The nuclear factor kappa B (NFκB) family of transcription factors is a key regulator of immune development, immune responses, inflammation, and cancer. The NFκB signaling system (defined by the interactions between NFκB dimers, IκB regulators, and IKK complexes) is responsive to a number of stimuli, and upon ligand-receptor engagement, distinct cellular outcomes, appropriate to the specific signal received, are set into motion. After almost three decades of study, many signaling mechanisms are well understood, rendering them amenable to mathematical modeling, which can reveal deeper insights about the regulatory design principles. While other reviews have focused on upstream, receptor proximal signaling (Hayden MS, Ghosh S. Signaling to NF-κB. Genes Dev 2004, 18:2195-2224; Verstrepen L, Bekaert T, Chau TL, Tavernier J, Chariot A, Beyaert R. TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme. Cell Mol Life Sci 2008, 65:2964-2978), and advances through computational modeling (Basak S, Behar M, Hoffmann A. Lessons from mathematically modeling the NF-κB pathway. Immunol Rev 2012, 246:221-238; Williams R, Timmis J, Qwarnstrom E. Computational models of the NF-KB signalling pathway. Computation 2014, 2:131), in this review we aim to summarize the current understanding of the NFκB signaling system itself, the molecular mechanisms, and systems properties that are key to its diverse biological functions, and we discuss remaining questions in the field. WIREs Syst Biol Med 2016, 8:227-241. doi: 10.1002/wsbm.1331 For further resources related to this article, please visit the WIREs website.

A hallmark of the inflammatory response to pathogen exposure is the production of tumor necrosis factor (TNF) that coordinates innate and adaptive immune responses by functioning in an autocrine or paracrine manner. Numerous molecular mechanisms contributing to TNF production have been identified, but how they function together in macrophages remains unclear. Here, we pursued an iterative systems biology approach to develop a quantitative understanding of the regulatory modules that control TNF mRNA synthesis and processing, mRNA half-life and translation, and protein processing and secretion. By linking the resulting model of TNF production to models of the TLR-, the TNFR-, and the NFkB signaling modules, we were able to study TNF's functions during the inflammatory response to diverse TLR agonists. Contrary to expectation, we predicted and then experimentally confirmed that in response to lipopolysaccaride, TNF does not have an autocrine function in amplifying the NFkB response, although it plays a potent paracrine role in neighboring cells. However, in response to CpG DNA, autocrine TNF extends the duration of NFkB activity and shapes CpG-induced gene expression programs. Our systems biology approach revealed that network dynamics of MyD88 and TRIF signaling and of cytokine production and response govern the stimulusspecific autocrine and paracrine functions of TNF.

Formation of the nuclear envelope (NE) around segregated chromosomes occurs by the reshaping of the endoplasmic reticulum (ER), a reservoir for disassembled nuclear membrane components during mitosis. In this study, we show that inner nuclear membrane proteins such as lamin B receptor (LBR), MAN1, Lap2β, and the trans-membrane nucleoporins Ndc1 and POM121 drive the spreading of ER membranes into the emerging NE via their capacity to bind chromatin in a collaborative manner. Despite their redundant functions, decreasing the levels of any of these trans-membrane proteins by RNAi-mediated knockdown delayed NE formation, whereas increasing the levels of any of them had the opposite effect. Furthermore, acceleration of NE formation interferes with chromosome separation during mitosis, indicating that the time frame over which chromatin becomes membrane enclosed is physiologically relevant and regulated. These data suggest that functionally distinct classes of chromatin-interacting membrane proteins, which are present at nonsaturating levels, collaborate to rapidly reestablish the nuclear compartment at the end of mitosis.

Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called "stripping." To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen-deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be "caught in the act of stripping" because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.transcription factor | binding kinetics | intrinsically disordered proteins | nuclear export | hydrogen-deuterium exchange S tress-response transcription factors turn on hundreds of genes, and their regulation requires robust activation as well as rapid and complete cessation of the ensuing response. A good example is the NFκB family of transcription factors, which responds to a large number of extracellular stress stimuli, including factors controlling inflammation and the immune response (1-3). Aberrant regulation of NFκB results in numerous disease states, including cancer (1, 4). The IκB family of inhibitors keeps NFκB in the cytoplasm (in the "off" state) (5). IκBα is the main temporally regulated IκB. When a stress signal is received, IκBα is degraded rapidly, releasing NFκB, which enters the nucleus, binds to κB DNA sites, and up-regulates gene expression (Fig. 1A). In a classic negative feedback loop, the promoter upstream of the IκBα gene is strongly up-regulated by NFκB. We previously showed that in vitro IκBα rapidly accelerates the dissociation of NFκB from many different DNA sequences containing the κB motif in a folding-upon-binding event (6, 7). Thus, removal of NFκB(RelA/p50) from its target sites is kinetically determined, a process we call "molecular stripping" (8). The kinetic control of transcription factor-DNA interactions represents a paradigm shift because these interactions typically are described with equilibrium-binding models (9, 10) and thus would require the formulation of novel models based on stochastic rates.For IκBα to strip NFκB from DNA, a ternary NFκB-DNA-IκBα complex must form at least transiently. A very transient NFκB-DNA-IκBα complex was indeed observed in stopped-flow fluorescence experiments (11). At high concentrations, signals corresponding to a ternary NFκB-DNA-IκBα complex were also observed by NMR (12, 13). Together the stopped-flow and NMR data showed that IκBα binds to the NFκB-DNA comple...

Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry–based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9–driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an 'activator-induced inhibitor' regulatory motif. Within the NFkB signalling pathway, a key negative feedback regulator is IkBa. We show here that, contrary to current understanding, NFkB-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NFkB signalling to identify IkBa molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of IkBa and the IkBa-NFkB complex, as well as the free IkBa half-life, as key determinants of post-induction repression of NFkB and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required 'activator-induced inhibitor' relationship.

The innate immune response is largely initiated by pathogen-responsive activation of the transcription factor IRF3. Among other target genes, IRF3 controls the expression of IFN-β, which triggers the activation of the transcription factor ISGF3 via the IFNAR. IRF3 and ISGF3 have been reported to control many of the same target genes and together, control the antimicrobial innate-immune program; however, their respective contributions and specificities remain unclear. Here, we used genomic technologies to characterize their specificity in terms of their physical DNA-binding and genetic function. With the use of ChiP-seq and transcriptomic measurements in WT versus ifnar(-/-) versus ifnar(-/-)irf3(-/-) macrophages responding to intracellular dsRNA, we confirmed the known ISGF3 DNA-binding motif and further specified a distinct IRF3 consensus sequence. The functional specificity of IRF3 is particularly pronounced in cytokine/chemokine regulation; yet, even in the control of IFN-β, that specificity is not absolute. By mathematically modeling IFN-β production within an abstracted tissue layer, we find that IRF3 versus ISGF3 specificity may be critical to limiting IFN-β production and ISGF3 activation, temporally and spatially, but that partial overlap in their specificity is tolerable and may enhance the effectiveness of the innate-immune response.