Roles and Regulation of Sphingosine Kinase 2 in Cancer

The two mammalian sphingosine kinases, SK1 and SK2, produce the bioactive signalling lipid sphingosine-1-phosphate (S1P), which generally promotes cell survival, proliferation and migration. In line with this, SK1 is often found to be upregulated in human cancers, and overexpression of SK1 leads to neoplastic transformation of cells and tumour growth. However, despite generating the same product, most evidence to date suggests that SK2 acts in an opposing manner to promote cell death. In contrast, knockout mouse models indicate that there is at least some functional redundancy between the two SK isoforms, and targeting SK2 via genetic or pharmacological approaches in cancer models results in reduced tumour growth. Clearly, the roles of SK2 are poorly understood, but it is apparent that these unique and complex functions of SK2 are largely dictated by its differential subcellular localisation. SK1 is generally a cytoplasmic protein, but it can also be translocated to the plasma membrane where it mediates cell survival, proliferation and oncogenic signalling through the production of S1P. SK2, however, has been reported to localise to the nucleus, endoplasmic reticulum and mitochondria, and at these locations it appears to possess anti-proliferative and pro-apoptotic functions. SK2 has also been reported to localise to the plasma membrane, but its specific roles here have not been well characterised. Therefore, the main aims of this study were to explore the roles of SK2 in cancer, and to characterise novel mechanisms that regulate SK2 subcellular localisation, such as interacting proteins and post translational modifications, in order to gain a better understanding of this complex enzyme and the potential benefits of targeting SK2 in cancer. To explore the roles of SK2 in cancer, we examined the expression of SK2 in various human tumour samples using publically available datasets, and found that SK2 shows statistically significant upregulation in many cancers, but only to modest levels up to 2.5- fold over normal tissues. As high-level SK2 overexpression has been previously shown to cause cell death, we explored the effects of low, close to physiological levels of SK2 overexpression. By engineering a series of human and mouse cell lines overexpressing graded levels of SK2, we found that low-level SK2 overexpression increased cell survival and proliferation, and activated oncogenic signalling pathways. Notably, low-level SK2 overexpression (5- to 10-fold over endogenous levels) was sufficient to induce neoplastic transformation of mouse fibroblasts, resulting in efficient tumour formation in vivo. These findings coincided with decreased nuclear localisation and increased plasma membrane localisation of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis. Furthermore, the Pitson laboratory previously identified a novel SK2-interacting protein, cytoplasmic dynein 1 intermediate chain 2 (IC-2), through a yeast two-hybrid screen, and characterising this interaction formed another part of these studies. We confirmed that SK2 interacts physiologically with the dynein complex in cells via the IC subunit, and being a retrograde-directed transport motor complex, we found that dynein mediates the translocation of SK2 away from the plasma membrane. Interestingly, although IC-2 was identified in the yeast two-hybrid screen, SK2 interacts more robustly with the highly-related IC-1 isoform, which is abundantly expressed in the brain. Strikingly, we found that IC-1 is downregulated 17-fold in glioblastoma multiforme (GBM) patient samples, which correlated with poorer survival of patients with this form of brain tumour. In line with a role for dynein in transporting SK2, low IC-1 expression in GBM cells coincided with more SK2 localised to the plasma membrane, where we had found it to accumulate in an oncogenic setting. Re-expression of IC-1 in these cells reduced plasma membrane localised-SK2 and extracellular S1P formation, and notably, decreased tumour growth and tumour-associated angiogenesis in vivo. Thus, these findings demonstrate a novel tumour-suppressive function of dynein IC-1, and uncover new mechanistic insights into SK2 regulation. Through previous mass spectrometric analyses performed by the Pitson laboratory, it is evident that SK2 contains multiple uncharacterised phosphorylation sites that are not shared with SK1. We explored the function of one such site, Ser363, and found it to potentially regulate nuclear localisation of SK2. Furthermore, we identified SK2 as a bona fide substrate of glycogen synthase kinase 3 (GSK3) in vitro and in cells, involving residues Ser437 and Ser441, and we found that other phosphorylation events may act to regulate SK2 catalytic activity. Overall, the studies outlined here have revealed a previously unreported role for SK2 in driving oncogenesis, and have described the characterisation of novel mechanisms that regulate the subcellular localisation of SK2. Therefore, these findings support the use of SK2 inhibitors as promising anti-cancer therapeutic agents. Furthermore, as the opposing functions of SK2 are largely dictated by it subcellular localisation, these findings may also assist in the development of new strategies to target oncogenic SK2 in cancer.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

Pharmacological Prevention of Atrial Fibrillation after Cardiac Surgery with and without the Use of Nonsteroidal Anti-inflammatory Drugs

Purpose: Atrial fibrillation after cardiac surgery (AFACS) is the most common complication following open heart procedures, with significantly increased morbidity and mortality associated with its occurrence1. A previous study done at Baptist Hospital of Miami (BHM) showed a decrease in the incidence of AFACS from 36% to 22% after implementation of a prevention protocol that includes guideline-directed therapy with a beta-blocker and amiodarone. The protocol also includes nonsteroidal anti-inflammatory drugs (NSAIDs) modeled after a study published in 2004 that showed that NSAIDs lowered the incidence of postoperative atrial fibrillation by 65% when they were used in the postoperative period7. In recent months, cardiothoracic surgery providers and nephrology specialists have begun to avoid the postoperative use of NSAIDs in this patient population due to the concern for acute kidney injury (AKI)/renal failure. The purpose of this study was to assess if there is a difference in the incidence of AFACS and AKI after cardiac surgery in patients who received metoprolol, amiodarone and NSAIDs compared to those who received only the combination of metoprolol and amiodarone. Methods: This was a multi-centered, IRB-approved, retrospective study of adult patients admitted to either BHM or South Miami Hospital (SMH) between 5/1/2019 and 12/20/2019. Patients were included if they underwent an open-heart procedure and received at least one of the medications from the postoperative atrial fibrillation prevention protocol. Patients in Cohort A included those who received NSAIDS in addition to a standard prophylactic regimen consisting of a beta blocker and amiodarone, and Cohort B included patients who received the standard regimen only. The primary endpoint was incidence of new-onset AFACS within the first 7 days after cardiac surgery, and secondary endpoints included incidence of AKI and length of stay in the intensive care unit (ICU) and hospital. Results: A total of 215 patients were screened for inclusion and exclusion criteria, with 140 patients being included in the final analysis of the primary and secondary outcomes. Baseline demographics were not statistically different between Cohorts, except for the average baseline serum creatinine on hospital admission (0.92 vs 1.1, p Conclusion: When administered after an open-heart procedure, NSAIDs did not impact the incidence of postoperative atrial fibrillation, incidence of AKI or length of stay. Given the risks of acute renal failure after cardiac surgery and the lack of benefit in the prevention of atrial fibrillation, these results support the removal of NSAIDs from the postoperative atrial fibrillation prevention protocol

The Diverse Roles of γδ T Cells in Cancer: From Rapid Immunity to Aggressive Lymphoma

γδ T cells are unique players in shaping immune responses, lying at the intersection between innate and adaptive immunity. Unlike conventional αβ T cells, γδ T cells largely populate non-lymphoid peripheral tissues, demonstrating tissue specificity, and they respond to ligands in an MHC-independent manner. γδ T cells display rapid activation and effector functions, with a capacity for cytotoxic anti-tumour responses and production of inflammatory cytokines such as IFN-γ or IL-17. Their rapid cytotoxic nature makes them attractive cells for use in anti-cancer immunotherapies. However, upon transformation, γδ T cells can give rise to highly aggressive lymphomas. These rare malignancies often display poor patient survival, and no curative therapies exist. In this review, we discuss the diverse roles of γδ T cells in immune surveillance and response, with a particular focus on cancer immunity. We summarise the intriguing dichotomy between pro- and anti-tumour functions of γδ T cells in solid and haematological cancers, highlighting the key subsets involved. Finally, we discuss potential drivers of γδ T-cell transformation, summarising the main γδ T-cell lymphoma/leukaemia entities, their clinical features, recent advances in mapping their molecular and genomic landscapes, current treatment strategies and potential future targeting options

The Diverse Roles of γδ T Cells in Cancer: From Rapid Immunity to Aggressive Lymphoma

γδ T cells are unique players in shaping immune responses, lying at the intersection between innate and adaptive immunity. Unlike conventional αβ T cells, γδ T cells largely populate non-lymphoid peripheral tissues, demonstrating tissue specificity, and they respond to ligands in an MHC-independent manner. γδ T cells display rapid activation and effector functions, with a capacity for cytotoxic anti-tumour responses and production of inflammatory cytokines such as IFN-γ or IL-17. Their rapid cytotoxic nature makes them attractive cells for use in anti-cancer immunotherapies. However, upon transformation, γδ T cells can give rise to highly aggressive lymphomas. These rare malignancies often display poor patient survival, and no curative therapies exist. In this review, we discuss the diverse roles of γδ T cells in immune surveillance and response, with a particular focus on cancer immunity. We summarise the intriguing dichotomy between pro- and anti-tumour functions of γδ T cells in solid and haematological cancers, highlighting the key subsets involved. Finally, we discuss potential drivers of γδ T-cell transformation, summarising the main γδ T-cell lymphoma/leukaemia entities, their clinical features, recent advances in mapping their molecular and genomic landscapes, current treatment strategies and potential future targeting options

The Diverse Roles of γδ T Cells in Cancer: From Rapid Immunity to Aggressive Lymphoma

γδ T cells are unique players in shaping immune responses, lying at the intersection between innate and adaptive immunity. Unlike conventional αβ T cells, γδ T cells largely populate non-lymphoid peripheral tissues, demonstrating tissue specificity, and they respond to ligands in an MHC-independent manner. γδ T cells display rapid activation and effector functions, with a capacity for cytotoxic anti-tumour responses and production of inflammatory cytokines such as IFN-γ or IL-17. Their rapid cytotoxic nature makes them attractive cells for use in anti-cancer immunotherapies. However, upon transformation, γδ T cells can give rise to highly aggressive lymphomas. These rare malignancies often display poor patient survival, and no curative therapies exist. In this review, we discuss the diverse roles of γδ T cells in immune surveillance and response, with a particular focus on cancer immunity. We summarise the intriguing dichotomy between pro- and anti-tumour functions of γδ T cells in solid and haematological cancers, highlighting the key subsets involved. Finally, we discuss potential drivers of γδ T-cell transformation, summarising the main γδ T-cell lymphoma/leukaemia entities, their clinical features, recent advances in mapping their molecular and genomic landscapes, current treatment strategies and potential future targeting options

Informed consent and assent guide for paediatric clinical trials in Europe

Objective Clinical trial sponsors spend considerable resources preparing informed consent (IC) and assent documentation for multinational paediatric clinical trial applications in Europe due to the limited and dispersed patient populations, the variation of national legal and ethical requirements, and the lack of detailed guidance. The aim of this study was to design new easy-to-use guide publicly available on European Medicines Agency's, Enpr-EMA website for all stakeholders. Methods Current EU legal, ethical and regulatory guidance for paediatric clinical trials were collated, analysed and divided into 30 subject elements in two tables. The European Network of Young Person's Advisory Group reviewed the data and provided specific comments. A three-level recommendation using 'traffic light' symbols was designed for four age groups of children, according to relevance and the requirements. Results A single guide document includes two tables: (1) general information and (2) trial-specific information. In the age group of 6-9 years old, 92% of the trial-specific subject elements can be or should be included in the IC discussion. Even in the youngest possible age group (2-5 years old children), the number of elements considered was, on average, 52%. Conclusion The EU Clinical Trial Regulation (2014) does not contain specific requirements exclusively for paediatric clinical trials. This work is the first to extensively collate all the current legal, regulatory and ethical documentation on the IC process, together with input from adolescents. This guide may increase the ethical standards in paediatric clinical trials. Young people and researchers gathered together to synthesise and rate the advice from all the EU systems they could find about paediatric clinical trials to create a simpler, patient-led, framework for information to aid meaningful trial consent discussions.Peer reviewe

DNM1 encephalopathy: A new disease of vesicle fission.

ObjectiveTo evaluate the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling.MethodsWe reviewed phenotypic data of 21 patients (7 previously published) with DNM1 mutations. We compared mutation data to known functional data and undertook biomolecular modeling to assess the effect of the mutations on protein function.ResultsWe identified 19 patients with de novo mutations in DNM1 and a sibling pair who had an inherited mutation from a mosaic parent. Seven patients (33.3%) carried the recurrent p.Arg237Trp mutation. A common phenotype emerged that included severe to profound intellectual disability and muscular hypotonia in all patients and an epilepsy characterized by infantile spasms in 16 of 21 patients, frequently evolving into Lennox-Gastaut syndrome. Two patients had profound global developmental delay without seizures. In addition, we describe a single patient with normal development before the onset of a catastrophic epilepsy, consistent with febrile infection-related epilepsy syndrome at 4 years. All mutations cluster within the GTPase or middle domains, and structural modeling and existing functional data suggest a dominant-negative effect on DMN1 function.ConclusionsThe phenotypic spectrum of DNM1-related encephalopathy is relatively homogeneous, in contrast to many other genetic epilepsies. Up to one-third of patients carry the recurrent p.Arg237Trp variant, which is now one of the most common recurrent variants in epileptic encephalopathies identified to date. Given the predicted dominant-negative mechanism of this mutation, this variant presents a prime target for therapeutic intervention

High Efficacy and Drug Synergy of HDAC6-Selective Inhibitor NN-429 in Natural Killer (NK)/T-Cell Lymphoma

NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies

Comparing proteolytic fingerprints of antigen-presenting cells during allergen processing

<span>Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein</span><span class="searchterm">antigens</span><span>. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for</span><span class="searchterm">proteolytic</span><span>processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall</span><span class="searchterm">proteolytic</span><span>activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine</span><span class="searchterm">antigenic</span><span>peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.</span>(VLID)219508