Animal models to study the role of INK4b loss in human acute myeloid leukemia

研究 INK4b 缺失在人类急性髓系白血病中的作用的动物模型

• 批准号: 7733426
• 负责人: Linda Wolff
• 金额: $ 68.02万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733426
• 关键词: Acute Myelocytic Leukemia; Acute T Cell Leukemia; Affect; Animal Model; Apoptotic; CDKN2A gene; Cell Cycle; Cell Lineage; Cells; Classification; Collaborations; Colon Carcinoma; Cyclin-Dependent Kinase Inhibitor; DNA; Development; Disease; Dysmyelopoietic Syndromes; Erythroid; Exons; Family member; Gene Deletion; Gene Expression; Genes; Genetically Engineered Mouse; Goals; Hematopoiesis; Human; Hypermethylation; In Vitro; Incidence; Infection; Insertional Mutagenesis; Inverse Polymerase Chain Reaction; Investigation; Knock-out; Laboratories; Ligation; MYB gene; Malignant Neoplasms; Mediating; Messenger RNA; Modeling; Mus; Myelogenous; Myeloid Cells; Myeloid Leukemia; Myeloid Progenitor Cells; Northern Blotting; Numbers; Oncogenes; Pathway interactions; Patients; Polymerase Chain Reaction; Production; Property; Proto-Oncogene Protein c-kit; Proto-Oncogene Proteins c-myb; Rate; Research Personnel; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Role; Site; Stem cells; System; Technology; Tissues; Tumor Suppressor Proteins; Undifferentiated; Wild Type Mouse; Work; base; granulocyte; human GATA1 protein; improved; interest; leukemia; macrophage; malignant breast neoplasm; man; member; novel; progenitor; research study; stem

Using p15Ink4b-deficient mice, our initial studies of p15Ink4b provided evidence that the gene is a tumor suppressor for myeloid leukemia. Recently we developed an improved model that more closely resembles the disease in man by deleting the gene specifically in myeloid lineage cells. The new model employing the Cre-loxP system allows conditional deletion of the gene. We successfully accomplished the tissue specific deletion by crossing INK4b(exon 2) floxed mice with LysMCre mice and showed by PCR technology that Ink4b was deleted in common myeloid progenitors. When these mice were injected with retrovirus they were found to be highly susceptible to acute myeloid leukemia with a 50% incidence as compared to wild-type mice which had a 5% incidence. The retrovirus used for these studies was one we constructed and is efficient in inducing myeloid leukemia. It has been used by numerous other investigators for looking for cooperating genes in genetically engineered mice. Sixty-five percent of the leukemias were positive for both granulocyte and macrophage markers suggesting that they were derived from undifferentiated progenitor cells. Of these, 36 % also had the stem cell marker Sca-1. The others were positive for macrophage markers only. Since retroviral insertional mutagenesis was utilized in conjunction with the Ink4b knock-out for the induction of acute myeloid leukemia, we were able to screen for retrovirus insertion sites that might collaborate with the loss of the p15Ink4b gene. Insertion sites of the retrovirus were cloned from leukemia DNA using inverse PCR and ligation-mediated PCR. A total of 34 acute myeloid leukemias were examined and a total of 164 retroviral integration sites have been identified. Common integration sites that were found in more than one leukemia included c-myb, sox4, gfi1 and stk10. c-Myb was found to be the most frequently activated gene based upon PCR identification of insertion sites and northern blot analysis. There was evidence of activation of c-myb in 23% of the leukemias. This is a much higher rate of activation than has been shown for other retroviral induced leukemias in mice and therefore possibly showing specific collaboration with the loss of p15Ink4b. This is very interesting because human c-Myb, which was implicated in human cancer for many years, was recently shown to be involved in translocations and duplications in T acute lymphocytic leukemia. It is a very important oncogene in human cancer and also is implicated in breast cancer and colon cancer. Interestingly, mRNA for myb and sox4 was demonstrated to be increased in leukemias which retroviruses at these sites. Furthermore, we have been able to show that infection of c-kit+ stem / progenitor cells from p15Ink4bfl/fl LysM-Cre mice with retroviruses expressing c-myb or sox4 results in an increased number of myeloid colonies in vitro. p15INK4b is a member of the INK4 family member of cyclin-dependent kinase inhibitors. However, since INK4b is the only member that is inactivated in acute myeloid leukemia, we have been interested in determining if there is any other function specific to the myeloid lineage that can be assigned to this gene. For these experiments we used the two knock-out models described above. Initial investigations of hematopoiesis in Ink4b- deficient mice showed that they have greater numbers of bi-potent granulocyte-macrophage progenitors (GMP) and this was found to be intrinsic to the cells. Interestingly, Ink4b-deficient granulocyte-macrophage progenitors did not cycle more frequently than wild-type progenitors and showed no differences in apoptotic potential. However, Ink4b was shown to affect differentiation of common myeloid progenitor cells. The resulting imbalance was shown to favor the production of granulocyte-macrophage progenitors 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. Since GATA-1 and PU.1 instruct erythroid and myeloid cell fates and inhibit each others transcriptional function we set out to determine if expression of these genes or other genes involved in lineage choice were altered in common myeloid progenitors from In4b-deficient compared to wild-type mice. RT-PCR results showed that PU.1 levels were unaffected while GATA-1, GATA-2, and GATA-3 were down-regulated in common myeloid progenitors. Whether p15Ink4b affects the common myeloid progenitor differentiation through its known capacity to interact with Cdk4/6 or by participating in an unrelated pathway is currently unclear.

使用P15INK4B缺陷型小鼠，我们对P15INK4B的最初研究提供了证据，证明该基因是髓样白血病的肿瘤抑制剂。最近，我们开发了一个改进的模型，该模型通过在髓样谱系中专门删除基因，与人类中的疾病更相似。采用CRE-LoxP系统的新模型允许有条件地缺失基因。我们通过将Ink4b（外显子2）用lysmcre小鼠越过Ink4b（外显子2）成功完成了组织特异性缺失，并通过PCR技术表明Ink4b在常见的髓样祖细胞中被删除。当这些小鼠注射逆转录病毒时，发现它们非常容易受到急性髓样白血病的影响，与发生率为5％的野生型小鼠相比，发病率为50％。用于这些研究的逆转录病毒是我们构建的，并且有效诱导髓样白血病。它已被许多其他研究人员用于寻找基因工程小鼠中的合作基因。粒细胞和巨噬细胞标志物的白血病中有65％均为阳性，这表明它们来自未分化的祖细胞。其中，36％的人也具有干细胞标记SCA-1。其他仅对巨噬细胞标记阳性。由于逆转录病毒插入诱变与Ink4b敲除一起用于诱导急性髓样白血病，因此我们能够筛选逆转录病毒插入部位，这些逆转录病毒插入部位可能与P15INK4B基因的丧失一起进行合作。使用逆PCR和连接介导的PCR从白血病DNA克隆逆转录病毒的插入位点。总共检查了34个急性髓样白血病，总共确定了164个逆转录病毒整合位点。 在多个白血病中发现的常见整合位点包括C-MYB，SOX4，GFI1和STK10。根据插入位点和北印迹分析的PCR鉴定，发现C-MYB是最常见的基因。有23％的白血病中C-MYB激活的证据。这比其他逆转录病毒诱导的小鼠的白血病所显示的激活速率要高得多，因此可能显示出与p15ink4b损失的特定协作。这很有趣，因为最近与人类癌症有关的人类C-MYB最近被证明与T急性淋巴细胞性白血病有关的易位和重复。这是人类癌症中非常重要的癌基因，也与乳腺癌和结肠癌有关。 有趣的是，在这些部位逆转录病毒的白血病中显示了MYB和SOX4的mRNA增加。此外，我们已经能够证明来自p15INK4BFL / FL LYSM-CRE小鼠的C-KWIT+ Stem /祖细胞的感染，其逆转录病毒表达C-MYB或SOX4会导致体外的髓样菌落数量增加。 P15INK4B是Cyclin依赖性激酶抑制剂的Ink4家族成员的成员。但是，由于INK4B是唯一在急性髓细胞性白血病中灭活的成员，因此我们感兴趣地确定是否存在可以分配给该基因的其他功能。对于这些实验，我们使用了上面描述的两个敲除模型。对Ink4b缺乏小鼠中造血的初步研究表明，它们具有更多的双功率粒细胞 - 巨噬细胞祖细胞（GMP），并且发现这对细胞是固有的。有趣的是，Ink4b缺陷型粒细胞巨噬细胞祖细胞比野生型祖细胞的循环频率不高，并且凋亡潜力没有差异。 但是，INK4B被证明会影响常见髓样祖细胞的分化。结果的失衡表明有利于粒细胞 - 巨噬细胞祖细胞的产生，而牺牲了红细胞祖细胞。这项工作证明了在红细胞/髓样分叉的细胞命运期间，Ink4b在细胞周期的新作用。由于GATA-1和PU.1指导红细胞和髓样细胞的命运，并抑制彼此的转录功能，因此与野生型小鼠相比，来自IN4B缺陷型的普通髓样祖细胞的这些基因或其他基因的表达是否会改变。 RT-PCR的结果表明，PU.1水平不受影响，而GATA-1，GATA-2和GATA-3在常见的髓样祖细胞中被下调。当前尚不清楚P15INK4B是否通过已知与CDK4/6相互作用的能力或参与无关的途径来影响常见的髓样祖细胞分化。