The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokineindependent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1-and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs. (Blood. 2011;117(25):6866-6875)

Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative diseases.

Limited options provided by the current standard of care for the patients suffering from myeloproliferative diseases (MPDs) prompted an extensive search for the underlying molecular mechanisms of these disorders. Recent discovery of a single activating mutation (V617F) in JAK2 kinase gene associated with the development of the polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) opened up a possibility to develop highly targeted therapies against these debilitating ailments. To that end, we engineered cytokine-independent Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F -dependent proliferation. We confirmed the ability of hit compounds to inhibit proliferation of JAK2-dependent tumor cell lines using UKE-1 and SET-2 cells carrying the V617F JAK2 mutation. A FACS-based phosphoSTAT5 assay was then used to demonstrate that the hits directly targeted mutant JAK2. JAK3 activity of each compound was evaluated in IL-2-dependent CTLL-2 cell line using phosphoSTAT5 FACS and proliferation assays. To avoid hits with nonspecific antiproliferative activity, the hits were tested in JAK2-independent MOLT4, A549 and H1299 cell lines. Compound hits with the desirable properties were further evaluated for their ability to inhibit JAK2, JAK3 and other kinases in the context of T cell, B cell, or mast cell activation using a variety of cell-based assays as well as in the in vitro biochemical assays. We identified a number of compounds that potently inhibit growth of the two V617F mutant cell lines with EC50s varying from 20 to 500 nM, but do not affect proliferation of control cell lines MOLT4, A549 and H1299 to the same degree. These compounds induce strong and highly specific suppression of STAT5 phosphorylation with IC50s of 10 to 200 nM in SET-2 and V617F JAK2 expressing Ba/F3 cells. One of the hits with the desirable biological and pharmacokinetic profiles was further evaluated in V617F JAK2 Ba/F3 engraftment mouse model where it demonstrated significant extension of survival at 150 and 200 mg/kg bid. Such potent JAK2 inhibitors could become the basis for the next generation of compounds targeting JAK2-dependent myeloproliferative diseases.