.

Regulation and dysregulation of CARD14 signalling and its physiological consequences in inflammatory skin disease.

CARD14 is a scaffold molecule predominantly expressed in keratinocytes and genetic variants in the CARD14 gene confer an increased risk of inflammatory skin disease. Due to its association with common skin diseases psoriasis and atopic dermatitis, the biological function of CARD14 is of relevant interest to human health. CARD14 recruits BCL10 and MALT1 to form the CARD-BCL10-MALT1 complex, which modulates NF-κB and MAPK signalling pathways, yet little is known about how CARD14 is regulated or activated in the context of the innate immune response and in chronic inflammation. This review summarises the current understanding of the molecular function and regulatory mechanisms of CARD14 and highlights recent findings in human disease and murine mouse models.

IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes.

Epidermal growth factor receptor (EGFR) and MEK inhibitors (EGFRi/MEKi) are beneficial for the treatment of solid cancers but are frequently associated with severe therapy-limiting acneiform skin toxicities. The underlying molecular mechanisms are poorly understood. Using gene expression profiling we identified IL-36γ and IL-8 as candidate drivers of EGFRi/MEKi skin toxicity. We provide molecular and translational evidence that EGFRi/MEKi in concert with the skin commensal bacterium Cutibacterium acnes act synergistically to induce IL-36γ in keratinocytes and subsequently IL-8, leading to cutaneous neutrophilia. IL-36γ expression was the combined result of C. acnes-induced NF-κB activation and EGFRi/MEKi-mediated expression of the transcription factor Krüppel-like factor 4 (KLF4), due to the presence of both NF-κB and KLF4 binding sites in the human IL-36γ gene promoter. EGFRi/MEKi increased KLF4 expression by blockade of the EGFR/MEK/ERK pathway. These results provide an insight into understanding the pathological mechanism of the acneiform skin toxicities induced by EGFRi/MEKi and identify IL-36γ and the transcription factor KLF4 as potential therapeutic targets.

Clinical and Genetic Heterogeneity of CARD14 Mutations in Psoriatic Skin Disease.

The CARD: BCL10: MALT1 (CBM) complex is an essential signaling node for maintaining both innate and adaptive immune responses. CBM complex components have gained considerable interest due to the dramatic effects of associated mutations in causing severe lymphomas, immunodeficiencies, carcinomas and inflammatory disease. While MALT1 and BCL10 are ubiquitous proteins, the CARD-containing proteins differ in their tissue expression. CARD14 is primarily expressed in keratinocytes. The CARD14-BCL10-MALT1 complex is activated by upstream pathogen-associated molecular pattern-recognition in vitro, highlighting a potentially crucial role in innate immune defense at the epidermal barrier. Recent findings have demonstrated how CARD14 orchestrates activation of the NF-κB and MAPK signaling pathways via recruitment of BCL10 and MALT1, leading to the upregulation of pro-inflammatory genes encoding IL-36γ, IL-8, Ccl20 and anti-microbial peptides. Following the identification of CARD14 gain-of function mutations as responsible for the psoriasis susceptibility locus PSORS2, the past years have witnessed a large volume of case reports and association studies describing CARD14 variants as causal or predisposing to a wide range of inflammatory skin disorders. Recent publications of mouse models also helped to better understand the physiological contribution of CARD14 to psoriasis pathogenesis. In this review, we summarize the clinical, genetic and functional aspects of human and murine CARD14 mutations and their contribution to psoriatic disease pathogenesis.

Orphan receptor IL-17RD tunes IL-17A signalling and is required for neutrophilia.

Interleukin-17A, the prototypical member of the interleukin-17 cytokine family, coordinates local tissue inflammation by recruiting neutrophils to sites of infection. Dysregulation of interleukin-17 signalling has been linked to the pathogenesis of inflammatory diseases and autoimmunity. The interleukin-17 receptor family members (A-E) have a broad range of functional effects in immune signalling yet no known role has been described for the remaining orphan receptor, interleukin-17 receptor D, in regulating interleukin-17A-induced signalling pathways. Here we demonstrate that interleukin-17 receptor D can differentially regulate the various pathways employed by interleukin-17A. Neutrophil recruitment, in response to in vivo administration of interleukin-17A, is abolished in interleukin-17 receptor D-deficient mice, correlating with reduced interleukin-17A-induced activation of p38 mitogen-activated protein kinase and expression of the neutrophil chemokine MIP-2. In contrast, interleukin-17 receptor D deficiency results in enhanced interleukin-17A-induced activation of nuclear factor-kappa B and interleukin-6 and keratinocyte chemoattractant expression. Interleukin-17 receptor D disrupts the interaction of Act1 and TRAF6 causing differential regulation of nuclear factor-kappa B and p38 mitogen-activated protein kinase signalling pathways.

Mal mediates TLR-induced activation of CREB and expression of IL-10.

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-κB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam(2)Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFR-associated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory genes.