Discovery and evaluation of ZT55, a novel highly-selective tyrosine kinase inhibitor of JAK2V617F against myeloproliferative neoplasms

Abstract

BackgroundThe JAK2-STAT signaling pathway plays a critical role in myeloproliferative neoplasms (MPN). An activating mutation in JAK2 (V617F) is present in ~ 95% of polycythemia vera, essential thrombocythemia, and primary myelofibrosis cases. This study aims to explore the selective JAK2V617F inhibitor, evaluate the efficacy and possible mechanism of ZT55 on MPN.MethodsHTRF assays were conducted to evaluate the selective inhibition of ZT55 for JAKs. Cell apoptosis, proliferation, and cycle arrest assays were performed to examine the effect of ZT55 on HEL cell line with JAK2V617F mutation in vitro. Western analysis was used to monitor the expression and activity of proteins on JAK2/STAT pathway. A mice xenograft model was established to evaluate the antitumor efficacy of ZT55 in vivo. Peripheral blood samples from patients with the JAK2V617F mutation were collected to estimate the effect of ZT55 on erythroid colony formation by colony-forming assay.ResultsWe found that ZT55 showed a selective inhibition of a 0.031 μM IC50 value against JAK2. It exhibited potent effects on the cellular JAK-STAT pathway, inhibiting tyrosine phosphorylation in JAK2V617F and downstream STAT3/5 transcription factors. ZT55 inhibited the proliferation of the JAK2V617F-expressing HEL cell line, leading to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis. Notably, ZT55 also significantly suppressed the growth of HEL xenograft tumors in vivo. Further evaluation indicated that ZT55 blocked erythroid colony formation of peripheral blood hematopoietic progenitors from patients carrying the JAK2V617F mutation.ConclusionThese results suggest that ZT55 is a highly-selective JAK2 inhibitor that can induce apoptosis of human erythroleukemia cells by inhibiting the JAK2-STAT signaling.Electronic supplementary materialThe online version of this article (10.1186/s13046-019-1062-x) contains supplementary material, which is available to authorized users.

Isotalatizidine, a C19-diterpenoid alkaloid, attenuates chronic neuropathic pain through stimulating ERK/CREB signaling pathway-mediated microglial dynorphin A expression

“…The protein concentration in the cell and tissue lysates were determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Carlsbad, CA, USA), and 20 μg of proteins per sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bands were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp., Bedford, MA, USA) that was then blocked with 5% bovine serum albumin (BSA) in tris-based saline-Tween 20 (TBST) at room temperature for 1 h. After incubating overnight with the primary antibodies against p-p38, p-ERK, p-JNK, p-CREB, p38, ERK, JNK, CREB, dynorphin A (all diluted 1:1000), β-tubulin, and GAPDH (1:5000 each) at 4°C, the blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies [31]. The membrane was developed using enhanced chemiluminescence reagents (Perkinelmer, USA), and the bands were visualized with Tanon 2000 Imaging system (Beijing, China) and their intensities were quantified using ImageJ Software (NIH, USA).…”

Network Pharmacology and Molecular docking Analysis on Mechanisms and Molecular Targets of LongChaiJiangXue Formula for Treatment of Polycythemia Vera

“…On the base of the components-targets-pathways network, STAT3, IL-6, JAK2, and VEGFA have been verified as powerful targets of LCJX in the management of PV in previous study [28,41]. Since over 90% of patients suffered from PV could be found JAK2V617F gene mutation [42], the JAK2/STAT3/HIF1A/VEGFA signaling pathway with a high degree, involving core targets such as JAK2 and STAT3, is supposed to be closely concerned with the pharmacological effect of LCJX act on PV.…”

“…LCJX includes multiple components effective for PV. For instance, isoliquiritigenin in RB inhibits the JAK2/STAT3 pathway to enhance apoptosis [27] since the JAK2/STAT3 pathway is the principal mechanism in PV [28]. Effect of anti-oxidation injury and controlling weight by puerarin through JAK2/STAT3 pathways were also discovered [29,30].…”

Somatically acquired mutations in primary myelofibrosis: A case report and meta‑analysis

“…Some clinical signs of the disease, such as anemia and splenomegaly, and the risk of transformation to AML have been shown to be associated with JAK2 V617F mutational status or the JAK2 V617F allele burden (51). As the most important mutation, JAK2 V617F was present in over half of the patients with PMF, and serves as a potential molecular target for therapeutic intervention (51), such as through the development of ruxolitinib, an inhibitor of JAK2, which has transformed therapy for myelofibrosis (52).…”