[Show abstract][Hide abstract] ABSTRACT: A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients.
PLoS ONE 01/2012; 7(5):e37207. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polycythemia vera (PV) is a myeloproliferative disorder characterized by increased red cell mass and splenomegaly in the absence of secondary causes [Tefferi A., Spivak J.L., Polycythemia vera: scientific advances and current practice. Semin Hematol 2005;42(4):206-20.]. Recently, several laboratories have discovered that the vast majority of patients with PV carry a single, activating mutation (V617F) in the pseudokinase domain of Janus kinase 2 (Jak2) [Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, et al., Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005;280(24):22788-92; James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature 2005;434(7037):1144-8; Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779-90; Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7(4):387-97.]. This discovery has spurred interest in developing therapies for PV via inhibition of Jak2. We induced polycythemia in mice by administering high dose recombinant erythropoietin (Epo) and determined that administration recapitulates almost all of the major and minor diagnostic features of human PV. We then tested a selective, small molecule inhibitor of Jak2 (Jak2i) and showed that this treatment prevents polycythemia. This prevention of polycythemia was accompanied by lower hematocrits, reduced spleen sizes and reductions in Stat5 phosphorylation (pStat5). Surprisingly, Epo rapidly (<1h) induces mobilization of activated erythroid precursors into the blood, thus allowing drug-response relationships to guide discovery. We conclude that inhibition of Jak2 prevents polycythemia in mice, and furthermore present this model as an efficient tool for the discovery of drugs that effectively treat human PV.
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.