Lymphoma microenvironment has a crucial role in lymphoma initiation, progression and drug resistance. Fibroblastic reticular cells (FRCs) participate dynamically in the cytokine microenvironment of the lymph node paracortex as well as the regulation of cell trafficking and access of T-cells into the paracortex. The FRC niche also participates in the cytokine microenvironment of lymphomas providing homing and survival signals including CCL19, CCL21, IL-7, CXCL12 and hedgehog (Hh) ligands. Hh signaling is evolutionary conserved signaling pathway that serves several physiological and development processes mediated by externally secreted Hh ligands. We previously demonstrated that the canonical Hh ligand-PTCH1-SMO-GLI axis is functional and contributes to cell survival, proliferation and enhances chemo tolerance in DLBCL. We further showed that GLI1, Hh signaling transcription factor is aberrantly activated in ∼87% of DLBCL and this activation is in part mediated by sonic Hh ligands secreted by stromal cells. Although the importance of GLI1 in tumor development is well recognized, the molecular mechanisms controlling the transcriptional activity of GLI1 is limited. To identify regulatory components that participate in the transcriptional activity of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immunoprecipitation of endogenous GLI1. We identified the inhibitor of NF-ĸB kinase, IKKβ as a novel GLI1-binding protein. In addition, we found that the kinase activity of IKKβ is critical to recruit and phosphorylate GLI1 in the C-terminal fragment. We further showed that in DLBCL cells stimulated with TNFα, IKKβ phosphorylates GLI1 and restricts the binding between GLI1 with HECT-type E3 ubiquitin ligase (ITCH) resulting in GLI1 stability and increased oncogenic potential. Inhibition of IKKβ-mediated GLI1 phosphorylation restored the binding between GLI1 and ITCH and promoted ubiquitination and degradation of GLI1. Collectively, these results indicate that IKKβ regulates the transcriptional activity of GLI1 by phosphorylating GLI1 and modulating the binding between GLI1 and ITCH. In DLBCL, canonical NF-κB and GLI1 mediated Hh signaling pathways are strongly responsive with tumor microenvironment; our study will help to understand the cross talk between these two pathways which could represents a novel therapeutic approach for DLBCL.
Citation Format: Nitin K. Agarwal, Kranthi Kunkalla, Francisco Vega. The inhibitor of NF-ĸB kinase, IKKβ, regulates the stability of GLI1 transcription factor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3605. doi:10.1158/1538-7445.AM2014-3605