The Impact of the HydroxyMethylCytosine epigenetic signature on DNA structure and function

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.

MALT1 Degradation with a Proteolysis-Targeting Chimera for the Treatment of Activated B-Cell Type Diffuse Large B-Cell Lymphoma

Introduction ABC-DLBCL is characterized by chronic activation of NFκB, which is dependent on the CARD11-BCL10-MALT1 (CBM) complex. MALT1 activates NFκB transcription factors via distinct protease and scaffold functions. Clinical allosteric MALT1 protease inhibitors are in development, however these agents partially preserve NFκB activation via MALT1 scaffold function. In addition, MALT1 protease is critical for regulatory T cell (Treg) function, and its inactivation is associated with increased inflammatory cytokine expression by T-effector cells. However, overt inflammation is not seen with MALT1 knockout. It is therefore possible that MALT1 protease inhibition with preserved scaffold function will lead to autoimmune toxicities. As an alternative strategy, we developed MALT1-directed proteolysis targeting chimera (PROTAC) compounds which consist of a MALT1-binding domain linked to an IMiD-based cereblon (CRBN) binding domain, which induce ubiquitination and proteasomal degradation of MALT1. Here we present preliminary studies of a lead compound, PS-II-115, to assess feasibility of this approach for ABC-DLBCL. Results To assess MALT1 protein degradation, OCI-Ly3 cells were treated with DMSO or PS-II-115 10 µM for 24 hours and immunoblots of cell lysates were performed. PS-II-115 induced degradation of MALT1 (degradation ±SEM 73.94 ±9.8% vs DMSO, N = 3). No significant degradation of CRBN neosubstrates IKZF1, IKZF3, or GSPT1 was seen. To assess effects downstream of MALT1, we treated OCI-Ly3 cells with PS-II-115 for 23.5 hours followed by stimulation with PMA/IO for 30 minutes, and immunoblots were performed. We found increased IκB, an inhibitor of NFκB which is degraded following MALT1 scaffold mediated IKK activation, in PS-II-115 treated cells (IkB 206% compared to DMSO), suggesting that MALT1 degradation inhibits MALT1 scaffold-mediated NFκB activation pathways. To measure MALT1 protease inhibition, we used the GloSensor split luciferase method, in which a chimeric protein is cleaved by MALT1 resulting in functional luciferase. Raji cells expressing this construct were treated with DMSO or PS-II-115 for 23.5 hours followed by stimulation with PMA/IO for 30 minutes, and GloSensor assay was performed. PS-II-115 induced dose-dependent inhibition of MALT1 protease activity compared to DMSO (IC 50 1.203 µM, 95% CI 0.374-4.230, N=4). To assess growth inhibition, ABC-DLBCL and GCB-DLBCL cell lines were treated with DMSO or PS-II-115 for 96 hours, and CellTiter Glo assay was performed. GCB-DLBCLs are not dependent on chronic NFκB activation and were expected to be resistant to PS-II-115. PS-II-115 induced growth inhibition of ABC-DLBCL cell lines (OCI-Ly3 IC 50 2.54 µM, OCI-Ly10 IC 50 9.65 µM, TMD8 IC 50 1.87 µM, N=4) more potently than GCB-DLBCL cell lines (OCI-Ly1 IC 50 34.6 µM, OCI-Ly7 IC 50 >50 µM, N=4), suggesting the effect of PS-II-115 is based on MALT1 rather than off-target effects. We then assessed the impact of PS-II-115 on T cell phenotypes. Human T cells were isolated from healthy donors' peripheral blood. Cells were stimulated with CD3/CD28 beads and treated with DMSO, PS-II-115, or an allosteric MALT1 inhibitor for 48 or 96 hours and flow cytometry was performed. We found a significant decrease in CD4+ FOXP3+ CD25+ CD127- Tregs among cells treated with PS-II-115 1 µM or allosteric inhibitor compared to DMSO at 48 hours (PS-II-115 3.89%, allo inh 3.85%, DMSO 9.72% Tregs among CD4+ T cells, P<0.005). We also found decreased pro-inflammatory CD4+ CD45RA+ CCR7- terminal effector T cells expressing RA (TEMRA) among cells treated with PS-II-115 20 µM for 96 hours compared to DMSO (35.67% vs 56.97% TEMRA among CD4+ T cells, p<0.05). The allosteric inhibitor did not affect TEMRA cells. Conclusion We describe a MALT1-directed PROTAC which induces (1) degradation of MALT1 with preservation of other IMiD-associated CRBN neosubstrates, (2) blockade of MALT1 scaffold mediated NFκB activation pathways, (3) MALT1 protease inhibition, (4) selective growth inhibition of ABC-DLBCL cell lines, (5) decrease in Tregs, and (6) decrease in CD4+ TEMRA cells. These results provide proof of principle that MALT1 degradation using a PROTAC compound is a feasible strategy against ABC-DLBCL, and may have distinct effects on T-effector phenotypes compared to MALT1 protease inhibition. Further studies are needed to elucidate immunologic effects of MALT1 degradation and to validate findings in vivo. Disclosures Fontan: Janssen Pharmaceuticals: Current Employment. Gray: Gatekeeper: Consultancy, Current holder of individual stocks in a privately-held company; Syros: Consultancy, Current holder of individual stocks in a privately-held company; Petra: Consultancy, Current holder of individual stocks in a privately-held company; C4: Consultancy, Current holder of individual stocks in a privately-held company; Allorion: Consultancy, Current holder of individual stocks in a privately-held company; Jengu: Consultancy, Current holder of individual stocks in a privately-held company; B2S: Consultancy, Current holder of individual stocks in a privately-held company; Inception: Consultancy, Current holder of individual stocks in a privately-held company; EcoCys: Consultancy, Current holder of individual stocks in a privately-held company; Soltego: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Takeda: Research Funding; Astellas: Research Funding; Taiho: Research Funding; Janssen: Research Funding; Kinogen: Research Funding; Her2IIc: Research Funding; Deerfield: Research Funding; Sanofi: Research Funding. Melnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees.

Functional Evaluation and Mechanism Study of Sting Inhibitor H-151 in Diffuse Large B-Cell Lymphoma

Objective: Diffuse large B-cell lymphoma (DLBCL) is a subtype of non-Hodgkin's lymphoma with a higher incidence rate and is highly heterogeneous.The current first-line treatment effect is limited. After entering the relapse/refractory treatment, the second-line treatment lacks an efficient solution. Explore DLBCL Potential therapeutic targets are imperative. The cGAS/STING pathway can promote or inhibit tumorigenesis. The specific tumor type,genetic background,pathway activation level and microenvironment may determine the tumor's response to STING agonists or inhibitors. The disorder of cGAS/STING pathway function can lead to many diseases such as tumors and autoimmune diseases, but it has not been reported in DLBCL. This study found for the first time that STING is heterogeneously expressed in DLBCL and is related to tumor cell proliferation. The STING inhibitor H-151 can kill DLBCL tumor cells. At the same time, multi-omics methods are used to further screen the specific DLBCL population sensitive to H-151, provide a theoretical basis for the improvement of the prognostic model of DLBCL and the choice of targeted therapy drugs. Methods Immunofluorescence staining to detect the expression of cGAS/STING on tissue microarrays in patients with DLBCL; Confocal microscopy imaging technology for localization and quantitative analysis of intracellular cGAS/STING expression; Whole-exome sequencing analysis of mutant genes related to drug sensitivity and pathway;The combination of quantitative proteomics and transcriptome sequencing to compare the biological characteristics of cell lines in different response groups, analyze gene expression differences and related activation pathways;Western Blotting to detect the expression of related proteins;CCK8 and flow cytometry were used to detect cell proliferation and apoptosis;NOD-SCID mice and Hu-PBMC mice were used to construct DLBCL tumor-bearing mouse models to explore the effect of STING inhibitors in vivo. Results The cGAS/STING pathway-related proteins are expressed heterogeneously in real-world clinical DLBCL samples and DLBCL cell lines, and the expression of cGAS/STING pathway-related proteins is positively correlated with the proliferation level of DLBCL cell lines(Figure A). Based on this, we speculate the increased expression of cGAS/STING pathway-related proteins may potentially promote the proliferation of tumor cells. The STING inhibitor H-151 can inhibit the proliferation and induced apoptosis of some DLBCL tumor cells in a concentration-dependent manner.The IC50 of H-151 has a certain positive correlation with the expression of cGAS/STING pathway-related proteins, which suggests that the up-regulation of cGAS/STING pathway-related proteins may be potentially related to the resistance of DLBCL to H-151(Figure B).Whole transcriptome sequencing combined with single-cell transcriptome sequencing (scRNA-seq) analyzes potential subgroups related to H-151 sensitivity and related activation pathways(Figure C-E).. We also use whole exome sequencing to screen potential targets for assessing H-151 sensitivity We found that celllines with ITIH6, NPIPA5, NPY2R and TUBGCP3 mutations are more sensitive to the treatment of H-151, while cell lines with SH2B2 and LILRB5 mutations were less responsive to H-151,while the related gene mutations in the C-MYC pathway may be potentially related to the resistance of DLBCL cells to H-151, so as to screen potentially sensitive target populations for the effective use of H-151(Figure F). Conclusion This study found that the cGAS/STING pathway is heterogeneously expressed in DLBCL and is related to tumor cell proliferation, and the STING inhibitor H-151 has anti-DLBCL effects. The anti-tumor mechanism of STING inhibitor H-151 was further explored by using multi-omics methods, and the sensitivity evaluation system of H-151 was established.Thus providing new ideas for precise and efficient treatment of DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Conduction system pacing in heart failure patients

Direct conduction system pacing delivers more physiological cardiac activation and can potentially correct intraventricular conduction disturbances and restore normal activation pathways. Permanent pacing that maintains cardiac electromechanical synchrony is essential in heart failure and reduced left ventricular ejection fraction. Conduction system pacing has recently emerged as an alternative to right ventricular pacing and biventricular resynchronization therapy. In this article, we review conduction system pacing in heart failure patients.

Gene Expression Analysis in Four Dogs With Canine Pemphigus Clinical Subtypes Reveals B Cell Signatures and Immune Activation Pathways Similar to Human Disease

Pemphigus is a group of autoimmune-mediated mucocutaneous blistering diseases characterized by acantholysis. Pemphigus has also been recognized in dogs and shares similar clinical characteristics and variants with human pemphigus. While relationships between human and canine pemphigus have been reported, gene expression patterns across species have not been described in the literature. We sought to perform gene expression analysis of lesional skin tissue from four dogs with various forms of pemphigus to examine gene expression during spontaneous disease in dogs. We found increased T and B cell signatures in canine pemphigus lesions compared to controls, as well as significant upregulation of CCL3, CCL4, CXCL10, and CXCL8 (IL8), among other genes. Similar chemokine/cytokine expression patterns and immune infiltrates have been reported in humans, suggesting that these genes play a role in spontaneous disease. Direct comparison of our dataset to previously published human pemphigus datasets revealed five conserved differentially expressed genes: CD19, WIF1, CXCL10, CD86, and S100A12. Our data expands our understanding of pemphigus and facilitates identification of biomarkers for prediction of disease prognosis and treatment response, which may be useful for future veterinary and human clinical trials.

Time-Averaged Wavefront Analysis Demonstrates Preferential Pathways of Atrial Fibrillation, Predicting Pulmonary Vein Isolation Acute Response

Electrical activation during atrial fibrillation (AF) appears chaotic and disorganised, which impedes characterisation of the underlying substrate and treatment planning. While globally chaotic, there may be local preferential activation pathways that represent potential ablation targets. This study aimed to identify preferential activation pathways during AF and predict the acute ablation response when these are targeted by pulmonary vein isolation (PVI). In patients with persistent AF (n = 14), simultaneous biatrial contact mapping with basket catheters was performed pre-ablation and following each ablation strategy (PVI, roof, and mitral lines). Unipolar wavefront activation directions were averaged over 10 s to identify preferential activation pathways. Clinical cases were classified as responders or non-responders to PVI during the procedure. Clinical data were augmented with a virtual cohort of 100 models. In AF pre-ablation, pathways originated from the pulmonary vein (PV) antra in PVI responders (7/7) but not in PVI non-responders (6/6). We proposed a novel index that measured activation waves from the PV antra into the atrial body. This index was significantly higher in PVI responders than non-responders (clinical: 16.3 vs. 3.7%, p = 0.04; simulated: 21.1 vs. 14.1%, p = 0.02). Overall, this novel technique and proof of concept study demonstrated that preferential activation pathways exist during AF. Targeting patient-specific activation pathways that flowed from the PV antra to the left atrial body using PVI resulted in AF termination during the procedure. These PV activation flow pathways may correspond to the presence of drivers in the PV regions.

Complement Alternative and Mannose-Binding Lectin Pathway Activation Is Associated With COVID-19 Mortality

BackgroundThe SARS-CoV-2 infection triggers excessive immune response resulting in increased levels of pro-inflammatory cytokines, endothelial injury, and intravascular coagulopathy. The complement system (CS) activation participates to this hyperinflammatory response. However, it is still unclear which activation pathways (classical, alternative, or lectin pathway) pilots the effector mechanisms that contribute to critical illness. To better understand the immune correlates of disease severity, we performed an analysis of CS activation pathways and components in samples collected from COVID-19 patients hospitalized in Grenoble Alpes University Hospital between 1 and 30 April 2020 and of their relationship with the clinical outcomes.MethodsWe conducted a retrospective, single-center study cohort in 74 hospitalized patients with RT-PCR-proven COVID-19. The functional activities of classical, alternative, and mannose-binding lectin (MBL) pathways and the antigenic levels of the individual components C1q, C4, C3, C5, Factor B, and MBL were measured in patients’ samples during hospital admission. Hierarchical clustering with the Ward method was performed in order to identify clusters of patients with similar characteristics of complement markers. Age was included in the model. Then, the clusters were compared with the patient clinical features: rate of intensive care unit (ICU) admission, corticoid treatment, oxygen requirement, and mortality.ResultsFour clusters were identified according to complement parameters. Among them, two clusters revealed remarkable profiles: in one cluster (n = 15), patients exhibited activation of alternative and lectin pathways and low antigenic levels of MBL, C4, C3, Factor B, and C5 compared to all the other clusters; this cluster had the higher proportion of patients who died (27%) and required oxygen support (80%) or ICU care (53%). In contrast, the second cluster (n = 19) presented inflammatory profile with high classical pathway activity and antigenic levels of complement components; a low proportion of patients required ICU care (26%) and no patient died in this group.ConclusionThese findings argue in favor of prominent activation of the alternative and MBL complement pathways in severe COVID-19, but the spectrum of complement involvement seems to be heterogeneous requiring larger studies.

Targeted RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Temporal Arteries From Giant Cell Arteritis Cases Reveals Viral Signatures

Background and ObjectivesVaricella zoster virus (VZV) antigen has been detected in temporal arteries (TAs) of individuals with giant cell arteritis (GCA), the most common systemic vasculitis in older adults. Thus, we explored the contribution of VZV to GCA pathogenesis.MethodsFormalin-fixed, paraffin-embedded TA sections from biopsy-positive GCA participants with VZV antigen (GCA/VZV-positive; n = 20) and without (GCA/VZV-negative, n = 20) and from normal participants with VZV antigen (control/VZV-positive, n = 11) and without (control/VZV-negative, n = 20) were analyzed by targeted RNA sequencing of the whole human transcriptome (BioSpyder TempO-Seq). Ingenuity pathway analysis and R-computational program were used to identify differentially expressed genes and pathways between groups.ResultsCompared with control/VZV-negative TAs, GCA/VZV-negative and GCA/VZV-positive TAs were significantly enriched for human transcripts specific for pathways involved in viral infections, including viral entry, nuclear factor kappa B activation by viruses, and other pathogen-related immune activation pathways. Similarly, human gene sets supporting viral infection were found in control/VZV-positive TAs that showed no morphological signs of inflammation, suggesting that the enriched pathways were not nonspecific signatures of infiltrating immune cells. All GCA TAs and control/VZV-positive TAs showed enrichment of transcripts involved in vascular remodeling, including smooth muscle cell migration.DiscussionThe detection of viral and immune activation pathways in GCA TAs supports a role for virus infection in GCA pathogenesis. In addition, the detection of viral pathways in control/VZV-positive TAs, along with vascular remodeling pathways, suggests that these samples may represent early infection with progression to clinical disease, depending on host and other environmental factors.