Jeffrey D. Kearns

A Fourth IκB Protein within the NF-κB Signaling Module

An assay method incorporating at least two different chemiluminescent compounds for detection and/or quantitation of at least two substances in a test sample is described. The synthesis of chemiluminescent reagents or conjugates for use in such methods as well as kits incorporating such reagents are also disclosed. The assays have particular application in the field of clinical diagnostics.

IκBε provides negative feedback to control NF-κB oscillations, signaling dynamics, and inflammatory gene expression

NF-κB signaling is known to be critically regulated by the NF-κB–inducible inhibitor protein IκBα. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-κB activity. We report integrated experimental and computational studies that demonstrate that another IκB isoform, IκBɛ, also provides negative feedback on NF-κB activity, but with distinct functional consequences. Upon stimulation, NF-κB–induced transcription of IκBɛ is delayed, relative to that of IκBα, rendering the two negative feedback loops to be in antiphase. As a result, IκBɛ has a role in dampening IκBα-mediated oscillations during long-lasting NF-κB activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-κB activity and NF-κB–mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of IκB–NF-κB signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway.

Encoding NF-κB temporal control in response to TNF: distinct roles for the negative regulators IκBα and A20

TNF-induced NF-kappaB activity shows complex temporal regulation whose different phases lead to distinct gene expression programs. Combining experimental studies and mathematical modeling, we identify two temporal amplification steps-one determined by the obligate negative feedback regulator IkappaBalpha-that define the duration of the first phase of NF-kappaB activity. The second phase is defined by A20, whose inducible expression provides for a rheostat function by which other inflammatory stimuli can regulate TNF responses. Our results delineate the nonredundant functions implied by the knockout phenotypes of ikappabalpha and a20, and identify the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses.

A homeostatic model of IκB metabolism to control constitutive NF-κB activity

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease‐associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF‐κB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IκB, until an external stimulus triggers IκB degradation through an IκB kinase‐dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady‐state regulation of the NF‐κB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF‐κB‐bound and ‐unbound IκB proteins that are critical for accurate computational predictions of steady‐state IκB protein levels and basal NF‐κB activity. Simulations reveal a homeostatic NF‐κB signaling module in which differential degradation rates of free and bound pools of IκB represent a novel cross‐regulation mechanism that imparts functional robustness to the signaling module.

Control of RelB during dendritic cell activation integrates canonical and noncanonical NF-κB pathways

The NF-κB protein RelB controls dendritic cell (DC) maturation and may be targeted therapeutically to manipulate T cell responses in disease. Here we report that RelB promoted DC activation not as the expected RelB-p52 effector of the noncanonical NF-κB pathway, but as a RelB-p50 dimer regulated by canonical IκBs, IκBα and IκBɛ. IκB control of RelB minimized spontaneous maturation but enabled rapid pathogen-responsive maturation. Computational modeling of the NF-κB signaling module identified control points of this unexpected cell type–specific regulation. Fibroblasts that we engineered accordingly showed DC-like RelB control. Canonical pathway control of RelB regulated pathogen-responsive gene expression programs. This work illustrates the potential utility of systems analyses in guiding the development of combination therapeutics for modulating DC-dependent T cell responses.

Kinetic control of negative feedback regulators of NF-κB/RelA determines their pathogen- and cytokine-receptor signaling specificity

Mammalian signaling networks contain an abundance of negative feedback regulators that may have overlapping (“fail-safe”) or specific functions. Within the NF-κB signaling module, IκBα is known as a negative feedback regulator, but the newly characterized inhibitor IκBδ is also inducibly expressed in response to inflammatory stimuli. To examine IκBδs roles in inflammatory signaling, we mathematically modeled the 4-IκB-containing NF-κB signaling module and developed a computational phenotyping methodology of general applicability. We found that IκBδ, like IκBα, can provide negative feedback, but each functions stimulus-specifically. Whereas IκBδ attenuates persistent, pathogen-triggered signals mediated by TLRs, the more prominent IκBα does not. Instead, IκBα, which functions more rapidly, is primarily involved in determining the temporal profile of NF-κB signaling in response to cytokines that serve intercellular communication. Indeed, when removing the inducing cytokine stimulus by compound deficiency of the tnf gene, we found that the lethality of iκbα−/− mouse was rescued. Finally, we found that IκBδ provides signaling memory owing to its long half-life; it integrates the inflammatory history of the cell to dampen NF-κB responsiveness during sequential stimulation events.

UV as an amplifier rather than inducer of NF-κB activity

Inflammatory activation of NF-kappaB involves the stimulus-induced degradation of the NF-kappaB-bound inhibitor IkappaB via the IkappaB kinase (IKK). In response to UV irradiation, however, the mechanism and function of NF-kappaB activation remain unclear. Using a combined biochemical, genetic, and computational modeling approach, we delineate a dual requirement for constitutive IKK-dependent and IKK-independent IkappaB degradation pathways in conjunction with UV-induced translational inhibition. Interestingly, we find that the high homeostatic turnover of IkappaB in resting cells renders the NF-kappaB system remarkably resistant to metabolic stresses, but the two degradation pathways critically and differentially tune NF-kappaB responsiveness to UV. Indeed, in the context of low chronic inflammation that accelerates NF-kappaB-bound IkappaB degradation, UV irradiation results in dramatic NF-kappaB activation. Our work suggests that the human health relevance of NF-kappaB activation by UV lies with cellular homeostatic states that are associated with pathology rather than with healthy physiology.

Stabilization of RelB Requires Multidomain Interactions with p100/p52

The NF-κB family member RelB has many properties not shared by other family members such as restricted subunit association and lack of regulation by the classical IκB proteins. We show that the protein level of RelB is significantly reduced in the absence of p100 and reduced even more when both p100 and p105 are absent. RelB stabilizes itself by directly interacting with p100, p105, and their processed products. However, RelB forms complexes with its partners using different interaction modes. Although the C-terminal ankyrin repeat domain of p105 is not involved in the RelB-p105 complex formation, all domains and flexible regions of each protein are engaged in the RelB-p100 complex. In several respects the RelB-p52 and RelB-p100 complexes are unique in the NF-κB family. The N-terminal domain of p100/p52 interacts with RelB but not RelA. The transcriptional activation domain of RelB, but not RelA, directly interacts with the processing region of p100. These unique protein-protein contacts explain why RelB prefers p52 as its dimeric partner for transcriptional activity and is retained in the cytoplasm as an inhibited complex by p100. This association-mediated stabilization of RelB implies a possible role for RelB in the processing of p100 into p52.

MM-151 overcomes acquired resistance to cetuximab and panitumumab in colorectal cancers harboring EGFR extracellular domain mutations

An oligoclonal antibody overcomes drug resistance in colorectal cancers with EGFR mutations. The more the merrier Monoclonal antibodies against the epidermal growth factor receptor (EGFR), which drives cancer growth, are frequently used in colorectal cancer. Unfortunately, the cancers commonly develop drug-resistant mutations, and the monoclonal antibodies become ineffective. To overcome this problem, Arena et al. used an oligoclonal mixture of monoclonal antibodies called MM-151, which binds multiple parts of the EGFR molecule at once, so that the cancer cannot develop resistance by mutating one site at a time. The authors demonstrated that this approach is effective in both preclinical models and patients who were resistant to other anti-EGFR therapies, paving the way for further clinical development of the oligoclonal antibody. The anti–epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab are used to treat RAS wild-type colorectal cancers (CRCs), but their efficacy is limited by the emergence of acquired drug resistance. After EGFR blockade, about 20% of CRCs develop mutations in the EGFR extracellular domain (ECD) that impair antibody binding and are associated with clinical relapse. We hypothesized that EGFR ECD–resistant variants could be targeted by the recently developed oligoclonal antibody MM-151 that binds multiple regions of the EGFR ECD. MM-151 inhibits EGFR signaling and cell growth in preclinical models, including patient-derived cells carrying mutant EGFR. Upon MM-151 treatment, EGFR ECD mutations decline in circulating cell-free tumor DNA (ctDNA) of CRC patients who previously developed resistance to EGFR blockade. These data provide molecular rationale for the clinical use of MM-151 in patients who become resistant to cetuximab or panitumumab as a result of EGFR ECD mutations.

Enhanced Targeting of the EGFR Network with MM-151, an Oligoclonal Anti-EGFR Antibody Therapeutic

Although EGFR is a validated therapeutic target across multiple cancer indications, the often modest clinical responses to current anti-EGFR agents suggest the need for improved therapeutics. Here, we demonstrate that signal amplification driven by high-affinity EGFR ligands limits the capacity of monoclonal anti-EGFR antibodies to block pathway signaling and cell proliferation and that these ligands are commonly coexpressed with low-affinity EGFR ligands in epithelial tumors. To develop an improved antibody therapeutic capable of overcoming high-affinity ligand-mediated signal amplification, we used a network biology approach comprised of signaling studies and computational modeling of receptor–antagonist interactions. Model simulations suggested that an oligoclonal antibody combination may overcome signal amplification within the EGFR:ERK pathway driven by all EGFR ligands. Based on this, we designed MM-151, a combination of three fully human IgG1 monoclonal antibodies that can simultaneously engage distinct, nonoverlapping epitopes on EGFR with subnanomolar affinities. In signaling studies, MM-151 antagonized high-affinity EGFR ligands more effectively than cetuximab, leading to an approximately 65-fold greater decrease in signal amplification to ERK. In cell viability studies, MM-151 demonstrated antiproliferative activity against high-affinity EGFR ligands, either singly or in combination, while cetuximab activity was largely abrogated under these conditions. We confirmed this finding both in vitro and in vivo in a cell line model of autocrine high-affinity ligand expression. Together, these preclinical studies provide rationale for the clinical study of MM-151 and suggest that high-affinity EGFR ligand expression may be a predictive response marker that distinguishes MM-151 from other anti-EGFR therapeutics. Mol Cancer Ther; 14(7); 1625–36. ©2015 AACR.