Published OnlineFirst May 3, 2017; DOI: 10.1158/1535-7163.MCT-16-0889 Small Molecule Therapeutics Molecular Cancer Therapeutics The Multi-kinase Inhibitor Debio 0617B Reduces Maintenance and Self-renewal of Primary Human AML CD34þ Stem/Progenitor Cells Maximilien Murone1, Ramin Radpour2, Antoine Attinger1, Anne Vaslin Chessex1, Anne-Laure Huguenin2, Christian M. Schurch€ 3, Yara Banz3, Saumitra Sengupta4, Michel Aguet5, Stefania Rigotti1, Yogeshwar Bachhav1,Fred eric Massiere 1, Murali Ramachandra4, Andres McAllister1, and Carsten Riether2,6 Abstract Acute myelogenous leukemia (AML) is initiated and main- Furthermore, phospho-STAT5 (pSTAT5) signaling is increased in þ tained by leukemia stem cells (LSC). LSCs are therapy-resistant, primary CD34 AML stem/progenitors. STAT3/STAT5 activation cause relapse, and represent a major obstacle for the cure of AML. depends on tyrosine phosphorylation, mediated by several Resistance to therapy is often mediated by aberrant tyrosine upstream TKs. Inhibition of single upstream TKs did not eliminate kinase (TK) activation. These TKs primarily activate downstream LSCs. In contrast, the multi-TK inhibitor Debio 0617B reduced þ signaling via STAT3/STAT5. In this study, we analyzed the poten- maintenance and self-renewal of primary human AML CD34 tial to therapeutically target aberrant TK signaling and to eliminate stem/progenitor cells in vitro and in xenotransplantation experi- LSCs via the multi-TK inhibitor Debio 0617B. Debio 0617B has a ments resulting in long-term elimination of human LSCs and unique profile targeting key kinases upstream of STAT3/STAT5 leukemia. Therefore, inhibition of multiple TKs upstream signaling such as JAK, SRC, ABL, and class III/V receptor TKs. We of STAT3/5 may result in sustained therapeutic efficacy of demonstrate that expression of phospho-STAT3 (pSTAT3) in AML targeted therapy in AML and prevent relapses. Mol Cancer Ther; blasts is an independent prognostic factor for overall survival. 16(8); 1497–510. Ó2017 AACR. Introduction they are resistant to existing therapeutic interventions resulting in high rates of relapse (6, 7). As a consequence, LSC numbers or LSC Acute myelogenous leukemia (AML) originates from onco- gene signatures in blasts are negative predictors for survival (8, 9). gene-transformed hematopoietic stem/progenitor cells known as AML pathogenesis is based on serial acquisition and accumu- leukemia stem cells (LSC; ref. 1). LSCs self-renew by symmetric lation of various mutations in transcription factors (TF) as well as cell division or divide asymmetrically into a stem cell and into growth factor receptor tyrosine kinases (RTK), such as FMS-related differentiated cells (2). Phenotypically, human LSCs in AML are a tyrosine kinase 3 (FLT3; refs. 10, 11). STAT proteins are TFs very heterogeneous population (3). Therefore, LSCs are primarily involved in cytokine signaling. STATs are activated through tyro- characterized by their function and capacity to induce leukemia in sine phosphorylation, typically through cytokine receptor–asso- xenotransplantation experiments (1, 4, 5). From a clinical per- ciated Janus kinases (JAK), cytoplasmic tyrosine kinases (TKs; SRC spective, LSCs are of fundamental interest in the therapy of AML as and ABL families of kinases), or subsets of RTKs (12). Cytoplasmic SRC, JAK, and ABL TKs act upstream of STAT family of TFs 1Debiopharm International S.A., Lausanne, Switzerland. 2Tumor Immunology, (12, 13). The STAT3/STAT5 pathway is crucial for oncogenesis Department of Clinical Research, University of Bern, Bern, Switzerland. 3Institute in several cancer entities including leukemia (14–17). An activat- of Pathology, University of Bern, Bern, Switzerland. 4Aurigene Discovery Tech- JAK2V617F fi 5 ing mutation in JAK2 ( ) was identi ed in patients with nologies Limited, Bangalore, Karnataka, India. Swiss Federal Institute of Tech- myeloproliferative neoplasms (18). Constitutive phosphoryla- nology (EPFL), Lausanne, Switzerland. 6Department of Medical Oncology, Inselspital, University Hospital and University of Bern, Bern, Switzerland. tion of JAK2, STAT3, and/or STAT5 has been reported in AML blasts and is associated with a shorter time to relapse and poor Note: Supplementary data for this article are available at Molecular Cancer outcome (19–22). Treatment of primary human AML cells with Therapeutics Online (http://mct.aacrjournals.org/). JAK2 and FLT3 inhibitors has resulted in reduced proliferation M. Murone and R. Radpour contributed equally to this article. (23). Recently, it has been demonstrated that JAK/STAT signaling þ Current address for M. Murone: Cellestia Biotech AG, Hochbergerstrasse 60C, is increased in CD34 AML LSCs compared with normal cord Basel 4057, Switzerland. blood or peripheral blood stem cells, suggesting that JAK/STAT Corresponding Author: Carsten Riether, Department of Medical Oncology, signaling supports AML LSC growth and survival (24). Further- University Hospital and University of Bern, Murtenstrasse 35, Bern 3008, more, pSTAT5 signaling is increased and essential for the main- Switzerland. Phone: 413-1632-0956; Fax: 413-1632-3297; E-mail: tenance of leukemic stem/progenitor cells (25). [email protected] Therefore, targeting key TKs upstream of pSTAT3/pSTAT5 may doi: 10.1158/1535-7163.MCT-16-0889 be a promising strategy to treat AML. A phase II study with the Ó2017 American Association for Cancer Research. JAK2 inhibitor ruxolitinib in relapsed/refractory AML patients www.aacrjournals.org 1497 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst May 3, 2017; DOI: 10.1158/1535-7163.MCT-16-0889 Murone et al. showed partial clinical efficacy with complete remission (CR) in 3 Experiments were performed one to two times. Details on repli- of 38 patients (26). However, in all patients, leukemia relapsed cates are indicated in the figure legends. Power calculation was not after a short time period, indicating that inhibiting a single performed to predetermine sample size. upstream TK may not be sufficient to eliminate LSCs. We therefore Human xenograft experiments were approved by the local analyzed the potential of combined inhibition of several key TKs experimental animal committee of the Canton of Bern and upstream of STAT3/STAT5 to treat AML. The multi-TK inhibitor performed according to Swiss laws for animal protection. (TKI), Debio 0617B, has a unique profile targeting key kinases Experimental studies using the disseminated MOLM-13-Luc upstream of STAT3/STAT5, including JAK, SRC, ABL, and a subset model were approved by the Ethics Committee for Animal Exper- of RTKs, class III (FLT3, c-KIT, CSF1R, PDGFRa, and PDGFRb) and imentation of Baden-Wuerttemberg. The experimental protocol V (VEGFR1-3) RTKs (27), which are also known to play a role in was registered by the Regierungspr€asidium Freiburg (G-11/11). STAT3 activation (15). So far, Debio 0617B has documented Experimental studies using MV-4-11 rat model were approved efficacy in STAT3-driven solid tumors in vitro and in vivo (27). by the Commission Nationale de l'Experimentation Animale de In this study, we demonstrate that pSTAT5 signaling is France. þ increased in primary AML CD34 stem/progenitor cells com- pared with bulk leukemia blasts and that nuclear pSTAT3 is an AML patients and construction of tissue microarray independent negative prognostic factor in AML. The multi-TKI Case selection and construction of the tissue microarray (TMA) Debio 0617B reduced maintenance and self-renewal of primary used in the current study was described previously (29). Briefly, þ in vitro human CD34 AML stem/progenitor cells and in xeno- bone marrow hematoxylin and eosin (H&E) slides were reviewed transplantation experiments resulting in elimination of human to confirm the diagnosis and to identify the most representative LSCs, without affecting nonmalignant hematopoietic stem/pro- and blast-rich areas. Punches (0.6 mm) of these areas were then genitor cells. removed from the formalin-fixed, paraffin-embedded (FFPE) tissue block using a fully automated arraying device (TMA Grand- Materials and Methods master, 3D Histech), and a TMA block containing 222 cases was assembled. Cell lines NB4, HT-93, and MV-4-11 cells were kindly provided by Prof. Jurg€ Schwaller, Department of Biomedicine, University Hospital Reagents for treatment Basel (Basel, Switzerland) and Prof. Mario Tschan, Institute of The compounds Debio 0617B (27), sunitinib, quizartinib Pathology, University of Bern (Bern, Switzerland), who obtained (AC220), dasatinib, saracatinib, and ruxolitinib as well as the the cell lines from ATCC and DSMZ in 2015 and 2016. Conse- vehicles EAD-Lip1 and Kleptose HPB solution were provided by quently, no additional authentication was performed by the Debiopharm. authors. All cell lines were tested mycoplasma-free. Cell lines were grown in FCS-containing medium recommended by ATCC Kinome scan ¼ (https://www.lgcstandards-atcc.org/?geo_country ch) with Glu- KINOMEscan was performed as described in ref. 30. Com- taMAX supplemented 100 U/mL penicillin, and 100 mg/mL of pound activity is described as %Ctrl. %Ctrl was calculated as fi streptomycin in a humidi ed atmosphere of 95% air and 5% CO2 at 37C. Media were routinely changed every 3 days. test compound signal À positive control signal  100 negative control signal À positive control signal Patients and healthy controls Peripheral blood samples and bone marrow aspirates were fl obtained from untreated AML patients at diagnosis