Preclinical studies of Flonoltinib Maleate,a novel JAK2/FLT3 inhibitor,in treatment of JAK2V617F-induced myeloproliferative neoplasms

Janus kinase 2 (JAK2) hyperactivation by JAK2^V617F 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 JAK2^V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2^V617F 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.Subject terms: Myeloproliferative disease, Targeted therapies     

Usefulness of JAK2V617F mutation in distinguishing idiopathic erythrocytosis from polycythemia vera

Idiopathic erythrocytosis (IE) is a primary erythrocytosis not fulfilling the criteria for polycythemia vera (PV) diagnosis. In order to verify the relationship between IE and PV, we screened JAK2V617F mutation in a consecutive series of 11 IE and, for comparison, in 15 PV. JAK2V617F mutation was screened by both cDNA sequencing and mutation specific PCR in both peripheral blood and bone marrow samples. All 11 IE tested negative for JAK2V617F mutation, which, conversely, occurred in 11/15 (73.3%) PV. Our results demonstrate that JAK2V617F is absent in IE and may represent a useful molecular marker for distinguishing IE from PV.     

Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera.

After accounting for misdiagnosis and treatment effect, allele-specific (AS)-PCR detects the JAK2V617F mutation in >95% of polycythemia vera (PV) patients. Using database inquiry, we identified 6 of a total 220 cases with PV that were JAK2V617F-negative (prevalence=3%). Of these, five cases ( approximately 80%) were found to harbor one of the two JAK2 exon 12 mutations (F537-K539delinsL or N542-E543del) in bone marrow (BM) and/or peripheral blood cells. Similar screening of six additional cases - three each with idiopathic erythrocytosis (IE) or otherwise unexplained erythrocytosis (UE) - did not reveal either JAK2V617F or JAK2 exon 12 mutations. We found JAK2 exon 12 mutations in PV cases to be readily detected by both DNA sequencing and AS-PCR, regardless of whether BM or peripheral blood cells were used as the source for DNA. Although erythroid hyperplasia was the predominant histologic feature on BM examination, megakaryocyte abnormalities and reticulin fibrosis were noted in most PV patients harboring exon 12 mutations. However, similar BM morphologic changes can also be seen in some JAK2V617F-positive PV cases; therefore, distinct genotype-phenotype association cannot be established.     

Switch in X-inactivation in a JAK2 V617F-negative case of polycythemia vera with two acquired X-autosome translocations

We report a JAK2 V617F-negative case of polycythemia vera with two acquired balanced X-autosome translocations and no history of previous exposure to chemo/radiotherapy. The patient's first clone carried a novel translocation t(X;15)(q24;q13) as a sole abnormality. The second clone exhibited an additional translocation, t(X;20)(q13;q13.3), which is a rare recurrent abnormality in myeloid malignancies. This is the first report of a hematological disorder with both X chromosomes being translocated. Late replication studies revealed a switch in X-inactivation from the X chromosome involved in t(X;15) (first clone) to the X chromosome involved in the t(X;20)(q13;q13.3) (second clone). The inactivation of the translocated X chromosomes could provide potential for the inactivation of the adjacent autosomal regions, resulting in epigenetic gene silencing.     

A refined diagnostic algorithm for polycythemia vera that incorporates mutation screening for JAK2V617F

Historically, red cell mass (RCM) measurement has been promoted as an accurate indicator of the body’s red cell content. Both the Polycythemia Vera Study Group and a committee sponsored by the World Health Organization have endorsed its inclusion in their diagnostic criteria without supporting evidence of diagnostic accuracy from a systematic study. Over the years, it has become evident that RCM measurement is a cumbersome and costly test and, as a result, it has been abandoned by many hematologists in practice. The recent discovery that a somatic JAK2-activating mutation, JAK2^V617F, is almost invariably associated with polycythemia vera further supports the use of diagnostic algorithms that are based on biologic parameters in place of traditional diagnostic criteria that are based on RCM measurement. Therefore, a contemporary approach to the diagnosis of polycythemia vera starts with peripheral blood mutation screening for JAK2^V617F as well as measurement of serum erythropoietin. The results of these tests, along with the clinical scenario, determine the need for further investigation, including bone marrow examination.     

Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden.

The aim of this study was to determine whether the burden of JAK2(V617F) allele correlated with major clinical outcomes in patients with polycythemia vera (PV). To this end, we determined JAK2 mutant allele levels in granulocytes of 173 PV patients at diagnosis. The mean (+/-s.d.) mutant allele burden was 52% (+/-29); 32 patients (18%) had greater than 75% mutant allele. The burden of JAK2(V617F) allele correlated with measurements of stimulated erythropoiesis (higher hematocrit, lower mean cell volume, serum ferritin and erythropoietin levels) and myelopoiesis (higher white cell count, neutrophil count and serum lactate dehydrogenase) and with markers of neutrophil activation (elevated leukocyte alkaline phosphatase and PRV-1 expression). As compared to those with less than 25% mutant allele, patients harboring greater than 75% JAK2(V617F) allele were at higher relative risk (RR) of presenting larger spleen (RR 4.7; P<0.001) or suffering from pruritus (RR 3.1; P<0.001). In these patients, the risk of requiring chemotherapy (RR 1.8; P=0.001) or developing major cardiovascular events (RR 7.1; P=0.003) during follow up were significantly increased. We conclude that a burden of JAK2(V617F) allele greater than 75% at diagnosis points to PV patients with high-risk disease.     

Characterization of murine JAK2V617F-positive myeloproliferative disease

The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.     

JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment.     

TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations.

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM), FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations.     

真性红细胞增多症患者JAK2基因突变的临床意义

真性红细胞增多症（polycythemia vera,PV）为一种以红细胞增多为特征的骨髓增殖性肿瘤（myeloproliferative neoplasm,MPN）。PV的发病机制尚未阐明,可能和JAK2基因的功能获得性体细胞突变相关,JAK2基因突变为PV提供了分子学诊断标准。JAK2基因突变和等位基因突变负荷可推测PV患者的临床特征和发展趋势。JAK2基因突变的发现促进了分子靶向治疗的发展,JAK2抑制剂ruxolitinib已应用于临床,治疗效果和安全性均良好。JAK2等位基因突变负荷与白细胞增多、转化为骨髓纤维化（myelofibrosis,MF）密切相关,高JAK2等位基因突变负荷可能为PV患者预后不良的危险因素。本文对PV患者JAK2基因突变的临床意义进行综述。