Finding therapeutic targets in FLT3-ITD AML using a systems biology approach

白血病是一种血液细胞癌，当干细胞或未成熟的血液细胞受到一系列DNA突变的袭击时，就会出现白血病。结果是细胞开始激活不正常的基因，或者基因被改变产生异常蛋白质，或者根本没有蛋白质。如果需要这种蛋白质来开启或关闭其他基因，后果可能是毁灭性的。其原因是，在血细胞发育过程中，基因开启或关闭的精细平衡秩序现在被打乱了。在早期阶段，单个突变可能没有什么影响，许多看起来正常的人在他们的血细胞中已经携带了一些突变。然而，额外的突变会产生多米诺骨牌效应。首先，一些靶基因被突变去调控。这可能导致干细胞生长过度，但在其他方面都很正常，仍然可以形成正常的血细胞。随着时间的推移，会发生额外的变化，打破平衡，从一个细胞生长得有点过度，到血细胞发育停止，细胞变成恶性细胞。这些细胞不会发育成正常的血细胞，而是形成白血病细胞，不断生长，直到最终占据整个身体。急性髓性白血病（AML）是成人最常见的急性白血病。尽管支持治疗有所改善，但老年AML患者的预后通常仍然很差。人们早就知道，AML不能仅仅归类为一种疾病，而是高度异质性的，涉及不同的基因突变和高度可变的临床结果。FLT3-ITD突变是一种促进生长的突变，也是最具破坏性的突变之一。大约25%的病例会发生这种情况，它会产生一种无法关闭的持续活跃的蛋白质，这种蛋白质会告诉细胞无限期地生长。发生这种突变的临床预后是可怕的，以FLT3-ITD蛋白为靶点的药物治疗很快导致耐药和复发。Bonifer/Cockerill小组最近开展了一系列实验，强调了FLT3-ITD AML中基因调控是如何改变的，并偏离了正常细胞。这是由于现代技术可以同时观察许多基因。我们已经发现了一个可能对FLT3-ITD AML的发展和维持至关重要的基因网络。其中包括转录调节因子RUNX1和AP-1，它们控制FLT3-ITD aml特异性蛋白的异常表达。我们现在与Heidenreich实验室合作，他们开发了人类FLT3-ITD AML的体内模型，以及来自弗吉尼亚大学的John Bushweller实验室，他开发了直接针对RUNX1的新药。我们提出的工作将以我们的结果为基础，旨在(i)确定新的治疗靶点，（ii）了解不同药物对哪些基因有影响，（iii）在aml小鼠模型中使用优化的药物，为在临床试验中测试这些新分子做准备。在这项工作中，我们将使用抑制性RNA分子来阻断FLT3-ITD AML中异常表达的蛋白质的产生。我们将使用这种筛选来确定哪些异常表达的基因对这些AML细胞的生长至关重要。一旦我们确定了控制基因调控网络的基因和途径，我们将使用特定的试剂和化学抑制剂来阻断AML网络中的这些点，并阻断白血病的发展。这些将包括(a)特异性阻断dna结合转录调节因子RUNX1结合的药物，(b)作为AP-1转录调节因子家族所有成员的主要抑制因子的FOS蛋白的缩短版本，以及(3)临床批准的FLT3-ITD和MAPK信号抑制剂的组合，我们预测这将比使用单一药物的治疗更有效。

Leukaemia is a blood cell cancer that arises when stem cells or immature blood cells are hit by a series of mutations in their DNA. The consequence is that the cell starts to activate genes that are not normally active, or genes are altered to make abnormal proteins, or no protein at all. If such a protein is required to switch other genes on or off, the consequences can be devastating. The reason for this is that the finely balanced order in which genes are switched on or off during blood cell development is now disturbed. In the early stages, a single mutation may have little effect, and many apparently normal people already carry some mutations in their blood cells. However, additional mutations create a domino effect. First, some target genes are de-regulated by the mutations. This can lead to stem cells that grow more than they should, but are otherwise quite normal and still can form normal blood cells. Over time additional changes occur that tip the balance from a cell that grows a bit too much, to cells where blood cell development grinds to a halt and the cells become malignant. Such cells do not develop into normal blood cells, but form leukemic cells that keep growing and growing until they finally take over the body. Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults. Despite improvements in supportive care, outcome typically remains poor for older AML patients. It has long been known that AML cannot be classified as just one disease but is highly heterogeneous, involving different genetic mutations and highly variable clinical outcomes. The FLT3-ITD mutation is a growth-promoting mutation, and one of the mutations that has the most devastating effects. It occurs in about 25% of all cases, and generates a continuously active protein that cannot be switched off, which tells cells to grow indefinitely. The clinical prognosis of having such a mutation is dire, and treatment with drugs targeting the FLT3-ITD protein soon results in the development of drug resistance and relapse. The Bonifer/Cockerill group has recently embarked on a series of experiments which highlighted how gene regulation is altered in AML with FLT3-ITD and deviates from normal cells. This was made possible by modern technology that looks at many genes simultaneously. We have uncovered a network of genes which are likely to be essential for the development and maintenance of FLT3-ITD AML. These include the transcriptional regulators RUNX1 and AP-1 which control the abnormal expression of FLT3-ITD AML-specific proteins. We have now teamed up with the Heidenreich lab who developed an in vivo model of human FLT3-ITD AML and the lab of John Bushweller from the University of Virginia who has developed novel drugs that target RUNX1 directly. Our proposed work will build on our results and is designed to (i) identify new targets for therapy, (ii) understand which genes are affected by different drug and (iii) use optimized drugs in mouse models of AMLs to prepare the stage to test these novel molecules in a clinical trial. In this work we will use inhibitory RNA molecules to block the production of proteins that are aberrantly expressed in FLT3-ITD AML. We will use this screen to identify which of the abnormally expressed genes are vital to the growth of these AML cells. Once we have identified genes and pathways that control the gene regulatory network we will use specific reagents and chemical inhibitors to block these points in the AML network, and block leukaemia development. These will include (a) a drug that than specifically block the binding of the DNA-binding transcriptional regulator RUNX1, (b) A shortened version of the FOS protein which acts as a dominantly acting repressor of all members of the AP-1 family of transcriptional regulators, and (3) combinations of clinically approved inhibitors of FLT3-ITD and MAPK signalling, which we predict will be more effective than therapies using single agents.