MODULATION OF ANTIGEN RECEPTOR AND ONCOGENIC SIGNALING TO NF-κB BY C9ORF9

The adaptive immune system utilizes tightly regulated signaling networks to fight off pathogens while remaining controlled enough to avoid harmful conditions like autoimmunity and cancer. While many of the steps in antigen receptor signaling are well characterized, some of the mechanisms and proteins that are active in these pathways remain unknown. This thesis identifies a potential modulator of NF-κB signaling in the antigen receptor pathways. A protein-protein interaction screen was conducted to identify proteins which interact with the scaffolding molecule CARD11. Secondary screening of the initial hits identified C9orf9 as a potential modulator of signaling in the pathways of interest. Knockdown studies identified a specific role for C9orf9 in antigen receptor-to-NF-κB signaling, but not in antigen receptor-to-NFAT signaling or TNF-to-NF-κB signaling. Significantly, these knockdown studies also confirmed the presence of two isoforms of C9orf9 in Jurkat T cells. While it was not possible to separate the functions of the two isoforms in Jurkat T cells, knockdown of both isoforms led to changes in phosphorylation and activation of pathway proteins, which could explain the role of C9orf9 in modulating NF-κB activity. Studies in 293T cells showed that C9orf9 interacts with several members of the CARD11 signaling complex, including Bcl10. Interestingly, C9orf9 has the ability to enhance the signaling capabilities of hyperactive CARD11 variants as well as Bcl10. This was true regardless of the mechanism of action of the hyperactive CARD11 variants. A knockout mouse was generated using CRISPR/Cas9 technology in the hope that the removal of C9orf9 would lead to a defect in B and/or T cell development or activation. While this was not observed, C9orf9 knockout mice were born slightly out of Mendelian ratios in a pattern that mimics the one seen in Bcl10 knockout mice. This observation, as well as the ability of C9orf9 to associate with Bcl10, suggests that the ability of C9orf9 to modulate NF-κB signaling may hinge on the interplay between the two isoforms of C9orf9 and Bcl10. This work introduces C9orf9 into the pantheon of antigen receptor signaling proteins and suggests further questions to understand how antigen receptor signaling is regulated