A Fourth Outcome: DNA Damage and the Differentiation of B Cells

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DESCRIPTION (provided by applicant): During an immune response, B cells undergo rapid proliferation and remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, genotoxic stress associated with the GC reaction also promotes most B cell malignancies. We recently discovered that ATM, activated by AID-dependent DNA double stranded breaks (DSBs) during IG class switch recombination (CSR) in GC B cells, signals through LKB1 to inactivate CRTC2, a known transcriptional co-activator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation into antibody (Ab)-secreting plasma cells while opposing lymphomagenesis. Defects in this pathway were identified in pilot studies of human B cell lymphoma samples by ATM or LKB1 repression or by a recently identified somatic mutation or genetic polymorphism in CRTC2. These pathway alterations are predicted to result in increased GC B cell proliferation and impaired plasma cell differentiation, which will be tested here in vitro and in vivo. Our data show a new outcome for the DNA damage response (DDR) using B lymphocytes as the model system. It is known that DNA damage activates a cellular DDR, which determines 3 main cell fates: 1) transient cell cycle arrest with DNA repair and cycle reentry, 2) permanent exit from the cell cycle (senescence), or 3) apoptosis. Here, we propose to define key molecular determinants and the significance of an unexpected fourth outcome for DNA damage, which is to drive precursor cell maturation, in this case from a GC B cell to an Ab-secreting plasma cell. In a sense, this new outcome is a form of cell senescence, in that a cell with potentially tumorigenic DNA damage is forced out of a rapidly dividing precursor pool to protect the host from cancer. However, this fourth DDR option differs significantly from senescence by coupling with differentiation, which leads to an essential new function, Ab production against infectious agents. The pathway we identified is DSB-initiated ATM->LKB1->"X"->"Y"/CRTC2-> target gene expression that controls the transition from a GC B cell to a plasma cell. In Aim 1, we will identify ~85 kDa LKB1 direct target phosphoprotein "X" by candidate elimination from the 14 member AMPK family and/or by biochemistry- mass spectrometry analysis, followed by shRNA and over-expression studies in a unique human GC B cell differentiation system. In Aim 2, we will identify CREB-independent CRTC2-interacting "Y" factor(s) that control a gene program that mediates DSB-induced differentiation into plasma cells. These two identification and function aims are essential to complete this novel signaling pathway and to link with Aim 3 studies. In Aim 3, we provide pre-clinical and clinical relevance by analysis of a unique LKB1 B-lineage knockout (KO) mouse and we determine whether pathway defects in human GC B cell lymphomas result from inherited or somatic alterations. Overall, we dissect a new and unexpected fourth outcome for DNA damage- cell differentiation.