Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation.

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations.

Delivery of loaded MR1 monomer results in efficient ligand exchange to host MR1 and subsequent MR1T cell activation.

MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.

Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML.

Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in preclinical studies and clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. In this study, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that cotargeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. Although IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complementary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via noncanonical MyD88-independent pathways. Genomic and proteomic analyses revealed that IRAK1 and IRAK4 preserve the undifferentiated state of MDS/AML LSPCs by coordinating a network of pathways, including ones that converge on the polycomb repressive complex 2 complex and JAK-STAT signaling. To translate these findings, we implemented a structure-based design of a potent and selective dual IRAK1 and IRAK4 inhibitor KME-2780. MDS/AML cell lines and patient-derived samples showed significant suppression of LSPCs in xenograft and in vitro studies when treated with KME-2780 as compared with selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for cotargeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.

The Rheumatoid Arthritis and MUScle (RAMUS) Study: Protocol for an observational single-arm study of skeletal muscle in patients with rheumatoid arthritis receiving tofacitinib.

People with rheumatoid arthritis (RA) are disproportionately affected by sarcopenia, the generalised loss of muscle strength and mass, consequently facing an increased risk of falls, functional decline and death. Currently, there are no approved pharmacological treatments for sarcopenia. RA patients who start tofacitinib (a Janus kinase inhibitor) develop small increases in serum creatinine that are not explained by renal function changes and could reflect sarcopenia improvement. The RAMUS Study is a proof of concept, single-arm observational study in which patients with RA who commence tofacitinib according to routine care will be offered participation according to eligibility criteria. Participants will undergo lower limb quantitative magnetic resonance imaging, whole-body dual energy x-ray absorptiometry, joint examination, muscle function testing and blood tests at three time points: prior to starting tofacitinib and 1 and 6 months afterwards. Muscle biopsy will be performed before and 6 months after starting tofacitinib. The primary outcome will be lower limb muscle volume changes following treatment initiation. The RAMUS Study will investigate whether muscle health improves following tofacitinib treatment for RA. Identifying a potential pharmacological treatment for sarcopenia could have important implications for individuals with RA and for older people in general. ISRCTN registry ID: 13364395.

Rheumatoid sarcopenia: loss of skeletal muscle strength and mass in rheumatoid arthritis.

Sarcopenia, a disorder that involves the generalized loss of skeletal muscle strength and mass, was formally recognized as a disease by its inclusion in the International Classification of Diseases in 2016. Sarcopenia typically affects older people, but younger individuals with chronic disease are also at risk. The risk of sarcopenia is high (with a prevalence of ≥25%) in individuals with rheumatoid arthritis (RA), and this rheumatoid sarcopenia is associated with increased likelihood of falls, fractures and physical disability, in addition to the burden of joint inflammation and damage. Chronic inflammation mediated by cytokines such as TNF, IL-6 and IFNγ contributes to aberrant muscle homeostasis (for instance, by exacerbating muscle protein breakdown), and results from transcriptomic studies have identified dysfunction of muscle stem cells and metabolism in RA. Progressive resistance exercise is an effective therapy for rheumatoid sarcopenia but it can be challenging or unsuitable for some individuals. The unmet need for anti-sarcopenia pharmacotherapies is great, both for people with RA and for otherwise healthy older adults.

Closed Collaborative Surgical Intensive Care Unit Modeling and Its Association With Trauma Patient Outcomes.

INTRODUCTION: The optimization of intensive care unit (ICU) care impacts clinical outcomes and resource utilization. In 2017, our surgical ICU (SICU) adopted a "closed-collaborative" model. The aim of this study is to compare patient outcomes in the closed-collaborative model versus the previous open model in a cohort of trauma surgical patients admitted to our adult level 1 trauma center. METHODS: A retrospective review of trauma patients in the SICU from August 1, 2015 to July 31, 2019 was performed. Patients were divided into those admitted prior to August 1, 2017 (the "open" cohort) and those admitted after August 1, 2017 (the "closed-collaborative" cohort). Demographic variables and clinical outcomes were analyzed. Trauma severity was assessed using injury severity score (ISS). RESULTS: We identified 1669 patients (O: 895; C: 774). While no differences in demographics were observed, the closed-collaborative cohort had a higher overall ISS (O: 21.5 ± 12.14; C: 25.10 ± 2.72; P < 0.0001). There were no significant differences between the two cohorts in the incidence of strokes (O: 1.90%; C: 2.58%, P = 0.3435), pulmonary embolism (O: 0.78%; C: 0.65%; P = 0.7427), sepsis (O: 5.25%; C: 7.49%; P = 0.0599), median ICU charges (O: $7784.50; C: $8986.53; P = 0.5286), mortality (O: 11.40%; C: 13.18%; P = 0.2678), or ICU length of stay (LOS) (O: 4.85 ± 6.23; C: 4.37 ± 4.94; P = 0.0795). CONCLUSIONS: Patients in the closed-collaborative cohort had similar clinical outcomes despite having a sicker cohort of patients. We hypothesize that the closed-collaborative ICU model was able to maintain equivalent outcomes due to the dedicated multidisciplinary critical care team caring for these patients. Further research is warranted to determine the optimal model of ICU care for trauma patients.

Paradoxical Effects of Endoplasmic Reticulum Aminopeptidase 1 Deficiency on HLA-B27 and Its Role as an Epistatic Modifier in Experimental Spondyloarthritis.

OBJECTIVE: We undertook this study to examine the functional basis for epistasis between endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 in experimental spondyloarthritis (SpA). METHODS: ERAP1-knockout rats were created using genome editing and bred with HLA-B27/human ß2 -microglobulin-transgenic (HLA-B27-Tg) rats and HLA-B7-Tg rats. The effects of ERAP1 deficiency on HLA allotypes were determined using immunoprecipitation and immunoblotting, flow cytometry, allogeneic T cell proliferation assays, and gene expression analyses. Animals were examined for clinical features of disease, and tissue was assessed by histology. RESULTS: ERAP1 deficiency increased the ratio of folded to unfolded (ß2 m-free) HLA-B27 heavy chains, while having the opposite effect on HLA-B7. Furthermore, in rats with ERAP1 deficiency, HLA-B27 misfolding was reduced, while free HLA-B27 heavy chain dimers on the cell surface and monomers were increased. The effects of ERAP1 deficiency persisted during up-regulation of HLA-B27 and led to a reduction in endoplasmic reticulum stress. ERAP1 deficiency reduced the prevalence of arthritis in HLA-B27-Tg rats by two-thirds without reducing gastrointestinal inflammation. Dendritic cell abnormalities attributed to the presence of HLA-B27, including reduced allogeneic T cell stimulation and loss of CD103-positive/major histocompatibility complex class II-positive cells, were not rescued by ERAP1 deficiency, while excess Il23a up-regulation was mitigated. CONCLUSION: ERAP1 deficiency reduced HLA-B27 misfolding and improved folding while having opposing effects on HLA-B7. The finding that HLA-B27-Tg rats had partial protection against SpA in this study is consistent with genetic evidence that loss-of-function and/or reduced expression of ERAP1 reduces the risk of ankylosing spondylitis. Functional studies support the concept that the effects of ERAP1 on HLA-B27 and SpA may be a consequence of how peptides affect the biology of this allotype rather than their role as antigenic determinants.

CD5 Deficiency Alters Helper T Cell Metabolic Function and Shifts the Systemic Metabolome.

Metabolic function plays a key role in immune cell activation, destruction of foreign pathogens, and memory cell generation. As T cells are activated, their metabolic profile is significantly changed due to signaling cascades mediated by the T cell receptor (TCR) and co-receptors found on their surface. CD5 is a T cell co-receptor that regulates thymocyte selection and peripheral T cell activation. The removal of CD5 enhances T cell activation and proliferation, but how this is accomplished is not well understood. We examined how CD5 specifically affects CD4+ T cell metabolic function and systemic metabolome by analyzing serum and T cell metabolites from CD5WT and CD5KO mice. We found that CD5 removal depletes certain serum metabolites, and CD5KO T cells have higher levels of several metabolites. Transcriptomic analysis identified several upregulated metabolic genes in CD5KO T cells. Bioinformatic analysis identified glycolysis and the TCA cycle as metabolic pathways promoted by CD5 removal. Functional metabolic analysis demonstrated that CD5KO T cells have higher oxygen consumption rates (OCR) and higher extracellular acidification rates (ECAR). Together, these findings suggest that the loss of CD5 is linked to CD4+ T cell metabolism changes in metabolic gene expression and metabolite concentration.

TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

The deubiquitinase USP15 modulates cellular redox and is a therapeutic target in acute myeloid leukemia.

Ubiquitin-specific peptidase 15 (USP15) is a deubiquitinating enzyme implicated in critical cellular and oncogenic processes. We report that USP15 mRNA and protein are overexpressed in human acute myeloid leukemia (AML) as compared to normal hematopoietic progenitor cells. This high expression of USP15 in AML correlates with KEAP1 protein and suppression of NRF2. Knockdown or deletion of USP15 in human and mouse AML models significantly impairs leukemic progenitor function and viability and de-represses an antioxidant response through the KEAP1-NRF2 axis. Inhibition of USP15 and subsequent activation of NRF2 leads to redox perturbations in AML cells, coincident with impaired leukemic cell function. In contrast, USP15 is dispensable for human and mouse normal hematopoietic cells in vitro and in vivo. A preclinical small-molecule inhibitor of USP15 induced the KEAP1-NRF2 axis and impaired AML cell function, suggesting that targeting USP15 catalytic function can suppress AML. Based on these findings, we report that USP15 drives AML cell function, in part, by suppressing a critical oxidative stress sensor mechanism and permitting an aberrant redox state. Furthermore, we postulate that inhibition of USP15 activity with small molecule inhibitors will selectively impair leukemic progenitor cells by re-engaging homeostatic redox responses while sparing normal hematopoiesis.

IRAK1 and IRAK4 as emerging therapeutic targets in hematologic malignancies.

PURPOSE OF REVIEW: Cell intrinsic and extrinsic perturbations to inflammatory signaling pathways are a hallmark of development and progression of hematologic malignancies. The interleukin 1 receptor-associated kinases (IRAKs) are a family of related signaling intermediates (IRAK1, IRAK2, IRAK3, IRAK4) that operate at the nexus of multiple inflammatory pathways implicated in the hematologic malignancies. In this review, we explicate the oncogenic role of these kinases and review recent therapeutic advances in the dawning era of IRAK-targeted therapy. RECENT FINDINGS: Emerging evidence places IRAK signaling at the confluence of adaptive resistance and oncogenesis in the hematologic malignancies and solid tissue tumors. Preclinical investigations nominate the IRAK kinases as targetable molecular dependencies in diverse cancers. SUMMARY: IRAK-targeted therapies that have matriculated to early phase trials are yielding promising preliminary results. However, studies of IRAK kinase signaling continue to defy conventional signaling models and raise questions as to the design of optimal treatment strategies. Efforts to refine IRAK signaling mechanisms in the malignant context will inspire deliberate IRAK-targeted drug development and informed combination therapy.

Making Medical Treatment Decisions for Unrepresented Patients in the ICU. An Official American Thoracic Society/American Geriatrics Society Policy Statement.

Background and Rationale: ICU clinicians regularly care for patients who lack capacity, an applicable advance directive, and an available surrogate decision-maker. Although there is no consensus on terminology, we refer to these patients as "unrepresented." There is considerable controversy about how to make treatment decisions for these patients, and there is significant variability in both law and clinical practice.Purpose and Objectives: This multisociety statement provides clinicians and hospital administrators with recommendations for decision-making on behalf of unrepresented patients in the critical care setting.Methods: An interprofessional, multidisciplinary expert committee developed this policy statement by using an iterative consensus process with a diverse working group representing critical care medicine, palliative care, pediatric medicine, nursing, social work, gerontology, geriatrics, patient advocacy, bioethics, philosophy, elder law, and health law.Main Results: The committee designed its policy recommendations to promote five ethical goals: 1) to protect highly vulnerable patients, 2) to demonstrate respect for persons, 3) to provide appropriate medical care, 4) to safeguard against unacceptable discrimination, and 5) to avoid undue influence of competing obligations and conflicting interests. These recommendations also are intended to strike an appropriate balance between excessive and insufficient procedural safeguards. The committee makes the following recommendations: 1) institutions should offer advance care planning to prevent patients at high risk for becoming unrepresented from meeting this definition; 2) institutions should implement strategies to determine whether seemingly unrepresented patients are actually unrepresented, including careful capacity assessments and diligent searches for potential surrogates; 3) institutions should manage decision-making for unrepresented patients using input from a diverse interprofessional, multidisciplinary committee rather than ad hoc by treating clinicians; 4) institutions should use all available information on the patient's preferences and values to guide treatment decisions; 5) institutions should manage decision-making for unrepresented patients using a fair process that comports with procedural due process; 6) institutions should employ this fair process even when state law authorizes procedures with less oversight.Conclusions: This multisociety statement provides guidance for clinicians and hospital administrators on medical decision-making for unrepresented patients in the critical care setting.

Can quantitative MRI be used in the clinical setting to quantify the impact of intra-articular glucocorticoid injection on synovial disease activity in juvenile idiopathic arthritis?

BACKGROUND: Juvenile idiopathic arthritis (JIA), the most common chronic rheumatic disease of childhood, is characterised by synovitis. Clinical assessments of synovitis are imperfect, relying on composite and indirect measures of disease activity including clinician-reported measures, patient-reported measures and blood markers. Contrast-enhanced MRI is a more sensitive synovitis assessment technique but clinical utility is currently limited by availability and inter-observer variation. Improved quantitative MRI techniques may enable future development of more stringent MRI-defined remission criteria. The objective of this study was to determine the utility and feasibility of quantitative MRI measurement of synovial volume and vascularity in JIA before and twelve weeks after intra-articular glucocorticoid injection (IAGI) of the knee and to assess the acceptability of MRI to participating families. METHODS: Children and young people with JIA and a new episode of knee synovitis requiring IAGI were recruited from the Great North Children's Hospital in Newcastle upon Tyne. Quantitative contrast-enhanced MRI was performed prior to and twelve weeks after IAGI, in addition to standard clinical assessment tools, including the three-variable clinical juvenile arthritis disease activity score (cJADAS) and active joint count. RESULTS: Eleven young people (5 male, median age 13 years, range 7-16) with JIA knee flare were recruited and 10 completed follow-up assessment. Following IAGI, the median (interquartile range) cJADAS improved from 8.5 (2.7) to 1.6 (3.9), whilst the median synovial volume improved from 38.5cm3 (82.1cm3) to 0.0cm3 (0.2cm3). Six patients presented with frank synovitis outside normal limits on routine MRI reporting. A further three had baseline MRI reports within normal limits but the quantitative measurements identified measurable synovial uptake. Post-IAGI quantitative measurements highlighted significant improvements in 9 patients. CONCLUSIONS: IAGI led to a marked reduction in synovial volume, with quantitative MRI identifying more patients with an improved synovial volume than routine qualitative clinical reporting. Improvements in cJADAS scores were more variable with the patient/parent global assessment component contributing most to the scores. Further work is indicated, exploring the utility of quantitative MRI in the assessment of less accessible joints and comparing the impact of different treatment modalities.

Multi-centre national audit of juvenile localised scleroderma: describing current UK practice in disease assessment and management.

OBJECTIVE: To describe current United Kingdom practice in assessment and management of patients with juvenile localised scleroderma (JLS) compared to Paediatric Rheumatology European Society (PRES) scleroderma working party recommendations. METHODS: Patients were included if they were diagnosed with JLS and were under the care of paediatric rheumatology between 04/2015-04/2016. Retrospective data was collected in eleven UK centres using a standardised proforma and collated centrally. RESULTS: 149 patients were included with a median age of 12.5 years. The outcome measures recommended by the PRES scleroderma working party were not utilised widely. The localised scleroderma cutaneous assessment tool was only used in 37.6% of patients. Screening for extracutaneous manifestations did not meet recommendations that patients with head involvement have regular screening for uveitis and baseline magnetic resonance imaging (MRI) brain: only 38.5% of these patients were ever screened for uveitis; 71.2% had a MRI brain. Systemic treatment with disease-modifying anti-rheumatic drugs (DMARDs) or biologics was widely used (96.0%). In keeping with the recommendations, 95.5% of patients were treated with methotrexate as first-line therapy. 82.6% received systemic corticosteroids and 34.2% of patients required two or more DMARDs/biologics, highlighting the significant treatment burden. Second-line treatment was mycophenolate mofetil in 89.5%. CONCLUSION: There is wide variation in assessment and screening of patients with JLS but a consistent approach to systemic treatment within UK paediatric rheumatology. Improved awareness of PRES recommendations is required to ensure standardised care. As recommendations are based on low level evidence and consensus opinion, further studies are needed to better define outcome measures and treatment regimens for JLS.

Association Between Quantitative Gait and Balance Measures and Total Daily Physical Activity in Community-Dwelling Older Adults.

Background: Total daily physical activity is associated with a wide range of adverse health outcomes. We examined the extent to which quantitative measures of gait and balance abilities were associated with total daily physical activity, controlling for a variety of potential covariates. Methods: Participants (n = 608) were older adults participating in the Rush Memory and Aging Project, a community-based cohort study of aging. Objective measures of total daily physical activity were derived from a wearable device. Gait and balance abilities were objectively quantified using a body-fixed sensor. We also collected measures of other motor functions, cognitive and psychosocial factors, and chronic health. We employed linear regression models to identify facets of mobility significantly associated with total daily physical activity, and tested for independence of these associations when all significant covariates were considered together in a final model. Results: Three gait and balance measures were independently associated with total daily physical activity (p < .01), together accounting for approximately 16% of its variance. Other motor measures, cognitive and psychosocial factors, and chronic health accounted for 8.8%, 4.9%, and 6.4% of the variance, respectively, when considered in isolation. Considered together in a single model, all significant covariates accounted for approximately 21% of the variance in physical activity. Conclusions: Gait and balance measures from a body-fixed sensor are strongly associated with objectively measured total daily physical activity in older adults. However, given the importance of physical activity to many health outcomes, further work is needed to more completely characterize the factors that may influence physical activity.

Novel substituted phenoxyalkyl pyridinium oximes enhance survival and attenuate seizure-like behavior of rats receiving lethal levels of nerve agent surrogates.

Novel substituted phenoxyalkyl pyridinium oximes, previously shown to reactivate brain cholinesterase in rats treated with high sublethal dosages of surrogates of sarin and VX, were tested for their ability to prevent mortality from lethal doses of these two surrogates. Rats were treated subcutaneously with 0.6mg/kg nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate) or 0.65mg/kg nitrophenyl ethyl methylphosphonate (NEMP; VX surrogate), dosages that were lethal within 24h to all tested rats when they received only 0.65mg/kg atropine at the time of initiation of seizure-like behavior (about 30min). If 146mmol/kg 2-PAM (human equivalent dosage) was also administered, 40% and 33% survival was obtained with NIMP and NEMP, respectively, while the novel Oximes 1 and 20 provided 65% and 55% survival for NIMP and 75 and 65% for NEMP, respectively. In addition, both novel oximes resulted in a highly significant decrease in time to cessation of seizure-like behavior compared to 2-PAM during the first 8h of observation. Brain cholinesterase inhibition was slightly less in novel oxime treated rats compared to 2-PAM in the 24h survivors. The lethality data indicate that 24h survival is improved by two of the novel oximes compared to 2-PAM. The cessation of seizure-like behavior data strongly suggest that these novel oximes are able to penetrate the blood-brain barrier and can combat the hypercholinergic activity that results in seizures. Therefore this oxime platform has exceptional promise as therapy that could both prevent nerve agent-induced lethality and attenuate nerve agent-induced seizures.

Solid-state covalent capture of CO2 by using N-heterocyclic carbenes.

Capture me! The first report of an N-heterocyclic carbene (NHC) as a solid-state carbon capture reagent is presented. Experimental and theoretical measurements demonstrate the ability of the NHC to react rapidly and stoichiometrically with CO2 at low partial pressures.