Oncogenes and tumor suppressors in murine acute myeloid leukemia

我们对p15Ink4b的初步研究提供了证据，证明该基因是p15Ink4b缺陷小鼠骨髓性白血病的肿瘤抑制因子。在逆转录病毒诱导的白血病发生方案中使用小鼠表明，Ink4b缺陷杂合的小鼠对髓性白血病的易感性增加。小鼠接种了MOL4070LTR逆转录病毒，这是我们实验室最近构建和生产的一种独特的髓系疾病诱导病毒。逆转录病毒通过插入诱变提供了协同事件。这证明了一个Ink4b等位基因的缺失提供了对白血病的易感性。缺乏两个等位基因的小鼠并不比缺乏一个等位基因的小鼠更易感，这增加了在这些Ink4b缺失小鼠中存在相反力量的可能性。这可能是由于在小鼠的多个组织中缺失。为了帮助我们理解为什么Ink4b-/-小鼠并不比Ink4b-/+小鼠更容易患白血病，我们基于Cre-loxP系统开发了Ink4b的条件敲除。当将这些小鼠与LysMCre小鼠杂交以特异性删除髓系细胞时，这些小鼠对逆转录病毒诱导的白血病高度敏感。丢失该基因的纯合子小鼠有50%的髓系疾病发病率。Ink4b在很大比例的髓系疾病中表达缺失，Ink4b缺陷小鼠AML易感性增加，这表明该CDKI在维持正常骨髓形成方面具有重要功能。尽管如此，对于Ink4b的缺失如何导致骨髓疾病或Ink4b在骨髓细胞形成中的潜在作用知之甚少。我们最近利用Ink4b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，但含有更多的双强效粒细胞-巨噬细胞祖细胞（GMP），这被发现是p15Ink4b细胞固有的。有趣的是，Ink4b-/-祖细胞并不比野生型祖细胞周期更频繁，在凋亡潜能上也没有差异。然而，Ink4b的缺失被证明会影响共同髓系祖细胞（CMP）的分化。由此产生的不平衡有利于髓系祖细胞（gmp）的产生，而牺牲了红系祖细胞。这项工作证明了Ink4b在红细胞/髓细胞分叉的细胞命运中具有一种新的细胞周期无关的作用。我们实验室的另一个研究领域是检测人类AML亚型INK4b位点的超甲基化。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AML的所有细胞遗传学亚型中都是高甲基化的。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）的比较显示，与其他类型的白血病相比，所有inv（16）白血病的甲基化水平都非常低。令人惊讶的是，INK4b在inv(16)+ AML样本中的表达水平很低，与其他亚型相当。对INK4b沉默的另一种机制的研究确定，INK4b表达的缺失是由inv（16）编码的CBF&#946；-SMMHC（&#946；核心结合因子的亚基，融合在框架内的平滑肌肌球蛋白重链）引起的。沉默表现为无法激活INK4b RNA的正常表达。CBF&#946；-SMMHC被证明从INK4b启动子中新确定的核心结合因子（CBF）位点取代RUNX1。重要的是，本研究1)证实了编码肿瘤抑制因子p15INK4b的基因是CBF&#946；-SMMHC的靶标，这一发现与白血病发生过程有关；2)表明，在含有inv（16）的AML患者中，使用低甲基化药物无法预测白血病中INK4b位点的重新表达

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. This 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 these Ink4b null mice. This may be due to the deletion throughout multiple tissues of the mouse. 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. These mice, when crossed with LysMCre mice for specific deletion in myeloid lineage cells, are highly susceptible to retrovirus-induced leukemia. Mice homozygous for loss of the gene have a 50% incidence of myeloid disease. <P> 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 (GMP) 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). The resulting imbalance favors the production of myeloid progenitor cells, GMPs at the expense of erythroid progenitors. This work demonstrates a novel cell cycle-independent role for Ink4b during cell fate at the erythroid/myeloid bifurcation. <P> 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 CBF&#946;-SMMHC (&#946; 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. CBF&#946;-SMMHC was shown to displace RUNX1 from a newly determined core binding factor (CBF) site in the promoter of INK4b. Importantly, this study 1) establishes that the gene encoding the tumor suppressor p15INK4b is a target of CBF&#946;-SMMHC, a finding relevant to the leukemogenesis process and 2) indicates that in patients with inv(16)-containing AML, re-expression from the INK4b locus in the leukemia would not be predicted to occur using hypomethylating drug