Analysis of the deregulation mechanism on cell growth and differentiation induced by mutant ELT3

在约20%的急性髓系白血病(AML)患者中发现了Flt3基因的内部串联重复(ITD)，它与AML患者的白细胞增多和预后不良密切相关。此外，我们在Flt3的TK结构域中新发现了D835(D835-mt)的激活突变。这种突变在大约7%的AML病例中被发现。突变的表达Flt3的32D细胞在没有IL3或FL的情况下增殖，突变的flt3被结构性激活。在这项研究中，我们阐明了JM结构域在Flt3激活中的作用。突变的Flt3不仅含有ITD结构域，而且含有延长或缩短的JM结构域，与JM结构域中的酪氨酸残基无关。这些突变的Flt3是结构性酪氨酸磷酸化和激活的信号转导分子，如SHC、MAP激酶和Stat5a。值得注意的是，将缺失激酶和C-末端的截短的Flt3/ITD结构域与Wt-flt3共转染32D细胞，导致了自主增殖。在这些细胞中，截短的Flt3/ITD与Wt-Flt3形成异源复合体，Wt-Flt3被结构性酪氨酸磷酸化。这些结果表明，Flt3JM结构域在受体激活中起重要作用，长度突变的JM结构域诱导配体非依赖的受体激活，但也以反式方式激活Wt-Flt3。此外，已知32D细胞在G-CSF的反应下分化为成熟的中性粒细胞，而表达Flt3/ITD的细胞在G-CSF作用下不分化为成熟的中性粒细胞。然而，用PKC调节剂和分子伴侣抑制剂治疗恢复了GCSF分化为成熟中性粒细胞的能力。

Internal tandem duplication (ITD) of the FLT3 gene (FLT3/ITD) is found in approximately 20% of acute myeloid leukemia (AML) cases, and is strongly associated with the leukocytosis and poor prognosis in AML patients. In addition, we newly identified activating mutations of D835 (D835-Mt) within a TK domain of FLT3. This mutation was found in approximately 7% of AML cases. Mutant FLT3-expressing 32D cells proliferated without IL3 or FL, and mutant FLT3 was constitutively activated. In this study, we elucidated the role of the JM domain in the activation of FLT3. Mutant FLT3 with not only ITD but also an elongating or shortening JM domain transformed 32D cells regardless of the tyrosine residues in the JM domain. These mutant FLT3s were constitutively tyrosine phosphorylated and activated signal-transduction molecules such as SHC, MAP kinase and STAT5a. Notably, co-transfection of the truncated FLT3/ITD lacking kinase and C-terminal domains with the Wt-FLT3 into 32D cells resulted in autonomous proliferation. In these cells, truncated FLT3/ITD generated a hetero-complex with Wt-FLT3, and Wt-FLT3 was constitutively tyrosine phosphorylated. These findings indicated that the FLT3 JM domain plays an important role in receptor activation, and that the length-mutated JM domain induces ligandindependent receptor activation but also activates Wt-FLT3 in a trans-manner.Furthermore, 32D cells are known to differentiate into mature neutrophils in response to G-CSF, while FLT3/ITD-expressing did not differentiate into mature neutrophils when treated with G-CSF. However, treatment with PKC modulator and inhibitors for molecular chaperon recovered the ability to differentiate into mature neutrophils by GCSF.