JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation

The JAK2V617F constitutively activated tyrosine kinase is found in most patients with myeloproliferative neoplasms. While examining the interaction between JAK2 and PRMT5, an arginine methyltransferase originally identified as JAK-binding protein 1, we found that JAK2V617F (and JAK2K539L) bound PRMT5 more strongly than did wild-type JAK2. These oncogenic kinases also acquired the ability to phosphorylate PRMT5, greatly impairing its ability to methylate its histone substrates, and representing a specific gain-of-function that allows them to regulate chromatin modifications. We readily detected PRMT5 phosphorylation in JAK2V617F-positive patient samples, and when we knocked down PRMT5 in human CD34+ cells using shRNA, we observed increased colony formation and erythroid differentiation. These results indicate that phosphorylation of PRMT5 contributes to the mutant JAK2-induced myeloproliferative phenotype.     

伴有JAK2 V617F突变的慢性中性粒细胞白血病1例并文献复习

目的:探讨伴有JAK2基因V617F突变的慢性中性粒细胞白血病(CNL)的实验室特征。方法:通过骨髓涂片进行细胞形态学分析诊断;采用实时荧光定量PCR方法检测BCR-ABL融合基因;实时荧光定量PCR方法及DNA测序方法检测JAK2 V617F突变;RHG显带进行常规染色体核型分析。结果:细胞形态学诊断为慢性中性粒细胞白血病;BCR/ABL融合基因为阴性;存在JAK2 V617F杂合突变;染色体分析结果为正常核型。结论:JAK2 V617F突变在慢性中性粒细胞白血病中很少见,具有该突变的CNL患者有较长的生存期,JAK2 V617F突变可能提示预后良好。     

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.     

伴有JAK2 V617F突变的慢性中性粒细胞白血病1例并文献复习

目的：探讨伴有JAK2基因V617F突变的慢性中性粒细胞白血病（CNL）的实验室特征。方法：通过骨髓涂片进行细胞形态学分析诊断;采用实时荧光定量PCR方法检测BCR-ABL融合基因;实时荧光定量PCR方法及DNA测序方法检测JAK2 V617F突变;RHG显带进行常规染色体核型分析。结果：细胞形态学诊断为慢性中性粒细胞白血病;BCR/ABL融合基因为阴性;存在JAK2 V617F杂合突变;染色体分析结果为正常核型。结论：JAK2 V617F突变在慢性中性粒细胞白血病中很少见,具有该突变的CNL患者有较长的生存期,JAK2 V617F突变可能提示预后良好。     

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC₅₀) of <1 n, and had 30–50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL–JAK2 fusion gene; IC₅₀=11–120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.     

JAK2抑制剂在肿瘤治疗中的应用价值

JAK家族是JAK-STAT信号传导通路中的非受体型酪氨酸蛋白激酶,JAK2-STAT3作为JAK-STAT通路中的一个重要信号轴,它在肿瘤中的持续性激活可以通过影响细胞的生长、凋亡、周期等起到促进肿瘤发生发展的作用.JAK2突变,尤其是JAK2V617F突变的发现引发了JAK2抑制剂的研究热潮,为肿瘤的治疗提供了新的方向.JAK2抑制剂能削弱肿瘤细胞的恶性生物学行为,在有JAK2V617F突变的血液系统肿瘤以及JAK2-STAT3信号异常的实体肿瘤中都具有一定的治疗价值.     

The two faces of myeloproliferative neoplasms: Molecular events underlying lymphoid transformation

Multipotent haematopoietic stem cells pass through stages of differentiation with the progressive loss of developmental options leading to the production of terminally differentiated mature blood cells. This process is regulated by soluble cytokines binding to a ligand specific cell surface receptor on a precursor cell. Key to signal transduction are tyrosine kinase proteins which can be divided into two sub families, the receptor protein tyrosine kinases which are transmembrane receptors and retain an intact catalytic kinase domain and the cytoplasmic tyrosine kinases which bind to cytokine receptors. Abnormalities of tyrosine kinase proteins are well recognised in myeloid malignancies, mutation in the cytoplasmic tyrosine kinase JAK2 (V617F) is key in the pathogenesis of myeloproliferative neoplasms, and translocations involving ABL key in the development of chronic myeloid leukaemia. However tyrosine kinase mutations are increasingly recognised to play a role in the pathogenesis of a wider range of haematological cancers. This review focuses on the role of deregulated tyrosine kinase genes either as part of novel fusion proteins involving FGFR1, PDGFRα, PDGFRβ, JAK2 and ABL, or as a consequence of point mutation in JAK1 or JAK2 in the development of precursor T and B lymphoid malignancies or mixed myeloid/lymphoid disorders. We also set out some of the postulated mechanisms which underlie the association of tyrosine kinase mutations with the development of lymphoid malignancy.     

p27 Kip1 inhibits HER2/neu-mediated cell growth and tumorigenesis.

HER2/neu, a receptor tyrosine kinase oncogene, promotes mitogenic growth and transformation of cancer cells. We previously identified that its oncogenic signals down-regulate the cyclin-dependent kinase inhibitor p27 Kip1, which is defined as a haplo-insufficient tumor suppressor. Here, we applied the human p27 gene as a novel anticancer agent for HER2/neu-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. Overexpression of p27 inhibits HER2/neu-activated CDK2 activity, cell proliferation, and transformation. Most significantly for clinical application, p27 expression in HER2/neu-overexpressing cells can be regulated in vivo and reduce the tumor volume in a tumor model. The findings demonstrate the applicability of employing p27 in HER2/neu-associated cancer gene therapy.     

Advances in the molecular characterization of Philadelphia-negative chronic myeloproliferative disorders

PURPOSE OF REVIEW: The identification and characterization of somatic disease alleles have greatly improved our understanding of the molecular pathogenesis of myeloproliferative disorders. This review focuses on recent studies investigating the role of activated tyrosine kinase signaling in the Philadelphia chromosome negative myeloproliferative disorders. RECENT FINDINGS: Previously identified tyrosine kinase mutations in chronic myeloid leukemia and other myeloproliferative disorders suggested the possibility that polycythemia vera, essential thrombocythemia and primary myelofibrosis are also caused by activated tyrosine kinases. Recent studies identified an activating mutation in the JAK2 tyrosine kinase (JAK2V617F) in most patients with polycythemia vera and in approximately half of those with essential thrombocythemia and primary myelofibrosis. More recently, activating mutations in the thrombopoietin receptor and in JAK2 exon 12 have been identified in JAK2V617F negative myeloproliferative disorders. SUMMARY: The discovery of activated tyrosine kinases in the majority of patients with polycythemia vera, essential thrombocythemia and primary myelofibrosis has diagnostic and pathogenetic implications. Subsequent studies are needed to elucidate the cause of myeloproliferative disorders without known disease alleles and to determine if inhibition of JAK2 signaling has therapeutic efficacy in the treatment of polycythemia vera, essential thrombocythemia and primary myelofibrosis.     

A novel CyclinE/CyclinA-CDK Inhibitor targets p27 degradation,cell cycle progression and cell survival: Implications in cancer therapy

p27^Kip1 (p27) binds and inhibits the cyclin E- or cyclin A-associated cyclin-dependent kinases (CDKs)2 and other CDKs, and negatively regulates G1–G2 cell cycle progression. To develop specific CDK inhibitors, we have modeled the interaction between p27 and cyclin A-CDK2, and designed a novel compound that mimics p27 binding to cyclin A-CDK2. The chemically synthesized inhibitor exhibited high potency and selective inhibition towards cyclin E/cyclin A-CDK2 kinase in vitro but not other kinases. To facilitate permeability of the inhibitor, a cell penetrating peptide (CPP) was conjugated to the inhibitor to examine its effect in several cancer cell lines. The CPP-conjugated inhibitor significantly inhibited the proliferation of cancer cells. The treatment of the inhibitor resulted in the increased accumulation of p27 and p21^Cip1/Waf1 (p21) and hypo-phosphorylation of retinoblastoma protein (Rb). The degradation of p27, mediated through the phosphorylation of threonine-187 in p27, was also inhibited. Consequently, exposure of cells to the inhibitor caused cell cycle arrest and apoptosis. We conclude that specific cyclinE/cyclin A-CDK2 inhibitors can be developed based on the interaction between p27 and cyclin/CDK to block cell cycle progression to prevent tumor growth and survival.     

JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders.

A mutation in the JH2 pseudokinase domain of the Janus kinase 2 gene (JAK2 V617F) has been described in chronic myeloproliferative disorders (MPD). We screened 79 acute myeloid leukemia (AML) cell lines and found five positive for JAK2 V617F (HEL, MB-02, MUTZ-8, SET-2, UKE-1), 4/5 with histories of MPD/MDS. While SET-2 expressed both mutant (mu) and wild-type (wt) JAK2, remaining positives carried homo-/hemizygous JAK2 mutations. Microsatellite analysis confirmed losses of heterozygosity (LOH) affecting the JAK2 region on chromosome 9p in MB-02, MUTZ-8 and UKE-1, but also in HEL, the only JAK2mu cell line lacking any reported MPD/MDS history. All five JAK2mu cell lines displayed cytogenetic hallmarks of MDS, namely losses of 5q or 7q, remarkably in 4/5 cases affecting both chromosomes. Our combined FISH and microsatellite analysis uncovered a novel mechanism to supplement mitotic recombination previously proposed to explain JAK2 LOH, namely chromosome deletion with/without selective JAK2mu amplification. Confirming the importance of the mutated JAK2 protein for growth and prevention of apoptosis, JAK2mu cell lines displayed higher sensitivities to JAK2 inhibition than JAK2wt cell lines. In summary, JAK2 V617F cell lines, derived from patients with history of MPD/MDS, represent novel research tools for elucidating the pathobiology of this JAK2 mutation.     

The JAK2V617F tyrosine kinase mutation has no impact on overall survival and the risk of leukemic transformation in myelofibrosis

The prevalence of JAK2V617F tyrosine kinase mutation differs between various variants of myelofibrosis with the higher detection rate for patients with post-polycythemia vera myelofibrosis (post-PV MF; 91%) if compared to primary myelofibrosis (PMF; 45%) and post-essential thrombocythemia myelofibrosis (post-ET MF; 39%). The impact of V617F point mutation and its allele burden on overall survival (OS) and the risk of leukemic transformation (LT) has been the subject of several studies, but the results were ambiguous. Our study included 77 patients with the following variants: 42 patients with PMF (55%), 16 with post-ET MF (21%) and 19 with post-PV MF (24%). Median age at diagnosis for the entire cohort was 61?years (range 19-81), with 53% of female. A total of 42 patients were JAK2V617F positive, giving an overall frequency of 55%; the median allele burden was 22% (range 2-96%). The JAK2V617F point mutation was detected in 21 patients with PMF (50%), 14 with post-PV MF (88%) and 7 with post-ET MF (37%). Lower JAK2V617F allele burden was more frequently detected in PMF patients, whereas higher allele burden was predominantly seen in post-PV/ET MF group. There was no significant difference between V617F-positive and V617F-negative patients in terms of studied parameters in PMF as well as in post-PV/ET MF subgroup. No significant difference was also demonstrated when the above-mentioned subpopulations were analyzed according to JAK2V617F allele burden, except higher leukocyte count in post-PV/ET MF patients with higher allele burden (14.3?×?10(9)/L vs. 6.2?×?10(9)/L; p?=?.03). Median follow-ups for V617F-positive and V617F-negative patients were 16.6?months (range 3.6-206.4) and 36.4?months (range 2.5-142.1), respectively. The presence of JAK2V617F mutation did not affect OS and the risk of LT development.     

A potential role for HSP90 inhibitors in the treatment of JAK2 mutant-positive diseases as demonstrated using quantitative flow cytometry

The V617F mutation of the JAK2 tyrosine kinase is found in a majority of patients with myeloproliferative disorders. Flow cytometry assays for quantitation of phosphorylated and total protein for JAK2, STAT5, and heat shock proteins (HSPs) were developed to facilitate the study of the JAK/STAT pathway. A cell line homozygous for V617F (HEL) was treated with inhibitors of JAK2 tyrosine kinase activity and the HSP90 inhibitor 17-AAG. 17-AAG reduced HSP90 levels, but increased HSP70 levels. Phospho-STAT5, total STAT5, and total AKT levels were also reduced by17-AAG treatment. Further, phospho-JAK2, total JAK2, and cell viability were reduced to a greater extent by 17-AAG than by the pan-JAK kinase family inhibitor JKII or the JAK2-specific inhibitor AG490, and these inhibitors failed to synergize with 17-AAG. Flow-cytometry-based assays for JAK/STAT signaling pathway and HSPs are likely to have broad clinical utility for monitoring patients with abnormalities in the JAK2 pathway.     

Involvement of cell cycle regulatory proteins and MAP kinase signaling pathway in growth inhibition and cell cycle arrest by a selective cyclooxygenase 2 inhibitor, etodolac, in human hepatocellular carcinoma cell lines

Recent studies have shown that selective cyclooxygenase-2 (COX-2) inhibitors induce growth inhibition and cell cycle arrest in hepatocellular carcinoma (HCC) cell lines. However, the mechanism by which COX-2 inhibitors regulate the cell cycle and whether or not growth signal pathways are involved in the growth inhibition remain unclear. In this study, we investigated the mechanisms of growth inhibition and cell cycle arrest by etodolac, a selective COX-2 inhibitor, in HCC cell lines, HepG2 and PLC/PRF/5, by studying cell cycle regulatory proteins, and the MAP kinase and PDK1-PKB/AKT signaling pathways. Etodolac inhibited growth and PCNA expression and induced cell cycle arrest in both HCC cell lines. Etodolac induced p21WAF1/Cip1 and p27Kip1 expression and inhibited CDK2, CDK4, CDC2, cyclin A and cyclin B1 expression, but did not affect cyclin D1 or cyclin E. HGF and 10% FBS induced ERK phosphorylation, but phosphorylation of p38, JNK and AKT was down-regulated by etodolac. PD98059, a selective inhibitor of ERK phosphorylation, induced growth inhibition, the expression of p27Kip1 and cell cycle arrest. In conclusion, p21WAF1/Cip1, p27Kip1, CDK2, CDK4, CDC2, cyclin A, cyclin B1 and the MAP kinase signaling pathway are involved in growth inhibition and cell cycle arrest by a selective COX-2 inhibitor in HCC cell lines.     

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27KiP1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.     

Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF-->mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase [MEK]-->ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF-->MEK-->ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF-->MEK-->ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.