The Activated B-Cell (ABC) Like subtype of Diffuse Large B-Cell Lymphoma (DLBCL) is known for its dependence on constitutive NF- B activity. Mutated forms of the scaffolding protein CARD11 have been identified from ABC DLBCL patient samples. A high-throughput protein-protein interaction screen identified the protein C9orf9 as a potential modulator of oncogenic CARD11 activity. The proposed studies seek to characterize the mechanism of action of C9orf9 in the TCR-to-NF- B pathway, including characterizing the phenotype when C9orf9 is knocked down in Jurkat cells, confirming the location of C9orf9 in the TCR-to-NF- B pathway, and identifying critical regions or residues of C9orf9. Furthermore, the role and mechanism of C9orf9 in the DLBCL line OCI-Ly3 will be elucidated. To achieve these aims, short hairpin RNAs will be used to knock down C9orf9 in Jurkat and an NF- B luciferase reporter will be used to compare TCR-to-NF- B signaling in C9orf9 knockdown Jurkats and in a control Jurkat line. Hairpin resistant C9orf9 cDNAs will be used to rescue any phenotype in the C9orf9 knockdown lines and demonstrate TCR-to-NF- B pathway specificity. C9orf9 is hypothesized to act downstream of PKC / and upstream of the IKK complex. This location will be confirmed by stimulating C9orf9 Jurkat knockdown lines and a control line using CD3/ CD28 antibodies. Samples will be collected over a time course and Western blots probed for phospho- PKC and the I B substrate of the IKK complex. If the hypothesized location of C9orf9 is correct, phospho- PKC levels will be consistent between the C9orf9 knockdown lines and the control Jurkat line. Conversely, if C9orf9 acts above the IKK complex as predicted, I B will fail to be degraded in C9orf9 knockdown lines and will be detectable at higher levels than in control Jurkat cells. The functional region of C9orf9 will be identified by testing a series of deletion an mutation constructs in HEK293T cells for their ability to activate the NF- B luciferase reporter, where constructs that lack the functional region will not activate the reporter. Kinetic associatio of C9orf9 and CARD11 will be measured using Bioluminescence Resonance Energy Transfer (BRET). If C9orf9 and CARD11 associate in a stimulation dependent manner, this interaction will be measured as an increase in BRET signal in Jurkat cells. Stimulation-dependent associations of CARD11 and its cofactors will be measured by immunoprecipitation and Western blot in C9orf9 Jurkat knockdown lines and in a control Jurkat line. If C9orf9 is required for binding of a known cofactor with CARD11, less of that co-factor will be detectable in Western blots of co-immunoprecipitation assays using C9orf9-deficient cells. Finally, the role of C9orf9 in the OCI-Ly3 DLBCL cell line will be elucidated. If C9orf9 is required for OCI-Ly3 survival, shRNA infected OCI-Ly3 cells will show rapid cell death as compared to OCI-Ly3 cells infected with the non-target hairpin. The proposed studies seek to confirm C9orf9 as a critical part of the CARD11-to-NF- B pathway in ABC DLBCL and as a novel therapeutic drug target for DLBCL treatment.
Diffuse Large B Cell Lymphoma (DLBCL) accounts for 30-40% of all Non-Hodgkin's Lymphomas. The Activated B Cell-Like subtype is the least treatable form of DLBCL and requires constitutive NF- B activity for survival. The proposed research seeks to identify previously unknown regulators of key components of the NF- B pathway and may provide a new target for the treatment of ABC DLBCL.
|Pedersen, Sarah M; Chan, Waipan; Jattani, Rakhi P et al. (2015) Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181. Mol Cell Biol 36:794-808|