Inhibition of p53 function, either through mutation or inhibition by viral transforming proteins, correlates strongly with the oncogenic potential of the cell. Here, we describe a unique mechanism of p53 inactivation that involves the interaction of p53 with the p65 subunit of NF-kB. The inactive p53 complex is induced in HTLV-1-transformed and ATL leukemic cells. This mechanism of p53 inhibition may occur in other human cancers. We initially demonstrated that wild-type p53 is stabilized and transcriptionally inactive in HTLV-transformed cells. The viral transcriptional activator Tax plays a role in both the stabilization and inactivation of p53. p53 is hyperphosphorylated at serines 15 and 392 in HTLV-1-transformed cells and phosphorylation of p53 at these specific residues inactivates p53 by blocking its interaction with basal transcription factors. In T-lymphocytes, Tax-induced p53 inactivation is dependent upon NF-kB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-kB. Further, the p65 subunit of NF-kB is critical and uniquely involved in the Tax-induced p53 inhibition pathway. Using chromatin immunoprecipitation assays we have determined that in HTLV-1-transformed cells, p53 and p65 form a complex on the inactive MDM2 promoter. Consistent with reduced transcription activity, TFIID binding is not observed. These studies provide evidence that the divergent NF-kB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels which include post-translational modification of p53. Further studies determined that p53 inhibition by Tax required a unique IKKbeta complex that required AKT activation. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant resulted in increased Tax-mediated NF-kB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT siRNA prevented NF-kB activation and inhibition of p53. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kB and p53. In more recent studies, we examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is """"""""druggable"""""""" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kB suggests a promising strategy for the treatment of HTLV-1-transformed cells. We further extended these studies to determine if the mechanism of p53 inactivation in HTLV-1 transformed cells could be more generalized in human cancers. To this end we examine the NCI-60 cells. Of the 60 cell lines, 9 had a wild type but impaired p53 protein. In 8 of the 9 cell lines which include renal carcinoma, melanoma, glioblastoma and ovarian carcinoma cells, 9AA treatment increased p53 transcriptional activity as measured by p53-promoter reporter assays and quantification of mRNA levels of p53 responsive genes MDM2 and p21(WAF1). Transcriptional activation of p53 resulted in reduced cell viability and increased apoptosis. Further we demonstrated that 9AA induced cell death is blocked when siRNA to p53 is used, linking reactivation of p53 activity to cell death. Similar to HTLV-1-transformed cells, 9AA reduced NF-kB activity and AKT activity in a melanoma cell line, LOX-IMVI. However, 9AA treatment did not affect NF-kB and/or AKT activity in the remaining NCI-60 cell lines tested. These results indicate that 9AA is effective at reactivating p53 function by various mechanisms depending on the cell type providing a promising therapeutic strategy. Using tumor cell lines we have identified drugs which target the p53 and NF-kB pathways. Our efforts are no focused on the use of these chemotherapeutic agents for the treatment of ATL. In collaboration with Thomas Waldmann's group we are investigating the efficiacy of 9AA, AKT inhibitors and IKKbeta inhibitors in the ATL mouse model system. Current studies are underway to address pharmacokinetics, toxicity and efficacy of these inhibitors in the MET-1 NOD/SCID mouse model. More recently we have investigated alternative anticancer drug approaches. A new anticancer drug, GGTI-298, has been demonstrated to decrease cell viability in multiple transformed cell lines. The effects of GGTI-298 have been attributed to the inhibition of geranylgeranylated proteins, including the Rho family of small GTPases. Indeed, we found that small GTPases are active in HTLV-1-transformed cells and that these cells are preferentially sensitive to prenylation inhibitors, such as GGTI-298. We found that GGTI-298 decreased cell viability and induced apoptosis in HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-kB, a downstream target of Rho and Tax, suggesting a possible mechanism leading to cell death. These studies suggest that activation of small GTPases contribute to viral expression and cellular dysregulation of HTLV-1-transformed cells and that geranylgeranyl transferase inhibitors may provide a novel class of therapeutic agents for treatment of ATL.
