Non-Hodgkin lymphomas are a major cause of death in the United States, yet the molecular causes are not thoroughly understood. In many lymphomas including Diffuse Large B Cell Lymphoma (DLBCL), the scaffold protein CARD11 is mutated, and this can drive aberrant activation of the transcription factor NF-?B, as well as JNK and mTOR. In healthy lymphocytes, CARD11 connects antigen receptor engagement to NF-?B, JNK, and mTOR activation, but in lymphoma, this signaling can become dysregulated and lead to aberrant lymphocyte proliferation, activation, and survival. Downregulation of CARD11 signaling can therefore be useful in treating lymphoma, but there is currently not a complete mechanistic understanding of signal transduction through CARD11 in healthy and diseased lymphocytes. Recent mass spectrometry screens have identified QRICH1 as a novel negative regulator of CARD11 signaling to NF-?B. QRICH1 is a highly conserved protein without a known function. Mutations in QRICH1 have been identified in individuals with developmental delay disorders and chondrodysplasia, but QRICH1 has not been thoroughly studied on the molecular level. Some DLBCL cases have QRICH1 copy number losses, but QRICH1 has never before been implicated in immune signaling or linked to CARD11. In preliminary studies, QRICH1 physically interacts with CARD11 and can inhibit NF-?B activation by wild-type and oncogenic CARD11. The proposed work aims to 1) determine the mechanism by which QRICH1 regulates CARD11 signaling, and 2) characterize the role of QRICH1 in B cell proliferation and survival. In order to elucidate the mechanism of CARD11 inhibition by QRICH1, a deletion analysis will be performed to determine the region(s) of QRICH1 that are necessary and/or sufficient for inhibition, and the interactions between QRICH1, CARD11, and known CARD11 cofactors will be characterized. The signaling step(s) that QRICH1 acts on will be determined by comparing CARD11 signaling events in wild-type and QRICH1-deficient Jurkat T cells. The effect of QRICH1 on CARD11 signaling to JNK and mTOR will be resolved by comparing JNK and mTOR activation markers in wild-type and QRICH1-deficient Jurkat T cells. To characterize the role of QRICH1 in B cell proliferation and survival, QRICH1 will be knocked out in DLBCL-derived cell lines, and the effect on growth/proliferation will be quantified using a flow-cytometry based assay. QRICH1 knockout primary B cells with and without a CARD11 gain-of-function mutation will then be injected into Rag1-/- mice, and injected B cell proliferation and survival will be quantified by flow-cytometry. Finally, a QRICH1fl/fl mouse has been generated and will be crossed to Mb1-Cre for conditional knockout of QRICH1 in B cells. B cell expansion and activation will be quantified by flow-cytometry, and QRICH1fl/fl Mb1-Cre mice with and without a CARD11 GOF mutation will be monitored for B cell lymphoma. This work will advance knowledge of CARD11 signaling regulation, which can be used to develop new treatments to downregulate CARD11?s signaling output in cancer. It will also improve the molecular understanding of DLBCL, which can improve precision medicine strategies for patients.
Oncogenic CARD11 signaling is often present in Non-Hodgkin lymphomas, which kill 20,000 Americans each year. This work will thoroughly characterize a novel suppressor of normal and oncogenic CARD11 signaling and determine how loss of this suppressive protein contributes to the development of Diffuse Large B Cell Lymphoma (DLBCL). These findings can be used to develop novel pharmacological inhibitors of oncogenic CARD11 signaling and to improve treatment strategies for DLBCL patients.
