Discovering small molecules that overcome differentiation arrest in acute myeloid

描述（由申请人提供）：成人急性髓性白血病（AML）是一种毁灭性疾病，5年生存率仅为25%。AML缺乏新的治疗方法，目前的化疗标准在过去30年没有改变。AML治疗的一个成功案例是开发了促进白血病细胞成熟或分化的疗法。在一小部分（~10%）AML合并急性早幼粒细胞白血病（APL）患者中，全反式维甲酸（ATRA）和砷的分化治疗耐受性良好且非常有效，导致5年生存率接近80%。不幸的是，其余90%的急性髓性白血病患者无法进行分化治疗。同源盒蛋白HoxA9在早期造血过程中表达，对髓系细胞的正常发育至关重要。HoxA9的不适当表达已在大约70%的AML中被证实。此外，表达涉及MLL（混合谱系白血病）基因的融合癌蛋白的白血病亚群依赖于HoxA9的表达。这些观察结果使HoxA9及其下游靶点成为小分子探针抑制的有吸引力的候选者。白血病模型系统的不足和主要患者样本的有限可用性阻碍了研究。一种新的AML体外模型已经被开发出来，通过肿瘤蛋白HoxA9使原代小鼠髓系细胞处于未成熟状态。这些细胞允许识别生物学上相关的化合物，可以克服骨髓分化阻滞。可以识别出两种类型的分子：直接干扰由HoxA9建立的分化阻止机制的分子，以及能够以不依赖HoxA9的方式促进分化的分子。该检测细胞系是用内置的分化标记进行工程设计的，因为它表达的绿色荧光蛋白（GFP）来自仅在成熟细胞中有活性的启动子。因此，促进分化的化合物可以很容易地以高通量的方式通过分析细胞的绿色荧光来识别。二级和反筛分析将消除潜在的自身荧光化合物，并通过分析基因表达和细胞表面标记物表达的变化来确认对髓细胞分化的影响。急性髓系白血病促分化小分子的开发将是当前白血病化疗的重要进展。第1页/ 1

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) in adults is a devastating disease with a 5-year survival rate of only 25%. New treatments for AML are lacking, and the current standard of care for chemotherapy has not changed in the last thirty years. One success story in the treatment of AML has been the development of therapies which promote the maturation, or differentiation of the leukemic cells. In the small subset (~10%) of AML patients with acute promyelocytic leukemia (APL), differentiation therapy in the form of all-trans retinoic acid (ATRA) and arsenic are both well-tolerated and extremely effective, leading to 5-year survival rates approaching 80%. Unfortunately, differentiation therapy is not available for the remaining 90% of acute myeloid leukemia patients. The homeobox protein HoxA9 is expressed in early hematopoiesis and is critical to the normal development of cells along the myeloid lineage. The inappropriate expression of HoxA9 has been demonstrated in approximately 70% of AML. Furthermore, the subset of leukemias which express a fusion oncoprotein involving the MLL (mixed lineage leukemia) gene are dependent upon the expression of HoxA9. These observations make HoxA9 and its downstream targets attractive candidates for inhibition by small molecule probes. Research has been hindered by inadequate model systems of leukemia and the limited availability of primary patient samples. A novel in vitro model of AML has been developed whereby primary murine myeloid cells are arrested in an immature state by the oncoprotein HoxA9. These cells allow for the identification of biologically relevant compounds that can overcome myeloid differentiation arrest. Two types of molecules can be identified: those that directly interfere with the mechanism of differentiation arrest established by HoxA9, and those that are capable of promoting differentiation in a HoxA9-independent manner. The assay cell line is engineered with a built-in marker of differentiation as it expresses the green fluorescent protein (GFP) from a promoter which is active only in mature cells. Thus, compounds that promote differentiation can be readily identified in a high-throughput fashion by assaying cells for green fluorescence. Secondary and counterscreen assays will eliminate potentially autofluorescent compounds and will confirm the effect on myeloid differentiation by assaying for changes in gene expression and cell surface marker expression. The development of a small molecule capable of promoting differentiation in acute myeloid leukemia will be an important advancement in the current state of leukemia chemotherapy. Page 1 of 1 PUBLIC HEALTH RELEVANCE: Acute myeloid leukemia (AML) is a devastating disease and new treatments for AML are in desperate need. Traditional chemotherapy, which typically poisons rapidly dividing leukemia cells, is ultimately ineffective in 75% of cases. A novel model system of AML has been devised to identify novel compounds which trigger leukemic cells to resume the normal process of maturation, thereby losing their proliferative and leukemic potential. Page 1 of 1