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作用中起关键作用。在20-30％的急性白血病患者中，发现影响RUNX1或CBFB的突变或染色体易位（编码CBFBETA）。 在所有急性骨髓性白血病（AML）亚型M4EO的患者中都发现了CBFB和MYH11（编码平滑肌肌球蛋白重链，SMMHC）之间产生融合基因的16染色体INV（16）。在过去的16年中，我的实验室研究了Runx1和Cbfbeta在白血病和正常造血作用中的作用。我们通过靶向插入融合基因CBFB-MYH11在小鼠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，我们已经证明了正常造血的多个步骤，从造血干细胞开始。我们还进行了体外分析，以了解CBFBETA-SMMHC功能的分子水平机制，这对于为AML设计新的治疗方法至关重要。 我们的部分一直使用转基因小鼠模型来研究融合​​基因CBFB-MYH11及其相关的Runx1基因的白血病发生机理。在过去的一年中，我们能够生成数据，证明CBFB-MYH11和突变的Kit基因在白血病发生之间的合作。在人类白血病患者中，试剂盒突变相对常见，我们的数据表明，试剂盒突变有助于白血病的发展，因此是治疗的潜在靶标。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相互作用对于涉及runx1突变的白血病可能至关重要，例如由t（8; 21）在AML中产生的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具有（INV）16和t（8; 21）AML的潜在治疗应用，占所有AML病例的20-30％。 与NIH化学基因组学中心（NCGC）合作，我们在放大的发光近端同质测定法（Alpha）屏幕格式中开发了CBFBETA和RUNX1珠子的近距离测定，并对高通量筛选进行了优化。 在NCGC上使用该测定法筛选了总共243,398种化合物，这导致通过结构活性关系和曲线类别鉴定了137种推定抑制剂。 进行了验证性α和HTRF（均质时间分辨荧光）测定法，并通过Biacore进一步测试了表现出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。到目前为止，这些后续测试已经确定了70种潜在候选化合物。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铅命中。 重要的是，三种化合物中的至少一种减轻了小鼠CBF白血病模型中的白血病负担，其功效与细胞丁滨具有可比的功效和协同作用。 这些发现可能导致开发针对CBF白血病的靶向疗法。