Human Cancer Biology Inhibition of Phosphotyrosine Phosphatase 1B Causes Resistance in BCR-ABL-PositiveLeukemiaCellstotheABLKinaseInhibitorSTI571 Noriko Koyama,1Steffen Koschmieder,1SandhyaTyagi,1Ignacio Portero-Robles,1Jo« rg Chromic,1 Silke Myloch,1Heike Nu« rnberger,1Ta nja Ro s s m a ni t h , 1Wolf-Karsten Hofmann,2 Dieter Hoelzer,1and Oliver Gerhard Ottmann1 Abstract Protein tyrosine phosphatase 1B(PTP1B) is a negative regulator of BCR-ABL-mediated transfor- mation in vitro and in vivo. Toinvestigate whether PTP1B modulates the biological effects of the abl kinase inhibitor STI571in BCR-ABL-positive cells, we transfected Philadelphia chromosome ^ positive (Ph+) chronic myeloid leukemia cell-derived K562 cells with either wild-type PTP1B (K562/PTP1B), a substrate-trapping dominant-negative mutant PTP1B(K562/D181A), or empty vector (K562/mock). Cells were cultured with or without STI571and analyzed for its effects on proliferation, differentiation, and apoptosis. In both K562/mock and K562/PTP1B cells, 0.25 to 1 Amol/L STI571 induced dose-dependent growth arrest and apoptosis, as measured by a decrease of cell proliferation and an increase of Annexin V-positive cells and/or of cells in the sub-G1 apoptotic phase. Western blot analysis showed increased protein levels of activated caspase-3 and caspase-8 and induction of poly(ADP-ribose) polymerase cleavage. Low con- centrations of STI571promoted erythroid differentiation of these cells. Conversely, K562/D181A cells displayed significantly lower PTP1B-specific tyrosine phosphatase activity and were signifi- cantly less sensitive to STI571-induced growth arrest, apoptosis, and erythroid differentiation. Pharmacologic inhibition of PTP1B activity in wild-type K562 cells, using bis(N,N-dimethylhy- droxamido)hydroxooxovanadate, attenuated STI571-induced apoptosis. Lastly, comparison of the STI571-sensitive Ph+ acute lymphoblastic leukemia cell line SupB15 with a STI571-resistant subline revealed significantly decreased PTP1Bactivity and enhanced BCR-ABL phosphorylation in the STI571-resistant SupB15 cells. In conclusion, functional PTP1B is involved in STI571- induced growth and cell cycle arrest, apoptosis, and differentiation, and attenuation of PTP1B function may contribute to resistance towards STI571. Philadelphia chromosome–positive (Ph+) chronic myeloid nib) is a selective BCR-ABL tyrosine kinase inhibitor that has leukemia (CML) and acute lymphoblastic leukemia (ALL) have significantly improved the prognosis of CML. Whereas 80% of become model diseases for the effectiveness of targeted drug patients with newly diagnosed CML achieve a complete therapy. The p185Bcr-Abl and p210Bcr-Abl tyrosine kinase proteins cytogenetic response to STI571, acquired (secondary) resis- are formed by the reciprocal translocation between chromo- tance to STI571 occurs with an annual incidence of f4% somes 9 and 22 (Philadelphia chromosome). STI571 (imati- (1–3). Primary resistance to STI571 is considerably more frequent in patients with advanced CML or Ph+ ALL and secondary resistance develops rapidly in the majority of Authors’Affiliations: 1Division of Hematology, Department of Internal Medicine II, responding patients (4, 5). At relapse, most patients display 2 Johann Wolfgang Goethe University, Frankfurt, Germany and Department of enhanced BCR-ABL kinase activity, reflecting loss of kinase Hematology, Oncology and Transfusion Medicine, University Hospital Benjamin Franklin, Berlin, Germany inhibition by STI571 (6). Several mechanisms of resistance Received 12/1/04; revised 10/27/05; accepted 11/18/05. against STI571 have been reported in patients with CML, Grant support: Held und Hecker Stiftung (N. Koyama); Deutsche including point mutations in the tyrosine kinase domain Forschungsgemeinschaft grant KO2155/1-1 (S. Koschmieder); BMBF grant and amplification of BCR-ABL at the genomic or transcrip- 01GI9971, Nationales Genomforschungsnetz (NGFN), and Novartis Pharma AG, Germany (O.G. Ottmann). tional level (7, 8). However, neither mutation nor amplifica- The costs of publication of this article were defrayed in part by the payment of page tion of BCR-ABL is detected in a significant number of charges. This article must therefore be hereby marked advertisement in accordance patients (7, 8). with 18 U.S.C. Section 1734 solely to indicate this fact. Protein tyrosine phosphatases (PTP) encompass a large Note: N. Koyama and S. Koschmieder contributed equally to this work. S. Koschmieder is currently at Medizinische Klinik und Poliklinik A, University family of enzymes that catalyze the dephosphorylation of Hospital of Muenster, Muenster, Germany. tyrosyl-phosphorylated proteins. In conjunction with protein Requests for reprints: Oliver Gerhard Ottmann, Department of Hematology and tyrosine kinases, they regulate the level of protein tyrosine Oncology, University Hospital Frankfurt/Main, Theodor-Stern-Kai 7, 60590 phosphorylation and are important for fundamental physio- Frankfurt am Main, Germany. Phone: 49-69-6301-6365; Fax: 49-69-6301- logic processes including cell growth and differentiation (9). 6089; E-mail: [email protected]. F 2006 American Association for Cancer Research. PTPs have been shown to be implicated in a wide range of doi: 10.1158/1078-0432.CCR-04-2392 cancers including breast, prostate, and brain cancer, juvenile www.aacrjournals.org 2025 Clin Cancer Res 2006;12(7) April 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Human Cancer Biology myelomonocytic leukemia, myelodysplastic syndromes, acute PTP1B- or T-cell PTP-specific phosphatase activity, 300 Ag of cell lysate myeloid leukemia, and B-cell lymphomas (10–13). PTP1B is a proteins were immunoprecipitated with monoclonal antibodies against widely expressed nonreceptor PTP that has been shown to be human PTP1B, human T-cell PTP (both from Oncogen, Seattle, WA), a negative regulator of multiple tyrosine kinases, including or mouse immunoglobulin G (Dako, Hamburg, Germany) for 1 hour at 4jC followed by 30 AL of a 50% protein A agarose beads (Amersham, receptor tyrosine kinases such as the insulin receptor, epidermal Freiburg, Germany) for an additional hour. Immune complexes were growth factor receptor, and erythropoietin receptor, as well washed thrice with HEPES buffer (pH 7.0) and used for the tyrosine as of cytoplasmic tyrosine kinases such as Src family kinases phosphatase assays. Activity was determined within a linear range of (14–20). PTP1B was shown to bind to and dephosphorylate detection and the background in the presence of mouse immunoglob- BCR-ABL and to antagonize BCR-ABL-mediated signaling and ulin G was subtracted. Phosphatase activities were calculated using transformation in vivo and in vitro (14–17). Overexpression purified recombinant human CD45 provided in the kit as a control. of PTP1B antagonized BCR-ABL-mediated transformation of Cell cycle, apoptosis, and cell-surface marker analysis. After treatment Rat-1 fibroblasts, reducing their capacity to generate colonies with STI571, cells were pelleted, fixed in 70% methanol on ice for in soft agar and their ability to form tumors in nude mice (15). 1 hour, and resuspended in propidium iodide solution (0.01 mg/mL In addition, both PTP1B overexpression and inhibition of propidium iodide, 200 units/mL RNase, 1% Triton X-100, 0.1 mmol/L EDTA, 0.38 mmol/L sodium citrate). Cell cycle analysis was carried out BCR-ABL by STI571 induced erythroid differentiation of BCR- using a FACScan analyzer and CellQuest software (Becton Dickinson, ABL-positive K562 cells (15). Interestingly, BCR-ABL induces Heidelberg, Germany). For the analysis of apoptosis, cells were stained expression of PTP1B (14, 21), suggesting that PTP1B is using the Annexin V kit according to the instructions of the manufac- implicated in a negative feedback loop of BCR-ABL. Because turer (Roche). bis(N,N-Dimethylhydroxamido)hydroxooxovanadate STI571 preferentially binds to the unphosphorylated confor- (DMHV; Calbiochem, Bad-Soden, Germany) was used for inhibition mation of the ABL kinase domain (22), we hypothesized that of phosphatase activity of PTP1B. For cell-surface marker expression attenuation of PTP1B activity would enhance phosphorylation analysis, cells were stained with phycoerythrin-conjugated anti-glyco- of BCR-ABL and thereby promote abnormal phosphorylation phorin-A antibodies or an isotype-matched nonspecific control anti- of its downstream signals and STI571 resistance. body, and analyzed using FACScan (all from Becton Dickinson). We therefore investigated whether suppression of PTP1B Western blotting. Western blotting was essentially done as previ- ously described (23). The following antibodies were used: anti-PTP1B activity would cause STI571 resistance in p210Bcr-Abl positive (Calbiochem), anti-T-cell PTP (Calbiochem), anti-phospho-extracellu- K562 cells and whether PTP1B activity is altered in an STI571- Bcr-Abl lar signal-regulated kinase (ERK)-1/ERK2 (Promega, Madison, WI), anti- resistant subline of p185 positive SupB15 cells, a Ph+ ALL ERK1/ERK2, anti-Akt, anti-phospho-Akt, anti-c-ABL, antitubulin, cell line derived from STI571-sensitive wild-type SubB15 cells. anti-active caspase-8 (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-signal transducer and activator of transcription Materials and Methods 5 (STAT5), anti-phosphotyrosine (both from Upstate Biotechnology, Lake Placid, NY), anti-STAT5 (Transduction Laboratories, Lexington, Cells and reagents. K562 cells (Deutsche