The central theme of this project is to determine whether BCL-2 is critical for the maintenance of B cell lymphoma as well as its genesis. If required, BCL-2 members and the apoptotic pathway constitute an attractive, validated target to develop small molecule regulators. We will generate a conditional transgenic model of BCL-2 overexpression and assess the effects of eliminating BCL-2 on the maintenance of established lymphoma. Similarly, we will determine the role of the B cell receptor (BCR) complex and its downstream signaling pathways on maintenance by eliminating or even replacing a conditional anti-NP IgH knock-in allele in established lymphoma. The effects of chronic antigenic stimulation on the progression of lymphoma will also be assessed in this model. The genetic changes during B cell tumor progression will be defined by RNA expression profiling, spectral karyotyping (SKY), single nucleotide polymorphisms (SNP), or comparative genomic hybridization (CGH). Comparison of these genetic changes with those of human lymphomas will lead to refinement of the mouse model. Finally, we will identify small molecules that regulate BCL-2 family member function. Candidate molecules from conventional screens that bind to the BCL-2 """"""""groove"""""""" to displace BH3 domain partner proteins will be assessed for mechanism of action in our multiple mammalian cell based assays, yeast based assays, and purified mitochondria assays. Evidence indicates that the active conformations of many BCL-2 members, especially pro-apoptotic BAX and BAK, is a pore-forming oligomer, therefore, we will better define this intramembranous structure and its regulation. This will enable novel high throughput screens (HTS) that identify candidate small molecules by their ability to affect BCL-2 member activity using yeast based assays, epitope exposure assays by mammalian cytoblots, and pore activity assays. Candidate pro-apoptotic small molecules comprise a ?tool box"""""""" to further interrogate a genetically validated pathway and potential lead compounds to ultimately test in these B cell lymphoma models.
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