Metabolism-focused CRISPR screen unveils mitochondrial pyruvate carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer.

Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for the repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase [PARP]1/2) is a proven target with several PARP inhibitors (PARPis) currently in clinical use. Resistance to PARPi often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells. We surmised that alterations in metabolic pathways by cancer drugs such as Olaparib might be involved in the development of resistance to drug therapy. To test this hypothesis, we conducted a metabolism-focused clustered regularly interspaced short palindromic repeats knockout screen to identify genes that undergo alterations during the treatment of tumor cells with PARPis. Of about 3000 genes in the screen, our data revealed that mitochondrial pyruvate carrier 1 (MPC1) is an essential factor in desensitizing nonsmall cell lung cancer (NSCLC) lung cancer lines to PARP inhibition. In contrast to NSCLC lung cancer cells, triple-negative breast cancer cells do not exhibit such desensitization following MPC1 loss and reprogram the tricarboxylic acid cycle and oxidative phosphorylation pathways to overcome PARPi treatment. Our findings unveil a previously unknown synergistic response between MPC1 loss and PARP inhibition in lung cancer cells.

Bioactive adrenomedullin (bio-ADM) is associated with endothelial dysfunction in infants and children with complex congenital heart disease undergoing open-heart surgery on cardiopulmonary bypass.

OBJECTIVES: Children with congenital heart disease (CHD) undergoing cardiac surgery on cardiopulmonary bypass (CPB) are at risk for systemic inflammation leading to endothelial dysfunction associated with increased morbidity. Bioactive adrenomedullin (bio-ADM) is a peptide regulating vascular tone and endothelial permeability. The aim of this study was to evaluate the dynamics of plasma bio-ADM in this patient cohort and its role in capillary leak. METHODS: Plasma samples from 73 pediatric CHD patients were collected for bio-ADM measurement at five different timepoints (TP) in the pre-, intra-, and post-operative period. The primary endpoint was a net increase in bio-ADM levels after surgery on CPB. Secondary endpoints included association of bio-ADM levels with clinical signs for endothelial dysfunction. RESULTS: Bio-ADM levels increased after surgery on CPB from pre-operative median of 12 pg/mL (IQR [interquartile range] 12.0-14.8 pg/mL) to a maximum post-operative median of 48.8 pg/mL (IQR 34.5-69.6 pg/mL, p<0.001). Bio-ADM concentrations correlated positively with post-operative volume balance, (r=0.341; p=0.005), increased demand for vasoactive medication (duration: r=0.415; p<0.001; quantity: TP3: r=0.415, p<0.001; TP4: r=0.414, p<0.001), and hydrocortisone treatment for vasoplegia (bio-ADM median [IQR]:129.1 [55.4-139.2] pg/mL vs. 37.9 [25.2-64.6] pg/mL; p=0.034). Patients who required pleural effusion drainage revealed higher bio-ADM levels compared to those who did not (median [IQR]: 66.4 [55.4-90.9] pg/mL vs. 40.2 [28.2-57.0] pg/mL; p<0.001). CONCLUSIONS: Bio-ADM is elevated in children after cardiac surgery and higher levels correlate with clinical signs of capillary leakage. The peptide should be considered as biomarker for endothelial dysfunction and as potential therapeutic target in this indication.

The role of Proenkephalin A 119-159 in the detection of acute kidney injury after open thoracoabdominal aortic repair.

Background: Acute kidney injury (AKI) after open thoracoabdominal aortic aneurysm repairs (TAAA) is a common postoperative complication, associated with increased mortality and morbidity. Early detection and management of the kidney tissue damage remains of paramount importance. The aim of this prospectively conducted, observational trial was to evaluate the clinical applicability of Proenkephalin A 119-159 (penKid) for the detection of postoperative AKI. Patients and methods: Thirty-six patients, planned for elective open TAAA repairs from January 2019 to December 2022, were recruited in two German centres (University Hospital Aachen and Charité - University Hospital Berlin). Blood samples were collected pre-surgery (baseline), directly postoperatively and at 12, 24 and 48 hours after surgery. The penKid concentration in plasma was measured using the immunoluminometric sphingotest® assay kit and they were statistically tested for association with AKI and other clinical parameters. Results: Twenty-four patients (62%) developed moderate or severe AKI postoperatively (Stage 2 or 3 of the KDIGO classification) and they had a significantly increased risk for the development of acute respiratory distress syndrome (p=.023) or a fatal outcome (p=.035). Starting from the 12th hour after surgery, we found penKid correlating with AKI stage 2/3 (12 hour penKid mean in pmol/L: 93.9 vs. 43.1; c index .776, p=.0037) and renal replacement therapy (12 hour c index .779, p=.0035). Patients with multi-organ dysfunction syndrome had significantly increased penKid levels at all timepoints. Conclusions: We found penKid to be a promising biomarker for the early detection of postoperative AKI and in-hospital mortality after open TAAA repair, which may enable the early initiation of organ-protective strategies and reduction of further complications associated with AKI.

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma.

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.

Selenoprotein P deficiency is associated with higher risk of incident heart failure.

INTRODUCTION: Selenium deficiency has been associated with mortality, cardiovascular disease and worsened prognosis in heart failure (HF). In a recent population-based study, high selenium levels were shown to be associated with reduced mortality and reduced incidence of HF, but only in non-smokers. Here, we aimed to examine if selenoprotein P (SELENOP), a main selenium carrier protein, is associated with incident HF. MATERIALS AND METHODS: SELENOP concentrations were measured in plasma of 5060 randomly selected subjects from the population-based prospective cohort "Malmö Preventive Project" (n = 18240) using an ELISA approach. Exclusion of subjects with prevalent HF (n = 230) and subjects with missing data on co-variates included in the regression analysis (n = 27) resulted in complete data for 4803 subjects (29.1% women, mean age 69.6 ± 6.2 years, 19.7% smokers). Cox regression models adjusted for traditional risk factors were used to analyse SELENOP's association with incident HF. Further, subjects within the quintile with the lowest SELENOP concentrations were compared to subjects in the remaining quintiles. RESULTS: Each 1 standard deviation increment in SELENOP levels was associated with lower risk of incident HF (n = 436) during a median follow-up period of 14.7 years (hazard ratio (HR) 0.90; CI95% 0.82-0.99; p = 0.043). Further analyses showed that subjects in the lowest SELENOP quintile were at the highest risk of incident HF when compared to quintiles 2-5 (HR 1.52; CI95% 1.21-1.89; p = 2.5 × 10-4). CONCLUSION: Low selenoprotein P levels are associated with a higher risk of incident HF in a general population. Further studies are warranted.

USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

Plasma bioactive adrenomedullin predicts outcome after acute stroke in early rehabilitation.

An early and reliable prediction of outcomes after stroke is important for early effective stroke management and the adequate optimal planning of post-stroke rehabilitation and long-term care. Bioactive adrenomedullin (bio-ADM) is a 52-amino acid peptide that is an important peptide hormone in nervous system diseases. The aim of this study was to investigate the prognostic value of bio-ADM on outcomes after rehabilitation in patients with stroke. A total of 557 consecutive patients with a primary diagnosis of ischemic or hemorrhagic stroke (age 69.6-12.9 years, male 51.3%, ischemic stroke 72.5%), who were admitted to an in-patient early rehabilitation center directly after discharge from acute stroke hospital care, were enrolled in this prospective observational study. Plasma concentrations of bio-ADM were determined by using a chemiluminescence immunoassay (functional assay sensitivity 8 pg/ml). The early rehabilitation barthel index (ERBI) was used for the neurological assessment of the patients. The plasma bio-ADM level was analyzed in association with 6-month all-cause mortality as well as a composite outcome of all-cause mortality, unscheduled re-hospitalization, or transfer to a long-term care facility in a vegetative or minimally conscious state. Bio-ADM levels significantly increased in patients with ischemic stroke who died compared to surviving patients (40.4 pg/ml vs. 23.8 pg/ml, p < 0.001) or in those with composite outcomes compared to those with no events (36.9 pg/ml vs. 23.5 pg/ml, p < 0.001). Six-month all-cause mortality was higher in all patients with bio-ADM levels > 70 pg/ml (HR 4.83 [CI 2.28-10.2]). Patients with bio-ADM levels > 70 pg/ml also had higher rates of 6-month composite outcomes (HR 3.82 [CI 2.08-7.01]). Bio-ADM was an independent predictor of all-cause mortality and 6-month composite outcomes after adjusting for age, gender, and ERBI (adjusted OR 1.5; 95% CI 1.0-2.1; p = 0.047 and adjusted OR 1.48; 95% CI 1.1-2.0; p = 0.01, respectively). Bio-ADM may be a suitable novel biomarker to assess the outcomes of patients in rehabilitation after acute stroke. Elevated bio-ADM concentrations may have prognostic value for fatal and nonfatal events in patients with ischemic stroke during early rehabilitation.

Circulating dipeptidyl peptidase 3 and bio-adrenomedullin levels are associated with impaired outcomes in critically ill COVID-19 patients: a prospective international multicentre study.

Introduction: Dipeptidyl peptidase-3 (DPP3) is a protease involved in the degradation of several cardiovascular mediators. Adrenomedullin (bio-ADM) is a peptide essential for regulation of endothelial barrier function. In different shock-pathologies, both biomarkers are associated with disease severity, organ dysfunction and mortality. Associations with outcome in critically ill COVID-19 patients are unknown. The objectives of the present study were to investigate associations of bio-ADM and "circulating DPP3" (cDPP3) with short-term outcome in critically ill COVID-19 patients (n=80). Methods: A multicentre prospective cohort study was performed. The primary end-point was 28-day mortality. Secondary end-points included different severities of acute kidney injury (AKI). Results: cDPP3 levels were mainly associated with 28-day mortality; Area under the receiver operating characteristics (AUROCs) of 0.69 (0.56-0.82, p=0.023), 0.77 (0.64-0.90, p<0.001) and 0.81 (0.65-0.96, p<0.001) at admission, day 3 and day 7, respectively. In contrast, bio-ADM levels were mainly associated with AKI, with AUROCs of 0.64 (0.51-0.77, p=0.048), 0.75 (0.64-0.86, p<0.001) and 0.83 (0.74-0.93, p<0.001) for day 1, 3 and 7, respectively. Interestingly, patients with high levels of both cDPP3 and bio-ADM at day 7 had an additionally increased risk of 28-day mortality (hazard ratio 11.8; 95% CI 2.5-55.3, p<0.001). Conclusions: cDPP3 and bio-ADM responses were associated with short-term mortality and AKI in critically ill COVID-19 patients, respectively. These findings suggest that treatment with specific antibodies modulating cDPP3 or bio-ADM-related pathways may improve outcome of COVID-19.

Proenkephalin A as a marker for glomerular filtration rate in critically ill children: validation against gold standard iohexol GFR measurements.

OBJECTIVES: Accurate determination of glomerular filtration rate (GFR) is important. Several endogenous biomarkers exist for estimating GFR, yet, they have limited accuracy, especially in the paediatric population. Proenkephalin A 119-159 (PENK) is a novel and promising GFR marker, but its relation with age in children remains unknown. Also, the value of PENK has never been validated against measured GFR (mGFR) in children when compared to traditional GFR markers including serum creatinine (SCr), SCr-based estimated GFR (eGFR) and cystatin C (cysC). METHODS: Critically ill children and term-born neonates were included in this single-centre, prospective study. Iohexol-based mGFR, SCr, and cysC were determined in each patient. eGFR was calculated using the bedside Schwartz equation, incorporating SCr and height. Spearman correlation coefficients were calculated to determine the correlation between mGFR and PENK, SCr, cysC and eGFR. RESULTS: For 97 patients (56 children and 41 neonates), mGFR, SCr, cysC and PENK levels were available. PENK levels were higher in young children and decreased to adult PENK reference values around two years of age. PENK levels were highly correlated with mGFR (ρ=-0.88, p<0.001), and similar to mGFR-eGFR correlation (ρ=-0.87, p<0.001). For cysC and SCr the correlation with mGFR was lower (ρ=-0.77 and ρ=-0.46, respectively. Both p<0.001). CONCLUSIONS: The correlation of PENK with mGFR was as good as SCr-based eGFR-mGFR correlation. To determine the added value of PENK in paediatric clinical care and prior to implementation, PENK reference values are needed and the development and validation of a paediatric PENK-based eGFR equation is necessary.

Utility of Measuring Circulating Bio-Adrenomedullin and Proenkephalin for 30-Day Mortality Risk Prediction in Patients with COVID-19 and Non-COVID-19 Interstitial Pneumonia in the Emergency Department.

Background and Objectives: In order to accelerate the risk stratification of patients referred to the Emergency Department (ED) with interstitial pneumonia, it could be useful to provide new and effective laboratory tests for use. The aim of our study was to evaluate the prognostic role of two biomarkers, bio-adrenomedullin (Bio-ADM) and proenkephalin (penKid), in patients with interstitial pneumonia (IP) at ED admission. Materials and Methods: In 153 consecutive patients with IP, both from COVID-19 or non-COVID-19 etiology, we measured, in a prospective observational manner, penKid and Bio-ADM at ED admission and after 24 h. In order to evaluate patient outcomes, 30-day follow-ups were also performed. The endpoints were 24 h, 10-day, and 30-day mortality. Results: Both biomarkers were shown to be good predictors of adverse events at 30 days, with Bio-ADM outperforming penKid. Bio-ADM was linked with 24 h and 10-day patient mortality. Moreover, PenKid was related to parameters defining worsening kidney function. Conclusions: Both in patients with COVID-19 or non-COVID-19 interstitial pneumonia at ED admission, Bio-ADM and penKid were good predictors of patient mortality. To evaluate these two biomarkers could be considered to be useful during the first evaluation in the ED when integrated with clinical scores.

Effects of enrichment strategies on outcome of adrecizumab treatment in septic shock: Post-hoc analyses of the phase II adrenomedullin and outcome in septic shock 2 trial.

Purpose: Adrecizumab, a non-neutralizing antibody of adrenomedullin (ADM) was recently investigated regarding its potential to restore endothelial barrier function in septic shock patients with high plasma ADM levels. Circulating dipeptidyl peptidase 3 (cDPP3), a protease involved in the degradation of several cardiovascular mediators, represents another biological pathway strongly associated with outcome in septic shock, although unrelated to ADM. Therefore, the prognosis of patients with elevated cDPP3 may not be influenced by Adrecizumab. Also, time until initiation of treatment may influence efficacy. Objective: To evaluate effects of cDPP3-based enrichment on treatment efficacy of Adrecizumab. Materials and Methods: Post-hoc analysis of AdrenOSS-2, a phase-II, double-blind, randomized, placebo-controlled biomarker-guided trial of Adrecizumab. Results: Compared to the total study cohort [HR for 28-day mortality of 0.84 (95% CI 0.53;1.31), p = 0.439], therapeutic benefit of Adrecizumab tended to be more pronounced in the subgroup of 249 patients with low cDPP3 (<50 ng/mL); [HR of 0.61 (95% CI 0.34;1.08), p = 0.085]. Median duration to study drug infusion was 8.5 h. In the subgroup of 129 patients with cDPP3 <50 ng/mL and an early start of treatment (<8.5 h after septic shock diagnosis) HR for 28-day mortality vs. placebo was 0.49 (95% CI 0.21-1.18), p = 0.105. In multivariate interaction analyses corrected for baseline disease severity, both cDPP3, as well as the cDPP3 * treatment interaction term were associated with a reduced HR for 28-day mortality in the Adrecizumab treated group; p = 0.015 for cDPP3 in univariate analysis, p = 0.025 for the interaction term between cDPP3 and treatment group. In contrast, treatment timing was not significantly associated with 28-day mortality in multivariate interaction analyses. Discussion: In septic shock patients with high ADM levels, a further post-hoc enrichment strategy based on cDPP3 may indicate (with all the caveats to be considered for post-hoc subgroup analyses) that therapeutic efficacy is most pronounced in patients with lower cDPP3 levels.

Proenkephalin A 119-159 predicts early and successful liberation from renal replacement therapy in critically ill patients with acute kidney injury: a post hoc analysis of the ELAIN trial.

BACKGROUND: Renal replacement therapy (RRT) remains the key rescue therapy for critically ill patients with severe acute kidney injury (AKI). However, there are currently no tools available to predict successful liberation from RRT. Biomarkers may allow for risk stratification and individualization of treatment strategies. Proenkephalin A 119-159 (penKid) has been suggested as a promising marker of kidney function in the context of AKI, but has not yet been evaluated for RRT liberation in critically ill patients with AKI. METHODS: This post hoc analysis included 210 patients from the randomized clinical ELAIN trial and penKid levels were measured in the blood of these patients. Competing risk time-to-event analyses were performed for pre-RRT penKid at initiation of RRT and in a landmark analysis at day 3 after initiation of RRT. Competing risk endpoints were successful liberation from RRT or death without prior liberation from RRT. RESULTS: Low pre-RRT penKid levels (penKid ≤ 89 pmol/l) at RRT initiation were associated with early and successful liberation from RRT compared to patients with high pre-RRT penKid levels (subdistribution hazard ratio (sHR) 1.83, 95%CI 1.26-2.67, p = 0.002, estimated 28d-cumulative incidence function (28d-CIF) of successful liberation from RRT 61% vs. 45%, p = 0.022). This association persisted in the landmark analysis on day 3 of RRT (sHR 1.78, 95%CI 1.17-2.71, p = 0.007, 28d-CIF of successful liberation from RRT 67% vs. 47%, p = 0.018). For both time points, no difference in the competing event of death was detected. CONCLUSIONS: In critically ill patients with RRT-dependent AKI, plasma penKid appears to be a useful biomarker for the prediction of shorter duration and successful liberation from RRT and may allow an individualized approach to guide strategies of RRT liberation in critically ill patients with RRT-dependent AKI. TRIAL REGISTRATION: The ELAIN trial was prospectively registered at the German Clinical Trial Registry (Identifier: DRKS00004367) on 28th of May 2013.

Growth hormone concentration and risk of all-cause and cardiovascular mortality: The REasons for Geographic And Racial Disparities in Stroke (REGARDS) study.

BACKGROUND AND AIMS: Identifying individuals at elevated risk for mortality, especially from cardiovascular disease, may help guide testing and treatment. Risk factors for mortality differ by sex and race. We investigated the association of growth hormone (GH) with all-cause and cardiovascular mortality in a racially diverse cohort in the United States. METHODS: Among an age, sex and race stratified subgroup of 1046 Black and White participants from the REasons for Geographic And Racial Disparities in Stroke (REGARDS) study, 881 had GH available; values were log2 transformed. Associations with all-cause and cardiovascular mortality were assessed in the whole subgroup, and by sex and race, using multivariable Cox-proportional hazard models and C-index. RESULTS: The mean age was 67.4 years, 51.1% were women, and 50.2% were Black participants. The median GH was 280 (interquartile range 79-838) ng/L. There were 237 deaths and 74 cardiovascular deaths over a mean of 8.0 years. In multivariable Cox analysis, GH was associated with higher risk of all-cause mortality per doubling (hazard ratio [HR] 1.17, 95% confidence interval [CI] 1.09-1.25) and cardiovascular mortality (HR 1.21, 95% CI 1.06-1.37). The association did not differ by sex or race (interaction p > 0.05). The addition of GH to a model of clinical variables significantly improved the C-index compared to clinical model alone for all-cause and cardiovascular death. CONCLUSIONS: Higher fasting GH was associated with higher risk of all-cause and cardiovascular mortality and improved risk prediction, regardless of sex or race.

RNF4~RGMb~BMP6 axis required for osteogenic differentiation and cancer cell survival.

Molecular understanding of osteogenic differentiation (OD) of human bone marrow-derived mesenchymal stem cells (hBMSCs) is important for regenerative medicine and has direct implications for cancer. We report that the RNF4 ubiquitin ligase is essential for OD of hBMSCs, and that RNF4-deficient hBMSCs remain as stalled progenitors. Remarkably, incubation of RNF4-deficient hBMSCs in conditioned media of differentiating hBMSCs restored OD. Transcriptional analysis of RNF4-dependent gene signatures identified two secreted factors that act downstream of RNF4 promoting OD: (1) BMP6 and (2) the BMP6 co-receptor, RGMb (Dragon). Indeed, knockdown of either RGMb or BMP6 in hBMSCs halted OD, while only the combined co-addition of purified RGMb and BMP6 proteins to RNF4-deficient hBMSCs fully restored OD. Moreover, we found that the RNF4-RGMb-BMP6 axis is essential for survival and tumorigenicity of osteosarcoma and therapy-resistant melanoma cells. Importantly, patient-derived sarcomas such as osteosarcoma, Ewing sarcoma, liposarcomas, and leiomyosarcomas exhibit high levels of RNF4 and BMP6, which are associated with reduced patient survival. Overall, we discovered that the RNF4~BMP6~RGMb axis is required for both OD and tumorigenesis.

Associations of Plasma Bioactive Adrenomedullin Levels with Cardiovascular Risk Factors in BRCA1/2 Mutation Carriers.

Background Cardiovascular disease (CVD) is an important cause of morbidity and mortality in breast cancer survivors. Effective screening modalities to identify CVD risk are lacking in this population. Adrenomedullin (ADM) has been suggested as a biomarker for subclinical cardiac dysfunction in the general population. Levels of ADM have been proven to be responsive to lifestyle changes that lead to improved cardiovascular health. As BRCA1/2 mutation carriers are deemed to be at an increased risk for CVD, the aim of this study was to examine plasma ADM levels in a cohort of BRCA mutation carriers and to assess their association with cardiovascular risk factors. Methods Plasma ADM concentrations were measured in 292 female BRCA1/2 mutation carriers with and without a history of breast cancer. Subjects were classified into high versus low ADM levels based on the median ADM level in the entire cohort (13.8 pg/mL). Logistic regression models were used to estimate the odds ratios (OR) of having elevated ADM levels by several cardiovascular risk factors. Results Of all women (median age: 43 years), 57.5% had a previous diagnosis of breast cancer. The median time between diagnosis and study entry was three years (range: 0 - 32 years). Women presenting with metabolic syndrome had 22-fold increased odds of having elevated ADM levels (p < 0.001). Elevated ADM levels were associated with lower cardiorespiratory fitness (OR = 0.88, p < 0.001) and several parameters of obesity (p < 0.001). ADM levels were higher in women who have ever smoked (OR = 1.72, p = 0.02). ADM levels were not associated with a previous diagnosis of breast cancer (p = 0.28). Conclusions This is the first study in BRCA mutation carriers that has linked circulating ADM levels to traditional cardiovascular risk factors. The long-term clinical implications of these findings are yet to be determined.

Bioactive adrenomedullin for assessment of venous congestion in heart failure.

AIMS: Bioactive adrenomedullin (bio-ADM) is a vascular-derived peptide hormone that has emerged as a promising biomarker for assessment of congestion in decompensated heart failure (HF). We aimed to evaluate diagnostic and prognostic performance of bio-ADM for HF in comparison to amino-terminal pro-B-type natriuretic peptide (NT-proBNP), with decision thresholds derived from invasive haemodynamic and population-based studies. METHODS AND RESULTS: Normal reference ranges for bio-ADM were derived from a community-based cohort (n = 5060). Correlations with haemodynamic data were explored in a cohort of HF patients undergoing right heart catheterization (n = 346). Mortality and decision cutoffs for bio-ADM was explored in a cohort of patients presenting in the ER with acute dyspnoea (n = 1534), including patients with decompensated HF (n = 570). The normal reference range was 8-39 pg/mL. The area under the receiver operating characteristic curve (AUROC) for discrimination of elevated mean right atrial pressure (mRAP) and pulmonary arterial wedge pressure (PAWP) was 0.74 (95% CI = 0.67-0.79) and 0.70 (95% CI = 0.64-0.75), respectively, with optimal bio-ADM decision cutoff of 39 pg/mL, concordant with cubic spline analyses. NT-proBNP discriminated PAWP slightly better than mRAP (AUROC 0.73 [95% CI = 0.68-0.79] and 0.68 [95% CI = 0.61-0.75]). Bio-ADM correlated with (mRAP, r = 0.55) while NT-proBNP correlated with PAWP. Finally, a bio-ADM decision cutoff of 39 pg/mL associated with 30 and 90 day mortality and conferred a two-fold increased odds of HF diagnosis, independently from NT-proBNP. CONCLUSIONS: Bio-ADM tracks with mRAP and associates with measures of systemic congestion and with mortality in decompensated HF independently from NT-proBNP. Our findings support utility of bio-ADM as a biomarker of systemic venous congestion in HF and nominate a decision threshold.

USP28 enables oncogenic transformation of respiratory cells, and its inhibition potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K.

Oncogenic transformation of lung epithelial cells is a multistep process, frequently starting with the inactivation of tumour suppressors and subsequent development of activating mutations in proto-oncogenes, such as members of the PI3K or MAPK families. Cells undergoing transformation have to adjust to changes, including altered metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors. Here, we report that the ubiquitin carboxyl-terminal hydrolase 28 (USP28) enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes such as c-JUN, c-MYC, NOTCH and ∆NP63 at early stages of malignant transformation. USP28 levels are increased in cancer compared with in normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small-molecule inhibitor resets the proteome of transformed cells towards a 'premalignant' state, and its inhibition synergizes with clinically established compounds used to target EGFRL858R -, BRAFV600E - or PI3KH1047R -driven tumour cells. Targeting USP28 protein abundance at an early stage via inhibition of its activity is therefore a feasible strategy for the treatment of early-stage lung tumours, and the observed synergism with current standard-of-care inhibitors holds the potential for improved targeting of established tumours.

Pseudomonas aeruginosa post-translational responses to elevated c-di-GMP levels.

C-di-GMP signaling can directly influence bacterial behavior by affecting the functionality of c-di-GMP-binding proteins. In addition, c-di-GMP can exert a global effect on gene transcription or translation, for example, via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c-di-GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c-di-GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c-di-GMP under growth conditions otherwise characterized by low c-di-GMP levels caused a switch to a non-motile, auto-aggregative P. aeruginosa phenotype. This phenotypic switch became apparent before any c-di-GMP-dependent role on transcription, translation, or protein abundance was observed. Our results suggest that rising global c-di-GMP pools first affects the motility phenotype of P. aeruginosa by altering protein functionality and only then global gene transcription.

PTEN mutant non-small cell lung cancer require ATM to suppress pro-apoptotic signalling and evade radiotherapy.

BACKGROUND: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy. RESULTS: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model. CONCLUSION: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.

Dipeptidyl peptidase-3 is associated with severity of liver disease and circulatory complications in patients with cirrhosis.

BACKGROUND: Patients with cirrhosis suffer from a complex multiorgan disturbance and their prognosis is influenced by the development of portal hypertension and systemic circulatory dysfunction. Although non-invasive techniques such as transient elastography aid in early detection, there is an unmet need for reliable markers of these clinically significant complications. METHODS: We conducted an exploratory single-center study investigating dipeptidyl peptidase-3 (DPP3) concentrations in various vascular beds in a cohort of 48 patients with cirrhosis and 16 healthy controls. Liver vein catheterisation with sampling from femoral artery and femoral, renal and hepatic veins as well as measurement of hepatic pressure and liver function via indocyanine green and galactose elimination tests were performed. RESULTS: DPP3 concentrations were higher in cirrhotic patients compared to controls (12.6 vs. 7.4 ng/mL, p = 0.006) and increased according to the severity of cirrhosis. DPP3 associated with MELD-Na score, Child class, indocyanine green clearance, increased DPP3 with the increased hepatic venous pressure gradient (p = 0.015) as well as increased heart rate and reduced systemic vascular resistance. DPP3 concentrations predicted the presence of clinically significant portal hypertension in cirrhotic patients (AUROC 0.78, 95% CI 0.65-0.9). CONCLUSION: DPP3 is a promising marker for portal hypertension and systemic hemodynamic changes in cirrhosis.