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

A P Komarov,C Frangou,A L Osterman,E A Komarova,K Green,A Eroshkin,P S Baker,A A Chenchick,L R Novototskaya,A V Gudkov

doi:10.1038/cddis.2016.45

Abstract

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.

Highlights

In addition to its established role in autoimmunity and tumor surveillance,[1,2] the prototypic death receptor (DR) FAS has an important role in the pathogenesis of numerous diseases.[3,4,5,6] in the liver, high expression of FAS has been implicated in the pathogenesis of viral hepatitis, inflammatory hepatitis, Wilson's disease, alcoholic liver disease, and chemotherapy-induced liver damage.[7,8,9]
Computational prediction of drug–target interactions using network-driven shRNA data prioritization and integration allowed us to ‘reposition’ existing pharmacological agents for inhibition of two candidate targets, somatostatin receptor 5 (SSTR5) and PPP2R5A. These agents completely abrogated otherwise lethal liver failure induced by FAS agonistic Ab or concanavalin A (ConA) administration in mice demonstrating their potential for prevention or treatment of acute liver failure (ALF) and other conditions associated with DR-mediated apoptosis known to be involved in pathogenesis of neuronal,[18] cardiac,[19] pulmonary,[20] renal 21 and other diseases.[22,23]
Mouse models established to simulate and study apoptotic pathways leading to ALF in vivo include treatment with concanavalin A (ConA), galactosamine, LPS, TNF, or anti-FAS agonistic Ab JO2.12–14 From our primary screens and analyses outlined above, we identified 102 different shRNAs targeting potential mediators of DR-dependent apoptosis

Summary

Introduction

In addition to its established role in autoimmunity and tumor surveillance,[1,2] the prototypic DR FAS ( called CD95 or APO-1) has an important role in the pathogenesis of numerous diseases.[3,4,5,6] in the liver, high expression of FAS has been implicated in the pathogenesis of viral hepatitis, inflammatory hepatitis, Wilson's disease, alcoholic liver disease, and chemotherapy-induced liver damage.[7,8,9]. We established an RNAi screening strategy to systematically identify genetic modifiers of FAS- and TNF-mediated apoptosis for potential use as therapeutic targets in treatment of pathologies associated with the activation of DR-mediated apoptosis Using this approach, we identified both canonical components and novel factors that, upon RNAi-mediated knockdown, suppress FAS- and/or TNF-mediated apoptosis in vitro. Computational prediction of drug–target interactions using network-driven shRNA data prioritization and integration allowed us to ‘reposition’ existing pharmacological agents for inhibition of two candidate targets, SSTR5 and PPP2R5A These agents completely abrogated otherwise lethal liver failure induced by FAS agonistic Ab or ConA administration in mice demonstrating their potential for prevention or treatment of ALF and other conditions associated with DR-mediated apoptosis known to be involved in pathogenesis of neuronal,[18] cardiac,[19] pulmonary,[20] renal 21 and other diseases.[22,23]

Methods

Results

Conclusion