Abstract 4553: Design and validation of a screening strategy for the identification of novel inhibitors of wild type and mutant CARD11 mediated BCR/NF-κB signaling.

Abstract

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma and can be divided into three distinct subtypes. The activated B cell-like (ABC) subtype is the most aggressive and is characterized by constitutive activation of the NF-κB pathway. Within the NF-κB pathway, the scaffolding protein CARD11 plays a critical role by coordinating IκB kinase activity and downstream pathway activation. With mounting evidence that CARD11 is a promising therapeutic target for the treatment of ABC-DLBCL we developed and executed a compound screening strategy with the ability to identify B cell receptor (BCR)/NF-κB pathway inhibitors and triage them based on mechanism of action. We began to screen our compound library by using the TMD-8 (ABC-DLBCL) cell line engineered with an NF-κB-dependent luciferase reporter. In order to ‘bin’ the primary hits, different stimuli were evaluated for their ability to rescue the inhibition of BCR/NF-κB activity in TMD-8 cells. This strategy allowed the identification of 3 potential classes of inhibitors: 1) inhibitors up-stream of PKCβ, that could be rescued by PMA/ionomycin; 2) inhibitors down-stream of IKKβ; 3) inhibitors acting between CARD11 and IKKβ, whose activity could be rescued by TNFα but not by PMA/ionomycin. Following triage, a hit confirmation step was designed using 293T cells engineered with an NF-κB-luciferase reporter construct and the ability to express wild-type or mutant CARD11 under the control of a Tet-responsive promoter. Inhibitors up-stream of PKCβ were confirmed by the ability of PMA/ionomycin to rescue inhibition of the NF-κB reporter. Inhibitors acting downstream of IKKβ were confirmed by the inability of TNFa to rescue. Finally, inhibitors acting between CARD11 and IKKβ were confirmed by their ability to inhibit NF-κB reporter expression in CARD11 wild-type and mutant settings without rescue by PMA/ionomycin but with rescue by TNFα. Currently, the majority of pharmacological therapeutics in clinical trials target constitutive BCR activation upstream of CARD11. As a result, activating CARD11 mutations pose a threat to the success of these therapeutics. The 10% of ABC-DLBCL patients with somatic mutations in CARD11 are likely to be resistant to these therapies and mutation to CARD11 may prove to be a common mechanism of resistance in tumors that initially respond to treatment. Consequently, therapies targeting CARD11, including mutant CARD11, or downstream NF-κB signaling components represent a valuable strategy for the treatment of ABC-DLBCL. Citation Format: Julia Zhu, Joshua C. Murtie, Rita Greco, Hongyun Wang, Monsif Bouaboula, Marion Dorsch, Francisco Adrian. Design and validation of a screening strategy for the identification of novel inhibitors of wild type and mutant CARD11 mediated BCR/NF-κB signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4553. doi:10.1158/1538-7445.AM2013-4553