Clinical cancer research

Stabilization of Notch1 by the Hsp90 Chaperon is Crucial for T Cell Leukemogenesis

Zhaojing Wang, Yufeng Hu, Daibiao Xiao, Jingchao Wang, Chuntao Liu, Yisheng Xu, Xiaomeng Shi, Peng Jiang, Liang Huang, Peng Li, Hudan Liu and Guoliang Qing*

Abstract

Purpose: Notch1 deregulation is assuming a focal role in T-cell acute lymphoblastic leukemia (T-ALL). Despite tremendous advances in our understanding of Notch1 transcriptional programs, the mechanisms by which Notch1 stability and turnover are regulated remain obscure. The goal of the present study is to identify intracellular Notch1 (ICN1, the activated form of Notch1) binding partner(s) regulating its stability and activity. Experimental Design: We employed immunoaffinity purification to identify ICN1-associating partner and used co-immunoprecipitation to verify the endogenous protein interaction. Pharmacological or shRNA-mediated inhibition was applied in loss-of-function assays to assess the role of tentative binding partner(s) in modulating ICN1 protein stability as well as affecting T-ALL cell expansion in vitro and in vivo. Mechanistic analysis involved protein degradation and polyubiquitination assays. Results: We identify the Hsp90 chaperon as a direct ICN1-binding partner essential for its stabilization and transcriptional activity. T-ALL cells exhibit constitutive endogenous ICN1-Hsp90 interaction and Hsp90 depletion markedly decreases ICN1 levels. The Hsp90-associated E3 ubiquitin ligase Stub1 mediates the ensuring proteasome-dependent ICN1 degradation. Administration of 17-AAG or PU-H71, two distinct Hsp90 inhibitors, depletes ICN1, inhibits T-ALL cell proliferation and triggers dramatic apoptotic cell death. Systemic treatment with PU-H71 reduces ICN1 expression and profoundly inhibits murine T-ALL allografts as well as human T-ALL xenografts. Conclusions: Our findings demonstrate Hsp90 blockade leads to ICN1 destabilization, providing an alternative strategy to antagonize oncogenic Notch1 signaling with Hsp90 selective inhibitors.

·         DOI: 10.1158/1078-0432.CCR-16-2880 Published 31 January 2017

·         Received November 17, 2016.

·         Revision received January 6, 2017.

·         Accepted January 22, 2017.

·         Copyright ©2017, American Association for Cancer Research.