Oncogenes and tumor suppressors in murine acute myeloid

我们对p15INK4b的初步研究提供了证据，证明该基因对p15INK4b缺陷小鼠的髓系白血病具有肿瘤抑制作用。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，该突变允许通过丢失一个Ink4b等位基因来证明对白血病的易感性。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。为了帮助我们理解为什么Ink4b-/-小鼠不比Ink4b-/+小鼠更容易患白血病，我们基于Cre-loxP系统开发了一种有条件的Ink4b基因敲除。条件等位基因纯合的小鼠已经被产生，并被证明在正常水平上表达p15INK4b。这些小鼠已经与Mx1Cre小鼠杂交，在用PI-PC治疗小鼠后，Mx1Cre小鼠将允许缺失Ink4b的第二外显子。此外，带有条件性基因敲除等位基因的小鼠已经与LysMCre小鼠杂交，以寻找髓系中的特定缺失。目前正在分析小鼠对逆转录病毒诱导的白血病的易感性。在许多髓系疾病中，Ink4b的表达缺失，在Ink4b基因缺陷的小鼠中，AML的易感性增加，这暗示了这种CDKI在维持正常的骨髓生成中的重要作用。尽管如此，关于Ink4b缺失如何导致骨髓细胞疾病或Ink4b在髓系细胞形成中的潜在作用尚不清楚。我们最近使用Ink4b基因敲除小鼠(Ink4b-/-)研究了Ink4b在造血中的作用。来自Ink4b-/-小鼠的骨髓(BM)显示造血干细胞的频率没有变化，但含有更多的双能粒细胞-巨噬细胞前体细胞，这被发现是p15INK4b细胞所固有的。有趣的是，Ink4b-/-前体细胞并不比野生型前体细胞周期更频繁，并且在凋亡潜力方面没有显示出差异。然而，Ink4b的缺失被证明影响了普通髓系祖细胞的分化，导致了红系和髓系潜能的失衡。这项工作展示了Ink4b在分化过程中的一个新角色。在另一个研究领域，我们的实验室检查了人类AML亚型的INK4b基因的超甲基化。DNA甲基化抑制剂是治疗人类AML和MDS的有希望的选择。到目前为止，还没有研究INK4b是否在所有细胞遗传学亚型的AML中都是高甲基化的。对AML中INK4b甲基化水平与三种最常见的细胞遗传学改变inv(16)、t(8；21)和t(15；17)进行比较，发现所有inv(16)白血病的甲基化水平显著低于其他类型。令人惊讶的是，INK4b在inv(16)+AML样本中的表达水平很低，与其他亚型的表达水平相当。对INK4b沉默的另一种机制的研究确定，INK4b表达的丧失是由inv(16)编码的CBFA-SMMHC(核心结合因子的一个亚单位，在框架内融合到平滑肌肌球蛋白重链)引起的。沉默表现为不能激活INK4b RNA的正常表达。CBFA-SMMHC取代了INK4b启动子中新确定的核心结合因子(CBF)位点的RUNX1。

Our initial studies on p15Ink4b provided evidence that the gene is a tumor suppressor for myeloid leukemia in mice using p15Ink4b-deficient mice. 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 Ink4b 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. To assist in our understanding of why Ink4b-/- mice are not more susceptible to leukemia than Ink4b-/+ mice we have developed a conditional knockout of Ink4b based on the Cre-loxP system. Mice, homozygous for the conditional allele, have been generated and shown to express p15Ink4b at normal levels. These mice have been crossed with Mx1Cre mice which will allow deletion of exon 2 of Ink4b following treatment of mice with pI-pC. In addition, mice with the conditional knockout allele have been crossed with LysMCre mice for specific deletion in the myeloid lineage. Mice are currently being analyzed for susceptibility to retrovirus-induced leukemia. The loss of Ink4b expression in a high proportion of myeloid based diseases and increased AML susceptibility in Ink4b-deficient mice implies an important function for this CDKI in the maintenance of normal myelopoiesis. Despite this, little is known about how loss of Ink4b contributes to myelopoietic diseases or of the potential role of Ink4b in myeloid cell formation.We have recently investigated the role of Ink4b in hematopoiesis using Ink4b knock-out mice (Ink4b-/-). Bone marrow (BM) from Ink4b-/- mice show no changes in the frequency of hematopoietic stem cells but contained greater numbers of bi-potent granulocyte-macrophage progenitors and this was found to be intrinsic to the p15Ink4b cells. Interestingly, Ink4b-/- progenitors did not cycle more frequently than wild-type progenitors and showed no differences in apoptotic potential. However, loss of Ink4b was shown to affect differentiation of the common myeloid progenitors (CMP) resulting in an imbalance between erythroid and myeloid potential. This work demonstrates a novel role for Ink4b during differentiation. In another area of research our laboratory has examined human AML subtypes for hypermethylation of the INK4b locus. Inhibitors of DNA methylation are promising options for the treatment of human AML and MDS. So far, it was not investigated whether or not INK4b is hypermethylated in all cytogenetic subtypes of AML. A comparison of levels of INK4b methylation in AML with the three most common cytogenetic alterations, inv(16), t(8;21) and t(15;17), revealed a strikingly low level of methylation in all leukemias with inv(16) compared to the other types. Surprisingly, the expression level of INK4b in inv(16)+ AML samples was low and comparable to that of the other subtypes. An investigation into an alternative mechanism of INK4b silencing determined that the loss of INK4b expression was caused by inv(16)-encoded CBFa-SMMHC (a subunit of the core binding factor fused in frame to the smooth muscle myosin heavy chain). The silencing was manifested in an inability to activate the normal expression of INK4b RNA. CBFa-SMMHC was shown to displace RUNX1 from a newly determined core binding factor (CBF) site in the promoter of INK4b.