B-cell survival and development controlled by the coordination of NF-κB family members RelB and cRel.

Targeted deletion of BAFF causes severe deficiency of splenic B cells. BAFF-R is commonly thought to signal to nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase dependent noncanonical NF-κB RelB. However, RelB-deficient mice have normal B-cell numbers. Recent studies showed that BAFF also signals to the canonical NF-κB pathway, and we found that both RelB and cRel are persistently activated, suggesting BAFF signaling coordinates both pathways to ensure robust B-cell development. Indeed, we report now that combined loss of these 2 NF-κB family members leads to impaired BAFF-mediated survival and development in vitro. Although single deletion of RelB and cRel was dispensable for normal B-cell development, double knockout mice displayed an early B-cell developmental blockade and decreased mature B cells. Despite disorganized splenic architecture in Relb(-/-)cRel(-/-) mice, generation of mixed-mouse chimeras established the developmental phenotype to be B-cell intrinsic. Together, our results indicate that BAFF signals coordinate both RelB and cRel activities to ensure survival during peripheral B-cell maturation.

A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide.

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.

IκBε is a key regulator of B cell expansion by providing negative feedback on cRel and RelA in a stimulus-specific manner.

The transcription factor NF-κB is a regulator of inflammatory and adaptive immune responses, yet only IκBα was shown to limit NF-κB activation and inflammatory responses. We investigated another negative feedback regulator, IκBε, in the regulation of B cell proliferation and survival. Loss of IκBε resulted in increased B cell proliferation and survival in response to both antigenic and innate stimulation. NF-κB activity was elevated during late-phase activation, but the dimer composition was stimulus specific. In response to IgM, cRel dimers were elevated in IκBε-deficient cells, yet in response to LPS, RelA dimers also were elevated. The corresponding dimer-specific sequences were found in the promoters of hyperactivated genes. Using a mathematical model of the NF-κB-signaling system in B cells, we demonstrated that kinetic considerations of IκB kinase-signaling input and IκBε's interactions with RelA- and cRel-specific dimers could account for this stimulus specificity. cRel is known to be the key regulator of B cell expansion. We found that the RelA-specific phenotype in LPS-stimulated cells was physiologically relevant: unbiased transcriptome profiling revealed that the inflammatory cytokine IL-6 was hyperactivated in IκBε(-/-) B cells. When IL-6R was blocked, LPS-responsive IκBε(-/-) B cell proliferation was reduced to near wild-type levels. Our results provide novel evidence for a critical role for immune-response functions of IκBε in B cells; it regulates proliferative capacity via at least two mechanisms involving cRel- and RelA-containing NF-κB dimers. This study illustrates the importance of kinetic considerations in understanding the functional specificity of negative-feedback regulators.

Expanding control of the tumor cell cycle with a CDK2/4/6 inhibitor.

The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.

A pathway switch directs BAFF signaling to distinct NFκB transcription factors in maturing and proliferating B cells.

BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

Glucose modulation of glucokinase activation by small molecules.

Small molecule activators of glucokinase (GK) were used in kinetic and equilibrium binding studies to probe the biochemical basis for their allosteric effects. These small molecules decreased the glucose K 0.5 ( approximately 1 mM vs approximately 8 mM) and the glucose cooperativity (Hill coefficient of 1.2 vs 1.7) and lowered the k cat to various degrees (62-95% of the control activity). These activators relieved GK's inhibition from glucokinase regulatory protein (GKRP) in a glucose-dependent manner and activated GK to the same extent as control reactions in the absence of GKRP. In equilibrium binding studies, the intrinsic glucose affinity to the activator-bound enzyme was determined and demonstrated a 700-fold increase relative to the apoenzyme. This is consistent with a reduction in apparent glucose K D and the steady-state parameter K 0.5 as a result of enzyme equilibrium shifting to the activator-bound form. The binding of small molecules to GK was dependent on glucose, consistent with the structural evidence for an allosteric binding site which is present in the glucose-induced, active enzyme form of GK and absent in the inactive apoenzyme [Kamata et al. (2004) Structure 12, 429-438]. A mechanistic model that brings together the kinetic and structural data is proposed which allows qualitative and quantitative analysis of the glucose-dependent GK regulation by small molecules. The regulation of GK activation by glucose may have an important implication for the discovery and design of GK activators as potential antidiabetic agents.

Assay development and data analysis of receptor-ligand binding based on scintillation proximity assay.

In this paper, we described the optimization of a generic binding assay to measure ligand-receptor interactions for peroxisome proliferator-activated receptors (PPARs). The assay is based on scintillation proximity assay, in which a protein is coated on scintillant-incorporated beads, and a radiolabeled ligand stimulates the beads to emit a signal by binding to the immobilized protein. An intrinsic binding affinity of unlabeled ligands is determined by competitive displacement of the radioligand. The protein coating and ligand binding are achieved in one step by simply mixing ligands, protein and beads in sequence. No additional steps of pre-coating and washing of beads are required. Protein is captured on beads effectively by electrostatic interactions, thus no affinity labeling of protein is required. In data analysis, ligands are grouped into two classes based on their binding affinities. For tight binding ligands, an equation is derived to accurately determine the binding affinity. Otherwise a general equation applies. This quantitative and high throughput assay provides a tool to screen a large library of molecules in search of potent ligands.