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ObjectivesTo identify key disease pathways driving conventional dendritic cell (cDC) alterations in Systemic Sclerosis (SSc).MethodsTranscriptomic profiling was performed on peripheral blood CD1c+ cDCs (cDC2s) isolated from 12 healthy donors and 48 SSc patients with all major disease subtypes. Differential expression analysis comparing the different SSc subtypes and healthy donors was performed to uncover genes dysregulated in SSc. To identify biologically relevant pathways, a gene co-expression network was built using Weighted Gene Correlation Network Analysis. We validated the role of key transcriptional regulators using ChIP-sequencing and in vitro functional assays.ResultsWe identified 17 modules of co-expressed genes in cDC2s that correlated with SSc subtypes and key clinical traits including auto-antibodies, skin score, and occurrence of interstitial lung disease. A module of immune regulatory genes was markedly down regulated in patients with the diffuse SSc subtype characterized by severe fibrosis. Transcriptional regulatory network analysis performed on this module predicted NR4A (nuclear receptor 4A) subfamily (NR4A1, NR4A2, NR4A3) genes as the key transcriptional mediators of inflammation. Indeed, ChIP-sequencing analysis supported that these NR4A members target numerous differentially expressed genes in SSc cDC2s. Inclusion of NR4A receptor agonists in culture-based experiments provided functional proof that dysregulation of NR4As affects cytokine production by cDC2s and modulates downstream T-cell activation.ConclusionsNR4A1, NR4A2 and NR4A3 are important regulators of immunosuppressive and fibrosis-associated pathways in SSc cDC2s. Thus, the NR4A family represent novel potential targets to restore cDC homeostasis in SSc.KEY MESSAGESWhat is already known about this subject?CD1c+ conventional dendritic cells (cDC2s) are implicated as key players in Systemic Sclerosis (SSc), but key molecular mechanisms underlying their dysregulation were unknown.What does this study add?Transcriptomic analysis and network analysis identified modules of coexpressed genes in cDC2s that correlated with SSc subtypes and key clinical traits.The NR4A (nuclear receptor 4A) subfamily (NR4A1, NR4A2, NR4A3) genes act as master regulators of key immune regulatory genes dysregulated in SSc cDC2s, as shown by multi-omics integration analysis using transcriptomics and targeted ChIP-sequencing.Pharmacological activation of NR4As inhibits pro-inflammatory cytokine production and CD4+ T-cell activation by cDC2s.How might this impact on clinical practice or future developments?NR4As are attractive candidates for novel treatment options to attenuate pro-inflammatory and pro-fibrotic responses in SSc patients.

Background:Type I interferons (IFNs) promote the expansion of subsets of CD1c+ conventional dendritic cells (CD1c+ DCs), but the molecular basis of CD1c+ DCs involvement in conditions not associated without elevated type I IFNs remains unclear.Methods:We analyzed CD1c+ DCs from two cohorts of non-infectious uveitis patients and healthy donors using RNA-sequencing followed by high-dimensional flow cytometry to characterize the CD1c+ DC populations.Results:We report that the CD1c+ DCs pool from patients with non-infectious uveitis is skewed toward a gene module with the chemokine receptor CX3CR1 as the key hub gene. We confirmed these results in an independent case–control cohort and show that the disease-associated gene module is not mediated by type I IFNs. An analysis of peripheral blood using flow cytometry revealed that CX3CR1+ DC3s were diminished, whereas CX3CR1− DC3s were not. Stimulated CX3CR1+ DC3s secrete high levels of inflammatory cytokines, including TNF-alpha, and CX3CR1+ DC3 like cells can be detected in inflamed eyes of patients.Conclusions:These results show that CX3CR1+ DC3s are implicated in non-infectious uveitis and can secrete proinflammatory mediators implicated in its pathophysiology.Funding:The presented work is supported by UitZicht (project number #2014-4, #2019-10, and #2021-4). The funders had no role in the design, execution, interpretation, or writing of the study.

Single nucleotide polymorphisms (SNP) near theERAP2gene are associated with autoimmune conditions such asCrohn’s disease, andbirdshot chorioretinopathy, as well as protection against lethal infections, including theBlack Death. Due to high linkage disequilibrium (LD), a great number of trait-associated SNPs are correlated withERAP2expression, however their functional mechanisms remain unidentified. We used genome editing and functional genomics to identify causal variants that remain obscured by LD. We demonstrate by reciprocal allelic replacement thatERAP2expression is directly controlled by the genotype of splice region SNP rs2248374. However, we demonstrate that autoimmune disease-risk SNPs located near the downstreamLNPEPgene promoter are independently associated withERAP2expression. Allele-specific conformation capture assays revealed long-range chromatin contacts between theLNPEPpromoter region and theERAP2promoter and showed that interactions were stronger in patients carrying the alleles that increase susceptibility to autoimmune diseases. Replacing the disease-associated SNPs in theLNPEPpromoter by reference sequences loweredERAP2expression. These findings show that clustered GWAS signals associated with diverse autoimmune conditions and lethal infections act in concert to control ERAP2 expression and that disease-associated variants can convert a gene promoter region into a potent enhancer of a distal gene.

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., Unbiased flow cytometry profiling in non‐Hodgkin orbital lymphoma and idiopathic orbital inflammation identified and independently replicated diminished pDC and cDC2 populations in blood and tissue.

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., MicroRNA profiling in non‐Hodgkin orbital lymphoma and idiopathic orbital inflammation identified and independently replicated the increased expression of a pan‐inflammatory cluster of microRNAs accompanied by B cell and neutrophil dysfunction.

BackgroundInflammatory subsets of CD1c+ conventional dendritic cells (CD1c+ DCs) are promoted by type I interferons (IFN), but the molecular basis for CD1c+ DCs involvement in conditions not driven by type I IFNs is unknown. MethodsOur objective was to use RNA-sequencing of blood CD1c+ DCs and high-dimensional flow cytometry of two cohorts of autoimmune uveitis patients and healthy donors to characterize the CD1c+ DCs population of type I IFN-negative autoimmune uveitis. ResultsWe report that the CD1c+ DCs pool from patients with autoimmune uveitis (n=45) is skewed towards a transcriptional network characterized by surface receptor genes CX3CR1, CCR2, and CD36. We confirmed the association of the transcriptional network with autoimmune uveitis by RNA-sequencing in another case-control cohort (n=35) and demonstrated that this network was governed by NOTCH2-RUNX3 signaling. Unbiased flow cytometry analysis based on the transcriptional network identified blood CD1c+ DC subsets that can be distinguished by CX3CR1 and CD36 surface expression. A CD36+CX3CR1+CD1c+ DC subset within the novel DC3 population was diminished in peripheral blood of patients, while CD1c+ DCs expressing CD36 and CX3CR1 accumulate locally in the inflamed eye. The CD36+CX3CR1+CD1c+ DC subset showed a differential capacity to produce cytokines, including TNF-alpha, IL-6, and VEGF, but not IL-23. ConclusionThese results show that CD1c+ DC subsets defined on the basis of surface expression of CD36 and CX3CR1 are linked to type I IFN-negative human autoimmune uveitis and show a differential capacity to secrete proinflammatory mediators that drive its pathophysiology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/468816v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@14f8f2borg.highwire.dtl.DTLVardef@406a5eorg.highwire.dtl.DTLVardef@1075804org.highwire.dtl.DTLVardef@1238bdc_HPS_FORMAT_FIGEXP M_FIG C_FIG

CD200 receptor 1 (CD200R) is an inhibitory immunoreceptor that suppresses Toll-like receptor (TLR)–induced cytokine production through the adaptor protein Dok2 and the GTPase activating protein (GAP) p120-RasGAP, which can be cleaved during mild cellular stress. We found that in the presence of cleaved p120-RasGAP, CD200R lost its capacity to inhibit phosphorylation of ribosomal S6 protein (rpS6), suggesting the reduced activity of mammalian target of rapamycin complex 1 (mTORC1). Furthermore, treatment of human peripheral blood mononuclear cells (PBMC) with interferon-α (IFN-α) resulted in increased amounts of cleaved p120-RasGAP. Upon pretreatment of cells with increasing concentrations of IFN-α, CD200R switched from inhibiting to potentiating the TLR7- and TLR8-induced expression of the gene encoding IFN-γ, a cytokine that is important for innate and adaptive immunity and is implicated in systemic lupus erythematosus (SLE) pathogenesis. PBMC from patients with SLE, a prototypic type I IFN disease, had an increased abundance of cleaved p120-RasGAP compared to that in cells from healthy controls. In a subset of SLE patients, CD200R stopped functioning as an inhibitory receptor or potentiated TLR-induced IFNG mRNA expression. Thus, our data suggest that type I IFN rewires CD200R signaling to be proinflammatory, which could contribute to the perpetuation of inflammation in patients with SLE.

Objective biomarkers that can predict a severe disease course of autoimmune uveitis are lacking, and warranted for early identification of high-risk patients to improve visual outcome. The need for non-steroid immunomodulatory therapy (IMT) to control autoimmune uveitis is indicative of a more severe disease course. We used aptamer-based proteomics and a bioinformatic pipeline to uncover the serum protein network of 52 treatment-free patients and 26 healthy controls, and validation cohorts of 114 and 67 patients. Network-based analyses identified a highly co-expressed serum signature (n=85 proteins) whose concentration was consistently low in controls, but varied between cases. Patients that were positive for the signature at baseline showed a significantly increased risk for IMT during follow-up, independent of anatomical location of disease. In an independent cohort (n=114), we established robust risk categories that confirmed that patients with high levels of the signature at diagnosis had a significantly increased risk to start IMT during follow-up. Finally, we further validated the predictive power of the signature in a third cohort of 67 treatment-naive North-American patients. A serum protein signature was highly predictive for IMT in human autoimmune uveitis and may serve as an objective blood biomarker to aid in clinical-decision making.Abstract Figure

Purpose Classical alleles of the human leukocyte antigen (HLA) complex have been linked to specific entities of pediatric noninfectious uveitis, yet genetic predisposition encoded by the HLA super-locus across the patient population remains understudied. Methods We performed next-generation full-length sequencing of HLA-A, HLA-B, HLA-C, HLA-DPB1, HLA-DQB1, and HLA-DRB1 in 280 cases. Dense genotype data from 499 Dutch controls from Genome of the Netherlands were imputed using an HLA-specific reference panel (n = 5225 samples from European ancestry). Cases and controls were compared using logistic regression models adjusting for sex. Results In total, 179 common and rare alleles were detected. Considering all cases and controls, HLA-DQB1*04:02 and HLA-DRB1*08:01 were identified as the principal HLA association, which was mainly driven by 92 cases with juvenile idiopathic arthritis-associated uveitis (JIA-U). The HLA-DQB1*04:02-HLA-DRB1*08:01 haplotype was also the primary association for the phenotypically similar idiopathic chronic anterior uveitis without arthritis (CAU). Also, HLA-DQB1*05:03 was an independent risk allele for CAU, but not in JIA-U. Analysis of 185 cases with other forms of uveitis revealed HLA-wide associations (P < 2.79 × 10−4) for HLA-DRB1*01:02, HLA-DRB1*04:03, and HLA-DQB1*05:03, which could be primarily attributed to cases with panuveitis. Finally, amino acid substitution modeling revealed that aspartic acid at position 57 that distinguishes the risk allele HLA-DQB1*05:03 (for CAU and panuveitis) from nonrisk alleles, significantly increased the binding capacity of naturally presented ligands to HLA-DQ. Conclusions These results uncovered novel shared HLA associations among clinically distinct phenotypes of pediatric uveitis and highlight genetic predisposition affecting the antigen presentation pathway.

OBJECTIVE: Childhood uveitis is a visual-threatening inflammatory eye disease commonly attributed to juvenile idiopathic arthritis (JIA). The pathogenesis is poorly understood, which makes clinical management challenging. We analyzed soluble mediators in ocular fluid (aqueous humor; AqH) and serum of children with JIA associated uveitis (JIA-U) and common childhood uveitis to identify potential biomarkers and investigate the ocular micro-environment of this sight-threatening eye disease. METHODS: AqH (n=73) and paired serum (n=66) samples were analyzed for 51 soluble inflammatory mediators by multiplex immunoassay. Twenty-one children with JIA-U were compared with 15 with chronic anterior uveitis without arthritis (CAU), 29 with non-infectious idiopathic uveitis (IU), and 8 non-inflammatory controls. For visualization of the joint effect of multiple mediators, we exploited the radial coordinate visualization method (Radviz(TM) ). Also, optimal biomarker level cutoffs were determined. RESULTS: The levels of interleukin-29/interferon-λ1 (IL-29/IFN-λ1) were decreased (P

PurposeKeratoconus (KC) is an eye condition that can lead to a severe vision loss and may warrant a corneal grafting procedure. Meta-analyses of genome wide association studies have identified several genes that confer risks for differences in corneal curvature, corneal thickness, and developing keratoconus. Currently, there is limited evidence of a functional role for the identified loci in the affected corneal tissues.MethodsWe investigated the gene expression profiles of 4 GWAS confirmed risk loci and several related pathways that function in cellular ageing and cell cycle control in corneal tissue of a discovery and replication cohort comprising in total 27 keratoconus patients, 16 healthy controls, and 21 diseased controls (failed corneal grafts).ResultsWe confirmed the MTOR gene locus as differentially expressed in KC corneas in a discovery cohort Next, we replicated these results in a second cohort and found evidence of increased expression of various mTORC1 pathway signature genes, namely MTOR itself (P=0.040), AKT1 (P=0.028), IGF1R (P=0.022) and RAPTOR (P=0.007).ConclusionsGene expression profiling in cornea tissues revealed robust up-regulation of the mTORC1 pathway in KC and substantiates a potential role for this pathway in its pathogenesis. Functional implications should be further studied since biomarkers for disease activity are needed and selective targeting of the mTOR pathway is a promising treatment concept.

Birdshot Uveitis (Birdshot) is a rare eye condition that affects HLA-A29-positive individuals and could be considered a prototypic member of the recently proposed ‘MHC-I (major histocompatibility complex class I)-opathy’ family. Genetic studies have pinpointed the endoplasmic reticulum aminopeptidase (ERAP1) and (ERAP2) genes as shared associations across MHC-I-opathies, which suggests ERAP dysfunction may be a root cause for MHC-I-opathies. We mapped the ERAP1 and ERAP2 haplotypes in 84 Dutch cases and 890 controls. We identified association at variant rs10044354, which mediated a marked increase in ERAP2 expression. We also identified and cloned an independently associated ERAP1 haplotype (tagged by rs2287987) present in more than half of the cases; this ERAP1 haplotype is also the primary risk and protective haplotype for other MHC-I-opathies. We show that the risk ERAP1 haplotype conferred significantly altered expression of ERAP1 isoforms in transcriptomic data (n = 360), resulting in lowered protein expression and distinct enzymatic activity. Both the association for rs10044354 (meta-analysis: odds ratio (OR) [95% CI]=2.07[1.58–2.71], P = 1.24 × 10(−7)) and rs2287987 (OR[95% CI]: =2.01[1.51–2.67], P = 1.41 × 10(−6)) replicated and showed consistent direction of effect in an independent Spanish cohort of 46 cases and 2103 controls. In both cohorts, the combined rs2287987-rs10044354 haplotype associated with Birdshot more strongly than either variant alone [meta-analysis: P=3.9 × 10(−9)]. Finally, we observed that ERAP2 protein expression is dependent on the ERAP1 background across three European populations (n = 3353). In conclusion, a functionally distinct combination of ERAP1 and ERAP2 are a hallmark of Birdshot and provide rationale for strategies designed to correct ERAP function for treatment of Birdshot and MHC-I-opathies more broadly.

Purpose The cause of noninfectious uveitis (NIU) is poorly understood but is considered to be mediated by a complex interplay between genetic, environmental, and-relatively unexplored-epigenetic factors. MicroRNAs (miRNAs) are noncoding small RNAs that are important epigenetic regulators implicated in pathologic signaling. Therefore, we mapped the circulating miRNA-ome of NIU patients and studied miRNA perturbations within the broader context of the immune system. Methods We designed a strategy to robustly identify changes in the miRNA profiles of two independent cohorts totaling 54 untreated patients with active and eye-restricted disease and 26 age-matched controls. High-resolution miRNA-ome data were obtained by TaqMan OpenArray technology and subsequent RT-qPCR. Flow cytometry data, and proteomic data spanning the cellular immune system, were used to map the uveitis-miRNA signature to changes in the composition of specific leukocyte subsets in blood. Results Using stringent selection criteria, we identified and independently validated an miRNA cluster that is associated with NIU. Pathway enrichment analysis for genes targeted by this cluster revealed significant enrichment for the PI3K/Akt, MAPK, FOXO, and VEGF signaling pathways, and photoreceptor development. In addition, unsupervised multidomain analyses linked the presence of the uveitis-associated miRNA cluster to a different composition of leukocyte subsets, more specifically, CD16+CD11c+HLA-DR- cells. Conclusions Together, this study identified a unique miRNA cluster associated with NIU that was related to changes in leukocyte subsets demonstrating systemic changes in epigenetic regulation underlying NIU.

Objectives: Uveitis is a visually-debilitating disorder that affects up to 30% of children with the most common forms of juvenile idiopathic arthritis (JIA). The disease mechanisms predisposing only a subgroup of children to uveitis are unknown. To identify genetic susceptibility loci for uveitis in JIA, we conducted a genome-wide association study totalling 522 JIA cases. Methods: Two cohorts of JIA patients with ophthalmological follow-up were separately genotyped and then imputed using a genome-wide imputation reference panel, and an HLA-specific reference panel used for imputing amino acids and HLA types in the major histocompatibility complex (MHC). After imputation, we performed genome-wide and MHC-specific analyses. We used a reverse immunology approach to model antigen presentation at 13 common HLA-DRB1 allotypes. Results: We identified the amino acid serine at position 11 (serine-11) in HLA-DRB1 as associated to increased risk of uveitis (OR = 2.60, p = 5.43 x 10 -10 ). We found the serine-11 signal to be specific to females (p females = 7.61 x 10 -10 , p males = 0.18). Serine-11 resides in the YST-motif in the peptide binding groove of the HLA-DRB1 protein; all three amino acids are in perfect linkage disequilibrium and show identical association to disease. Quantitative prediction of binding affinity revealed that discernable peptide-binding preferences distinguish HLA-DRB1 allotypes with the YST-motif. Conclusion: Our findings highlight a genetically distinct, sexually-dimorphic feature of JIA-uveitis compared to JIA without uveitis in HLA-DRB1. The association indicates the potential involvement for antigen presentation by HLA-DRB1 in the development of uveitis in JIA.

Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was

Childhood uveitis is a vision-threatening inflammatory eye disease commonly attributed to juvenile idiopathic arthritis (JIA). The pathogenesis is poorly understood, which makes clinical management challenging. We analyzed soluble mediators in ocular fluid (aqueous humor [AqH]) and serum from children with JIA-associated uveitis and common childhood uveitis to identify potential biomarkers and investigate the ocular microenvironment of this sight-threatening eye disease.AqH (n = 73) and paired serum (n = 66) samples were analyzed for 51 soluble mediators of inflammation by multiplex immunoassay. Twenty-one children with JIA-associated uveitis were compared to 15 children with chronic anterior uveitis without arthritis, 29 children with noninfectious idiopathic uveitis, and 8 children with noninflammatory conditions (controls). For visualization of the joint effect of multiple mediators, we used the radial coordinate visualization (Radviz) method. Optimal biomarker level cutoffs were also determined.The levels of interleukin-29 (IL-29)/interferon-λ1 (IFNλ1) were decreased (P 0.001) and the levels of latency-associated peptide and osteoprotegerin were increased (P = 0.002 and P = 0.001, respectively) in samples of AqH, but not serum, from patients with JIA-associated uveitis. Multivariate analysis correcting for disease activity and treatment revealed that intraocular levels of IL-29/IFNλ1 were specifically decreased in patients with JIA-associated uveitis as compared to those with idiopathic uveitis. Indeed, JIA-associated uveitis patients and idiopathic uveitis patients showed distinct profiles of intraocular soluble mediators. IL-29/IFNλ1 showed a high area under the curve value (0.954), with 23.5 pg/ml as the optimal cutoff value.We identified IL-29/IFNλ1 as an intraocular biomarker for JIA-associated uveitis, which suggests that aberrant IFNλ signaling might be important in JIA-associated uveitis and distinct from other forms of childhood uveitis.

The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53. ispartof: Cell Death & Disease vol:6 issue:7 ispartof: location:England status: published

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.

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.

The CD38 molecule, with its high expression on Multiple Myeloma (MM), is considered a suitable target for antibody therapy of MM. We developed daratumumab (DARA), a human CD38 monoclonal antibody (mAb) with direct and Fc-mediated cell killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011), and antibody-dependent cellular phagocytosis (ADCP) by macrophages (both murine and human). In addition, DARA induces apoptosis upon secondary cross-linking and modulates CD38 enzymatic function. DARA is currently in phase I, II and III clinical evaluation in patients with MM. Besides DARA, several other anti-CD38 mAb are in development; SAR650894 (SAR; clone 38SB19; Sanofi-Aventis) for MM and other CD38+ hematological malignancies, MOR03087 (MOR; Morphosys) for relapsed/refractory MM and Ab79 (Millenium/Takeda) which is in preclinical development. Similar mechanisms of action (MoA) are described for these mAb; nevertheless direct comparison studies would be critical for differentiation among these antibodies. . In this study, the efficacy of these anti-CD38 mAb was directly compared to DARA with respect to binding, apoptosis, CD38 ectoenzyme activity, and the induction of ADCC, ADCP and CDC. Surrogate antibodies of MOR, SAR and Ab79 were generated on the basis of protein sequences, as published in their corresponding patents families, and were attached to the backbone of DARA. Binding to CD38 expressing Daudi tumor cells was assessed by flow cytomery. All CD38 antibodies showed similar EC50 (~0.1 µg/mL) and maximal binding, except MOR which showed a lower apparent affinity (~0.3 µg/mL). Previously, CD38 amino acid residues Q272 and S274 were reported as critical for DARA binding. ELISA analyses using CD38 point mutants revealed MOR, SAR and Ab79 not to be affected by mutation of these residues. All CD38 mAb were equally potent in inducing ADCC of Daudi cells (40-60% lysis, 0.02 µg/mL), in classic Cr51-release ADCC assay using human PBMC effector cells (E:T ratio 100:1). Important differences were observed with respect to induction of CDC. SAR was unable to induce CDC in Daudi cells at concentrations up to 30 µg/mL, while DARA induced more than 80% lysis at concentrations above 1 µg/mL. Ab79 and MOR induced CDC, yet maximum lysis was 50% and 20%, respectively. Evaluation of Annexin V/propidium iodide (AnnV/PI) staining and activation of caspase-3 showed that only SAR induced AnnV/PI+ in Ramos cells (~40%) after 48 h exposure without Fc crosslinking, but did not activate caspase-3. In the presence of Fc crosslinking antibodies, all anti-CD38 mAb induced AnnV/PI+, caspase-3 mediated apoptosis. In enzyme activity assays using purified CD38 protein, SAR inhibited generation of cGDPR (indicative of the combined CD38 cyclase activity generating fluorescent cGDPR and hydrolase activity converting cGDPR into GDPR)more effectively than DARA (~70% vs. ~20% inhibition at 30 µg/mL). Ab79 had a modest effect on CD38 enzyme activity (~10% inhibition). MOR did not affect CD38 enzyme activity at all. The capacity to induce ADCP was only tested for DARA, MOR and Ab79 using mouse macrophages (mφ) as effector cells and Daudi target cells. mφ, isolated and matured from bone marrow cells, and calcein-AM labelled target cells (E:T ratio 1:1) were cultured in the presence of 0.0006-5 µg/mL antibody for 4 h. Non-phagocytosed target cells and mφ were collected and ADCP was evaluated by flow cytometry. All CD38 mAb induced mφ-mediated phagocytosis, as observed by a concentration dependent increase in the number of double positive mφ and killing of target cells. Ab79 was as effective as DARA (EC50 ~0.01 µg/mL) in ADCP induction, whereas MOR was less effective (EC50 0.04 µg/mL). In summary, DARA and surrogate mAb of MOR, SAR and Ab79 showed similar binding to cells and induced similar amounts of ADCC. Differences between these mAb involved the ability to directly induce apoptosis, to inhibit the enzymatic activity of CD38 and to induce ADCP. The most striking difference was observed for the ability to induce CDC, the MoA which is currently believed the most important mechanism of MM cell killing in the clinic. DARA efficiently induced high levels of CDC at low concentrations, whereas the other CD38 mAb were unable or less capable to induce CDC. Disclosures Lammerts van Bueren: Genmab: Employment, warrents Other. Jakobs:Genmab: Employment, warrents Other. Kaldenhoven:Genmab: Employment, warrents Other. Roza:Genmab: Employment, warrents Other. Hiddingh:Genmab: Employment. Meesters:Genmab: Employment, warrents Other. Voorhorst:Genmab: Employment, warrents Other. Gresnigt:Genmab: Employment, warrents Other. Wiegman:Genmab: Employment, warrents Other. Ortiz Buijsse:Genmab: Employment, warrents Other. Andringa:Genmab: Employment, warrents Other. Overdijk:Genmab: Employment, warrents Other. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. de Weers:Genmab: Employment, warrents Other. Parren:Genmab: Employment, inventor on patents regarding daratumumab Patents & Royalties, stock and warrents Other.