Rapid TNFR1-dependent lymphocyte depletion in vivo with a selective chemical inhibitor of IKKβ

Abstract

The transcription factor NF-B plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKK␤, an Ikappa IntroductionNF-B plays a central role in inflammation and apoptosis, making it a primary target for inhibition for treatment of inflammatory disease and cancer. 1,2 NF-B consists of subunits that associate in dimers. 3,4 The commonly described dimer forms are p50 (NF-B1) with RelA (p65) and p52 (NF-B2) with RelB. IB␣ holds the p50/RelA complex in the cytoplasm in an inactive form. NF-B nuclear translocation is regulated by upstream signaling events initiated primarily by proinflammatory stimuli, resulting in activation of IKK. 5 IKK contains 3 related subunits: ␣, ␤, and ␥. IKK␤ is the subunit responsible for phosphorylating IB␣, resulting in its ubiquitination and subsequent proteasomal degradation. [6][7][8] Released NF-B translocates to the nucleus to initiate transcription of response genes, which include proinflammatory and antiapoptotic genes. 9 In addition to phosphorylating IB␣, IKK␤ also phosphorylates the transcription factor FOXO3a, which is also degraded by the proteasome. 10 FOXO3a that is not phosphorylated by IKK␤ can translocate to the nucleus where it upregulates expression of genes that control cell cycle progression [11][12][13] and induce apoptosis. 14,15 NF-B activation has been associated with several human immune modulated diseases including arthritis, asthma, diabetes, stroke, inflammatory bowel disease, and atherosclerosis. 16 In addition, IKK␤ and NF-B have been shown to play a direct role in regulating the development of insulin resistance 17,18 and in severe muscle wasting. 19 Recently, it has been demonstrated that IKK␤ and NF-B play a key role in inflammation-associated cancer and tumor progression. [20][21][22][23][24] These data suggest that inhibition of IKK␤ in vivo with a specific small molecule may be a potential therapeutic approach for several human diseases.In addition to controlling inflammatory response and apoptosis, IKK␤ and NF-B are required for normal lymphocyte development. Mice deficient in the p65 (RelA) subunit of NF-B or IKK␤ die during fetal development through a TNF-dependent mechanism. 6,25,26 Generation of p65-or IKK␤-deficient mice on a TNF␣-or TNFR-deficient background rescues mice from embryonic lethality. [27][28][29] Studies evaluating the requirement for NF-B in hematopoiesis using transplantation of fetal liver stem cells from p65-or IKK␤-deficient mice into lethally irradiated hosts have revealed a specific requirement for these molecules in development of T cells, B cells, and common lymphoid progenitors but not myeloid cells or stem cells and a role in regulating the production of granulocytes. [30][31][32] We identified ML120B, a specific small chemical inhibitor of IKK␤ that protects against cartilage and bone destruction in adjuvant and collagen-induced models of arthritis. 33 Here we demonstrate that IKK␤ inhib...

Opposing functions of IKKβ during acute and chronic intestinal inflammation

“…3C). The latter indicates increased neutrophil numbers in the colon after ML120B treatment, which is consistent with reports of blood neutrophilia caused by IKK␤ inhibition (15,20). These results, together with the cytokine analyses ( Fig.…”

“…The attenuation of STAT3 activation was not related to any direct effects of ML120B on epithelial STAT3 signaling (Fig. 2C), consistent with a previous report that ML120B is a highly specific IKK␤ inhibitor (20). These results suggest that inhibition of NF-B indirectly attenuates epithelial STAT3 signaling, resulting in loss of cytoprotective HSP70 in IECs.…”

“…To exclude the possibility that the observed late healing phenotype might have been caused by increased early damage induced by increased apoptosis in IKK␤-deficient enterocytes (13), we treated WT mice after completion of DSS administration with a highly specific IKK␤ inhibitor, ML120B (20), or vehicle for 5 days (Fig. 1F).…”

Compensatory IKKα activation of classical NF-κB signaling during IKKβ inhibition identified by an RNA interference sensitization screen

“…Two structurally related small-molecule inhibitors of IK⌲␤, MLX105. and MLN120B block the NF-B pathway and are toxic for ABC DLBCL cell lines (5,8,9). The specificity of these inhibitors is highlighted by their lack of toxicity for cells without NF-B pathway activation and by the fact that their toxicity for ABC DLBCL cells can be prevented by expressing constitutively active forms of NF-B (5).…”

“…Likewise, IKK␣ acted as an I B␣ kinase during CD40 and CD27 stimulation of a Burkitt lymphoma cell line (12), and during lymphotoxin ␣ stimulation of fibroblasts (24). Although IKK␤ is required for the maintenance of all mature B cells (8,25,26), the role of IKK␣ in B lymphocyte development is more restricted. B-cell-intrinsic IKK␣ kinase activity is required for the development of germinal center B cells and their differentiation into long-lived plasma cells (27).…”