Functional genetics-directed identification of novel pharmacological inhibitors of FAS- and TNF-dependent apoptosis that protect mice from acute liver failure.

shRNA-mediated gene-silencing technology paired with cell-based functional readouts reveals potential targets directly, providing an opportunity to identify drugs against the target without knowing the precise role of the target in the pathophysiological processes of interest. By screening a lentiviral shRNA library targeting for major components of human signaling pathways and known drug targets, we identified and validated both canonical as well as 52 novel mediators of FAS and TNF ligand-induced apoptosis. Presence of potential therapeutic targets among these mediators was confirmed by demonstration of in vivo activity of siRNAs against four identified target candidates that protected mice from acute liver failure (ALF), a life-threatening disease with known involvement of death receptor (DR)-mediated apoptosis. Network-based modeling was used to predict small-molecule inhibitors for several candidate apoptosis mediators, including somatostatin receptor 5 (SSTR5) and a regulatory subunit of PP2A phosphatase, PPP2R5A. Remarkably, pharmacological inhibition of either SSTR5 or PPP2R5A reduced apoptosis induced by either FASL or TNF in cultured cells and dramatically improved survival in several mouse models of ALF. These results demonstrate the utility of loss-of-function genetic screens and network-based drug-repositioning methods for expedited identification of targeted drug candidates and revealed pharmacological agents potentially suitable for treatment of DR-mediated pathologies.

Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53.

The anti-cancer agent paclitaxel (Taxol) stabilizes microtubules leading to G2/M cell cycle arrest and apoptotic cell death. In order to analyse the molecular mechanisms of Taxol-induced cytotoxicity, we studied the involvement of mitogen-activated protein kinases (MAPK) ERK and p38 as well as the p53 pathways in Taxol-induced apoptosis. The human breast carcinoma cell line MCF7 and its derivatives, MCF7/HER-2 and MDD2, were used in the study. We found that Taxol treatment strongly activated ERK, p38 MAP kinase and p53 in MAP kinase MCF7 cells prior to apoptosis. PD98059 or SB203580, specific inhibitors of ERK and p38 kinase activities, significantly decreased apoptosis, leaving the surviving cells arrested in G2/M. These inhibitors did not significantly affect Taxol-induced alterations in the cell cycle regulatory proteins Rb, p53, p21/Waf1 and Cdk-2. In addition, inactivation of p53 did not affect cellular sensitivity to Taxol killing. However, cells with inactivated p53, unlike cells harboring wild type p53, failed to arrest in G2/M after treatment with Taxol and continued to divide or go into apoptosis. Our data show that both ERK and p38 MAP kinase cascades are essential for apoptotic response to Taxol-induced cellular killing and are independent of p53 activity. However, p53 may serve as a survival factor in breast carcinoma cells treated with Taxol by blocking cells in G2/M phase of the cell cycle.