Oncogenes and tumor suppressors in murine acute myeloid

我们的实验室已经开始对p15INK4b进行系统的分析，在人类急性髓系白血病(AML)中，p15INK4b比任何其他具有细胞周期调节特性的肿瘤抑制基因更容易失活。P15INK4b是一种细胞周期蛋白依赖的激酶抑制因子，与其他Ink4b家族成员一样，它能与早期的G1期细胞周期蛋白依赖性蛋白激酶4和6结合，从而阻止它们与D细胞周期蛋白的相互作用。我们最近提供的证据表明，该基因是小鼠髓系白血病的肿瘤抑制基因。首先，逆转录病毒诱导的粒单核细胞表型髓系白血病被发现在该基因的启动子区域有超甲基化。在一些白血病在原发状态下没有高甲基化的情况下，肿瘤细胞在同基因小鼠移植后发生甲基化。TaqMan实时RT-PCR显示，表观遗传学改变与原发和移植白血病中表达减少相关。此外，用甲基化抑制剂5-aza-2‘脱氧胞苷处理具有Ink4b超甲基化的白血病细胞株，导致p15INK4b mRNA表达增加。在p15INK4b缺陷小鼠身上进行的第二种类型的研究提供了关于Ink4b的肿瘤抑制作用的最令人信服的数据。在逆转录病毒诱导的白血病方案中使用这些小鼠表明，Ink4b缺乏杂合子的小鼠对髓系白血病的易感性增加。用本实验室新近构建和生产的一种独特的髓系致病病毒MOL4070LTR型逆转录病毒接种小鼠。逆转录病毒通过插入突变提供了协同事件，从而证明了一个等位基因缺失对白血病的易感性。两个等位基因缺失的小鼠并不比一个等位基因缺失的小鼠更容易患上髓系白血病，这增加了Ink4b基因缺失小鼠患髓系白血病的相反力量的可能性。我们的实验室已经证明，在造血和免疫环境中发现的细胞因子(如干扰素、转化生长因子β和IL-6)治疗后，Ink4b在mRNA水平上的表达上调。这是细胞在分化过程中被指示停止生长的重要方式。作为对干扰素β诱导小鼠髓系细胞系M1中p15INK4b mRNA表达机制的研究的一部分，我们确定干扰素共有序列结合蛋白(ICSBP)/IRF-8和Ets样蛋白PU.1都能够激活Ink4b启动子的转录。我们的数据表明，为了稳定PU.1/ICSBP结合复合体，这两种因子都需要与DNA结合。Ink4b启动子的这种调节显然是髓系特有的，因为ICSBP和PU.1都是髓系承诺因子。有趣的是，这项关于Ink4b转录调控的研究至少部分解释了ICSBP在ICSBP缺陷小鼠中表现出的肿瘤抑制活性。这些小鼠发展为慢性粒细胞白血病(CML)样疾病，这与高比例的人类AML和CML缺乏ICSBP转录本的发现是一致的。DNA甲基化抑制剂是治疗人类AML和MDS的有希望的选择。这在一定程度上是因为INK4B的超甲基化在这些疾病中非常普遍，并已被证明是可逆的。随着逆转异常甲基化的治疗变得越来越有效，能够识别对这种治疗更有可能产生反应的AML亚群变得越来越重要。尽管根据细胞遗传学异常对AML进行了分类，但还没有试图将INK4b的表观遗传失活与已知的易位联系起来。我们实验室最近的一项研究表明，基于细胞遗传学异常的AML亚型在INK4B基因座上甲基化的频率差异很大。最值得注意的是在所有inv(16)白血病中缺乏甲基化，足以减少表达。这与t(15；17)和t(8；21)相关白血病形成对比，在分析的AML中，分别有88%和57%的患者发现11%至43%的甲基化水平。我们正在进行的研究旨在确定与Ink4b失活协同的基因，确定p15INK4b对正常造血的影响，并通过转录因子和甲基化进一步研究Ink4b转录的调节。

Our laboratory has embarked on a systematic analysis of p15INK4b, which in human acute myeloid leukemia (AML) is inactivated more frequently than any other tumor suppressor gene with cell cycle regulatory properties. p15Ink4b is a cyclin-dependent kinase inhibitor and like other Ink4b family members binds to early G1 cyclin-dependent kinases 4 and 6, thereby preventing their interaction with D cyclins. We have recently provided evidence that the gene is a tumor suppressor for myeloid leukemia in mice. First, retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation within the gene's promoter region. In some cases where leukemias did not have hypermethylation in the primary state, the neoplastic cells developed methylation following transplantation in syngeneic mice. The epigenetic change correlated with decreased expression in primary and transplanted leukemias as shown by TaqMan real-time RT-PCR. Furthermore, treatment of a leukemia cell line that had Ink4b hypermethylation, with the methylation inhibitor 5-aza-2' deoxycytidine, resulted in an increase in p15Ink4b mRNA expression. A second type of study carried out in p15Ink4b-deficient mice provided the most convincing data regarding the tumor suppressor role of Ink4b. Use of the mice in a retrovirus-induced leukemogenesis protocol showed that mice heterozygous for Ink4b deficiency had increased susceptibility to myeloid leukemia. Mice were inoculated with retrovirus MOL4070LTR, a unique myeloid disease-inducing virus, which was recently constructed and produced in our laboratory. The retrovirus provided the cooperating events through insertional mutagenesis that allowed the demonstration of the susceptibility to leukemia provided by loss of one allele. Mice deficient in both alleles were not more susceptible than those deficient in one allele, raising the possibility that there are opposing forces to the development of myeloid leukemia in Ink4b null mice. Our laboratory has shown that Ink4b expression at the mRNA level is up-regulated following treatment with cytokines such as interferon (IFN), TGFbeta, and IL-6 that are found in hematopoietic and immune environments. This is an important way that cells are instructed to growth arrest during differentiation. As a part of an investigation into the mechanisms involved in IFNbeta-induced expression of p15Ink4b mRNA in the murine myeloid cell line M1, we determined that both the interferon consensus sequence binding protein (ICSBP)/IRF-8 and the Ets-like protein PU.1 were able to activate transcription from the Ink4b promoter. Our data suggest that binding of both factors to DNA is required in order to stabilize a PU.1/ICSBP binding complex. This regulation of the Ink4b promoter is apparently myeloid-specific, because both ICSBP and PU.1 are myeloid commitment factors. Interestingly, this study on transcriptional regulation of Ink4b provides, at least in part, an explanation for the tumor suppressor activity of ICSBP demonstrated in ICSBP-deficient mice. These mice develop a chronic myelogenous leukemia (CML)-like disease and this is consistant with the finding that a high percentage of human AMLs and CMLs lack ICSBP transcripts. Inhibitors of DNA methylation are promising options for the treatment of human AML and MDS. This is in part because hypermethylation of INK4B is so prevalent in these diseases and has been shown to be reversible. As treatments for reversing aberrant methylation become more effective, it becomes increasingly important to be able to identify those subgroups of AML that would be the more likely to respond to such treatment. Despite categorization of AML based upon cytogenetic abnormalities, there has been no attempt to correlate epigenetic inactivation of INK4b with known translocations. A recent study from our laboratory shows that AML subtypes based on their cytogenetic abnormalities vary dramatically in their frequency of methylation at the INK4B locus. Most notable is the lack of methylation, sufficient to reduce expression, in all leukemias with inv(16). This is in contrast to t(15;17) and t(8;21) associated leukemias, where levels of methylation of 11% to 43 % were found in 88% and 57 %, respectively, of the AML analyzed. Our ongoing studies are aimed at determining genes that collaborate with inactivation of Ink4b, determining the effects of p15Ink4b on normal hematopoiesis, and investigating further regulation of Ink4b transcription through transcription factors and methylation.