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Targeting STAT5 Or STAT5-Regulated Pathways

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Targeting STAT5 Or STAT5-Regulated Pathways Published OnlineFirst August 28, 2018; DOI: 10.1158/0008-5472.CAN-18-0195 Cancer Molecular Cell Biology Research Targeting STAT5 or STAT5-Regulated Pathways Suppresses Leukemogenesis of Phþ Acute Lymphoblastic Leukemia Valentina Minieri1, Marco De Dominici1, Patrizia Porazzi1, Samanta A. Mariani2, Orietta Spinelli3, Alessandro Rambaldi3,4, Luke F. Peterson5, Pierluigi Porcu6, Marja T. Nevalainen7, and Bruno Calabretta1 Abstract Combining standard cytotoxic chemotherapy with BCR- expression of proapoptotic BIM protein. The resulting apoptosis ABL1 tyrosine kinase inhibitors (TKI) has greatly improved the of STAT5-silenced Phþ BV173 cells was rescued by silencing of upfront treatment of patients with Philadelphia chromosome- BIM or restoration of BCL2 expression. Treatment of Phþ ALL positive (Phþ) acute lymphoblastic leukemia (ALL). However, cells, including samples from relapsed/refractory patients, with due to the development of drug resistance through both BCR- the PIM kinase inhibitor AZD1208 and/or the BCL2 family ABL1–dependent and -independent mechanisms, prognosis antagonist Sabutoclax markedly suppressed cell growth and remains poor. The STAT5 transcription factor is activated by leukemogenesis ex vivo and in mice. Together, these studies BCR-ABL1 and by JAK2-dependent cytokine signaling; there- indicate that targeting STAT5 or STAT5-regulated pathways may fore, inhibiting its activity could address both mechanisms of provide a new approach for therapy development in Phþ ALL, resistance in Phþ ALL. We show here that genetic and pharma- especially the relapsed/TKI-resistant disease. cologic inhibition of STAT5 activity suppresses cell growth, induces apoptosis, and inhibits leukemogenesis of Phþ cell Significance: Suppression of STAT5 by BCL2 and PIM lines and patient-derived newly diagnosed and relapsed/TKI- kinase inhibitors reduces leukemia burden in mice and resistant Phþ ALL cells ex vivo and in mouse models. STAT5 constitutes a new potential therapeutic approach against silencing decreased expression of the growth-promoting PIM-1 Phþ ALL, especially in tyrosine kinase inhibitor-resistant kinase, the apoptosis inhibitors MCL1 and BCL2, and increased disease. Cancer Res; 78(20); 5793–807. Ó2018 AACR. Introduction hematopoietic stem cell transplantation (HSCT) is an effective consolidation therapy for patients with Phþ ALL who have Philadelphia chromosome-positive (Phþ) acute lymphoblas- achieved a complete response (CR) after induction of remission tic leukemia (ALL) is characterized by the t(9;22) translocation chemotherapy (6). However, 10% to 20% of patients fail to that generates the p190- and, less frequently, the p210-BCR-ABL1 achieve CR and allogeneic HSCT is only available for patients fusion protein, both of which have constitutive tyrosine kinase with suitable matched donors. In addition, the odds of treatment- activity (1, 2). The Philadelphia chromosome is the most com- related mortality as well as relapse are high (7). The outcome of mon cytogenetic abnormality in adult patient with ALL, occurring patients with Phþ ALL has improved significantly with the intro- in about 20% to 30% of the cases (3, 4). duction of imatinib and second-generation TKI as a first-line The incidence of Phþ ALL increases with age, with up to 50% therapy, especially when used in combination with chemotherapy being diagnosed in patients older than 60 years (5). Allogeneic (8). However, resistance to TKI develops rapidly in most patients with Phþ ALL and the 5-year overall survival is <50% (9–11). Thus, inhibiting the BCR-ABL1 kinase with TKI fails to eradicate 1Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas þ 2 most Ph ALL clones due to BCR-ABL1-dependent and -inde- Jefferson University, Philadelphia, Pennsylvania. The Queen's Medical fi Research Institute, Centre for Inflammation Research, The University of pendent mechanisms of resistance (12). Collectively, these nd- fi Edinburgh, Scotland, United Kingdom. 3Hematology and Bone Marrow ings indicate that additional pathways need to be identi ed and Transplant Unit, Ospedale Papa Giovanni XXIII, Bergamo, Italy. 4Universita' targeted for an effective treatment of Phþ ALL. Statale Milano, Italy. 5Department of Internal Medicine, University of Michigan, STAT5 has a pivotal role in promoting cell survival and the 6 Ann Arbor, Michigan. Department of Medical Oncology, Thomas Jefferson subsequent B-cells' expansion during early B-cell development. 7 University, Philadelphia, Pennsylvania. Department of Pathology, Medical CD19-Cre-mediated deletion of STAT5A/B in the B-cell compart- College of Wisconsin Cancer Center, Milwaukee, Wisconsin. ment impairs IL7-activated survival pathways, blocking B-cell dif- Note: Supplementary data for this article are available at Cancer Research ferentiation at the pre-pro-B stage (13–15). Of interest, the defective Online (http://cancerres.aacrjournals.org/). B-cell development induced by genetic deletion of STAT5 was Corresponding Author: Bruno Calabretta, Thomas Jefferson University, 233 S. rescued by restoring expression of STAT5-regulated BCL2 (16). 10th Street, Bluemle Life Science Bld. Suite 630, Philadelphia, PA 19107. Phone: In malignant precursor B cells, deregulated JAK-STAT5 activity 215-503-4522; E-mail: [email protected] may allow survival of leukemic cells independently of stroma- doi: 10.1158/0008-5472.CAN-18-0195 derived cytokine signals (17). The STAT5 pathway is constitutively Ó2018 American Association for Cancer Research. active in Phþ ALL and in a subset of B-ALL that contains activating www.aacrjournals.org 5793 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 28, 2018; DOI: 10.1158/0008-5472.CAN-18-0195 Minieri et al. mutations in JAK1 or JAK2 (18–20). Importantly, STAT5 can be with IL3 (10 ng/mL), IL6 (10 ng/mL), IL7 (10 ng/mL), Flt3-L activated in Phþ leukemia cells either indirectly through JAK2 (20 ng/mL), and stem cell factor (30 ng/mL; ProSpec). phosphorylation or directly by BCR-ABL1 because STAT5 is a Relapsed/TKI-resistant Phþ ALL samples were assessed for the known substrate of BCR-ABL1 (21), and an intact STAT5 signaling presence of ABL1 TKD mutations by Sanger sequencing; briefly, is required for maintenance of BCR-ABL1-driven leukemias (19). RNA was extractedwithRNAeasy Plus Mini Kit (Qiagen), cDNA was Furthermore, STAT5 is a marker of disease progression in Phþ reverse transcribed by using Superscript III (Thermo Fisher Scien- chronic myeloid leukemia (CML), based on correlation of high tific). The ABL1 TKD was PCR-amplified with a forward primer STAT5 mRNA levels with TKI resistance and advanced disease located on BCR exon 1 (p190 FW: 50-CTCGCAACAGTCCTTC- stages, irrespective of the presence of tyrosine kinase domain GACA-30) or with a forward primer located on exon 12 (P210 FW: (TKD) BCR-ABL1 mutations (22, 23). 50-CTGCAGATGCTGACCAACTC-30) and a common reverse prim- Together, these data suggest that STAT5 itself or STAT5- er (TKD REV: 50-CCTGCAGCAAGGTACTCACA-30). Gel purified regulated pathways could serve as rational targets not only to PCR products were sequenced in both directions using TKD REV circumvent the BCR-ABL1-dependent TKI resistance of Phþ ALL primer or TKD FW primer (50-CCCACTGTCTATGGTGTGTCC-30). but also to suppress growth-promoting STAT5 signals activated through BCR-ABL1-independent mechanisms. In this study, using Cell viability genetic and pharmacologic approaches, we show that STAT5 is Cell viability was assessed by MTT assay in 96-multiwell plates. critical for the growth of Phþ ALL cell lines and of newly Cells were seeded at a concentration of 150,000 or 300,000 diagnosed and relapsed/TKI-resistant patient-derived Phþ ALL cells/mL (depending on length of the treatment and cell line fi cells ex vivo and in mice. Moreover, we found that the growth- growth rate) and then treated with DMSO (Ctrl) or the speci c promoting effects of STAT5 depend on changes in the expression/ drug. At the time point of interest, 100 mL of cell cultures were activity of PIM-1, BIM, and BCL2 and that these proteins can serve transferred in 96-multiwell plates and incubated with 10 mLof ex vivo 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- as therapeutic targets and in xenografts of patient-derived Phþ ALL cells. lium bromide (MTT; Sigma Aldrich) at 37 C for 3 hours. Then, formazan crystals were dissolved with 0.1 M HCl in 2-propanol and absorbance was measured at 570 nm using a mQUANT Materials and Methods microplate scanning spectrophotometer (BioTek Instruments) Cell lines, Phþ primary ALL samples, and cell cultures and KC4 V.3.4 software. The SUP-B15 cell line (Phþ ALL) was purchased from ATCC; the Apoptosis Z181 cell line (Phþ ALL) was kindly provided by Dr. Z. Estrov (M. Cells were incubated at 100,000/mL with 1 mL of the CellEvent D. Anderson Cancer Center, Houston, TX); the BV173 cell line (Phþ Caspase 3/7 Green Detection Reagent (Thermo Fisher Scientific) CML-lymphoid blast crisis; ref. 24) was kindly provided by Dr. N. for 25 minutes at 37 C and analyzed by flow cytometry using a Donato (NIH). EBV-immortalized B cells GM03798 and GM12878 488 nm excitation laser. were purchased from the Coriell Institute (Camden, NJ). All these For GFP-positive cell lines, apoptosis was measured by Annexin cell lines and the TKI-resistant T315I-BV173 derivative line (25) V staining: 100,000 cells were washed in Annexin V Binding Buffer were cultured in Iscove's Modified Dulbecco's Medium (Corning) (10 mmol/L HEPES, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2, supplemented with 10% heat-inactivated FBS (Biowest), 1% pen- pH 7.4), spun and resuspended in 50 mL of Annexin V Binding icillin–streptomycin (Thermo Fisher Scientific), and 1% L-gluta- Buffer, then incubated with 1.5 mL of Cy 5.5-Annexin V (BD mine (Thermo Fisher Scientific) at 37 C, 5% CO . The Ph-like ALL 2 Biosciences) for 15 minutes at room temperature. Samples were MUTZ-5 and MHH-CALL-4 cell lines were kindly provided by Dr.
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