Abstract LB-225: E2F1 potentiates proteasome inhibition-mediated cell death in leukemia cells through induction of Noxa

Abstract

Abstract While proteasome inhibitors exhibit significant activity in multiple myeloma and lymphoma, the proteasome inhibitor (PI) bortezomib displays little single-agent activity in patients with acute myeloid leukemia (AML). However, the ability of PIs to inhibit NF-κB, critical for AML stem cell survival, has sustained interest in this class of agents in AML. Indeed, treatment with bortezomib in combination with Idarubicin and Cytarabine has shown significant activity in patients with AML. Here we demonstrate that the transcriptional factor E2F1 plays an important role in determining the activity of the PIs bortezomib or carfilzomib in AML cells. Specifically, overexpression of human E2F1 in U937 cells using a tetracycline inducible system dramatically enhanced cell death mediated by bortezomib or carfilzomib in association with increased cytochrome c and AIF release into the cytosol and caspse-3 activation reflected by cleavage of caspase-3 and PARP. Of note, E2F1 overexpression by itself triggered a modest increase in cell death. Conversely, knockdown of E2F1 using shRNA significantly diminished bortezomib- or carfilzomib-induced apoptosis. Importantly, synergistic lethality between bortezomib and the histone deacetylase inhibitor (HDACI) vorinostat was largely abrogated by E2F1 knockdown, and conversely, significantly enhanced by E2F1 over-expression. These findings argue for a critical functional role for E2F1in anti-leukemic synergism between PIs and HDACIs. Significantly, over-expression of E2F1 markedly increased, whereas knockdown of E2F1 significantly diminished Noxa protein levels in cells exposed to bortezomib or carfilzomib. Furthermore, immunoprecipitation analysis revealed that E2F1 over-expression induced a pronounced increase in PI-mediated Noxa binding to Mcl-1, an effect that correlated with Bim binding to Mcl-1, suggesting displacement of Bim from Mcl-1 by Noxa. To determine what role Noxa might play in enhancement of PI-induced apoptosis by E2F1, Noxa was knocked down with shRNA in cells displaying inducible enforced expression of E2F1. Notably, knockdown of Noxa abrogated the ability of E2F1 to enhance bortezomib or carfilzomib lethality, thus demonstrating that Noxa plays a key functional role in E2F1-mediated sensitization of cells to proteasome inhibitors. Collectively, these findings argue that E2F1 is an important determinant of the sensitivity of AML cells to PIs through modulation of Noxa protein levels. They also suggest that AML cells with low E2F1 activity might be intrinsically resistant to PIs. Such findings also raise the possibility that agents capable of activating E2F1 e.g., by disrupting pRb/E2F1 interactions, might enhance the activity of PIs in leukemia and potentially other hematological malignancies. Citation Format: Mohamed Rahmani, Mandy Mayo Aust, Steven Grant. E2F1 potentiates proteasome inhibition-mediated cell death in leukemia cells through induction of Noxa. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-225. doi:10.1158/1538-7445.AM2014-LB-225