Targeting PIM Kinases Impairs Survival of Hematopoietic Cells Transformed by Kinase Inhibitor–Sensitive and Kinase Inhibitor–Resistant Forms of Fms-Like Tyrosine Kinase 3 and BCR/ABL Myriam Adam, 1 Vanda Pogacic, 2 Marina Bendit, 2 Richard Chappuis, 1 Martijn C. Nawijn, 3 Justus Duyster, 4 Casey J. Fox, 5 Craig B. Thompson, 5 Jan Cools, 6 and Juerg Schwaller 1,2 1 Department of Pathology, Geneva Medical School, Geneva, Switzerland; 2 Department of Research, University Hospital Basel, Basel, Switzerland; 3 Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam, the Netherlands; 4 Department of Internal Medicine III, Technical University of Munich, Munich, Germany; 5 Abramson Family Cancer Research Institute, Philadelphia; and the 6 Center for Human Genetics and Flanders Interuniversity Institute for Biotechnology, University of Leuven, Leuven, Belgium Abstract Previous studies have shown that activation of the signal transducer and activator of transcription 5 (STAT5) plays an essential role in leukemogenesis mediated through constitu- tive activated protein tyrosine kinases (PTK). Because PIM-1 is a STAT5 target gene, we analyzed the role of the family of PIM serine/threonine kinases (PIM-1 to PIM-3) in PTK- mediated transformation of hematopoietic cells. Ba/F3 cells transformed to growth factor independence by various oncogenic PTKs (TEL/JAK2, TEL/TRKC, TEL/ABL, BCR/ABL, FLT3-ITD, and H4/PDGFBR) show abundant expression of PIM-1 and PIM-2. Suppression of PIM-1 activity had a negligible effect on transformation. In contrast, expression of kinase-dead PIM-2 mutant (PIM-2KD) led to a rapid decline of survival in Ba/F3 cells transformed by FLT3-ITD but not by other oncogenic PTKs tested. Coexpression of PIM-1KD and PIM-2KD abrogated growth factor–independent growth of Ba/F3 transformed by several PTKs, including BCR/ABL. Targeted down-regulation of PIM-2 by RNA interference (RNAi) selectively abrogated survival of Ba/F3 cells trans- formed by various Fms-like tyrosine kinase 3 (FLT3)– activating mutants [internal tandem duplication (ITD) and kinase domain] and attenuated growth of human cell lines containing FLT3 mutations. Interestingly, cells transformed by FLT3 and BCR/ABL mutations that confer resistance to small-molecule tyrosine kinase inhibitors were still sensitive to knockdown of PIM-2, or PIM-1 and PIM-2 by RNAi. Our observations indicate that combined inactivation of PIM-1 and PIM-2 interferes with oncogenic PTKs and suggest that PIMs are alternative therapeutic targets in PTK-mediated leukemia. Targeting the PIM kinase family could provide a new avenue to overcome resistance against small-molecule tyrosine kinase inhibitors. (Cancer Res 2006; 66(7): 3828-35) Introduction A high percentage of acute leukemias express functional class III receptor protein tyrosine kinase (PTK) Fms-like tyrosine kinase 3 (FLT3). Approximately 25% of acute myeloid leukemia (AML) patients have activating FLT3 mutations, either internal tandem duplications (ITD) or mutations in the activation loop of the tyrosine kinase domain (1). Overexpression and/or activating point mutations have also been found in acute lymphoblastic leukemia (ALL) from infants associated with rearrangement of the MLL gene. Activating mutations of the FLT3 tyrosine kinase are the most common somatic genetic alterations in acute leukemia, and there is evidence that patients with FLT3-ITD may be associated with poorer prognosis. The biological effects of activating FLT3 mutations have been tested in several in vitro and in vivo experimental systems, indicating that activating FLT3 mutants provide growth factor–independent proliferation and survival to hematopoietic cells in vitro and in vivo (2). Several new compounds with activity against FLT3 by compet- ing with ATP binding have been recently characterized (3). These drugs were shown to be selectively cytotoxic to cells either transfected with FLT3-ITD or to primary AML samples carrying FLT3-ITD. In vivo application of three different compounds (CEP- 701,PKC-412,andMLN-518)hasbeenshowntoprolongsurvivalof mice transplanted either with FLT3-ITD–expressing marrow or FLT3-ITD–transfected Ba/F3 cells (4). However, these compounds show limited activity against activation loop FLT3 mutations (5, 6), and resistance to these inhibitors can develop rapidly (7, 8). The molecular mechanisms of hematopoietic transformation by mutated FLT3 are not well understood. Significant differences have been observed in downstream signaling either induced by wild type (WT) or by constitutive active mutated FLT3. First, restoration of the apoptosis inhibitor Bcl-XL was critical for survival of hematopoietic cells by FLT3-ITD but not WT-FLT3. Second, hematopoietic cells transformed by activating FLT3 mutants showed constitutive activation of the signal transducer and activation of transcription 5 (STAT5), whereas transfectants with WT-FLT3 showed only weak or transient activation of STAT5 when exposed to FLT3 ligand (9). Moreover, STAT5 phosphoryla- tion and activation is inhibited by selective FLT3 PTK inhibitors. These findings suggest that regulation of antiapoptotic proteins like Bcl-XL in conjunction with activation of STAT5 may be critical for the leukemogenic effects of oncogenic FLT3 mutants. Constitutive activation of STAT5 has been shown to be a critical mediator for transformation by several PTK fusion genes, including BCR/ABL, FLT3 , or TEL/JAK2 associated with chronic myeloproliferative disorders (10). Several downstream STAT5 target genes (such as SOCS1, BCL-XL , and PIMs ) were shown to play a role in cancer. The PIM family of serine/threonine kinases Note: J. Cools is a postdoctoral researcher of the ‘‘FWO-Vlaanderen.’’ Requests for reprints: Juerg Schwaller, Department of Research, Basel University Hospital, ZLF, Lab 318, Hebelstrasse 20, CH-4031 Basel, Switzerland. Phone: 41-61-265- 3504; Fax: 41-22-37-24920; E-mail: J.Schwaller@unibas.ch. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2309 Cancer Res 2006; 66: (7). April 1, 2006 3828 www.aacrjournals.org Research Article Research. on February 10, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from