SUMMARY Non-steroidal anti-inflammatory drugs (NSAIDs) exert their anti-cancer effects through cyclooxygenase-2 (COX-2)-dependent and -independent mechanisms. Here we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-α (RXRα). We identified an N-terminally-truncated RXRα (tRXRα) in several cancer cell lines and primary tumors, which interacted with the p85α subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-α (TNFα) promoted tRXRα interaction with the p85α, activating PI3K/AKT signaling. When combined with TNFα, Sulindac inhibited TNFα-induced tRXRα/p85α interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRα but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRα-dependent AKT activation and tRXRα tumor growth in animals.

Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy.

The orphan nuclear receptor Nur77 is an immediate-early response gene whose expression is rapidly induced by various extracellular stimuli. The aims of this study were to study the role of Nur77 expression in the growth and survival of colon cancer cells and the mechanism by which Nur77 expression was regulated. We showed that levels of Nur77 were elevated in a majority of human colon tumors (9/12) compared to their nontumorous tissues and that Nur77 expression could be strongly induced by different colonic carcinogens including deoxycholic acid (DCA). DCA-induced Nur77 expression resulted in up-regulation of antiapoptotic BRE and angiogenic VEGF, and it enhanced the growth, colony formation, and migration of colon cancer cells. In studying the mechanism by which Nur77 was regulated in colon cancer cells, we found that β-catenin was involved in induction of Nur77 expression through its activation of the transcriptional activity of AP-1 (c-Fos/c-Jun) that bound to and transactivated the Nur77 promoter. Together, our results demonstrate that Nur77 acts to promote the growth and survival of colon cancer cells and serves as an important mediator of the Wnt/β-catenin and AP-1 signaling pathways.

TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-kB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-kB-regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation. ImmunoHorizons, 2020, 4: 93-107.