MOLECULAR PATHOGENESIS OF CHROMOSOME 16 INVERSION IN HUMAN LEUKEMIA

人类白血病 16 号染色体倒转的分子发病机制

• 批准号: 8565516
• 负责人: Paul Liu
• 金额: $ 125.82万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 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/8565516
• 关键词: Accounting; Acute Myelocytic Leukemia; Adult; Adult Acute Myeloblastic Leukemia; Affect; American; Benzodiazepines; Biochemical; Biological Assay; Blood; Blood Cells; Bone Marrow Cells; CBFB gene; Cell Line; Cell Therapy; Cells; Cellular biology; Chemicals; Childhood Acute Lymphocytic Leukemia; Chromosomes, Human, Pair 16; Code; Collection; Complement; Core-Binding Factor; DNA Sequence; Data; Development; Diagnosis; Disease; Drug Delivery Systems; Embryo; Employee Strikes; Episome; FLT3 gene; Frequencies; Genes; Genetic; Genome; Genomics; Goals; Hematological Disease; Hematopoiesis; Human; Human Development; KIT gene; Laboratories; Lead; Libraries; MAP Kinase Gene; MYH11 gene; Malignant Neoplasms; Mediating; Methods; Modeling; Molecular; Morbidity - disease rate; Mus; Mutate; Mutation; Nucleotides; PKC412; Pathogenesis; Pathway Analysis; Phenotype; Play; Proteins; Proto-Oncogene Proteins c-akt; Publications; Publishing; RUNX1 gene; Reporter; Role; Signal Pathway; Source; Stem cells; Survival Rate; Technology; Transactivation; Transcription factor genes; Transgenic Mice; Transgenic Organisms; Translating; Transplantation; Variant; Zebrafish; cell type; cellular transduction; clinical practice; clinically relevant; drug development; fusion gene; genome sequencing; high throughput screening; human disease; improved; in vitro Model; induced pluripotent stem cell; interest; killings; kinase inhibitor; leukemia; leukemogenesis; mortality; mouse model; outcome forecast; stem cell technology; transcription factor; vector

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for approximately 24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia. Our section has been developing clinically relevant transgenic mouse models to study human CBF leukemia. We previously generated mice that express the human CBFB-MYH11 fusion gene, which is one of the most common causes of AML. During the last several years we have been generating transgenic mice that harbor CBFB-MYH11 and other common mutations in AML, such as activating mutations of KIT and internal tandem duplication of FLT3 (FLT3-ITD), to demonstrate and analyze their cooperation during leukemogenesis. In the past year we generated data demonstrating cooperation between CBFB-MYH11 and the mutated KIT gene for leukemogenesis in a mouse model. KIT mutations are among the most common secondary mutations in CBF AML and are associated with poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11 for leukemogenesis. In this study, we transduced wild type (WT) and conditional Cbfb-MYH11 knockin (KI) mouse bone marrow (BM) cells with KIT D816V/Y mutations, and the transduced cells were then transplanted to adult healthy recipient mice. We found that 60% and 80% of mice transplanted with KI BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, while no control mice did. Results from limiting dilution transplantations indicate higher frequencies of leukemia initiating cells in KIT-induced leukemia. Signaling pathway analysis revealed that p44/42 MAPK and Stat3, but not AKT and Stat5, were strongly phosphorylated in the leukemia cells. Finally, leukemia cells carrying KIT D816 mutations were sensitive to the kinase inhibitor PKC412. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis. These findings were published recently (Zhao et al., Blood 119:1511-21, 2012). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-year survival rate of approximately 50%. We hypothesize that the interaction between RUNX1 and CBFβ, the proteins encoded by RUNX1 and CBFB respectively, is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput screen methods to quantify the RUNX1-CBFβ interaction and screened a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFβ directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued pre-leukemic phenotype in a RUNX1-ETO (another CBF fusion gene in leukemia) transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFβ interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose. These results have been published recently (Cunningham et al., Proc Natl Acad Sci USA 109:14592-7, 2012). A new direction of the lab is the development of human induced pluripotent stem cells (iPSCs) to model human disease. The utility of iPSCs as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. We conducted the first whole-genome sequencing of three human iPSC lines, which were derived from two cell types of an adult donor by episomal vectors. The vector sequence was not detected in the genomes of the deeply sequenced iPSC lines. We identified 1058-1808 heterozygous single nucleotide variants (SNVs), but no copy number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but 50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers or other diseases. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during iPSC induction. These findings have been published earlier this year (Cheng et al., Cell Stem Cell, 10:337-44, 2012).

核心结合因子（CBF）白血病，即那些影响转录因子基因RUNX 1或CBFB的易位或倒位的白血病，约占成人急性髓性白血病（AML）的24%和儿科急性淋巴细胞白血病的25%。 我们部门一直在开发临床相关的转基因小鼠模型来研究人类CBF白血病。我们先前产生了表达人CBFB-MYH 11融合基因的小鼠，这是AML最常见的原因之一。 在过去的几年中，我们一直在生产携带CBFB-MYH 11和AML中其他常见突变的转基因小鼠，如KIT的激活突变和FLT 3的内部串联重复（FLT 3-ITD），以证明和分析它们在白血病发生过程中的合作。 在过去的一年中，我们产生的数据表明CBFB-MYH 11和突变的KIT基因在小鼠模型中的白血病发生之间的合作。KIT突变是CBF AML中最常见的继发性突变，与预后不良相关。因此，重要的是要验证KIT突变与CBFB-MYH 11合作白血病的发生。在这项研究中，我们转导野生型（WT）和条件性Cbfb-MYH 11敲入（KI）小鼠骨髓（BM）细胞与KIT D816 V/Y突变，并将转导的细胞移植到成年健康受体小鼠。我们发现，分别移植了表达D816 V或D816 Y KIT的KI BM细胞的小鼠中，有60%和80%在9个月内死于白血病，而对照小鼠则没有。有限稀释移植的结果表明，在KIT诱导的白血病中，白血病起始细胞的频率较高。信号通路分析显示，p44/42 MAPK和Stat 3，而不是AKT和Stat 5，在白血病细胞中强烈磷酸化。最后，携带KIT D816突变的白血病细胞对激酶抑制剂PKC 412敏感。我们的数据为突变的KIT和CBFB-MYH 11在白血病发生过程中的合作提供了明确的证据。这些发现最近发表（Zhao et al.，Blood 119：1511-21，2012）。 目前CBF白血病的治疗与显著的发病率和死亡率相关，5年生存率约为50%。 我们假设RUNX 1和CBF（分别由RUNX 1和CBFB编码的蛋白质）之间的相互作用对CBF白血病至关重要，可以作为药物开发的靶点。 我们开发了高通量筛选方法来定量RUNX 1-CBF相互作用，并筛选了243，398种化合物的文库。 Ro 5 -3335是从筛选中鉴定的苯二氮卓类药物，能够直接与RUNX 1和CBF相互作用，在报告基因测定中抑制RUNX 1/CBFB依赖的反式激活，并抑制斑马鱼胚胎中的runx 1依赖的造血。 Ro 5 -3335优先杀死人CBF白血病细胞系，拯救RUNX 1-ETO（白血病中的另一种CBF融合基因）转基因斑马鱼中的白血病前表型，并降低小鼠CBFB-MYH 11白血病模型中的白血病负荷。 因此，我们的数据证实，RUNX 1-CBF相互作用可以靶向白血病治疗，我们已经确定了一个有前途的先导化合物用于此目的。 这些结果最近已经发表（Cunningham等人，Proc Natl Acad Sci USA 109：14592-7，2012）。 该实验室的一个新方向是开发人类诱导多能干细胞（iPSC）来模拟人类疾病。iPSC作为研究疾病的模型和作为细胞治疗来源的效用取决于其基因组的完整性。尽管最近发表了iPSC中DNA序列变异的文章，但整个基因组的这种变化的真正范围尚不清楚。我们进行了三个人iPSC系的第一次全基因组测序，这三个人iPSC系通过附加型载体来源于成年供体的两种细胞类型。在深度测序的iPSC系的基因组中未检测到载体序列。我们在每个iPSC系中鉴定了1058-1808个杂合单核苷酸变体（SNV），但没有拷贝数变体。 这些SNV中有6到12个在每个iPSC系的编码区内，但其中50%是同义变化，其余的没有选择性地富集与癌症或其他疾病相关的已知基因。因此，我们的数据表明，附加体介导的重编程在iPSC诱导过程中不是固有的致突变性。这些发现已于今年早些时候发表（Cheng et al.，Cell Stem Cell，10：337-44，2012）。