Dissection of Molecular Pathways Downstream of HOXA9 & Discovery of New Drug Trgt

对小鼠和人类的研究表明，主要的HOX基因可能整合了骨髓增生异常和白血病的活性。 几种不同的致癌蛋白和分子通路在高危人类骨髓增生异常综合征(MDS)和 促进其进展为急性髓系白血病(AML)。这项提案中总结的研究表明 HOXA9的上调诱导了多能骨髓干细胞的转化途径，最初导致 三系发育不良和染色体不稳定，最终导致具有复杂核型的高危AML和 无论接受何种治疗，结果都很差。目前还不可能描绘HOXA9下游的分子通路， 阻碍确定可作为设计小分子抑制剂的特定靶点的关键蛋白质的努力 MDS的治疗和防止进展为AML。像HOXA9这样的转录因子尚未被证明是 被小分子抑制的靶标，因此描绘关键的下游通路对于设计 有针对性的治疗。这一项目开发的新机遇是开发小抑制rna(Sirna)。 下调人类急性髓系白血病细胞株HOXA9水平的策略，因此下调细胞生物学后果 关键的HOXA9介导的通路是可以定义的。初步数据证明我们有能力“压低”HOXA9水平 降至人AML细胞系原有水平的10%以下。我们还表明，HOXA9的下调显著 抑制这些细胞的生长，提示HOXA9在维持改变的干细胞的增殖能力方面有作用 在积累了额外的基因后，会导致MDS，并最终促进白血病的转化 异常现象。我们的基本假设是，我们将在HOXA9中确定下游目标 MDS中造血细胞发育障碍和白血病转化为 急性髓系白血病，包括那些作为酶的功能，并可以有利地被小分子抑制。这个 这些靶点的识别将通过结合使用微阵列和 染色质免疫沉淀技术。HOXA9靶子的意义将通过“击倒”来验证 蛋白质水平与siRNA的关系，并评估功能抑制是否也抑制髓系细胞中异常细胞的生长 线型系统。一个积极的结果将提供原则上的证据，证明这些蛋白质是可用的合适的靶标。 可在项目1中快速测试的药物或小分子抑制剂的开发。

Studies in mice and humans suggest major HOX genes may integrate myelodysplastic and leukemogenic activities of several different oncogenic proteins and molecular pathways in high risk human myelodysplastic syndrome (MDS) and promote its progression to acute myeloid leukemia {AML). Studies summarized in this proposal suggest that upregulation of HOXA9 elicits a transforming pathway in pluripotent bone marrow stem cells, leading initially to trilineage dysplasia and chromosomal instability and culminating in high risk AML with complex karyotypes and very poor outcomes regardless of therapy. It has not been possible to delineate molecular pathways downstream of HOXA9, impeding efforts to identify key proteins that could be specific targets for the design of small molecule inhibitors for treatment of MDS and prevention of progression to AML. Transcription factors like HOXA9 have not been proven to be targets for inhibition by small molecules, so delineation of crucial downstream pathways is critical for the design of targeted therapies. The new opportunity to be exploited in this project is development of small inhibitory RNA (siRNA) strategies to "knock-down" HOXA9 levels in human AML cell lines, so cell biologic consequences of down-regulating crucial HOXA9-mediated pathways can be defined. Preliminary data attest to our ability to "knock-down" HOXA9 levels to less than 10% of preexisting levels in human AML cell lines. We also show that down-regulation of HOXA9 markedly retards growth of these cells, suggesting an effect of HOXA9 in maintaining proliferative capacity of altered stem cells that give rise to MDS and eventually contribute to leukemic transformation, after accumulation of additional genetic abnormalities. Our fundamental hypothesis is that we will identify downstream targets within the HOXA9 pathway that contribute to disordered hematopoietic cell development in MDS and leukemic transformation to AML, including those that function as enzymes and can be advantageously inhibited by small molecules. The identification of these targets will be addressed by a combination of gene expression analysis using microarrays and chromatin immunoprecipitation techniques. The significance of HOXA9 targets will be validated by "knocking-down" protein levels with siRNA and assessing whether function inhibition also retards aberrant cell growth in a myeloid cell line model system. A positive result would provide proof-of-principle that such proteins are suitable targets for available drugs or the development of small molecule inhibitors that could be rapidly tested in Project 1.