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. 2017 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 downstream 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 2017 American Association for Cancer Research. have shown promises in solid tumors (10–12) as well as

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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 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 and con- salmon sperm DNA–saturated protein G agarose (Millipore) siderable side-effects (19). Thus, attempts to seek alternative for 1 hour at 4C. Eluted DNA was quantified by CFX Connect approaches to Notch1 inhibition become imperative for effec- Real-Time PCR System (Bio-Rad) using specific primers (Sup- tive T-ALL therapies. plementary Table S1). In the current study, we identify Hsp90 as a novel ICN1- binding partner crucial for its stabilization and oncogenic Mice function. Inhibition of Hsp90 leads to Stub1-mediated ICN1 Animal procedures were approved by the Animal Experimenta- polyubiquitination and subsequent degradation by the 26S tions Ethics Committee of Guangzhou Institutes of Biomedicine proteasome. Two different categories of Hsp90 inhibitors, and Health, Chinese Academy of Sciences (Guangzhou, China). geldanamycin analogues and purine analogues, exhibit marked cytotoxicities against Notch1-dependent T-ALL cells as well as Human T-ALL xenograft 6 murine models. These findings suggest Hsp90 inhibition as a A total of 2.5 10 CUTLL1 cells were tail vein injected into 6- fi potential alternative approach and also a new horizon of anti- to 8-week-old NOD-SCID IL2Rg-de cient (NSI) mice (Guangz- Notch1 strategy to benefit patients with T-ALL and other hou Institutes of Biomedicine and Health, Chinese Academy of Notch1-addicted malignancies. Sciences, Guangzhou, China) that had been subjected to 100 cGy whole-body irradiation (Precision X-ray). PU-H71 (75 mg/kg, Materials and Methods thrice weekly) or vehicle (10 mmol/L PBS) was intraperitoneally injected into NSI mice two days after engraftment. T-ALL devel- Cell lines and reagents opment in vivo was monitored by periodic blood drawings and SIL-ALL, HPB-ALL, DND41, and CUTTL1 cells were kindly flow cytometry analysis of human CD8 and CD45. After four provided by Warren Pear (University of Pennsylvania, Phila- weeks of treatment, mice were sacrificed and assessed for disease delphia, PA; April, 2011). 293T, MOLT-4, JURKAT, and CCRF- progression. CEM cells were purchased from ATCC (June, 2012). All cell lines were maintained as described previously (3), authenti- Murine T-ALL transplant cated using the variable number of tandem repeats (VNTR) PCR Murine T-ALL cells driven by KrasG12D/Notch1L1601P mutations assay, cultured for fewer than 6 months after resuscitation, and were described by Chiang and colleagues (4). A total of 2.5 106

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murine T-ALL cells were injected into 6- to 8-week-old half- inhibitor (26). As shown in Fig. 2A, administration of 17-AAG lethally irradiated (450 cGy) C57BL/6 mice (Beijing HFK Biosci- dose dependently depleted ICN1 expression in all the cell lines ence). Five days later, mice were intraperitoneally injected with examined (Fig. 2A). Similar results were obtained when PU-H71, PU-H71 (50 mg/kg, thrice weekly) or vehicle (10 mmol/L PBS). a purine analogue Hsp90 inhibitor (27), was applied in CUTLL1, After three weeks of treatment, mice were sacrificed and assessed HPB-ALL, and JURKAT cells. Again, PU-H71 induced downregu- þ for leukemia progression by flow cytometry analysis of GFP as lation of ICN1 protein in a time-dependent manner in these þ þ well as CD4 CD8 cells in the bone marrow and spleen. cells (Fig. 2B). The effect of Hsp90 blockade on ICN1 decrease was not only restricted to Notch1-mutant cells (SIL-ALL, CUTLL1, Statistical analysis HPB-ALL, and MOLT-4), but also applied to JURKAT cells which Significance analysis between groups were performed using express wild-type Notch1. To exclude potential drug-associated Student's t-test, with P < 0.05 considered significant. nonspecific effects, we infected HPB-ALL or CUTLL1 cells with lentiviruses expressing Hsp90b-specific shRNAs (#1, #2, or #3) or Results a control shRNA. Hsp90b shRNA #2 and #3, which exhibited a Identification of Hsp90 as a novel ICN1-binding protein profound silencing effect, markedly downregulated ICN1 expres- To identify novel partners of ICN1, we generated 293T cells sion in both T-ALL cell lines (Fig. 2C), validating that Hsp90 is expressing Flag-tagged ICN1. Flag-bound immunoprecipitates required for aberrant ICN1 accumulation in this context. from these cells were resolved by SDS-PAGE and visualized by The implication of Hsp90 in ICN1 regulation prompted us to silver staining (Fig. 1A). Potential specific bands were excised and investigate whether its inhibition affects Notch1 transcriptional subjected to LC/MS-MS analysis, which led to the identification of activities. We analyzed five representative Notch1 targets in Hsp90 as a protein associated with ICN1 (Fig. 1B; Supplementary CUTLL1 cells by qPCR. Gapdh and Actin, two constitutively Table S3). expressed genes not regulated by Notch1, were included as To validate the mass spectrometry analysis, we enforced the negative controls. Compared with the mock treatment, admin- expression of Flag-ICN1 and HA-Hsp90 in 293T cells for recip- istration of PU-H71 significantly decreased the transcription of rocal immunoprecipitation and confirmed associations between representative Notch1 targets (Fig. 2D). In support of this both proteins (Fig. 1C). Moreover, coimmunoprecipitation using notion, ChIP revealed that the administration of PU-H71 in SIL-ALL and CUTLL1 cell lysates validated the specific interaction SIL-ALL cells significantly decreased the occupancy of ICN1 on between endogenous ICN1 and Hsp90 in T-ALL cells (Fig. 1D). To the Hes1 promoter (Fig. 2E). The Actin promoter was used as a define the precise region(s) for Hsp90–ICN1 interaction, we negative control. These findings argue that Hsp90 plays a expressed full-length HA-tagged Hsp90 in combination with crucial role in mediation of transcriptional activation by onco- respective Flag-tagged fragments of ICN1 in 293T cells (Fig. genic Notch1 in T-ALL cells. 1E). The N-terminal region of ICN1 (amino acids 1761–2155) containing the RAM and ANK domains did not interact with Hsp90 inactivation causes proteasome-mediated ICN1 Hsp90, whereas the C-terminal region (amino acids 2156–2555) degradation interacted as strongly as the full-length protein (Fig. 1E, lanes 1– We postulate that stabilization of ICN1 might be a result of 4). As the C-terminal region of ICN1 contains the TAD domain, protection from degradation by physical association with the the PEST domain, and a linker peptide (amino acids 2425–2442; multicomponent Hsp90 chaperone complex. To test this and for simplicity, we named it H) between these two domains, we further delineate the kinetics of ICN1 degradation, we per- expressed different mutants of these interacting modules with formed protein stability assays in two T-ALL cell lines (CUTLL1 Hsp90 and determined their contribution to the association and SIL-ALL) that express high levels of ICN1 and are extremely between the two proteins. Interestingly, deletion of either the sensitive to g-secretase inhibitor treatment. In the g-secretase TAD domain (H-PEST, 2425–2555) or the PEST domain (TAD-H, inhibitor DAPT-pretreated CUTLL1 cells, administration of 2215–2442) alone did not impair the interaction with Hsp90 17-AAG markedly accelerated the turnover of ICN1 protein (Fig. 1E, lanes 6 and 8). Yet lack of the linker peptide H (TAD, (Fig. 3A, left). Time-course analysis revealed that pharmaco- 2215-2424 or PEST, 2443-2555) resulted in complete loss of logic inhibition of Hsp90 significantly shortened the half-life of protein interaction (Fig. 1E, lanes 5 and 7). To evaluate the endogenous ICN1 from 13.8 hours to 7.4 hours (Fig. 3A, right). importance of the peptide H in mediating ICN1–Hsp90 interac- Similar results were obtained in SIL-ALL (Fig. 3B) as well as tion, we synthesized a Flag-tagged peptide H as well as a control T6E, a murine T-ALL cell line (data not shown). Moreover, peptide (amino acids 1861–1888 of ICN1) and examined their Hsp90 inhibition also significantly destabilized ectopically interaction with Hsp90. As expected, the Flag-tagged peptide H expressed ICN1 upon cycloheximide pretreatment to block but not the control peptide efficiently pulled down Hsp90 from protein synthesis; time-course analysis revealed that it signif- lysates of 293T cells expressing HA-tagged Hsp90 (Fig. 1E, lanes 9 icantly shortened the half-life of exogenous ICN1 from 16.3 and 10). Altogether, these results provide strong evidence sup- hours to 8.5 hours (Fig. 3C). Addition of the 26S proteasome porting that Hsp90 is a direct ICN1-binding partner, and amino inhibitor (MG132) completely blocked ICN1 destabilization in acids 2425–2442 within ICN1 are responsible for the interaction response to 17-AAG in multiple T-ALL cell lines (Fig. 3D), between ICN1 and Hsp90. further corroborating that Hsp90 regulates ICN1 expression predominantly by preventing proteasomal degradation. In Hsp90 inhibition depletes ICN1 and impairs Notch1 support of this notion, we observed that 17-AAG treatment transcriptional activities significantly enhanced ICN1 polyubiquitination in 293T cells To assess potential roles of Hsp90 in regulating Notch1, we (Fig. 3E, lanes 2 and 3). Collectively, these results suggest that treated multiple T-ALL cell lines (SIL-ALL, CUTLL1, HPB-ALL, acceleration of ICN1 degradation upon Hsp90 inactivation is MOLT-4, and JURKAT) with 17-AAG, a well-characterized Hsp90 strictly dependent on the ubiquitin–proteasome pathway.

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Figure 1. ICN1 interacts with the Hsp90 chaperone. A, Total cell extracts prepared from 293T cells expressing Flag-tagged ICN1 or vector alone were subjected to immunoprecipitation using anti-Flag beads. Proteins were resolved by SDS-PAGE and visualized by silver staining. B, LC/MS-MS spectrometry of the puriﬁed Flag-ICN1–associated peptides corresponding to Hsp90. C, Flag-tagged ICN1 and/or HA-tagged Hsp90 were overexpressed in 293T cells and subjected to reciprocal coimmunoprecipitation (co-IP) to detect protein interaction. D, Coimmunoprecipitation with anti-ICN1 antibody to detect interaction between endogenous Hsp90 and ICN1. E, Schematic presentation of various ICN1 truncations and potential Hsp90-binding region (termed as Peptide H) used in Hsp90-binding assays. 293T cells expressing HA-tagged Hsp90 and/or various Flag-tagged ICN1 truncations were subjected to coimmunoprecipitation using anti-Flag beads (lanes 1–8). A Flag epitope followed by a tentative Hsp90-binding peptide (Peptide H) or a control peptide (Peptide Ctrl) was synthesized (ABclonal) and subjected to coimmunoprecipitation using anti-Flag beads (lane 9 and 10).

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Figure 2. Hsp90 inactivation downregulates ICN1 expression and its transcriptional activity. A–C, ICN1 proteins were analyzed by immunoblot upon Hsp90 inhibition. ICN1 band intensities were measured by ImageJ and relative intensities are shown. A, Multiple T-ALL cells were treated with increasing concentrations of 17- AAG for 24 hours. B, T-ALL cells were treated with PU-H71 (1 mmol/L) for the indicated time points. C, Hsp90 was depleted using lentiviruses expressing shRNAs targeting Hsp90b or GFP (Ctrl). Transduced cells were purified upon puromycin (2 mg/mL) treatment for 48 hours. Before selection, initial transduction efficiencies of shGFP and shHsp90 were about 60% and 50%, respectively. D, CUTLL1 cells were treated with PU-H71 (1 mmol/L) or DMSO for 16 hours. Gene expression was quantified by qPCR. Data shown represent the means (SD) of triplicate (, P < 0.01). E, ChIP was performed in SIL-ALL cells treated with DMSO or 1 mmol/L PU-H71 for 16 hours. Eluted DNAs were analyzed by qPCR with primers flanking the Hes1 CSL–binding site. Data shown represent the means (SD) of triplicates (, P < 0.01; n.s., not significant). All the experiments were repeated at least three times.

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The ubiquitin E3 ligase Stub1 negatively regulates ICN1 degradation contributes to the antitumor effects of Hsp90 inhi- The observation that enhanced ICN1 polyubiquitylation bition in T-ALL cells, we overexpressed ICN1 in HPB-ALL cells, upon 17-AAG treatment prompted us to identify the E3 ligase and found that ectopic ICN1 expression partially, but significant- that mediates ICN1 degradation. In this regard, the Hsp90- ly, rescued 17-AAG–induced growth inhibition (Fig. 5B), arguing associated ubiquitin E3 ligase Stub1 (Stip1 homology and U- that ICN1 degradation is an important route that mediates the box containing protein 1, also called CHIP) is recruited to cytotoxic effects of Hsp90 inhibition. induce proteasomal degradation of misfolded or aggregated To translate the effects of Hsp90 deficiency in vitro into an in vivo molecules (17). We depleted Stub1 by a specificshRNAand setting, we assessed leukemia burden in T-ALL xenograft–bearing then subjected these cells to 17-AAG treatment. In comparison mice with or without PU-H71, which had shown great efficacies in with the mock treatment, Stub1 depletion largely prevented 17- various preclinical tumor models (15, 34–36), and is currently AAG–induced ICN1 degradation in HPB-ALL and MOLT-4 cells under clinical examination (27). For this, CUTLL1 cells were (Fig.4A).Consistentwithpriorfindings that Stub1 reduces intravenously injected into immunode ficient NSI mice. Engrafted chaperone efficiency and induces substrate degradation (17, mice were randomized and respectively treated with 75 mg/kg 28, 29), ectopic expression of Stub1 markedly diminished PU-H71 or vehicle. This dose is well tolerated with minimal body endogenous ICN1 expression in 293T and multiple T-ALL cells weight loss (Supplementary Fig. S2A), consistent with previous even when Hsp90 inhibitors were absent (Fig. 4B). Exogenous reports that chronic PU-H71 (75 mg/kg) therapy is not associated Stub1 expression markedly accelerated Flag-tagged ICN1 deg- with significant toxicities (27, 34). The percentage of human þ þ radation upon 17-AAG treatment of 293T cells (Fig. 4C); time- CD45 CD8 leukemic cells was significantly reduced in bone course analysis revealed that it significantly shortened the half- marrow and spleen of each PU-H71–treated mouse (Fig. 5C; life of exogenous ICN1 from 9.0 hours to 4.2 hours (Fig. 4D). Supplementary Fig. S2B), resulting in bones with more reddish Similar to Fbw7, a well-characterized ICN1 E3 ligase (30, 31), color and spleens with much smaller sizes (Fig. 5C; Supplemen- wild-type Stub1 significantly inhibited ICN1-induced luciferase tary Fig. S2C). Hematoxylin and eosin (H&E) staining showed activity. In contrast, Stub1 E3 ligase-inactive H260Q mutant or that, in comparison with vehicle treatment, PU-H71 significantly substrate-binding–deficientK30Amutantfailedtoinduceany reduced lymphoblastic leukemia burdens in bone marrow and noticeable effects (Fig. 4E). Coimmunoprecipitation demon- subsequent leukemia cell infiltration into spleens and livers strated a robust physical interaction of ICN1 and Stub1 when (Supplementary Fig. S3). Consistent with the in vitro results shown coexpressed in 293T cells (Fig. 4F). As such, only wild-type in Figs. 2B and 5A, splenocytes from PU-H71–treated mice Stub1, but not its inactivating H260Q or K30A mutant, resulted exhibited reduced ICN1 and PCNA (a marker indicating cell in robust ICN1 polyubiquitination, and Hsp90 inhibition by proliferation) IHC staining (Fig. 5D). As such, Hsp90 inactivation PU-H71 further enhanced polyubiquitination densities (Fig. significantly decreased Notch1 target gene expression in sorted þ 4G). In sum, these results identify Stub1 as the E3 ligase that is human CD45 splenocytes (Supplementary Fig. S2D), arguing largely responsible for ICN1 degradation via the ubiquitin– that PU-H71 functions, in part, through Notch1 downregulation. proteasome pathway upon Hsp90 inhibition in T-ALL cells. Administration of PU-H71 did not substantially improve overall survival (Supplementary Fig. S2E), suggesting that PU-H71, as a fi Hsp90 inhibition induces T-ALL cell apoptosis in vitro and single agent, is insuf cient for a prolonged survival in our disease impedes xenograft growth in vivo model. Most likely, a drug combination with a standard chemo- To determine the biological outcomes of Hsp90 inhibition in T- therapy would achieve a more desirable outcome. ALL, we first assessed the effect of 17-AAG or PU-H71 on survival of multiple T-ALL cell lines that harbor Notch1 gain-of-function PU-H71 impedes T-ALL leukemia progression in a murine mutations and rely on Notch1 activity for efficient expansion. In allograft model T-ALL cell lines, we tested 17-AAG- or PU-H71–induced robust Notch1 gain-of-function mutations at the heterodimerization apoptosis (Supplementary Fig. S1A) and effectively decreased cell domain are weak alleles to elicit leukemogenesis but capable of viability in a dose-dependent manner (Fig. 5A). Notch1 activation accelerating T-ALL development in K-rasG12D transgenic back- was shown to sustain aerobic glycolysis (the Warburg effect) in T- ground (4). We obtained these primary T-ALL cells driven by ALL cells (32, 33). Consistently, we found that Hsp90 pharma- K-rasG12D and Notch1L1601P mutants (Notch1L1601P is expressed cologic inhibitors, which caused ICN1 depletion, markedly inhib- in MigR1 virus with GFP as a surrogate marker) and also found ited glucose uptake and subsequent lactate secretion in HPB-ALL that Hsp90 antagonism decreased ICN1 levels (Fig. 6A) and its and CUTLL1 cells (Supplementary Fig. S1B). To test whether ICN1 transcriptional activity (Supplementary Fig. S4A). These murine

Figure 3. Hsp90 inhibition accelerates ICN1 proteasomal degradation. A–C, Time course analysis of ICN1 levels by immunoblot in CUTLL1 (A) or SIL-ALL cells (B). Cells were pretreated with 5 mmol/LDAPTfor2hours,followedbyDMSOor1mmol/L 17-AAG treatment for indicated time points. C, Exogenous ICN1 was analyzed in 293T cells. 293T cells expressing ectopic Flag-tagged ICN1 were pretreated with cycloheximide (CHX, 100 mg/mL) for 2 hours. Cells were harvested at the indicated time points upon DMSO or 17-AAG (1 mmol/L) treatment. ICN1 band intensities were quantiﬁed, normalized to b-actin, and then normalized to t ¼ 0 controls. Data shown are averages of three independent experiments. D, Indicated T-ALL cells were treated with DMSO or 17-AAG (1 mmol/L) for 24 hours. Before harvest, cells were treated with MG132 (10 mmol/L) for 5 hours as indicated. ICN1 levels were analyzed by immunoblot with b-actin as a loading control. E, In vivo ICN1 polyubiquitination assay. 293T cells were transfected with plasmids expressing HA-tagged ubiquitin and Flag-tagged ICN1, followed by DMSO or 17-AAG (1 mmol/L) treatment for 16 hours. MG132 (10 mmol/L) was added 5 hours prior to harvest; cells were subsequently lysed in RIPA buffer (24). Ubiquitin-conjugated ICN1 proteins were immunoprecipitated with Flag-tagged antibody and subjected to immunoblot with ubiquitin antibody (lane 1–3). ICN1 and polyubiquitination from input extract (lanes 4–6) were shown on the right.

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T-ALL cells were then intravenously injected into half-lethally identified in ovarian cancer, breast cancer, lung carcinoma, and irradiated C57BL/6 mice for a secondary transplant. After three cancers of the pancreas and prostate (39). Although not yet FDA weeks of either vehicle or 50 mg/kg PU-H71 treatment, mice were approved, the clinical development of Hsp90 inhibitors is making sacrificed and assessed for in vivo leukemia cell expansion (Fig. steady progress. There are currently more than twenty active þ 6B). PU-H71 treatment significantly reduced the GFP leukemic clinical trials involving Hsp90 inhibitors. Conceivably, these cell percentages in the bone marrows and spleens (Fig. 6C and D). Hsp90 inhibitors would act as potential, alternative therapeutic þ þ Murine T-ALL cell population characterized by CD4 CD8 stain- regimens to benefit patients with Notch-dependent malignancies. ing were markedly reduced in PU-H71–treated mice compared More interestingly, we have identified the particular ICN1 with the vehicle-treated group (Fig. 6E and F). Moreover, PU-H71 sequence (peptide H), which mediates the interaction between treatment ameliorated splenomegaly (Fig. 6G) and leukemia cell ICN1 and Hsp90. In principle, cellular delivery of this peptide infiltration into spleens (Fig. 6H). Again, the Notch1 targets were would block ICN1–Hsp90 interaction and lead to selective ICN1 þ significantly downregulated in GFP splenocytes from PU-H71– degradation, which holds great promise for therapeutic purposes injected mice (Supplementary Fig. S4B). Altogether, these results in Notch-addicted tumors. show that pharmacologic inactivation of Hsp90 reduces leukemia Stub1 is a cochaperone that interacts with Hsp70/90 and burden in vivo and provide proof-of-concept in administration of substrates. Stub1 remodels Hsp90 machinery and induces Hsp90 inhibitors as a potential therapeutic regimen for Notch1- proteasome-mediated substrate degradation when Hsp90 is addicted T-ALLs. inactivated (17). Consistently, we demonstrate that 17-AAG– induced ICN1 turnover is largely blocked when Stub1 is silenced. It is well supported that Stub1 modulates protein Discussion triage decisions that regulate the balance between protein Much progress has been made in identifying transcriptional folding and degradation for charperone substrates. When over- oncogenic programs activated by Notch1 during T-ALL patho- expressed, Stub1 negatively regulates Hsp90 chaperone func- genesis. Yet not much is known about the molecular mechanisms tion evidenced by inhibiting the glucocorticoid receptor, a sustaining the aberrant Notch1 activities, especially those critical well-characterized Hsp90 client (29). Similarly, here we show for Notch1 stabilization. In the current study, we show that Hsp90 enhanced expression of Stub1 promotes ICN1 polyubiquitina- is responsible for aberrant ICN1 accumulation in T-ALL cells. tion and subsequent protein degradation, suggesting a critical Upon Hsp90 inhibition, the ubiquitin E3 ligase Stub1 mediates role of Stub1 in the triage decision of ICN1 protein folding or ICN1 polyubiquitination and subsequent proteasome-depen- degradation. This observation raises a possibility that manip- dent degradation. These data describe a previously unsuspected ulation of Stub1 levels may affect T-ALL cell growth. Indeed, pathway, amenable to pharmacologic manipulation, which med- depletion of Stub1 significantly enhanced, whereas its over- iates ICN1 stability. expression inhibited T-ALL cell growth in vitro (Supplementary Conventional chemotherapy is still the mainstay treatment Fig. S5). Considering the negative roles of Stub1 in regulation guideline for patients with T-ALL and overall prognosis of T-ALL of oncogenic substrates, it is unsurprising that Stub1 could play remains unsatisfied (2, 37, 38). Effective targeted therapies are a tumor-suppressive role (40–42). Whether manipulation of currently lacking. Inhibition of Notch by g-secretase inhibitors Stub1 offers a therapeutic opportunity in cancer treatment have not achieved impressive efficacy and yielded considerable awaits more vigorous in vivo investigation using complemen- side-effects (19). In this report, we show that Hsp90 inhibition tary disease models. depletes ICN1 expression and two distinct Hsp90 inhibitors (17- As inhibition of Hsp90 simultaneously downregulates mul- AAG and PU-H71) demonstrate efficacy in Notch1-dependent T- tiple oncogenic client proteins crucial for cell viability and ALL cells and murine models. These effects were associated with tumor development, it is likely that the effects of 17-AAG and dose-dependent, potent in vitro and in vivo inhibition of Notch1 PU-H71 result from inhibition of multiple target proteins in activation (via ICN1 degradation) and downstream target expres- addition to ICN1. Several oncogenic Hsp90 substrates, includ- sion. In addition to T-ALL, aberrant Notch activation has been ing AKT (43), JAK (36), and Tyk2 (44, 45), also play important

Figure 4. Stub1 promotes ICN1 ubiquitination and degradation. A, HPB-ALL and MOLT-4 cells were transduced with lentivirus carrying shRNAs against the control GFP or Stub1. Transduced cells were purified upon puromycin (2 mg/mL) treatment for 48 hours. Before selection, initial transduction efficiency of shGFP or shStub1 was about 50%. Cells were subsequently treated with 17-AAG (1 mmol/L) for 24 hours. ICN1 and Stub1 protein levels were assessed by immunoblotting with b-actin as a loading control. B, 293T, HPB-ALL and CUTLL1 cells were infected with lentiviruses expressing pCDH-Stub1 or empty vector for 48 hours. Initial transduction efficiencies of pCDH and pCDH-Stub1 were about 40% and 30%, respectively. After 2 mg/mL puromycin selection for 48 hours, ICN1 and Stub1 protein levels were assessed by immunoblot with b-actin as a loading control. C, 293T cells were transfected with Myc-tagged Stub1 and/or Flag-tagged ICN1 for 48 hours. Cells were pretreated with cycloheximide (100 mg/mL) for 2 hours, followed by 17-AAG treatment (1 mmol/L) for indicated time points. ICN1 and Stub1 protein levels were assessed by immunoblotting with b-actin as a loading control. D, ICN1 band intensities were quantified, normalized to b-actin, and then normalized to t ¼ 0 controls. Data shown are averages of three independent experiments. E, Hes1 promoter luciferase activity was determined in 293T cells expressing Flag-ICN1, Flag-ICN1/Myc-Stub1 (WT or H260Q, K30A mutant) or Flag-ICN1/Myc-Fbw7. Data shown represent the means (SD) of triplicates (, P < 0.01; , P < 0.001; n.s., means not significant). These experiments were repeated at least three times. F, ICN1 and Stub1 protein interaction. Lysates from 293T cells overexpressing Flag-tagged ICN1 and Myc-tagged Stub1 were subjected to immunoprecipitation using anti-Flag antibody, and the coprecipitated Stub1 was detected by anti-Myc antibody. G, In vivo ICN1 polyubiquitination assay. 293T cells were transfected with plasmids expressing HA-tagged ubiquitin, Flag-tagged ICN1, and Myc-tagged Stub1 (wild-type or mutants) for 12 hours, followed by PU-H71 (1 mmol/L) or DMSO treatment for 16 hours. MG132 (10 mmol/L) was included 5 hours prior to harvest; cells were subsequently lysed in RIPA buffer (24). Ubiquitin-conjugated ICN1 proteins were immunoprecipitated with Flag-tag antibody and subjected to immunoblot with ubiquitin antibody.

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Figure 5. Hsp90 inhibition exhibits antileukemia effects in T-ALL cells and human T-ALL xenografts. A, Assessment of T-ALL cell viability. Notch1-dependent T-ALL cell lines were treated with 17-AAG or PU-H71 at the various concentrations for 24 hours. Cell viability was determined using CCK8 assay kit. Data shown represent the means (SEM) of triplicates (, P < 0.01). These experiments were repeated at least three times. B, HPB-ALL cells transduced with pCDH or pCDH-ICN1 were subjected to DMSO or 17-AAG (1 mmol/L) treatment as indicated. Initial transduction efficiencies of pCDH and pCDH-ICN1 were about 40% and 25%, respectively. After puromycin (2 mg/mL, 48 hours) selection, viable cells were counted and plotted as shown. Data shown represent the means (SEM) of triplicates (, P < 0.01). ICN1 overexpression was confirmed by immunoblotting. C, In vivo CUTLL1 cell expansions in vehicle or PU-H71–treated mice were presented as human CD45þCD8þ percentages in the bone marrow (BM) or spleen. Representative bone and spleen images from treated or untreated cohorts are shown at the bottom. D, IHC staining of ICN1 and PCNA in the spleens from vehicle or PU-H71–treated mice. Representative images are shown (top, 200; bottom, 1,000). Relative ICN1 or PCNA staining was quantified and presented as mean SD, 5 mice per group (, P < 0.01).

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Figure 6. PU-H71 reduces tumor burden in a murine T-ALL allograft. A, Murine T-ALL cells derived from T-ALL mice driven by K-rasG12D and Notch1L1601P mutants were treated with PU-H71 (1 mmol/L) for the indicated time points. ICN1 levels were analyzed by immunoblotting and relative band intensities are shown. B, Schematic presentation of secondary transplant of T-ALL cells derived from T-ALL mouse model driven by K-rasG12D and Notch1L1601P mutants. Five days post-transplantation, PBS (n ¼ 5) or PU-H71 (n ¼ 5) was administered thrice weekly. After three weeks of treatment, mice were sacrificed and analyzed for T-ALL progression. C and D, Leukemia penetrations in the spleen and bone marrow were assessed by flow cytometry analysis of GFP fluorescence. Representative plots (C)andGFPþ percentages from all mice (D)areshown.E and F, Leukemia penetrations in the spleen and bone marrow were analyzed by flow cytometry analysis of CD4þCD8þ populations. Representative plots (E)andCD4þCD8þ percentages from all mice (F)are shown. G, Spleen weights (top) and representative spleen images (bottom) are presented. H, Representative spleen H&E stains are shown (top, 200; bottom, 1,000). , P < 0.05; , P < 0.01.

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roles in T-ALL pathogenesis. By adding an important new Administrative, technical, or material support (i.e., reporting or organizing member in this list, our data provide a rationale for immediate data, constructing databases): J. Wang, C. Liu, Y. Xu, X. Shi, P. Jiang, L. Huang clinical development of Hsp90 inhibitors in treating T-ALL and Study supervision: G. Qing other Notch1-addicted malignancies. Acknowledgments We thank Drs. Warren Pear (University of Pennsylvania, Philadelphia, PA) Disclosure of Potential Conﬂicts of Interest and Bin Li (Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shang- No potential conﬂicts of interest were disclosed. hai, China) for generous provision of reagents.

Authors' Contributions Grant Support Conception and design: G. Qing This work was supported by grants from the National Natural Science Development of methodology: Z. Wang, Y. Hu Foundation of China (81470332, to H. Liu; 81372205, to G. Qing). Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the payment provided facilities, etc.): Z. Wang, Y. Hu, D. Xiao, P. Li of page charges. This article must therefore be hereby marked advertisement Analysis and interpretation of data (e.g., statistical analysis, biostatistics, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. computational analysis): Z. Wang, Y. Hu, D. Xiao, H. Liu, G. Qing Writing, review, and/or revision of the manuscript: Z. Wang, Y. Hu, H. Liu, Received November 17, 2016; revised January 6, 2017; accepted January 22, G. Qing 2017; published OnlineFirst January 31, 2017.

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

Zhaojing Wang, Yufeng Hu, Daibiao Xiao, et al.

Clin Cancer Res Published OnlineFirst January 31, 2017.

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