Therapeutic Effects of a TANK-Binding Kinase 1 Inhibitor in Germinal Center-Driven Collagen-Induced Arthritis.

OBJECTIVE: The production of class-switched high-affinity autoantibodies derived from organized germinal centers (GCs) is a hallmark of many autoimmune inflammatory diseases, including rheumatoid arthritis (RA). TANK-binding kinase 1 (TBK-1) is a serine/threonine kinase involved in the maturation of GC follicular helper T (Tfh) cells downstream of inducible costimulator signaling. We undertook this study to assess the therapeutic potential of TBK-1 inhibition using the small-molecule inhibitor WEHI-112 in antibody-dependent models of inflammatory arthritis. METHODS: Using the models of collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum-transfer-induced arthritis (STIA), we determined the effectiveness of WEHI-112 at inhibiting clinical and histologic features of arthritis in C57BL/6 and DBA/1 mice. We used immunohistochemistry to characterize GC populations during CIA development, and we used enzyme-linked immunosorbent assays to determine levels of Ig autoantibodies in WEHI-112-treated mice compared to vehicle-treated mice. RESULTS: WEHI-112, a tool compound that is semiselective for TBK-1 but that also has activity against IKKε and JAK2, abolished TBK-1-dependent activation of interferon (IFN) regulatory factor 3 and inhibited type I IFN responses in vitro. In vivo, treatment with WEHI-112 selectively abrogated clinical and histologic features of established, antibody-dependent CIA, but had minimal effects on an antibody-independent model of AIA or on K/BxN STIA. In keeping with these findings, WEHI-112 reduced arthritogenic type II collagen-specific IgG1 and IgG2b antibody production. Furthermore, WEHI-112 altered the GC Tfh cell phenotype and GC B cell function in CIA. CONCLUSION: We report that TBK-1 inhibition using WEHI-112 abrogated antibody-dependent CIA. As WEHI-112 failed to inhibit non-antibody-driven joint inflammation, we conclude that the major effect of this compound was most likely the targeting of TBK-1-mediated mechanisms in the GC reaction. This approach may have therapeutic potential in RA and in other GC-associated autoantibody-driven inflammatory diseases.

Design, Synthesis, and Biological Activity of 1,2,3-Triazolobenzodiazepine BET Bromodomain Inhibitors.

A number of diazepines are known to inhibit bromo- and extra-terminal domain (BET) proteins. Their BET inhibitory activity derives from the fusion of an acetyl-lysine mimetic heterocycle onto the diazepine framework. Herein we describe a straightforward, modular synthesis of novel 1,2,3-triazolobenzodiazepines and show that the 1,2,3-triazole acts as an effective acetyl-lysine mimetic heterocycle. Structure-based optimization of this series of compounds led to the development of potent BET bromodomain inhibitors with excellent activity against leukemic cells, concomitant with a reduction in c-MYC expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors.

Colorectal Cancer Cell Line Proteomes Are Representative of Primary Tumors and Predict Drug Sensitivity.

BACKGROUND AND AIMS: Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. METHODS: Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. RESULTS: Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The 5 proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling, and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared with genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. CONCLUSIONS: Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared with genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine.

Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells.

The ER Ca²+ sensor STIM1 and the Ca²+ channel Orai1 are key players in store-operated Ca²+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca²+ entry by about 50-60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca²+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca²+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCeta is required for Ca²+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca²+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca²+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca²+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.

STIM1- and Orai1-dependent store-operated calcium entry regulates human myoblast differentiation.

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca(2+)](in) involving store-operated Ca(2+) entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca(2+) sensor activating Orai Ca(2+) channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca(2+)](in) and intracellular Ca(2+) store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K(+) channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion.

Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic beta-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose-stimulated secretion to the levels observed in isolated cells but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca(2+) activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca(2+) activity were corrected by transient removal of Ca(2+) or prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion, we show that dispersed pancreatic beta-cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca(2+) transients that activate downstream Ca(2+) effectors, whereas engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.

STIM1 knockdown reveals that store-operated Ca2+ channels located close to sarco/endoplasmic Ca2+ ATPases (SERCA) pumps silently refill the endoplasmic reticulum.

Stromal interaction molecule (STIM) proteins are putative ER Ca2+ sensors that recruit and activate store-operated Ca2+ (SOC) channels at the plasma membrane, a process triggered by the Ca2+ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca2+ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca2+ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca2+ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca2+ concentration above physiological values, but no cytosolic Ca2+ signals were detected during store refilling even at very high Ca2+ concentrations. [Ca2+](ER) measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca2+](ER) levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca2+ entry, because Ca2+ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol.