MOLECULAR PATHOGENESIS OF CHROMOSOME 16 INVERSION IN HUMAN LEUKEMIA

转录因子RUNX1和CBFbeta在白血病发生和正常造血中起关键作用。影响RUNX1或Cbfb(编码CBFbeta)的突变或染色体易位在20%-30%的急性白血病患者中被发现。在所有急性髓系白血病(AML)M4Eo亚型患者中都发现了16号染色体倒位inv(16)，它产生Cbfb和MYH11(编码平滑肌肌球蛋白重链，SMMHC)之间的融合基因。在过去的16年里，我的实验室一直在研究RUNX1和CBFbeta在白血病发生和正常造血中的作用。我们通过在小鼠ES细胞中定向插入融合基因Cbfb-MYH11建立了人AML小鼠模型，证实了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RUNX1和CBFbeta是正常造血的多个步骤所必需的，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，这将是设计新的AML治疗方法的关键。 本课题组一直在利用转基因小鼠模型研究融合基因Cbfb-MYH11及其相关基因RUNX1在白血病发生机制中的作用。在过去的一年里，我们能够产生数据，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与RUNX1之间的相互作用对Cbfb-MYH11的致白血病功能至关重要。同样，RUNX1-CBFbeta相互作用可能在涉及RUNX1突变的白血病中起关键作用，例如AML中由t(8；21)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对占所有AML病例20-30%的(Inv)16和t(8；21)AML都有潜在的治疗应用。 在与NIH化学基因组中心(NCGC)的合作下，我们开发了一种基于CBFbeta和RUNX1微珠的放大发光均相分析(Alpha)筛查格式的邻近分析，并对其进行了优化，以进行高通量筛选。NCGC用这一方法筛选了243,398个化合物，通过构效关系和曲线分类鉴定了137个可能的抑制剂。进行了确认性Alpha和HTRF(均相时间分辨荧光)分析，结果一致的候选化合物进一步通过Biacore测试来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个潜在的候选化合物。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铅命中。重要的是，在小鼠CBF白血病模型中，这三种化合物中至少有一种降低了白血病负担，与阿糖胞苷具有相似的疗效和协同作用。这些发现可能导致针对CBF白血病的靶向治疗的发展。

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.