Collaborative Pathways that Lead to Leukemia

导致白血病的协同途径

• 批准号: 8349258
• 负责人: Peter Aplan
• 金额: $ 37.04万
• 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/8349258
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Alleles; Animal Model; Apoptosis; Biological Assay; Bone Marrow; Candidate Disease Gene; Cell Proliferation; Cells; Collaborations; Data; Dysmyelopoietic Syndromes; Epigenetic Process; FLT3 gene; Gene Cluster; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Genome; Genotype; Hematopoietic; In Vitro; Induced Mutation; Insertional Mutagenesis; Lead; Length; Lesion; MYCN gene; Malignant - descriptor; Manuscripts; Modeling; Mus; Mutate; Mutation; NUP98 gene; Pathway interactions; Patients; Point Mutation; Preparation; Proteins; Publishing; Reporting; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Running; Sampling; Series; Site; Small Interfering RNA; Stem cells; TGF Beta Signaling Pathway; TOP1 gene; Transcript; Transgenes; Universities; Work; abstracting; chromatin immunoprecipitation; clinically relevant; knock-down; leukemia; overexpression; research study; self-renewal

In collaboration with Dr. Donald Small (Johns Hopkins University), we have crossed mice that express a NUP98-HOXD13 (NHD13) transgene and develop myelodysplastic syndrome (MDS) with mice that express a FLT3 internal tandem duplication and develop a myeloproliferative disesase (MPD). Both of these abnormalities were initially identified in patients with acute myeloid leukemia (AML), underscoring the clinical relevance of this experiment. Although very few of the NHD13 or FLT3 only mice developed AML, 100% of the mice with both NHD13 and FLT3 alleles devleoped AML, demonstrating that these two genetic lesions collaborated with one another quite powerfully. A careful study of these mice has revealed a surprising deletion that is highly specific for leukemic mice. Parts of this study have been presented in abstract form in Dec. 2009, and a manuscript describing the findings in detail is in preparation. Retroviral insertional mutagenesis (RIM) has proven to be a valuable whole-genome screen for the identification of genes involved in malignant transformation. Common insertion sites (CIS) are regions that have retroviral integrations in more than one leukemic sample, and have been biologically selected as dominant clones, and are thought to harbor genes important for malignant transformation. We previously described the use of RIM to identify a number of genes which collaborate with a NUP98-HOXD13 to produce a leukemic clone. We have used the same approach to identify genes that collaborate with a CALM-AF10 fusion to produce acute leukemia. We identified 19 common insertion sites, including Zeb2, Nf1, Mn1, Evi1, Ift57, Mpl, Plag1, Kras, Erg, Vav1, and Gata1. Of note, over 25% of the mice had retroviral integrations near Zeb2, a transcriptional co-repressor in the TGF-beta signaling pathway, leading to over-expression of the Zeb2-transcript. 91% of mice with Zeb2 insertions developed B-lineage ALL suggesting that Zeb2 activation promotes the transformation of CALM-AF10 hematopoietic precursors toward B-lineage leukemias. Over half of the mice with Zeb2 integrations also had Nf1 integrations, suggesting cooperativity among CALM-AF10, Zeb2 and Ras pathway mutations. We then searched for Nras, Kras, and Ptpn11 point mutations in this series of CALM-AF10 leukemic mice infected with the replication competent retrovirus . Three mutations were identified, all of which occurred in mice with Zeb2 integrations, consistent with the hypothesis that Zeb2 and Ras pathway activation promotes B-lineage leukemic transformation in concert with CALM-AF10. In addition, targeted "knock-down" of Zeb2 led to decreased proliferation of cells with a CALM-AF10 fusion. This data was published in 2010. The prior experiments used either gene targeting or RIM to experimentally induce mutations that we suspect are leukemogenic in combination with an NHD13 or CALM-AF10 transgene. As a complementary approach, we searched for spontaneous (ie, not induced by RIM or gene targeting) mutations that might collaborate with the NHD13 or CALM-AF10 fusions. We searched for mutations of Runx1, Npm1, Tp53, Flt3, Kit, Nras, Kras, and Cbl. We thought this was an important study to help validate murine AML models, because there are no examples of spontaneous N/Kras mutations associated with murine AML. We studied 22-26 mice with each transgene. We found no mutations of Runx1, Npm1, Tp53, or Kit. 25-30 % of both NHD13 and CALM-AF10 mice had Nras or Kras mutations. Intriguingly, almost 30% of the CALM-AF10 mice had Flt3 mutations, inlcuding length mutations, whereas none of the NHD13 mice had Flt3 mutations. One potential explanation for the lack of Flt3 mutations in the NHD13 mice is as follows. Both the NHD13 and CALM-AF10 fusions lead to overexpression of HOXA-cluster genes, specifically HOXA7/9/10, in the bone marrow of clinically healthy (pre-leukemic) mice. However, one distinction is that the CALM-AF10 mice overexpress Meis1, whereas the NHD13 mice downregulate Meis1. Therefore, the lack of Flt3 mutations in the NHD13 mice may be due to the lack of Meis1 expression in the NHD13 mice, as Meis1 is reported to drive Flt3 expression. Given that Nras, Kras, and Flt3 mutations have been shown to enhance proliferation, these results support a working hypothesis that predicts AML cells have one mutation which impairs differentiation (such as NHD13 or CALM-AF10), and a second, complementary mutation which enhances proliferation or inhibit apoptosis. We have used gene expression arrays to compare and contrast genes that are differentially expressed between WT BM, NHD13 leukemias, and CALM-AF10 leukemias. Intriguingly, we find that NMYC, a gene not previously implicated in AML, is markedly overexpressed in a subset of CALM-AF10 leukemias. Preliminary experiments suggest that simultaneous overexpression of the CALM-AF10 and NMYC proteins in vitro lead to increased proliferation and immortalization of murine BM cells. In addition, we find that a subset of NHD13 leukemias spontaneously upregulate Meis1, and Flt3. These studies support recently published data which suggests that Flt3 is downstream of Meis1; we are currently searching for flt3 mutations in this subset of samples. An abstract describing these findings was presented in 2010, and a manuscript describing the findings is in preparation. In collaboration with Dr. Paul Meltzer, we have used multiplex PCR and deep sequencing to identify mutations in candidate genes in a set of 152 mouse leukemias. The genotypes of the leukemias are NHD13, CALM-AF10, NUP98-PHF23, and Lin28b; 24 genes commonly mutated in AML and/or T-ALL, including Nras, Kras, Tp53, Notch1, Runx1, Kit, and Flt3 are assayed. The initial samples have been run and the data acquired; the data is currently being assembled and interpreted.

通过与Donald Small博士（Johns Hopkins University）合作，我们跨越了表达NUP98-HOXD13（NHD13）转基因的小鼠，并与表达FLT3内部串联复制的小鼠发展并发展骨髓增生性综合征（MDS） ）。这两种异常最初均在急性髓样白血病（AML）的患者中鉴定出来，强调了该实验的临床相关性。尽管只有NHD13或FLT3中很少有小鼠开发了AML，但100％具有NHD13和FLT3等位基因的小鼠分离了AML，这表明这两种遗传病变彼此相当有力地合作。对这些小鼠的仔细研究表明，令人惊讶的缺失对白血病小鼠具有很高的特异性。这项研究的一部分已于2009年12月以抽象的形式介绍，并准备了描述发现的手稿。 事实证明，逆转录病毒插入诱变（RIM）是鉴定与恶性转化有关的基因的宝贵全基因组筛选。常见的插入位点（CIS）是在多个白血病样品中具有逆转录病毒整合的区域，并已被生物学选择为主要克隆，并且被认为具有对恶性转化重要的基因。我们先前描述了RIM的使用来鉴定许多与NUP98-HOXD13合作以产生白血病克隆的基因。我们已经使用了相同的方法来识别与Calm-AF10融合合作以产生急性白血病的基因。我们确定了19个常见的插入位点，包括ZEB2，NF1，MN1，EVI1，IFT57，MPL，PLAG1，KRAS，ERG，VAV1和GATA1。值得注意的是，超过25％的小鼠在Zeb2附近具有逆转录病毒整合，这是TGF-beta信号通路中的转录共抑制剂，导致Zeb2转录过度表达。 91％的带有ZEB2插入的小鼠出现了B-linege，所有小鼠均表明Zeb2激活促进了平静AF10造血前体向B-Linege白血病的转化。超过一半的具有ZEB2整合的小鼠也具有NF1整合，这表明CALM-AF10，ZEB2和RAS途径突变之间的合作性。然后，我们在这一系列的Calm-AF10白血病小鼠中搜索了NRA，KRAS和PTPN11点突变，感染了复制能力的逆转录病毒。鉴定出三个突变，所有突变发生在具有ZEB2整合的小鼠中，这与Zeb2和Ras途径激活激活的假设一致，促进了B-Linege Leukemic Transcation与Calm-AF10共同促进B-linege Leukemic Transformation。此外，针对Zeb2的目标“击倒”导致细胞的增殖减少，而AF-10融合融合。该数据于2010年发布。先前的实验使用基因靶向或边缘来实验诱导我们怀疑白血病与NHD13或Calm-AF10转基因相结合的突变。作为一种互补的方法，我们搜索了可能与NHD13或Calm-AF10融合的自发性（IE，不是由RIM或基因靶向）突变引起的。我们搜索了RUNX1，NPM1，TP53，FLT3，KIT，NRAS，KRAS和CBL的突变。我们认为这是一项重要的研究，可以帮助验证鼠AML模型，因为没有与鼠AML相关的自发N/KRAS突变的例子。我们研究了每个转基因的22-26只小鼠。我们没有发现Runx1，NPM1，TP53或套件的突变。 NHD13和Calm-AF10小鼠的25-30％具有NRAS或KRAS突变。 有趣的是，几乎30％的平静AF10小鼠具有FLT3突变，嵌入了长度突变，而NHD13小鼠均无FLT3突变。 NHD13小鼠缺乏FLT3突变的一种潜在解释是如下。 NHD13和CALM-AF10融合都导致Hoxa簇基因（特别是Hoxa7/9/10）过表达，该基因在临床健康（白细胞学前）小鼠的骨髓中。但是，一个区别是平静的AF-10小鼠过表达MEIS1，而NHD13小鼠下调Meis1。因此，NHD13小鼠中缺乏FLT3突变可能是由于NHD13小鼠中缺乏MEIS1表达所致，因为据报道MEIS1驱动FLT3表达。鉴于NRA，KRAS和FLT3突变已显示出增殖的增殖，因此这些结果支持了一个工作假设，该假设可以预测AML细胞具有一个突变，会损害分化（例如NHD13或Calm-AF10），第二个互补突变增强了互补突变。增殖或抑制凋亡。 我们已经使用基因表达阵列比较和对比基因，这些基因在WT BM，NHD13白血病和Calm-AF-10白血病之间差异表达。有趣的是，我们发现NMYC是以前与AML相关的基因，在Calm-Af-10 Leukemias的一部分中明显过表达。初步实验表明，同时在体外对CALM-AF10和NMYC蛋白的过度表达会导致鼠BM细胞的增殖和永生化增加。此外，我们发现NHD13白血病的子集自发上调meis1和flt3。这些研究支持最近发布的数据，这表明FLT3是MEIS1的下游。我们目前正在此样品子集中搜索FLT3突变。描述这些发现的摘要在2010年提出，并准备了描述这些发现的手稿。 与Paul Meltzer博士合作，我们使用了多重PCR和深层测序来鉴定一组152个小鼠白血病中候选基因中的突变。白血病的基因型是NHD13，Calm-AF10，NUP98-PHF23和Lin28B；分析了24个通常在AML和/或T-ALL中突变的基因，包括NRA，KRAS，TP53，NOTCH1，RUNX1，KIT和FLT3。初始样本已运行并获取数据；数据当前正在组装和解释。