No abstract

Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure-activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.

No abstract

Small molecule tyrosine kinase inhibitors have had dramatic clinical efficacy in chronic myelogenous leukemia. The identification of a specific activating mutation (V617F) in the gene encoding Janus Kinase 2 (JAK2) in a large proportion of patients with myeloproliferative disorders (MPDs) suggests that a kinase inhibitor might have similar clinical utility. CEP-701 is an orally-active inhibitor of receptor tyrosine kinases including FLT-3, and is currently in phase II clinical trials in acute myeloid leukemia (AML) for patients with FLT-3 mutations. The ability of CEP-701 to inhibit JAK2 and suppress the growth of cells from patients with MPDs was evaluated. In in vitro kinase assay CEP-701 inhibited wild type JAK2 (IC50 l nM). In addition, CEP-701 inhibited: constitutive JAK2/STAT5 signaling in human HEL92.1.7 erythroleukemia cell line homozygous for the V617F mutation (IC50 10 nM); growth of HEL92.1.7 cells in vitro (IC50 of 30–100 nM depending upon assay and serum conditions); and growth of HEL92.1.7 xenografts in nude mice. To test whether these observations could extend to clinical samples from MPD patients, peripheral blood cells from patients with MPDs were collected and CD34+ cells were isolated using immunomagnetic separation. Cells were cultured in the presence of stem cell factor, interleukin 3 and interleukin 6 for 5 days, followed by the addition of erythropoietin for up to 5 days. Between days 7 and 10, cells were incubated in the presence of CEP-701 (up to 300 nM) and proliferation was assayed after 24 to 72 hours using the tetrazolium dye XTT. Flow cytometry and morphological examination confirmed that culture conditions strongly favored erythroid proliferation, with loss of expression of CD45 and the stepwise expression of high levels of CD71 and glycophorin A; cell expansion was more than 100-fold. Concentrations of ≤100 nM CEP-701 inhibited (> 50%) the growth of cells from 2/4 patients with polycythemia vera (PV), 10/11 with essential thrombocythemia (ET), and 2/2 with agnogenic myeloid metaplasia (AMM), as well as one AML sample from a patient with a prior history of PV. The JAK2 V617F mutation was detected by allele specific PCR and restriction analysis in mononuclear cells from 6 of 11 ET samples (6/10 inhibited), 3 of 4 PV samples (2/2 inhibited) and 1 of 2 AMM samples, as well as the AML sample. In contrast, CD34+ cells from 4 normal bone marrows cultured identically were not significantly inhibited. Biochemical analysis confirmed that in maturing erythroid cells CEP-701, at 30–100 nM, strongly inhibited JAK2/STAT5 and AKT signaling. In addition, CEP-701 suppressed expression of BclxL and cyclin D1/D2, the downstream targets of the JAK2/STAT5 pathway involved in proliferation of progenitor cells. These results demonstrate that in primary cells derived from patients with MPDs, CEP-701, in clinically achievable concentrations, inhibits constitutive JAK2/STAT5 signaling driven by the wild type and V617F JAK2 and inhibits the proliferation of cells from patients with MPDs. CEP-701 holds promise as a therapeutic agent for patients with MPDs.