The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).

INTRODUCTION AND OBJECTIVES:Psychosocial stressors related to the coronavirus-19 (COVID-19) pandemic increased alcohol consumption. The effect on patients with alcohol-related liver diseases remains unclear.MATERIALS AND METHODS:Hospitalizations at a tertiary care center due to alcohol-related liver disease from March 1 through August 31 in 2019 (pre-pandemic cohort) and 2020 (pandemic cohort) were reviewed retrospectively. Differences in patient demographics, disease features, and outcomes were estimated in patients with alcoholic hepatitis utilizing T-tests, Mann-Whitney tests, Chi-square and Fisher Exact Tests and Anova models and logistic regression models in patients with alcoholic cirrhosis.RESULTS:146 patients with alcoholic hepatitis and 305 patients with alcoholic cirrhosis were admitted during the pandemic compared to 75 and 396 in the pre-pandemic cohort. Despite similar median Maddrey Scores (41.20 vs. 37.45, p=0.57), patients were 25% less likely to receive steroids during the pandemic. Patients with alcoholic hepatitis admitted during the pandemic were more likely to have hepatic encephalopathy (0.13; 95% CI:0.01, 0.25), variceal hemorrhage (0.14; 95% CI:0.04, 0.25), require oxygen (0.11; 95% CI:0.01, 0.21), vasopressors (OR:3.49; 95% CI:1.27, 12.01) and hemodialysis (OR:3.70; 95% CI:1.22, 15.13). On average, patients with alcoholic cirrhosis had MELD-Na scores 3.77 points higher (95% CI:1.05, 13.46) as compared to the pre-pandemic and had higher odds of experiencing hepatic encephalopathy (OR:1.34; 95% CI:1.04, 1.73), spontaneous bacterial peritonitis (OR:1.88; 95% CI:1.03, 3.43), ascites (OR:1.40, 95% CI:1.10, 1.79), vasopressors (OR:1.68, 95% CI:1.14, 2.46) or inpatient mortality (OR:2.00, 95% CI:1.33, 2.99) than the pre-pandemic.CONCLUSIONS:Patients with alcohol-related liver disease experienced worse outcomes during the pandemic.

Background: Coxiella burnetii is the causative agent of Q fever, a human disease that can be acquired from livestock. Diseases caused by this organism have caused great losses in livestock and human health. No vaccine is approved for use in the United States, and formalin-inactivated whole-cell vaccines pose a significant manufacturing risk for biocontainment. A subunit vaccine using recombinant peptides from C. burnetii would be safer and less resource-intensive to produce. This study used reverse vaccinology to expand our prediction sets of T-cell epitopes for the major histocompatibility complex (MHC) Class I and II alleles of cattle, sheep, and goats. Thereafter, the present results were compared with those from our previous prediction sets for mice and humans. Results: Small ruminant breed representation for the United States was ensured by querying whole genome sequences on the National Center for Biotechnology Information database. Consequently, twenty-two sheep MHC Class I, seventeen goat MHC Class I, and one goat MHC Class II alleles were added to the analyses, resulting in a total of fifty-six sheep MHC Class I, eighteen goat MHC Class II, and twenty-seven goat MHC Class II alleles. Predicted interactions of C. burnetii proteome-derived peptides with each MHC allele were categorized as strong, weak, or non-binding based on bioinformatic scores. Interspecies comparisons resulted in 256 peptides of interest for MHC Class II presentation and 766 peptides of interest for MHC Class I presentation. Of these, 51 peptides were predicted to bind with both classes of MHC alleles, of which 33 were newly identified. Conclusion: The high scoring T-cell epitope predictions identified in this study provide grounds for prioritizing subunit candidates to further develop a safe and perhaps broadly effective C. burnetii vaccine.

We present the case of a 61-year-old woman who presented with acutely worsening right upper quadrant pain and was found to be in acute liver failure with Klebsiella pneumoniae bacteremia. Despite aggressive intensive care management, the patient ultimately died of refractory shock attributed to sepsis and fulminant liver failure. On autopsy, she was found unexpectedly to have diffuse intrahepatic cholangiocarcinoma with metastases to regional lymph nodes and intravascular spread to the lungs. The case highlights a rare instance where intrahepatic cholangiocarcinoma presents with acute liver failure and discusses key intensive care management principles of this clinical syndrome.

Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N 1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.

Objective The coronavirus disease 2019 pandemic led to changes in individuals' behaviors and healthcare delivery. We examined the impact of these changes on the rates and clinical course of acute pancreatitis (AP). Methods Hospitalizations for AP from March 1 through August 31 in 2019 (baseline group) and the same period in 2020 (pandemic group) were retrospectively reviewed. Univariate and multivariate analyses were used for demographics and outcomes. Results Two hundred eighty subjects (315 admissions) were identified in 2019 and 237 subjects (264 admissions) in 2020. Subjects in the pandemic group were more likely to have systemic inflammatory response syndrome (40% vs 25%, P < 0.01), pancreatic necrosis (14% vs 10%, P = 0.03), and persistent organ failure (17% vs 9%, P = 0.01) compared with prepandemic. There was no difference in etiology of AP. A multivariable model indicates that increased comorbidities, prior pancreatitis, pancreatic necrosis, and prescription of opiates at discharge were associated with 30-day readmissions during the pandemic. Conclusions Fewer patients were admitted for AP during the pandemic, suggesting that patients with milder symptoms avoided hospital interaction. Practices followed during the pandemic, especially avoidance of hospitalization and improved efficiency of hospital management, may reduce the burden of pancreatitis care in the future.

This Perspective discusses the published data and recent developments in the research area of bromodomains in parasitic protozoa. Further work is needed to evaluate the tractability of this target class in the context of infectious diseases and launch drug discovery campaigns to identify and develop antiparasite drugs that can offer differentiated mechanisms of action.

INTRODUCTION:Delayed graft function (DGF) following renal transplantation is a manifestation of acute kidney injury (AKI) leading to poor long-term outcome. Current treatments have limited effectiveness in preventing DGF. Interleukin-18 (IL18), a biomarker of AKI, induces interferon-γ expression and immune activation. GSK1070806, an anti-IL18 monoclonal antibody, neutralizes activated (mature) IL18 released from damaged cells following inflammasome activation. This phase IIa, single-arm trial assessed the effect of a single dose of GSK1070806 on DGF occurrence post donation after circulatory death (DCD) kidney transplantation.METHODS:The 3 mg/kg intravenous dose was selected based on prior studies and physiologically based pharmacokinetic (PBPK) modeling, indicating the high likelihood of a rapid and high level of IL18 target engagement when administered prior to kidney allograft reperfusion. Utilization of a Bayesian sequential design with a background standard-of-care DGF rate of 50% based on literature, and confirmed via extensive registry data analyses, enabled a statistical efficacy assessment with a minimal sample size. The primary endpoint was DGF frequency, defined as dialysis requirement ≤7 days post transplantation (except for hyperkalemia). Secondary endpoints included safety, pharmacokinetics and pharmacodynamic biomarkers.RESULTS:GSK1070806 administration was associated with IL18-GSK1070806 complex detection and increased total serum IL18 levels due to IL18 half-life prolongation induced by GSK1070806 binding. Interferon-γ-induced chemokine levels declined or remained unchanged in most patients. Although the study was concluded prior to the Bayesian-defined stopping point, 4/7 enrolled patients (57%) had DGF, exceeding the 50% standard-of-care rate, and an additional two patients, although not reaching the protocol-defined DGF definition, demonstrated poor graft function. Six of seven patients experienced serious adverse events (SAEs), including two treatment-related SAEs.CONCLUSION:Overall, using a Bayesian design and extensive PBPK dose modeling with only a small sample size, it was deemed unlikely that GSK1070806 would be efficacious in preventing DGF in the enrolled DCD transplant population.TRIAL REGISTRATION:NCT02723786.

COVID-19 positive LT patients to date. Methods: We identified a cohort of LT recipients who received a positive COVID-19 test (COVID1) between 1/1/2020 and 3/30/2021. We evaluated mortality outcomes and predictive risk factors for death. We used univariate and multivariate models to analyze the variable-outcome associations. The primary outcome was in-hospital mortality. Survival analysis methods were used to estimate cumulative incidences of death and mechanical ventilation, and to calculate the associations of COVID-19 infection and demographic and clinical factors to mortality in LT patients. Results: 34 sites account for 6.3 million patients in the Enclave, of whom 2.1 million are COVID1. A total of 300 post-LT patients with COVID-19 were included in the analysis. The in-hospital mortality rate in LT COVID1 patients was approximately 7%. Hyperlipidemia, coronary artery disease (CAD) and chronic kidney disease (CKD) were common comorbidities in COVID1 LT recipients, but significantly more common in those who died. In univariate analysis, age, albumin level, CKD, CAD, hyperlipidemia, sepsis, acute kidney injury (AKI), prednisone use and mechanical ventilation were statistically significant in predicting death (p, 0.001). In multivariate analysis, only age, albumin level, prednisone use and mechanical ventilation remained statistically significant (p50.02). Conclusion: In this study of a US cohort of COVID1 LT recipients using the N3C database, we found that mortality rate in post-LT patients with COVID-19 infection is approximately 7%. In our final predictive model, age, mechanical ventilation, and low albumin level were associated with higher risk of death. In contrast, prednisone use was associated with reduced mortality. Those factors are not only predictive, but also potentially prognostic and thus may assist with the management of COVID1 LT patients.

Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1; also known as NSD2) is a SET domain-containing histone lysine methyltransferase. A chromosomal translocation occurs in 15-20% of multiple myeloma patients and is associated with increased production of WHSC1 and poor clinical prognosis. To define the substrate requirements of NSD2, we established a platform for the large-scale production of recombinant polynucleosomes, based on authentic human histone proteins, expressed in E. coli, and complexed with linearized DNA. A brief survey of methyltransferases whose substrate requirements are recorded in the literature yielded expected results, lending credence to the fitness of our approach. This platform was readily 'codified' with respect to both position and extent of methylation at histone 3 lysines 18 and 36 and led to the conclusion that the most readily discernible activity of NSD2 in contact with a nucleosome substrate is dimethylation of histone 3 lysine 36. We further explored reaction mechanism, and conclude a processive, rather than distributive mechanism best describes the interaction of NSD2 with intact nucleosome substrates. The methods developed feature scale and flexibility and are suited to thorough pharmaceutical-scale drug discovery campaigns.

Chronic kidney disease and associated comorbidities (diabetes, cardiovascular diseases) manifest with an accelerated ageing phenotype, leading ultimately to organ failure and renal replacement therapy. This process can be modulated by epigenetic and environmental factors which promote loss of physiological function and resilience to stress earlier, linking biological age with adverse outcomes post-transplantation including delayed graft function (DGF). The molecular features underpinning this have yet to be fully elucidated. We have determined a molecular signature for loss of resilience and impaired physiological function, via a synchronous genome, transcriptome and proteome snapshot, using human renal allografts as a source of healthy tissue as an in vivo model of ageing in humans. This comprises 42 specific transcripts, related through IFNγ signalling, which in allografts displaying clinically impaired physiological function (DGF) exhibited a greater magnitude of change in transcriptional amplitude and elevated expression of noncoding RNAs and pseudogenes, consistent with increased allostatic load. This was accompanied by increased DNA methylation within the promoter and intragenic regions of the DGF panel in preperfusion allografts with immediate graft function. Pathway analysis indicated that an inability to sufficiently resolve inflammatory responses was enabled by decreased resilience to stress and resulted in impaired physiological function in biologically older allografts. Cross-comparison with publically available data sets for renal pathologies identified significant transcriptional commonality for over 20 DGF transcripts. Our data are clinically relevant and important, as they provide a clear molecular signature for the burden of "wear and tear" within the kidney and thus age-related physiological capability and resilience.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix 9 (bHLH) transcription factor acting downstream of Notch and is required for the differentiation of sensory hair cells in the inner ear and the specification of secretory cells during the intestinal crypt cell regeneration. Motivated by the observations that the upregulation of Atoh1 gene expression, through genetic manipulation or pharmacological inhibition of Notch signaling (e.g. γ-secretase inhibitors, GSIs), induces ectopic hair cell growth in the cochlea of the inner ear and partially restores hearing after injuries in experimental models, we decided to identify small molecule modulators of the Notch-Atoh1 pathway, which could potentially regenerate hair cells. However, the lack of cellular models of the inner ear has precluded the screening and characterization of such modulators. Here we report using a colon cancer cell line LS-174T, which displays Notch inhibition-dependent Atoh1 expression as a surrogate cellular model to screen for inducers of Atoh1 expression. We designed an Atoh1 promoter-driven luciferase assay to screen a target-annotated library of ~6000 compounds. We further developed a medium throughput, real-time quantitative RT-PCR assay measuring the endogenous Atoh1 gene expression to confirm the hits and eliminate false positives from the reporter-based screen. This strategy allowed us to successfully recover GSIs of known chemotypes. This LS-174T cell-based assay directly measures Atoh1 gene expression induced through Notch-Hes1 inhibition, and therefore offers an opportunity to identify novel cellular modulators along the Notch-Atoh1 pathway.

Introduction: Orthotopic liver transplantation (OLT) can be associated with significant bleeding requiring multiple blood product transfusions. Rotational thromboelastometry (ROTEM) is a point-of-care device that has been used to monitor coagulation during OLT. Whether it reduces blood loss/transfusions during OLT remains controversial. Materials and Methods: We aim to compare ROTEM with conventional coagulation tests (aPTT, PT, INR, platelet count, fibrinogen) to guide transfusion of platelets, cryoprecip-itate, and fresh frozen plasma (FFP) during OLT over 3 years. Thirty-four patients who had transfusions guided by ROTEM were compared to 34 controls who received transfusions guided by conventional coagulation tests (CCT). Intraoperative blood loss, type/ amount of blood products transfused, and direct costs were compared between the two groups. Results: The ROTEM group had significantly less intra-operative blood loss (2.0 vs. 3.0 L, p = 0.04) and fresh frozen plasma (FFP) transfusion (4 units vs. 6.5 units, p = 0.015) compared to the CCT group (2.0L vs. 3.0L, p = 0.04). However, total number of patients transfused cryoprecipitate was increased in ROTEM (n = 25;73%) as compared to CCT (n = 19; 56%), p = 0.033. The direct cost of blood products plus testing was reduced in the ROTEM group ($113,142.89 vs. $127,814.77). Conclusion. In conclusion implementation of a ROTEM-guided transfusion algorithm resulted in a reduction in intra-operative blood loss, FFP transfusion and a decrease in direct cost during OLT. ROTEM is a useful and safe point of care device in OLT setting.

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communication and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediate a long-range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.

Background The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti‐inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl‐tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. Methods and Results Consistent with its ability to inhibit prolyl‐tRNA synthetase, halofuginone elicited a general control nonderepressible 2–dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l‐proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2–dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell–derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin‐1‐mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α‐dependent manner. Conclusions Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communication and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediate a long-range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.

Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.