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Stabilization of Notch1 by the Hsp90 Chaperone Is Crucial for T-Cell

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Stabilization of Notch1 by the Hsp90 Chaperone Is Crucial for T-Cell Published OnlineFirst January 31, 2017; DOI: 10.1158/1078-0432.CCR-16-2880 Biology of Human Tumors Clinical Cancer Research Stabilization of Notch1 by the Hsp90 Chaperone is Crucial for T-Cell Leukemogenesis Zhaojing Wang1,2,3, Yufeng Hu3, Daibiao Xiao2, Jingchao Wang2, Chuntao Liu3, Yisheng Xu4, Xiaomeng Shi4, Peng Jiang5, Liang Huang6, Peng Li7, Hudan Liu1,2, and Guoliang Qing1,2 Abstract Purpose: Notch1 deregulation is assuming a focal role in T-cell Results: We identify the Hsp90 chaperone as a direct ICN1- acute lymphoblastic leukemia (T-ALL). Despite tremendous binding partner essential for its stabilization and transcrip- advances in our understanding of Notch1 transcriptional pro- tional activity. T-ALL cells exhibit constitutive endogenous grams, the mechanisms by which Notch1 stability and turnover ICN1–Hsp90 interaction and Hsp90 depletion markedly are regulated remain obscure. The goal of the current study is to decreases ICN1 levels. The Hsp90-associated E3 ubiquitin identify intracellular Notch1 (ICN1, the activated form of ligase Stub1 mediates the ensuring proteasome-dependent Notch1) binding partner(s) regulating its stability and activity. ICN1 degradation. Administration of 17-AAG or PU-H71, two Experimental Design: We employed immunoaffinity purifi- distinct Hsp90 inhibitors, depletes ICN1, inhibits T-ALL cell cation to identify ICN1-associating partner(s) and used coim- proliferation, and triggers dramatic apoptotic cell death. Sys- munoprecipitation to verify the endogenous protein interac- temic treatment with PU-H71 reduces ICN1 expression and tion. Pharmacologic or short hairpin RNA–mediated inhibition profoundly inhibits murine T-ALL allografts as well as human was applied in loss-of-function assays to assess the role of T-ALL xenografts. tentative binding partner(s) in modulating ICN1 protein sta- Conclusions: Our findings demonstrate Hsp90 blockade bility as well as affecting T-ALL cell expansion in vitro and in leads to ICN1 destabilization, providing an alternative strategy vivo. Mechanistic analysis involved protein degradation and to antagonize oncogenic Notch1 signaling with Hsp90-selective polyubiquitination assays. inhibitors. Clin Cancer Res; 1–13. Ó2017 AACR. Introduction variety of tumors, including T-ALL, a malignant disorder of thymocyte progenitors (2). The central role of Notch1 in T-cell Notch1 is a highly conserved transmembrane receptor that transformation was realized upon the identification of activating elicits signaling transduction required for cell proliferation, sur- mutations in the Notch1 gene present in about 60% of T-ALL cases vival, and differentiation. Normally, transmembrane ligands in (3). Once obtaining these activating mutations, Notch1 signaling adjacent cells stimulate Notch1 receptor by inducing step-wise is potentiated by either eliciting ligand-independent activation or intramembrane proteolysis to produce a transcriptional effector prolonging ICN1 half-life. These genetic lesions remarkably ICN1, which specifically turns on target gene expression (1). enhance ICN1 transcriptional programs and the expression of Aberrantly activated Notch1 signaling has been implicated in a downstream genes that promote leukemogenesis such as c-Myc (4–7). Tremendous efforts have been paid in identifying down- stream targets regulated by Notch1 signaling, whereas less atten- 1Zhongnan Hospital of Wuhan University, Wuhan, PR China. 2Medical Research tion is gained to understand the upstream mechanisms sustaining Institute, Wuhan University, Wuhan, PR China. 3School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR aberrant Notch1 activities, particularly those involved in Notch1 China. 4Protein Facility, Center of Biomedical Analysis, Tsinghua University, stabilization. Beijing, PR China. 5School of Life Sciences, Tsinghua University, Beijing, PR Hsp90 is an abundant, highly conserved molecular chaperone China. 6Department of Hematology, Affiliated Tongji Hospital, Tongji Medical crucial for correct folding and maturation of a variety of cellular College, Huazhong University of Science and Technology, Wuhan, PR China. proteins that regulates cell survival, proliferation, and apoptosis. 7South China Institute for Stem Cell Biology and Regenerative Medicine, The increased expression of Hsp90, which is observed in many Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, fl Guangzhou, PR China. tumor types, re ects the efforts of malignant cells to maintain homeostasis in a hostile environment as well as tolerate altera- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). tions from numerous genetic lesions that often result in aberrant accumulations of oncoproteins. This dependence on Hsp90 Z. Wang and Y. Hu contributed equally to this article. appears to be a vulnerability of cancer cells and has led to the Corresponding Author: Guoliang Qing, Medical Research Institute, Wuhan development of drugs aimed at depleting the molecular chaper- University, 185 East Lake Rd, Wuhan 430071, PR. China. Phone: 8627-6875- one and degrading cancer proteome, leading to loss of tumor 0015; Fax: 8627-6875-5692; E-mail: [email protected] cell viability (8). Several Hsp90 inhibitors are currently being doi: 10.1158/1078-0432.CCR-16-2880 tested in both preclinical models and clinical settings (9). Many Ó2017 American Association for Cancer Research. have shown promises in solid tumors (10–12) as well as www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst January 31, 2017; DOI: 10.1158/1078-0432.CCR-16-2880 Wang et al. tested for mycoplasma contamination every 3 months using Translational Relevance MycoAlert (Lonza). T-cell acute lymphoblastic leukemias (T-ALL) are aggressive pcDNA3-Flag-ICN1 was obtained from Warren Pear (Univer- and heterogeneous hematologic tumors resulting from the sity of Pennsylvania, Philadelphia, PA). pcDNA3-HA-Hsp90b was malignant transformation of T-cell progenitors. The major a gift from William Sessa (Yale University, New Haven, CT. challenges in the treatments of T-ALLs are dose-limiting toxi- Addgene plasmid # 22487; ref. 20), and the Myc-tagged Stub1 cities of chemotherapeutics and drug resistance. Notch1 sig- constructs (wild-type or mutants) were provided by Bin Li (Insti- naling is one of the prominent oncogenic pathways in T-ALLs tut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, that reciprocally present an ideal malignancy for studying China; ref. 21). For the experiments in which Hsp90b (or Stub1) Notch1-driven cancer. Anti-Notch1 strategy has emerged as was silenced in T-ALL cells, short hairpin RNA (shRNA) sequences a promising targeted therapy for T-ALL treatments. Yet Notch were obtained from the collection of the RNAi Consortium (22) inhibition by g-secretase inhibitor has not achieved impressive and cloned into a lentiviral vector PLKO.1. For ICN1 (or Stub1) therapeutic efficacy, raising the urge for an alternative overexpression in T-ALL cells, the protein coding sequence was approach to Notch1 inhibition. We here demonstrate intra- cloned into a lentiviral vector pCDH. Primers used for shRNAs cellular Notch1 protein stability is dependent on active Hsp90 and molecular cloning are listed in Supplementary Table S1. chaperone. Specific Hsp90 inhibitors accelerate Notch1 deg- Chemicals and antibodies used in the study are listed in Supple- radation, leading to T-ALL cell growth inhibition in vitro as well mentary Table S2. as in disease models. These findings rationalize Hsp90 antago- fi nists as a treatment strategy for patients with T-ALL or other Identi cation of ICN1-interacting partners Notch1-dependent tumors. 293T cells stably expressing Flag-tagged ICN1 were lysed for 30 minutes and subjected to centrifugation at 12,000 Â g for 15 minutes. The resulting supernatant was incubated with antibody against Flag epitope (Sigma) at 4C for 4 hours, followed by addition of protein G-agarose (Roche; 4C overnight). Immuno- – hematologic malignancies (13 15). Inhibition of Hsp90 leads to precipitated proteins were washed and eluted, followed by SDS- degradation of its client proteins through the ubiquitin-depen- PAGE and Coomassie blue staining (23). Gel bands were excised dent proteasome pathway (16). Downregulation of many Hsp90 and digested with trypsin overnight. The resulting peptides were substrates is in large part dependent on Stub1 activity, the U-box analyzed by the Protein Facility, Center of Biomedical Analysis at ubiquitin E3 ligase that interacts with the Hsp90 chaperone Tsinghua University (Beijing, China). complex to favor substrate degradation (17). Current treatment guidelines for patients with T-ALL include Chromatin immunoprecipitation only conventional chemotherapy and overall prognosis of Chromatin immunoprecipitation (ChIP) was performed as T-ALL remains unsatisfied (18). Anti-Notch–targeted therapy described previously (24, 25). Briefly, SIL-ALL cells were fixed by g-secretase inhibitors, which inhibit Notch proteolytic pro- with 1% paraformaldehyde at room temperature for 10 min- cessing and subsequent activation, has been launched for a long utes. Precleared chromatin was immunoprecipitated with anti- time. Unfortunately, clinical application of these inhibitors body against cleaved Notch1 (Val1744) for 16 hours and then has been hampered owing to their limited efficacies
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