Frequent over-expression and mutation of B-cell receptor (BCR) signaling molecules contributes to the pro survival phenotype and aggressive nature of diffuse large B-cell lymphoma (DLBCL). To understand the mechanisms that underlay the deregulation of this important pathway and ultimately improve the outcome of patients with this often refractory and relapse prone disease, we developed a therapeutic research program to investigate the role of and target DNA secondary structures within BCR regulatory genes. We propose that promoter G-quadruplex (G4) DNA structures regulate expression of BCR-related genes and can be targeted to silence oncogenic signaling. In preliminary studies, we discovered the presence of G4 forming sequences Nithin BCR signaling genes, CARD11 and MYDBB. We screened the NCI Diversity Set library of compounds 3nd identified small molecules that stabilize these structures, lower mRNA levels, and sensitize DLBCL cells to standard chemotherapy. Here, we will use these G4 stabilizing compounds as molecular tools to define the 11echanism by which these DNA structures regulate the BCR pathway in DLBCL cell line and patient derived :ells in vitro. We will determine the subsequent effects on BCR signaling by performing phospho-proteomics to 3ssess repression of downstream BCR molecules including NF-KB and ERK. We will then translate these studies into a DLBCL patient derived xenograft mouse model to demonstrate the extent of silencing CARD11 md MYDBB expression on tumor growth and chemosensitivity. We will also test our hypothesis that G4s play a ole in the BCR mutations induced by the antibody diversification enzyme, activation induced cytidine foaminase (AID). In support of this idea, we found a genome wide enrichment for G4 forming sequences Nithin AID hotspots that overlay with oncogene mutations in patient samples. We will now define the nvolvement of G4 structures in the mechanism for AID induced CARD11 and MYDBB mutations by evaluating he recognition and binding of AID to G4 structures with binding assays, spectroscopy, and ChlP-sequencing. Ne will perform functional assays where the G4 sequences are used as bait in an established GFP mutation eporter assay in the presence of AID to assess how G4s impact mutation frequency. Our findings will provide nsight into the mechanisms leading to the aberrant BCR signaling and demonstrate the CARD11 and MYDBB 34s can serve as therapeutic targets for inhibiting these oncogenic signals and tumor growth. Transcriptionally egulating BCR signaling genes has high potential for uncovering a treatment breakthrough for the clinical nanagement of patients with BCR-dependent DLBCL. This work will also establish models for continued nvestigations and accumulate data for a competitive R01 application.
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