FLT3 Expressing Leukemias Are Selectively Sensitive to Inhibitors Of

Vol. 9, 4483–4493, October 1, 2003 Clinical Cancer Research 4483

FLT3 Expressing Leukemias Are Selectively Sensitive to Inhibitors of the Molecular Chaperone Heat Shock Protein 90 through Destabilization of Signal Transduction-Associated Kinases

Qing Yao, Ritsuo Nishiuchi, Quanzhi Li, for therapy of FLT3-expressing leukemias, including the Ashish R. Kumar, Wendy A. Hudson, and mixed lineage leukemia fusion gene leukemias. John H. Kersey1 INTRODUCTION University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Acute leukemias are known to have overexpression or mutations of oncoproteins resulting from distinct cellular genetic alterations. Examples are the oncoproteins that result from ABSTRACT gene fusions involving transcription factors, such as MLL2 or Purpose: We conducted studies to evaluate the hypoth- signal transduction-associated kinases, such as FLT3, RAF, and esis that FLT3 is a client of heat shock protein (Hsp) 90 and AKT. These oncoproteins are of great interest as potential inhibitors of Hsp90 may be useful for therapy of leukemia. molecular targets for specific chemotherapy. Rarely, leukemia Experimental Design: The effects of the Hsp90-inhibitor results from a single genetic alteration; an example is leukemia 17-allylamino-17-demethoxygeldanamycin (17-AAG) on cell induced by the Bcr-Abl oncoprotein, where the early phase of growth, expression of signal transduction kinases, apoptosis, the disease can be reversed by inhibition of the fusion onco- FLT3 phosphorylation and interaction with Hsp90 was de- protein. However, most leukemias have more than one genetic termined in FLT3؉ human leukemias. alteration. As a result, therapeutic agents that disrupt more than Results: We found that FLT3 is included in a multipro- one oncoprotein in the leukemia cells are of potential interest for tein complex that includes Hsp90 and p23. 17-AAG inhib- multitargeted molecular therapy. ited FLT3 phosphorylation and interaction with Hsp90. Hsps (including Hsp90 and related molecules) are attrac- -FLT3؉ leukemias were significantly more sensitive to the tive molecular targets because they are known to act as chap Hsp90 inhibitors 17-AAG and Herbimycin A in cell growth erones that prevent the degradation of a number of important assays than FLT3-negative leukemias. Cells transfected with cellular oncoproteins including receptor and nonreceptor ki- FLT3 became sensitive to 17-AAG. Cell cycle inhibition and nases (1, 2). Hsp90 client proteins are brought into a multipro- apoptosis were induced by 17-AAG. Cells with constitutive tein Hsp90/p23/Hsp70/Hop/Hsp40 complex (3). Ansamycin an- expression of FLT3, as a result of internal tandem duplica- tibiotics, such as geldanamycin, specifically inhibit Hsp90 tion, were the most sensitive; cells with wild-type FLT3 were function. The structure-function relationship of geldanamycin intermediate in sensitivity, and FLT3-negative cells were the and Hsp90 is well established (4, 5). Geldanamycin treatment of least sensitive. 17-AAG resulted in reduced cellular mass of cells results in a loss of p23 protein from Hsp90 complex (6) and FLT3, RAF, and AKT. The mass of another Hsp, Hsp70, induces a degradation of client proteins (7, 8). ϩ was increased. The expression level of MLL-AF4 fusion FLT3 leukemias, including MLL fusion gene leukemias, protein was not reduced by 17-AAG in human leukemia were chosen for this study because they are relatively well cells. characterized at the molecular level, generally have more than Conclusions: FLT3؉ leukemias are sensitive to 17-AAG one genetic abnormality (9), frequently have a poor prognosis and Herbimycin A. 17-AAG inhibits leukemia cells with using conventional chemotherapy (10), and because the role of either FLT3-internal tandem duplication or wild-type FLT3 in Hsp90 inhibition is not well defined. At the molecular ϩ FLT3, in part through destabilization of client kinases in- level, FLT3 MLL fusion gene leukemia cells often express an cluding FLT3, RAF, and AKT. 17-AAG is potentially useful MLL fusion oncoprotein, frequently express high levels of FLT3 (11), and occasionally have mutations of FLT3 (12). In this study, we evaluated the hypothesis that some or all of the FLT3ϩ leukemia cell lines, including MLL fusion gene leukemia cell lines, require the chaperone function of Hsp90 and, as Received 1/15/03; revised 6/12/03; accepted 6/12/03. a result, will be sensitive to Hsp90 inhibitors. One of these The costs of publication of this article were defrayed in part by the ansamycin family of Hsp90 inhibitors, 17-AAG, is already in payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported in part by a grant (CA87053) from the National Cancer Institute, and William Kennedy research fellow of the National Childhood Cancer Foundation (to A. R. K.). 2 The abbreviations used are: MLL, mixed lineage leukemia; Hsp, heat 1 To whom requests for reprints should be addressed, at the Cancer shock protein; HMA, Herbimycin A; 17-AAG, 17-allylamino-17-deme- Center, University of Minnesota, MMC 86, 420 Delaware Street, Min- thoxygeldanamycin; FLT3, Fms-like tyrosine kinase 3; ITD, internal neapolis, MN 55455. Phone: (612) 625-4659; Fax: (612) 626-3069; tandem duplication; PI, propidium iodide; FL, FLT3-ligand; wt, wild- E-mail: [email protected]. type; PARP, poly(ADP-ribose) polymerase; FBS, fetal bovine serum.

clinical trials in adult solid tumors (13–15). Here we show that PCR for the ITD and D835 Mutation of the FLT3 Gene. treatment of FLT3ϩ leukemias with 17-AAG results in: (a) the PCR was performed on genomic DNA as published previously degradation of RAF, AKT, and both wt and mutant FLT3; and (16). (b) cell growth arrest and apoptosis. Immunoblotting. Cells were lysed by lysis buffer [50 mM Tris (pH7.4), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 1% SDS], and the cell lysate was clarified by centrifugation. MATERIALS AND METHODS Twenty ␮g of cell lysate of each sample were electrophoresed Reagents. 17-AAG was kindly provided by the Devel- by 10% SDS-PAGE, transferred to a nitrocellulose membrane, opmental Therapeutics Branch of the Cancer Therapy Evalua- and immunoblotted with different antibodies, such as anti-FLT3 tion Program/National Cancer Institute/NIH (Bethesda, MD). polyclonal antibody (s-18; Santa Cruz Biotechnology Biotech- HMA was obtained from Sigma (St. Louis, MI). HMA and nologies, Santa Cruz, CA), anti-Hsp70 monoclonal antibody, 17-AAG were stored in the dark at 4°C and reconstituted in anti-RAF monoclonal antibody, anti-AKT polyclonal antibody DMSO before use. (BD PharMingen), anti-actin monoclonal antibody (Sigma), and Cell Culture and Growth Assay. Human leukemia cell anti-AF4 monoclonal antibody. Antihuman AF4 monoclonal lines are RS 4;11 (MLL-AF4, established previously in our antibody was produced in our laboratory using AF4-GST fusion laboratory), Kid92, SEMK2 (MLL-AF4, gifts from Dr. Finbarr protein as immunogen. For detection, the blots were incubated E. Cotter, Department of Hematology and Oncology, Institute of with horseradish peroxidase-conjugated anti-IgG antibody (Pro- Child Health, London, United Kingdom), 1E8, LAZ221, U937 mega) and developed using the enhanced chemiluminescence (obtained from Dr. Tucker W. Lebien, University of Minnesota, detection system (Amersham Pharmacia Biotech, Arlington Minneapolis, MN), Nalm 6 (obtained from Dr. Jun Minowada, Heights, IL). Hayashibara Biochemical Laboratories, Inc., Okayama, Japan), Analysis of FLT3 Phosphorylation. To assess FLT3 MV 4;11 (MLL-AF4, obtained from Dr. Carolyn A. Felix, phosphorylation, cells were washed three times with serum-free University of Pennsylvania), Molm 13 (MLL-AF9), KOPN-8, RPMI 1640 and placed in the same medium overnight. Cells NALM20, KLM-2 and p30/OHKUBO (obtained from Dr. Yo- were then stimulated with human FL (100 ng/ml) at 37°C for 10 shinobu Matsuo, Hayashibara Biochemical Laboratories, Inc., min, or treated with 17-AAG (37°C; 6 h) before FL stimulation ϫ Okayama, Japan). All of the cell lines were grown in RPMI (37°C; 10 min). Cells were washed twice with 1 PBS, lysed in 1640 tissue culture medium (Life Technologies, Inc., Grand IP-1 lysis buffer [20 mM Tris (pH 7.4), 1% NP40, 1 mM EDTA, Island, NY) with 10% FCS, 100 IU penicillin/ml, and 100 units 50 mM NaCl, 25 mM NaF, 4 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and a protein inhibitor mixture streptomycin/ml in 5% CO2 at 37°C. Before HMA or 17-AAG treatment, cells were seeded in 96-well plates at a density of tablet prepared according to manufacture’s recommendation (Roche Applied Science, Indianapolis, IN)] at 4°C for 15 min 10,000 cells/well. The cells were treated with increasing doses and centrifuged at 15,000 ϫ g at 4°C for 15 min. The protein of HMA or 17-AAG as indicated. The IC for cell growth was 50 concentration of the supernatants was determined using BCA determined using Nonradioactive Cell Proliferation kit (Pro- protein assay kit (Pierce, Rockford, IL). Protein (1.5 mg) from mega, Madison, WI) after 72 h of treatment. cell lysates were precleared with 5 ␮l of a 50% slurry of Protein Detection of Cell Surface FLT3. Cells were incubated A-Sepharose CL-4B (Amersham Pharmacia Biosciences, Pisca- with phycoerythrin-conjugated CD135 (anti-FLT3; BD Phar- taway, NJ) for 1 h and then incubated with 5 ␮l of anti-FLT3 Mingen, San Diego, CA) for 30 min at 4°C. The cells were rabbit polyclonal antibody (s-18; Santa Cruz Biotechnology washed twice in PBS/0.1% BSA and analyzed on a FACS Biotechnologies) overnight at 4°C. Thirty ␮l of Protein A beads Calibur using CellQuest-Pro software (Becton Dickinson, were added and incubated for 4 h. This complex was washed Mountain View, CA). three times with IP-1 lysis buffer, and 30 ␮l of SDS sample Cell Cycle Analysis. One million cells were suspended buffer was added to each sample. The samples were then elec- in 1-ml solution containing 50 mg/ml PI, 0.1% sodium citrate, trophoresed, transferred to nitrocellulose membranes, and im- and 0.1% Triton X-100. The PI-stained samples were analyzed munoblotted with anti-FLT3 polyclonal antibody (s-18; Santa within 24 h. The cell cycle distribution and apoptosis were Cruz Biotechnology) or anti-phosphotyrosine monoclonal anti- determined by the analysis of nuclear DNA content using body (4G10; Upstate Biotechnology Inc., Lake Placid, NY). CellQuest-Pro software (Becton Dickinson). Immunoprecipitation of FLT3-Hsp90 Complex. Cells Detection of Apoptosis. Cells cultured under the various were lysed with IP-2 lysis buffer [20 mM Tris (pH 7.4), 0.02% conditions were harvested and labeled using the CaspaTag NP40, 1 mM EDTA, 50 mM NaCl, 20 mM sodium molybdate, 4 caspase activity kit (Serologicals Corporation, Norcross, GA). mM sodium orthovanadate, and a protein inhibitor mixture tablet Briefly, harvested cells were incubated with FAM-VAD-FMK prepared according to manufacture’s recommendation (Roche that irreversibly binds to caspases-1, 2, 3, 4, 5, 6, 7, 8, and 9. Applied Science)]. Antibody resin was prepared by incubating After two washes, PI was added, and cells were analyzed on a anti-Hsp90 monoclonal antibody (H9010) or anti-p23 mono- FACS Calibur flow cytometer. Green fluorescence signal clonal antibody (JJ3) with a 50% slurry of Protein A-Sepharose (Caspatag) was measured on the FL-1 channel, whereas red CL-4B (Amersham Pharmacia Biosciences) in 1ϫ Tris-buffered fluorescence (PI) was measured on the FL-3 channel. Dot-plots saline (pH 8.0) for1hatroom temperature. The H9010- or of log FL-1 versus log FL-3 were generated using the JJ3-conjugated resin was washed three times in IP-2 lysis buffer. CellQuest-Pro software. Clarified cell lysate was incubated with 30 ␮l antibody resin at

Table 1 Correlation between molecular genetic and immunophenotypic features of leukemias, and their response to 17-AAG, FLT3 and Hsp90 expression 17-AAG FLT3 expression Hsp90 a b Leukemia cell lines Phenotype MLL fusion protein IC50 (nM) (geo. mean) expression FLT3-ITD leukemias Molm 13 CD10Ϫ, CD11bϩ, CD19Ϫ MLL-AF9 31 Ϯ 3 10 1.00 MV 4;11 CD10Ϫ, CD11bϪ, CD19Ϫ MLL-AF4 40 Ϯ 2 2 0.78 FLT3-wild-type leukemias SEMK2 CD10Ϫ, CD11bϪ, CD19ϩ MLL-AF4 350 Ϯ 25 183 2.25 RS 4;11 CD10Ϫ, CD11bϪ, CD19ϩ MLL-AF4 700 Ϯ 52 16 4.75 Kid92 CD10Ϫ, CD11bϪ, CD19ϩ MLL-AF4 770 Ϯ 65 54 4.02 HPB-Null CD10ϩ, CD11bϪ, CD19ϩ None 470 Ϯ 36 21 1.28 KOPN-8 CD10ϩ, CD11bϪ, CD19ϩ MLL-ENL 490 Ϯ 41 16 1.31 LAZ221 CD10ϩ, CD11bϪ, CD19ϩ None 540 Ϯ 32 28 2.23 P30/OHKUBO CD10ϩ, CD11bϪ, CD19ϩ None 575 Ϯ 43 20 2.39 NALM20 CD10ϩ, CD11bϪ, CD19ϩ None 1100 Ϯ 87 18 2.31 NALM16 CD10ϩ, CD11bϪ, CD19ϩ None 1800 Ϯ 61 28 1.76 KM3 CD10ϩ, CD11bϪ, CD19ϩ None 2200 Ϯ 132 8 N/A THP-1 CD10Ϫ, CD11bϩ, CD19Ϫ MLL-AF9 800 Ϯ 48 12 2.71 FLT3-negative leukemias KLM-2 CD10ϩ, CD11bϪ, CD19ϩ None 1660 Ϯ 165 0 N/A 1E8 CD10ϩ, CD11bϪ, CD19ϩ None 2100 Ϯ 196 0 2.25 Nalm 6 CD10ϩ, CD11bϪ, CD19ϩ None 2400 Ϯ 287 0 1.87 U937 CD10Ϫ, CD11bϪ, CD19Ϫ None 4500 Ϯ 321 0 15.58 a Each value presents the mean Ϯ SE of three experiments. b Each value was determined by the ratio of Hsp90 to actin.

4°C for 2 h. The resins were washed with IP-2 lysis buffer five solid tumors (13, 14). One of three similar experiments for times at 4°C and immunoblotted with anti-FLT3 polyclonal FLT3ϩ human leukemia cell lines (RS 4;11 and SEMK2) and antibody (s-18; Santa Cruz Biotechnology), anti-Hsp90 (H9010) FLT3-negative phenotype-matched (CD19ϩ) B-lineage leuke- monoclonal antibody, and anti-p23 (JJ3) monoclonal antibody. mia cell lines (Nalm 6 and 1E8) is shown in Fig. 1A and Table

Statistic Analysis. The relationship between the expres- 1. We found that the IC50 values for cell proliferation at 72 h sion of FLT3 and the IC50 of 17-AAG was examined using the were 700 Ϯ 52 nM (RS 4;11), 350 Ϯ 25 nM (SEMK2), 2400 Ϯ nonparametric Spearman’s rank correlation coefficient. This 287 nM (Nalm 6), and 2100 Ϯ 196 nM (1E8; Table 1; Fig. 2). method gives an idea of the strength of the relationship between Therefore, the two FLT3ϩ leukemia cells were more sensitive the two variables using rank of the data. Spearman’s rank than the leukemia cells without FLT3. Interestingly, monocytic- correlation coefficient was deemed to be the most appropriate macrophage Molm 13 leukemia cells, which contain both FLT3- measure of the relationship between the two variables. Data ITD and MLL-AF9, were significantly more sensitive than were analyzed using SAS software. FLT3-negative CD10Ϫ/CD19Ϫ-matched U937 (monocytic- macrophage leukemia cells) and were the most sensitive of all of RESULTS the lines tested (Fig. 1B; Fig. 2). Molm-13 is known to have both

Characteristics of the Cell Lines Studied. Human acute an ITD of FLT3 andawtFLT3 allele (16, 17). The IC50 values leukemia cell lines with or without FLT3 expression and with or for cell proliferation at 72 h were 31 Ϯ 3nM (Molm 13) and without an MLL fusion protein (MLL-AF4 or MLL-AF9) were 4500 Ϯ 321 nM (U937; Table 1; Fig. 2). chosen for study. Cell lines were tested for FLT3 expression and This raised the question as to whether FLT3 mutations mutation as described in “Materials and Methods.” As shown in were present in the other cell lines that were under study. We Table 1, leukemia cell lines were grouped by the presence of: (a) used PCR to detect ITD of the FLT3 gene. In the cell lines FLT3-ITD; (b) FLT3-wt; and (c) FLT3-negative. FLT3-nega- tested, Molm 13 has the ITD in one allele and MV 4;11 has the tive leukemia cell lines were generally relatively mature with a ITD in both alleles, whereas none of the other cell lines were CD10ϩ CD19ϩ phenotype, and none had an MLL fusion pro- found to have ITD in the FLT3 gene (Table 1). We also tested tein. FLT3-wt leukemia cell lines were either: (a) CD10- with an the cell lines for other FLT3 mutations and found that none have MLL fusion protein; or (b) CD10ϩ with or without an MLL the Asp mutation in position 835 that is also known to result in fusion protein. Both FLT3-ITD leukemia cell lines were CD10Ϫ constitutive activation of FLT3 (16). with an MLL fusion protein. FLT3 expression levels that dem- To address whether the sensitivity to 17-AAG in different onstrated by flow cytometry and Hsp90 expression levels de- leukemia cell lines is determined by the relative expression termined by immunoblotting are shown in Table 1. levels of the FLT3-wt and constitutively active form of FLT3- 17-AAG Inhibits Cell Proliferation of FLT3؉ Leuke- ITD, additional leukemia cell lines were compared for cell mias. We conducted cell growth experiments with 17-AAG; growth and expression levels of cell surface FLT3. As shown in 17-AAG is an ansamycin that binds to and inhibits the function Fig. 2 and Table 1, Molm 13 and MV 4;11, which express of Hsp90 and is currently undergoing a Phase I clinical trial in FLT3-ITD, are the most sensitive to 17-AAG. KLM2, 1E8,

Fig. 1 Effect of 17-AAG/HMA on cell growth. A and C, FLT3ϩMLL-AF4 leukemia cell lines, RS 4;11, SEMK2, with phenotype- matched (CD19ϩ) B-lineage leukemia cell lines, Nalm 6 and 1E8. B and D, FLT3ϩMLL- AF9 monocytic-macrophage leukemia cell line Molm 13 with phenotype-matched leukemia cell line U937. All cell lines were exposed for 72 h to various concentrations of 17-AAG (A and B) or HMA (C and D). Each point represents the mean of six determinations from one of three similar experiments; bars, ϮSE.

310 Ϯ 60 nM (Baf3) and 90 Ϯ 11 nM (Baf3-FLT3-wt, Baf3- FLT3-ITD). These results provide direct demonstration that FLT3 increases sensitivity of cells to 17-AAG. We also tested Hsp90 expression levels in most of FLT3- ITD, FLT3-wt, and FLT3-negative cell lines. As shown in Table 1, Hsp90 expression levels were similar in all of the cell lines, except U937. The reduced sensitivity of U937 to 17-AAG might be caused by its high Hsp90 expression level. However, the overall sensitivity to 17-AAG was not significantly determined by Hsp90 expression levels. HMA Inhibits Cell Proliferation of FLT3؉ Leukemia Similar to 17-AAG. The cell lines were treated with a second ansamycin Hsp90 inhibitor, HMA. As described in “Materials and Methods” cells were treated with a range of HMA concentrations for 72 h. Results in Fig. 1C represent one of three similar experiments demonstrating dose-response data for the Fig. 2 IC50 of 17-AAG for different cell lines. The IC50 of 17-AAG of all of the listed cell lines were evaluated as described in “Materials and four leukemia cell lines. The IC50 values for cell proliferation at Methods.” Cell lines were grouped into three categories, FLT3-ITD, 72 h were 65 Ϯ 5nM (RS 4;11), 40 Ϯ 3nM (SEMK2), 873 Ϯ FLT3-wt, and FLT3-negative. 28 nM (Nalm 6), and 674 Ϯ 57 nM (1E8). In summary, the two FLT3-wt leukemia cell lines, RS 4;11 and SEMK2, were significantly more sensitive than the FLT3-negative leukemia cell NALM6, and U937, which do not express FLT3, are the least lines, Nalm 6 and 1E8. sensitive to 17-AAG. Intermediate sensitivity was observed with To determine whether the HMA has effects on a leukemia cells expressing FLT3-wt. In an analysis of the relationship cell line with FLT3-ITD, Molm-13 was compared with the phenotype-matched U937, which does not express FLT3-ITD or between the level of FLT3 expression and IC50 of 17-AAG, all of the cell lines except those with FLT3-ITD were compared the MLL-AF9 fusion proteins. Results shown in Fig. 1D dem- using Spearman’s rank correlation coefficient. The value for the onstrate dose-response data for the cell lines. The IC50 values correlation coefficient was Ϫ0.5358 with a P of 0.02, demon- for cell proliferation at 72 h were 25 Ϯ 2nM (Molm 13) and strating a significant correlation between the expression of 7800 Ϯ 512 nM (U937). FLT3-wt and sensitivity to 17-AAG. Therefore, FLT3-ITD leukemia cells (Molm 13) were the To directly evaluate the effects of FLT3-wt and FLT3-ITD most sensitive, FLT3-wt leukemia cells (RS 4;11 and SEMK2) on 17-AAG sensitivity, cell growth experiments were performed were the intermediate, and FLT3-negative (Nalm 6, 1E8 and on wt murine Baf3 cells, and cells transfected with FLT3-wt and U937) were the least sensitive to HMA. Thus, the sensitivity to

FLT3-ITD. The IC50 values for cell proliferation at 72 h were HMA was FLT3 dependent, as seen with 17-AAG.

Fig. 3 Effects of 17-AAG on cell cycle distribution for FLT3ϩMLL-AF4 and FLT3ϩMLL-AF9 leukemia cells. RS 4;11 and 1E8 were treated with 0, 700, 1000, and 1500 nM 17-AAG for 24 h. Molm 13 and U937 were treated with 0, 30, 60, and 100 nM 17-AAG for 24 h. Cell cycle distribution was analyzed by fluorescence-activated cell sorter. Each experiment was repeated in triplicate; bars, ϮSE.

17-AAG Causes Cell Growth Arrest and Apoptosis in erone, and whether the MLL-AF4 and MLL-AF9 fusion onco- FLT3؉ Leukemia Cells. Inhibition of cell proliferation or/ proteins will be sensitive to Hsp90 inhibitors. This question is and induction of apoptosis were evaluated as possible mecha- raised in part because the cell lines that were most sensitive to nisms of the cell growth inhibition by 17-AAG. We evaluated 17-AAG express both FLT3 and MLL fusion proteins. cell cycle arrest in FLT3ϩ leukemia cells treated with varied To directly study possible effects of 17-AAG on levels of concentrations of 17-AAG for RS 4;11, 1E8, Molm 13, and the MLL-AF4 protein, immunoblotting was conducted using U937 by staining them with PI and analyzing the DNA content FLT3-wt cell lines, RS 4;11 and SEMK2, after the incubation using the CellQuest-Pro program as described in “Materials and with varied concentration of 17-AAG. As shown in Fig. 6, no Methods.” A representative experiment (n ϭ 3) is shown in Fig. change was noted in the mass of MLL-AF4 fusion protein in the 3. After 24 h, treatment with 17-AAG resulted in a significant presence of 17-AAG. Therefore, the immunoblotting experi- accumulation of cells in G1 phase and fewer cells in S phase in ment provides evidence that the MLL fusion proteins are not both FLT3-wt leukemia cells (RS 4;11) and FLT3-ITD leuke- directly dependent on Hsp90 in their role in the pathogenesis of mia cells (Molm 13) as compared with phenotype-matched cells the leukemia. 1E8 and U937, respectively (Fig. 3). Other evidence that MLL fusion proteins are not important For the induction of apoptosis, activation of caspases is a targets for 17-AAG is found in the leukemia cell lines that key event required in this process. To evaluate the induction of express wt FLT3 but do or do not express MLL fusion proteins. apoptosis by 17-AAG treatment, we analyzed activation of As shown in Table 1, there is no relationship between 17-AAG caspases and degradation of PARP, a caspase substrate. RS 4;11 sensitivity and expression of MLL fusion genes. and Molm 13 cells were treated with varied concentrations of 17-AAG Inhibits FLT3, RAF, and AKT Protein Ki- 17-AAG. Fig. 4 shows that both RS 4;11 and Molm 13 devel- nases, and Increases Hsp70 Expression in FLT3؉ Leukemia oped apoptosis by 17-AAG after 48 h of treatment by immuno- Cells. Previous studies have demonstrated that inhibitors of fluorescence using flow cytometry. Viable cells were decreased Hsp90 reduce the total cellular mass of several protein kinases from 91% (0 nM) to 69% (5000 nM) for RS 4;11 and 90% (0 nM) in solid tumor cells. RAF and AKT have been found to be to 12% (1000 nM) for Molm 13, respectively. Apoptotic and reduced in quantity after Hsp90 inhibition in several solid tumor dead cells were increased from 7% to 23% for RS 4;11 and 8% lines (5, 18, 19). Apoptosis in a tandemly duplicated FLT3- to 75% for Molm 13, respectively. In Fig. 5, cleaved PARP was transformed leukemia cell line was also induced by Hsp90 also observed in both RS 4;11 and Molm 13 cells in a dose- inhibitors (20). In our study, we evaluated the molecular effects dependent fashion after 48 h of treatment with 17-AAG by of 17-AAG on FLT3, RAF, and AKT protein expression in immunoblotting. Both immunofluorescence and immunoblot- 17-AAG-sensitive RS 4;11, HPB-Null, Molm 13, and MV 4;11 ting experiments showed consistently that Molm 13 (FLT3- leukemia cell lines. The amount of FLT3, as well as RAF and ITD) cells were more sensitive to 17-AAG than RS 4;11 (FLT3- AKT, decreased after 17-AAG treatment in a dose-dependent wt) cells. manner for all four of the cell lines after 24 h by immunoblotting MLL Fusion Protein Is Not an Important Target for (Fig. 7). Hsp90 Inhibitors. One important question is whether the Another Hsp, Hsp70, is known to be a participant in the MLL fusion oncoproteins require Hsp90 as a molecular chap- Hsp90 chaperone complexes (21). 17-AAG has been reported to

Fig. 5 17-AAG induces cleaved PARP in RS 4;11 and Molm 13 cell lines. RS 4;11 cells were exposed to 0, 1000, 2000, and 5000 nM 17-AAG, and Molm 13 cells were exposed to 0, 100, 500, and 1000 nM 17-AAG. After 48 h treatment of 17-AAG, 20 ␮g cell lysates were subjected to SDS-PAGE and Western blot to determine PARP and cleaved PARP.

Fig. 4 Treatment with 17-AAG causes apoptosis. RS 4;11 with 0, 1000, 2000, and 5000, nM 17-AAG and Molm 13 with 0, 100, 500, and 1000 nM 17-AAG were cultured for 48 h. Apoptosis was analyzed in RS 4;11 and Molm 13 cells by flow cytometry as described in “Materials and Methods.” The X-axis represents labeling with Caspatag (measured Fig. 6 17-AAG has no effect on MLL-AF4 expression. RS 4;11 and on FL-1), and the Y-axis represents labeling with PI (measured on FL-3). SEMK2 cells were treated with 0, 500, 1000, and 5000 nM of 17-AAG Dot-plots were divided into four quadrants. The percentage of cells for 48 h. Whole cell lysates were analyzed by immunoblotting with an falling in the lower-left (viable) cells, upper-right (caspase positive dead AF4 antibody. cells), and lower-right (caspase positive live cells) are shown.

and HPB-Null cells was only seen after FL stimulation (Fig. increase the level of Hsp70 in human colon cells (18). We 9A). Basal phosphorylation of FLT3 in MV 4;11 was not af- examined the effect of 17-AAG on the amount of Hsp70 in fected by FL stimulation. FLT3ϩ leukemia cells that are 17-AAG sensitive. As shown in To distinguish the effect of FL on FLT3-wt cell lines (RS Fig. 7, Hsp70 expression increased after 24 h treatment with 4;11 and HPB-Null) and FLT3-ITD cell line (Molm 13), cell 17-AAG in a dose-dependent manner for RS 4;11, HPB-Null, growth of these cell lines was determined in the presence or Molm 13, and MV 4;11 leukemia cell lines. absence of FBS and in the presence or absence of FL (Fig. 10). 17-AAG Inhibits Cell Surface FLT3. We tested FLT3 In the presence of FBS, the cell growth of both the FLT3-wt and expression in all of the cell lines in the study by immunofluo- the FLT3-ITD cell lines was not affected by FL stimulation. rescence using flow cytometry. We next evaluated whether the This is likely explained by the existence of the trace of cyto- Hsp90 inhibitor, 17-AAG, inhibited the cell surface expression kines, including FL, in FBS. In the absence of FBS, the cell of FLT3 in FLT3ϩ leukemia cell lines. Fig. 8 shows that growth of FLT3-wt cell lines (RS 4;11 and HPB-Null) was 17-AAG resulted in significant reduction in the level of expres- enhanced by FL, whereas the cell growth of the FLT3-ITD cell sion of FLT3 on cell surface with a shift of the peak to the left, line (Molm 13) was not affected by FL. Therefore, FLT3 signal in a dose-dependent manner, in SEMK2, RS 4;11, and Molm 13. is required for cell growth of FLT3-wt cells, and the FLT3-ITD 17-AAG Inhibits Phosphorylation of FLT3 in Leukemia cells harboring constitutive activation of FLT3 are largely in- Cells. To evaluate the level of FLT3 phosphorylation in leu- dependent of FL. kemia cell lines, FLT3 basal phosphorylation was measured The effect of 17-AAG on FL-stimulated and constitutive using antiphosphotyrosine immunoblotting. Ligand-independ- phosphorylation of FLT3 was additionally tested (Fig. 9B). ent FLT3 phosphorylation was observed in MV 4;11, Molm 13, Pretreatment of 17-AAG inhibited ligand-induced phosphoryl- SEMK2, and RS 4;11, whereas FLT3 phosphorylation in THP-1 ation in RS 4;11 and HPB-Null cells in a dose-dependent

Fig. 7 Effects of 17-AAG on protein amount of FLT3, RAF, AKT, and Hsp70 in RS 4;11, HPB-Null, Molm 13, and MV 4;11 leukemia cell lines. RS 4;11 and HPB-Null cells were exposed to 0, 500, 1000, 2000, and 5000 nM 17-AAG for 24 h. Molm 13 and MV 4;11 cells were exposed to 0, 100, 500, 1000, and 2000 nM 17-AAG for 24 h. Cell lysates containing 20 ␮g of total protein were as- sayed for FLT3, RAF, AKT, Hsp70, and actin by immunoblotting analysis.

manner. Basal phosphorylation in MV 4;11 cells was also in- both FLT3-wt and FLT3-ITD are associated with Hsp90 and hibited by 17-AAG in a dose-dependent manner. p23 complex. 17-AAG resulted in reduced cellular FLT3-wt and To determine whether 17-AAG modulated the expression FLT3-ITD in a dose-dependent manner. Both FLT3 and p23 of FLT3-wt and FLT3-ITD proteins, the membranes were were also found to be released from the Hsp90 complex. Our stripped and reprobed with anti-FLT3 antibody. There was a results do not permit us to determine the extent to which decrease in the expression of both FLT3-wt and FLT3-ITD reduction in band intensity is because of a decrease in total proteins in cells treated with 17-AAG. Therefore, the inhibition FLT3 and/or release of FLT3 from the Hsp90 complex. of FLT3 phosphorylation by 17-AAG is largely caused by the decrease of total amount of FLT3 protein in cells. FLT3 Forms a Molecular Complex with Hsp90 and DISCUSSION p23. Because we had found that exposure to 17-AAG results The major observations in these studies are that FLT3 is a in a significant decrease in FLT3 protein detected in cell lysate client protein of Hsp90, and FLT3ϩ human leukemias are se- by immunoblotting (Fig. 7) and on the cell surface by immu- lectively sensitive in vitro to the Hsp90 inhibitor, 17-AAG. nofluorescence (Fig. 8), we asked whether FLT3 has a direct 17-AAG was found to be effective at concentrations that are interaction with Hsp90. The molecular interaction between attainable with reportedly acceptable tolerance in vivo in hu- Hsp90 and FLT3-wt was studied using SEMK2 leukemia cells mans (13–15). The leukemia cells that were most sensitive were as described in “Materials and Methods.” Fig. 11A shows that those that express FLT3-ITD followed by those that express the FLT3-wt molecule was immunoprecipitated by either anti- FLT3-wt. Hsp90 antibody or anti-p23 antibody. This result provides direct Because there is a strong concordance between FLT3 and evidence that FLT3-wt, Hsp90, and p23 are in the same molec- MLL fusion gene expression, and all of the MLL fusion gene ular complex. leukemia cell lines examined to date also express FLT3 protein Because the FLT3-ITD expression level in either MV 4;11 (Table 1), it was important to determine whether the MLL or Molm 13 is very low, we were unable to use immunopre- fusion oncoprotein is an Hsp90 client and a possible target for cipitation methods to demonstrate the interaction of FLT3-ITD Hsp90 inhibitors. Our studies provide evidence that MLL fusion and Hsp90 in human leukemia cell lines. As an alternative proteins are not sensitive to Hsp90 inhibitors. We found that: (a) approach we compared the effect of 17-AAG on the interaction the amount of MLL fusion proteins was not reduced after of FLT3 with Hsp90 in FLT3-wt and FLT3-ITD transfected 17-AAG treatment as seen in immunoblotting assays; (b) cells. Two murine cell lines, Baf3/FLT3-wt and Baf3/FLT3- FLT3-wt leukemia cell lines have similar sensitivity to 17-AAG ITD, were chosen because of abundantly expressed FLT3-wt whether they are MLL fusion gene positive or negative; and (c) and FLT3-ITD. Baf3/FLT3-wt and Baf3/FLT3-ITD cells were FLT3-wt and FLT3-ITD transformed Baf3 cells were more treated with 0, 100, 500, and 1000 nM 17-AAG for 6 h. Cell sensitive to 17-AAG than nontransformed Baf3 cells. These lysates were incubated with anti-Hsp90-conjugated Protein A results are consistent with a report from others; Dias et al. (22) resin to immunoprecipitate Hsp90. Fig. 11B demonstrates that found that FLT3-wt-transformed HL-60 cells were sensitive to

Fig. 9 A, FLT3 tyrosine phosphorylation in leukemia cell lines. MV4; 11, Molm 13, THP-1, SEMK2, RS 4;11, and HPB-Null leukemia cells were serum-starved overnight and either unstimulated (Ϫ) or stimulated (ϩ) with 100 ng/ml of FL. FLT3 protein was immunoprecipitated followed by immunoblot analysis. B, inhibition of FLT3 phosphorylation by 17-AAG. RS 4;11 and HPB-Null leukemia cells were serum starved overnight and incubated with 0, 1000, and 5000 nM 17-AAG for 6 h before FL (100 ng/ml) stimulation for 10 min. MV 4;11 leukemia Fig. 8 Inhibition of FLT3 receptors on cell surface by 17-AAG. Cells cells were treated with 0, 50, 100, and 500 nM 17-AAG for 6 h. Cell of SEMK2 and RS 4;11 were treated with 0, 500, 1000, and 5000 nM lysates were prepared and subjected to immunoprecipitation with anti- 17-AAG and Molm 13 cells were treated with 0, 50, 100, and 500 nM for FLT3 antibody. Immunoblotting was performed on all samples with a 48 h. The cells of SEMK2 (A), RS 4;11 (B), and Molm 13 (C) were FLT3 antibody to detect total FLT3 and an anti-phospho-tyrosine anti- analyzed by staining with phycoerythrin-conjugated antihuman FLT3 body to detect phosphorylated FLT3. antibody (CD135-PE). The shaded areas show control cells, whereas the blank areas show the cells treated with varied concentrations of 17-AAG. MLL fusion genes. To date there is no evidence that the fusion gene alters FLT3 expression. geldanamycin, another ansamycin family member. Whereas it is Recent studies with a variety of FLT3 kinase inhibitors possible that total mass as reflected in immunoblotting assays have demonstrated effects in the presence of mutated but gen- does not necessarily reflect functions of the MLL fusion pro- erally not wt FLT3 (23–27). Our results suggest that both teins, it is likely that the functions of MLL fusion proteins are FLT3-wt and FLT3-ITD are important targets for the Hsp90 also not sensitive to Hsp90 inhibition. inhibitor, 17-AAG. In all of the FLT3-wt cell lines we tested, A likely explanation for the association between MLL only one, THP-1, is an acute myelogenous leukemia cell line, fusion protein expression and FLT3 expression is that cells with and it has the same sensitivity to 17-AAG as the acute lympho- cell stage-specific tyrosine kinases undergo malignant transfor- blastic leukemia cell lines tested. This suggests that the type of mation by the fusion gene. FLT3 is an excellent example of such leukemia (acute myelogenous leukemia or acute lymphoblastic a cell-stage specific tyrosine kinase. Examples of this associa- leukemia) may not be important to the sensitivity to 17-AAG. tion are: (a) FLT3 is expressed very early in the Pro B lympho- Several lines of evidence indicate that in FLT3ϩ leukemias, the cyte cells that are transformed by MLL-AF4; and (b) FLT3 FLT3 oncoprotein is important in the response to 17-AAG. expressing cells in the monocyte lineage that are transformed by First, we found a direct correlation between the expression of

Fig. 11 A, FLT3 directly associates with Hsp90. SEMK2 cell lysates were immunoprecipitated by mouse IgG, anti-Hsp90 antibody, and anti-p23 antibody as described in “Materials and Methods.” B, 17-AAG treatment destabilizes multimolecular complexes containing both FLT3-wt and FLT3-ITD proteins. Baf3-FLT3-wt and Baf3-FLT3-ITD cells were treated with 0, 100, 500, and 1000 nM 17-AAG for 6 h. Hsp90 complexes were immunoprecipitated as described in “Materials and Fig. 10 The effect of FL on the growth of FLT3-expressing leukemia Methods.” The immunoprecipitates were immunoblotted with anti- cells. RS 4;11, HPB-Null, and Molm 13 were serum-starved overnight FLT3 antibody, anti-Hsp90 antibody, and anti-p23 antibody. and then grown under the following conditions: (f) FBS absent, (Ⅺ) FBS absent and FL absent, (Œ) FBS present, and (‚) FBS present and FL present. Cell growth was measured at 24, 48, and 72 h. 17-AAG has an effect on both FLT3-wt and FLT3-ITD by inhibition of their phosphorylation and dissociation from Hsp90. FLT3 and the response to 17-AAG. Leukemias tested that were Previous studies demonstrated that the Hsp90 inhibitor, relatively resistant to 17-AAG (1E8, Nalm 6, and U937) were HMA, inhibited the growth of murine cells transfected with a all FLT3 negative, whereas the sensitive cells (SEMK2, RS4;11, mutant FLT3 (FLT3-ITD/32D; Ref. 29) and also that FLT3-ITD HPB-Null, Molm 13, and MV 4;11) were all FLT3-positive. formed a complex with Hsp90 (20). We found that: (a) 17-AAG Second, we directly demonstrated a decrease in both FLT3-wt inhibited the growth of both FLT3-wt and FLT3-ITD leukemia and FLT3-ITD cell surface expression in SEMK2, RS 4;11, and cells; (b) FLT3-wt formed a complex with Hsp90 and p23 in Molm 13 cell lines after 48 h treatment of 17-AAG by immu- human leukemia cells; (c) both FLT3-wt and FLT3-ITD formed nofluorescence. Third, Molm 13 and MV 4;11 cell lines, which complexes with Hsp90 and p23 in Baf3 cells transfected with were reported previously (16, 17, 28) and confirmed by our FLT3-wt and FLT3-ITD; and (d) 17-AAG induced the dissoci- studies to have ITD of FLT3 resulting in constitutive activation ation of p23 and FLT3 from Hsp90 complex. Our results pro- of FLT3, were found to be most sensitive to 17-AAG. Fourth, vide direct evidence that FLT3, like other kinases, RAF (30),

AKT (31), and Bcr-Abl (32), is a client of Hsp90. Both FLT3-wt important biologic activities with geldanamycin. Cancer Chemother. and FLT3-ITD proteins apparently require Hsp90 to act as a Pharmacol., 42: 273–279, 1998. molecular chaperone to maintain their biological functions. 6. Whitesell, L., and Cook, P. Stable and specific binding of heat shock We found that 17-AAG resulted in a reduction of total protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells. Mol. Endocrinol., 10: 705–712, 1996. amount of FLT3, RAF, and AKT. Whereas previous studies 7. Dasgupta, G., and Momand, J. Geldanamycin prevents nuclear trans- have demonstrated that ligand binding of FLT3 results in acti- location of mutant p53. Exp. Cell Res., 237: 29–37, 1997. vation of the downstream signal transducers and activators of 8. Tikhomirov, O., and Carpenter, G. Geldanamycin induces ErbB-2 transcription, RAF/RAS/mitogen-activated protein kinase and degradation by proteolytic fragmentation. J. Biol. Chem., 275: 26625– phosphatidylinositol 3Ј-kinase/AKT kinase cascades (33–35), 26631, 2000. there is no evidence that FLT3 controls the total amount of RAF 9. Ayton, P. M., and Cleary, M. L. Molecular mechanisms of leuke- and AKT kinases. Most likely, 17-AAG inhibits the Hsp90 mogenesis mediated by MLL fusion proteins. Oncogene, 20: 5695– chaperone function on the kinases independently, with the pos- 5707, 2001. sibility that the total cellular effects of 17-AAG become syner- 10. Kersey, J. H., Wang, D., and Oberto, M. Resistance of t(4;11) (MLL-AF4 fusion gene) leukemias to stress-induced cell death: possible gistic. mechanism for extensive extramedullary accumulation of cells and poor We found the effect of 17-AAG to be less in FLT3-wt cell prognosis. Leukemia (Baltimore), 12: 1561–1564, 1998. lines (RS 4;11 and HPB-Null) than FLT3-ITD cell lines (Molm 11. Armstrong, S. A., Staunton, J. E., Silverman, L. B., Pieters, R., den 13 and MV 4;11). FLT3-ITD cell lines were much more sensi- Boer, M. L., Minden, M. D., Sallan, S. E., Lander, E. S., Golub, T. R., tive to 17-AAG. The reason for these differences is not clear and and Korsmeyer, S. J. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat. Genet., 30: may be multiple. One possibility is that the downstream signals 41–47, 2002. of FLT3-wt and FLT3-ITD are not identical. Previous studies 12. Christiansen, D. H., and Pedersen-Bjergaard, J. Internal tandem have indicated that Flt3-ITD led to strong factor-independent duplications of the FLT3 and MLL genes are mainly observed in activation of signal transducers and activators of transcription 5 atypical cases of therapy-related acute myeloid leukemia with a normal (36) and up-regulation of the serine-threonine kinase Pim-2 karyotype and are unrelated to type of previous therapy. Leukemia (37). (Baltimore), 15: 1848–1851, 2001. Hsp90 inhibitors, such as 17-AAG, may be considered as 13. Wilson, R. H., Takimoto, C. H., Agnew, E. B., Morrison, G., Grollman, F., Thomas, R. R., Saif, M. W., Hopkins, J., Allerga, C., multitarget therapy, because any cellular protein that uses Hsp90 Grochow, L., Szabo, E., Hamilton, J. M., Brain, P., Monahan, B. P., as a molecular chaperone is likely to be a target for the inhibitor. Neckers, L., and Grem, J. L. Demethoxygeldanamycin (AGG) in adult This multitarget therapy is likely to be advantageous in the patients with advanced solid tumors. Proc. Am. Soc. Clin. Oncol., 82a, therapy of leukemias, which in most cases have more than one 2001. genetic mutation. 14. Munster, P. N., Tong, W., Schwartz, L., Larson, S., Kenneson, K., De La Cruz, A., Rosen, N., and Scher, H. Phase 1 trial of 17- 17-AAG is currently in Phase I clinical studies in breast, (allylamino)-17-demethoxygeldanamycin (17-AAG) in patients (pts) colon, and other solid tumors, and is reported to be well toler- with advanced solid malignancies. Proc. Am. Soc. Clin. Oncol., 83a, ated in these studies (13–15). Similar Phase I studies in leuke- 2001. mias expressing FLT3 are planned. 15. Banerji, U., O’Donnell, A., Scurr, M., Benson, C., Hanwell, J., Clark, S., Raynaud, F., Turner, A., Walton, M., Workman, P., and Judson, I. Phase 1 trial of the heat shock protein 90 (HSP90) inhibitor ACKNOWLEDGMENTS 17-allylamino 17-demethoxygeldanamycin 17aag). Pharmacokinetic (PK) profile and pharmacodynamic (PD) endpoints. Proc. Am. Soc. We thank Dr. David O. 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Qing Yao, Ritsuo Nishiuchi, Quanzhi Li, et al.

Clin Cancer Res 2003;9:4483-4493.

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