Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Department of Laboratory Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
PLoS One. 2014 Jan 3;9(1):e84746. doi: 10.1371/journal.pone.0084746. eCollection 2014.
The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),-16,+21,-22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.
该 gain of function 突变 JAK2-V617F 非常频繁地出现在骨髓增殖性肿瘤（MPN）中，并强烈暗示其在这些和其他血液恶性肿瘤的发病机制中起作用。在这里，我们报告了从一名 73 岁的女性急性髓系白血病（AML）转化为 MPN 的患者中建立了一个新的白血病细胞系 PVTL-1，该细胞系为 JAK2-V617F 纯合子。PVTL-1 阳性表达 CD7、CD13、CD33、CD34、CD117、HLA-DR 和 MPO，并具有复杂的核型异常，44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),-16,+21,-22,+mar1。JAK2 的序列分析仅显示突变等位基因编码 Jak2-V617F。PVTL-1 的增殖受到泛 Jak 抑制剂 Jak 抑制剂-1（JakI-1）或 dasatinib 的抑制，后者抑制 Src 家族激酶以及 BCR/ABL。一致地，Src 家族激酶 Lyn 被激活，磷酸化激活环中的 Y396，该磷酸化被 dasatinib 抑制，但不受 JakI-1 抑制。进一步用 JakI-1 和 dasatinib 进行分析表明，Jak2-V617F 磷酸化 STAT5 和 SHP2，而 Lyn 磷酸化 SHP1、SHP2、Gab-2、c-Cbl 和 CrkL，以诱导 SHP2/Gab2 和 c-Cbl/CrkL 复合物形成。此外，JakI-1 和 dasatinib 抑制 mTOR/p70S6K/4EBP1 通路，并降低 PVTL-1 细胞中 GSK3 的抑制性磷酸化，这与它们对这些细胞增殖和存活的影响相关。此外，通过其抑制剂 SB216763 抑制 GSK3 可减轻 dasatinib 诱导的细胞凋亡，但 JakI-1 则不然。综上所述，这些数据表明，通过 Lyn 以及 Jak2-V617F 使 GSK3 失活，以及通过 Jak2-V617F 使 STAT5 激活，可能抑制了 PVTL-1 细胞的凋亡。此外，还推测 mTOR/p70S6K/4EBP1 通路的激活可能介导了 Jak2-V617F 和 Lyn 从增殖信号的传递。PVTL-1 细胞可能为阐明 Jak2-V617F 表达的 MPN 向 AML 进化的分子机制以及开发针对这种难治性疾病的新疗法提供了一个有价值的模型系统。
