The role of RUNX1 in MLL-AF4 leukaemia

在过去的几十年里，我们对儿科急性淋巴细胞性白血病生物学的了解取得了进步，治疗方案的优化使存活率达到了85%。然而，MLL基因易位及其与不同配对基因融合所致的儿童白血病存活率较低。MLL-AF4融合蛋白引起的白血病是MLL重排白血病中最常见的一种，具有少量的协同突变和很短的潜伏期，这突显了MLL在白血病发生过程中的驱动作用。本项目的目标是阐明先前在实验室中发现的特定转录因子在MLL白血病发生中的作用。参与白血病发生的转录因子分为两类：(I)作为MLL-AF4融合蛋白下游靶点的转录因子；这些转录因子在MLL重排白血病中上调，并直接导致白血病发生；(Ii)在淋巴样细胞中正常表达的转录因子，但由于融合蛋白引起的染色质景观改变和表观遗传特征，其正常功能在白血病细胞中被取消。已识别的转录因子的结合模式将通过高通量方法进行研究，包括在含有MLL-AF4突变的扫描电子显微镜白血病细胞系中进行芯片测序。通过这种方式，我将调查在用小分子抑制剂治疗后是否会影响白血病的生长。此外，将通过在扫描电子显微镜细胞系中进行基因敲除实验来检验已识别的转录因子的作用，以确定对白血病生长的影响。此外，我将采用新开发的技术，包括CITE-SEQ和REAP-SEQ，它们将高通量单细胞RNA测序与细胞表面蛋白检测相结合。这将使我们能够检测不同的转录因子在原代MLL-AF4细胞中的表达，与正常B细胞和B细胞前体细胞相比，并将为MLL白血病的起源和疾病的分子生物学提供有价值的见解。

During the past decades advances in our understanding of the biology of paediatric acute lymphoblastic leukaemias and optimization of therapeutic protocols have led to survival rates up to 85%. However, childhood leukaemias caused by translocation of MLL (Mixed Lineage Leukaemia) gene and its fusion with different partner genes are characterized by poor survival rates. Leukaemias caused by MLL-AF4 fusion protein are the most frequent among the MLL rearranged leukaemias and are characterized by small number of cooperating mutations and a very short latency, which underlines the role of MLL as a driver in the process of leukaemogenesis.The goal of this project will be to elucidate the role of specific transcription factors, previously identified in the lab, to MLL leukaemogenesis. The transcription factors involved in leukaemogenesis fall into two classes: (i) transcription factors that are downstream targets of the MLL-AF4 fusion protein; these are upregulated in MLL rearranged leukaemias and contribute directly to leukaemogenesis; and (ii) transcription factors that are normally expressed in lymphoid cells, but their normal function is abrogated in leukaemia cells due to the altered chromatin landscape and epigenetic signatures caused by the fusion protein.The binding patterns of the identified transcription factors will be investigated by high-throughput methods including ChIP-sequencing in the SEM leukaemia cell line, which harbours the MLL-AF4 mutation. In this way I will investigate whether this is affected after treatment with small molecule inhibitors that abrogate leukaemic growth. In addition, the role of the identified transcription factors will be examined by performing knockdown experiments in the SEM cell line, in order to determine the effects on leukaemic growth. Furthermore, I will employ newly developed techniques, including CITE-Seq and REAP-Seq, that combine high-throughput single cell RNA-sequencing with cell surface protein detection. This will enable us to examine the expression of different transcription factors in primary MLL-AF4 cells, compared to normal B cells and B cell progenitors, and will provide valuable insight into the origin of MLL leukaemias and the molecular biology of the disease.