Janus kinase 2 (JAK2) hyperactivation by JAK2V617F mutation leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 could serve as a promising therapeutic strategy for MPNs. Here, we report that Flonoltinib Maleate (FM), a selective JAK2/FLT3 inhibitor, shows high selectivity for JAK2 over the JAK family. Surface plasmon resonance assays verified that FM had a stronger affinity for the pseudokinase domain JH2 than JH1 of JAK2 and had an inhibitory effect on JAK2 JH2V617F. The cocrystal structure confirmed that FM could stably bind to JAK2 JH2, and FM suppressed endogenous colony formation of primary erythroid progenitor cells from patients with MPNs. In several JAK2V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2V617F bone marrow transplantation mice. FM exhibited strong inhibitory effects on fibrosis of the spleen and bone marrow. Long-term FM treatment showed good pharmacokinetic/pharmacodynamic characteristics with high drug exposure in tumor-bearing tissues and low toxicity. Currently, FM has been approved by the National Medical Products Administration of China (CXHL2000628), and this study will guide clinical trials for patients with MPNs.
Myeloproliferative neoplasms (MPNs) are a family of clonal disorders of hematopoietic stem cells featuring a continuous proliferation of one or more lineage cells in the bone marrow (BM). Mutations of Janus kinase 2 (JAK2), predominantly JAK2V617F, are discovered in ~95% of patients with polycythemia vera and 50–60% of patients with essential thrombocythemia, as well as primary myelofibrosis (MF). These previous results suggest that JAK2 is an important therapeutic target in the treatment of JAK2V617F-induced MPNs.
Ruxolitinib, a JAK1/JAK2 inhibitor, is the first drug approved for the treatment of intermediate-2 and high-risk patients with MF and alleviates the splenomegaly and systemic symptoms in patients with MF. Despite these clinical benefits, ruxolitinib could cause severe anemia, thrombocytopenia, and granulocyte deficiency, apart from non-specific systemic symptoms, such as diarrhea and fatigue. These side effects may be related to the inhibition of JAK1, which is associated with IFN-γ resistance and cancer evasion. Fedratinib, a dual JAK2/FLT3 inhibitor approved in 2019 by the FDA for the treatment of MF, improved the selectivity of JAK2 over the JAK family and simultaneously inhibited FLT3. Fedratinib significantly inhibited disease progression compared to the best available therapies after long-term treatment. However, fedratinib is associated with vitamin B1 deficiency-related Wernicke’s encephalopathy. Therefore, the development of highly selective JAK2/FLT3 inhibitors with reduced toxicities to treat JAK2V617F-induced MPNs is clinically urgently needed.
JAK2 protein contains seven homology domains (JH1-7), of which JH1 (residues 836-1132) is the kinase domain, and JH2 (residues 543-824) is the pseudokinase domain. JH1 could activate JAK2 through auto-phosphorylation and block the catalytic activity of kinases by blocking ATP and downstream phosphorylation pathway signal transduction. The JH2 domain has no catalytic activity but could negatively regulate the activity of JH1 and inhibit the kinase activity of JAK2. The V617F mutation site is located above the N-terminus of the JH2 domain, and when the valine at position 617 is replaced by phenylalanine with a higher molecular weight, the prolongation of helix C and the interaction between F617 and the helix C phenylalanines 594 and 595 of JH2 domain is stable and activates the JH1 domain. Ruxolitinib and fedratinib are the most representative JAK kinase inhibitors for the treatment of MF, and both of them bind to the JAK2 JH1 domain. Recently, Bristol Myers Squibb Company (BMS) has developed a highly selective TYK2 inhibitor, named BMS-986165, which acts by binding to the TYK2 JH2 domain. BMS-986165 has been proven to greatly improve selectivity and reduce side effects. Therefore, the development of highly selective JAK2 inhibitors binding to the JAK2 JH2 (pseudokinase domain) may be of great significance in improving the selectivity for JAK2 over the JAK family for the treatment of JAK2V617F-driven patients.
Here, we reported that a highly selective JAK2 inhibitor, named Flonoltinib Maleate (FM), had an inhibitory effect on JH1, JH2, and JH2V617F of JAK2, and its crystal structure confirmed that FM could stably bind to the JAK2 JH2 domain, which may contribute to the selectivity for the JAK family. Furthermore, FM showed potent efficacy in cell lines as well as murine models of MPNs, additional to primary cells acquired from patients with MPNs. In various MPN models, FM could significantly reduce tumor burden, suppress disease progression, and extend the survival of mice. In addition, FM exhibited higher drug exposure in the tumor-bearing tissue than the plasma and showed good pharmacokinetic/pharmacodynamic (PK/PD) characteristics and low toxicity. Currently, FM has been approved by the National Medical Productions Administration of China (CXHL2000628), and this research will provide evidence for FM clinical trials in JAK2-driven MPN treatment.Preclinical studies of Flonoltinib Maleate