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Assembly of a transcriptional and post‐translational molecular interaction network in B cells, the human B‐cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre‐replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues. Synopsis We have assembled an interaction network specific to human B cells (the human B‐cell interactome or HBCI), containing protein–DNA and protein–protein interactions using an evidence integration approach. The integration of different interaction layers in one network allowed us to elucidate master regulator (MR) genes controlling specific cellular processes as well as transcriptional regulation of proteins in complexes whose availability must be regulated in context‐dependent manner. The latter is a poorly understood process, as transcriptional networks and protein–protein interaction networks are usually studied in isolation. We have developed a new algorithm called master regulator inference analysis (MARINa) for discovering MRs of specific phenotypes and applied it to the HBCI to infer MRs of germinal center (GC) formation. GCs are structures where antigen‐stimulated B cells highly proliferate, undergo somatic hypermutation of immunoglobulin genes, and are selected based on the production of high‐affinity antibodies. GC B cells (centroblasts) derive from naive B cells, from which they differ for the activation of genetic programs controlling cell proliferation, DNA metabolism, and pro‐apoptotic programs and for the repression of anti‐apoptotic, cell‐cycle arrest, DNA repair, and signal transduction programs from cytokines and chemokines. MARINa recovered known MRs of GC B cells and also revealed a new transcription factor module controlling their proliferation. In particular, we identified MYB and FOXM1 as being key MRs of GC B cells. Indeed, 80% of the genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a predicted complex regulating DNA pre‐replication, replication, and mitosis. We first tested whether MYB and FOXM1 may regulate each other as predicted in the HBCI, and show that MYB is a transcriptional activator of FOXM1, suggesting that they form a feed‐forward loop, involved in the synergistic activation of a large subset of GC‐specific genes. We then validated that common MYB/FOXM1 targets and other predicted MRs were affected by the silencing of either TF, using gene expression profiling. Furthermore, we showed that downregulated targets (AURKA, BUBR1, CCNB2, FANCI, MCM3, and PTTG1) and MRs (NFYB, E2F1, and E2F5) after MYB or FOXM1 silencing are indeed directly bound by them in their promoter region. Silencing of FOXM1 and MYB showed a decrease in proliferating cells and an increase in apoptotic cells, indicating that MYB and FOXM1 are necessary for viability and rapid proliferation of GC B cells. To gain more insight into the control of GC‐proliferation phenotype by MYB and FOXM1, we further examined specific targets involved in the formation of a predicted protein complex. Approximately half of MYB/FOXM1 targets cluster within a complex, including new interactions between pre‐replication and mitotic proteins. We experimentally validate that two mitotic kinases in the inferred complex, BUBR1 and AURKA, physically interact with MCM3, all of them being confirmed to be direct targets of FOXM1 and MYB. In summary, these results document that coordinated analysis of both transcriptional and post‐translational interactions in the HBCI can identify synergistic MRs of human phenotypes, as well as provide insight on the functional regulatory role of these proteins. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues. Assembly of a mixed interaction network specific to human B cells. Identification and validation of master regulators of germinal center reaction. MYB and FOXM1 are synergistic master regulators of proliferation in germinal center B cells and control a new protein complex involving replication and mitotic‐related genes.

To understand drug combination effect, it is necessary to decipher the interactions between drug targets-many of which are signaling molecules. Previously, such signaling pathway models are largely based on the compilation of literature data from heterogeneous cellular contexts. Indeed, de novo reconstruction of signaling interactions from large-scale molecular profiling is still lagging, compared to similar efforts in transcriptional and protein-protein interaction networks. To address this challenge, we introduce a novel algorithm for the systematic inference of protein kinase pathways, and applied it to published mass spectrometry-based phosphotyrosine profile data from 250 lung adenocarcinoma (LUAD) samples. The resulting network includes 43 TKs and 415 inferred, LUAD-specific substrates, which were validated at >60% accuracy by SILAC assays, including \"novel' substrates of the EGFR and c-MET TKs, which play a critical oncogenic role in lung cancer. This systematic, data-driven model supported drug response prediction on an individual sample basis, including accurate prediction and validation of synergistic EGFR and c-MET inhibitor activity in cells lacking mutations in either gene, thus contributing to current precision oncology efforts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy. Nephritis is a major cause of lupus morbidity. Putterman and colleagues use single-cell RNA sequencing on human renal and skin biopsies to describe the expression landscape associated with lupus nephritis.

We describe a computational protocol for the ARACNE algorithm, an information-theoretic method for identifying transcriptional interactions between gene products using microarray expression profile data. Similar to other algorithms, ARACNE predicts potential functional associations among genes, or novel functions for uncharacterized genes, by identifying statistical dependencies between gene products. However, based on biochemical validation, literature searches and DNA binding site enrichment analysis, ARACNE has also proven effective in identifying bona fide transcriptional targets, even in complex mammalian networks. Thus we envision that predictions made by ARACNE, especially when supplemented with prior knowledge or additional data sources, can provide appropriate hypotheses for the further investigation of cellular networks. While the examples in this protocol use only gene expression profile data, the algorithm's theoretical basis readily extends to a variety of other high-throughput measurements, such as pathway-specific or genome-wide proteomics, microRNA and metabolomics data. As these data become readily available, we expect that ARACNE might prove increasingly useful in elucidating the underlying interaction models. For a microarray data set containing ∼10,000 probes, reconstructing the network around a single probe completes in several minutes using a desktop computer with a Pentium 4 processor. Reconstructing a genome-wide network generally requires a computational cluster, especially if the recommended bootstrapping procedure is used.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ObjectivesIn lupus nephritis the pathological diagnosis from tissue retrieved during kidney biopsy drives treatment and management. Despite recent approval of new drugs, complete remission rates remain well under aspirational levels, necessitating identification of new therapeutic targets by greater dissection of the pathways to tissue inflammation and injury. This study assessed the safety of kidney biopsies in patients with SLE enrolled in the Accelerating Medicines Partnership, a consortium formed to molecularly deconstruct nephritis.Methods475 patients with SLE across 15 clinical sites in the USA consented to obtain tissue for research purposes during a clinically indicated kidney biopsy. Adverse events (AEs) were documented for 30 days following the procedure and were determined to be related or unrelated by all site investigators. Serious AEs were defined according to the National Institutes of Health reporting guidelines.Results34 patients (7.2%) experienced a procedure-related AE: 30 with haematoma, 2 with jets, 1 with pain and 1 with an arteriovenous fistula. Eighteen (3.8%) experienced a serious AE requiring hospitalisation; four patients (0.8%) required a blood transfusion related to the kidney biopsy. At one site where the number of cores retrieved during the biopsy was recorded, the mean was 3.4 for those who experienced a related AE (n=9) and 3.07 for those who did not experience any AE (n=140). All related AEs resolved.ConclusionsProcurement of research tissue should be considered feasible, accompanied by a complication risk likely no greater than that incurred for standard clinical purposes. In the quest for targeted treatments personalised based on molecular findings, enhanced diagnostics beyond histology will likely be required.

The authors have withdrawn this manuscript because the process for obtaining access to the data was updated. The following is a link to Immport (https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.immport.org%2Fresources%2Famp-ra-sle&data=02%7C01%7Cchaim.putterman%40einstein.yu.edu%7C8c23d666c911415479cc08d77cebf60b%7C04c70eb48f2648079934e02e89266ad0%7C1%7C0%7C637115225197330433&sdata=s%2FwD2d%2BTG28ZiQ3fKyQ1UTCE%2ByNz1m2VIE4z%2FZskTsc%3D&reserved=0), the permanent hosting location of the data presented in the article. This article is now published and can be found with PubMed ID 31110316 (https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F31110316&data=02%7C01%7Cchaim.putterman%40einstein.yu.edu%7C8c23d666c911415479cc08d77cebf60b%7C04c70eb48f2648079934e02e89266ad0%7C1%7C0%7C637115225197330433&sdata=%2FbvqYr6Kg0IyYIeK77sU2Kpo9hVPmAChQqJ%2BCdE0svM%3D&reserved=0) Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author Footnotes * This manuscript has been withdrawn.