| Popis: |
The rapid, exponential clonal expansion of lymphocytes necessary for an effective immune response to a pathogen is accompanied by a tremendous amount of genomic stress, such that these cells are pushed to the verge of genotoxic death. Studies performed by our lab and our collaborators have shown that upon activation, T cells display markers of spontaneous DNA damage and DDR, including DSBs, ?H2AX, phospho-p53, phospho-ATM, and phospho-Chk2, providing mechanistic insight into the distinct sensitivity activated lymphocytes display in response to chemotherapeutics and radiation. Nevertheless, aberrant inflammatory conditions and autoimmunity can and do occur. The goal of the research described in this dissertation was to explore the therapeutic potential associated with this unique trait of lymphocyte biology through the manipulation of DDR signaling molecules with small molecule inhibitors. Our lab and our collaborators have shown that the administration of the chemotherapeutic etoposide at temporal points chosen to target activated T cells during periods of maximal expansion can significantly ameliorate symptoms of EAE and the CD8 T cell-driven inflammatory disorder hemophagocytic lymphohistiocytosis (HLH) by selectively purging activated pathogenic T cells from the repertoire. Further, we and our collaborators have shown that this selective elimination of activated pathogenic cells is possible without the use of drugs that induce genotoxic damage. This is vital because while etoposide, and other topoisomerase II inhibitors, are highly efficacious in targeting activated lymphocytes, they also produce discrete DNA damage that often leads to secondary cancer development. Instead, we have found that it is possible to achieve the same effects in these models by taking advantage of the genomic stress that activated lymphocytes are under using small molecule inhibitors targeting the cell cycle checkpoint kinases Chk1/2 and Wee1 and the negative regulator of p53, MDM2. The checkpoint kinases targeted govern cell cycle arrest in S phase and at the G2/M checkpoint, and would normally function to prevent the cell from continuing to replicate its DNA in preparation for division and from entering into mitosis in response to DNA damage by the inhibitory phosphorylation of CDKs in order to guard from mutations resulting in dysfunction or oncogenesis in daughter cells. Inhibiting these kinases results in premature cell cycle progression in activated proliferating cells, DNA damage, and death due to mitotic catastrophe. The addition of an inhibitor of MDM2 allows significantly greater intracellular p53 accumulation, which has the effect of synergistically increasing the amount of apoptosis caused by these kinase inhibitors in activated T cells. Importantly, this combination of inhibitors (termed “p53 potentiation with checkpoint abrogation,” or PPCA) has very little or no impact on Tregs, naive T cells, quiescent memory T cells, and clearance of concurrent viral infections. |
