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: 8349971
• 负责人: Paul Liu
• 金额: $ 109.16万
• 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/8349971
• 关键词: AML1-ETO fusion protein; Accounting; Acute Lymphocytic Leukemia; Acute Myelocytic Leukemia; Acute leukemia; Affect; Affinity; Binding; Biological; Biological Assay; CBFbeta-MYH11 fusion protein; Chemicals; Childhood Acute Lymphocytic Leukemia; Chimeric Proteins; Chromosomal translocation; Chromosome abnormality; Chromosomes, Human, Pair 16; Collaborations; Core-Binding Factor; Cytarabine; Data; Development; Diagnosis; Fluorescence; Genes; Genomics; Goals; Hematopoiesis; Hematopoietic stem cells; Human; In Vitro; KIT gene; Kinetics; Knowledge; Lead; Modeling; Molecular; Molecular Genetics; Mus; Mutate; Mutation; Myosin Heavy Chains; Osteogenesis; Pathogenesis; Patients; Pharmaceutical Preparations; Play; RUNX1 gene; Recurrence; Role; Smooth Muscle Myosins; Structure-Activity Relationship; Testing; Therapeutic; Time; Transgenic Mice; Translating; United States National Institutes of Health; Zebrafish; base; design; embryonic stem cell; follow-up; fusion gene; high throughput screening; improved; in vivo; inhibitor/antagonist; leukemia; leukemogenesis; luminescence; mouse model; novel; novel therapeutic intervention; small molecule libraries; t(8; 21)(q22; q22); tissue/cell culture; transcription factor

Transcription factors RUNX1 and CBFbeta play key roles in leukemogenesis and normal hematopoiesis. Mutations or chromosome translocations affecting RUNX1 or CBFB (which encodes CBFbeta) are found in 20-30% of patients with acute leukemia. A chromosome 16 inversion inv(16) that generates a fusion gene between CBFB and MYH11 (which encodes the smooth muscle myosin heavy chain, SMMHC) is found in all patients with acute myeloid leukemia (AML) subtype M4Eo. My lab has studied RUNX1 and CBFbeta for their roles in leukemogenesis and normal hematopoiesis for the last 16 years. We have established a mouse model of human AML by targeted insertion of the fusion gene CBFB-MYH11 in mouse ES cells, which demonstrated the critical role of CBFB-MYH11 in leukemogenesis. Using transgenic mouse and zebrafish models we have demonstrated that RUNX1 and CBFbeta are required for multiple steps of normal hematopoiesis, starting from the hematopoietic stem cells. We have also conducted in vitro analysis to understand the molecular level mechanisms of CBFbeta-SMMHC function, which will be critical for designing new therapeutic approaches for AML. Our section has been using transgenic mouse models to study the mechanism of leukemogenesis by the fusion gene CBFB-MYH11 and its related RUNX1 gene. In the past year we were able to generate data demonstrating cooperation between CBFB-MYH11 and mutated KIT gene for leukemogenesis. KIT mutations is relatively common in human patients with leukemia, and our data suggest that KIT mutations contribute to leukemia development and are therefore potential targets for therapy. We also provided evidence that the interaction between CBFB-MYH11 and RUNX1 is critical for the leukemogenic function of CBFB-MYH11. Likewise, RUNX1-CBFbeta interaction might be critical for leukemia involving RUNX1 mutations, such as the AML1-ETO (also known as RUNX1-ETO) fusion gene generated by t(8;21) in AML. Thus, inhibitors of CBFbeta - RUNX1 interaction may have potential therapeutic applications for both (inv)16 and t(8;21) AML, which account for 20-30% of all AML cases. In collaboration with the NIH Chemical Genomics Center (NCGC), we developed a CBFbeta and RUNX1 bead-based proximity assay in Amplified Luminescence Proximity Homogenous Assay (ALPHA) Screen format and optimized it for high throughput screening. A total of 243,398 compounds were screened with this assay at NCGC, which led to the identification of 137 putative inhibitors by Structure-Activity Relationships and Curve Class. Confirmatory ALPHA and HTRF (homogeneous time resolved fluorescence) assays were performed and candidate compounds showing consistent results were further tested by Biacore to characterize the kinetics and binding affinity of the compounds. These follow-up tests have so far identified 70 potential candidate compounds. Three related lead hits have been confirmed in tissue culture cells and in our zebrafish model. Importantly, at least one of the three compounds reduced leukemia burden in a mouse CBF leukemia model, with comparable efficacy to and synergistic with cytarabine. These findings may lead to the development of targeted therapy for CBF leukemias.

转录因子 RUNX1 和 CBFbeta 在白血病发生和正常造血中发挥关键作用。在 20-30% 的急性白血病患者中发现了影响 RUNX1 或 CBFB（编码 CBFbeta）的突变或染色体易位。 在所有急性髓性白血病 (AML) 亚型 M4Eo 患者中都发现了 16 号染色体倒位 inv(16)，该基因在 CBFB 和 MYH11（编码平滑肌肌球蛋白重链，SMMHC）之间产生融合基因。我的实验室在过去 16 年里一直在研究 RUNX1 和 CBFbeta 在白血病发生和正常造血中的作用。我们通过将融合基因CBFB-MYH11靶向插入小鼠ES细胞中，建立了人类AML的小鼠模型，这证明了CBFB-MYH11在白血病发生中的关键作用。使用转基因小鼠和斑马鱼模型，我们证明从造血干细胞开始，RUNX1 和 CBFbeta 是正常造血多个步骤所必需的。我们还进行了体外分析，以了解 CBFbeta-SMMHC 功能的分子水平机制，这对于设计 AML 的新治疗方法至关重要。 我们课题组一直在利用转基因小鼠模型来研究融合​​基因CBFB-MYH11及其相关RUNX1基因导致白血病的机制。在过去的一年中，我们能够生成数据，证明 CBFB-MYH11 和突变的 KIT 基因在白血病发生中的合作。 KIT 突变在人类白血病患者中相对常见，我们的数据表明 KIT 突变有助于白血病的发展，因此是潜在的治疗靶点。我们还提供了证据表明 CBFB-MYH11 和 RUNX1 之间的相互作用对于 CBFB-MYH11 的白血病功能至关重要。 同样，RUNX1-CBFbeta 相互作用可能对于涉及 RUNX1 突变的白血病至关重要，例如 AML 中 t(8;21) 生成的 AML1-ETO（也称为 RUNX1-ETO）融合基因。因此，CBFbeta - RUNX1相互作用的抑制剂可能对(inv)16和t(8;21) AML具有潜在的治疗应用，这两种AML病例占所有AML病例的20-30%。 我们与 NIH 化学基因组中心 (NCGC) 合作，采用放大发光邻近均质测定 (ALPHA) 筛选格式开发了基于 CBFbeta 和 RUNX1 珠的邻近测定，并对其进行了优化以实现高通量筛选。 NCGC 通过该测定总共筛选了 243,398 种化合物，通过构效关系和曲线类别鉴定了 137 种假定的抑制剂。 进行了验证性 ALPHA 和 HTRF（均质时间分辨荧光）测定，并通过 Biacore 进一步测试了显示一致结果的候选化合物，以表征化合物的动力学和结合亲和力。迄今为止，这些后续测试已鉴定出 70 种潜在的候选化合物。在组织培养细胞和我们的斑马鱼模型中已经证实了三个相关的先导化合物。 重要的是，这三种化合物中至少有一种可以减轻小鼠 CBF 白血病模型中的白血病负担，其功效与阿糖胞苷相当并具有协同作用。 这些发现可能会促进 CBF 白血病靶向治疗的发展。