실시간 반정량 PCR, JAK2 MutaScreenTM kit를 이용한 JAK2 V617F 유전자 변이 선별검사의 유용성

채효진1,이제훈,임지향,김명신,김용구,한경자,이종욱,민우성,정승원,조병식,조석구 대한진단검사의학회 2009 Annals of Laboratory Medicine Vol.29 No.3

Background : Real-time PCR for quantification of JAK2 V617F has recently been introduced and used to evaluate the importance of mutant allele burden in both diagnosis and disease progression in myeloproliferative diseases (MPDs). We evaluated the usefulness of JAK2 MutaScreenTM kit that uses a real-time semiquantitative PCR method and has been designed to screen JAK2 V617F mutant allele burden. Methods : Forty MPD patients were included in this study. We screened JAK2 V617F and determined the mutant allele burden using JAK2 MutaScreenTM kit. The mutant allele burden was estimated by six-scaled standards of JAK2 V617F mutant allele (2%, 5%, 12.5%, 31%, 50%, and 78%). For evaluation of test performance, an allele-specific PCR (AS-PCR) was carried out in all samples by using Seeplex JAK2 Genotyping kit. We assessed the clinical differences in distinct disease entities of MPDs according to JAK2 V617F mutant allele burden. Results : JAK2 V617F mutation was detected in 30 cases, including 10 of 11 cases (91%) of polycythemia vera (PV), 13 of 20 cases (65%) of essential thrombocythemia (ET), and 2 of 3 cases (67%) of chronic idiopathic myelofibrosis (CIMF). The concordance rate between the two tests was 95% (38/40). JAK2 V617F mutant allele burden was greater than 50% in 17 cases, and 10 of them (59%) were PV. In contrast, mutant allele burden was less than 50% in 13 cases and 11 of them (85%) were ET. Conclusions : JAK2 MutaScreenTM kit that utilizes a real-time semi-quantitative PCR method is a useful tool for diagnosing MPDs precisely. It can be used to assess the grade of mutant allele burden as well as to screen JAK2 V617F simultaneously. 배경 : 최근 JAK2 V617F 유전자 변이에 실시간 정량 PCR법 을 이용한 검사법이 소개되어 MPDs의 진단과 질환의 진행에 있어 유전자 변이량 측정의 중요성 평가에 사용되고 있다. 저자 들은 JAK2 V617F 유전자 변이 선별을 위해 새로이 고안된 실 시간 반정량 PCR법인 JAK2 MutaScreenTM kit의 유용성을 평 가하였다. 방법 : 40예의 MPDs 환자를 대상으로 JAK2 MutaScreenTM kit를 이용하여 JAK2 V617F 선별 및 유전자 변이량을 측정하 였다. JAK2 V617F 유전자 변이량 측정에는 6단계의 표준물질 을 사용하였다(2%, 5%, 12.5%, 25%, 31%, 50%, 78%). 검사 수 행능의 평가를 위하여 동일한 모든 검체에 대하여 Seeplex JAK2 Genotyping kit을 이용한 AS-PCR법으로 시행한 검사 결과와 비교하였다. 또한 각 MPDs의 질환군과 유전자 변이량에 따른 임상적 특성의 차이를 분석하였다. 결과 : 30예에서 JAK2 V617F 유전자 변이가 검출되었으며 각 질환별 양성률은 PV 91% (10/11), ET 65% (13/20), CMIF 67% (2/3)였다. 두 검사방법 간의 일치율은 95%였다(38/40). JAK2 V617F 유전자 변이량이 50% 이상인 17예 중 10예가 PV였으며, JAK2 유전자 변이량이 50% 이하인 13예 중에서는 11예가 ET였 다(85%). 결론 : 실시간 반정량 PCR법을 이용한 검사법인 JAK2 V617F MutaScreenTM kit는 유전자 변이 검출 및 유전자 변이량의 동 시 측정이 가능하므로 보다 정밀한 MPDs의 진단에 유용한 검 사방법으로 생각된다.

JAK2V617F 돌연변이를 보인 비정형만성골수성백혈병 1예

김주연,이세련,남명현,윤수영,임채승,이창규,김병수,조윤정,김영기,이갑노 대한진단검사의학회 2011 Laboratory Medicine Online Vol.1 No.4

Atypical chronic myeloid leukemia (aCML) is a rare leukemic disorder that shows myelodysplastic and myeloproliferative features simultaneously. The Janus kinase 2 gene V617F mutation (JAK2V617F) in aCML has been the source of much controversy. Some JAK2V617F positive cases have been reported but others observed no JAK2V617F mutation in aCML as defined by WHO classification. Recently, we experienced a case of aCML with JAK2V617F mutation with typical myelodysplastic/myeloproliferative features in peripheral blood and bone marrow aspirates. The karyotype was normal and no BCR/ABL1, PDGFRA or PDGFRB gene rearrangement was noted with FISH analysis. JAK2V617F mutation of the case was identified with amplification refractory mutation system PCR and direct sequencing. We also studied JAK2V617F mutation status in 3 additional cases of previously diagnosed aCML in our institution, but no mutation was identified.Atypical chronic myeloid leukemia (aCML) is a rare leukemic disorder that shows myelodysplastic and myeloproliferative features simultaneously. The Janus kinase 2 gene V617F mutation (JAK2V617F) in aCML has been the source of much controversy. Some JAK2V617F positive cases have been reported but others observed no JAK2V617F mutation in aCML as defined by WHO classification. Recently, we experienced a case of aCML with JAK2V617F mutation with typical myelodysplastic/myeloproliferative features in peripheral blood and bone marrow aspirates. The karyotype was normal and no BCR/ABL1, PDGFRA or PDGFRB gene rearrangement was noted with FISH analysis. JAK2V617F mutation of the case was identified with amplification refractory mutation system PCR and direct sequencing. We also studied JAK2V617F mutation status in 3 additional cases of previously diagnosed aCML in our institution, but no mutation was identified.

The discovery of 2,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 2 (JAK2) inhibitors versus JAK1 and JAK3

Lee, S.M.,Yoon, K.B.,Lee, H.J.,Kim, J.,Chung, Y.K.,Cho, W.J.,Mukai, C.,Choi, S.,Kang, K.W.,Han, S.Y.,Ko, H.,Kim, Y.C. Elsevier/Pergamon 2016 Bioorganic & medicinal chemistry Vol.24 No.21

Members of the Janus kinase (JAK) family are potential therapeutic targets. Abnormal signaling by mutant JAK2 is related to hematological malignancy, such as myeloproliferative neoplasms (MPNs), and tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC). We discovered a potent and highly selective inhibitor of JAK2 over JAK1 and -3 based on the structure of 4-(2,5-triazole)-pyrrolopyrimidine. Among all triazole compounds tested, 2,5-triazole regioisomers more effectively inhibited JAK2 kinase activity than isomers with substitutions of various alkyl groups at the R<SUB>2</SUB> position, except for methyl-substituted 1,5-triazole, which was more potent than the corresponding 1,4- and 2,5-triazoles. None of the synthesized 1,4-isomers inhibited all three JAK family members. Compounds with phenyl or tolyl group substituents at the R<SUB>1</SUB> position were completely inactive compared with the corresponding analogues with a methyl substituted at the R<SUB>1</SUB> position. As a result of this structure-activity relationship, 54, which is substituted with a cyclopropylmethyl moiety, exhibited significant inhibitory activity and selectivity (IC<SUB>50</SUB>=41.9nM, fold selectivity JAK½ 10.6 and JAK3/2 58.1). Compound 54 also exhibited an equivalent inhibition of wild type JAK2 and the V617F mutant. Moreover, 54 inhibited the proliferation of HEL 92.1.7 cells, which carry JAK2 V617F, and gefitinib-resistant HCC827 cells. Compound 54 also suppressed STAT3 phosphorylation at Y705.

Clinical features and outcomes of JAK2 V617F-positive polycythemia vera and essential thrombocythemia according to the JAK2 V617F allele burden

A-Jin Lee,Sang-Gyung Kim,Jun Yeb Nam,Jaehum Yun,류헌모,Sung Hwa Bae 대한혈액학회 2021 Blood Research Vol.56 No.4

Background JAK2 mutation status is a well-known risk factor for thrombosis in patients with myeloproliferative neoplasms. However, the clinical usefulness of JAK2 V617F allele burden is under investigation. Methods We retrospectively evaluated the impact of the JAK2 V617F allele burden on clinical characteristics and outcomes of JAK2 V617F-positive polycythemia vera (PV) and essential thrombocythemia (ET). The JAK2 V617F allele burden was measured using sequencing. Results Altogether, 127 patients with JAK2 V617F mutation (PV, N=61; ET, N=66) were included in this study. JAK2 V617F allele burdens were positively correlated with white blood cell counts, hemoglobin values, lactate dehydrogenase levels, and platelet counts. The median values of JAK2 V617F allele burden in patients with PV and ET were 58% and 30%, respectively. A JAK2 V617F allele burden of ≥30%, older age, and a higher hemoglobin level were risk factors for thrombotic events in ET. In patients with PV, older age was the only thrombotic risk factor. The 8-year probabilities of overall survival (OS) were 82.9% in all patients. A high JAK2 V617F allele burden (≥58%) was associated with poor OS in patients with PV. For the patients with ET, the difference in 8-year OS based on the JAK2 V617F allele burden was not significant. Conclusion The JAK2 V617F allele burden was correlated with hematologic parameters and clinical outcomes. Assessing the JAK2 V617F allele burden can be helpful in predicting the thrombotic risk and disease course in patients with JAK2 V617F-positive PV and ET.

JAK2 Loss Arising From Tumor-Spread-Through-Air-Spaces (STAS) Promotes Tumor Progression by Suppressing CD8+ T Cells in Lung Adenocarcinoma: A Machine Learning Approach

Soohwan Choi,Hyung Suk Kim,Kyueng-Whan Min,Yung-Kyun Noh,Jeong-Yeon Lee,Ji-Yong Moon,Un Suk Jung,Mi Jung Kwon,Dong-Hoon Kim,Byoung Kwan Son,Jung Soo Pyo,Sun Kyun Ro 대한의학회 2024 Journal of Korean medical science Vol.39 No.2

Background: Tumor spread through air spaces (STAS) is a recently discovered risk factor for lung adenocarcinoma (LUAD). The aim of this study was to investigate specific genetic alterations and anticancer immune responses related to STAS. By using a machine learning algorithm and drug screening in lung cancer cell lines, we analyzed the effect of Janus kinase 2 (JAK2) on the survival of patients with LUAD and possible drug candidates. Methods: This study included 566 patients with LUAD corresponding to clinicopathological and genetic data. For analyses of LUAD, we applied gene set enrichment analysis (GSEA), in silico cytometry, pathway network analysis, in vitro drug screening, and gradient boosting machine (GBM) analysis. Results: The patients with STAS had a shorter survival time than those without STAS (P < 0.001). We detected gene set-related downregulation of JAK2 associated with STAS using GSEA. Low JAK2 expression was related to poor prognosis and a low CD8+ T-cell fraction. In GBM, JAK2 showed improved survival prediction performance when it was added to other parameters (T stage, N stage, lymphovascular invasion, pleural invasion, tumor size). In drug screening, mirin, CCT007093, dihydroretenone, and ABT737 suppressed the growth of lung cancer cell lines with low JAK2 expression. Conclusion: In LUAD, low JAK2 expression linked to the presence of STAS might serve as an unfavorable prognostic factor. A relationship between JAK2 and CD8+ T cells suggests that STAS is indirectly related to the anticancer immune response. These results may contribute to the design of future experimental research and drug development programs for LUAD with STAS. Background: Tumor spread through air spaces (STAS) is a recently discovered risk factor for lung adenocarcinoma (LUAD). The aim of this study was to investigate specific genetic alterations and anticancer immune responses related to STAS. By using a machine learning algorithm and drug screening in lung cancer cell lines, we analyzed the effect of Janus kinase 2 (JAK2) on the survival of patients with LUAD and possible drug candidates. Methods: This study included 566 patients with LUAD corresponding to clinicopathological and genetic data. For analyses of LUAD, we applied gene set enrichment analysis (GSEA), in silico cytometry, pathway network analysis, in vitro drug screening, and gradient boosting machine (GBM) analysis. Results: The patients with STAS had a shorter survival time than those without STAS (P < 0.001). We detected gene set-related downregulation of JAK2 associated with STAS using GSEA. Low JAK2 expression was related to poor prognosis and a low CD8+ T-cell fraction. In GBM, JAK2 showed improved survival prediction performance when it was added to other parameters (T stage, N stage, lymphovascular invasion, pleural invasion, tumor size). In drug screening, mirin, CCT007093, dihydroretenone, and ABT737 suppressed the growth of lung cancer cell lines with low JAK2 expression. Conclusion: In LUAD, low JAK2 expression linked to the presence of STAS might serve as an unfavorable prognostic factor. A relationship between JAK2 and CD8+ T cells suggests that STAS is indirectly related to the anticancer immune response. These results may contribute to the design of future experimental research and drug development programs for LUAD with STAS.

마그놀롤의 HDF세포에서 Nrf2-SOCS3-Jak2-STAT3에 의한 UVB 유래 염증데미지 조절

남영선(Young sun Nam),지주리(Juree Ji) 한국생명과학회 2020 생명과학회지 Vol.30 No.10

본 연구는 magnolol에 의한 UVB 유도 세포 손상의 복구를 조사하였다. 우리는 약물재배치를 위해 STAT3 기작을 분석하였고, magnolol HDF 세포에서 세포 생존력을 향상시키며, STAT3의 억제제인 것을 확인하였다. IL-6, UVB 및 IFNγ로 처리 된 HDF 세포는 Jak2 및 인산화 된 STAT3 (p-STAT3)의 높은 발현을 나타냈다. Magnolol의 처리는 UVB 유도 세포에서 Jak2 및 p-STAT3의 발현을 감소시킬 수 있었다. 또한, UVB- 손상된 세포 성장은 용량 의존적 방식으로 재 활성화 및 magnolol 과의 상관 관계가 상당히 증가되었다. UVB 처리 된 HDF 세포에 대한 AG490 (Jak2 억제제) 처리와 비교하여, 세포 증식이 유의하게 증가 하였다. 우리는 AG490 및 magnolol 이 TNF-α 농도를 감소시키는 것을 확인했다. Western blot (단백질 수준)은 오직 magnolol 처리 된 세포에서만 Jak2 및 p-STAT3 발현의 감소를 나타냈고, Jak2, p-STAT3 및 SOCS3의 발현은 또한 magnolol 처리한 세포에서만 증가하였다. 세포를 magnolol 및 ML385 (NRF2 억제제)로 동시 처리시 세포 증식 및 NRF2 발현을 감소시켰다. MMP9의 양은 magnolol 및 ML385 로의 처리에 의해 증가되었다. 종합적으로, 이들 결과는 NRF2, SOCS3, Jak2 및 STAT3의 발현을 조절함으로써 UVB 손상 후 세포를 회복시키는데 있어 magnolol의 가능성을 입증한다. This study investigated the repair of UVB-induced cell damage by magnolol. We performed a drug-repurposing screen, and, in the STAT3 reporter gene assay, magnolol was identified as a suppressor of STAT3 that improves the cell viability of HDF cells. HDF cells treated with IL-6, UVB, and IFNγ showed the highest expression of Jak2 and phosphorylated STAT3 (p-STAT3), and magnolol was able to decrease the expression of Jak2 and p-STAT3 in UVB-induced cells. Moreover, UVB-damaged cell growth increased significantly in correlation with both reactivation and with magnolol in a dose-dependent manner. Compared with AG490 (a Jak2 inhibitor) treatment of UVB-treated HDF cells, cell proliferation increased significantly. We confirmed that AG490 and magnolol reduced TNF-α concentrations, and Western blotting (protein level) showed decreases in Jak2 and p-STAT3 expression in only the magnolol-treated cells. The expression of Jak2, p-STAT3, and SOCS3 also increased only after treatment with magnolol. Cells were treated with magnolol and ML385 (an NRF2 inhibitor), and these secondary metabolites reduced cell proliferation and NRF2 expression. The amount of MMP9 was also increased by cotreatment with magnolol and ML385. Collectively, these results demonstrate the potential of magnolol for repairing cells after UVB-induced damage by regulating the expression of NRF2, SOCS3, Jak2, and STAT3.

JAK-STAT pathway and myogenic differentiation

Jang, You-Na,Baik, Eun Joo Landes Bioscience 2013 JAK-STAT Vol.2 No.2

<P>Myogenic differentiation plays an important role in muscle regeneration and is regulated by two transcription factor families, MRFs and MEF2, which induce differentiation of myoblasts through expression of the muscle-specific gene, <I>myogenin</I>. In addition, many intracellular signaling pathways are also involved in myogenic differentiation, including p38 MAPK, ERK/MAPK and PI3K/AKT. The JAK-STAT pathway is activated by various cytokines and positively or negatively regulates the differentiation of myoblasts. JAK1 plays a notable role in proliferation; whereas, JAK2 and JAK3 function mainly in differentiation. The STATs, molecules downstream of JAK, regulate myogenesis. With JAK1, STAT1 promotes proliferation, while STAT3 has a dual effect on proliferation and differentiation. The JAK-STAT negative regulator, SOCS, is also associated with myogenesis; although, its role is controversial. In this review, we will discuss the role of the JAK-STAT pathway on myogenic differentiation.</P>

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

Singdong, Roongrudee,Siriboonpiputtana, Teerapong,Chareonsirisuthigul, Takol,Kongruang, Adcharee,Limsuwanachot, Nittaya,Sirirat, Tanasan,Chuncharunee, Suporn,Rerkamnuaychoke, Budsaba Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.10

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9 mutations in 100 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET.

Development of JAK inhibitors for the treatment of immune‑mediated diseases: kinase‑targeted inhibitors and pseudokinase‑targeted inhibitors

Hyung‑Ook Kim 대한약학회 2020 Archives of Pharmacal Research Vol.43 No.11

JAKs are a family of intracellular tyrosinekinases consisting of four members, JAK1, JAK2, JAK3,and TYK2. They are key components of the JAK-STATpathway that transmit signals of many cytokines involvedin the pathogenesis of numerous immune-mediated diseasesand have been major molecular targets in developing newdrugs for the treatment of such diseases. Some small-moleculeinhibitors of JAKs have been approved by the FDA forrheumatoid arthritis, psoriatic arthritis, and inflammatorybowel disease. Now, newer JAK inhibitors with isoformselectivityamong the four different JAKs are being developed,with the aim of improving clinical outcomes comparedwith earlier developed drugs with pan-JAK inhibition. Mostof these selective inhibitors target the kinase domains ofJAKs, functioning through the traditional inhibition modeof kinases; but recently those that target their pseudokinasedomains, allosterically inhibiting the enzymes, have beenunder development. In this review, key characteristics, efficacy,and safety of FDA-approved and representative drugsin late stages of development are briefly described in orderto provide clinical implications with respect to JAK inhibitorselectivity and future development perspectives. The recentdevelopment of pseudokinase-targeted inhibitors of JAKsis also included.

JAK2 regulation by licochalcone H inhibits the cell growth and induces apoptosis in oral squamous cell carcinoma

Oh, Ha-Na,Oh, Keon Bong,Lee, Mee-Hyun,Seo, Ji-Hye,Kim, Eunae,Yoon, Goo,Cho, Seung-Sik,Cho, Young Sik,Choi, Hyun Woo,Chae, Jung-II,Shim, Jung-Hyun Elsevier 2019 Phytomedicine Vol.52 No.-

<P><B>Abstract</B></P> <P><B>Background</B></P> <P>Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations.</P> <P><B>Purpose</B></P> <P>The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms.</P> <P><B>Study Design</B></P> <P>We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway.</P> <P><B>Methods</B></P> <P>To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis.</P> <P><B>Results</B></P> <P>According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity <I>in vitro</I> by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade.</P> <P><B>Conclusion</B></P> <P>LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>