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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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，即具有影响转录因子基因 RUNX1 或 CBFB 的易位或倒位的白血病，约占成人急性髓系白血病 (AML) 的 24% 和儿童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。我们之前培育了表达人类 CBFB-MYH11 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，我们一直在培育携带 CBFB-MYH11 和 AML 中其他常见突变的转基因小鼠，例如 KIT 的激活突变和 FLT3 的内部串联重复 (FLT3-ITD)，以证明和分析它们在白血病发生过程中的合作。去年，我们生成的数据证明了 CBFB-MYH11 和突变的 KIT 基因在小鼠模型中的白血病发生中的合作。 KIT 突变是 CBF AML 最常见的继发突变之一，与不良预后相关。因此，验证 KIT 突变是否与 CBFB-MYH11 共同促进白血病发生非常重要。在这项研究中，我们转导了带有 KIT D816V/Y 突变的野生型 (WT) 和条件性 Cbfb-MYH11 敲入 (KI) 小鼠骨髓 (BM) 细胞，然后将转导的细胞移植到成年健康受体小鼠中。我们发现，移植表达 D816V 或 D816Y KIT 的 KI BM 细胞的小鼠分别有 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，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，白血病细胞中 p44/42 MAPK 和 Stat3 被强烈磷酸化，但 AKT 和 Stat5 则没有。最后，携带 KIT D816 突变的白血病细胞对激酶抑制剂 PKC412 敏感。我们的数据为突变的 KIT 和 CBFB-MYH11 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 白血病的治疗与显着的发病率和死亡率相关，5 年生存率约为 50%。我们假设 RUNX1 和 CBFβ（分别由 RUNX1 和 CBFB 编码的蛋白质）之间的相互作用对于 CBF 白血病至关重要，并且可以作为药物开发的目标。我们开发了高通量筛选方法来量化 RUNX1-CBFβ 相互作用，并筛选了 243,398 种化合物的文库集合。 Ro5-3335 是一种从筛选中鉴定出的苯二氮卓类药物，能够直接与 RUNX1 和 CBFβ 相互作用，在报告基因检测中抑制 RUNX1/CBFB 依赖性反式激活，并抑制斑马鱼胚胎中依赖 runx1 的造血作用。 Ro5-3335优先杀死人CBF白血病细胞系，挽救RUNX1-ETO（白血病中的另一种CBF融合基因）转基因斑马鱼的白血病前表型，并减少小鼠CBFB-MYH11白血病模型中的白血病负担。因此，我们的数据证实了 RUNX1-CBFβ 相互作用可以作为白血病治疗的目标，并且我们已经为此目的确定了一种有前途的先导化合物。这些结果最近已发表（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。我们对三个人类 iPSC 系进行了首次全基因组测序，这些系通过附加型载体衍生自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该载体序列。我们在每个 iPSC 系中鉴定出 1058-1808 个杂合单核苷酸变异 (SNV)，但没有拷贝数变异。其中 6 到 12 个 SNV 位于每个 iPSC 系的编码区内，但其中 50% 是同义变化，其余的未选择性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 诱导过程中并不具有固有的诱变性。这些研究结果已于今年早些时候发表（Cheng et al., Cell Stem Cell, 10:337-44, 2012）。