Whole Transcriptome Analysis Reveals Heterogeneity in B Cell Memory Populations in Patients With Juvenile Idiopathic Arthritis-Associated Uveitis.

Purpose: Patients with juvenile idiopathic arthritis (JIA) are prone to developing chronic anterior uveitis (JIA-U+). Although several risk factors for JIA-U+ have been identified, the underlying etiology is poorly understood. Histopathological studies demonstrate B cell infiltrates in eye tissues of patients with JIA-U+. Methods: We performed transcriptome profiling of peripheral blood CD19-positive B cells taken from 14 cases with JIA-U+, 13 JIA cases without uveitis (JIA-U-), and five healthy controls. Deconvolution-based estimation was used to determine the immune cell fractions for each sample. Results: Deconvolution results revealed that naive B cells made up on average 71% of the CD19-positive cell fractions analyzed. Differential expression analysis identified 614 differentially expressed genes (DEGs) between the groups at nominal significance and six genes at a false discovery rate of 5% (FDR < 0.05). Head-to-head comparison of all JIA-U- versus JIA-U+ revealed no DEGs in the CD19+ B cell pool (FDR < 0.05). However, principal component analysis based on a panel of key genes for B cell subsets revealed that JIA-U+ cases bifurcate into distinct clusters, characterized by markedly disparate expression for genes associated with specific memory B cell populations. CIBERSORT analysis of the overall transcriptome of the new uveitis cluster identified an increased proportion of memory B cells. Conclusion: These data show that JIA-U- and JIA-U+ have a globally similar transcriptome considering the global peripheral CD19-positive B cell pool. However, heterogeneity in B cell memory genes among cases with uveitis suggests a role for specific memory B cell subsets in the etiology of JIA-U+.

cDC2 and plasmacytoid dendritic cells diminish from tissues of patients with non-Hodgkin orbital lymphoma and idiopathic orbital inflammation.

Non-Hodgkin orbital lymphoma (NHOL) and idiopathic orbital inflammation (IOI) are common orbital conditions with largely unknown pathophysiology. To investigate the immune cell composition of these diseases, we performed standardized 29 parameter flow cytometry phenotyping in peripheral blood mononuclear cells of 18 NHOL patients, 21 IOI patients, and 41 unaffected controls. Automatic gating by FlowSOM revealed decreased abundance of meta-clusters containing dendritic cells in patients, which we confirmed by manual gating. A decreased percentage of (HLA-DR+ CD303+ CD123+ ) plasmacytoid dendritic cells (pDC) in the circulation of IOI patients and decreased (HLA-DR+ CD11c+ CD1c+ ) conventional dendritic cells (cDC) type-2 for IOI patients were replicated in an independent cohort of patients and controls. Meta-analysis of both cohorts demonstrated that pDCs are also decreased in blood of NHOL patients and highlighted that the decrease in blood cDC type-2 was specific for IOI patients compared to NHOL or controls. Deconvolution-based estimation of immune cells in transcriptomic data of 48 orbital biopsies revealed a decrease in the abundance of pDC and cDC populations within the orbital microenvironment of IOI patients. Collectively, these data suggest a previously underappreciated role for dendritic cells in orbital disorders.

A pan-inflammatory microRNA-cluster is associated with orbital non-Hodgkin lymphoma and idiopathic orbital inflammation.

Non-Hodgkin orbital lymphoma (NHOL) and idiopathic orbital inflammation (IOI) are common orbital conditions with largely unknown pathophysiology that can be difficult to diagnose. In this study we aim to identify serum miRNAs associated with NHOL and IOI. We performed OpenArray® miRNA profiling in 33 patients and controls. Differentially expressed miRNAs were technically validated across technology platforms and replicated in an additional cohort of 32 patients and controls. We identified and independently validated a serum miRNA profile of NHOL that was remarkably similar to IOI and characterized by an increased expression of a cluster of eight miRNAs. Pathway enrichment analysis indicated that the miRNA-cluster is associated with immune-mediated pathways, which we supported by demonstrating the elevated expression of this cluster in serum of patients with other inflammatory conditions. The cluster contained miR-148a, a key driver of B-cell tolerance, and miR-365 that correlated with serum IgG and IgM concentrations. In addition, miR-29a and miR-223 were associated with blood lymphocyte and neutrophil populations, respectively. NHOL and IOI are characterized by an abnormal serum miRNA-cluster associated with immune pathway activation and linked to B cell and neutrophil dysfunction.

53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers.

Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors.

BACKGROUND: The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. RESULTS: Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. CONCLUSIONS: These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.