Research Article Potent and Selective Inhibition of Polycythemia by the Quinoxaline JAK2 Inhibitor NVP-BSK805 Fabienne Baffert 1 , Catherine H. Régnier 1 , Alain De Pover 1 , Carole Pissot-Soldermann 2 , Gisele A. Tavares 3 , Francesca Blasco 4 , Josef Brueggen 1 , Patrick Chène 1 , Peter Drueckes 3 , Dirk Erdmann 1 , Pascal Furet 2 , Marc Gerspacher 2 , Marc Lang 2 , David Ledieu 5 , Lynda Nolan 5 , Stephan Ruetz 1 , Joerg Trappe 3 , Eric Vangrevelinghe 2 , Markus Wartmann 1 , Lorenza Wyder 1 , Francesco Hofmann 1 , and Thomas Radimerski 1 Abstract The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism un- derlying chronic myeloproliferative neoplasms. Strikingly, the JAK2 V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2 V617F and JAK2 wild-type enzymes by a novel sub- stituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP- BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2 V617F -bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leuke- mic cell spreading and splenomegaly in a Ba/F3 JAK2 V617F cell-driven mouse mechanistic model. Further- more, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945–55. ©2010 AACR. Introduction The discovery of an acquired activating point mutation in the pseudokinase domain of JAK2 in patients suffering from chronic myeloproliferative neoplasms (cMPN; refs. 1–3) has drawn a lot of attention to this kinase. For the first time, there is a molecular understanding of the un- derlying disease mechanism and, equally of importance, the mutated JAK2 is a druggable target for therapeutic in- tervention (4, 5). The JAK2 valine 617 to phenylalanine mutation is found in nearly every patient with polycythe- mia vera (PV) as well as in approximately every second patient suffering from essential thrombocythemia and pri- mary myelofibrosis (6). Interestingly, in the remainder of V617F-negative PV patients, mutations were discovered in JAK2 exon 12, also affecting the pseudokinase domain (7). In Down's syndrome–associated acute lymphoblastic leukemia, yet another JAK2-activating pseudokinase point mutation was identified, affecting arginine 683 (8, 9). Aberrant JAK2 signaling can also be brought about by mutations in receptors. For instance, W515L/K muta- tions in the thrombopoietin receptor account for ∼5% to 10% of V617F-negative essential thrombocythemia and primary myelofibrosis cases (10, 11). Loss of negative feed- back regulation as a consequence of suppressor of cyto- kine signaling silencing or mutation has been described in certain lymphomas (12). Furthermore, chromosomal translocations that involve the JAK2 kinase, such as the t(9;12) TEL-JAK2 fusion in rare cases of T-cell acute lym- phoblastic leukemia, can also cause constitutive kinase activation (13). Thus, the patient population that is antici- pated to benefit from JAK2 inhibitor treatment is fairly well defined by a series of alterations that confer depen- dency on JAK2 signaling. Encouragingly, clinical trials in patients suffering from cMPNs are already under way with first-generation JAK inhibitors (14). Here, we describe the discovery of a novel substituted quinoxaline, termed NVP-BSK805, as a potent and selec- tive ATP-competitive inhibitor of JAK2. The binding Authors' Affiliations: 1 Disease Area Oncology, 2 Global Discovery Chemistry, 3 Center for Proteomic Chemistry, 4 Metabolism and Pharmacokinetics, and 5 Preclinical Safety Clinical Pathology, Novartis Institutes for BioMedical Research, Basel, Switzerland Note: Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Current address for L. Wyder: Actelion Pharmaceuticals, Ltd., Allschwil, Switzerland. Corresponding Authors: F. Hofmann and T. Radimerski, Disease Area Oncology, Novartis Institutes for BioMedical Research, Klybeckstrasse 141, 4057 Basel, Switzerland. Phone: 41-61-696-2064; Fax: 41-61-696- 3835. E-mail: francesco.hofmann@novartis.com and thomas.radimerski@ novartis.com doi: 10.1158/1535-7163.MCT-10-0053 ©2010 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 1945 on June 11, 2020. © 2010 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst June 29, 2010; DOI: 10.1158/1535-7163.MCT-10-0053