Aberrant signal transduction resulting from dysregulated phosphorylation is a hallmark of human cancer. Targeted cancer therapies are predominantly inhibitors of oncogenic kinases. While these drugs have had some clinical success, an alternative approach ? inactivation of kinase signaling by stimulating endogenous phosphatases ? has received minimal attention. Recently, our group has developed a novel series of small molecule activators of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, as a promising therapeutic approach for the treatment of cancers. PP2A is a heterotrimeric complex whose substrate specificity is dependent on one of 23 different regulatory subunits that can bind to form over 60 distinct holoenzyme complexes. Broad inhibition of holoenzyme formation using specific PP2A inhibitors, specifically the overexpression of the small T antigen of the SV40 tumor virus, renders cells resistant to SMAP therapy. This suggests that the formation of PP2A trimeric complexes is necessary for SMAP activity. However, from these studies the specific role of individual regulatory subunits in regulating the biological effects of SMAP activation of PP2A remains unclear. Preliminary data generated by targeted knockdown of a series of PP2A regulatory subunits demonstrated that specific subunit knockdown of PR72 attenuated SMAP activity in both cellular and in vivo models systems. Specifically, targeted reduction of PR72 but not B55 and B56 regulatory subunit family members resulted in blunted induction of PARP cleavage in SMAP treated cells. Additionally, stable PR72 knockdown resulted in abrogation of SMAP activity in a H358 xenograft model. Collectively, this data suggests that the PR72 regulatory subunit plays a critical role in small molecule mediated activation of PP2A's tumor suppressive activity. Here we propose to test the role of a single PP2A regulatory subunit, PR72, in mediating PP2A-dependent apoptosis using gain and loss of function experiments. The three interconnected specific aims are specifically directed at (1) determining the dependence on PR72 for PP2A-mediated cell death, (2) determining the signaling events regulated by the PR72 holoenzymes, and (3) defining the effect of SMAP binding to PP2A on PR72 holoenzyme stability.